Chromatography of Alkaloids

- JOURNAL OF CHROMATOGRAPHY LIBRARY VOLUME 23B chromatography of alkaloids part B: gas-liquid chromatography and high...

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JOURNAL OF CHROMATOGRAPHY LIBRARY VOLUME 23B

chromatography of alkaloids part B: gas-liquid chromatography and high-performance liquid chromatography

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JOURNAL OF CHROMATOGRAPHY L!6RARY

- volume 23B

chromatograph y of alkaloids part B: gas-liquid chromatography and high-performance liquid chromatography R. Verpoorte and

A. Baerheim Svendsen Department of Pharmacognosy, State University Leyden, Leyden, The Netherlands

E LSEVl ER Amsterdam - Oxford - New York

- Tokyo

1984

ELSEVIER SCIENCE PUBLISHERS B.V. Molenwerf 1 P.O. Box 21 1,1000 AE Amsterdam, The Netherlands

Distributors for the United States and Canada: ELSEVIER SCIENCE PUBLISHING COMPANY INC.

52, Vanderbilt Avenue New York, NY 10017

Library of Coagreir Cataloging in Publication Data (Revised f o r part B)

Baerheim-Svendsen, A. Chromatography of alkalcias.

(Journal of ChrODIatOgrapkii l i b r v y ; v. 23-23B) Includes b i b l i o g r y h i c a l references and indexes.

--

p t . B. Contents: p t . A. Thin-layer chromatography Oae-liquid chromatography a n d high-perfomence l i q u i d chromatography. 1. Alkaloids--Analysis. 2. Chromatographic analysis. I. Verpoorte, R. 11. T i t l e . 111. Seriee. QD421.Bl25 1983 547.7'2C46 82-20976 ISBN 0-444-42145-9 (U.S. : pt. A ) ISBN 0-444-42265-X (U.S. : pt. B)

ISBN 044442265-X (Vol. 238) ISBN 044441616-1 (Series) 0 Elsevier Science Publishers B.V.. 1984 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science Publishers B.V./Science & Technology Division, P.O. Box 330,1000 AH Amsterdam, The Netherlands. Special regulations for readers in the USA - This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside of the USA, should be referred to the publisher. Printed in The Netherlands

CONTENTS Journal o f Chromatography L i b r a r y

.................

..

............

vii

GAS-LIQUID CHROMATOGRAPHY

........................................................................ Chapter 1. Packed columns ...................................................... Chapter 2 . C a p i l l a r y columns ...................................................

Preface

5

I General p a r t

Chapter 3

. Derivatization o f

a l k a l o i d s f o r gas chromatography

..................

9 15 21

I 1 Special p a r t

. P y r r o l i z i d i n e a l k a l o i d s ............................................. . P y r i d i n e a l k a l o i d s .................................................. Chapter 6 . P i p e r i d i n e a l k a l o i d s ................................................ Chapter 7 . Q u i n o l i z i d i n e a l k a l o i d s ............................................. Chapter 8 . Tropine a l k a l o i d s ................................................... Chapter 9 . Pseudotropine a l k a l o i d s .............................................

Chapter 4

Chapter 5

. .................................................. Chapter 11. Acronychia a1 kaloids ................................................ Chapter 12 . Cactus a l k a l o i d s .................................................... Chapter13.Ephedra a l k a l o i d s ................................................... Chapter 14 . Opium a1 k a l o i ds ..................................................... Chapter 15 . Aporphine a l k a l o i d s ................................................. Chapter 16 . Isoquinoline r e l a t e d a l k a l o i d s . , .................................... Chapter 17 . Terpenoid i n d o l e a l k a l o i d s and simple i n d o l e a l k a l o i d s .............. Chs.pter 18 . Ergot a l k a l o i d s ..................................................... Chapter 19 . s o i ~ ~a l m k a l o i d s ................................................... Chapter 20 . Xanthine a l k a l o i d s .................................................. Chapter 21 . Cephalotaxus a l k a l o i d s . , ............................................ Chapter22 . Imidazole a l k a l o i d s ................................................. Chapter 10 Cinchona a l k a l o i d s

29 33 53 55 61 73 87 93 97 103

111 147 151 155 173 185 187 213 217

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I General p a r t Chapter 1 General aspects o f HPLC o f a l k a l o i d s

. .

Chapter 2 HPLC analysis o f various a l k a l o i d s

................................ ..................................

223 235

I 1 Special p a r t

. P y r r o l i d i n e . p y r r o l i z i d i n e . p y r i dine. p i p e r i d i n e and quinol i z i d i n e a l k a l o i d s ........................................................... Chapter 4 . Tropane a l k a l o i d s : 4.1 Tropine a l k a l o i d s ............................ 4.2 Pseudotropine a l k a l o i d s ...................... Chapter 5 . Q u i n o l i n e a l k a l o i d s : Cinchona a l k a l o i d s ............................. Chapter 6 . Phenylethylamines and i s o q u i n o l i n e a l k a l o i d s ........................

Chapter 3

241 249 260 269 287

..................................................... .............. Chapter 9 . Ergot a l k a l o i d s ..................................................... Chapter 10. Steroidal a l k a l o i d s ................................................ Chapter 11 . Xanthine a l k a l o i d s ................................................. Chapter 12 . Diterpene a l k a l o i d s ................................................ Chapter 13 . Colchicine and r e l a t e d a l k a l o i d s ................................... Chapter 14 . Imidazole a l k a l o i d s ................................................ Chapter 15 . Quaternary ammonium compounds ...................................... Appendix .......................................................................... Subject index ..................................................................... Compound index .................................................................... .

Chapter 7 Opium a l k a l o i d s

Chapter 8 . Terpenoid i n d o l e a l k a l o i d s and simple i n d o l e a l k a l o i d s

29 7 331 357 381 387 415 417 421 425 432 437 443

JOURNAL OF CHROMATOGRAPHY LIBRARY A Series of Books Devoted to Chromatographic and Electrophoretic Techniques and their Applications Although complementary t o the Journal o f Chromatography, each volume in the library series is an important and independent contribution in the field of chromatography and electrophoresis. The library contains no material reprinted from the journal itself. Volume 1

Chromatography of Antibiotics (see also Volume 2 6 ) by G.H. Wagman and M.J. Weinstein

Volume 2

Extraction Chromatography edited by T. Braun and G. Ghersini

Volume 3

Liquid Column Chromatography. A Survey of Modern Techniques and Applications edited by Z. Deyl, K. Macek and J. Janak

Volume 4

Detectors in Gas Chromatography by J. SevEik

Volume 5

Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods (see also Volume 27) by N.A. Parris

Volume 6

Isotachophoresis. Theory, Instrumentation and Applications by F.M. Everaerts, J.L. Beckers and Th.P.E.M. Verheggen

Volume 7

Chemical Derivatization in Liquid Chromatography by J . F . Lawrence and R.W. Frei

Volume 8

Chromatography of Steroids by E. Heftmann

Volume 9

HPTLC - High Performance Thin-Layer Chromatography edited by A. Zlatkis and R.E. Kaiser

Volume 1 0

Gas Chromatography of Polymers

by V.G. Berezkin, V.R. Alishoyev and I.B. Nemirovskaya Volume 11

Liquid Chromatography Detectors by R.P.W. Scott

Volume 1 2

Affinity Chromatography by J. Turkova

Volume 1 3

Instrumentation for High-Performance Liquid Chromatography edited by J.F.K. Huber

Volume 1 4

Radiochromatography. The Chromatography and Electrophoresis of Radiolabelled Compounds by T.R. Roberts

Volume 15

Antibiotics. Isolation, Separation and Purification edited by M.J. Weinstein and G.H. Wagman

Volume 1 6

Porous Silica. Its Properties and Use as Support in Column Liquid Chromatography by K.K. Unger

Volume 1 7

75 Years of Chromatography - A Historical Dialogue edited by L.S. Ettre and A. Zlatkis

Volume 18A Electrophoresis. A Survey of Techniques and Applications. Part A: Techniques edited by Z. Deyl Volume 1 8 B Electrophoresis. A Survey of Techniques and Applications. Part B: Applications edited by Z. Deyl Volume 19

Chemical Derivatization in Gas Chromatography by J. Drozd

Volume 20

Electron Capture. Theory and Practice in Chromatography edited by A. Zlatkis and C.F. Poole

Volume 2 1

Environmental Problem Solving using Gas and Liquid Chromatography by R.L. Grob and M.A. Kaiser

Volume 22A Chromatography. Fundamentals and Applications of Chromatographic and Electrophoretic Methods. Part A: Fundamentals edited by E. Heftmann Volume 22B

Chromatography. Fundamentals and Applications of Chromatographic and Electrophoretic Methods. Part B: Applications edited by E. Heftmann

Volume 23A Chromatography of Alkaloids. Part A: Thin-Layer Chromatography by A. Baerheim Svendsen and R. Verpoorte Volume 23B Chromatography of Alkaloids. Part B: Gas-Liquid High-Performance Liquid Chromatography by R. Verpoorte and A. Baerheim Svendsen

Chromatography and

Volume 24

Chemical Methods in Gas Chromatography by V.G. Berezkin

Volume 25

Modern Liquid Chromatography of Macromolecules by B.G. Belenkii and L.Z. Vilenchik

Volume 26

Chromatography of Antibiotics Second, Completely Revised Edition by G.H. Wagman and M.J. Weinstein

Volume 27

Instrumental Liquid Chromatography. A Practical Manual o n High-Performance Liquid Chromatographic Methods Second, Completely Revised Edition by N.A. Parris

Volume 28

Microcolumn High-Performance Liquid Chromatography by P. Kucera

Volume 29

Quantitative Column Liquid Chromatography. A Survey of Chemometric Methods by S.T.Balke

GAS-LIQUID

CHROMATOGRAPHY

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CONTENTS Preface

.........

...............

5

I. GENERAL PART

........................ ......................

Chapter 1. Packed columns Chapter 2. C a p i l l a r y columns

Chapter 3. D e r i v a t i z a t i o n o f a l k a l o i d s f o r gas chromatography

......

9 15 21

11. SPECIAL PART 11.1. P y r r o l i z i d i n e , p y r i d i n e , p i p e r i d i n e and q u i n o l i z i d i n e a l k a l o i d s

................... .....................

Chapter 4. P y r r o l i z i d i n e a l k a l o i d s Chapter 5. Pyridine a l k a l o i d s

.................... Q u i n o l i z i d i n e a1 k a l o i d s . . . . . . . . . . . . . . . . . . .

29 33

Chapter 6. P i p e r i d i n e a l k a l o i d s

53

Chapter 7.

55

11.2. Tropane a l k a l o i d s Chapter 8. Tropine a l k a l o i d s

...................... ...................

Chapter 9. Pseudotropine a l k a l o i d s 11.3. Q u i n o l i n e a l k a l o i d s Chapter 10. Cinchona a l k a l o i d s

.....................

Chapter 11. Acronychia a1 k a l o i d s

....................

61 73 87 93

11.4. Phenylethylamine and i s o q u i n o l i n e a l k a l o i d s

...................... ..................... Opium a l k a l o i d s . . . . . . . . . . . . . . . . . . . . . . Aporphine a l k a l o i d s . . . . . . . . . . . . . . . . . . . . Isoquinoline r e l a t e d a l k a l o i d s . . . . . . . . . . . . . . .

Chapter 12. Cactus a l k a l o i d s Chapter 13. Ephedra a1 k a l o i ds

97 103

Chapter 14.

111 147

Chapter 15. Chapter 16.

11.5. I n d o l e a l k a l o i d s

... ......................

15 1

Chapter 17. Terpenoid i n d o l e a l k a l o i d s and simple i n d o l e a l k a l o i d s

155

Chapter 18. Ergot a l k a l o i d s

173

11.6. S t e r o i d a l a l k a l o i d s ( G l y c o a l k a l o i d s ) Chapter 19. soianum a l k a l o i d s

.....................

185

11.7. Xanthine a l k a l o i d s Chapter 20. Xanthine a1 k a l o i d s

.....................

187

11.8. Miscellaneous a l k a l o i d s Chapter 21. Cephalotaxus a l k a l o i d s Chapter 22. Imidazole a l k a l o i d s

...................

....................

213 217

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6

PREFACE Most n a t u r a l l y o c c u r r i n g a l k a l o i d s are f a i r l y high-molecular-weight compounds. As such they are u s u a l l y o n l y s l i g h t l y v o l a t i l e o r n o n - v o l a t i l e . The a p p l i c a t i o n o f gas chromatography t o the a n a l y s i s o f a l k a l o i d s i s , therefore, l i m i t e d . When working with compounds o f h i g h molecular weight and low v o l a t i l i t y i n gas chromatography, i t i s o f t e n necessary t o increase t h e i r v o l a t i l i t y o r t o reduce t h e i r p o l a r i t y by converting them t o special derivatives. Usually i t i s a l s o necessary t o work w i t h gas chromatographic column w i t h a low percentage o f s t a t i o n a r y phase, i n order t o be able t o operate a t r e l a t i v e l y moderate column temperatures 1

.

The i n j e c t i o n o f an a l k a l o i d o r a mixture o f a l k a l o i d s i n t o t h e i n j e c t i o n p o r t o f the gas chromatograph may be o f v i t a l importance f o r the f u r t h e r course o f the analysis. One has t o pay a t t e n t i o n t o the f a c t t h a t the substance t o be analyzed may be i n many cases degraded during the analysis due t o c a t a l y t i c e f f e c t s o f t h e metal p a r t s o f the gas chromatograph, e s p e c i a l l y t o the i n j e c t i o n port, where the temperature has t o be r e l a t i v e l y h i g h t o o b t a i n an i n s t a n t and complete evaporation o f the compounds. The h i g h temperature o f the i n j e c t i o n p o r t r e q u i r e d f o r the evaporation o f the a l k a l o i d s may sometimes l e a d t o decomposition. such as dehydration, h y d r o l y s i s o r t r a n s e s t e r i f i c a t i o n . Atropine can under c e r t a i n conditions be dedydrated t o apoatropine. The degree o f dehydration has been found t o be associated w i t h the amount o f glass wool on t h e t o p o f the column materi a l . Diacetylmorphine i s e l u t e d as a sharp w e l l - d e f i n e d peak when chromatographed alone. I n mixtures w i t h codeine, morphine o r o t h e r phenolic o r a l c o h o l i c substances t r a n s e s t e r i f i c a t i o n s take place i n the i n j e c t i o n p o r t , g i v i n g r i s e t o several new e s t e r s not.present i n t h e o r i g i n a l solution. 6-0-Acetylmorphine gives peaks o f morphine, 6-0-acetylmorphine and d i acetylmorphine. 3-0-Acetylmorphine 2 o r no decomposition

.

i s more s t a b l e and may be gas chromatographed w i t h l i t t l e

To prevent an undesirable degradation o f the compounds t o be analyzed, glass columns have mostly been used f o r gas chromatography o f a l k a l o i d s because they a r e i n d i f f e r e n t t o the compounds. Possible c a t a l y t i c decomposition o f s e n s i t i v e compounds and adsorption phenomena caused by metal columns, e . g . copper, aluminium and s t a i n l e s s s t e e l , may. however, be e l i m i n a t e d i n some cases by a simple coating o f the t u b i n g m a t e r i a l w i t h the s t a t i o n a r y phase used i n the analysis. The decomposition and adsorption phenomena d i ~ a p p e a r ~ ' Codeine ~. and 4 noscapine3 and ephedrine were successfully gas chromatographed on coated, packed metal c o l umns w i t h o u t decomposition o r adsorption. REFERENCES

1 E.C. Horning, E.A. M o s c a t e l l i and C.C. Sweeley, Chem. rnd. (London), (1959) 751. 2 E. Brochmann-Hanssen and A. Baerheim Svendsen, J . P h a n n . sci., 5 1 (1962) 1095. 3 J.E. Arnold and H.t1. Fales. J . cas Chromatop.. 4 (1965) 131. 4 A.M.J.A. Duchateau and A. Baerheim Svendsen, Pharm. weekbl., 107 (1972) 377.

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1. GENERAL PART

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9

Chapter I

PACKED COLUMNS

............................................... ........... ...............

1.1. Deactivation o f s o l i d support 1.1.1. Deactivation o f s o l i d support by a c i d and a l k a l i n e washing 1.1.2. Deactivation o f s o l i d support by chemical d e a c t i v a t i o n 1.1.3. Deactivation o f s o l i d support by chemical bonding o f s t a t i o n a r y phase. 1.1.4. Deactivation o f s o l i d support by precoating w i t h small amounts o f a p o l a r s t a t i o n a r y phase 1.2. Coating o f s o l i d support w i t h s t a t i o n a r y phase 1.3. References

............................................... .............................. ..................................................................

10

10 10 11 13 13 14

A successful gas chromatography depends t o a g r e a t e x t e n t upon the q u a l i t y o f the columns used. For packed columns Roman e t a l . ' showed t h a t the q u a l i t y o f such columns i s p r o p o r t i o n a l t o the care bestowed upon the preparation procedure. I n comparing some commercially a v a i l able s o l i d supports the authors found t h a t poor gas chromatographic performance was o f t e n caused by adsorptive s i t e s on the surface o f the s o l i d support, e.g. by incomplete deactivat i o n o f the support. However, the q u a l i t y could be improved by more c a r e f u l , e v e n t u a l l y r e peated, d e a c t i v a t i o n o f the support.

Most authors p r e f e r t o make t h e i r columns themselves. I n the l i t e r a t u r e a number o f recommendations f o r preparation o f packed columns f o r h i g h b o i l i n g , s l i g h t l y v o l a t i l e compounds are given. Most o f them are based on empirical observations, made when performing gas chromatography t o solve s p e c i f i c a n a l y t i c a l problems. When s a t i s f a c t o r y r e s u l t s were achieved, no need f o r f u r t h e r evaluation o f the column was found t o be necessary, and the procedure used was t h e r e f o r e assumed t o be g e n e r a l l y applicable. When adopted by others, t h e y became gradu a l l y transformed i n t o some k i n d o f magic! For gas chromatography o f a l k a l o i d s low load packed columns a r e u s u a l l y used. I n such cases " t a i l i n g " , caused by a c t i v e adsorptive s i t e s , i s o f t e n observed. With g a s - l i q u i d chromatography a separation i s achieved by the d i f f e r e n c e s i n the p a r t i t i o n c o e f f i c i e n t s o f the various compounds between the gaseous mobile phase and t h e s t a t i o n a r y l i q u i d phase. However, i n g a s - l i q u i d chromatography w i t h low l o a d packed columns, t h e g a s - l i q u i d p a r t i t i o n e q u i l i b r i u m i s influenced by the p r o p e r t i e s o f t h e s o l i d support, because o f a c t i v e adsorptive s i t e s on the surface o f the support, and inhomogeneous c o a t i n g o f the support, which leaves p a r t s o f t h e support uncoated. I n both cases adsorption o f the solutes t o the support m a t e r i a l can take place. The e f f e c t i s e s p e c i a l l y troublesome w i t h small sample sizes. Various techniques have been developed t o d e a c t i v a t e the a c t i v e s i t e s on the s o l i d support as w e l l as t o assure a homogeneous coating. The d e a c t i v a t i o n has mostly been achieved by a c i d and a l k a l i n e washing, o f t e n followed by chemical d e a c t i v a t i o n , l a s t l y by precoating o f i t w i t h a very small amount o f a p o l a r s t a t i o n a r y phase.

R8lenne.r p. 14

10 1.1. Deactivation o f s o l i d support

1.1.1. D e a c t i v a t i o n of s o l i d s u p p o r t by a c i d and a l k a l i n e washing

The methods which have mostly been used t o deactivate the a c t i v e adsorptive s i t e s on s o l i d supports o f diatomaceous e a r t h type include a c i d washing and a l k a l i n e washing. Acid washing o f such support material was recomnended by James and Martin2. They suspected m e t a l l i c oxides t o be a t l e a s t p a r t l y responsible f o r the adsorptive properties. Washing w i t h concentrated hydrochloric a c i d followed by r i n s i n g w i t h water u n t i l n e u t r a l , was intended t o remove these m e t a l l i c oxides. Several o t h e r authors have c l e a r l y shown t h a t t h e a c i d washing i s an importa n t step p r i o r t o s i l a n i z a t i o n . I n some cases an a l k a l i n e washing has been a p p l i e d i n a d d i t i o n t o the a c i d one, as a pretreatment f o r s i l a n i z a t i o n . The combination was thought t o be more e f f e c t i v e f o r removing amphoteric ions, such as aluminium, than a c i d washing alone. Holmes and Stack 3 prepared packing material w i t h low adsorptive properties i n t h i s way, so a l s o Brochmann-Hanssen and Baerheim Svendsen f o r t h e i r analysis o f barbiturates, sympathomimetic amines and a1 k a l ~ i d s ~ - ~ . Comparative studies t o i n v e s t i g a t e the influence o f a c i d and a l k a l i n e washing on a s o l i d support, such as Chromosorb W, p r i o r t o s i l a n i z a t i o n and coating w i t h the s t a t i o n a r y phase by a) a c i d washing, b ) a c i d washing followed by a l k a l i n e washing and c ) a c i d washing followed by a l k a l i n e washing and then again a c i d washing, were c a r r i e d o u t by Meilink7. He recomnended the f o l l o w i n g procedure t o achieve optimum deactivation: Suspend Chromosorb W i n hydrochloric a c i d (25 X), f i l t e r o f f a f t e r two days by suction and r i n s e the Chromosorb w i t h d i s t i l l e d water u n t i l a l l a c i d i s removed. Remove most o f the water by washing with methanol, f i l t e r and d r y the product i n a r o t a r y evaporator under reduced pressure a t 90°C. Suspend the a c i d washed m a t e r i a l i n 1 mole per l i t e r KDH i n methanol. F i l t e r o f f a f t e r 15 hours and r i n s e w i t h methanol u n t i l f r e e o f KOH. Dry as described above. Treat the a c i d and a l k a l i washed material once more with hydrochloric a c i d as described above, r i n s e i t w i t h water and dry i t as described above. 1.1.2. D e a c t i v a . t i o n of s o l i d s u p p o r t b y chemical d e a c t i v a t i o n

An a c i d and base washing o f the s o l i d support i s u s u a l l y followed by a chemical deactivat i o n . mostly i n t h e form o f s i l a n i z a t i o n . Horning e t a1.’ used gaseous dimethyl d i c h l o r o s i l a n e (DMDCS) f o r t h i s purpose on t h e a c i d washed support. Horning e t a1.’ emphasized t h a t a c i d washing was essential. Bohemen e t a1.l’ pointed out the r i s k t h a t DMDCS s i l a n i z a t i o n might leave a c t i v e c h l o r i n e groups behind, which i n t h e i r t u r n could be converted t o hydroxyl groups when brought i n t o contact w i t h water. This problem w i l l n o t be encountered i f hexamethyldisilazane (HMDS) i s used as s i l a n i z i n g reagent. According t o Bohemen e t a1.l’ a c i d prewashing i s not necessary f o r HMDS s i l a n i z a t i o n . Brochmann-Hanssen and Baerheim S v e n d ~ e n ~ obtained ’~’~ w e l l deactivated support on treatment w i t h HMDS; n e i t h e r a c i d nor base washing were, however, omitted. Sawyer and Barr” found HMDS s i l a n i z a t i o n very e f f e c t i v e f o r d e a c t i v a t l o n purposes, although a s l i g h t residual adsorpt i v e a c t i v i t y was l e f t . Compared w i t h DMDCS s i l a n i z a t i o n , a disadvantage o f HMDS should be mentioned. McMartin and Street“ found t h a t a support which had been w e l l deactivated by HMDS was r e a c t i v a t e d a t

11 temperatures above 26OoC. A l k a l i n e treatment o f the s o l i d support d i r e c t l y p r i o r t o s i l a n i z a t i o n w i t h DMDCS seems n o t t o be advantageous. The presence o f f r e e hydroxyl groups on t h e support, obtained by a c i d washings, seems t o be essential f o r the s i l a n i z a t i o n r e a c t i o n w i t h DMDCS. According t o McMartin and Street" the a c i d washing can be omitted when DMDCS s i l a n i z a t i o n i s performed on "wet" o r "damp" s o l i d support. They assumed t h a t hydrochloric acid, released i n the react i o n o f DMDCS w i t h water, replaces the a c i d washing. They also supposed t h a t p o l y n e r i z a t i o n o f DMDCS takes place i n t h e presence o f water, r e s u l t i n g i n a chemically bonded polysiloxane l a y e r on the support. Ifhigher concentrations o f water i n the support than about 5 X are used, excessive formation o f gaseous hydrochloric a c i d w i l l be t h e r e s u l t . M e i l i n k 7 preferred the procedure i n v o l v i n g acid, base and a c i d washing t o o b t a i n a best possible deactivation o f the support. Recommended procedure f o r s i l a n i z a t i o n w i t h DMDCS: Suspend the acid, base and again a c i d washed s o l i d support i n DMDCS i n toluene (5 % v/v), t r e a t the suspension i n an u l t r a s o n i c bath f o r 5 minutes and f i l t e r o f f the s o l v e n t a f t e r 24 hours. Wash w i t h d r i e d toluene and d r i e d methanol under exclusion o f water and d r y t h e support material i n a r o t a r y evaporator under reduced pressure a t 90°C. Bohemen e t a1.l'

recommended the f o l l o w i n g procedure f o r s i l a n i z a t i o n w i t h HMDS:

Dry the support under vacuum a t 15OoC. Cover 25 g o f t h i s sample, while s t i l l warm. w i t h a mixture o f 80 m l o f l i g h t petroleum (b.p. 60-80°C) and 15 m l hexamethyldisilazane. Heat the mixture on a steam-bath and r e f l u x f o r 1 hr. Use a d r y i n g tube o f calcium sulphate a t the condenser e x i t . A f t e r r e f l u x i n g , add 2 m l o f n-propanol; t h i s helps m a t e r i a l l y by w e t t i n g t h e support, and a1 though i t reacts w i t h unchanged hexamethyldisilazane t o form SiMejDPr,

this

i n t u r n reacts with hydroxyl groups i n the same way as t h e parent silazane. A f t e r 30 hr. heat the mixture again and r e f l u x f o r several hours. Wash the support w i t h l i g h t petroleum (2 x 50 ml), then n-propanol (1 x 50 m l ) , and then again w i t h l i g h t petroleum ( 2 x 50 m l ) . F i n a l l y , f i l t e r o f f the support and dry i t f o r 2 hr. on a steam-bath i n an atmosphere o f n i t r o g e n . 1.1.3.

D e a c t i v a t i o n of solid s u p p o r t b y chemical bonding of s t a t i o n a r y p h a s e

A c t i v e groups on t h e support can also react w i t h other than s i l a n i z i n g agents. I n t h a t way small amounts o f s t a t i o n a r y phases can be chemically bonded t o t h e s o l i d support as esters o f the ( 4 1 - 0 4 ) type. According t o Grushka and Kikta13 such esters are l i a b l e t o hydrolysis. Chemical bonds o f the (-Si-0-Si-R)

type. a r i s i n g from s i l a n i z a t i o n w i t h organosilanes, a r e

more stable. The main advantage o f gas chromatographic packings w i t h chemically bonded s t a t i o n a r y phases, compared t o p h y s i c a l l y coated ones, i s t h e i r greater thermal s t a b i l i t y . The 14 upper temperature l i m i t l i e s about 80-90' above t h a t o f the corresponding nonbonded ones Two types o f reaction procedures have been described. I n t h e f i r s t one t h e coupling react i o n i s brought about a t an elevated temperature. Aue e t a l ? s t a t e d t h a t polyethylene

.

glycol 20M (PEG 20M) could n o t be f u l l y washed o f f a support w i t h methanol and methylene c h l o r i d e a f t e r heating t o 280°C. The remaining PEG 20M l a y e r was t o o t h i n t o be measured with a n a l y t i c a l combustion techniques. Hastings and Aue16 demonstrated t h a t chemical bonding o f PEG t o support m a t e r i a l leads t o w e l l deactivated products. These "polymer deactivated" products proved t o be good supports when they were p h y s i c a l l y coated w l t h conventional s t a t i o n -

Reference8p. 14

12 a r y phases. The s t a t i o n a r y phase t h a t was used, then determined t h e c h a r a c t e r i s t i c s o f the packing, and n o t t h e u l t r a - t h i n , non-extractable PEG-film. Moseman17 and W i n t e r l i n and Moseman18 used PEG deactivated supports coated with qV-210 f o r gas chromatography o f pesticides. They s t a t e d t h a t the PEG deactivated support was superior t o non-deactivated materials; no comparison was, however, made w i t h s i l a n i z e d supports. The method o f chemical bonding was modified by Daniewski and Aue". They r e f l u x e d the support i n a s o l u t i o n o f PEG instead o f dry heating the PEG coated support. The coupling r e a c t i o n can a l s o be r e a l i z e d by using a chlorosilane as an intermediate t o "activate" the support surface. Chlorosilanes r e a c t e a s i l y w i t h t h e a c t i v e s i t e s on the supp o r t . When t r i c h l o r o m e t h y l s i l a n e i s used, always a t l e a s t one a c t i v e c h l o r i n e gmup i s l e f t , 14 which i n i t s t u r n can r e a c t w i t h alcohols. such as polyethylene glycols. I n t h i s way, Mori prepared Chromosorb W (AW) w i t h chemically bonded PEG, r e s u l t i n g i n a loading o f 4.2 % with PEG 20M and o f 2.0 % w i t h PEG 3000. Both thermally and chlorosi lane-mediated PEG-bonded supports can be used as gas chromatographic packings without f u r t h e r coating14s17. Because the chemically bonded PEG molecules are thought t o be arranged on the support surface l i k e " b r i s t l e s o f a brush", Mori14 concluded t h a t the r a t e o f mass t r a n s f e r should be increased, making chemically bonded s t a t i o n a r y phases very s u i t a b l e f o r gas chromatographic separations. Recently Street e t a1." acylated diatomaceous e a r t h w i t h benzoyl c h l o r i d e i n p y r i d i n e as a pretreatment f o r coating. They reported t h a t a marked reduction i n adsorption could be obtained, enabling p o l a r compounds, such as morphine, t o be gas chmmatographed i n nanogram amounts without d e r i v a t i z a t i o n . A 1000-fold improvement i n chromatographic c a p a b i l i t y could be obtained, compared t o the best conventional c m e r c i a l packing. Recomnended procedure f o r thermal bonding o f PEG 20M: Coat acid-base-acid washed Chranosorb W w i t h 5 % (w/w) PEG 20M using t h e f i l t r a t i o n technique o f Horning e t a1.8 and f i l l a glass gas chromatographic column w i t h the m a t e r i a l . Flush the column w i t h nitrogen (60 ml/min) f o r 6 hours and heat a t 28OoC f o r 15 hours w i t h a n i t r o gen flow o f 3.5 ml/min. Empty the column and r i n s e t h e packing material thoroughly w i t h methanol and methylene c h l o r i d e respectively, followed by e x t r a c t i o n with methylene c h l o r i d e f o r 6 hours i n a Soxhlet apparatus. Dry the support i n a r o t a r y evaporator a t 6OoC and coat i t w i t h t h e s t a t i o n a r y phase t h a t was chosen. Recomnded procedure f o r chlorosilane-mediated bonding o f PEG 4000: Suspend 18 g acid-base-acid washed Chromosorb W i n a mixture o f 50 m l d r i e d toluene and 25 m l t e t r a c h l o r o s i l a n e (TCS), t r e a t i t f o r 5 min. i n an u l t r a s o n i c bath t o remove a i r , f i l t e r o f f t h e s o l u t i o n a f t e r 4 hours a t room temperature, r i n s e w i t h d r i e d toluene under exclusion o f water t o remove the TCS completely. While i t i s s t i l l excluded f r o m atmospheric moisture, suspend the TCS t r e a t e d support i n a s o l u t i o n o f 8 g PEG 4000 i n 75 m l d r i e d toluene f o r 48 hours a t 5OoC. F i l t e r the PEG s o l u t i o n o f f and r i n s e the mass with toluene and methylene c h l o r i d e successively, followed by e x t r a c t i o n w i t h methylene c h l o r i d e i n a Soxhlet apparatus. Dry t h e mass i n a r o t a r y evaporator a t 6OoC.

13 1.1.4.

D e a c t i v a t i o n of s o l i d support b y p r e c v a t i n g w i t h small amounts of a polar s t a t i o n a r y phase

To diminish any eventual r e s i d u a l adsorptive a c t i v i t y o f the support t h a t may s t i l l be present a f t e r deactivation by a c i d and a l k a l i n e washing, and by chemical procedures. Bohemen e t al.lOintroduced a precoating w i t h 0.1 X PEG 400. The PEG molecules a r e thought t o be adsorbed t i g h t l y t o t h e r e s i d u a l a c t i v e s i t e s o f the support. This precoating procedure was a l so used by Brochmann-Hanssen and Baerheiq Svendsen' i n t h e i r gas chromatographic studies on a l k a l o i d s . They p r e f e r r e d t o apply PEG 9000 0.1 X (w/w). The higher molecular weight was used as higher temperatures were employed. Kabot and Ettre'l recomnended t o apply the main stat i o n a r y phase by means o f a solvent " i n which the precoated p o l a r phase (PEG) i s insoluble". Lipsky and Landowne'O dissolved both t h e p o l a r phase (PEG) and the non p o l a r phase i n the same solvent and c a r r i e d o u t both precoating and coating simultaneously. They found t h a t t h i s procedure reduced o r eliminated t a i l i n g due t o non-linear s o r p t i o n isotherms. and a t t r i buted the e f f e c t t o deactivation o f the support. The bonding of PEG t o t h e a c t i v e adsorptive s i t e s on t h e support can take place i n two d i f f e r e n t ways. F i r s t , some chemical bonding may occur when the packing i s used a t higher temperatures. This e f f e c t i s comparable w i t h the thermal bonding o f PEG as described by Aue e t a1.l5. On the o t h e r hand, strong hydrogen bonding may be involved, as s t a t e d by Evans e t They used amine antioxidants as deactivators. These substances may be expected t o combat o x i d a t i v e degradation o f t h e s t a t i o n a r y phase i n a d d i t i o n t o t h e i r d e a c t i v a t i n g prope r t i e s . They suspended the support, which had not been deactivated by any previous s i l a n i z ation, i n a s o l u t i o n o f the compounds. The s o l u t i o n was suctioned o f f , b u t the amine a n t i oxidant molecules remained bonded t o the a c t i v e s i t e s o f the support. The support was then coated w i t h a s t a t i o n a r y phase dissolved i n a solvent, which d i d n o t displace t h e amine a n t i -

d3.

oxidant from the support. I n t h a t way t h e amine a n t i o x i d a n t served as a d e a c t i v a t o r between the s t a t i o n a r y phase and the support. 1.2. Coating o f s o l i d support w i t h s t a t i o n a r y phase Since Horning e t a1.* gas chromatographed several kinds o f compounds on s o l i d support t h i n l y coated w i t h a s t a t i o n a r y phase, t h e use o f such packing m a t e r i a l has beccine increasi n g l y comnon f o r gas chromatography o f high b o i l i n g , s l i g h t l y v o l a t i l e compounds. The reason i s obvious: The r e t e n t i o n times a r e d i s t i n c t l y shortened, f a c i l i a t i n g gas chromatography o f such compounds a t r e l a t i v e l y low column temperatures. However, w i t h a decrease i n t h e percentage o f s t a t i o n a r y phase, t h e r i s k o f g e t t i n g uncoated areas on t h e surface o f the support increases. According t o Bohemen e t a1.l' a support w i t h l e s s than 5 5, o f s t a t i o n a r y phase g r e a t l y enhances t h e influence o f t h e s o l i d support on the gas chromatographic performance. A frequently used coating procedure f o r packing m a t e r i a l s w i t h a low percentage o f s t a t i o n ary phase was described by Horning e t a1.8. It i s generally c a l l e d the " f i l t r a t i o n technique". A f t e r the s o l i d support has been suspended i n a s o l u t i o n o f the s t a t i o n a r y phase, enough o f the s o l u t i o n i s f i l t e r e d o f f t o ensure t h a t the support on d r y i n g w i l l contain t h e desired amount o f s t a t i o n a r y phase. Because o n l y small amounts o f the s o l u t i o n are l e f t a f t e r f i l t r a t i o n , great differences i n t h e concentration o f the s t a t i o n a r y phase i n t h e moist mass during the evaporation w i l l not occur. McMartin and Street" used another technique t o o b t a i n the same r e s u l t : They r i n s e d the

Referencesp. 14

14

support with a s o l u t i o n o f the s t a t i o n a r y phase and d r i e d i t on a h o t p l a t e , w h i l e s t i r r i n g g e n t l y w i t h a glass rod. However, they d i d n o t i n d i c a t e how a d e f i n i t e percentage o f s t a t i o n a r y phase on the support could be obtained. Parcher and UroneZ4 prepared packinq m a t e r i a l by " s o l u t i o n coating" and f l u i d i z e d drying. The percentage o f s t a t i o n a r y phase on the support 25 depended on the concentration o f the s o l u t i o n used. A v e r i l l coated s o l i d supports, when packed i n gas chranatographic columns, by passing a s o l u t i o n o f the s t a t i o n a r y phase through the column. Although reproducible r e s u l t s were obtained when the coating was c a r r i e d o u t i n e x a c t l y the same manner, no d e t a i l s o f the percentage o f s t a t i o n a r y phase on the support were given. Recomnended coating procedure: Suspend a c i d washed, s i l a n i z e d s o l i d support (Chromosorb W) i n a s o l u t i o n o f t h e s t a t i o n ary phase i n a s u i t a b l e solvent t o o b t a i n the desired percentage o f s t a t i o n a r y phase a f t e r evaporation o f the solvent. Treat the suspension f o r 5 minutes i n an u l t r a s o n i c bath t o r e move a i r from the support and evaporate the s o l v e n t i n a r o t a r y evaporator a t the b o i l i n g p o i n t o f the solvent u n t i l p a r t i c l e s o f the support begin t o s t i c k together. S t i r t h e mass g e n t l y w i t h a glass r o d under continous heating i n a c u r r e n t o f a i r . Care must be taken n o t t o damage the p a r t i c l e s o f the support. Continue evaporation i n t h i s way u n t i l t h e mass has g o t flowing properties. Continue the evaporation i n the r o t a r y evaporator, and r a i s e t h e temperature gradually t o about 100°C and heat a t t h a t temperature f o r 30 minutes.

1.3. REFERENCES R. Roman, C.H. Yates and F.F. M i l l a r , J . Chromatogr. s c i . , 15 (1977) 555. A.T. James and A. J.P. Martin, Biochem. J . , 50 (1952) 679. W.L. Holmes and E. Stack, Biochim. Biophys. d c t a , 56 (1962) 163. E. Brochmann-Hanssen and A. Baerheim Svendsen, J . Pharm. s c i . , 51 (1962) 318. E. Brochmann-Hanssen and A. Baerheim Svendsen, J . Pharm. S c i . , 51 (1962) 938. E. Brochmann-Hanssen and A. Baerheim Svendsen. J . Pharm. S c i . 51 (1962) 1095. J.W. Meilink, Gas Chromatography and C a r d e n o l i d e s , Thesis, State U n i v e r s i t y o f Leiden, The Netherland, 1980. 8 E.C. Horning, E.A. M o s c a t e l l i and C.C. Sweeley, Chem. r n d . ( L o n d o n ) , (1959) 751. 9 E.C. Horning, K.C. Maddock, K.V. Anthony and W.J.A. Vandenheuvel Anal. Chem., 35 (1963)

1 2 3 4 5 6 7

526. 10 J. Bohemen, S.H. Langer, R.H. P e r r e t and J.H. Purnell, J . Chem. SOC., (1060) 2444. 11 D.T. Sawyer and J.K. Barr, Anal. Chem., 34 (1962) 1518. 12 C. McMartin and H.V. Street. J . Chromatogr., 22 (1966) 274. 13 E. Grushka and E.J. Kikta, Anal. Chem., 49 (1977) 1004 A. 14 s. Mori. J . C h r m a t o g r . , 135 (1977) 261. 15 W.A. Aue, R.C. Hastings and S. Kapital, J . Chromatogr., 77 (1973) 299. 16 R.C. Hastings and W.A. Aue, J . Chromatogr.. 89 (1974) 369. 17 R.F. Moseman, J . Chromatogr., 166 (1978) 397. 18 W.L. W i n t e r l i n and R.F. Moseman, J . Chromatogr., 153 (1978) 409. 19 M.M. Daniewski and W.A. Aue, J . Chromatogr., 147 (1978) 119. 20 H.V. Street, W. V y c u d i l i k and G. Machata, J . Chromatogr., 168 (1979) 117. 21 F.J. Kabot and L.S. E t t r e , J. as Chromatogr., 2 (1964) 21. 22 S.R. Lipsky and R.A. Landowne, Anal. Chem., 33 (1961) 818. 23 M.B. Evans, R. Newton and J.D. Carmi, J . c h r o m a t o g r . , 166 (1978) 101. 24 J.F. Parcher and P. Urone, J . Gas Chromatogr., 2 (1964) 184. 25 w. A v e r i l l , J . Gas C h r m a t o g r . , 1 (1963) 34.

16

Chapter 2 CAPILLARY COLUMNS

........................................................... ..................................................................

2.1. C a p i l l a r y columns 2.2. References

15 19

2.1. CAPILLARY COLUMNS

Since Desty e t a 1 . l introduced glass c a p i l l a r y columns i n the pas chromatonraphic analys i s o f petroleum products many s c i e n t i s t have been involved i n developing procedures t o prepare h i g h q u a l i t y glass c a p i l l a r y columns. This was achieved by leaching conventional nlass, followed by h i g h temperature s i l y l a t i o n . Since most chromatographic separations o f a l k a l o i d s on c a p i l l a r y columns employ temperat u r e p r o g r a m i n g and o f t e n r e l a t i v e l y h i g h temperatures, the s t a b i l i t y o f a coated column a t higher temperatures i s o f the utmost importance. Depolymerization o f the s t a t i o n a r y phase can take place because o f i m p u r i t i e s i n t h e s t a t i o n a r y phase i t s e l f , o r i n t h e q l a s s w a l l (metal s a l t s , s i l a n o l groups, s t r a i g h t siloxane bridqes) o r i n t h e a c t u a l s i l i c a surface structure . Chemical bonding o f s t a t i o n a r y phases has been shown t o increase t h e s t a b i l i t y o f the s t a t i o n a r y phase compared w i t h conventionally coated f i l m s . A chemically bonded phase may be regarded as one t h a t i s n o t e x t r a c t a b l e by solvents t h a t do n o t a t t a c k the Dhase. Conventional columns can be basic, they can be made i n any i n t e r n a l diameter, they can be whiskered, and they can, therefore, be coated e f f e c t i v e l y w i t h any phase. Fused s i l i c a columns have obvious advantages, b u t do n o t have these p o s s i b i l i t i e s . The fused s i l i c a c a p i l l a r y columns do n o t solve a l l t h e problems. The a c t i v i t y o f s i l i c i u m based c a p i l l a r y columns (glass o r fused s i l i c a ) i s due t o the t r a c e o f metal ions, s i l a n o l qroups and siloxane bridges. Whereas a leached soda-lime column i s always basic, fused s i l i c a columns are always a c i d i c . I t may, therefore, be concluded t h a t fused s i l i c a and leached glass columns coated w i t h polysiloxanes (OV-1, OV-101, OV-73 and SE-54) and polyethylene q l y c o l s (Superox 2011) provide a good choice f o r c a p i l l a r y gas chromatography o f a l k a l o i d s . The q u a l i t y o f fused s i l i c a and 2 leached glass columns i s p r a c t i c a l l y equal . I n a number o f papers t h e p r e p a r a t i o n o f glass c a p i l l a r i e s f o r the analysis o f a l k a l o i d s has been described, as w e l l as t h e i n a c t i v a t i o n o f such glass c a p i l l a r i e s , and the chemical bonding o f s t a t i o n a r y phases on glass c a p i l l a r i e s 3 ' 4,596 The i n j e c t i o n o f the sample o f s l i g h t l y v o l a t i l e compounds, such as a l k a l o i d s , i n c a p i l l a r y gas chromatography can be c a r r i e d out i n d i f f e r e n t ways dependinq upon t h e k i n d o f column used. Verzele e t a1.2 s t a t e d t h a t the " c o l d on column i n j e c t i o n " and t h e " f a l l i n g needle i n j e c t i o n " were the best a l t e r n a t i v e s f o r q u a n t i t a t i v e gas chromatoqraphy since they were applicable t o h i g h temperature c a p i l l a r y gas chromatoqraphy. The sampling system i n c a p i l l a r y gas chromatography has been d e a l t w i t h i n a s e r i e s o f Although the h i g h temperature s t a b i l i t y o f many c a p i l l a r y columns w i t h non e x t r a c t a b l e s t a t i o n a r y phases has e l i m i n a t e d t h e need f o r d e r i v a t i z a t i o n o f many compounds, d e r i v a t i z a -

References p. 19

16

t i o n can o f t e n improve the s p e c i f i c i t y and the s e n s i t i v i t y . O e r h a t i z a t i o n can be c a r r i e d out especially by converting polar compounds i n t o non-polar compounds - before the i n j e c t i o n o f the sample, o r by means o f f l a s h heater derivatization". D e r i v a t i z a t i o n may i n many cases increase the r e s o l u t i o n and the differences i n r e t e n t i o n time o f a compound, and the d e r i v a t i v e may give extra information during the i d e n t i f i c a t i o n o f unknown compounds. Oncolumn d e r i v a t i z a t i o n i n gas chromatography has been described i n a number o f papers10y11' 12.13

-

C a p i l l a r y columns f o r the gas chromatography o f a l k a l o i d s were f i r s t described by Massing i l l and Hodgkins14. They i n v e s t i g a t e d a number o f a l k a l o i d s on t h r e e d i f f e r e n t s t a t i o n a r y phases: Apiezon L, SE-30 and QF-1: Apiezon L 100 f e e t by 0.01 i n c h I.D., temperature p r o g r a m i n g 100-200°C, lZ°C/min SE-30 200 f e e t by 0.01 inch I.D., temperature programming 100-250°C, lZ°C/min QF-1 100 f e e t by 0.01 inch I.D., The r e s u l t s are shown i n Table 2.1.

temperature p r o g r a m i n g 10O-25O0C, lZ°C/min

TABLE 2.1 RETENTION DATA OF ALKALOIDS ON CAPILLARY COLUMNS14 M.W. = Molecular Weight, t = r e t e n t i o n time, tR(rel)= N.R = no response, ft = f e l t

100 f t QF-1 A1 k a l o i d

M.W.

tR

(min) Tropi none Tropine Nicotine Anabasine (Neonicotine) Ephedrine Gramine Caffeine Bufotenine P i locarpine Isopilocarpine Procaine Homatropine Atropine P i perine Cinchonidine Cinchonine Scopolamine Ajmaline Strychnine Papaverine Aspidospenine Berberine Conessi ne Brucine Colchicine

tR( r e 1 ) (Nic = 1)

r e l a t i v e r e t e n t i o n time, Nic = n i c o t i n e ,

200 f t SE-30 tR (min)

%( re1 ) (Nic = 1)

100 f t Apiezon tR (min)

tR( r e 1 ) (Nic = 1)

139 141 162 162

1.58 1.33 2.00 4.08

0.79 0.67 1.00 2.04

3.42 3.17 4.17

0.82 0.76 1.00

1.33 1.66 3.33 10.08

0.40 0.50 1.00 3.03

165 174 194 204 208 208 236 275 209 285 294 294 303 326 334 339 352 353 356 394 399

2.33 2.42 8.17 11.50

1.17 1.21 4.09 5.75

5.25 6.83 11.58

1.25 1.64 2.77

6.00 4.00 N.R.

1.80 1.20

22.42

5.40

15.00 11.75

3.60 2.82

18.33 18.58

4.40 4.46

16.08 N.R. N.R. N.R. N. R. N.R. N. R. N.R.

3.85

11.42 9.08 7.58

9.75 N.R. N.R. U.P. N.R. N.R. N.R. N.R. N.R.

5.71 4.54 3.79

4.88

L

N.R.

The Apiezon L c a p i l l a r y column was useful, o n l y f o r a l k a l o i d s w i t h molecular weights about 175. A t 125OC (isothermal) it separated n i c o t i n e and anabasine by s i x minutes, b u t t a i l i n g was pronounced. The SE-30 c a p i l l a r y column resolved the lower molecular weight (< 200) a l -

17

k a l o i d s very w e l l , b u t the higher molecular weight (- 300) a l k a l o i d s were n o t so w e l l r e solved. T a i l i n g was apparent f o r most o f the samples, b u t was n o t pronounced. The QF-1 capi l l a r y column gave good r e s o l u t i o n o f tropine-tropinone, nicotine-anabasine. and a t m p i n e homatropine-scopolamine mixtures. Generally the peaks were extremely sharp w i t h p r a c t i c a l l y no t a i l i n g . The QF-1 c a p i l l a r y column o u t - p e r f o n e d the o t h e r c a p i l l a r y columns and gave good r e s o l u t i o n o f a l k a l o i d s w i t h molecular weights up t o 303. However, a comparison o f the separation o f atropine-homatropine-scopolamine on a 6 f e e t by 1/8 inch 1 % packed QF-1 c o l umn w i t h t h a t o f t h e 100 f e e t QF-1 c a p i l l a r y column showed t h a t t h e packed column gave b e t t e r resolution. Since the f i r s t paper o f M a s s i n g i l l and Hodgkins14 appeared, t h e use o f c a p i l l a r y columns i n the analysis o f a l k a l o i d s has been l i m i t e d . A few examples o f a p p l i c a t i o n s a r e mentioned below. Harke and Drews15 used a 50 m long s t a i n l e s s s t e e l c a p i l l a r y column, 0.5 mn I.O., coated with Ucon LEI 550 X (polyethylene g l y c o l ) and KOH f o r the separation o f tobacco a l k a l o i d s . A t y p i c a l chromatogram o f a test-mixture containing 3-pyridyl -n-propyl keton, n i c o t i n e . norn i c o t i n e , myosmine, anabasine and n i c o t y r i n e i s shown i n Fiqure 5.1 (Tobacco a l k a l o i d s ) .

To i d e n t i f y tobacco a l k a l o i d s and t h e i r mammalian metabolites, P i l o t t i e t a1.16 made use o f c a p i l l a r y gas chromatography-mass spectrometry using glass c a p i l l a r y columns ( 33 m by

0.4 mn I.D.) coated w i t h Emulphor-0, o r a c a p i l l a r y (9.6 m by 0.2 mn I.D.)

coated w i t h OV-101.

The gas chromatographic data are sumnarized i n Table 5.10 (Tobacco a l k a l o i d s ) . Bohn e t al.17 used glass c a p i l l a r y gas chromatography i n i n v e s t i g a t i o n s o f i l l i c i t h e r o i n samples and obtained good separation o f heroin, 6-O-monoacetylmorphine, c a f f e i n e on a 12 m by 0.3 n I.D.

acetylcodeine and

glass c a p i l l a r y coated w i t h T r i t o n X 303 (Merck) and tem-

perature programming from 200°C t o 25OoC. Dow and Ha1118 used a combination o f c a p i l l a r y gas chromatography and mass spectrometry t o estimate n i c o t i n e i n plasma by s e l e c t i v e i o n monitoring. The c a p i l l a r y was 20 m by 0.3 mn

I.D.,

coated w i t h SP 1000 and the temperature 16OoC.

For c a p i l l a r y gas chromatography o f a l k a l o i d s and o t h e r h i g h b o i l i n g compounds s t a i n l e s s s t e e l o r b o r o s i l i c a t e glass c a p i l l a r i e s have mostly been used. Verzele e t a l . ' ,

i n a study on

high temperature q u a n t i t a t i v e glass c a p i l l a r y chromatographic a n a l y s i s o f p i p e r i n e and quinine-quinidine, found t h a t untreated s o f t glass gave b e t t e r r e s u l t s than b o r o s i l i c a t e glass. Some occasional t a i l i n g could be removed by the a n a l y s i s o f quinine-quinidine by sodium c h l o r i d e dendrite deposition. With OV-1,

OV-17,

OV-225, Superox-4,

RSL-702 and RSL-903 good

peaks were obtained. The r e s o l u t i o n o f quinine and q u i n i d i n e was zero on OV-1,

b u t improved

w i t h increasing p o l a r i t y o f the s t a t i o n a r y phase and was complete o n l y on RSL-903 (a h i g h l y p o l a r polyaromatic sulfone), the most p o l a r phase o f the series. A 30 m by 0.3 n I.D. dendrite column coated w i t h 0.15

ym

sodium

l a y e r o f RSL-903 was used. A moving needle i n j e c t o r

proved t o be the b e s t choice i n order t o o b t a i n accurate q u a n t i t a t i v e r e s u l t s . One v l o f the s o l u t i o n t o be analyzed was introduced on the needle p o i n t and the sample i n j e c t e d i s o t h e r m a l l y a t 28OoC. Figure 2.1 and 2.2. o f such a l k a l o i d s i n a

Cinchona

show chromatograms o f some pure Cinchona a l k a l o i d s and

bark pharmaceutical preparation. F o r the q u a n t i t a t i v e assay,

piperine, e l u t i n g s h o r t l y a f t e r quinine, was used as an i n t e r n a l standard. The isothermal a n a l y s i s was a p p l i e d t o the assay o f quinine i n s o f t drinke, and o f quini n e and q u i n i d i n e i n pharmaceutical preparations, w i t h good r e s u l t s . By m u l t i p l e a n a l y s i s the standard d e v i a t i o n f o r quinine i n s o f t d r i n k s was found t o be 1.97 %, and f o r q u i n i n e - q u i n i -

References p. 19

18 FIGURE 2.1 GLASS CAPILLARY

GAS CHROMATOGRAPHY OF QUININE AND RELATED ALKALOIDS~ on a 30 in by 0 . 3 mn I . D . s o f t glass c a p i l l a r y w i t h sodium dendrite deposition and RSL-903 as stationary phase; temperature 280OC; f a l l i n g needle i n j e c t i o n . 1 = cinchonine, 2 = cinconidine,"3 = quinidine and 4 = quinine.

b

5

min

FIGURE 2 . 2 GLASS CAPILLARY GAS CHROMATOGRAPHY OF ALKALOIDS FROM A CINCHONA BARK PHARHACEUTICAL PREPARA-

TION~. GLC conditions and peak numbers as given i n Figure 2 . 1 . Unnumbered peaks u n i d e n t i f i e d .

19 dine i n pharmaceutical preparations 1.07 % and 0.90 % r e s p e c t i v e l y . For the assay o f p i p e r i n e i n pepper, a 25 m by 0.5 nun 1.0. glass c a p i l l a r y deactivated by h i g h temperature s i l a n i z a t i o n and coated w i t h O V - 1 was used. The samples were i n j e c t e d by the on-column i n j e c t i o n technique a t 100°C, as described by Grob and Grob 5r.l'.

The standard

d e v i a t i o n o f the whole procedure, sample preparation and chromatographic analysis, was 2.5 %. Verzele e t a1.'

concluded t h a t the " c o l d on-column'' i n j e c t i o n , a p p l i e d f o r t h e p i p e r i n e

assay, and the " f a l l i n g needle" i n j e c t i o n , a p p l i e d f o r the quinine-quinidine assay, are the best a l t e r n a t i v e a t the moment f o r q u a n t i t a t i v e c a p i l l a r y gas chromatography. They are app l i c a b l e t o h i g h temperature c a p i l l a r y gas chromatography, b u t they a l s o have t h e i r l i m i t a tions. Floberg e t a1."

applied glass c a p i l l a r y gas chromatography f o r the analysis o f theophyl-

l i n e and c a f f e i n e i n plasma. The a l k a l o i d s were analyzed as such, o r a f t e r d e r i v a t i z a t i o n . Severson e t a1

a l s o used glass capi1.laries f o r the separation and q u a n t i f i c a t i o n o f t o -

bacco alkaloids, whereas Edlund"

separated morphine, 6-0-monoacetylmorphine and codeine

from plasma samples a f t e r d e r i v a t i z a t i o n . He stated, however, t h a t degradation w i l l always occur during gas chromatography on the columns used, since no a b s o l u t e l y i n e r t column i s a v a i l a b l e . I n the cases studied by him, the degradation was very reproducible, so t h a t quant i t a t i v e analysis could be c a r r i e d out i n s p i t e o f degradation. Gas chromatography on glass c a p i l l a r i e s was a l s o a p p l i e d by Neumann und GlogerZ3 i n " f i n g e r p r i n t i n g " i l l i c i t h e r o i n samples d i r e c t l y , and a f t e r d e r i v a t i z a t i o n . The development o f the c o l d on-column i n j e c t i o n technique and fused s i l i c a column w i t h non-extractable s t a t i o n a r y phases opened new ways i n the analysis o f u n d e r i v a t i z e d drugs, such as a l k a l o i d s . PlotczykZ4 a p p l i e d the c o l d on-column i n j e c t i o n technique and fused s i l ca 25 c a p i l l a r y columns f o r the analysis o f i . a . cocaine, codeine and quinine. Demedts e t a l . introduced fused s i l i c a c a p i l l a r y columns i n the t o x i c o l o g i c a l a n a l y s i s o f i l l i c i t heroin samples. With a NP-detector and permanent d e a c t i v a t i o n o f the column w i t h polysiloxane, ex c e l l e n t r e s u l t s were obtained f o r heroin, down t o t h e low nanogram range, w i t h o u t d e r i v a t i zation. REFERENCES

1 D.H. Desty, A. Goldup and B.H.F. Whyman, J . rnst. P e t r o l . , 45 (1959) 287. 2 M. Verzele, G. Redant, S. Quereshi and P. Sandar, J . C h r o m a t o g r . , 199 (1980) 105. 3 L. Blomberg, K. Markiedes and T. Kannman, P r o c . F o u r t h rnt. symp. G a s c h r o m a t o y r . 1 9 8 1 , 73. 4 M.L. Lee, B.H. Wright and K.D. B a r t l e , i b i d . , 505. 5 H. Tausch, J. Kainzbauer and F. Schneider, i b i d . , 335 6 S.R. Lipsky and W.J. McMurray, i b i d . , 109. 7 K. Grob J r . , i b i d . , 185. 8 F. Munari and S. Trestianu, i b i d . , 349. 9 G. Schomburg, i b i d . , 371. 10 A.S. Christophersen and K.E. Rasmussen, J . C h r o m a t o g r . , 174 (1979) 454. 11 K.E. Rasmussen, J . C h r o m a t o g r . , 114 (1975) 250. 12 K.E. Rasmussen, J . C h r o m a t o g r . , 120 (1976) 491. 13 G. Brugaard and K.E. Rasmussen, J . C h r o m a t o g r . , 147 (1978) 476. 14 J.L. Massingill J r . and J.E. Hodgkins, d n a l . C h e m . , 37 (1965) 952. 15 H.-P. Harke and C.-J. Drews, F r e z e n i u s ' z. A n a l . C h e m . , 242 (1968) 248. 16 R . P i l o t t i , C.R. Enzell, Fr.H. McKennis, E.R. Bowman, E. Dufva and B. Holmstedt, B e i t r . T a b a k s f o r s c h . , 8 (1975/76) 339. 17 G. Bohn, E. Schulte and W. Audick, A r c h . K r i m i n o l . , 160 (1977) 27. 18 J. Dow and K. H a l l , J . C h r o m a t o g r . , 153 (1978) 52. 19 K. Grob and K. Grob J r . , J. C h r o m a t o g r . , 151 (1978) 311. 20 S. Floberg, B. Lindstrom and G. Ldnnerholm, J . C h r o m a t o g r . , 221 (1980) 166.

20

21 R.F. Severson, K.L. McOuffie, R.F. Arrendale, G.R. Gwynn, J.F. Chaplin and A.W. Johnson, J . Chromatogr., 211 (1981) 111. 22 P.O. Edlund, J. Chromatogr., 206 (1981) 117. 23 H. Neumann and M. Gloger, Chromatographia, 16 (1982) 261. 24 L.L. Plotczyk, J . Chramatogr., 240 (1982) 349. 25 P. Demedts. M. van den Heede. J. van der Verren and A. Heyndrickx, J . Anal. T o x i c u l . , 6 (1982) 30.

21

Chapter 3

DERIVATIZATION OF ALKALOIDS FOR GAS CHROMATOGRAPHY

......................... .................................................................

3.1. 3.2.

O e r i v a t i z a t i o n o f a l k a l o i d s f o r gas chromatography References

3.1.

DERIVATIZATION OF ALKALOIDS FOR GAS CHROMATOGRAPHY

21 24

Owing t o the low v o l a t i l i t y and thermal i n s t a b i l i t y o f many a l k a l o i d s , d e r i v a t i z a t i o n i s performed t o g i v e them b e t t e r gas chromatographic properties. D e r i v a t i z a t i o n i s , however, a l s o done t o g i v e a l k a l o i d s b e t t e r detection p r o p e r t i e s and f o r q u a n t i t a t i v e determinations.

TABLE 3.1 DERIVATIZATION REACTIONS AND REAGENTS USED BY GAS CHROMATOGRAPHY OF ALKALOIDS Oerivatization

Reagent

A1 k a l o i d

TOBACCO ALKALOIDS Hydrogenation t pentafluoro-

1

Perfluoropropionic anhydride

n ic o t ine

T r i c h l o r o e t h y l chloroformate

nicotine

N ,N-bi s ( t r i m e t h y l s i l y l ) t r i f l u o r o -

p y r r o l i z i d i n e a1 k a l o i d s

propylation

Trichloroethylchlorocarbamate

2

deri vatization PYRROLIZ IDINE ALKALOIDS Trimethyl s i l y l a t i o n

3

ace tami de TROPINE ALKALOIDS Trimethyl s i l y l a t i o n

N ,0-b i s ( t r imet h y l s i1y 1 )ace tami de

scopol ami ne4*',

tropic

8

acid', scopol i n e tropane a1 k a l o i d s 6

Hexamethyldisi lazane N-methyl -N-trimethyl s i l y l tri f 1uoroacetami de

tropine, scopol ine, atropine, scopolamine, 5 meteloidine

Heptafl uorobutyla ti on

Heptafl uorobutyric anhydride

scopoline'

PSEUDOTROPINE ALKALOIDS Methyl a t i on

Diazomethane Dimethylformamide

+

dimethyl-

acetal Trimethylsi l y l a t i o n

benzoyl ecgoni ne

11

N-methyl-N-trimethylsilyl t r i f l uoroacetamide

ecgonine, pseudoecgonine, 5 benzoyl ecgoni ne

N,O-bi s ( t r i m e t h y l s i 1y l )acetami de

cis- and trans-cinnamoyl-

cocaine, ecgonine, benzoylecgonine

Retenner p. 21

I0

22

TABLE 3.1 (continued) Derivatization Acyl a t i o n

Reagent f l u o r o b u t y r i c anhydride

Reduction and 0-acylation

Alkaloid

Hexafluoroisopropanol t heptaecgonine, benzoyl15 ecgoni ne

LiA1H4 t pentafluoroprupionic o r 14

h e p t a f l uoropropioni c anhydride

cocaine

Methylation

Trimethylani 1i n i um hydroxide

qui n i d i ne 17

Trime thy1 s i 1y l a t ion

N-methyl-N-trimethylsilyl t r i -

CINCHONA ALKALOIDS

f l uoroacetamide o r b i s ( t r i methylsi l y l ) tri fluoroacetamide

cinchona a l k a l o i d s

T r i f l u o r u a c e t i c anhydride

mescaline

Acetone d e r i v a t i z a t i o n

Acetone

ephedrine, pseudo-

T r i fluoroacetyl p r o p y l a t i o n

N - t r i f l uoroacetyl -L-propyl

16

CACTUS ALKALOIDS Trifluoroacetylation

18

EPHEDRA ALKALOIDS ephedri ,el9 '20 ch 1 o r i de

ephedrines, pseudo-

'22

ephedrines" N-( R)-a-phenyl b u t y r y l -

N-(R)-a-phenyl b u t y r i c anhydride

0- t r i m e t h y l s i1y l a t ion

t N,O-bis(trimethylsily1 )acetamide ephedrines:_pseudo-

Oxidation

Sodi um metaper iodate

ephedri nes 24,25 ephedrine

Acetic anhydride

,,orphi

OPIUM ALKALOIDS Acetyl a t ion

2,6

2 7 332 939

morphine-N-oxide,

F1 uoroacetylation

nor-

T r i fluoroaceti c a c i d

morphine, pseudomorphi ne, 39 codeine 37 morphine, codeine

T r i f l u o r o a c e t i c anhydride

morphine3' '41 349, morphineN-oxide, nor-morphine, pseudomorphine, codeine, 39

T r i f l uoroacetylimidazole Propi onyl a t i o n

Propionic anhydride

Pentafluoropropylation

Pentafl uoropropionic anhydride

nor- co de ine morphine 51

6-056 acetylmorphi ne morphine36 945353957, 6-0-acetylmorphine

Heptafl uorobutylation

57 52,54

Heptafluorobutyric anhydride

6-0-acetylmorphine

Heptafl uorobutyri c a c i d

morphine, codeine37, apomorphine46

Heptafluorobutyrylimidazole

morphine 55

23 TABLE 3.1 (continued) Derivatization Trimethylsilylation

Reagent

A1 k a l o i d

Hexamethyldisi lazane Hexamethyldisilazane t

morphine"

29 t r i m e t h y l c h l o r o s i 1ane morphine N,O-bis(trimethy1si 1y l )acetami de morphine30933y38s40*44 apomorphi ne34y35

N ,0-bi s ( t r i m e t h y l s i 1y l )acetami de t trimethylchlorosilane N.0-bi s( t r i m e t h y l s i 1y l ) - t r i

-

morphine31

fluoroacetamide t t r i m e t h y l chlorosilane

morphineN-oxide,

nor-morphine,

pseudomorphine, codeine, nor-codeine 39 Pentaf 1uorobenzyl a t i o n

Pentafl uorobenzoyl bromide

morphine5'

APORPHINE ALKALOIDS Trimethyl s i l y l a t i o n

N,O-bis(trimethylsily1 )acetamide t trimethylsilylimidazole t

trimethylchlorosilane

aporphines, tetrahydroberber-

T r i f 1uoroace t i c anhydri de

demethylated tetrahydroberber58 ines aporphines 59

ines, demethylated aporphines,

Trifluoroacetylation AMARYLLIOACEAE ALKALOIDS Trimethyl s i l y l a t i o n

N,O-bis( trimethylsily1)acetamide

haryllidaceae alkaloids E r y t h r i n a a l k a l o i d s6 1

60

INDOLE ALKALOIDS Trimethyl s i l y l a t i o n

Hexamethyldisi lazane

hydroxyl s u b s t i t u t e d N,N-di me thy1 tryptami nes62

N-methyl -N-trimethyl s i l y l t r i f l uo roace tami de

v i ncamine65*66

N,O-bis(trimethy1 s i l y l ) f l u o r o acetamide

hydroxyl s u b s t i t u t e d Vinca67

a1 k a l oids

N ,0- b is ( t r i m e t hy 1s i 1y l )ace tami de Hydrolysis and methylation

Diazomethane

68 physostigmine 68 a jmal ine reserpine, r e s c i nnamine63

o f the acids formed ERGOT ALKALOIDS Trimethylsi l y l a t i o n

N.0-bis( t r i m e t h y l s i l y l )acetamide

LSD70'71y72, l y s e r g i c a c i d a m i d e ~ ~agroclavine ~, 76

N.0-bis(trimethy1 s i l y l ) t r i fluoroacetami de

References p. 24

LSD74

-

24

TABLE 3.1 (continued) Derivatization

Reagent

Alkaloid

T r i methyl s i 1y l d i e t h y l mine t ergmetrine

t r i m e t h y l s i l y l imidazole N,O-bis( t r i m e t h y l s i l y l ) tri

75

-

fluoroacetamide t t r i m e t h y l s i l y l d i e t h y l amine t t r i m e t h y l

-

T r i f l u o r o a c e t i c anhydride

agroclavine76 76 agroclavine

Methyl sulphate t sodium hydride

solanine, demissi ne

s i lylchlorosilane Tr if 1uoroacetyl a t i on STEROIDAL ALKALOIDS Methyl a t i o n

77

XANTHINE ALKALOIDS Methyl a t i o n

Trimethylanil inium hydroxide

theophyl 1ine78s813 92 78

t h e o b r a i ne Ethylation

Ethyl i o d i d e

theophyl 1ine

Butylation

Tetra -n-butyl ammon ium hydroxide

xanthines7’, 1i,87,94

95 theophyl-

Dimethyl fonnamide t d i -n-butylacetal

theophyl 1ineE2

Butyl i o d i d e

theophyl 1ineE3

Tet ramethyl amon ium hydro x i de t butyl iodide

theophyl l i n e ”

(N,N-dimethylacetami d e ) t e t r a methylammonium hydroxide t b u t y l Propylation

theophyll inegl ’93 iodide . . Dimethylfonnamide t dipropylacetal t h e o p h y l l i n e86 Tetrapropylammonium hydroxide

theophyl l i n e80

Pent y l a t i on

Pentyl i o d i d e

theophyll i n e

Pentafl uorobenzyll a t i o n

Pentafl uorobenzyl bromide

theophyl caffeine, t h e o b r m i n e95

Pentafl uorobenzyl c h l o r i d e

theophyl 1i,ego

Heptafl uorobutyri c anhydride

p ilocarpine

84

IMIDAZOLE ALKALOIDS Acyl a t ion 3.2. REFERENCES

1 L. Neelakantan and H.B. Kostenbauder, Anal. Chem., 46 (1974) 452. P. Hartvig, N.-0. Ahnfelt, M. Hammarlund and J. Vessman, J . Chrmatogr., 173 (1979) 127. P. Stengl, H. Wiedenfeld and E. Rider, Dtsch. dpoth.-ztg., 122 (1982) 851. J.J. Windheuser, J.L. S u t t e r and A. S a r r i f , J . Pham. S c i . , 6 1 (1972) 1311. H.W. Liebisch, H. Bernasch and H. R. Schutte, z. Chem., 13 (1973) 496. W.J. G r i f f i n , H.P. Brand and J.G. Dare, J.Pharm. S c i . , 64 (1975) 1821. W.F. Bayne, F.T. Tao and N. C r i t l o g o , J . Pharm. S c i . , 64 (1975) 288. B. Gober, U. Timm and H. Dohnert. Z e n t r a l b l . Pharm., Pharmakother. Laboratoriumsdiagn.,

2 3 4 5 6 7 8

116 (1977) 13. 9 F. Fish and W.D.C. Wilson, J . Chromatogr., 40 (1969) 164. 10 J. E. Wallace, H. E. Hamil ton, D.E. King, D. J. Bason, H.A. Schwertner and S.C. A n a l . Chem., 40 (1976) 34.

Harris,

26

11 12 13 14 15

S. Koontz, D. Besemer, N. Mackey and R. P h i l l i p s , J . Chromatogr., 85 (1973) 75. Moore, J. d s s o c . off. Anal. Chem., 56 (1973) 1199. J.M. Moore, J. Chromatogr., 101 (1974) 215. J.W. Blake, R.S. Ray, J.S. Noonan and P.W. Murick, Anal. Chem., 46 (1974) 288. J.M.

J.I.

Javaid, H. Dekirmenjian. E.G.

Brunngraber and J.M.

Davis, J. C h r m a t o g r . , 110 (1975)

141. 16 17 18 19 20

E. Smith, S . Barkan, B. Ross, M. Marienthal and J . Levine, J . Pharm. s c i . , 62 (1973) 1151. K.K. Midha and C. Charette, J. Pharm. s c i . , 63 (1974) 1244. G. van Peteghem, A. Heyndrickx and W. van Zele, J . Pharm. S c i . , 69 (1980) 118. E. Brochmann-Hanssen and A. Baerheim Svendsen. J . Pharm. S c i . , 51 (1962) 938. K. Yamasaki, K. F u j i a , M. Sakamoto, K. Okada, M. Yoshia and 0. Tanaka, Chem. Pharm. B u l l . ,

92 (1974) 2898; C . A . , 82 (1975) 103221 V. 21 A.H. Beckett and B. Testa, J . Chromatogr.. 69 (1972) 285. 22 A.H. Beckett and B. Testa, J . Pharm. Pharmacol., 25 (1973) 382. 23 M.T. G i l b e r t and Ch.J.W. Brooks, Biomed. Mass S p e c t r m . , 4 (1977) 226. 24 M. E l e f a n t , L. Chafetz and J.M. Talmage, J . Pharm. S c i . , 56 (1967) 1181. 25 L. Vuorinen and J. Halmenkoski, Farm. d i k a k . , 81 (1972) 185. 26 M.W. Anders and G.J. Mannering, Anal. Chem.. 34 (1962) 730. 27 S.J. Mule, Anal. Chem., 36 (1964) 1907. 28 E. Brochmann-Hanssen and A. Baerheim Svendsen, J . Pharm. S c i . , 51 (1962) 1095. 29 G.E. M a r t i n and J.S. Swinehart, Anal. Chem., 38 (1966) 1789. 30 N. Ikekawa, K. Takayama, E. Hosoya and T. Oka, Anal. Biochem., 28 (1969) 156. 31 G.R. Wilkinson and E.L. Way, Biochem. Pharmacwl., 18 (1969) 1435. 32 J.E. Wallace, J.D. Biggs and K. Blum, C l i n . Chem. A c t a , 36 (1972) 85. 33 D. Eskes. A.M.A. Verwey and A.H. Witte, B u l l . Narc., 25 (1973) 41. 34 R.V. Smith and A.W. S t o c k l i n s k i . J. Chromatogr., 77 (1973) 419. 35 R.V. Smith and A.W. S t o c k l i n s k i , A n a l . Chem., 47 (1975) 1321. 36 S.Y. Yeh, J. Pharm. S c i . , 62 (1973) 1827. 37 W.O.R. Ebbighausen, J . Mowat and P. Vestergaard, J . Pharm. S c i . , 62 (1973) 146. 38 P.A. Clarke and R.L. F o l t z , C l i n . Chem. (Winston-Salem, N . C . ) , 4 (1974) 465. 39 H. Kaneshina, Y. Kinoshita. M. Mori, T. Yamagishi, S. Honma and H. Mitubashi, Shoyakugaku z a s s b i , 28 (1974) 127; C . A . , 83 (1975) 152416 S . 40 R. Truhaut, A. Esmailzadeh, J. Lebbe, J.-P. Lafarge and N y y e n Phu Lich, Ann. B i d . C l i n . ( P a r i s ) , 32 (1974) 429. 41 S.P. Sobol and A.R. Sperling, F o r e n s i c S c i . Symp. ( A m e r i c . Chem. Soc.), G . D a v i e s , e d i t . , 1974, 170. 42 E.P.J. van der Slooten and H.J. van der Helm, F o r e n s i c S c i . , 6 (1975) 83. 43 J.E. Wallace, H.E. Hamil ton, K. Blum and C. P e t t y , A n a l . Chem., 46 (1974) 2107. 44 G.R. Nakamura and L.E. Way, Anal. Chem., 47 (1975) 775. 45 B. Dahlstrom and L. Paalzow, J . Pharm. Pharmacol., 27 (1975) 172. 46 D.M. Baaske, J.E. Keiser and R.V. Smith, J . Chromatoyr., 140 (1977) 5 7 . 47 S.Y. Yeh and R.L. McQuinn, J. Pharm. S c i . , 64 (1975) 1237. 48 K.E. Rasmussen. J. Chromatour.. 120 (19761 491. 49 P.P. Hipp, M.R: Eveland, E.R. Meyer,’W.R.’Sherman and T.J. Ciceru, J . Pharmacol. EX^. T h e r . , 196 (1976) 642. 50 W.J. Cole, J . Parkhouse and Y.Y. Yousef, J . Chromatogr., 136 (1977) 409. 51 G. Brugaard and K.E. Rasmussen, J. Chromatogr., 147 (1978) 476. 52 J.M. Moore and M. Klein, J . Chromatoqr., 154 (1978) 76. 53 E.R. G a r r e t t and T. Gurkan. J . Pharm; S c i . . 67 (1978) 1512. 54 J. Moore, J . Chromatoyr., 147 (1978) 327. 55 A.S. ChristoDhersen and K.E. Rasmussen. J . Chromatour.. 168 (19791 , 216. 56 G.-Machata a i d W. Vycudilik, J. A n a l . & i c o l . , 4 (i980) 318: 57 P.O. Edlund, J . Chromatogr.. 206 (1981) 117. 58 J.L. Cashaw, K.D. McMurtrey, L.R. Meyerson and V.E. Davis, A n a l . Biochem., 74 (1976) 343. 59 J.F. Green, G.N. Jham, J.L. Neumeyer and P. Vouras, J. Pharm. S c i . , 69 (1980) . . 936. 60 D.S. M i l l i n g t o n , D.E. Games and A;H. Jackson, Proc. I n t e r n a t . Symp. Gas Chromatogr. Mass S p e c t r o m . , 1972, 277. 61 D.S. M i l l i n g t o n , D.H. Steinman and K.L. Rinehart, Jr.. J . ~ J L Chem. SOC., 96 (1974) 1909. 62 B. Holmstedt, W.J.A. Vandenheuvel, W.L. Gardiner and E.C. Horning, A n a l . Biochem., 8 (1964) 151. 63 G. Settimj, L. D i Simone and M.R. Del Giudice, J . Chromatogr., 116 (1976) 263. 64 G.P. Forni, J. Chromatogr., ,176 (1979) 129. 65 H. Laufen, W. Juhran, W. F l e i s s i g , R. Glitz, F. Scharpf and G. Bartsch, A r z n e i m . - F o r s c h . , 27 (1977) 1255. 66 H.-0. Hoppen, R. Heuer and G. Seidel, Biomed. Mass s p e c t r o m . , 5 (1978) 133. 67 M. Gazdag, K. M i h a l y f i and G. Szepesi. F r e s e n i u s ‘ z . A n a l . Chem., 309 (1981) 105. 68 F.W. Teare and S. I . Borst, J . Pharm. P h a r m a m l . , 21 (1969) 277.

26

69 M. Lerner, B U ~ L Narc., 19 (1967) 39. 70 M. Lerner and M.D. Katsiaficas, B u l l . Narc., 21 (1969) 47. 71 J . Jane and 6.6. Wheals, J . Chromatogr.. 84 (1973) 181. 72 K. Bailey, 0. Verner and 0. Legault, J . ASSOC. off. Anal. Chem., 56 (1973) 88. 73 A.R. Sperling, J. Pharm. S c i . , 12 (1974) 265. 74 D. Sondack, J . pharm. s c i . , 63 (1974) 584. 75 K.D. Barrow and F.R. Quigley, J . Chromatogr., 105 (1975) 393. 76 S.F. Herb, Th.J. F i t r p a t r i c k and S.F. Osman. J . Agric. ~ o o dChem., 23 (1975) 520. 77 E. Brochmann-Hanssen and T.O. Oke. J . Pharm. Sci., 58 (1969) 370. 78 M. Kowblansky, B.M. Scheinthal, G.D. C r a v e l l o and L. Chafetz. J . Chromatogr., 76 (1973) 467. 79 V.P. Shah and S . Riegelman, J . Pharm. S c i . , 63 (1974) 1283. .80 L.J. Dusci, P. Hackett and I.A. McDonald, J. Chromatogr.104 (1975) 147. 81 A. Arbin and P.-0. Edlund, Acta Pharm. s u e c . , 11 (1974) 249. 82 G.F. Johnson, W.A. Dechtiaruk and H.M. Solomon, C l i n . Chem. (Winston-Salem, N . C . ) , 21 (1975) 144. 83 W.A. Dechtiaruk, G.F. Johnson and H.M. Solomon, C l i n . Chem. (Winston-Salem, N . C . ) , 21 (1975) 1038. 84 A. Arbin and P.-0. Edlund, Acta.Pharm. S u e c . , 12 (1975) 119. 85 J . Zuidema, J.E.C.P.M. L i c h t , J. Prins and F.W.H.M. Merkus, Pharm. weekbl., 111 (1976) 570. 86 D. P e r r i e r and E. Lear, C l i n . C h e m . (Winston-Salem, N.c.), 22 (1976) 898. 87 Ch.J. Least, G.F. Johnson and H.M. Solomon, C l i n . Chem. (Winston-Salem), N . c . ) , 22 (1976) 765 88 D.C. Bailey, H.L. Davis and G.E. Johnson, J . Chromatogr., 121 (1976) 263. 89 H.A. Schwertner, Th.M. Ludden and J.E. Wallace, Anal. Chem., 48 (1976) 1875. 90 J.O. Lowry, L.J. Williamson and V.A. Raisys, J . Chromatogr., 143 (1977) 83. 91 H. Kinsun, M.A. Moulin, R. Venezia, D. Laloum and M.C. Bigot, C l i n . Chim. A c t a , 84 (1978) 315. 92 C.A. Pranskevitch, J . I . Swihart and J.J. Thoma, J . Anal. T o x i c o l . , 2 (1978) 3. 93 6. Vinet and L. Zizian, C l i n . Chem. (Winston-Salem, N.C.), 25 (1979) 156. 94 S. Floberg, 6. Lindstrom and G. Lonnerholm, J. Chromatogr., 221 (1980) 166. 95 W.F. Mayne, L.-C. Chu and F.T. Tao, J . Pharm. S c i . , 65 (1976) 1724.

II. SPECIAL PART

This Page Intentionally Left Blank

29

11.1.

PYRROLIZIDINE, PYRIDINE, P I P E R I D I N E AND QUINOLIZIOINE ALKALOIDS

Chapter 4 PYRROLIZIDINE ALKALOIDS

....... ....................

................................... ...................................

4.1. P y r r o l i z i d i n e a l k a l o i d s 4.2.

References

29

32

4.1 PYRROLIZIDINE ALKALOIDS I n a systematic study on gas chromatography o f p y r r o l i r i d i n e a l k a l o i d s , Chalmers e t a l .

1

emphasized the importance o f using a s i l a n i z e d a l l - g l a s s system and w e l l deactivated support m a t e r i a l t o reduce t h e p o s s i b i l i t i e s o f adsorption and decomposition d u r i n g gas chromatography. On a 6 ft. long s i l a n i z e d glass column packed w i t h 4 % SE-30 on GasChrom P, t h e none s t e r a l k a l o i d s could be gas chromatographed a t 14OoC, and the e s t e r s w i t h monocarboxylic acids and the macrocyclic d i e s t e r a l k a l o i d s a t 205OC. The s t r u c t u r e s o f some p y r r o l i z i d i n e a l k a l o i d s are given i n Figure 4.1. FIGURE 4 . 1

PY RROLIZIDINE ALKALOIDS I = l-Methoxymethyl-1,2-dehydropyrrolizidine, t r i n e , I V = monocrotaline, V = senecionine

I 1 = i t s 7phydroxy d e r i v a t i v e , 111 = h e l i o -

4’

11’

3‘

2’

RmcH20cH3 CH2OCOC-CH-CH3 Hb

H3C

OH

I I CH3-CH-C-C-CH3 I I I CO H O

I

CO

>o

OpJCH2

m

hCH3

CH3 O H 7’

6’

CH3-CH=C-

5’

I

C’

CM2-

13‘

12’

CH-C-CH3

co

I co

O m C H 2 -

O

I

I

P

1’

30

The retention times o f the non-ester alkaloids and derivatives are listed in Table 4.1 and those o f the esters with monocarboxylic acids in Table 4.2. In Table 4.3 the retention times of the macrocyclic diesters are given. TABLE 4.1 RETENTION TIMES, tR, OF NON-ESTER PYRROLIZIDINE ALKALOIDS AND DERIVATIVES ON A 4 % SE-30 PACKED COLUMN ON GAS CHROM P AT 14OoC1

A1 kaloid

B.p.('C/mm)

1-Methylenepyrrolizidine Heliotridane (lp-methyl-8wpyrrolizidine Anhydroplatynecine 7p-Hydroxy-1-methyl ene-8a-pyrrolzidi ne Desoxyretronecine (7~-hydroxy-1-methyl1 ,2-dehydro-8a-pyrrol izidi ne) 7p-Hydroxy-1-methylene-8p-pyrrolizidine Retronecanol (7D-hydroxy-lp-rnethy1-8a-

pyrrol i zidi ne) Hydroxyhel iotridane (7a-hydroxy-lp-methylb-pyrrolizidine) 7a-Hydroxy-l-methyl-1,2-dehydro-8a-pyrrolizidine 1-Methoxymethy 1 1 ,Z-dehydr o - b - pyrro1 i zi d i ne 1-Methoxymethyl- 1,2-epoxypyrrol i z i di ne Isoretronal ( lp-hydroxymethyl-8a-pyrrol i zidi ne) Supinidi ne (l-hydroxymethyl-l,2-dehydro-8apyrrolizidine) 1- Hydroxymethy 1 - 1 ,2-epoxy-8a-pyrro1 i z i di ne

-

M.p.(OC)

t.(min)

41/0.1

35-36

1.6 i.8 2.4 3.3

62/0.03

79-80 34-36

3.4 4.4

98-98.5

4.5

92/0.5 61.5-62.5 114/3.5 67-68 loo/10 53/0.1 115-16/1.5 39-40

4.5 4.5 5.4 6.4 7.4

115/150

---. 194/750

1691760 .--

140/30

90/0.1 80/0.04

29-30

7710.4

36-38

7.4 8.3

7p-Hydroxy-l-methoxymethyl-l,2-dehydro-8a-

pyrrol izidine

9.0

7p-Acetoxy-l-methox)rmethyl-1,2-dehydro-b-

15.0

pyrrol izidine Retronecine (7p-hydroxy-1hydroxymethyl-1,2dehydro-8a-pyrrolizidine)

He1 iotri dine ( 7a-hydroxy-1-hydroxymethyl-1 ,2dehydro-8a-pyrrolizidine) Platynecine (7p-hydroxy-lp-hydroxymethyl8a-pyrrolizidine

117-118

15.0

117-118

15.0

148-148.5

15.0

TABLE 4.2 RETENTION TIMES OF PYRROLIZIDINE ESTERS WITH MONO CARBOXYLIC ACIDS ON A 4 % SE-30 PACKED COLUMN ON GAS CHROM P AT 2O5OC1 Alkaloid 7-Angelylretronecine 7-Angelylheliotridine Heleurine Supinine Heliotrine Indicine Retronecine trachelanthate Retronecine viridiflorate Rinderine Echi nat i ne Europine Sarracine

M.p. (OC) 76-77 116-117 67-68 148-149 128 97-98 100-101 109-110 (N-oxide 171) 45-46

tR(min) 4.2 4.8 8.0 8.8

12.4 14.3 14.3 14.3 15.6 15.6 18.2 29.1

31 TABLE 4.2 (continued) A1 k a l o i d

-

Echi umine Lasi ocarpi ne Echimi d i ne Heliosupine L a t i f o l ine

M. p. (OC)

tR(min)

99-100 96.5-97 ( p i i r a t e 142-143) ( p i c r a t e 103-106) 102-103

35.6 46.4 47.6 50.6 55.0

TABLE 4.3 RETENTION TIMES OF MACROCYCLIC DIESTER ALKALOIDS ON A 4 % SE-30 PACKED COLUMN ON GAS CHROM P

AT 2O5OC1 A1 kal o i d Retusine Fulvine Crispatine Monocrotaline Senecionine Seneci phyl 1ine Platyphyl 1i n e Integerrimine Spectabi 1 i n e Senk ir k ine Jacobine Scel e r a t i n e Jacoz ine Jacol i n e Rosmari nine Jaconine Ret r o r s ine R i ddel 1iine Retusamine Otoseni ne Grantianine

M . P . [OC)

174-175 213.5-214 137-138 202-203 245 217 129 172.5 185.5-186 198 228 178 228 221 209 147 219-220 198 174.5 22 1 209-209.5

t,(mi

n1

13:9 15.5 15.6 19.5 20.6 21.1 24.0 24.3 27.2 31.6 34.0 34.6 35.5 36.6 37.1 40.4 40.7 41.9 50.3 51.0 57.0

Stengl e t a1.2 c a r r i e d o u t an i n v e s t i g a t i o n o f the l i v e r t o x i c p y r r o l i z i d i n e a l k a l o i d s i n Symphytum o f f i c i n a l e by means o f gas chromatography. The a l k a l o i d m i x t u r e was e x t r a c t e d i n a Soxhlet apparatus w i t h methanol, the e x t r a c t p u r i f i e d by repeated a c i d i c aqueous and a1 k a l i n e organic s o l v e n t e x t r a c t i o n and column chromatography on F l o r i s i l . A f t e r d e r i v a t i z a t i o n w i t h n - b u t y l b o r o n i c a c i d and N,N-bis(trimethylsily1)trifluoroacetamide were gas chromatographed on a 1.8 m by 2 rnrn I . D .

the a l k a l o i d s

glass column packed w i t h 4 % SE-30 on Vara-

p o r t (80-100 mesh). F i v e a l k a l o i d s , lycopsamine, intermedine, acetyllycopsamine,

acetyl-

intermedine and symphytine were i d e n t i f i e d and t h e i r amounts q u a n t i t a t i v e l y determined. The gas chromatographic separation o f the a l k a l o i d d e r i v a t i v e s i s shown i n Table 4.4.

References p. 32

32

TABLE 4.4 RETENTION TIMES

OF

2

Si'hfPHYTrhV OFFICINALE ALKALOIDS

a f t e r d e r i v a t i z a t i o n w i t h n-butylboronic a c i d and N,N-bis(trimetylsily),)trifluo&oacetamide on a packed column w i t h 4 X SE-30 and temperature programming from 200 C t o 300 C

tR (set)

tR(rel)

7 79 Symphyti ne Acetyl 1ycopsami ne 576 Acetyl i ntermedi ne 598 Lycopsamine 545 Intenedine 56 2 I n t . standard (Cholesterol TMS)1116

0.698 0.516 0.536 Oi489 0.504 1.000

TABLE 4.5 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY

OF PYRROLIZIOINE ALKALOIDS

Col umn

S o l i d support

Stat.phase %

Temperature

glass, 6 f t x 6 mn I . D .

Gas Chrom P

SE-30

4

glass, 1.8 m x 2 mn I.D.

Varaport

SE-30

4

14OoC non e s t e r a l k . 205'C e s t e r a l k . 200-300°C p r .

Comp.Prep.

Abbreviations: a l k = a l k a l o i d ( s ) , p r = (temperature) programming 4.2 REFERENCES

1 A.H. Chalmers. C.C.J. Culvenor and L.W. Smith, J. Chromatogr., 20 (1965) 270. 2 P. Stengl, W. Wiedenfeld and E. Rider, Dtsch. Apoth.-ztg., 122 (1982) 851.

Ref.

1 2

33

Chapter 5

PYRIDINE ALKALOIDS

........................................................... ......................................... ........................................... .................................................... ......................................... ........................................................

5.1. Tobacco a l k a l o i d s 5.1.1. A l k a l o i d s i n tobacco samples 5.1.2. A l k a l o i d s i n tobacco smoke 5.1.3. N i c o t i n e i n u r i n e 5.1.4. N i c o t i n e i n blood and t i s s u e 5.1.5. Miscellaneous 5.1.5.1. N i c o t i n e i n residues on foods 5.1.5.2. Biosynthesis o f n i c o t i n e a l k a l o i d s 5.1.5.3. Airborne n i c o t i n e 5.2. References

............................... ........................... ........................................... ..................................................................

33 38

40 42 44 47 47 48 48 50

5.1 TOBACCO ALKALOIDS The f i r s t group o f a l k a l o i d s t h a t was subjected t o gas chromatographic a n a l y s i s was the very v o l a t i l e tobacco alkaloids. They were gas chromatographed by Quin' i n 1958 on packed columns w i t h p o l y g l y c o l s as s t a t i o n a r y phases. Since then a g r e a t number o f papers have been published o f tobacco a l k a l o i d s i n connection w i t h

1) studies on the a l k a l o i d a l composition o f various types o f tobacco, 2) studies on the a l k a l o i d s i n tobacco smoke and p a r t i c u l a t e m a t t e r o f tobacco smoke, 3) studies on the biosynthesis o f tobacco a l k a l o i d s , 4) studies on the metabolism o f tobacco a l k a l o i d s i n man,

5 ) determination o f n i c o t i n e i n blood, t i s s u e and urine, as w e l l as i n 6 ) residues i n food. I n v e s t i g a t i o n s by means o f gas chromatography have a l s o been conducted t o

7) study the thermal decomposition o f tobacco a l k a l o i d s . To increase t h e s e n s i t i v i t y o f the method, d e r i v a t i z a t i o n o f n i c o t i n e has been done w i t h the use o f an e l e c t r o n capture detector. Also, m u l t i p l e i o n detection, employing deuterated a l k a l o i d s as i n t e r n a l standards, has been used f o r t h e same purpose.

A number o f d i f f e r e n t s t a t i o n a r y phases have been used, mostly on a s o l i d support impregnated w i t h potassium hydroxide; from very p o l a r ones, such as polyglycols, t o non-polar ones, such as s i l i c o n e rubber. Packed columns have mostly been used, b u t good separations have a l s o been achieved w i t h c a p i l l a r y columns. Many i n v e s t i g a t i o n s have been c a r r i e d o u t o f gas chromatography o f tobacco a l k a l o i d s i n order t o study the v o l a t i l e compounds o f the smoke, i.e. thermal d e c a p o s i t i o n products o f a l k a l o i d s and o t h e r substances i n tobacco. This chapter i s mainly concerned w i t h t h e qas chromatography of t h e a l k a l o i d s themselves and t h e i r metabolites i n man, a l s o t o some e x t e n t w i t h t h e d e c m p o s i t i o n products o f the a l k a l o i d s . I n h i s f i r s t p u b l i c a t i o n on gas chromatography o f tobacco a l k a l o i d s Q u i n l used packed columns w i t h polyethylene glycol, polypropylene g l y c o l and polybutylene g l y c o l as s t a t i o n a r y phases on alkali-washed f i r e b r i c k . The percentage o f s t a t i o n a r y phase was 25. The p o l y g l y c o l columns e x h i b i t e d good s e l e c t i v i t y f o r the a l k a l o i d s , making the separation o f most o f the members o f complex mixtures possible, as shown i n Table 5.1.

Rtferences p. 60

34

TABLE 5.1 SEPARATION

OF TOBACCO ALKALOIDS ON POLYGLYCOL COLUMNS, 25 % ON ALKALI-HASHED

FIREBRICK^

Columns and conditions L i q u i d phase Temperature Helium f l o w (ml/min) 3-Pyridyl methyl ketone 3-Pyridyl e t h y l ketone 3-Pyridyl n-propyl ketone Nicotine Nor-nicotine Myosmine Anabasine Metani c o t i n e N i c o t y r i ne Cot in ine

Pol y e t h y l ene glycol 190% 48 4.3 5.3 6.6 5.2 12.3 13.4 13.816.5 19.4

85

Polypropylene glycol 190°c 45 Retention times (min) 4.3 6.1 8.1 8.6 16.1 16.4 19.4 23.5 21.0 79

Polybutylene glycol 180OC

50 3.1 5.0 5.0

7.0 8.2 14.3 14.7 18.1 20.9 18.3 63

To c o n t r o l whether o r n o t the s t r u c t u r e o f the a l k a l o i d s had been a l t e r e d during the gas chromatography, Quin i s o l a t e d the compounds e l u t e d f r o m the column and showed t h a t i n a l l cases the c o l l e c t e d and s t a r t i n g compounds were i d e n t i c a l . The method was applied f o r the analysis o f the a l k a l o i d s i n tobacco

To overcome

d i f f i c u l t i e s associated w i t h the wide b o i l i n g range o f the a l k a l o i d a l m i x t u r e and the r e l a t i v e l y massive amount o f n i c o t i n e present, t h e gas chromatography was c a r r i e d o u t under three sets o f conditions: 1 ) A t 140-150°C on a 1 m by 6 mn column f o r the a l k a l o i d s emerging before n i c o t i n e ; 2) a t 190°C on a 1 m by 10 mm column f o r the a l k a l o i d s imnediately f o l l o w i n g n i c o t i n e . The wide diameter permitted a l a r g e n i c o t i n e l o a d t o be placed on t h e column w i t h o u t an asymnetric, t a i l i n g peak r e s u l t i n g ; 3) a t 190°C on a 1 m by 6 nun column f o r the h i g h e r boi 1i n g a1 kaloids. 4 For r o u t i n e determinations o f n i c o t i n e and n o r n i c o t i n e i n tobacco samples Q u i n and Pappas extracted the a l k a l o i d s w i t h benzene-chloroform ( 1 : l ) f r o m tobacco made a l k a l i n e w i t h sodium hydroxide. N i c o t i n e was determined on a 1 m long polypropylene g l y c o l column 25 % a t 190°C and n o r n i c o t i n e on a 1 m long polybutylene g l y c o l column 25 % a t 18OoC. The n o r n i c o t i n e determination requires a column s p e c i a l l y selected t o g i v e good r e s o l u t i o n from anabasine. Myosmine. which my be present i n some tobacco samples t o an e x t e n t t h a t may i n t e r f e r e w i t h t h e n o r n i c o t i n e determination. can be detected by using a 2 m l o n g polyethylene g l y c o l column operated a t 15OOC. The method described i s most u s e f u l when the amount o f a l k a l o i d s exceeds about 0.2% o f the tobacco. The r e p r o d u c i b i l i t y o f the method i s good, as can be seen i n Table L.

I n connection w i t h studies on t h e thermal decomposition o f tobacco a l k a l o i d s Kobashi5 i n vestigated the separation and i d e n t i f i c a t i o n o f bases o f n i c o t i n e and p y r i d i n e . For the n i c o t i n e bases and r e l a t e d h i g h b o i l i n g p y r i d i n e bases (see Table 5.3) the separation was c a r r i e d out using s i l i c o n e grease and polyethylene g l y c o l 6000 columns t r e a t e d w i t h potassium hydroxide. The r e t e n t i o n times o f these compounds r e l a t e d t o n i c o t i n e on the columns mentioned are given i n Table 5.3,

together w i t h the b o i l i n g p o i n t s o f each compound. Kobashi and Watanabe6 c a r r i e d o u t determinations o f p y r i d i n e and n i c o t i n e homologs using

packed columns w i t h polyethylene g l y c o l 1500 and 6000 and support p r e t r e a t e d w i t h potassium

36 hydroxide. The determinations o f the a l k a l o i d s ' c a l i b r a t i o n curves, showing the r e l a t i o n between the r a t i o o f peaks' areas and t h a t o f the a l k a l o i d s ' weight were made t o w i t h i n 1.5 % error. TABLE 5.2 ALKALOIDAL CONTENT OF SEVERAL TOBACCOS4 Nicotine % Sample

Burley

Bright

Commercia1 C i qa r e t t e

Cigar F i l t e r

1 2 3 4 5

2.04 2.06 2.06 1.93 1.93

1.68 1.72 1.68 1.67 1.67

1.28 1.34 1.24 1.25 1.31

0.59 0.62 0.56 0.59 0.62

Average Std. dev.

2.00 0.07

1.68 0.02

1.29 0.04

0.60 0.03

Nornicotine % Nicotiana s i l v e s t r i s

1 2 3

1.53 1.56

Average

1.55

Cherry Red Nicotiana tabacum

1.41 1.41 1.41 1.41

TABLE 5.3 RELATIVE RETENTION TIMES OF N I C O T I N E BASES AND HIGH BOILING PYRIDINE BASES5 Re1a t i ve r e t e n t i o n times Compound a-AminoDvri d i ne B-Pyri dyi methyl ketone 8-Pyridyl n-propyl ketone N i co t i ne N-methyl anabasine B-hinopyridine D i hydrometanicotine Norni c o t i ne Myosmine Anabasi ne Anatabine a,a'-Oipyridyl Metanicotine Nicotyrine a,B' - D i p y r i dyl N-methyl nicotineamide Cot in ine

B. p. (OC) 211 218 245 247 121/7 mn Hg 250 141/15 mn Hg 267 118/3.2 mm Hg 146/15 nun Hg 145/10 nun Hg 2 72 110/1.5 inn Hg 280 137/4 mn Hg 210/6 nun Hg

PEG 6000

S i l i c o n e grease

0.89 0.88

0.27 0.39

2.30 2.47 2.59 4.60 2.78 2.87 3.47 5.50 10.30 14.20

1.24 1.26 1.72 1.60 1.37 1.64 1.45 1.83 1.73 3.10

I n a paper on the mechanism o f demethylation o f n i c o t i n e , Craig e t a l . '

used gas chromato-

graphy t o separate and estimate the a l k a l o i d a l products f r o m the r e a c t i o n mixture. They obt a i n e d good separation on a 2 m long column using PEG 20M 5.6 % as s t a t i o n a r y phase on F i r e b r i c k , a t 200°C. The r e t e n t i o n times o f the i n d i v i d u a l a l k a l o i d s a r e given i n Table 5.4.

References p. 60

36

TABLE 5.4 RETENTION TIMES OF INDIVIDUAL TOBACCO ALKALOIDS ON A 2 M LONG PEG 20M 5.6 % COLUMN AT 200°C; QUIN: PEG 20M, 190°C, NICOTINE 5.2 MIN; KOBASHI: PEG 6000, 220°C, NICOTINE 10.6 M I N 7

3-Pyridyl e t h y l ketone 3-Pyridyl n-propyl ketone Nicotine N-Methyl myosmi ne Norni c o t i ne Myosmine Ana ba s ine Metanicotine Nicotyrine N-Methyl n ic o t i neami de Cotinine

Time (min) 17.28 21.92 17.28 25.76 39.68 42.88 43.52 45.92 58.88 179.36 239.20

Ratio 1.00 1.26 1.oo 1.49 2.30 2.49 2.52 2.66 3.42 10.47 13.84

Quin r a t i o 1.01 1.27 1.00

Kobashi r a t i o 1.24 1.00

2.37 2.57 2.65 3.17 3.73 12.3 16.4

2.30 2.47 2.57 2.87 3.47 10.3 14.2

Weeks e t a1 .8 canpared the separation o f n i c o t i n e , nornicotine, myosmine. anabasine and anatabine on three gas chromatographic packed columns using SE-30, Versamid and OC 550 as stationary phases. Relative r e t e n t i o n times. e f f e c t i v e p l a t e values and r e s o l u t i o n are l i s t ed i n Table 5.5. TABLE 5.5 RELATIVE RETENTION TIME ( t R ) , EFFECTIVE PLATE VALUES (N) AND RESOLUTION (R) OF TOBACCO ALKALOIDS~.

2.44 m long columns packed w i t h 10 % OC 550, 5 % SE-30 o r 10 % Versamid 900 on Chromosorb W. column temperatures 17OoC. 190°C and 17OoC and c a r r i e r gas f l o w 40, 30 and 40 ml/min respectively. ~~

A1 kal o i d Nicotine Nornicotine Myosmine Anabasine Anatabine

DC '550 tR N R 1.00 2532 1.55 2320 1.87 2.03 2511 0 * 6 4 2.50 2000 2*18

-

7:::

SE-30 R tR N 1. 0 1390 l.!l 1366 1'57

1.41

1302

~~~

Versamid 900 tR N

Nicotine Myosmine Nornicotine Anabasine Anatabine

1. 0 1.i7 2.15 2.60 3.28

1258 1290 1330 1642 1580

R 9*0 Oe5' 1'78

3'28

Massingill and Hodgkins' i n a study on t h e gas chromatographic separation o f a l k a l o i d s , used four packed columns w i t h JXR (dimethylpolysiloxane), SE-52. XE-60 and Epon 1001 r e s i n as s t a t i o n a r y phases. and c a p i l l a r y columns w i t h QF-1. SE-30 and Apiezon L. N i c o t i n e and anabasine were w e l l separated on SE-52, JXR and XE-60 packed columns. On the c a p i l l a r y c o l umns n i c o t i n e and anabasine were w e l l separated on the Apiezon L column a t 125°C. b u t t a i l i n g was pronounced. The SE-30 c a p i l l a r y colunn resolved the two a l k a l o i d s mentioned, w i t h sane t a i l i n g ; the 100 f o o t QF-1 column gave good r e s o l u t i o n w i t h sharp peaks and p r a c t i c a l l y no t a i l i n g . Although t h e 100-foot QF-1 c a p i l l a r y column out-performed the o t h e r c a p i l l a r y c o l umns and gave good r e s o l u t i o n o f the a l k a l o i d s w i t h molecular weight o f up t o 303, a comparison o f the separation o f i . a . atropine-hanatropine-scopolamine showed t h a t the packed columns gave b e t t e r r e s o l u t i o n (a 6 f o o t x 1/8 inch 1 % QF-1 packed colunn and a 100 f o o t QF-1 c a p i l l a r y column). Harke and Drewsl' separated n i c o t i n e , nornicotine, anabasine and n i c o t y r i n e on a 50 m long

by 0.5 mm s t e e l c a p i l l a r y coated w i t h Ucon LB 550X (= polypropylene g l y c o l ) and KOH. However, myosmine and n o r n i c o t i n e could n o t be separated under the conditions given. A t y p i c a l chromatogram i s shown i n Figure 5.1. FIGURE 5.1

GAS CHROMATOGRAM OF TOBACCO ALKALOIDS~~ 1 = Pyridyl-n-propylketon, 2 = Nicotine, 3 = Nornicotine and Myosmine, 4 = Anabasine, 5 = N icotyrine

I

I

I

I

0

5

10

15

I

20 min

TO be able t o determine n i c o t i n e i n sub-picomole q u a n t i t i e s Neelakantan and Kostenbauer 11 used an electron-capture detector i n connection w i t h d e r i v a t i z a t i o n o f n i c o t i n e : N i c o t i n e was hydrogenated c a t a l y t i c a l l y t o y i e l d N-methyl-4- ( 3 ' - p i p e r i d y l )-n-butylamine

(= octahydro-

n i c o t i n e ) . the two secondary amino-functions o f which were t r e a t e d w i t h p e r f l u o r o p r o p i o n i c anhydride t o g i v e the d i - p e n t a f l uoropropionyl octahydronicotine. The p e r f l u o r o p r o p i o n y l der i v a t i v e o f octahydronicotine provides an e l e c t r o n capture d e r i v a t i v e which represents a means o f detecting n i c o t i n e i n amounts o f approximately 1/100th o f the minimum q u a n t i t y of n i c o t i n e t h a t i s detectable by

FID.

For the analysis o f n i c o t i n e i n t h e picogram range, H a r t v i o e t a1.l'

prepared t h e 6 - t r i -

chloroethylcarbamate d e r i v a t i v e o f n i c o t i n e , which has an e x c e l l e n t s e n s i t i v i t y i n t h e e l e c tron-capture detector. The adsorptive p r o p e r t i e s o f the derivat+ve are lower than those o f n i c o t i n e during gas chromatography. However, the formation

Gf

the corresponding o l e f i n due

t o p a r t i a l thermal dehydrohalogenation i n the i n j e c t o r and on the column i s a disadvantage o f the procedure. N i c o t i n e i s subjected t o r e a c t i o n a t 90°C w i t h t r i c h l o r o e t h y l chloroformate i n the presence o f p y r i d i n e t o form the carbamate, i n which the p y r r o l i d i n e r i n g i s opened. On heat treatment (i.a. i n the i n j e c t o r ) the carbamate p a r t i a l l y formed the corresponding o l e f i n . For q u a n t i t a t i v e determination N-n-propylnornicotine was used as an i n t e r n a l standard. The p r e c i s i o n a t the 30 ng/ml l e v e l was

RaCamncei p. 60

?

8.8 X (n = 7).

38 5.1.1.

A l k a l o i d s i n tobacco samples

4 I n 1962 Quin and Pappas used gas chromatography f o r the r o u t i n e determination o f n i c o t i n e and n o r n i c o t i n e i n tobacco samples. N i c o t i n e was determined on a 1 m long propylene g l y c o l column 25 X a t 190°C. and n o r n i c o t i n e on a 1 m long polybutylene g l y c o l column 25 % a t 180°C. The n o r n i c o t i n e determination required a s p e c i a l l y selected column t o g i v e r e s o l u t i o n f r o m anabasine. Myosmine, which may be present i n some tobacco samples t o an e x t e n t t h a t may i n t e r f e r e w i t h the n o r n i c o t i n e determination, was detected by using a 2 m long polyethylene g l y c o l column operated a t 15OoC. Yasumatsu and Murayama13 used polyethylene g l y c o l 20 M as s t a t i o n a r y phase f o r the determination o f n i c o t i n e i n tobacco samples. The a l k a l o i d s were e x t r a c t e d w i t h 0.5 N HC1 containi n g i s o q u i n o l i n e as an i n t e r n a l standard. The mixture o f the e x t r a c t and an equivalent volume o f N NaOH was i n j e c t e d i n t o the i n j e c t o r o f t h e gas chromatograph f i t t e d w i t h a soda l i m e tube. The n i c o t i n e content was determined from the peak heights o f n i c o t i n e and i s o q u i n o l i n e . The r e l a t i v e standard d e v i a t i o n was 1.9 %. I n a paper published i n 1971, Yasumatsu and Murayama14 improved the technique and the gas chromatographic conditions f o r the determination o f n i c o t i n e . Bush15 determined the f o u r most important tobacco a l k a l o i d s ( n i c o t i n e , n o r n i c o t i n e , anabasi n e and anatabine) using a 10 % DC 550 packed column on Chromosorb 60-80. and using isoquinol i n e as an i n t e r n a l standard. The a l k a l o i d s were e x t r a c t e d w i t h benzene-chloroform ( 9 : l ) a f t e r treatment o f the tobacco sample (1 g) w i t h bariumhydroxide and water. The organic phase was concentrated and used f o r the gas chromatographic determination. Because o f the g r e a t d i f f e r ences i n t h e amounts o f the minor a l k a l o i d s and n i c o t i n e i n most tobacco samples, two e x t r a c tions, each w i t h an appropriate amount o f i n t e r n a l standard, were r e q u i r e d f o r a complete assay. The p r e c i s i o n o f the q u a n t i t a t i v e analysis on tobacco samples o f d i f f e r e n t a l k a l o i d s i s given i n Table 5.6. TABLE 5.6 PRECISION OF QUANTITATIVE ALKALOID ANALYSIS ON TOBACCO SAMPLES OF DIFFERENT ALKALOID COMPOSITION~~ Sample N i c o t i n e (mg/g)

1

20.14

0.19

9.79 !0.26 (264 2 4.48).

2 3

Nornicotine (ug/g) 393

lo3

(47.6 1268

:12.0 ? 2

1.53). 45.6

Anabasine (ug/g) 65

lo3

!3.0 trace

n o t detected

Anatabine (ug/g) 408

7.0

(2.39

2

35.3

?

0.13).

lo3

1.40

A q u a n t i t a t i v e comparison o f a l k a l o i d a n a l y s i s using gas chromatography and steam d i s t i l -

l a t i o n i s given i n Table 5.7. A rapid, accurate and reproducible method t o determine very low n i c o t i n e l e v e l s i n tobacco

(0.03 19

-

3 X ) was developed by L y e r l y and Greenel'.

Samples o f 1 g tobacco were t r e a t e d w i t h

sodium hydroxide s o l u t i o n and the a l k a l o i d s e x t r a c t e d w i t h chloroform. N i c o t i n e i n t h e c h l o r o form was determined by gas chromatography on a Castorwax-KOH packed column, using n-hexadecane as an i n t e r n a l standard. The r e s u l t s obtained agreed q u i t e w e l l w i t h the r e s u l t s from o t h e r methods, e s p e c i a l l y the p i c r a t e method. To determine c e r t a i n a l k a l o i d s , o t h e r than n i c o t i n e , i n tobacco samples (nornicotine,

39 TABLE 5.7 QUANTITATIVE COMPARISON OF ALKALOID ANALYSIS USING Sample

GAS CHROMATOGRAPHY AND STEAM DISTILLATION15

Gas chromatography Nicotine (ms/s)

Tobacco 20.4 Tobacco t low-level exogenous a l k a l o i d s 30.6 Tobacco t h i g h - l e v e l exogenous a l k a l o i d s 40.0

!0.2 ? 0.9

t

0.8

Norni c o t i n e Anabasi ne (pg/g) (pg/g) 642

!32

709

20

!5 304 !7

1120

35

549

Anatabi ne Summation (pg/g) (mg)

? 768 t

109

541

!11

954

n i c o t y r i n e , anatabine, anabasinelmyosmine) Burns and C o l l i n ”

Steam d i s t i l d i s t i 11a t i o n T o t a l (mg)

8

21.69

20.25

16

32.38

31.40

!18

42.62

40.80

developed a method u s i n g a

pacKed column w i t h Carbowax 20 M and KOH as s t a t i o n a r y phase. No separation was obtained f o r anabasine and myosmine. Samples of 2 g tobacco were e x t r a c t e d by Soxhlet e x t r a c t i o n w i t h methanol. Q u i n a l d i n e ( = 2-Methylquinoline) was added as an i n t e r n a l standard. The amounts o f a l k a l o i d s were expressed as nornicotine. A t y p i c a l chromatogram o f a tobacco e x t r a c t i s shown i n Figure 5.2. FIGURE 5.2

GAS CHROMATOGRAM OF A TYPICAL TOBACCO EXTRACT17 1 = Nicotine, 2 = unknown, 3 = i n t e r n a l standard, 4 = n o r n i c o t i n e , 5 t 6 = anabasine t myosmine, 7 = s o l v e n t impurity, 8 = n i c o t y r i n e , 9 = anatabine

?

20

’h

1

I

1

r

15

10

5

0

When t h e major a l k a l o i d i n tobacco samples i s nor-nicotine,

t h e commonly used s t e a m - d i s t i l -

l a t i o n method and automated procedures r e s u l t i n poor estimates o f n i c o t i n e and n o r n i c o t i n e . Rosa18 therefore developed a pyrolysis-gas chromatographic method, whereby p y r o l y s i s was carr i e d o u t w i t h a Victoreen p y r o l y z e r f i t t e d t o t h e gas chranatograph. N i c o t i n e i s r e l a t i v e l y v o l a t i l e and r e a d i l y released by p y r o l y s i s , even a t 100°C. Nornicotine, being l e s s v o l a t i l e , showed maximum release by p y r o l y s i s a t 30OoC. The pyrolysis-gas chromatography was c a r r i e d out w i t h ca. 1 mg o f tobacco. The r e s u l t s obtained w i t h t h e method are presented i n Figure 5.3.

References p. 60

40

FIGURE 5.3

QUALITATIVE

CHROMATOGRAPHY OF THREE TOBACCO

OF PYROLYSIS-GAS

COMPARISON

Ca. 1 mg of tobacco t i s s u e was used t o produce each pyrogram; 1

-

CULTIVARP

Nicotine, 2 = Neophytadiene,

3 = Nornicotine.

ONIBOW

__?

5

r__l

min

5

5

The pyrolysis-gas chromatography o f f e r s a r e l i a b l e and r a p i d means o f e s t i m a t i n g nornicot i n e i n tobacco w i t h o u t e x t r a c t i o n . Although M a s s i n g i l l and Hodgkins2 had demonstrated i n 1965 t h a t tobacco a l k a l o i d s could be analyzed on c a p i l l a r y columns, several years passed before c a p i l l a r y gas chromatography developed a method f o r t h e o f tobacco a1 k a l o i d s became more commonly used. Severson e t a l . l9 determination o f a l k a l o i d s i n tobacco samples and f r e s h tobacco leaves, i n v o l v i n g a b r i e f s o n i f i c a t i o n - e x t r a c t i o n step w i t h subsequent c a p i l l a r y gas chromatographic separation and q u a n t i f i c a t i o n . The method permitted the screening o f numerous samples using a NitrogenPhosphorus-detector,

g i v i n g a l i n e a r and very reproducible response. However, when such a

detector was replaced by another, each was found t o g i v e a r e l a t i v e response against concent r a t i o n , so t h a t a new c a l i b r a t i o n curve had t o be s e t up. As l i t t l e as 25 mg o f ground t o bacco was extracted, w i t h 1 m l 0.05 N methanolic potassium hydroxide containing the i n t e r n a l standard (0.25 mg/ml o f 2,3'

D i p y r i d y l ) , by s o n i f i c a t i o n , and w i t h 1 p l samples i n j e c t e d . A

glass c a p i l l a r y coated w i t h Carbowax (temp. p r o g r a m i n g 170-200°C - 2OC/ TM min) and a 15 m x 0.25 mm I.D. glass c a p i l l a r y coated w i t h Superox (temp. programming 13035 m x 0.25 mn 1.0.

2OO0C 5.1.2.

- 4'C/min)

were used.

A l k a l o i d s i n tobacco smoke

Q u i n Z g 3was the f i r s t t o apply gas chromatography f o r the analysis o f n i c o t i n e a l k a l o i d s i n tobacco smoke. He used t h r e e sets o f conditions t o analyse t h e many a l k a l o i d s present i n a wide b o i l i n g range. L y e r l y and Greenel'

determined n i c o t i n e , menthol and some o t h e r non-alkaloidal c o n s t i t u e n t s

i n tobacco smoke. Single c i g a r e t t e s were smoked through a Cambrigde f i l t e r , and t h e f i l t e r

41 placed i n a s p e c i a l l y designed holder, which was heated w h i l e a l l o w i n g c a r r i e r gas t o f l o w through i t . The vapors were c o l l e c t e d and deposited on the chromatographic column. The procedure permits a puff-by-puff

analysis. Jacin e t a1."

developed a method f o r the determina-

t i o n o f n i c o t i n e i n tobacco samples, and i n t h e p a r t i c u l a t e m a t t e r o f smoke. I n the l a t t e r case t h e Cambridge f i l t e r , through which t h e smoke o f f i v e c i g a r e t t e s had been smoked, was e x t r a c t e d w i t h benzene-chlorofon (9:1),

and the e x t r a c t was gas chromatographed on a packed

column w i t h neopentyl g l y c o l adipate as s t a t i o n a r y phase. Comparison w i t h a spectrophotometric method showed t h a t t h e gas chromatographic method was a t l e a s t as good as t h e spectmphotometr i c method. I t was a l s o very simple and rapid. using a packed column w i t h 20 % Apiezon L on Chromosorb, found n i c o -

Anastasov e t al.",

t i n e , nornicotine, myosmine, anabasine and metanicotine, as w e l l as a number o f p y r i d i n e bases, i n tobacco smoke.

A r a p i d method f o r the d e t e n i n a t i o n o f n i c o t i n e i n the p a r t i c u l a t e matter o f tobacco smoke was developed by Yasumatsu et'a1.22. o f tobacco 25 %

PEG

2.34-2.52

I n a propyl alcohol e x t r a c t o f p a r t i c u l a t e matter

smoke r e t a i n e d on a glass f i b e r f i l t e r , the n i c o t i n e content was determined on a

20 M column on C e l i t e 545 a t 190°C. Analysis o f a p l a i n and a f i l t e r c i g a r e t t e gave and 1.07-2.15

mg n i c o t i n e , r e s p e c t i v e l y . Also, Kusama e t al.23 used gas chromato-

graphy t o determine n i c o t i n e (and t a r ) i n c i g a r e t t e smoke, using a column o f 5 % Castor wax on D i a s o l i d L 60-80 mesh, 1.5 m l o n g by 3 mm, a t 16OoC. The i n t e r n a l standard was n-hexadecane. There were no s i g n i f i c a n t d i f f e r e n c e s between the a n a l y t i c a l r e s u l t s o f n i c o t i n e i n c i g a r e t t e smoke obtained by gas chromatography and t h a t by a UV-method. However, the c o e f f i c i e n t s o f v a r i a t i o n o f the r e s u l t s obtained by gas chromatography were considerably lower than those obtained by UV-spectrophotometry. Ohnishi e t al.24 determined the n i c o t i n e i n c i g a r e t t e f i l t e r s and i n the main-stream smoke adsorbed by a f i l t e r by e x t r a c t i o n w i t h isopropyl alcohol c o n t a i n i n g n-hexadecane as an i n t e r nal standard. A packed glass column, 2 m long by 3 mm w i t h Castor wax 5 % as s t a t i o n a r y phase on D i a s o l i d L 60-80 mesh a t column temperature 150°C,

was used. Also, RandolphE5 used extrac-

t i o n o f Cambridge f i l t e r s w i t h dry isopropyl alcohol t o determine the n i c o t i n e content o f the p a r t i c u l a t e matter o f smoke. He used a packed column w i t h Carbowax 20 M-polyphenylether on Gas Chrom Q t r e a t e d w i t h 2 % KOH. The r e t e n t i o n times o f various tobacco a l k a l o i d s are given i n Table 5.8.

No i n t e r n a l standard was used by the q u a n t i t a t i v e determinations. Recov-

e r i e s o f n i c o t i n e over a wide range o f concentrations averaged 98 %, when n i c o t i n e was added t o the standard blended c i g a r e t t e . TABLE 5.8 RETENTION TIMES OF VARIOUS TOBACCO ALKALOIDS 6 f t long packed column (Carbowax 20 M 7 %, Polyphenyl ether (6 r i n g ) 3 %, KDH 2 % on Gas Chrom Q 80-100 mesh) a t 190°C25 N i c o t ine Anabasine Nor-nicotine

2 min 50 sec 6 min 51.6 sec 6 min 0.8 sec

Hollweg e t a1 .26 determined simultaneously n i c o t i n e and water i n tobacco smoke condensates. For the n i c o t i n e determination a packed column, 1.8 m long by 2 mm I.D.

w i t h 10 % Carborax

20 M on Chromosorb WHP 60-80 mesh was used. Since t h e column was prepared w i t h o u t a d d i t i o n o f

References p. SO

42

a l k a l i , i t l a s t e d longer. No t a i l i n g o f the n i c o t i n e peak was observed, when small amounts o f the sample were injected. Good r e p r o d u c i b i l i t y was achieved.

5.1.3. N i c o t i n e i n u r i n e Several i n v e s t i g a t i o n s have been c a r r i e d o u t t o study the content o f n i c o t i n e i n urine, i n blood and i n b i o l o g i c a l t i s s u e i n general. McNiven e t a l . 2 7 e x t r a c t e d the u r i n e a t pH 1 w i t h methylene c h l o r i d e t o remove n e u t r a l and a c i d i c m a t e r i a l , and a t pH 11 w i t h the same solvent t o i s o l a t e the n i c o t i n e . Back e x t r a c t i o n w i t h aqueous h y d r o c h l o r i c a c i d followed by b a s i f i c a t i o n and e x t r a c t i o n w i t h a c e t o n i t r i l e gave a s o l u t i o n c o n t a i n i n g the n i c o t i n e . I t was gas chromatographed on an SE-30 column a t 2OO0C using 3-methyl-3-phenylpiperidine as an i n t e r n a l standard. Recovery ranged f r o m 60 t o 95 %. Beckett and TriggsZ8 were i n t e r e s t e d i n t h e determination o f b o l i t e c o t i n i n e i n urine. An a c i d i f i e d u r i n e was e x t r a c t e d with p u r i t i e s , the u r i n e was made a l k a l i n e w i t h sodium hydroxide and d i e t h y l ether. Cotinine was e x t r a c t e d from u r i n e w i t h methylene

n i c o t i n e and i t s main metad i e t h y l e t h e r t o remove i m the n i c o t i n e e x t r a c t e d w i t h chloride a f t e r basification

o f the sample w i t h ammonia. On concentration o f the s o l u t i o n , t h e gas chromatography was c a r -

r i e d o u t on a packed column t r e a t e d w i t h KOH using Carbowax 20 M as s t a t i o n a r y phase. A r e l a t i v e recovery o f 95-100 % f o r the two a l k a l o i d s was obtained w i t h respect t o t h e i r i n t e r n a l standard, chlorophentermine f o r n i c o t i n e and l i g n o c a i n e f o r c o t i n i n e , which were added t o the u r i n e a t the s t a r t o f the assay procedure. Typical chromatograms a r e given i n Figure 4. FIGURE 5.4 CHROMATOGRAMS OF TOBACCO ALKALOIDS FROM U R I N E ~ ~ a ) 1 = d i e t h y l ether, 2 = n i c o t i n e , 3 = chlorophentermine b ) 1 = methylene c h l o r i d e , 2 = lignocaine, 3 = c o t i n i n e

.c L 1

1 5

Reprinted by permission from Nature, 211 (1966) 1415. Copyright 1966 Macmillan Journals Limited.

The same experimental conditions were used by Beckett e t a1.*'

min

to

b

f o r f u r t h e r studies o f

n i c o t i n e metabolism i n man. However, phendimetrazine was used as the i n t e r n a l standard f o r n i c o t i n e , and lignocaine f o r c o t i n i n e . For the determination o f n i c o t i n e i n urine, Can0 e t al?' used packed columns o f d i f f e r e n t

43

kinds using f i r e b r i c k , Chromosorb and Gas Chrom Q as s o l i d support KOH and Apiezon, Versamid 900

-

a l l t r e a t e d w i t h 2-6 %

and Ucon 50 HB 2000 Polar as s t a t i o n a r y phases. F i r e b r i c k

columns gave symmetrical peaks, b u t the number o f t h e o r e t i c a l p l a t e s was low; Chromosorb W gave high numbers o f plates, b u t t a i l i n g peaks. Best r e s u l t s were obtained w i t h t h e Gas Chrom Q

-

KOH (6 % )

-

Ucon 50 HB 2000 P o l a r column. For the a p p l i c a t i o n o f the gas chromatographic

method f o r determination o f n i c o t i n e i n urine, the sample (30 m l ) was made a c i d and e x t r a c t e d w i t h d i e t h y l ether, t h e n b a s i f i e d w i t h NaOH (pH 12.5) and extracted w i t h d i e t h y l e t h e r , A f t e r concentration, the e t h e r s o l u t i o n was used f o r gas chromatographic determination. Recovery: 90 %

:3 %.

The same method was used by Can0 e t al.31 f o r the determination o f n i c o t i n e i n a i r and i n the u r i n e o f smokers. A i r was pumped through 30 m l s u l p h u r i c a c i d 2N and 30 m l u r i n e was used f o r one analysis, which was c a r r i e d out as described above. The method described by Can0 e t a130*31was m o d i f i e d by Dumas e t al.32,

the l e n g t h o f the

column was reduced frm 1.5 m t o 1 m. Two column temperatures were used, 120°C and 18OoC, f o r the determination o f n i c o t i n e and c o t i n i n e , r e s p e c t i v e l y . For the e x t r a c t i o n o f t h e u r i n e samples (20 ml) chloroform was used i n s t e a d o f d i e t h y l ether. Recovery was, f o r n i c o t i n e , 90

!4 %.Dumas e t

al.33 a l s o developed a micro-method f o r the same determination i n u r i n e

and blood. Samples o f 3 m l blood were t r e a t e d w i t h ammonium o x a l a t e and f i l t e r e d . 1 m l plasma was made a l k a l i n e and e x t r a c t e d with chloroform three times, the chloroform e x t r a c t was evaporated and the residue solved i n 10 p1 o f a s o l u t i o n o f 5 ng diphenylaminelul ( i n t e r n a l standard) i n e t h y l acetate. The s o l u t i o n was used f o r the gas chromatographic assay. A s p e c i f i c N-detector was used and the s e n s i t i v i t y was 0.2 ng f o r n i c o t i n e and 1 ng f o r c o t i n i n e . The recovery i s given i n Table 5.9. TABLE 5.9 RECOVERY OF NICOTINE

AND COTININE”

Cot ini ne found nq

Nicotine Sample

added nq

found nq

%

added nq

%

~

I I1 111

20 40 40

17.5 38.08 36.84

87.5 95.2 91.1

20 40 40

18.8 38.8 38.0

94 97.2 95.2

Tausch e t a1.34 found t h a t gas chromatography o f trace amounts o f n i c o t i n e and i t s metab o l i t e s r e q u i r e d c a p i l l a r y columns o f very h i g h inertness against b a s i c compounds. Boros i l i c a t e glass columns contain r e l a t i v e l y h i g h amounts o f B203 and A1203, which a c t as strong Lewis acids. By leaching the columns w i t h aqueous HC1, such compounds can be removed. Subsequent h i g h temperature s i l y l a t i o n and coating w i t h a non p o l a r s i l i c o n e gum phase l i k e O V - 1 o r SE-30 yilelded columns o f low a d s o r p t i v i t y f o r i.a. tobacco a l k a l o i d s . The columns were h i g h l y thermostable and l o n g - l i v e d unless water o r l a r g e amounts o f i m p u r i t i e s were i n t r o duced i n t o the c a p i l l a r y . To o b t a i n s u f f i c i e n t l y i n e r t columns f o r r o u t i n e a n a l y s i s o f such r e l a t i v e l y impure e x t r a c t s , the a c i d i c leaching was followed by fanning o f a BaC03 intermediate l a y e r . This pretreatment permitted coating w i t h polyethylene g l y c o l phases l i k e Carbowax 20

M o r Pluronic

F 68. These columns e x h i b i t e d besides s a t i s f a c t o r y i n e r t n e s s a considerable

higher d u r a b i l i t y i n r o u t i n e a n a l y s i s o f plasma and u r i n e e x t r a c t s .

Refereme#p. 60

44

5.1.4.

N i c o t i n e i n blood and t i s s u e

Schievelbein and G r ~ n d k edeveloped ~~ an assay f o r t h e determination o f n i c o t i n e i n blood and tissue. The proteins were removed from blood samples by t r e a t i n g with t r i c h l o r o a c e t i c acid. The f i l t r a t e was made a l k a l i n e w i t h NaOH. MgO and NaCl were added and i t was d i s t i l l e d t o dryness. The d i s t i l l a t e was collected. made a c i d and the solvent evaporated. NaOH-solution and a c e t o n i t r i l e were added and the a c e t o n i t r i l e s o l u t i o n used f o r gas chromatography. Tissue samples were homogenized i n hydrochloric acid-methanol before e x t r a c t i o n o f the n i c o t i n e . Gas chromatography was c a r r i e d o u t on a packed column using polyglycol 4000 as s t a t i o n a r y phase on C e l i t e 60-100 mesh a t 190°C. From the method, n i c o t i n e can be estimated i n concentrations o f 0.01 pg/ml blood respectively f o r g tissue by using 5 g o f material. The use o f greater amounts o f material increases the s e n s i t i v i t y . A method f o r the determination o f submicrogram amounts o f n i c o t i n e i n blood was developed by Burrows e t al.36. Samples o f 10 m l heparinised blood were used f o r an analysis, where, a f t e r a d d i t i o n o f YaOH. the a l k a l o i d was i s o l a t e d by steam d i s t i l l a t i o n . From solvent p a r t i t i o n and column chranatographic clean-up on alumina, a n i c o t i n e s o l u t i o n i n ethanol was obtained, which was used f o r the gas chromatographic assay on Carbowax 20 M 8 % columns on Chromosorb W t r e a t e d w i t h 2 % KOH. Q u i n o l i n e was used as an i n t e r n a l standard and n i c o t i n e down t o 1 ng was determined by t h i s method. The method developed by Burrows e t a1.36 was modified by Falkman e t a1.37 t o improve t h e o v e r a l l s e n s i t i v i t y o f the method and t o decrease t h e l e v e l o f i n t e r f e r i n g co-extractives. The blood sample was a l k a l i n i z e d w i t h NaOH and s t e a m - d i s t i l l e d , t h e d i s t i l l a t e made a c i d w i t h sulphuric a c i d and p u r i f i e d by e x t r a c t i o n w i t h dichloromethane, then made a l k a l i n e w i t h NaOH and extracted w i t h dichlormethane t o i s o l a t e the n i c o t i n e . The a l k a l o i d was back e x t r a c t e d w i t h sulphuric a c i d and, a f t e r a d d i t i o n o f NaOH, extracted w i t h benzene. The benzene s o l u t i o n was used f o r the gas chromatographic analysis, which was c a r r i e d o u t under the same conditions as described by Burrows e t al.36. The o v e r a l l recovery increased f r o m 55 t o over 80 X compared w i t h the method o f Burrows e t a l . I n one paper, Isaac and Rand38 described a method f o r the determination o f n i c o t i n e i n plasma. By using an a l k a l i flame i o n i z a t i o n detector the s e n s i t i v i t y o f t h e method was i m proved t o 1 ng/ml o f n i c o t i n e i n a 2.5 m l sample. Modaline was used as an i n t e r n a l standard. The a l k a l o i d was extracted from the b a s i f i e d plasma (NaOH) w i t h d i e t h y l ether, and t h i s ext r a c t was used f o r the gas chromatographic assay on a packed column w i t h 13 % KOH and 6.5 % Carbowax 20 M as s t a t i o n a r y phase on Varaport 30. Due t o the poor r e p r o d u c i b i l i t y o f the method developed by Isaac and Rand3*, Feyerabend e t al.39 developed a b e t t e r method f o r the determination o f n i c o t i n e i n b i o l o g i c a l f l u i d s . Nicotine was extracted from a l k a l i n i z e d (NaOH) plasma i n t o d i e t h y l ether. This was concent r a t e d by evaporation, and a f t e r a c i d back e x t r a c t i o n i t was re-extracted i n t o n-heptane (Ndetector) o r dichloromethane (FID) before the gas c h r m t o g r a p h i c analysis. Q u i n o l i n e was used as an i n t e r n a l standard. The method i s r a p i d and enables concentrations o f 0.1 ng/ml-' to be measured. The Isaac and Rand38 d i r e c t e x t r a c t i o n procedure,improved by Feyerabend e t al.39 was again modified by Feyerabend and Russel4'. The e x t r a c t i o n procedure was s i m p l i f i e d and the reprud u c i b i l i t y o f the method improved. The i n t e r n a l standard ( q u i n o l i n e ) was added t o the samples (3 ml) and these made a l k a l i n e and extracted w i t h d i e t h y l ether. The solvent was evaporated

45

t o small b u l k and then e x t r a c t e d w i t h d i l u t e a c i d . An excess o f a l k a l i was added and the n i c o t i n e e x t r a c t e d w i t h b u t y l acetate. An a l i q u o t was gas chromatographed on a packed column w i t h 10 % Apiezon and 10 % KOH on Chromosorb a t 220°C u s i n g a n i t r o g e n - d e t e c t o r . Q u a n t i t a t i o n r e l i e d on t h e comparison o f peak areas and t h e c a l i b r a t i o n curve was l i n e a r over t h e concentrat i o n range, 0.5 t o 100 ng/ml-'.

N i c o t i n e concentrations as low as 0.1 ng/ml-l c o u l d be

measured. I n a d d i t i o n , a d i r e c t m i c r o - e x t r a c t i o n technique a p p l i e d t o o n l y 100 111 o f sample was developed: An aqueous s o l u t i o n o f q u i n o l i n e (0.075 pg/ml) as i n t e r n a l standard, sodium hydroxide (5M. 400 p l ) and d i - i s o p r o p y l e t h e r (50 u l ) were added t o samples (100 p l ) i n a Dreyer tube. A f t e r a g i t a t i o n on a Vortex mixer ( 1 min) the tube was c e n t r i f u g e d ( 1 min) and 5 p1 o f t h e organic l a y e r was i n j e c t e d o n t o t h e gas chromatograph. The method y i e l d s an accurate r e s u l t i n 5 min. Hengen and Hengen 41 developed a method f o r t h e determination o f n i c o t i n e and c o t i n i n e i n 1 m l samples o f plasma. N i c o t i n e was e x t r a c t e d f r o m a l k a l i n i z e d plasma (NaOH) w i t h d i e t h y l ether, and c o t i n i n e from the same sample w i t h dichldromethane. Modaline was used as i n t e r n a l standard f o r n i c o t i n e , l i d o c a i n e f o r c o t i n i n e . A n a l y t i c a l recovery o f n i c o t i n e added t o the plasma was 80

6 %, f o r c o t i n i n e 95

5

%.The

i n t e r n a l standards were d i r e c t l y added t o t h e

plasma t o monitor e x t r a c t i o n losses. The s e n s i t i v i t y was such t h a t l e s s than 0.1 ug o f n i c o t i n e and 0.1 pg o f c o t i n i n e could be detected per l i t e r . Day-to-day r e p r o d u c i b i l i t y f o r n i c o t i n e was w i t h i n 14 % and w i t h i n 6 % f o r c o t i n i n e . Narrow peaks f o r the gas chromatographed compounds were obtained on the very s t a b l e SP-2250 column ( 3 %) on Supelcoport a t 155OC f o r n i c o t i n e and 190°C f o r c o t i n i n e . P i l o t t i e t a l . 4 2 c a r r i e d o u t studies on the i d e n t i f i c a t i o n o f tobacco a l k a l o i d s , t h e i r mamnalian m e t a b o l i t e s avd r e l a t e d compounds by gas chromatography-mass spectrometry u s i n g packed columns (SE-30, SE-52 and Carbowax 20 M

0 and 9.6 m

-

+

KOH) and c a p i l l a r y columns (33 m

-

Emulphor

OV-101). Various p y r i d i n e compounds, e i t h e r i d e n t i f i e d o r i m p l i e d as i n t e r m e d i -

ates i n t h e mamnalian metabolism o f n i c o t i n e present i n tobacco o r tobacco smoke, were s t u d i e d by GC-MS. P r e l i m i n a r y GC-MS experiments on t h e determination o f n i c o t i n e u s i n g c a p i l l a r y columns i n combination w i t h m u l t i p l e i o n d e t e c t i o n (MID) employing deuterated n i c o t i n e as i n t e r n a l standard were reported. The gas chromatographic data o f t h e compounds i n v e s t i g a t e d a r e given i n Table 5.10. Dow and H a l l 4 3 a l s o used c a p i l l a r y column combined gas chromatography-mass spectrometry. They developed a method f o r the e s t i m a t i o n o f n i c o t i n e i n plasma by s e l e c t i v e i o n monitoring.

A glass c a p i l l a r y coated w i t h SP-1000 was attached d i r e c t l y t o a mass spectrometer. which was operated i n the S I M ( s e l e c t i v e i o n m o n i t o r i n g ) mode. N i c o t i n e could be determined down t o 3 m l samples o f plasma, The e x t r a c t i o n procedure was a m o d i f i c a t i o n o f the method des c r i b e d by Feyerabend e t a1.39, which allowed t h e d i r e c t a d d i t i o n o f the i n t e r n a l standard ( q u i n o l i n e ) t o plasma p r i o r t o e x t r a c t i o n . The c a l i b r a t i o n curve was constructed b y p l o t t i n g the r a t i o o f t h e peak heights o f t h e m/e 84 i o n o f n i c o t i n e and t h e m / e 129 i o n o f q u i n o l i n e a g a i n s t the concentration o f n i c o t i n e (ng/ml). This p l o t was l i n e a r over t h e c o n c e n t r a t i o n range 5-100 ng n i c o t i n e / m l . The method i s s e n s i t i v e and s p e c i f i c w i t h o u t the need f o r deuterated n i c o t i n e as i n t e r n a l standard. Kogan e t al.44 described a method f o r simultaneous determination o f n i c o t i n e and c o t i n i n e i n plasma using ketamine as i n t e r n a l standard. A f t e r b a s i f i c a t i o n o f t h e sample t h e a l k a l o i d s and the added i n t e r n a l standard were e x t r a c t e d with methylene c h l o r i d e , back-extracted i n t o

R e h r s n c a p. SO

46

acid, and then re-extracted i n t o methylene c h l o r i d e . Glass columns (1.8 m by 2 mm I.D.) packed w i t h 3 % SE-30 on Gas Chrom Q 100-120 mesh and temperature programming from 150 t o 2OO0C (24'C/min)

and n i t r o g e n detection were used. Detector response was l i n e a r over a range

o f 2 t o 50 ng/ml n i c o t i n e and 50 t o 500 ng c o t i n i n e TABLE 5.10 GAS CHROMATOGRAPHIC DATA OF TOBACCO ALKALOIDS AND THEIR MAMMALIAN METABOLITES4'

A B C D E

= = = = =

Retention Retention Retention Retention Retention

times times times times times

on on on on on

the the the the the

SE-30 column ( a ) = Mixture o f R,S- and S,S-diastereoisomers Carbowax (KOH) column Emulphor-0 glass c a p i l l a r y column ( b ) = P a r t l y decomposed i n t o n i c o t i n e d u r i n g GLC SE-52 column OV-101 glass c a p i l l a r y column(c)= Mixture o f enantiomers

Compound Ia Ib

Retention times

S-( -)-Nicotine

5-d2-S-( -)-Nicotine

I1 S- (-)-Nicotine isomethi odide hydroiodi de (a) 111 S-(-)-Nicotine-1'-oxide IV S- ( - ) - N ic o t ine- 1,l' -d io x i de (a ) S- (-)-Norni cot ine V VI V II

Myosmi ne 8-Nicotyrine

V I I I S-(-)-Anabasi ne IX

S- (-)-Methylanabasine

X

S-(-)-Cotinine

S- (-)-Cotinine methiodi de XI X I I S- (-)-Cotinine-1-oxi de X I 1 1 S- (-)-Norcotini ne

XIV

(5S,3R)-3-Hydroxycotinine

xv

D i hydrometan ic o t i ne

xv I

XV I I c i s-Metani c o t i n e X V I I I 4-( 3-Pyridyl ) b u t y r i c a c i d X I X Methyl 4-(3-pyridyl ) b u t y r a t e XX 4-(3-Pyridyl)-4-oxobutyric a c i d X X I Methyl 4-(3-pyridyl)-4-oxobutyrate

5.0

,. L

4.7/142OC

B

10.6

C(b)

9 .o/90°c 10.7 5.9/900& 5.0/100 C 13.2 5.6/ 100°C 10.8 4.5/1OO0C 5.3 5. 8/13OoC 7.0/213OC 23.2 3. 2/17OoC 5.4

A C A A C A C A C A B C D E

6.8/95'C

X X I I 4-(3-Pyridyl)-4-hydroxybutyric a c i d ( c ) X X I I I Methyl 4-(3-pyridyl)-4-hydroxybutyrate

XXV 4-(3-F?yridyl)-4-oxobutyramide X X V I A1 lohydroxycoti nine ( c )

A B

5.9/ 135OC 10.9/265OC 4.4/17OoC 5.7/14OoC 4.6/ 17OoC 4.9/105OC 9.9 4.3/ 110°C 13.5 3.8/11OoC

trans-Metanicotine

X X I V 5-(3-Pyridyl)tetrahydrofuran-2-one

6.0/85'& 5.3/135 C

(c)

A B D A D A

GC-MS GC-MS D i r e c t probe D i r e c t probe D i r e c t probe GC-MS

GC-MS GC-MS

GC-MS GC-MS GC-MS

D i r e c t probe D i r e c t probe GC-MS GC-MS GC-MS

C

A

GC-MS

C

A A

4.3/125OC 19.1

A C

5. 9/14OoC

A C

19.0 6.0/140°C 23.2

Mode o f recording MS

GC-MS D i r e c t probe GC-MS D i r e c t probe GC-MS D i r e c t probe GC-MS GC-MS

5.8

A C E

6.7/145OC 5.6/14OoC 6.8/2 13OC 3 .O/17O0C

A A B D

GC-MS GC-MS

41

TABLE 5.10 (continued) Compound

Retention times ~~~

XXVII X X V I I1

3-Pyridylacetic acid Methyl 3 - p y r i dylacetate

XXIX XXX

N-( 3 - p y r i d y l a c e t y l ) g l y c i n e Methyl N-(3-pyridylacetyl)glycinate

Mode o f recording MS ~~

3.3/85'C 7.9

A

5.7/15OoC

A

D i r e c t probe GC-MS

C

D i r e c t probe GC-MS

I n order t o detennine n i c o t i n e and c o t i n i n e i n low nanogram l e v e l s i n 1 g samples o f t i s s u e homogenates. t h a t are more complex b i o l o g i c a l matrices than b i o l o g i c a l f l u i d s , Thompson e t a1.45 used 12 m long fused s i l i c a c a p i l l a r y columns deactivated w i t h Carbowax 20 M and coated w i t h a dimethyl s i l i c o n e l i q u i d . Close s t r u c u r a l analogues, methylanabasine ( f o r n i c o t i n e ) and 1'-trideuteromethyl-nornicotine ( f o r c o t i n i n e ) were used as i n t e r n a l standards. N i c o t i n e and c o t i n i n e were e x t r a c t e d separately a f t e r a d d i t i o n o f NaOH t o the homogenate, w i t h e t h y l acetate ( n i c o t i n e ) and toluene ( c o t i n i n e ) . N i c o t i n e was gas chromatographed by 8O-15O0C and c o t i n i n e by 120-200°C

-

both 4'C/min.

For the n i c o t i n e determination a n i t r o g e n -

detector was used. Cotinine could o n l y be q u a n t i f i e d by chemical i o n i z a t i o n GC-MS methods. Jacob e t al.46 developed a method f o r the determination o f n i c o t i n e and c o t i n i n e i n plasma and u r i n e samples using s t r u c t u r a l analogues f o r both compounds as i n t e r n a l standards, N-ethyl n o r n i c o t i n e f o r n i c o t i n e and N- (2-methoxyethyl )-norni c o t i n e f o r c o t i n i n e . Glass columns (1.8 m f o r n i c o t i n e and 1.2 m f o r c o t i n i n e , by 2 mn I . D . )

packed w i t h 2 % Carbowax

20 M t 2 % KOH on Gas Chrom P 100-120 mesh o r 3 % SP-2250 DB on Supelcoport 100-120 mesh were used, and column temperatures 145OC f o r n i c o t i n e and 21OoC f o r c o t i n i n e . The a l k a l o i d s were e x t r a c t e d from the sample w i t h d i e t h y l ether ( n i c o t i n e ) o r butanol ( c o t i n i n e ) a f t e r a d d i t i o n o f NaOH, back e x t r a c t i o n i n t o a c i d and r e - e x t r a c t i o n i n t o d i e t h y l e t h e r ( n i c o t i n e ) o r methylc h l o r i d e ( c o t i n i n e ) . L i n e a r i t y i n t h e range o f 0-100 ng/ml n i c o t i n e and 0-1000 ng/ml c o t i n i n e was achieved by means o f a nitrogen-detector.

A special a p p l i c a t i o n o f gas chromatographic determination o f n i c o t i n e i n b i o l o g i c a l f l u i d s i s i t s determination i n the breast f l u i d o f non-lactating women. By using a canbinat i o n o f gas chromatography, mass spectrometry and a selected i o n recording technique, Petrak i s e t a l . 4 7 i d e n t i f i e d n i c o t i n e and i t s major m e t a b o l i t e c o t i n i n e i n the b r e a s t f l u i d o f non-lactating women smokers. As l i t t l e as 25 picograms could be measured by u s i n g t h e deuter2 ated variants, (5',5'-2H)-nicotine and (3.3- H)-cotinine, both as i n t e r n a l standards and as c a r r i e r s i n an inverse isotope d i l u t i o n method.

5.1.5.

M i scel laneous

5.1.5.1.

Nicotine i n residues on foods

Martin48 used ?as chromatonraphy t o determine n i c o t i n e residues on mustard oreen samples. Samples o f mustard green were analysed by t h e US o f f i c i a l method, m o d i f i e d by t h e author f o r gas chromatography on a packed DC-200 column 10 % on Gas Chrom

4. The i d e n t i t y o f the

n i c o t i n e peak was confirmed by TLC a f t e r t r a p p i n n the eluated comnound from the ?as chromatographic column. Recovery obtained on f i v e samples spiked a t the 1-3 ppm l e v e l ranned from

95 t o 97

%.

Reference8 p. 60

48

5.1.5.2.

Biosynthesis of n i c o t i n e a l k a l o i d s

4 Almost the same method as described by Quin and Pappas was used by Alworth e t al.49 f o r i n v e s t i g a t i o n s on the biosynthesis o f n i c o t i n e i n Nicotiana g l u t i n o s a . The assay o f n i c o t i n e was c a r r i e d o u t on the a e r i a l and r o o t sections o f the p l a n t separately. Polybutylene g l y c o l 10 % on F i r e b r i c k was used, a t column temperature 169OC. For studies on the i n t e r r e l a t i o n s s h i p among n i c o t i n e , nornicotine, anabasine and anatabine t ~ ~ almost the same during the biosynthesis i n Nicotiana g l u t i n o s a , Alworth and R a p ~ p o r used gas chromatographic conditions as reported above. On a polybutylene g l y c o l column the a l k a l oids mentioned were s a t i s f a c t o r i l y resolved. 5.1.5.3.

Airborne n i c o t i n e

A method f o r the determination o f . a i r b o r n e n i c o t i n e was developed by Crouse e t al.51.

The

a i r was taken up i n water by suction o f the a i r through a tube w i t h water. The s o l u t i o n obt a i n e d i n t h a t way was gas chromatographed d i r e c t l y on a 58 inch. x 3 nun 0.0. glass column packed w i t h 5 % polyphenyl e t h e r (6 r i n g ) , on Anakrom ABS 110-120 mesh a t 190°C using FIO. The minor airborne tobacco a l k a l o i d s , nornicotine, myosmine and anabasine, d i d n o t i n t e r f e r e w i t h n i c o t i n e . The standard curve was l i n e a r over a range o f 10-500 pg/ml w i t h a r e l a t i v e standard d e v i a t i o n o f 95 % f o r 10-50 pg/ml. TABLE 5.11 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY OF TOBACCO ALKALOIDS Col umn

Sol i d support mesh

Stat.phase

glass, 1 m x 6 nun 0.0.

Fib. BW Fib. BW Fib. BW

PEG PPG PBG PPG PBG PEG PEG PPG

glass, 1 m x 6 nun 0.0.

glass, 1 m x 10

mm 0.0.

glass, 1 m x 6 mm O.D. glass, 1 m x 10 mn 0.0. glass, 1 m x 6 mn 0.0. glass, 2 m x 6 mm O.D. glass, 1 m x 14 mn 0.0.

%

Temperature

Cap.Prep.

Ref.

20M 1025 1500

25 25 25

190°c 190°c 180OC

s.to.alk.

1

1025 1500 20 M 4000 1025

25 25 25 25 25

145OC 190°c 190°c 190°c 190°c

s.alk.to.sm.

2

Fib. BW Fib. BW Fib. BW

PPG 1025 PEG 20 M PPG 1025

25 25 25

190°c 190°c 190%

s.alk.to.sm.

3

Fib. BW 30-60 Fib. BW Fib. B W 30-60 Fib.

PPG PBG PEG PPG

25 25 25 25

190°c 18OoC 18OoC 18OoC

Fib. Fib. Fib. Fib. Fib.

BW BW BW BW BW

no i n f .

1025 1500 20 M 1025

n i .qnt. t o . noni.qnt.to. noni .my. s . n i noni pre.

.

.

Sil.gr. t KOH

PEG 6000

a1k.s.

5

alk.qnt.

6

t KOH

PEG 1500

14OoC

t KOH

glass, 2 m x 5

mn

Fib.

glass, 2.44 m x 3.5 mn

I.D.

CW AWS 60-80 CW AWS 60-80

PEG 6000 t KOH PEG 20 M

SE-30 Ver.

21oOc 5.6

20oOc

a1k.s.

7

5 10

1900c 17OoC

a1k.s.

8

49

TABLE 5.11 (continued) Column glass, 2.44 m x 3.5 mn

Solid support CW AWS 60-80

Stat.phase DC 550

1.0. cop., 6 ft x 1/8 in 0.0. GP 100-120 S.S. S.S. S.S. S.S.

cap. cap. cap. cap.

100 200 100 50

Dia S 80-100 Dia S 80-100 ft x 0.01 in 1.0. f t x 0.01 in I.D. ft x 0.01 in 1.0. m x 0.5 mm

glass, 2 m x 2 mn I.D.

CG 100-120

glass, 0.9 m x 2 mn I.D. CW 80-100 t

glass, 2.1 m x 2 mm 1.0. GZ 80-100 glass, 1.2 m x 2 mm I.D. GQ 80-100 t t

glass cap. 10 m x 0.77 mn 1.0. no inf. Cel 545 glass, 3.05 m x 2 mn I.D.CW AWS 60-80 s . s . , 3 ft x 1/8 in CW 60-80 Diat M 80-100 t

no inf. 6 ft

sup 80-100

no inf.

sup 80-100

t t

3 ft

JXR 1 SE-52 1 XE-60 1 QF-1 SE-30 Apiezon L Ucon LB 550 X t KOH OV-17 1'25 ov- 1 OV-17 3 OV-17 3 OV-17 Cab 20 M 0.6 terpht. a.

;

SE-30 PEG 20 M

t

glass, 4 m x 3 mn 1.0.

t t

glass cap. 35 m x 0.25 mn I . D .

%

10

DC 550 Castor KOH Cab 20 M KOH Cab 20 M Ppe 6 KOH Cab 20 M Ppe 6 KOH Cab

25 10 10 3 6 1

Temperature 17OoC 100-300°C 100-300°C 10O-25O0C 100-zoooc 100-250°C 100-250°C 195OC

Comp.Prep. a1k.s.

pr. pr. pr. pr. pr. pr. a1k.s.

10

ni .qnt.der.

11

ni .qnt.der.

12

2oooc 200-220°C 185OC 160'C

ni .qnt. alk.qnt.to. ni.qnt.to.

13 14 15 16

180°C

a1 k.qnt. to.

17

$$

.

,

210°c 225OC 2oooc

18 180-200°C pr. pyr.to.alk. 1°C/min

I ~ O - ~ O Opr. ~C

glass cap. 15 m x 0.25 mn 1.0.

I ~ O - ~ O Opr. ~C

GQ 80-100

cw Cel 545 Dias L 60-80 Dias L 60-80 GQ 80-100 t t

NGA Apiezon L PEG 20 M Castor Castor Cab 20 M Ppe 6 KOH Cab 20 M

glass, 1.8 m x 2 n I.D. CW HP glass, 12 ft x 3-4 m I . D . Ana ABS 100-110 SE-30 s . s . , 1 m x 1/8 in O.D. Dia S 80-100 Cab 20 M t KOH s . s . , 1.5 m 1/8 in Fib. C 22 AW Apiezon t KOH s . s . , 1.5 m x 1/8 in Fib. C 22 AW Ver t KOH s . s . , 2.1 m x 1/8 in CW AWS Ver t KOH s . s . , 1.5 m x 1/8 in GQ Uc.HB Po t KOH s . s . , 1.5 m x 1/8 i n 0.0. GQ Uc.HB Po t KOH

References p. 50

8

9

2 Chin

glass, 6 ft x 1/8 in no inf. no inf. glass, 1.5 m x 3 mm glass, 2 m x 3 mm s . s . , 6 ft x 1/8 in

Ref.

10

4'~/mi n 168OC

19 20

190°C 16OoC 15OoC 170-190°C

ni.qnt.to.sm. a1k.to.m. ni .qnt. to.sm. ni.qnt.to.sm. ni.qnt.to.sm. ni.qnt.spm.

21 22 23 24 25

no inf.

ni.to.sm.

26

ni.qnt.ur. ni .qnt.ur. cot. ant. ur.

27

ni.qnt.ur.

30 31

20

25 5 5 7 3

to.alk. qnt.

L

10

14.5 200°C 2 135OC 5 20goc 185OC

2" 2"

9ooc

28,29

13OoC

2.2

12oOc

ni.qnt.ur.

63.2

120°c

ni.cot.qnt.ur.

32

so TABLE 5.11 (continued) Col umn 5.5..

S o l i d support

1 m x 1/8 i n 0.0. GO 100-120

glass cap. 20-28 m x 0.32-0.34

mm 1.0.

glass, 1.8 m x 1/4 i n glass, 2 m x 4 mm 1.0.

Cel 60-100 CW AWS 80-100

glass, 2 m x 3 mn 1.0.

CW AWS 80-100

s.s..

5 f t x 1/8 i n

Uc.HB Po t KOH SE-52 PEG 4000 Cab 20 M t KOH Cab 20 M t KOH

Var 30 80-100

glass S, 6 ft x 6 mm

CW 80-100

glass S, 6 f t x 6 mn

CW 80-100

glass, 1.8 m x 2 mn I.0.Sup 100-120 glass. 1.7 m x 1 mn 1.0. GQ 100-120

X

Stat. phase

Cab 20 M t KOH Apiezon L t KOH Apiezon L t KOH SP-2250-DB

3.2

6

8 2 8 2 6.5 13 10 10 10 10 3

Cab 20 M KOH SE-30 t KOH SE-52 t KOH Emulphor 0 ov-101

8 2 3 2 5 2

t

GQ 100-120 GQ 100-120

cap. 33 m x 0.4 mn 1.0. cap. 9.6 m x 0.2 nwn

SP-1000

glass, 1.8 m x 2 m 1.0. GQ 100-120

SE-30

f . s i 1 . cap. 12 m x 0.2 mn 1.0.

Dim.si1 . l i q u .

glass, 1.2 m x 2

mn 1.0. GP 100-120

mn 1.0.

GP 100-120

glass, 2 in x 2 mm glass, 6 f t x 4 mn I.D. s . s . , 5 ft x 0.5 i n

GQ Fib. 60-80

s . s . , 5 f t x 114 i n

Fib. 60-80

SE-30 t KOH

SP 2250 DB t KOH Cab 20 M t KOH DC-200 PBG t KOH PBG

-

15OoC

ni.qnt.bl.

36

15OoC

ni.qnt.bl.

37

146OC

ni.qnt.pl.

38

17OoC

n i .qnt . b i f l . 3 9

22oOc

ni.qnt.bifl.40

155OC 190°c

ni.qnt.pl. 41 cot. qnt. p l .

142OC a l k . t met. 42 p l . MS 1g5-200' 6 Chin

glass cap. 20 m x 0.3 mm I.D.

glass, 1.8 m x 2

Tenperature Comp.Prep. Ref. 118-18OoC pr. ni.cot.qnt. 33 pl. ur. LO-C/min 4OUC p r . 30UC/min 8OoC D r . 10°C/min n i . t met 34 230'C' ni.bl.tis. 35 190°c

pr

160°C 3

2 2 3 2 2 2 10 10 10

158-2OO0C p r . 24 C/mgn 88-150 pr. 4 C/min ~ ~ o - ~ opr.o 4 Chin

~

ni.qnt.pl. 43 SIM n i . c o t . q n t . 44 PI. ni. c c o t . qnt. ti s .45

14OoC

ni.

21oOc

cot.

1 3 0 pr. ~~ 5 C6min 125 C 169OC

n i .qnt.brfl.47 MS n i res. food 48 alk. biosynth. 49

180-200°C

a l k . biosynth.50

190°c

alk. a i r .

q n t . p l . 46

.

t KOH

glass, 50 i n x 3 mn 0.0. Ana A8S 110-120

Ppe 6

5

51

5.2. REFERENCES

1 2 3 4 5

L.O. Quin, Nature, 182 (1958) 865. L.D. Quin. J. org. chem., 24 (1959) 911. L.D. Quin, J. Org. chem., 24 (1959) 914. L.D. Quin and N.A. Pappas, J. dgric. Food Chem., 10 (1962) 79. Y. Kobashi, Nippon m g a k u Zasshi, 82 (1961) 1262; C . A . , 58 (1963) 11411 e. 6 Y. Kobashi and M. Watanabe, Nippon Kagaku Zasshi, 82 (1961) 1265; c . d . , 58 (1963) 11676 n. 7 J.C. Craig, N.Y. Mary, N.L. Goldman and L. Wolf, J. m. Chem. S O C . , 86 (1964) 3866. 8 W.W. Weeks, D.L. Davis and L.P. Bush, J. Chromatogr., 43 (1969) 506. 9 J.L. M a s s i n g i l l Jr. and J.E. Hodgkins, Anal. Chem., 37 (1965) 952. 10 H.-P. Harke and C . 4 . Drews, Fresenius'Z. Anal. Chem., 242 (1968) 248. 11 L. Neelakantan and H.B. Kostenbauer, Anal. Chem.. 46 (1974) 452. 12 P. Hartvig, N.-0. Ahnfelt, M. Hammarlund and J. Vessman, J. Chromatogr., 173 (1979) 127.

51 13 N. Yasumatsu and T. Murayama, Hatano ~abakoShikenjo Hokoku, 6 8 (1969) 75; c . A . , 75 (1971) 1501 z. 14 N. Yasumatsu and T. Murayama, Hatano Tabako Shikenjo Hokoku, 70 (1971) 111; c . A . , 79 (1973) 2229 m. 15 L.P. Bush, J . Chromatoqr., 73 (1972) 243. 16 L.A. L y e r l y and G.H. Greene, B e i t r . Tabaksforsch., 8 (1976) 359. 17 D.T. Burns and E.J. C o l l i n , J . Chromatoqr., 133 (1977) 378. 18 N. Rosa. J . Chromatour.. 171 (1979) 419. 19 R. F. Severson, K. L. k D u f f i e , ' R . F . 'Arrendale, G.R. Gwynn, J.F. Chaplin and A.W. Johnson, J . Chromatoqr., 211 (1981) 111. 20 H. Jacin, J.M. Slanski and J. Moshy, A n a l . Chim. A c t a , 41 (1968) 347. 21 A. Anastasov, J.G. Mokhnachev and N.A. Sherstyanykh, Tabak(Moscow), 29 (1968) 54; c . d . , 71 (1969) 922 t . 22 N. Yasumatsu, 0. Yoshida and T. Murayama, Hatano ~abakoShikenjo Hokoku, 70 (1971) 119; C.A., 79 (1973) 2230 e. 23 M. Kusama, K. Watanabe, S. Ogihara and Y. Kobashi, Nippon Sembai Kosha Chuo Kenkyusho Kenkyu Hokoku, 114 (1972) 89; C . A . , 79 (1975) 134478 b. 24 A. Ohnishi, Y . Akinaga, K. Kobayashi, M. I s h i i , K. Maeda and M. Uehara, uippon sembai Kosha Chuo Kenkyusho Kenkyu Hokoku, 114 (1972) 97; C . A . , 79 (1973) 25 H. R. Randolph, TO^. sci. , 18 (1974) 137. 26 J . Hollweg, H.-J. Schumacher and F. Seehofer, B e i t r . Tabaksforsch. I n t e r n a t . , 11 (1981) 39 27 N.L. McNiven, K.H. Raisinghani, S. Patashnik and R.J. Dorfman, Nature, 208 (1965) 788. 28 A.H. Beckett and E.J. Triggs, 29 A.H. Beckett, J.W. Gort-ud and P. Jenner, 30 J.-P. Cano, J. Catalin, R. Bade, C. Dumas, A. V i a l a and R. G u i l l e m e , Ann. Pharm. F r . , 88 (1970) 581. 31 J.-P. Cano, J . Catalin, R. Badr$. C. Dumas, A. V i a l a and R. Guillerme, m. Pharm. F r . , 28 (1970) 633. 32 C. Dumas, A. Ourand, R. Badre, J.-P. Cano, A. V i a l a and R. Guillerme, Eur. J . T o x i c o l . , 8 (1975) 142. 33 C. Dumas, R. Badre, A. Viala, J.-P. Can0 and R. Guillerme, E u r . J. T o x i c o l . , 8 (1975) 280. 34 H. Tausch, J. Kainzbauer and F. Schneider, 4th Proeed. rnt. Symp. C a p i l l a r y Chromatoqr. 1981, 335. 35 H. Schievelbein and K. Grundke, Frsenius' 2. Anal. Chem., 237 (1968) 1. 36 I .E. Burrows, P.J. Corp, G.C. Jackson and B.F.J. Page, A n a l y s t , 96 (1971) 81. 37 S.E. Falkman, J.E. Burrows, R.A. Lundgren and B.F.J. Page, A n a l y s t , 100 (1975) 99. 38 P.F. Isaac and M.J. Rand, Nature, 236 (1972) 308. 39 C. Feyerabend, T. L e v i t t and M.A.H. Russell, J . Pharm. Pharmaml., 27 (1975) 434. 40 C . Feyerabend and M.A.H. Russell, J . Pharm. Pharmacol., 31 (1979) 73. 41 N. Hengen and M. Hengen, C l i n . Chem. (Winston-Salem, N . C . ) , 24 (1978) 50. 42 8 . P i l o t t i , C.R. Enzell, Fr. H. McKennis, E.R. Bowman, E. Dufva and B. Holmstedt, B e i t r . Tabaksforsch., 8 (1976) 339. 43 J. Dow and K. H a l l , J . Chromatogr., 153 (1978) 521. 44 M.J. Kogan, K. Verebey, J.H. Jaffee and S.J. Mule, J . Forensic s c i . , 26 (1981) 6. 45 J.A. Thompson, Ming-Shan Ho and D.R. Petersen, J . Chromatogr., 231 (1982) 53. 46 P. Jacob, 111, M. Wilson and N.L. Benowitz, J . Chromatoqr., 222 (1981) 61. 47 N . Petrakis, L.O. Gruenke, T.C. Beel, N. Castagnoli and J.C. Craig, Science, 199 (1978) 303. 48 R.J. Martin, J . dssoc. off. A n a l . Chem., 50 (1967) 939. 49 W.L. Alworth, R.C. De Selms and H. Rapoport, J . dm. Chem. S O C . , 86 (1964) 1608. 50 W.L. Alworth and H. Rapoport, Arch. Biochem. Biophys., 122 (1965) 45. 51 W.E. Crouse, L.F. Johnson and R.S. Marmor, B e i t r . 'Tabaksforsch., 10 (1980) 111.

62

TABLE 5.12 TOBACCO ALKALOIDS

-

LIST OF ABBREVIATIONS

ABS = acid, base washed, s i l a n i z e d alk alkaloid Ana = Anakrom anab = anabasine AW = a c i d washed b i = biological material b i f l = biological f l u i d b l = blood b r f l = breast f l u i d BW = base washed Cab = Carbowax cap = c a p i l l a r y Castor = Castorwax Cel = C e l i t e CG = Chromosorb G c i f = cigarette f i l t e r comp = compound cop = copper cot = cotinine der = d e r i v a t i v e Dia S = Diatoport Dias L = D i a s o l i d L D i a t = Diatomite Dim.si1. l i q u . = Dimethylsi l i c o n e 1i q u i d ECD = e l e c t r o n capture d e t e c t o r Fib = firebrick f . s i l = fuse1 s i l i c a GP = Gas Chrom P GQ = Gas Chrom Q GZ = Gas Chrom Z HP = h i g h performance I.D. = i n n e r diameter i d = identification i n = inch i s = i n t e r n a l standard JXR = dimethylpolysiloxane

met = m e t a b o l i t e MF = mass fragmentography MD mass spectrometry my = myosmine ni = nicotine no i n f . = no i n f o r m a t i o n noni nornicotine ND = n i t r o g e n d e t e c t o r NGA = neopentyl adipate O.D. = o u t s i d e diameter PBG = polybutylene g l y c o l PEG = polyethylene g l y c o l pl plasma PPG = polypropylene g l y c o l Ppe6 = polyphenylether ( 6 - r i n g ) pre = preparative pur = purification pyr = pyrolysis qnt = quantitative s = 'separation S = silanized s i l . g r . = s i l i c o n e grease SIM = s l e c t i v e i o n monitoring sm = smoke spm = smoke p a r t i c u l a t e matter s.s = s t a i n l e s s s t e e l Sup = Supelcoport terpht = terephtalic acid t i s = tissue t o = tobacco ur = u r i n e Uc HB Po = Ucon HB 2000 P o l a r Var = Varaport Ver = Versamid 900

Chapter 6 PIPERIDINE ALKALOIDS

....................................................... ...................................................

6.1 P i p e r n i g r m a l k a l o i d s C o n i m macuiatm a l k a l o i d s 6.3 References

6.2

53 54 54

...................................................................

6.1 PIPER NIGRUM ALKALOIDS Piperine ( 1 - p i p e r o y l p i p e r i d i n e ) . a l k a l o i d from P i p e r nigrum

L., belonged t o the group o f

a l k a l o i d s which, i n 1960, was gas chromatographed on a packed SE-30 column by L l o y d e t a l . C a p i l l a r y gas chromatography and c o l d on-column i n j e c t i o n was used by Verzele e t a1.'

1.

for

q u a n t i t a t i v e determination o f p i p e r i n e i n pepper and pepper e x t r a c t . Piperine was e x t r a c t e d from samples o f 70 mg ground pepper o r 10 mg pepper e x t r a c t w i t h dichloromethane c o n t a i n i n g the i n t e r n a l standard, tetrahydropiperine, and the dichloromethane e x t r a c t obtained was used f o r gas chromatography. A 25 m by 0.5 mn 1.0. glass c a p i l l a r y column, deactivated by hightemperature s i l y l a t i o n and coated w i t h OV-1, was used. The samples were i n j e c t e d a t an oven temperature o f 100°C, and the temperature was immediately increased t o 25OoC. Chromatography o f pure p i p e r i n e (0.435-0.027 mg) against a constant amount o f the i n t e r n a l standard gave a s t r a i g h t - l i n e c a l i b r a t i o n graph passing through the o r i g i n . Typical gas chromatograms are shown i n Figure 6.1.

Good r e s u l t s were obtained, w i t h a standard d e v i a t i o n f o r a pepper

sample ( n = 5) o f 1.03 %. FIGURE 6.1 GLASS CAPILLARY GC ANALYSIS WITH COLD ON-COLUMN INJECTION OF PIPERINE' A = pure p i p e r i n e and B = pepper e x t r a c t . I n t e r n a l standard (1) tetrahydropiperine, and p i p e r i n e ( 2 ) . Column: 25 m x 0.5 mn I.D. HTS-OV-1 column, 25OoC isothermal.

1

A

B

2

L

L

L

120

lbV250'

References p. 64

ISOTHERMAL

64

6.2 CONIUMMACULATUM ALKALOIDS Moll

3

gas chromatographed some Conium a l k a l o i d s and a number o f o t h e r p i p e r i d i n e bases.

A good separation was obtained on a packed column o f 30 % polyethylene g l y c o l 4000 on s i l i c a gel impregnated w i t h 20 % potassium hydroxide. Potassium hydroxide was used t o reduce adsorption t o the s o l i d support. The r e s u l t s a r e given i n Table 6.1. TABLE 6.1 RETENTION TIMES OF SOME P I P E R I D I N E BASES' 1.8 m long packed column ( S i l i c a gel, 20 % KOH t 30 % PEG 4000) Compound Piperidine Morpholine Coniine Conhydrine

130°C 6.75 17.25 15.25

-

16OoC 2.60 4.75 4.07 28.00

Compound

13OoC

2.6-Dimethyl p i p e r i d i n e y-Coni c e i ne N-Methylconi ine N-Methylconiceine

5.55 26.50 13.60 84.00

16OoC 2.25 7.25 4.00 16.60

F a i r b a i r n e t a l . 4 a p p l i e d gas chromatography t o q u a n t i t a t i v e assay o f the a l k a l o i d s i n conium macubturn L. The d i f f i c u l t y o f preparing a concentrated s o l u t i o n o f the v o l a t i l e

bases, w i t h o u t loss, was overcome by e x t r a c t i n g the bases i n t h e form o f t h e i r n o n - v o l a t i l e s a l t s and l i b e r a t i n g them in s i t u on the column. This was achieved by i n s e r t i n g a small amount o f soda l i m e a t the beginning o f the column and using

a s o l u t i o n o f the a l k a l o i d s t o be ana-

lysed i n 70 % methanol. Samples o f 25-30 g o f f r e s h f r u i t s ( = 25-100 mg a l k a l o i d ) were b a s i f i e d and e x t r a c t e d w i t h d i e t h y l ether. The d i e t h y l e t h e r l a y e r was e x t r a c t e d w i t h N HC1, and the aqueous l a y e r b a s i f i e d and e x t r a c t e d w i t h d i e t h y l e t h e r again. A f t e r a d d i t i o n o f 0.5 m l concentrated hydrochloric a c i d the d i e t h y l e t h e r was evaporated. When the excess o f hydroc h l o r i c a c i d had a l s o disappeared, the residue was dissolved i n 1 m l 70 % methanol and gas chromatographed. A packed column w i t h 25 % s i l i c o n e elastomer 301 on C e l i t e was used a t a column temperature o f l l O ° C .

The separation obtained was s u f f i c i e n t f o r q u a n t i t a t i v e assay

o f the main a l k a l o i d s , coniine, M-methylconiine and y-coniceine. The values obtained by gas chromatography were i n good agreement w i t h those obtained by spectrophotometry. TABLE 6.2 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY OF P I P E R I O I N E ALKALOIDS Column

S o l i d support mesh

CW 80-100 glass, 6 f t x 4 mm I . D . glass cap. S, 25 m x 0.5 mm I . D . glass, 1.8 m x 4 mm I . D . Sig 0.2-0.3 mm qlass, 6 f t

Cel 70-80

Stat.phase SE-30 ov-1 PEG 4000 + KOH S i .el.

% 2-3 30 20 25

Temperature

Comp. Prep.

Ref,

204OC 25OoC 13OoC

alk. s. pip.qnt.pm. con.alk.

1 2 3

llO°C

con.al k.

4

Abbreviati0ns:Cel = C e l i t e , con = Conium, CW = Chromosorb W, p i p = piperine, S = s i l a n i z e d , S i . e l = s i l i c o n e elastomer, S i g = s i l i c a gel. 6.3

REFERENCES

1 H.A.

Lloyd, H.M. Fales, P.F. Highet, W.J.A. VandenHeuvel and W.C. Wildman, J. 82 (1960) 3791. 2 M. Verzele, G. Redant and P. Sandra, J . Chromatogr., 199 (1980) 105. 3 F. Moll, N a t u r w i s s e n s c h a f t e n , 49 (1962) 450. 4 J.W. Fairbairn, E.J. Shellard and S . T a l a l a j , P l a n t a M e d . , 11 (1963) 92. SOC.,

Am.

Chem.

Chapter 7

PU I NOL I Z ID I N E ALKALOI DS 7.1 7.2

............................................................. ...................................................................

Lupine a l k a l o i d s References

55 59

7.1 LUPINE ALKALOIDS Ten o f t h e 45 a l k a l o i d s t h a t were gas chromatographed b y L l o y d e t a l . i n 19611 on a 2-3 % SE-30 on Chromosorb W column were l u p i n e a l k a l o i d s . The b i c y c l i c l u p i n i n e and t h e t r i c y c l i c s p a r t e i n e , a - i s o s p a r t e i n e and 1 3 - h y d r o x y s p a r t e i n e were chromatographed a t a t column temperat u r e o f 16OoC, t h e t r i c y c l i c c y t i s i n e , m e t h y l c y t i s i n e , m e t h y l c y t i s i n e - N - o x i d e and t h e t e t r a c y c l i c l u p a n i n e , 13-hydroxylupanine and m a t r i n e a t 204'C.The

r e t e n t i o n times o f the a l k a l o i d s

a r e l i s t e d i n Table 7.1. TABLE 7.1

RETENTION TIMES

OF LUPINE ALKALOIDS~

6 f t l o n s Dacked column (Chromosorb W , 2-3 % SE-30 a t 16OoC ( a ) and 204OC ( b ) Cyt is ine Met hy 1c y t is i n e Methylcytisine-N-oxide Lupanine 13-Hydroxylupanine Matrine Lupinine Sparteine a- I s o s p a r t e i ne 13-Hydroxyspartei ne Faugeras and Paris' Genista p i l o s a

L.

5.1 ( b ) 4.3 ( b ) 5.8 ( b ) 5.5 ( b ) 11.6 ( b ) 8.5 ( b ) 1.5 ( a ) 5.9 ( a ) 5.2 ( a ) 14.3 ( a )

a p p l i e d gas chromatography t o an i n v e s t i g a t i o n o f t h e a l k a l o i d s i n

f r u i t s . The a l k a l o i d s were e x t r a c t e d w i t h a l c o h o l c o n t a i n i n g t a r t a r i c a c i d

and w a t e r c o n t a i n i n g 5 7L s u l p h u r i c a c i d . A f t e r p u r i f i c a t i o n o f t h e aqueous a c i d i c e x t r a c t s b y e x t r a c t i o n w i t h d i e t h y l e t h e r and c h l o r o f o r m , t h e a l k a l o i d bases were e x t r a c t e d w i t h c h l o r o f o r m a f t e r a d j u s t m e n t o f t h e pH t o > 12. Gas chromatography demonstrated t h e presence o f t h r e e a l k a l o i d s , t w o o f which were i d e n t i f i e d , b y means o f t h e i r r e t e n t i o n times on a 10 % SE-30 column on C h r o m s o r b W, as c y t i s i n e and N - m e t h y l c y t i s i n e ( r e t e n t i o n t i m e s 6.2 m i n and 5.7 min r e s p e c t i v e l y on a 3 m l o n g column a t 245OC). I n another paper, Faugeras and P a r i s 3 i n v e s t i g a t e d t h e a1 k a l o i d s i n Genista acanthoclada DC. The p l a n t m a t e r i a l was s t a b i l i z e d i n b o i l i n g methanol and c o m p l e t e l y e x t r a c t e d w i t h t h e same s o l v e n t . A f t e r e v a p o r a t i o n o f t h e s o l v e n t , t h e r e s i d u e was t a k e n up i n w a t e r and t h e aqueous s o l u t i o n e x t r a c t e d w i t h d i e t h y l e t h e r a f t e r a d d i t i o n o f h y d r o c h l o r i c a c i d . A f t e r adjustment o f t h e pH t o 10 t h e a l k a l o i d bases were e x t r a c t e d w i t h d i e t h y l e t h e r and t h e ext r a c t o b t a i n e d used f o r t h e gas chromatographic a n a l y s i s , which was performed w i t h packed columns u s i n g SE-30 and SE-52 as s t a t i o n a r y phases. The r e t e n t i o n t i m e s o f t h e a l k a l o i d s found a r e g i v e n i n T a b l e 7.2.

References p. 59

66 TABLE 7.2 RETENTION TIMES OF ALKALOIDS I N

DC3

GENrsTd AcdNTnocrmd

1.5 m lona Dacked column fAeroDak, 5 % SE-30 and Chromosorb SE-30

W.

5 % SE-521 a t 205OC

SE-52

~~

Retami ne Cytisine Anagyrinr N-methyl c y t i s i n e

1.3 3.0 7.0

3.7 5.5 10.0

4 Cranmer and Mabry applied gas chromatography t o i n v e s t i g a t e the a l k a l o i d s i n s i x t e e n species belonging t o the genus B a p t i s i a . The a l k a l o i d s were e x t r a c t e d w i t h methylene c h l o r i d e made basic w i t h ammonium hydroxide. A f t e r concentration o f the s o l u t i o n the a l k a l o i d s were taken up i n aqueous c i t r i c a c i d s o l u t i o n and t h i s was e x t r a c t e d w i t h methylene c h l o r i d e t o remove i m p u r i t i e s . The pH o f the aqueous s o l u t i o n was then adjusted t o 10 w i t h ammonium hydroxide and the a l k a l o i d bases e x t r a c t e d w i t h methylene c h l o r i d e . A f t e r concentration o f the methylene c h l o r i d e e x t r a c t i t was gas chromatographed on a packed column o f 3 % XE-60 and 5 % DC 550 on Chromosorb W a t 220-223°C.

The r e t e n t i o n times are given i n Table 7.3.

TABLE 7.3 RETENTION TIMES OF SOME LUPINE ALKALOIDS4

1.3 m long packed column (Chromosorb W, 3 % XE-60 and 5 % DC-550) a t 220-223'C A1 k a l o i d

XE-60

An agy r i ne 12.1 Cytisine 4.90 Methylcyti s i n e 2.80 Lupanine 2.60 Hydroxylupanine 17.4 0.50 Lupinine Spartei ne 1.00 13-Hydroxysparteine 1.40 1.65 17-Oxysparteine Thermopsine 9.70 Baptifoline c. 30

DC-550 87.0 27.0 20.5 26.4 c. 200 c.2.0 4.6 12.3 14.1 54.0 275.0

FIGURE 7.1 CHEMICAL STRUCTURES OF SOME LUPINE ALKALOIDS

1) Cytisine, R = H N-Methylcytisine, R = CH3 2) Anagyrine, R = H Thermopsine, R = H B a p t i f o l i n e , R = OH

QgR II

0

1

3) Sparteine, R = H Hydroxysparteine, R = OH 4) Lupanine

3

0

2

57

Faugeras and Paris

5

c a r r i e d out an i n v e s t i g a t i o n o f the a l k a l o i d s o f S a r o t h a m u s c a t a l u n i -

cus Webb. by means o f gas chromatography. The a l k a l o i d s were e x t r a c t e d i n a Soxhlet apparatus

w i t h methanol, then w i t h methanol c o n t a i n i n g 0.15 % t a r t a r i c acid. A f t e r a d d i t i o n o f water the methanol was evaporated and the a c i d i c aqueous s o l u t i o n e x t r a c t e d w i t h d i e t h y l e t h e r t o remove i m p u r i t i e s . Sodium carbonate was added t o o b t a i n pH 9 and the a l k a l o i d bases were ext r a c t e d w i t h d i e t h y l e t h e r and chloroform. The d i e t h y l ether-chloroform e x t r a c t was concent r a t e d and gas chromatographed. Because o f the s l i g h t l y v o l a t i l e c i n e v e r i n e and catalauverine, a 30 cm long packed column w i t h 10 % SE-52 and temperature p r o g r a m i n g from 190°C t o 285OC was used f o r the gas chromatographic analysis. A gas chromatogram o f the a l k a l o i d s i s shown i n Figure 7.2. FIGURE 7.2 GAS CHROMATOGRAM OF ALKALOIDS IN sdRoT/fmvrrs

CAT~L~~~NTC~S~

30 cm long column (Aeropak 30, 10 % SE-52). temperature p r o g r a m i n g 190-285'C

m

1= 2 = 3 = 4 = 5 = 6 =

Sparteine Angustifoline Lupanine Hydroxylupanine Cineverine Catalauverine

1

d 5

I

10

15

1

20

__._._

25

35

1

I

1

1

LO L5 5 0 5 5

min

Gas chromatographic separation and i s o l a t i o n o f microgram amounts o f l u p i n e a l k a l o i d s combined w i t h mass spectrometry was used by Cho and Martin'

f o r the unambiguous i d e n t i f i c -

a t i o n o f 20 such a l k a l o i d s . The r e t e n t i o n times on a packed column o f 10 % QF-1 and the f i v e most abundant ions by spectrometry are l i s t e d i n Table 7.4. Owing t o the value o f l u p i n e seeds as a p r o t e i n source and the t o x i c i t y o f the a l k a l o i d s t h a t may be present even i n "sweet" l u p i n e seeds, Ruiz7 developed a gas chromatographic method t o analyse the a l k a l o i d s i n such seeds. The sample ( 2 g seed) was f i n e l y ground and extracted w i t h amoniacal chloroform, and the a l k a l o i d s removed from the chloroform w i t h a 0.1 N sulphuric acid. A f t e r b a s i f i c a t i o n the a l k a l o i d bases were e x t r a c t e d w i t h chloroform. The i n t e r n a l standard ( c a f f e i n e ) was added and the s o l v e n t evaporated. The residue was d i s solved i n ethanol and the s o l u t i o n obtained was gas chromatographed on a 8 % JXR packed column on Chrornosorb W. The r e t e n t i o n times o f the a l k a l o i d s are l i s t e d i n Table 7 . 5 . The a l k a l o i d s o f the overground p a r t s o f Lupinus a r g e n t e u s Pursh. var. s t e n o p h y l l u s (Rydb.) Davis were i n v e s t i g a t e d using gas chromatography by K e l l e r and Zelenski8. The pres-

References p. 69

68

TABLE 7.4 RETENTION TIMES AND THE FIVE MOST ABUNDANT IONS OF SOME LUPINE ALKALOIDS BY MS6 1.3 m l o n g packed column (Chromosorb W, 10 % QF-1) A1 kal o i d all’khydrolupanine Sparteine 13-Hydroxylupanine Lupinine Cytisine Angustifol i n e N-Methyl c y t i s i ne 4-Hydroxyl upani ne ( n u t t a l ine) Anagyri ne Lampro) obine Lupanine Thermopsine Isosophoramine a-Isolupanine 1 Oxolupanine AlIOehydrolupanine Matrine 17-Oxosparteine Rhombi f o l i n e 8-Ketosparteine

Mt

Five most abundant ions

246 234 264 169 190 234 204 264

246 137 152 83 190 193 58 264

134 234 264 152 146 112 204 136

148 98 246 138 147 194 146 134

245 193 55 169 134

244 264 248 244 244 248 262 246 248 248 244 248

98 138 136 98 244 136 262 98 248 97 58 98

244 264 248 244 243 248 150 97 96 248 98 150

97 83 149 97 149 149 234 246 247 98

146 97 150 146 245 247 110 245 150 110 146 151

55 97

tR(mi n)

55 160 263

247 136 165 97 160 150 205 150

46.1 7.0 64.0 4.4 42.2 41.3 40.2 44.1

243

59.5 39.4 41.5 53.5 54.5 40.0 67.0 36.2 46.2 35.6 46.1 32.4

55 247 96 148 98 263 121 137 223 160 55

TABLE 7.5 RETENTION TIMES OF LUPINE ALKALOIDS7 2 m long packed column (Chromosorb

W, 8 % JXR) 140-240°C temperature p r o g r a m i n g

A1 k a l o i d

t~ ( r e l )

Lupinine 0.371 Cyt is ine 1.468 Sparteine 1.611 Lupanine 1.800 Anagyrine 2.384 Caffeine ( i n t . s t a n d . ) 1 ence of sparteine,

-

t R = 11.31 min

p - i sosparteine, a5-dehydrol upanine, a-is01 upani ne, 1upanine, thermopsine

and anagyrine was suggested by gas chromatography. Gas chromatography-mass spectrometry confirmed these f i n d i n g s . On a 3 % OV-17 column on Gas Chrorn Q, sparteine and genisteine could n o t be separated. The r e t e n t i o n times r e l a t i v e t o sparteine were: Sparteine p5Isosparteine A -khydrolupanine a - I s o l upanine Lupanine Thermopsi ne Anagyrine

1.00 1.29 3.20 3.36 3.76 5.06 5.61

The a l k a l o i d s were extracted f r o m a i r - d r i e d p l a n t m a t e r i a l w i t h ethanol, the ethanol ext r a c t concentrated, a c i d i f i e d w i t h a c e t i c a c i d and e x t r a c t e d successively w i t h d i e t h y l e t h e r , e t h y l acetate and chloroform t o remove i m p u r i t i e s . The a c i d i c aqueous s o l u t i o n was made a l k a l i n e w i t h ammonia and e x t r a c t e d w i t h chloroform. A f t e r concentration t h i s chloroform ext r a c t was gas chromatographed on a packed 3 % OV-17 column on Gas Chrom Q by temperature

59

p r o g r a m i n g . Combined GLC-MS was c a r r i e d o u t on a 2 m l o n g b y 2 mn I . D .

g l a s s column packed

w i t h 3 % DV-17 on Gas Chrom Q and temperature programming, 16OoC t o 265OC a t 2'C/min. D a i l y e t a1.'

i n v e s t i g a t e d t h e c o n t e n t o f a l k a l o i d s i n t h e B u l g a r i a n C h a m a e c y t i s u s spe-

c i e s q u a l i t a t i v e l y by means o f gas Chromatography. The d r i e d ground p l a n t m a t e r i a l was ext r a c t e d with methanol and t h e methanol e x t r a c t c o n c e n t r a t e d . A f t e r a d d i t i o n o f h y d r o c h l o r i c a c i d , n e u t r a l and a c i d i c compounds were removed b y e x t r a c t i o n w i t h e t h y l e n e c h l o r i d e . The aqueous phase was made a l k a l i n e w i t h sodium c a r b o n a t e and t h e a l k a l o i d bases e x t r a c t e d w i t h e t h y l e n e c h l o r i d e . A f t e r removal o f t h e s o l v e n t t h e r e s i d u e was d i s s o l v e d i n methanol and analysed b y gas chromatography on a packed column o f 1.5 % SE-30 on Chranosorb W u s i n g temp e r a t u r e programming f r o m 150°C t o 25OoC. TABLE 7.6 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY OF LUPINE ALKALOIDS Column

S o l i d support

Stat.phase

%

Temperature

Comp.Prep.

Ref.

mesh

glass, 6 f t x 4 mm I.D. S.S. 3 m x 3 mm I.D. S.S. 1.5 m S . S . 1.5 m S.S. 6 f t x 0.25 i n S.S. 6 f t x 0.25 i n S . S . 30 cm

CW 80-100 CW 60-80 Ae r CW AWS CW AWS Ae r

SE-30 SE-30 SE-52 SE-30 XE-60 DC 550 SE-52

2-3 10 5 5

glass, 1.3 m x 4 mn glass, 2 m x 1.6 mn I.D.

CW AWS CW 100-120

QF-1 JXR

10 8

g l a s s , 2 m x 2 mm I.D.

GQ

OV-17

3

SE-30

1.5

2mx3mn

cw

cws

100-200

5"

10

16OoC and 204OC a1k.s. 245OC alk.pm. 205OC alk.pm. 205OC

1 2

220-223°C

alk.pm.

4

190-285°C p r . 4O~/min 23OoC 170-240°C p r . 4O~/min 125-2650 C p r . 4 O ~ / mni 150-250°C 2o0/mi n

alk.pm. alk.pm. alk.pm.

3

6

alk.pm.

alk.pm.

A b b r e v i a t i o n s : Aer = Aeropak 30, a l k = a l k a l o i d , CW = Chromosorb W, GQ = Gas Chrom Q, pm = p l a n t m a t e r i a l , S = s i l a n i z e d , s = s e p a r a t i o n , s.s = s t a i n l e s s s t e e l . 7.2 REFERLNCES

1 H.A. Llovd. H.M. Fales, P.F. H i q h e t , W.J.A. VandenHeuvel and W.C. Wildman, J. Am. C h e m . sot., 82" ( i 9 6 0 ) 3971. 2 G. Faugeras and M. P a r i s , C . R . d c a d . S c i . P a r i s , 258 (1964) 3113. 3 G. Faugeras and M. P a r i s , C . R . d a d . S c i . P a r i s , 264 (1967) 1290. 4 M.F. Cranmer and T.J. Mabry, P h y t o c h e m i s t r y , 5 (1966) 1133. 5 G. Faugeras and M. P a r i s , Ann. P h a r m . F r . , 26 (1968) 265. 6 Y.D. Cho and R.O. M a r t i n , A n a l . B i o c h e m . , 44 (1971) 49. 7 L.P. Ruiz, J r . , N.Z. d g r i c . R e s . , 2 1 (1978) 241. 8 W.J. K e l l e r and S.G. Z e l e n s k i , J . P h a r m . S c i . , 67 (1978) 430. 9 A. D a i l y , N. Kotsev, L. I l i e v a , H. Outschewska and N. M o l l o v , A r c h . P h a r m . ( W e i n h e i m , G e r . 311 (1978) 889.

This Page Intentionally Left Blank

61

11.2. TROPANE ALKALOIDS Chapter 8

TROPINE ALKALOIDS 8.1. Solanaceae a l k a l o i d s 8.2. References

............................

61

......................................

72

8.1. SOLdNdCEAE ALKALOIDS Atropine belongs t o the f i r s t group o f a l k a l o i d s t h a t was gas chromatographed by Lloyd e t al.'

using packed columns w i t h a support t h i n l y coated (2-3 % ) w i t h a non-polar, thenno2 i n t h e i r studies

stable s t a t i o n a r y phase (SE-30). Brochmann-Hanssen and Baerheim Svendsen

on gas chromatography o f a l k a l o i d s and a l k a l o i d a l s a l t s , used a s o l i d support which was a c i d and base washed, s i l a n i z e d (hexamethyldisilazane) and t r e a t e d w i t h 0.1 % polyethylene g l y c o l p r i o r t o coating w i t h the s t a t i o n a r y phase (SE-30) 1.15 % i n order t o prevent t a i l i n g o f the a l k a l o i d s during the gas chromatography (due t o adsorptive e f f e c t s o f the support m a t e r i a l ) . On a 2 m long glass column by 3 mm I.D.,

they obtained a good separation o f a t r o p i n e and

scopolamine. From gas chromatography o f e x t r a c t s o f crude drugs (Belladonna, Stramonium and Hyoscyamus) the r a t i o hyoscyaminelscopolamine could be roughly estimated. Atropine and scopolamine were, under c e r t a i n experimental conditions, dehydrated t o t h e i r apo-compounds, g i v i n g two peaks on the chromatogram: atropine/apoatropine and scopolamine/ aposcopolamine. The degree o f dehydration was found t o be associated w i t h the amount o f glass wool placed on the t o p o f the column packing and w i t h the temperature o f the i n j e c t i o n p o r t . When a f a i r l y l a r g e amount o f glass wool was used, the degree o f decomposition decreased as the i n j e c t i o n p o r t temperature was reduced, b u t could never be e n t i r e l y eliminated. When the amount o f glass wool was reduced t o a very small amount o r removed completely, no decomposit i o n took place even a t i n j e c t i o n p o r t temperatures o f 35OoC. 3 Vigneron and P e l t a p p l i e d gas chromatography f o r i n v e s t i g a t i o n s o f the a l k a l o i d s present i n some Solanaceae plants. With a 5 % SE-30 column they observed t h a t some dehydration o f a t r o p i n e and scopolamine took place, b u t i n t e r p r e t e d t h i s dehydration as being connected w i t h the solvent used f o r the e x t r a c t i o n and gas chromatography o f the a l k a l o i d s . When ethanol was used, more apo-alkaloids were found than when chloroform was used. From gas chranatography o f a three-month-old s o l u t i o n o f a t r o p i n e i n ethanol, a r e l a t i v e l y l a r g e amount o f apoatropine was observed on the chromatogram.

4

Achari and Newcombe studied the i n f l u e n c e o f the tubing m a t e r i a l (glass

-

stainless steel)

a f t e r s i l a n i z a t i o n o f the i n s i d e o f the tubing. The columns were packed w i t h XE-60 (10 %) and w i t h a mixture o f XE-60 (2.5 %) and QF-1 (2.5 %) on Chrumosorb W 80-100 mesh (HMDS). On the 10 % XE-60 s t a i n l e s s s t e e l column, scopolamine and hyoscyamine were n o t e l u t e d a t a l l - as

w i l l be seen from Table 8.1. I n a paper on gas chromatographic screening o f t o x i c o l o g i c a l e x t r a c t s f o r a1 kaloids, Parker e t a l . 5 separated hyoscyamine ( a t r o p i n e ) , scopolamine and homatropine on packed columns 4 o f s t a i n l e s s s t e e l w i t h o u t any problems, as mentioned by Achari and Newcombe They used

.

References p. 7 2

62

TABLE 8 . 1 COMPARISON OF THE EFFICIENCIES OF GLASS AND STEEL COLUMNS4 x = Compound n o t e l u t e d ; C o n d i t i o n s : Column l e n g t h 152 cm, 1.0. 5 mn; s u p p o r t Chromosorb W 80-100 mesh (HMDS); oven temp. 20OoC; i n j e c t i o n h e a t e r temp. 22OoC; c a r r i e r gas f l o w 50 m l / min. ~~

~~

~

Retention time (sec)

Compoun d

2.5 % QF-1 t 2.5 % XE-60

10 % XE-60 glass Tropine 3,6-Di hydroxytropane 3-Ti g l oy 1o x y t ropa ne Scopolamine Hyoscyamine 3,4-Di t i g l o y l o x y t r o p a n e

steel

10 38 28 269 99 250

21 85 59

glass

steel

5

26 78 73 558 302 48

14 14 222 165 222

X X

524

SE-30 ( 5 % ) and Carbowax 20 m ( 1 %) as s t a t i o n a r y phases on Chromosorb 60-80 mesh, a c i d washed. A s o l i d i n j e c t o r was employed t o p r e v e n t c o n t a m i n a t i o n o f t h e column b y n o n - v o l a t i l e compounds p r e s e n t i n t h e e x t r a c t s o f t h e b i o l o g i c a l m a t e r i a l i n v e s t i g a t e d . S a t i s f a c t o r y sepa r a t i o n o f t h e t r o p a n e a l k a l o i d s mentioned was o b t a i n e d a t temperatures above 210°C on t h e SE-30 column. A l t h o u g h a s a t i s f a c t o r y s e p a r a t i o n o f a t r o p i n e and scopolamine can be achieved on a nonp o l a r s t a t i o n a r y phase (SE-30), a b e t t e r s e p a r a t i o n can be o b t a i n e d on more p o l a r s t a t i o n a r y phases, as shown by Brochmann-Hanssen and Fontan7, see T a b l e 8.2. TABLE 8.2 RELATIVE RETENTION VALUES OF SOME TROPANE AND RELATED ALKALOIDS ON STATIONARY PHASES OF VARIOUS POLARITY7 SE-30 1 % 195'

XE-60 1 %

EGSS-Y 1 X

HI-EFF-8B 1 X

225'

220°

230'

230'

240'

-

0.45 1.13 0.29 0.36 1.00

0.46 1.10 0.28 0.31 1.00

0.46 1.12 0.30 0.32 1.00

5 .0

8.7

5.6

Compound Atropine Scopolamine Homatropine Cocaine Codeine

0.51 0.84 0.31 0.52 1.00

1.00

0.59 1.40 0.39 0.48 1.00

Codeine t i m e f m i n )

16.4

1.7

3.6

Also, M o f f a t e t a l .

s t u d i e d t h e s e p a r a t i o n o f a number o f b a s i c drugs, i n c l u d i n g a t r o -

pine, on s t a t i o n a r y phases o f d i f f e r e n t p o l a r i t y . They concluded t h a t a l o w p o l a r i t y phase, such as SE-30 o r OV-17,

s h o u l d be chosen as t h e " p r e f e r r e d " l i q u i d p h a s e ' f o r t h e GLC o f drugs.

The e f f i c i e n c i e s o f d i f f e r e n t s t a t i o n a r y phases on t h e s e p a r a t i o n o f some n a t u r a l t r o p a n e compounds and f o u r c l o s e l y r e l a t e d d i e s t e r s o f 3,6-dihydroxytropane d i i s o v a l e r y l and d i - 2 - m e t h y l b u t y l )

( d i t i g l o y l , disenecioyl,

was a l s o s t u d i e d b y A c h a r i and Newcombe4. The r e s u l t s ob-

t a i n e d a r e g i v e n i n Table 8.3 I n F i g u r e 8 . 1 a chromatogram i s g i v e n o f t h e s e p a r a t i o n o f some tropanes. The t e c h n i q u e was a p p l i e d t o t h e a n a l y s i s o f v a r i o u s m i n o r a l k a l o i d s i n samples o f t h e r o o t s o f two s p e c i e s o f D a t u m a f t e r f r a c t i o n a t i o n o f an a l k a l o i d a l e x t r a c t on a k i e s e l g u h r

63

TABLE 8.3 COMPARISON OF THE EFFICIENCIES OF DIFFERENT STATIONARY PHASES4 Column 137 cm long, I.D. 4 mm (glass); support Chromosorb W 80-100 mesh (HMDS); oven temp. 180' (a 200°, b 215', c 250'); carrier gas flow 50 ml/min. Compound

Tropi ne/pseudot ropi ne 3,6-Di hydroxytropane 3-Ti gl oyl oxytropane 3,6-Ditigloyloxytropane

SE-30 2 % 12 38 62 250

XE-60 2 % 7 24 26 272

3,6-Di senecioyloxytropane

241

269

Retention time (sec) Ve rs ami dc 5 % 19 47 38 165 16 1

QF-1 2.5 % 14.1 59 57 560 qq,-,a LL" 567 7n~b 90

PEGS 2.5 % 28 76 165 1143 1152

-1-

3,6-Di i sovaleryloxytropane 123 3.6 -Di - (2-methyl butyryl ) oxytropane 113 Scopolamine 236

95

66

-90 274

62 189

Hyoscyamine

109

118

180

87 695 217b 165

1068 1039 1596 965

column prior to the gas chromatographic determination of the alkaloids. The analytical results achieved were in good agreement with results obtained by partition chromatographic analysis of the scopolamine and hyoscyamine content in the aerial parts of D a t u m stramonium and d t r o p a belladonna (Table 8.4). FIGURE 8.1 GAS CHROMATOGRAM OF SOME TROPANES4

Gas chromatographic conditions as described in Table 8.3 for the 2 % XE-60 column Tropine/pseudotropine 3-Tigloyloxytropane = 3,6-Diisovaleryloxytropane/ 3,6-0i -2-methyl butyryl oxytropane 4 = 3,6-Ditigloyloxytropane/ 3,6-Di seneci oyl oxytropane 5 = 3,6-Oitigloyloxy-7-hydroxytropane

1 2 3

=

=

Reproduced from Planta Medica 19 (1971) 241, by permission of Hippocrates-Verlag.

References p. 7 2

64

TABLE 8.4 ANALYSIS OF STRAMONIUM, BELLADONNA AND BELLADONNA TINCTURE B.P.4 Stramoni um Partition GLC Total a l k a l o i d s Scopolamine Hyoscyamine Hyoscyami ne/ Scopolamine

0.258 0.082 0.176

0.260 0.078 0.182

0.465

0.428

Total a l k a l o i d s n o t l e s s than ( B r i t i s h Pharmacopoeia) 0.25

Be1ladonna P a r t i t i o n GLC 0.208

.O.OOO 0.208

Belladonna T i n c t u r e P a r t it i o n GLC

0.203 0.001 0.202

0.029 0.001 0.028

0.028 0.000 0.028

0.005 n o t l e s s than 0.30

0.028-0.032

I n a number o f i n v e s t i g a t i o n s the methyl phenyl s i l i c o n e OV-17 has s u c c e s s f u l l y been used f o r separation and a l s o f o r q u a n t i t a t i v e determinations o f tropane a1 k a l o i d s i n pharmaceutical preparations9, 10,119 12,13914915 Wilms e t a1.16 i n v e s t i g a t e d the changes i n the content o f a t r o p i n e and scopolamine d u r i n p the development o f A t r o p a belladonna from A p r i l t o September using a 3 % OV-17 co:umn.

Apo-

atropine was a l s o found by analysis i n m u n t s v a r y i n g from 2.3 % t o 4.4 % o f the t o t a l cont e n t of atropine, depending on the stage o f the development o f the p l a n t . Nothing was, however, mentioned about a possible dehydration o f a t r o p i n e t o apoatropine during the gas chromatography. Codeine was u t i l i z e d as an i n t e r n a l standard s i n c e homatropine, which has commonl y been used by detetminations o f a t r o p i n e and scopolamine, was e l u a t e d w i t h about the same r e t e n t i o n time as apoatropine under the chromatographic conditions employed. Other s t a t i o n a r y phases have a l s o proved t o be useful f o r gas chromatography o1 tropane a l k a l o i d s . For simultaneous q u a n t i t a t i v e determination o f a t r o p i n e and scopolamine, Solomon e t a1.l'

applied an SE-30 column (2.5 %). During studies on the metabolism o f tropane a l -

kaloids, whereby q u a n t i t a t i v e determinations o f minor a l k a l o i d s were c a r r i e d out, Achari and 4 Newcombe used columns w i t h SE-30 (2 %), XE-60 (2 %), Versamid ( 5 %), QF-1 (2.5 % ) and PEGS (2.5 % ) . The best r e s u l t s were obtained w i t h the XE-60 (2 % ) column. I n 1964 t h i s s t a t i o n a r y phase had been successfully employed by Frauendorf and Vogel"

i n a concentration o f 5 % f o r

i n v e s t i g a t i o n s o f the a l k a l o i d s i n e x t r a c t s o f the Duboisia species. As a1 ready observed by Brochmann-Hanssen and Baerheim Svendsen',

dehydration o f a t r o p i n e

and scopolamine may take place under c e r t a i n experimental conditions. I n t h e i r gas chromatographic studies on a t r o p i n e and scopolamine, Solomon e t a l .17 made the same observations. They a l s o found t h a t scopolamine hydrobromide was decomposed i n some o t h e r way, g i v i n g sev9 era1 peaks on the chromatogram. Zimnerer Jr. and Grady worked o u t an assay o f hyoscyamine ( a t r o p i n e ) and scopolamine i n pharmaceutical preparations and they found t h a t when u s i n g properly cured columns, no d e r i v a t i z a t i o n o f the a l k a l o i d s was necessary; standards assayed w i t h precisions o f 4.8 % f o r scopolamine, 6 p g / u n i t dose, and 2.5 % f o r hyoscyamine ( a t r o p i n e ) a t 100 pg/dose. Homatropine served as an i n t e r n a l standard. I n another paper, Grady and Z i m merer 5r.l'

stressed the importance o f the q u a l i t y o f the column. Improperly o r p a r t i a l l y

cured and conditioned columns o f t e n cause extensive t a i l i n g o f the a l k a l o i d s mentioned, and an a d d i t i o n a l problem can be p a r t i a l on-column dehydration. A c o l l a b o r a t i v e study o f the assay f o r atropine sulphate t a b l e t s , i n j e c t i o n and ophthalmic s o l u t i o n , and f o r scopolamine hydrobromide t a b l e t s and i n j e c t i o n (USP X V I I I ) i n n i n e d i f f e r e n t l a b o r a t o r i e s , where hom-

65

a t r o p i n e was added as i n t e r n a l standard, showed t h a t the method was accurate, r e l i a b l e , sens i t i v e , h i g h l y s p e c i f i c , r a p i d and reasonable precise. Santoro e t a l . l l

worked o u t a s i m i l a r method f o r the q u a n t i t a t i v e determination o f scopol-

amine i n pha rmaceut ica 1 preparations containing i. a. phenyl propanolamine and chlorpheni ramine a f t e r e x t r a c t i o n o f the f r e e base and using homatropine as an i n t e r n a l standard. The scopolamine content was l e s s than 9

X

o f the t o t a l belladonna a l k a l o i d s present i n the preparation.

To prevent dehydration and/or decomposition o f tropane a1 kaloids d u r i n g gas chromatography, Windheuser e t a1.I'

prepared the t r i m e t h y l s i l y l d e r i v a t i v e s o f the a l k a l o i d s by means o f

N,O-bis(trimethylsily1 )acetamide i n an N,N-dimethylformamide

s o l u t i o n o f the a l k a l o i d s . I n

t h i s way they c a r r i e d o u t q u a n t i t a t i v e determinations o f scopolamine and i t s a c i d i c and basic degradation products (scopoline, a t r o p i c acid, t r o p i c a c i d and aposcopolamine). Nieminen"

applied gas chromatography t o determine a t r o p i n e and scopolamine i n a number o f

pharmaceutical preparations: multicomponent t a b l e t s , suppositories, i n j e c t i o n s , ophthalmic s o l u t i o n s and asthma c i g a r e t t e s containing Stramonium leaves. The separation o f t h e a1 k a l o i d s from multicomponent preparations was achieved w i t h a C e l i t e column followed by l i q u i d - l i q u i d e x t r a c t i o n . Mestranol was used as an i n t e r n a l standard by the gas chromatography. The author d i d n o t observe any dehydration o r o t h e r decomposition o f the a l k a l o i d s d u r i n g the gas chromatographic analysis caused by glass wool placed on the top o f the column, as s t a t e d by o t h e r workers. Results o f analyses c a r r i e d o u t are given i n Table 8.5. TABLE 8.5

ANALYSIS

OF ATROPINE AND SCOPOLAMINE

IN PHARMACEUTICAL PREPARATIONS~' Amount s t a t e d on the l a b e l i n mq

Found ma

Dosage form

Declared cmposi t i o n

Tablet

Atropine sul phate Scopolamine h y d r o b r m i de Phenobarbi tone

0.400 0.210 40.0

0.397 0.218

Tablet

T o t a l a l k a l o i d s o f Belladonna leaves Ergotami ne t a r t r a t e Phenobarbi tone

0.200 0.6 40.0

0.205

Tablet

Belladonna e x t r a c t corresponding t o a t rop ine Opium e x t r a c t A1 bumin tannate Bismuth subsal i c y l a t e

Tablet

Be1ladonna e x t r a c t corresponding t o a t r o p i n e Aloe e x t r a c t Sap0 medicatus Resina s cammoniae Rheum e x t r a c t Frangula e x t r a c t

Injection

Atropine s u l phate Morphine hydrochl o r i de Glycerol Alcohol Sodium p y r o s u l p h i t e P u r i f i e d water

Ibfncnmp. 72

10.0 0.140 5.0 0.200 400.0 2.4 0.0336 40.0 10.0 20.0 20.0 16.0 0.250 20.0 50.0 120.0 1.0 ad 1 m l

0.142

0.0334

0.255

66

TABLE 8.5 (continued) Dosage form

Declared composition

Injection

Scopolamine hydrobromide Morphine hydrochloride Glycerol Alcohol Sodium pyrosulphite P u r i f i e d water

Ophthalmic solution

Atropine sulphate Preservatives P u r i f i e d water

Ophthalmic solution

Scopolamine hydrobromi de Preservatives P u r i f i e d water

Suppository

T o t a l a l k a l o i d s o f Belladonna leaves Ergotamine t a r t r a t e Caffeine Allylbarbituric acid Adeps solidus. Excip.

Amount s t a t e d on the l a b e l i n mg 0.600 20.0 50.0 120.0 1.0 ad 1 m l 5.0 ad

4.95

1 ml 2.5

ad

Found mg 0.602

2.45

1ml

0.250 2.0 100.0 100.0

0.247

q.s.

For the q u a n t i t a t i v e determination o f homatropine methylbromide i n t a b l e t s and e l i x i r s , Grabowski e t a1 hydrolyzed the capound, e x t r a c t e d the f r e e mandelic a c i d and determined i t q u a n t i t a t i v e l y as t r i m e t h y l s i l y l d e r i v a t i v e . A glass column packed w i t h 15 % QF-1 on Gas Chrom Q, a c i d washed and silanized, was used a t a column temperature o f 16OoC. Liebisch e t gas chromatographed tropane a l k a l o i d s and r e l a t e d compounds ( t r o p i n e . pseudotropine, a1

."

-

nortropine, scopol ine, ecgonine, pseudoecgonine, atropine, cochlearine, scopolamine, mete1 o i d i n e and benzoylecgonine) as t h e i r t r i m e t h y l s i l y l d e r i v a t i v e s , using N-methyl-N-trimethyl-

silyl-trifluoroacetamide as s i l y l a t i o n reagent. They u t i l i z e d a 3 % QF-1 column w i t h Gas ChmmQat 80°C f o r the tropanealkamines, 135OC f o r ecgonine and 185OC f o r the e s t e r a l k a l o i d s . For analysis o f the a l k a l o i d s present i n Cuboisia species, G r i f f i n e t al.23 converted the a1 kaloids a f t e r e x t r a c t i o n and p u r i f i c a t i o n i n t o t h e i r t r i m e t h y l s i l y l d e r i v a t i v e s t o prevent dehydration t o the apo-form. Q u a n t i t a t i v e determinations were c a r r i e d o u t on a 1.5 % SE-30 column on a c i d washed, s i l a n i z e d Chromosorb W , which was precoated w i t h t r a c e s o f Carbowax 2 4000 p r i o r t o SE-30, as proposed by Brochmann-Hanssen and Baerheim Svendsen

.

TABLE 8.6 ~. ANALYSIS OF SAMPLES OF DumrsrA , w m m o r ~ E sLEAVES 23 Percentage scopolamine Percentage hyoscyamine confidence l i m i t s p = 0.95 confidence l i m i t s p = 0.95 Sample Lower Upper Amount found Upper Lower Amount found E5/5 0.209 0.223 0.194 0.244 0.257 0.231 E5/15 0.487 0.507 0.468 0.213 0.225 0.201 E5/ 10 0.415 0.432 0.398 0.163 0.175 0.151 E5/13 0.552 0.575 0.531 0.121 0.134 0.109 F9/5 0.267 0.280 0.254 0.239 0.251 0.227 F9/15 0.351 0.367 0.333 0.169 0.180 0.157 F9/12 0.223 0.236 0.210 0.103 0.115 0.090 G2/6 0.283 0.300 0.265 0.093 0.110 0.075 0.078 0.091 0.065 62/16 0.414 0.431 0.398 6218 0.218 0.231 0.205 0.067 0.080 0.054 G2/ 11 0.372 0.387 0.357 0.104 0.116 0.092 J11/5 0.573 0.596 0.551 0.073 0.084 0.066 0.088 0.100 0.075 J12/3 0.586 0.611 0.564 J12/10 0.569 0.592 0.547 0.086 0.099 0.073 512114 0.587 0.611 0.565 0.220 0.208 0.232

67

The usefulness of gas chromatography i n t o x i c o l o g i c a l analysis of a l k a l o i d s

-

-

e s p e c i a l l y i n the f i e l d

was emphasized by Parker e t al.5, who a l s o included a t r o p i n e and scopolamine

i n t h e i r studies. Almost a t the same time Kazyak and KnoblockZ4 c a r r i e d out an i n v e s t i g a t i o n on gas chromatography o f a number of drugs i n t o x i c o l o g i a l analysis. Atropine and scopolamine were separated on an SE-30 (1 %) column a t temperatures above 200°C.

Kolb and P a t t Z 5 gas

chromatographed a t r o p i n e on SE-52 (2.5 % ) a t 200°C. A p o l a r s t a t i o n a r y phase (HI-EFF-8B = cyclohexanedimethanol succinate) was a p p l i e d by J a i n and K i r k z 6 f o r gas chromatography of several a l k a l o i d s i n c l u d i n g atropine. They a l s o describe an e x t r a c t i o n procedure f o r a l k a l o i d s from 0.5 m l blood samples w i t h acetone-diethyl e t h e r without any pH adjustment.

A r a p i d gas chromatographic method f o r the a n a l y s i s o f mixtures o f t r o p i n e and pseudot r o p i n e w i t h an absolute accuracy o f l e s s than 0.5 % was used by Van der V l i e s and Caron using 15 % Apiezon L on Chromosorb P p r e t r e a t e d w i t h potassiirm hydroxide.

27

,

A special a p p l i c a t i o n i n the f i e l d o f gas chromatography o f tropane a l k a l o i d s i s r e a c t i o n 28 gas chromatography, whereby the tropane s t r u c t u r e i n the a l k a l o i d s can be determined . By dehydration on a p l a t i n u m - f i r e b r i c k c a t a l y s t o f tropanol and a l k a l o i d s c o n t a i n i n g a tropan-

01 moiety, the same main products are formed, viz., among others, p y r r o l e , methylpyrrole, p y r r o l i d i n e , p y r i d i n e , p i p e r i d i n e and toluene. The gas chromatography was c a r r i e d o u t by the authors on a 20 % Carbowax column on base-washed f i r e b r i c k a t 14OoC. Bayne e t a1

."

developed a gas chromatographic-mass spectrometric method f o r the determi-

nation o f scopolamine i n plasma and u r i n e samples

-

s e n s i t i v e t o 50 pg/ml f o r a 4 m l sample.

A deuterated i n t e r n a l standard (scopolamine (N-CD3) hydrobromide hydrate) was used t o m i n i mize v a r i a b i l i t y i n absolute recovery i n the e x t r a c t i n g procedure. Scopoline and deuterated scopoline were formed from the base-catalyzed h y d r o l y s i s o f scopolamine and the i n t e r n a l standard, and were analyzed as the heptafluorobutyrates, using a gas chromatographic-mass spectrometric system by monitoring the m/e 138 and 141 fragments, r e s p e c t i v e l y . The heptaf l u o r o b u t y r y l d e r i v a t i v e o f scopoline was n o t used f o r i t s s e n s i t i v i t y towards e l e c t r o n capture detection, b u t because i t chromatographed w e l l on the picogram l e v e l and showed a favorable fragmentation p a t t e r n under electron-impact i o n i z a t i o n . The use o f deuterated i n ternal standard f o r s e l e c t i v e i o n monitoring negates the necessity f o r f i n d i n g an i n t e r n a l standard w i t h s i m i l a r p a r t i t i o n properties, b u t w i t h d i f f e r e n t r e t e n t i o n times, which i s r e quired i f electron-capture detection i s used. Further, the use o f s e l e c t i v e i o n monitoring i n t h e gas chromatographic-mass spectrometric method g r e a t l y reduces the problem o f b i o l o g i c a l i n t e r f e r e n c e as compared t o electron-capture detection. The gas Chromatography o f the heptafluorobutyryl d e r i v a t i v e s o f scopoline was carr i e d o u t e i t h e r on a 6 f e e t by 2 mm

I.D.

glass column packed w i t h 3 % OV-17 o r 1 % OV-225 on

Gas Chrom Q (200-120 mesh) a t 95OC. Wyatt e t a1.30 worked out a GLC assay for a t r o p i n e and scopolamine i n belladonna e x t r a c t . The e x t r a c t was solved i n 0.1 N s u l p h u r i c acid, homatropine hydrobromide was added t o t h i s s o l u t i o n as an i n t e r n a l standard, and i n t e r f e r i n g m a t e r i a l s were e x t r a c t e d from the a c i d i f i e d s o l u t i o n w i t h chloroform - and f i n a l l y a mixture o f chloroform and 2-propanol (10:3) i f there i s an emulsion problem. The a l k a l o i d s were subsequently e x t r a c t e d i n t o chloroform ( o r chloroform-2-propanol)

from the b a s i f i e d aqueous l a y e r (pH 9.5 phosphate b u f f e r was used

instead o f mineral a l k a l i t o minimize e s t e r cleavage) and the chloroform e x t r a c t s were f i l tered through anhydrous sodium sulphate ( p r e v i o u s l y washed w i t h chloroform). 87 % o f the a l k a l o i d s were recovered i n the f i r s t e x t r a c t , so t h a t two a d d i t i o n a l e x t r a c t i o n s gave s u f f i -

References p. I 2

c i e n t a l k a l o i d recovery. Pure sodium sulphate should be used since small amounts o f i m p u r i t i e s , such as calcium, were found t o catalyze on-column decomposition o f the a t r o p i n e sample w i t h gas chromatography. Two OV-17 columns were prepared: on the f i r s t , p r e f e r e n t i a l decomposition t o apoatropine was observed; on the second t h e a t r o p i n e peak was severely d i s t o r t e d only. Assay values are given i n Table 8.7. TABLE 8.7 CONTENT OF ATROPINE, SCOPOLAMINE AND TOTAL ALKALOIDS I N BELLADONNA EXTRACTS DETERMINED BY GLC30 k c l a r e d content: 12.5 mg/g o f t o t a l a l k a l o i d s Sample

Weighing

Pilular extract 1

A

2

A B C

D E

Powdered e x t r a c t

1

A

Atropine mg/g

12.69 12.59 12.57 12.48 12.25

12.59 12.65 12.64 12.44 12.34

A B C D E

0.29 0.29 0.30 0.29 0.29

0.31 0.30 0.30 0.28 0.29

(averaqe = 12.51 - 0.13 RSO = 1.07 % ) 11.21 11.20 11.18 11.20 11.12 11.18 11.30 11.22 11.30 11.17 (averaqe = 11.21 - 0.06 RSD = 0.49 %) 11.48 11.34 ii.36 ii.34 11.36 11.36 11.26 11.47 11.32 11.28 (averaqe = 11.36 0.07 RSD = 0.62 % )

(average = 0.29 t 0.01 RSD = 2.87 % ) 0.50 0.50 0.49 0.50 0.48 0.49 0.50 0.49 0.52 0.53 (average = 0.50 f 0.01 RSD = 2.59 % ) 0.20 0.20 0.18 0.22 0.18 0.22 0.20 0.21 0.22 0.19 faveraae = 0.20 i-0.02 RSD = 7.71 % )

11.64 11.58 11.74 11.76 11.91

0.11 0.09 0.09 0.10 0.12

-

2

Scopolamine mg/g

11.58 11.58 11.59 11.68 11.80

(averaqe = 11.69 0.11 RSD = 0.97 % )

-

0.11 0.11 0.09 0.11 0.12

T o t a l a l k a l o i d s mg/g

12.81 ? 0.14 (RSD = 1.08 %)

11.71 0.06 (RSO = 0.54 X )

11.56 2 0.07 (RSD = 0.63 %)

11.79 ? 0.12 (RSD = 1.02 % )

(average = 0.11 f 0.01 RSD = 11.80 %)

GSber e t a1.32 applied gas chromatography f o r an i n v e s t i g a t i o n o f the s t a b i l i t y o f scopolamine hydrobromide i n eye-drops. During h e a t - s t e r i l i s a t i o n o f such s o l u t i o n s , scopolamine and t r o p i c acid, as w e l l as aposcopolamine, may be formed. By converting these compounds i n t o t h e i r t r i m e t h y l s i l y l d e r i v a t i v e s w i t h N . 0 - b i s ( t r i m e t h y l s i l y 1 )acetamide as s i l y l a t i n g reagent. good r e s u l t s could be obtained, as can be seen i n t h e chromatogram i n Figure 8.2.

Atropine

was used as an i n t e r n a l standard and the gas chromatography was performed on a packed 2 m long column w i t h 10 % SE-30 on s i l a n i z e d Chromosorb W, w i t h temperature programing. The U.S.P.

XX33 introduced gas chromatography f o r the assay o f a t r o p i n e i n various a t r o -

pine sulphate containing preparations ( I n j e c t i o n , Ophthalmic s o l u t i o n , Ophthalmic ointment and Tablets) using a 1.8 m long by 2 mm

I.D. packed column w i t h 3 % o f a m i x t u r e o f phenyl-

and methylpolysiloxane ( 1 : l ) as s t a t i o n a r y phase on " s i l i c e o u s e a r t h f o r gas chromatography". Homatropine was used as an i n t e r n a l standard f o r the assay, which was c a r r i e d o u t a t a column temperature o f 225OC. Gas chromatography was a l s o a p p l i e d f o r the assay o f Belladonna l e a f , e x t r a c t and t i n c t u r e w i t h homatropine as an i n t e r n a l standard. However, i n t h i s case a s h o r t e r column

(1.2 m) and a column temperature o f 215OC were used.

Also the B r i t i s h Pharmacopoeia 1 9 8 0 ~describes ~ an assay f o r a t r o p i n e i n a t r o p i n e sulphate eye drops and t a b l e t s , as w e l l as i n Hyoscyamus d r y e x t r a c t . However, a t r o p i n e was s i l a n i z e d w i t h N,O-bis(trimethy1si 1yl)acetamide and t r i m e t h y l d i c h l o r o s i l a n e ( 4 : l ) and gas chromatographed on a 1.5 m l o n g by 4 inn I.D.

packed w i t h 3 % OV-17 on "diatomaceous support" AWS a t

2OO0C using homatropine as an i n t e r n a l standard. Majlat35 worked o u t a gas chromatographic assay o f a t r o p i n e and phenobarbital i n pharmac e u t i c a l preparations containing Valeriana 1i q u i d e x t r a c t . A f t e r e x t r a c t i o n o f atropine, i t was hydrolyzed and the f r e e t r o p i c a c i d s i l y l a t e d w i t h N . 0 - b i s ( t r i m e t h y l s i l y 1 )acetamide. The

r n 1.0. packed w i t h 1.5 % DV-101 on assay was performed on a glass column. 1 m long by 3 m s i l a n i z e d Gas Chrom P, 100-120 mesh, a t 14OoC, using s i l y l a t e d 2-naphtol as an i n t e r n a l standard.

FIGURE 8.2

GAS CHROMATOGRAM OF TROPINE ALKALOIDS AND DERIVATIVES AS TRIMETHYLSILYL DERIVATIVES3' Scopoline ( l ) , t r o p i c a c i d ( 2 ) , aposcopolamine ( 3 ) , a t r o p i n e ( 4 ) and scopolamine ( 5 ) ; column: 2 m long, 3 mn 1.0.;

10 % SE-30 on Chromosorb W; temperature p r o g r a m i n g 100-240°C

n I

2

Referencei p. I 2

6

20

'

I

24

'

1

28

*

1

32

.

I

36 min

70

TABLE 8.8 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY Column

S o l i d support mesh CW 80-100 glass, 6 f t x 4 mn glass, 6 f t x 3 mn GP ABS t PEG 100-140 s . s . , 5 ft x 1/8 i n Aer 100-120 glass, 1.52 m x 5 mn I.D.C,,S 80-100 s . s . , 1.52 x 5 mm I.D. glass, 1.52 m x 5 m l*D*CWS s . s . , 1.52 m x 5 mn D.

.

80-100

QF-1

OF

TROPINE ALKALOIDS

Stat.phase

Temperature

Comp.Prep.

204q 175OC

a l k . s. a l k . pm.

2

SE-30

5 175OC

a l k . pm.

3

XE-60

10 2oooc a l k . pm.

4

SE-30 SE-30

t

XE-60

%

2-3 1.15

2.5

t

1

2.5 200'

SE-30 NGS NGS t PVP SE-30 XE-60 EGSSY HI-EFF 8B SE-30 OV-17 OV-17 OV-17 OV-17 OV-17

tOX. 5 210°c 1 23OoC alk. 5. 1 t 1 2JOOC 1 195 C and 220°c 1 220°c alk. 5. 1 23OoC 1 23OoC and 24OoC 2 alk. t o x . 5 alk.qnt.prep. 3 210Oc 3 210Oc a l k . prep. 3 225OC a l k . prep. 3 2lOOC

OV-17 OV-17 SE-30 OV-17

3 3 30 3

glass, 3 f t x 3 nun GQ 100-120 6 f t x 0.19 i n I.D. CG AW 80-100

OV-17 SE-30

3 2.5

s . s . , 60 cm x 4.5 mm I . D . CWS 80-100 glass, 2.4 m x 4 mm GQ AWS 80-100

XE-60 OV-17

5 1

glass, 1.2 m x 4 mm I . D . D i a t S 80-100 glass, 1.52 m x 3.2 mn I.D. GQ AWS

SE-30 QF-1

3.5 15

215OC 16OoC

glass, 1.5 m x 4 mn I . D . GQ glass, 1.5 m x 4 mm I.D. CW AWS t PEG 80-100 glass, 6 f t x 4 mn 1.D. Ana ABS 100-120

QF-1 SE-30

3 1.5

85-b85' 194 C

QF-1-0065 SE-52 HI-EFF-8B Apiezon L

3 2.5

2oooc

15

glass, 2.25 m x 6 mn I.D. F i r . BW glass S, 1.8 m x 2 mn 1.0. GQ 100-120

Cab 20M OV-17 OV-225

20 3 1

glass, 1.2 m x 4 mn I . D . GQ AW 100-120 s . s . , 2 m x 3 mm I.D. CW AWS 80-100

OV-17 SE-30

3 10

5 ft glass, 3 f t glass, 3 f t glass, 3 f t

x x x x

0.093 2 mn 2 mn 2 mn

2 m l m

60

glass, un x glass, 60 cm x glass, 1.2 m x glass, 1.2 m x

3 4 4 4

mm mn mm mn

n I.D. CP CP CP

CG CG I.D. GQ I.D. GQ I.D. GQ I.D. GQ

CWS AWS AWS AWS

AW 80-80 80-100 80-100 80-100

AWS 80-100 AWS 80-100 AWS 80-100 AWS 80-100 AWS

2.8 m x 4 mn I.D. GQ 100-120 glass, 1.45 m x 3.8 mn GQ 80-100 glass, 1.45 m x 3.8 mn

GQ 80-100

Ref.

120-2300c

9 10

11

alk.qnt.pn/

12

alk.pm.

13

pr. alk,pm.

15

24OoC

6 C/mig

8

90-250 C p r . 120-275°C p r . 6 Chin110-210°C p r . 100-220°C p r .

alk.pn. alk.qnt.prep.

16 17

a l k . s. scop.decomp.pr. TMS d e r i v . a1 k .prep. a1k, prep. d e r i v.

18 19 20 21

TMS

glass, 1.2 m x 4 mn I.D.

glass, glass, glass, glass,

1.8 1.2 1.5 1m

CP BW 175-21Op

m x 2 mn I.D. SiEG m x 2 mn I.D. S i E G m x 4 mm 1.0. Diasup. AWS x 3 mm I.D.

GP S 100-120

PtM s i l ( 1 : l ) 3 PtM s i l ( 1 : l ) 3 OV-17 3 OV-101 1.5

pr

alk.TMS deriv. 22 a1 k.pm. 23 a l k . tox.

24 25 26 pseudotropine/ 27 132OC tropine 14OoC r e a c t i o n GLC 28 95OC scop. p l ur.as scopl.deriv. 29 GC-MS 215OC alk.pm.extr. 30 100-240°C p r . scop. decomp. 32 or. TMS 225OC a t r . qnt.prep. 33 215OC a l k . pm. 2oooc a t r . qnt.prep. 34 a t r . as trop.a.35 14OoC s i 1. der.

.

71

Abbreviations :

ABS = acid, base washed, s i l a n i z e d AW = a c i d washed Aer = Aeropak alk = alkaloid Ana = Anakrom a t r = atropine BW = base washed Cab = Carbowax CW = Chromosorb W decomp. pr. = decomposition product der = d e r i v a t i v e Dia S = Oiatoport S Diasup = diatomaceous support extr = extract Fir = firebrick GP = Gas Chrom P GQ = Gas Chrom Q I.D. = i n s i d e diameter

M s i l x = methylpolysiloxane p l = plasma pin = p l a n t m a t e r i a l P s i l x = phenylpolysiloxane prep = pharmaceutical preparation p r = (temperature) p r o g r a m i n g qnt = quantitative S = silanized s = separation sco = scopolamine scopl = scopoline S i E G = s i l i c e o u s e a r t h f o r GC s i l = silylated S.S. = stainless steel tox = toxicology TMS = t r i m e t h y l s i l y l tr0p.a = t r o p i c a c i d ur = urine

TABLE 8.9 SILYLATION OF SOME TRDPINE ALKALOIDS AND RELATED COMPOUNDS

1. With N. 0-bi s ( t r i m e t h y l s i l y l )acetamide To the sample (1.5 mg scopolamine, 0.5 mg scopoline, a t r o p i c a c i d ) add 400 p1 o f N,N-dimethylformamide and 400 p1 N,O-bis(trimethylsilyl)acetamide,

stopper t i g h t l y , and s w i r l un-

til complete d i ~ s o l u t i o n ~ ~ . Add 200 u l o f the s i l y l a t i n g agent t o the d r y sample (0.6 mg homatropine methylbromide o r 0.2 mg mandelic a c i d ) i n a cone-shaped v i a l , cover and a l l o w t o stand a t room temperature f o r 20 minutes w i t h occasional shaking 21

.

2. With N-methyl -N-trimethyl s i l y l -tri f l uoroacetamide Add a small excess o f N-methyl-N-trimethylsilyl-trifluoroacetamide t o the d r y f r e e base 22 o r t o the base i n benzene s o l u t i o n and heat t o 8OoC f o r a s h o r t t i m e

.

3. With hexamethyldisilazane Solve the dry f r e e base i n p y r i d i n e containing 5 % hexamethyldisilazane and a l l o w t o stand u n t i l next day

Reference8 p. I 2

23

.

12

8.2

REFERENCES

1 H.A.

Lloyd, H.M. Fales, P.F. Highet, W.J.A. VandenHeuvel and W.C. Wildman, J. AM. Chem. , 82 (1960) 3791. 2 E. Brochmann-Hanssen and A. Baerheim Svendsen, J. Pharm. S c i . , 5 1 .(1962) 1095. 3 C. Vigneron and J.M. P e l t , P l a n t . Med. P h y t o t h e r . , 2 (1968) 300. 4 R. Achari and F. Newcombe, P i a n t a Med., 19 (1971) 241 5 K.O. Parker, C.R. Fontan and P.L. K i r k , Anal. Chem., 35 (1963) 356. 6 E. Brochmann-Hanssen and C.R. Fontan, J. Chromatogr., 19 (1965) 296. 7 E. Brochmann-Hanssen and C.R. Fontan, J. Chromatogr., 20 (1965) 384. 8 A.C. Moffat, A.H. Stead and K.W. Smalldon, J. Chromatogr., 90 (1974) 19. 9 R.O. Z i n e r e r Jr. and L.T. Grady, J. Pharm. S c i . , 59 (1970) 87. 10 L.T. Grady and R.O. Zimmerer, J . Pharm. S c i . , 59 (1970) 1324. 11 R.S. Santoro, P.P. Progner, €.A. Ambush and D.E. Guttman, J . Pharm. S c i . , 62 (1973) 1346. 12 L.Z. Padula, A.L. Bandoni, R.V.D. Rondina and J.D. Coussio, P l a n t a Med., 29 (1970) 357. 13 G. Verzar-Petri and M.Y. Haggag. Herba Hung., 15 (1976) 87. 14 G. Verssr-Petri, M. Vincze Vermes, L. Horvgth, A . I . B a l i n t and T. Szarvas, A c t a Pharm. Hung., 45 (1975) 167. 47 (1977) 37. 15 G. Verzsr-Petri and Dinh Huynh K i e t . Acta Pharm. Hung. 16 J. Wilms, E. Riider and H. Katinq, Planta Med.. 3 1 (19773 249. 17 M. J. Solomon. F.A. Crane, B. L. uu Chu and E.S; Mika, J. 'Pharm. S c i . , 58 (1969) 264. 18 H. Frauendorf and H. Vogel, Fresenius' 2. Anal. Chem., 205 (1964) 460. 19 J.J. Windheuser, J.L. S u t t e r and A. S a r r i f , J. Pharm. S c i . , 6 1 (1972) 1311. 20 E. Nieminen, Z e n t r a l b l . Pharm. Pharmakother. L a b o r a t o r i u m s d i a g n . , 110 (1971) 1137. 21 B.F. Grabowski, 8.J. S o f t l y , B.L. Chang and W.G. Haney Jr., J . Pharm. sci., 62 (1973) 806. 22 H.W. Liebisch, H. Bernasch and H.R. Schitte, z. Chem., 13 (1973) 496. 23 W.J. G r i f f i n , H.P. Brand and J.G. Dare, J. Pharm. s c i . , 64 (1975) 1821. 24 L. Kazyak and E. Knoblock, Anal. Chem., 35 (1963) 1448. 25 H. Kolb and P.W. Patt, Arzneim.-Forsch., 15 (1965) 924. 26 N.C. J a i n and P.L. Kirk. Microchem. J., 12 (1967) 229. 27 C. van der V l i e s and B.C. Caron, J . Chromatogr., 12 (1963) 533. 28 C. Radecka and I.C. Nigam, J. Pharm. S c i . , 56 (1967) 1608. 29 W.F. Bayne, F.T. Tao and C. C r i t l o g o , J. Pharm. S c i . , 64 (1975) 288. 30 D.K. Wyatt, W.G. Richardson, B. McEwan, J.M. Woodside and L.T. Grady, J . Pharm. S c i . , 65 (1976) 680. 31 R.L. Ballbach, D.J. Brown and S.M. Walters, J . Pharm. S c i . , 66 (1977) 1553. 32 B. Gober, U. T i m and H. Diihnert, Z e n t r a l b l . Pharm. Pharmakother. L a b o r a t o r i u m s d i a g n . , 116 (1977) 13. SOC.

73

Chapter 9

PSEUWTROPINE ALKALOIDS

.................................................................. ......................................................................

9.1. Coca a l k a l o i d s 9.2. References 9.1.

73 84

COCA ALKALOIDS

The gas chromatography o f cocaine has mainly been d e a l t w i t h i n connection w i t h i n v e s t i g a t i o n s t o develop s e l e c t i v e and s e n s i t i v e methods f o r i t s detection and q u a n t i t a t i v e determin a t i o n i n pharmacological and t o x i c o l o g i c a l analysis. One study has so f a r been done on n a t u r a l l y o c c u r r i n g a l k a l o i d a l mixtures present i n the coca p l a n t (Erythroxylon coca Lam.) 1. The gas chromatography o f cocaine has mostly been c a r r i e d o u t on packed columns w i t h nonp o l a r s t a t i o n a r y phases. S i l i c o n e rubber SE-30 was u t i l i z e d by Lloyd e t al.', sen and Baerheim Svendsen3, Parker e t a1.4,

Brochmann-Hanssen and Fontan',

Brochmann-Hansas w e l l as by

Koontz e t a1.6, whereas SE-52 was used by Kolb and Patt7, DV-1 by Blake e t a1.8 and OV-101 and OV-25 by Moore'.

However, s t a t i o n a r y phases o f various p o l a r i t i e s have also been used

successfully, such as s i l i c o n e n i t r i l e gum (XE-60), (EGSS-Y) Fontan',

, and

polyester m e t h y l s i l i c o n e copolymer

c y c l ohexanedimethanol succinate p o l y e s t e r (HI-EFF-86)

by Brochmann-Hanssen and

and J a i n and Kirk";

neopentylglycol sebacate (NGSE) by Kolb and Patt', 11 neopentyl g l y c o l succinate (NGS) by Brochmann-Hanssen and Fontan

as w e l l as

.

1-2 m long packed columns w i t h 1-2 % s t a t i o n a r y phase and column temperatures o f about 175-24OoC were used t o o b t a i n reasonable r e t e n t i o n times f o r cocaine. For a gas chromatographic i d e n t i f i c a t i o n o f a l k a l o i d s , Brochmann-Hanssen and Fontan' recommended a combination o f two columns, one non-polar (SE-30) and one semi-polar (XE-60). Hammer e t a1.l'

recommended, f o r a d e f i n i t i v e GLC i d e n t i f i c a t i o n o f cocaine i n l a b o r a t o r i e s

where GC-MS apparatus i s available, an on column methylation o f cocaine by means o f t r i m e t h y l a n i l i n i u m hydroxide, whereby cocaine i s converted i n t o ecgonidine methyl ester, which appears as the major peak on the gas chromatogram on an OV-17 column, 2 f e e t l o n g a t 25OoC, i n addit i o n t o N,N-dimethylaniline,

a product derived f r o m t r i m e t h y l a n i l i n i u m hydroxide d u r i n g the

methyl at i on r e a c t i on. S c a r i n g e l l i 1 3 separated cocaine and a number o f o t h e r "caines" (Procaine, Tetracaine, Benzocaine) on a 2 f e e t long s i l i c o n e rubber column (10 % ) a t 220-225°C,

and w i t h temperature

programming, s t a r t i n g a t 75OC. He c o l l e c t e d the separated compounds and i d e n t i f i e d them by t h e i r special c r y s t a l t e s t s . Estimation o f cocaine was obtained by u s i n g benzocaine as an int e r n a l standard. For a quick detection o f cocaine i n mixtures w i t h o t h e r compounds, such as amphetamines, other "caines" etc.,

Cavallaro e t a1.14 a p p l i e d gas chromatography on t h r e e columns o f d i f f e r -

ent p o l a r i t y (SE-30, T r i t o n X 305 and OF-1). In t h i s way the i d e n t i t y o f t h e various compounds could be established v i a t h e i r r e t e n t i o n times. Detection and determination o f small amounts o f minor a l k a l o i d s and/or degradation products o f cocaine i n i l l i c i t cocaine samples may be o f importance i n t r a c i n g danestic and i n t e r n a t i o n a l clandestine cocaine routes.

developed a gas chromatoeraphic method f o r the

determination o f cis- and trans-cinnamoylcocaine i n i l l i c i t cocaine samples, i n which t h e

Referencesp. 84

74

cinnamylcocaine may be present i n concentrations o f 1 % o r l e s s o f the amount o f cocaine. The gas chromatographic analysis was c a r r i e d o u t a f t e r s i l y l treatment on packed columns w i t h 3 % OV-1 on Chromosorb W HP w i t h temperature programing, using eicosane as an i n t e r n a l standard.

A t y p i c a l chromatogram i s given i n Figure 9.1. FIGURE 9.1

GAS CHROMATOGRAM OF ILLICIT PERUVIAN COCA PASTE AFTER SILYL TREATMENT" Eicosane used as an i n t e r n a l standard. Packed glass column, 4 f t by 6 mm ID., 3 % OV-1 on Chromosorb W HP 100-120 mesh; temperature programming 190-280°C, 20°C/min

1 = Eicosane 2 = Cocaine 3 = cis-cinnamoylcocaine 4 = Trans-cinnamoylcocaine

l

0

~

l

2

~

L

l

'

6

l

'

6

l

'

1

'

10 min

An o f f i c i a l gas chromatographic method f o r q u a n t i t a t i v e determination o f cocaine hydroc h l o r i d e was described i n J. Assoc. O f f . Anal. Chem.,

6 1 (1978) 47316, whereby cocaine i s

extracted from a weakly basic aqueous s o l u t i o n , w i t h chloroform c o n t a i n i n g the i n t e r n a l standard, tetracosane. A 6 f e e t by 4 mn i n s i d e diameter glass column packed with 3 % O V - 1 on 100-120 mesh Chromosorb W HP was u t i l i z e d a t column temperature 225OC. I n pharmaceutical cocaine the h y d r o l y s i s products, benzoylecgonine and ecgonine may be present as i m p u r i t i e s . I n t o x i c o l o g i c a l studies these two compounds are found as the main metabolites o f cocaine. Several workers have gas chromatographed benzoylecgonine and ecgonine a f t e r d e r i v a t i z a t i o n , due t o the very poor gas chromatographic behaviour o f the two compounds. Moore"

proposed s i l y l a t i o n by means o f N.0-bis(trimethylsily1)acetamide

p r i o r t o GLC on 10 %

OV-101 and 3 % OV-25 columns, using temperature programming. When using the OV-25 column, the e l u t i o n order o f cocaine and the benzoylecgonine s i l y l d e r i v a t i v e was reversed, compared t o OV-101. The OV-25, therefore, was used as a confirmation o f the presence o f ecgonine and benzoylecgonine i n cocaine. OV-101 was the column o f one's f i r s t choice because the r e s o l u t i o n , s e n s i t i v i t y and the r e t e n t i o n times o f cocaine and i t s h y d r o l y s i s products were the most favorable, as w i l l be seen i n Table 9.1. Moore was able t o detect ecgonine and benzoylecgonine i n cocaine samples a t l e v e l s l e s s than 0.1 % and 0.3 %, respectively, as s i l y l d e r i v a t i v e s .

TABLE 9.1 RETENTION TIMES OF COCAINE, THE SILYL DERIVATIVES OF ECGONINE, BENZOYLECGONINE AND INTERNAL STANDARDS' Columns: 4 f t x 4 m I.O.,

10 % OV-101 on Chromosorb W HP; 6 f t x 4 m I.D..

3 % OV-25 on

Chromosorb Q. Compound

Retention time (min) OV-101

Hexadecane - i n t e r n a l standard Ecgonine s i l y l d e r i v a t i v e Eicosane - i n t e r n a l standard Cocaine Benzoylecgonine s i l y l d e r i v a t i v e Tetracosane - i n t e r n a l standard i n t e r n a l standard Triacontane

-

3.8 6.7

OV-25 2.8 5.2 25.8 24.5

19.7 22.2 26.7

30.2

Fish and Wilson17 worked o u t a method f o r the determination o f cocaine i n u r i n e by ext r a c t i o n o f the u r i n e w i t h d i e t h y l ether a f t e r a d d i t i o n o f h y d r o c h l o r i c a c i d t o remove i m p u r i t i e s , followed by a d d i t i o n o f sodium bicarbonate t o g i v e pH 8 (approx.) and f u r t h e r ext r a c t i o n o f the cocaine w i t h d i e t h y l ether, concentration o f the d i e t h y l e t h e r e x t r a c t and GLC. Benzhexol was u t i l i z e d as an i n t e r n a l standard. The r e p r o d u c i b i l i t y o f the gas chromatographic determination o f cocaine e x t r a c t e d from u r i n e i s seen i n Table 9.2. TABLE 9.2 REPRODUCIBILITY OF THE GAS CHROMATOGRAPHIC DETERMINATION OF COCAINE EXTRACTED FROM URINE17 Concentration (ug/ml) Actual Cocaine

The same authors"

3.25 13.0

Number o f determinations

Found 3.01 12.91

? 0.09 t 0.39

10 10

studied the e x c r e t i o n o f cocaine and i t s metabolites i n man; cocaine

was e x t r a c t e d from u r i n e w i t h d i e t h y l e t h e r and the benzoylecgonine w i t h chloroform, t o which the i n t e r n a l standard (5 a-cholestane) was added. Benzoylecgonine was converted i n t o i t s methyl e s t e r (cocaine) by means o f diazomethane, and the gas chromatography was c a r r i e d 17

out as described by t h e same authors i n t h e paper r e f e r r e d t o above

.

Wallace e t a l . l9 determined benzoylecgonine i n u r i n e a f t e r e x t r a c t i o n and methylation t o cocaine. Separate simultaneous determinations o f cocaine and benzoylecgonine were achieved by analyzing both a methylated (combined cocaine and benzoylecgonine) and a non-methylated (cocaine o n l y ) a l i q u o t o f the specimen e x t r a c t on a 1 m by 3 mm I.D.

OV-17 column on Supel-

coport 80-100 mesh a t 220°C. The recovery from b i o l o g i c a l specimens o f 93 and 65 % f o r cocaine and benzoylecgonine,

respectively, and 73 % conversion o f benzoylecgonine t o cocaine, p m -

vided detection l i m i t s o f 0.1 and 0.2 pg/ml f o r cocaine and benzoylecgonine,

respectively.

For the determinations of benzoylecgonine i n u r i n e Koontz e t a1.6 s a l t e d o u t t h e compound o f the u r i n e w i t h K2HP04 and KH2P04 i n t o 95 % ethanol, evaporated the ethanol e x t r a c t t o dryness and p u r i f i e d the benzoylecgonine by t h i n - l a y e r chromatography. The compound was then r e moved from the t h i n - l a y e r p l a t e , methylated w i t h dimethylformamide dimethyl acetal and determined by gas chromatography.

References p. 84

76

TABLE 9.3 RECOVERY OF COCAINE AND BENZOYLECGONINE FROM URINE1'

Benzoylecgoni ne Added t o u r i n e Amount determined vg/ml vdml *) 0.5 0.32 ? 0.05 1 0.69 f 0.05 2.5 1.56 f 0.08 5 3.14 2 0.40 10 6.29 t 1.23 20 13.56 f 0.54

Cocaine Added t o u r i n e d m l 0.25 0.5 1 2.5 5

Amount determined r g h l *) 0.20 ? 0.05 0.50 t 0.05 1.06 ? 0.09 2.18 f 0.24 4.63 ? 0.14

*) Mean o f quadruplet determination

f

standard d e v i a t i o n

To achieve a rapid, s e n s i t i v e and r e l a t i v e l y s p e c i f i c gas chromatographic screening procedure f o r cocaine, Blake e t a1 .8 reduced cocaine w i t h LiA1H4 t o 2-hydroxymethyl tropine, 0-acylated t h i s compound by t r e a t i n g i t w i t h pentafluoropropionic anhydride o r h e p t a f l uorob u t y r i c anhydride and chromatographed the d e r i v a t i v e formed using an OV-1 3 % on Chromosorb C HP 80-100 mesh column (4 f t by 4 mn I.D.) a t 15OoC and an e l e c t r o n capture detector which g r e a t l y increased the s e n s i t i v i t y o f the method. The procedure used by Blake e t a l . f o r the determination o f cocaine i n u r i n e involved a 2.5-fold concentration f a c t o r i n e x t r a c t i o n , and employing the pentafluoropropionic anhydride d e r i v a t i v e , a sample containing 39 ng/ml cocaine i n urine gave an average S/N r a t i o o f 1 4 : l f o r 1.0 v1 i n j e c t e d . Therefore, s e n s i t i v i t i e s o f 20-30 ng o f drug/ml o f the sample could be achieved without concentration through evaporation procedures. Javaid e t a1 ." applied p r a c t i c a l l y the same method t o determine q u a n t i t a t i v e l y cocaine and i t s metabolites benzoylecgonine and ecgonine. The method involved the formation o f acyl d e r i v a t i v e s which were separated on 3 X and 5 % O V - 1 columns and detected i n p i c m o l e quant i t i e s using an e l e c t r o n capture detector. Ecgonine and benzoylecqonine were d e r i v a t i z e d w i t h a mixture o f hexafluoroisopropanol and h e p t a f l u o r o b u t y r i c anhydride (1:Z). Cocaine was f i r s t reduced by LiA1H4 and then acylated by pentafluoropropionic anhydride. Benzoylecgonine, b u t n o t ecgonine, could a l s o be determined by reduction and subsequent acylation. This provided the basis f o r the determination o f cocaine, benzoylecgonine and ecgonine from the same sample. Cocaine could be determined i n u r i n e and plasma by t h i s method. The r e s u l t s obtained w i t h the method are given i n Table 9.4 TABLE 9.4 SIMULTANEOUS DETERMINATION

OF COCAINE,

ECGONINE AND BENZOYLECGONINE IN THE SAME SAMPLE"

Coca ine Ecgonine Benzoylecgoni ne

Concentration v / m l 'Calculated Defekin!d 0.25 0.21 0.1 0.12 3.0 2.9

*)

*) These values are the average o f three determinations Javaid e t al.'l extended t h e i r method described above" f o r the determination o f cocaine. t o i t s determination i n urine, plasma and red blood c e l l s o f volunteer subjects who were

given d i f f e r e n t doses o f cocaine intravenously. A t s l i g h t l y a1 k a l i n e pH, cocaine was e x t r a c t e d w i t h cyclohexane, reduced t o 2-hydroxymethyl t r o p i n e w i t h LiA1H4, a c y l a t e d w i t h pentafluorop r o p i o n i c anhydride and gas chromatographed using an e l e c t r o n capture detector. The recove r i e s from u r i n e (95-102 %), plasma (65-80 %) and r e d blood c e l l s (60-70

I) were

t h e same a t

the pH range o f 6.5 t o 9.5. Above pH 10 recoveries were g r e a t l y reduced, due t o chemical hyd r o l y s i s o f cocaine t o benzoylecgonine. Berry and Grove"

applied gas chromatography f o r cocaine base as a confirmatory t e s t f o r

i t s detection i n u r i n e samples a f t e r a simple pH adjustment and e x t r a c t i o n w i t h organic s o l vent. As a c o n f i n n a t i o n t e s t i n general

- especially

f o r TLC r e s u l t s f o r h o s p i t a l drug emerg-

encies as w e l l as f o r q u a l i t a t i v e and q u a n t i t a t i v e analysis o f n a r c o t i c drugs from b i o l o g i c a l 23 material - gas chromatography i s an i d e a l method

.

For the determination o f nine CNS drugs, i . a . cocaine, codeine, methadone, i n human plasma, Medzihradsky and D a h l ~ t r o mused ~ ~ gas chromatography. The drugs were e x t r a c t e d w i t h benzeneisopropanol ( 9 : l ) from 1 m l plasma a f t e r adjustment o f the pH t o 10. The benzene-isopropanol e x t r a c t was evaporated t o dryness and the residue dissolved i n 25-50 p1 aceton. 2 p1 o f the s o l u t i o n was i n j e c t e d i n t o the gas chromatograph. The recovery o f the drugs, 0.25-4 pg/ml. was 80-100 %, the lower l i m i t o f s e n s i t i v i t y 3 ng t o 6 ng. corresponding t o concentrations i n plasma o f 0.05 u g h 1 and 0.13 pg/ml respectively. To be able t o determine small q u a n t i t i e s o f cocaine i n blood plasma f o l l o w i n g i t s t o p i c a l a p p l i c a t i o n t o mucous membranes, Dvorchik e t a l . 2 5 developed a method using a n i t r o g e n sensit i v e flame i o n i z a t i o n detector. A l i q u o t s ( 1 o r 2 m l ) o f whole blood o r plasma were made a l k a l i n e w i t h carbonate b u f f e r and e x t r a c t e d w i t h benzene-isopropanol

(9:1),

t h e cocaine was

e x t r a c t e d back w i t h 0.01 N h y d r o c h l o r i c acid. the aqueous phase adjusted t o pH 7-7.3 and the cocaine base e x t r a c t e d w i t h benzene-isopropanol.

A f t e r evaporation t o dryness the residue

was dissolved i n aceton and the s o l u t i o n obtained used f o r gas chromatographic analysis: 20 ng o f cocaine from 1 m l whole blood o r plasma could be determined by t h i s method on a SP2100-DB 3 % column. The mean a n a l y t i c a l recovery was 65

%.

I n a comparison o f the methods employed f o r the detection o f i . a . cocaine and metabolites i n t o x i c o l o g i c a l analysis, Bastos and Hoffman"

d e a l t w i t h the e x t r a c t i o n procedure f r o m b i o -

l o g i c a l m a t e r i a l and the subsequent gas chromatography. They concluded t h a t cocaine may be reduced and a c y l a t e d before gas chromatographic analysis, b u t t h a t i t does n o t r e q u i r e derivat i z a t i o n . However, i t s metabolites, benzoylecgonine and ecgonine, do r e q u i r e e s t e r i f i c a t i o n w i t h dimethyl a c e t a l , N,N-dicyclohexylcarbodiimide, o r w i t h a s i m i l a r reagent. The r e s u l t i n a compounds are normally separated a t temperatures i n the range 215-240°C.

Chromatographic

columns of SE-30 on Chromosorb W AW and s i l a n i z e d have been used f o r the a n a l y s i s o f cocaine as w e l l as f o r the a l k y l d e r i v a t i v e s o f benzoylecgonine and ecgonine. The use o f O V - 1 has become popular f o r the analysis o f basic drugs, thus p r o v i d i n g an a l t e r n a t e system t o SE-30. Also, OV-17 has been used successfully. The r e s u l t s obtained by gas chromatography should be confirmed by a l t e r n a t i v e procedures. This could i n v o l v e performing the GC analysis under d i f f e r e n t operating conditions, o r employing techniques t h a t can be l i n k e d t o a GC system. Such techniques may range from m i c r o c r y s t a l t e s t s t o t h e more s o p h i s t i c a t e d i n f r a r e d spectrometry and mass spectrometry. Because o f the l i m i t e d s e n s i t i v i t y o f a conventional flame-ionization d e t e c t o r and i t s non-selective response t o co-extracted endogenous constituents, J a t l o w and Baileyz7 u t i l i z e d a n i t r o g e n detector f o r gas chromatographic assay o f cocaine i n plasma. They achieved the de-

Refemnees p. 84

termination o f cocaine i n plasma a t concentrations as low as 5 u g / l i t e r u s i n g benzoylecgonine-n-propylester as an i n t e r n a l standard. Despite the s e l e c t i v e response o f the n i t r o g e n detector, the authors found i t d e s i r a b l e t o use a back e x t r a c t i o n and clean-up step t o e l i m i n a t e i.a. phospholipids. The more comnon

"drugs o f abuse" and commonly used l o c a l anesthet-

i c s d i d n o t i n t e r f e r e , b u t serious sources o f i n t e r f e r e n c e were found t o be contaminants on glass ware, rubber stoppers and supposedly pure solvents. Kogan e t a1 .28 determined cocaine and i t s p r i n c i p a l metabolite i n man, benzoylecgonine, by using gas chromatography and (a) e l e c t r o n capture d e t e c t i o n f o r the determination o f benzoylecgonine from plasma as i t s pentafluorobenzylderivative, (b) FID f o r the determination o f benzoylecgonine and cocaine from u r i n e as t r i m e t h y l s i l y l d e r i v a t i v e s , and ( c ) n i t r o g e n detect i o n f o r the determination o f cocaine from plasma. The l i m i t s o f d e t e c t i o n f o r cocaine, underivatized, and benzoylecgonine as i t s pentafluorobenzyl d e r i v a t i v e i n plasma, were 10 and 5 ng/ml, respectively. I n u r i n e the s e n s i t i v i t y l i m i t s o f the s i l y l d e r i v a t i v e s o f cocaine and benzoylecgonine were 0.5 and 1.0 ug/ml, r e s p e c t i v e l y . The c o e f f i c i e n t o f v a r i a t i o n ranged between 0.9 and 2.2 % and the c o e f f i c i e n t o f determination was 0.99 f o r the method used. For the determination o f benzoylecgonine from plasma. t h e compound was e x t r a c t e d a f t e r b a s i f i c a t i o n (pH 9.5) w i t h ethanol-chloroform (20:80),

then i t was immediately reacted w i t h

pentafluorobenzyl bromide and the pentafluorobenzyl-benzoylecgonine

p a r t i t i o n e d i n t o a non-

p o l a r solvent (benzene) l e a v i n g the niure p o l a r i n t e r f e r i n g substances behind. This i s the important step i n the procedure. Chlorproethazine proved t o be a very r e l i a b l e i n t e r n a l standard; i t does n o t r e a c t w i t h the d e r i v a t i z i n g reagent, i s e a s i l y back-extracted i n t o acid, i s l i n e a r over a 100-fold conc e n t r a t i o n range and i s e a s i l y resolved on the gas chromatograph.

A method f o r simultaneous determination o f cocaine and benzoylecgonine i n u r i n e was developed by Von Minden and D'Amato'',

whereby the compounds were e x t r a c t e d f r o m u r i n e i n t o

ethanol-chloroform (25:75) followed by p r o p y l a t i o n o f benzoylecgonine using p r o p y l i o d i d e and a mixture o f trimethylphenylammoniumhydroxide (0.1 M i n methanol ) and tetramethylamnoniumhydroxide (25 % i n methanol) i n N,N-dimethylacetamide;

n-pentylbenzoylecgonine was u t i l i z e d

as an i n t e r n a l standard. Since the recovery from b i o l o g i c a l samples was 99 and 80 % f o r cocaine and benzoylecgonine. r e s p e c t i v e l y , and 98 % o f the benzoylecgonine was converted i n t o i t s n-propylester, the detection l i m i t o f 0.2 ug/ml i n 5 m l u r i n e was obtained.

A procedure f o r the simultaneous determination o f cocaine and benzoylecgonine i n u r i n e specimens was developed by J a i n e t a1 .30. The drug was e x t r a c t e d w i t h chloroform-isopropanol from u r i n e samples saturated w i t h a b i s a l t - b u f f e r . The organic e x t r a c t was evaporated t o dryness, and an a l i q u o t o f the residue i n j e c t e d onto t h e gas chromatograph t o determine t h e presence o f cocaine and the l o c a t i o n o f any extraneous peaks. Depending on the chromatogram obtained, benzoylecgonine was converted by on-column d e r i v a t i z a t i o n i n t o an a l k y l e s t e r , which was e l u t e d on the chranatogram undisturbed by o t h e r compounds present i n t h e e x t r a c t . The r e l a t i v e r e t e n t i o n times o f benzoylecgonine e s t e r s formed by on-column d e r i v a t i z a t i o n

f r o m the u t i l i z e d N.N-dimethyl formamide dimethyl -, d i e t h y l - . dipropyl-, d i b u t y l - , d i -terb u t y l , and dicyclohexyl-acetals are given i,n Table 9.5 To be able t o determine unchanged cocaine and benzoylecgonine excreted i n human u r i n e i n amounts t h a t are generally below the l i m i t s o f detection, J i n d a l and Vestergaard3'

used gas

chromatography-mass spectrometry and s t a b l e isotope l a b e l e d analogs (cocaine-d3 and benzoyl-

79

TABLE 9.5 RELATIVE RETENTION TIMES OF BENZOYLECGONINE ESTERS FORMED BY DIMETHYLFORMWIDE-DIALKYL ACETALS" Column: 3 f t by 2 mm I.D., Ester

3 % OV-17 on Chromosorb W HP 80-100 mesh a t 2OO0C Retention time (sec)

Methyl ( = cocaine) Ethyl Isopropyl n-Propyl ter-Bu t y l n-Butyl Cyclohexyl

R e l a t i v e r e t e n t i o n time

147 181 192 239 2 46 329 849

1.00 1.27 1.31 1.63 1.67 2.24 5.78

ecgonine-d3, both N-methylated-d3) as i n t e r n a l standards. The assay u t i l i z e d i o n focussing t o monitor i n the GLC-effluent the molecular ions o f cocaine and benzoylecgonine generated by electron-impact i o n i z a t i o n . The assay can measure 2 ng o f cocaine/ml and 5 ng o f benzoylecgonine/ml w i t h about 5 % precision. A magnetic sector, single-focussing mass spectrometer (LKB 9000) i n t e r f a c e d w i t h a gas chromatograph and equipped w i t h a m u l t i p l e i o n d e t e c t o r / peak matcher accessory (MID/PM) was used. The assay o f cocaine and benzoylecgonine i s sensit i v e , s p e c i f i c and a l s o applicable t o o t h e r body f l u i d s and tissues. I n a paper on chromatographic q u a n t i t a t i o n o f cocaine, Roberson3'

pointed o u t some prob-

lems concerning q u a n t i t a t i v e determinations w i t h i n t e r n a l standards. When i n j e c t i n g a chloroform s o l u t i o n o f cocaine hydrochloride and an i n t e r n a l standard i n t o the i n j e c t i o n p o r t w i t h a syringe, the chloroform i s immediately b o i l e d away, l e a v i n g a deposit o f cocaine hydrochlori d e and the i n t e r n a l standard on the i n s i d e o f the needle. Cocaine hydrochloride i s n o t readi l y soluble i n chloroform. The deposit i s p a r t i a l l y washed away on expulsion o f t h e remainder o f the s o l u t i o n by depression o f the plunger. The washing process i s n o t reproducible and gives r i s e t o variance i n the r e s u l t s . The i d e a l s i t u a t i o n i s , therefore, t h a t both analyte and i n t e r n a l standard are very r e a d i l y soluble i n the s o l v e n t t o be used; e v e n t u a l l y an i m proved r e p r o d u c i b i l i t y can be achieved by lowering the amounts o f cocaine hydrochloride i n the s o l u t i o n w h i l e keeping the concentration o f the i n t e r n a l standard ( r e a d i l y s o l u b l e i n chloroform) constant. Because methylecgonine has been found t o be a prominent u r i n a r y m e t a b o l i t e o f cocaine, i. a. a f t e r s t r e e t use o f cocaine, which p r i m a r i l y i n v o l v e s i n t r a n a s a l a p p l i c a t i o n and i n t r a -

venous i n j e c t i o n r a t h e r than ingestion, Ambre et.a1.33 developed a GC-MS method f o r the determination o f methylecgonine i n u r i n e . The i d e n t i f i c a t i o n o f t h i s m e t a b o l i t e i n t h e u r i n e i s an e f f i c i e n t and r e l i a b l e means o f d e t e c t i n g cocaine use. Samples o f 0.5 o r 2 m l u r i n e were adjusted t o pH 8.5-9 and phencyclidine was added as an i n t e r n a l standard. The sample was then e x t r a c t e d w i t h methylene ch1oride:isopropanol

(3:l).

the organic phase separated and

taken t o dryness. The residue was redissolved i n isopropanol o r ethanol and analyzed by GC-MS i n t h e selected i o n mode. A 74 cm long, 2 mm I.D.

packed glass column w i t h 2 % OV-101 on

Gas Chrom Q AWS and temperature p r o g r a m i n g 140-240°C,

15'C/min

was used.

For detection o f cocaine, a n a l y s i s o f methylecgonine has advantages over t h a t o f benzoylecgonine. Methylecgonine can be gas chromatographed d i r e c t l y on comnon gas chromatographic s t a t i o n a r y phases w i t h o u t d e r i v a t i z a t i o n , and i t s e a r l y e l u t i o n shortens a n a l y s i s time. However, because of i n t e r f e r e n c e of o t h e r compounds i n a u r i n e analysis, the combination o f GC

Ralerencsl p. 84

80

and MS i s indispensible. The assay s e n s i t i v i t y o f methylecgonine and cocaine by GC-MS using selected monitoring was found t o be 0.1 pg/ml when e x t r a c t i n g a 2 m l u r i n e sample. E x t r a c t i o n recovery a t 5 and 10 ug/ml was 51 % f o r methylecgonine and 89 % f o r cocaine. Graas and Watson34 developed a GC-MS-COM method f o r the determination o f benzoylecgonine i n u r i n e f o l l o w i n g a one-step e x t r a c t i o n and d e r i v a t i z a t i o n technique, an e x t r a c t i v e a1 k y l a t i o n technique. A 2 m l sample o f u r i n e was made basic w i t h sodium hydroxide c o n t a i n i n g t e t r a hexylammonium hydrogen sulphate (THA), and the i o n p a i r benzoylecgonine-THA was e x t r a c t e d i n t o a 5 m l s o l u t i o n o f iodoethane i n methylene c h l o r i d e . Using t h i s e x t r a c t i o n procedure, 70 % o f the benzoylecgonine was converted t o the e t h y l e s t e r . Residual amounts on THA caused gas chromatographic i n t e r f e r e n c e s r e s u l t i n g i n broad solvent peaks. Therefore, the THA residue was f i r s t dissolved i n toluene, then the benzoylecgonine was removed q u a n t i t a t i v e l y and w i t h o u t any major interferences from THA by a l i q u i d - l i q u i d e x t r a c t i o n i n t o hexane. The f i n a l volumes used were optimized so t h a t no f i n a l concentration step was necessary. L i n e a r i t y was demonstrated from 1 t o 50 pg/ml. Q u a n t i t a t i o n was accomplished by m o n i t o r i n g m u l t i p l e mass spectral fragments w i t h a p r e c i s i o n o f 6 % ( c o e f f i c i e n t o f v a r i a t i o n ) o r less. I n a c o l l a b o r a t i v e study on the q u a n t i t a t i v e determination o f cocaine hydrochloride i n powders and t a b l e t s t h a t a l s o contain other drugs ( f . a . c a f f e i n e , procaine hydrochloride and benzocaine) as w e l l as lactose, mannitol and starch, the f r e e cocaine base was e x t r a c t e d w i t h chloroform from an aqueous s o l u t i o n a f t e r a d d i t i o n o f %HP04. Tetracosane (n-C24)

was added

i n the e x t r a c t i n g chloroform as an i n t e r n a l standard. Since cocaine base and tetracosane are both r e a d i l y soluble i n chloroform, none o f the problems mentioned by Roberson3’ were encountered. The recoveries ranged from 98.7 t o 103 % f o r cocaine hydrochloride amounts ranoing 35 from 6 t o 100 %, w i t h a c o e f f i c i e n t o f v a r i a t i o n from 0.89 t o 3.16

.

For the assay o f cocaine i n E r y t h r o x y l o n coca Lam. fra three l o c a t i o n s i n Peru, Turner e t al.’ worked o u t a gas chromatographic method. The a l k a l o i d s were e x t r a c t e d by r e f l u x i n g the powdered l e a f samples (1.00 g) w i t h ethanol (40 m l ) f o r 15 minutes. The f i l t r a t e was evaporated t o dryness under reduced pressure i n a r o t a r y evaporator, the residue dissolved i n 20 m l chloroform and the a l k a l o i d s e x t r a c t e d i n t o 1.5 % aqueous c i t r i c acid. The pH o f the s o l u t i o n was adjusted t o 8.2 w i t h sodium bicarbonate and the a l k a l o i d bases e x t r a c t e d w i t h chloroform. A f t e r evaporation the residue was dissolved i n ethanol c o n t a i n i n g the i n t e r n a l standard

(androst-4-ene-3,17-dione) and gas chromatographed on a packed column o f 6 % OV-1 on Chromosorb W. A t y p i c a l chromatogram i s shown i n Figure 9.2 and the r e s u l t s o f the a n a l y s i s i n Table 9.6. TABLE 9.6

COCAINE

CONTENT IN

ERYTHROXYLON

c m Lam.’

Species

Content (%)

*) cuzco T r u j i 11o Tinqo Maria

0.60 0.60

0.57

Coefficient o f v a r i a t i o n (%) **)

0.03 0.03 0.01

*) Mean o f t r i p l i c a t e determinations **) Mean c o e f f i c i e n t o f v a r i a t i o n = 3.9

5.0 5.0 1.8

81 FIGURE 9.2 CHROMATOGRAM OF COCAINE

FROM COCA LEAVE+

Column: 2.4 m l o n g b y 2 mn 1.0. packed g l a s s column, 6 % O V - 1 on Chromosorb W AWS a t 22OoC

1 = I n t e r n a l standard (Androst-4-ene-3,17dione)

2 = Cocaine

min 30

10

20

0

C a p i l l a r y gas chromatography o f c o c a i n e has so f a r been s c a r c e l y done. C h r i s t o p h e r s e n and R a s m ~ s s e nanalyzed ~~ a number o f n a r c o t i c drugs, i.a. cocaine. on a g l a s s c a p i l l a r y column c o a t e d w i t h SE-30 u s i n g c o l d on-column i n j e c t i o n (5OOC). The oven t e m p e r a t u r e was p r o g r a m e d t o 25OoC w i t h a speed o f 5°C/min and cocaine was e l u t e d a t 198OC as a v e r y sharp peak. The same i n j e c t i o n t e c h n i q u e was a p p l i e d b y P l o t c ~ y kf o~ r ~a number o f u n d e r i v a t i z e d drugs. i . a . cocaine. He used f u s e d s i l i c a c a p i l l a r y columns w i t h n o n - e x t r a c t a b l e s t a t i o n a r y phases ( s i l oxane d e a c t i v a t e d c r o s s li n k e d SE-54) and o b t a i n e d e x c e l l e n t r e s u l t s . TABLE 9.7 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY OF PSEUDOTROPINE ALKALOIDS Column

S o l i d support mesh

Stat.phase

g l a s s S, 2.4 m x 2 mm 1.0. cw AWS 100-200 g l a s s , 6 f t x 4 mm 1.0. cw 80-100 CP ABS 100-140 g l a s s , 6 f t x 3 mn 1.0. t PEG CW AW 60-80 s . s . , 5 f t x 1/8 i n 0.0. CP AWS 80-100 g l a s s , 3 f t x 3 mm 1.0.

g l a s s , 6 f t x 1/4 i n

GQ 100-120

glass, 1 m

GP S 100-120

OV-1 SE-30 SE-30 SE-30 SE-30 XE-60 EGSSY HI-EFF 8 8 SE-30

References p. 84

Temperature

6 220°C 2-3 222OC 1. l! 5 175OC 20oOc 5 21oOc 1 175OC 1 220% 1 230;C 1 230TC 24OoC 2.5 2oooc

Comp.Prep.

Ref.

coc.qnt.prn.

1

COC.

2

COC.

3

coc.tox.id.

4

a1k.s.

5

coc.bzecg.tox. ur. -. .

6

SE-52 t Ver.

CW S 80-100

%

SE-52

:'5 15O-25O0C 2.5 15O-25O0C

7

82 TABLE 9.7 (continued) ~

~~~

X 1

Col umn glass, 1 m glass, 4 ft x 4 mn I.D.

Solid support

Stat.phase

CW S 80-100 CG HP 80-100

NGSE OV-1

glass, 4 ft x 4 mn 1.0.

CW HP 100-120

OV-101

10

OV-25

3

glass, 6 f t x 6 mn I . D .

GQ 100-120

glass 3 ft x 0.070 in 1.0. GP S 100-120 glass, 3 f t x 2 mm GQ S 100-120

HI-EFF-88 NGS GP AW 80-100 NGS t PVP glass, 6 f t x 2 mm I . D . CW 80-100 OV-17 glass, 2 f t x 1/4 in SE-30 glass, 1.8 m x 3.5 mn 1.0. CW S 80-100 SE-30 CW S 100-120 Triton X CW S 100-120 QF-1

glass, 4 ft x 6 n I . D .

CW HP 100-120

glass, 1.8 m x 4 mn 1.0. glass, 1 m x 3 mm 1.0. glass, 1 m x 3 mn I . D .

CW HP 100-120 OV-1 CW AWS 100-120 OV-17 Sup 80-100 OV-17

glass, 6 ft x 2 mm I . D .

CW HP 80-100

OV-17

glass, 10 f t

GQ 100-120

OV-225

glass S, 5 ft glass, 1.8 m x 4 n 1.0.

GQ GQ 100-120

OV-225 OV-1

glass, 3 ft x 2 mn 1.0.

CW AWS 100-120 SP-2100-DB

glass, 1.8 m x 2 mn I.D. glass, 1.8 m x 2 mm I.D.

GQ 100-120

OV-17

sup 80-100

OV-225 SE-30

GQ 80-100

sup 80-100 glass S, 1.8 m x 2 mm I . D . Sup 100-120

glass S, 6 ft x 2 mm I.D. GQ 80-100 glass S, 3 ft x 2 n I . D . CW HP 80-100 glass S, 1.8 m x 2 m I . D . GQ 100-200

180-260°C pr 3'~/min 170-26OoC pr 3'~/min 22O-25O0C 23OoC

Comp.Prep. a1k.s. coc.(deriv.) id.ur. COC.

bzecg

.

ecg.

Ref. 7 8 9

COC. tox. a1k.s.

10

COC.

12 13

11

23OoC

7 10

1 2 3

25OoC 200-225°C 190°c 190°c 195OC

s.i. "caines" COC. t

other drugs, pl.

14

cis-, trans-

15

SP-2250-DA

ci nm. coc. 16,3 coc.qnt. coc.qnt. ur. 17,18 2.9 185OC coc.becg. der. 19 3 22ooc qnt .ur. coc.ecg.bzecg. 2o 130-190°C qnt. ur. 5 llO°C COC. der.qnt. 21 ur. coc.id.ur. 2 190°c 22 COC. t other 24 3 210Oc drugs, id. 3 200-230°C pr coc.qnr.bl.pl. 25 30°/rnin 3 26OoC coc.qnt.pl. 27 becg. der. qnt. pl 3 25OoC 3 2oooc coc.bzecg.der. 28 qn t. ur. 25OoC coc. qnt. pl . 3 3 238-26OoC pr

SE-30

3

ov-22

UC W-98 OV-17 OV-1

GQ AWS 100-120 OV-101

glass, 1.8 m x 2 mm 1.0.

CW HP 100-120

f.sil. cap. 25 m x 0.32 nun 1.0.

1 1

150-2OO0C 150°C

OV-1

glass, 74 cm x 2 mm 1.0.

glass cap. 20 m x 0.35 mm 1.0.

3

Temperature

OV-17 SE-30 SE-54

2

.

12 C / r n i i 200-230 C pr 6OC/min 3.8 2oooc 3 2oooc 1.5 205OC 2 3

COC. bzecg. der. qnt. ur. coc.bzecg.der. ur. coc.bzecg. qnt. ur. 140-240°C pr meecg.qnt.ur. 150pin 255 C bzecg.der.qnt. ur. 5O-25O0C pr. COC. 50C/man 80-250 C pr 200C/rni n

COC.

29 3o

31

33 34 36 37

83 TABLE 9 . 8 PSEUDOTROPINE ALKALOIDS - LIST OF ABBREVIATIONS

ABS = a c i d , base washed, s i l a n i z e d alk = alkaloid AWS = a c i d washed, s i l a n i z e d b l = blood bzecg = benzoylecgonine CW = Chromosorb W cap = c a p i l l a r y CP = Chromosorb P cinm.coc. = cinnamylcocaine der = d e r i v a t i v e ecg = ecgonine f . s i l = fused s i l i c a ft = feet GP = Gas Chrom P GQ = Gas Chrom Q

HP = h i g h performance I.D. = i n s i d e diameter i d = identification meecg = methylecgonine p l = plasma pn = p l a n t m a t e r i a l p r = (temperature) programing prep = pharmaceutical preparation qnt = quantitative S = silanized s = separation s.s = stainless steel Sup = S u p e l c o p o r t tox = toxicology Ver = Versamid 900 u r = urine

TABLE 9 . 9 DERIVATIZATION OF SOME PSEUDOTROPINE ALKALOIDS 1 S i l y l a t i o n o f ecgonine and benzoylecgonine i n c o c a i n e w i t h N,O-bi s ( t r i m e t h y 1 s i l y l )acetamide

A 25 mg sample o f c o c a i n e h y d r o c h l o r i d e ( c o n t a i n i n g ecgonine a n d / o r b e n z o y l e c g o n i n e as i m p u r i t i e s ) i s p l a c e d i n a 1-ml g l a s s - s t o p p e r e d t e s t t u b e and 500 p1 o f t h e r e a g e n t i s added. The t u b e i s s t o p p e r e d l o o s e l y and h e a t e d a t 75OC f o r t e n m i n u t e s w i t h o c c a s i o n a l a g i t a t i o n . A f t e r d e r i v a t i z a t i o n i s complete, 3 - 4 p1 o f t h e s o l u t i o n a r e i n j e c t e d f o r gas c h r o m a t o g r a p h i c 9 analysis . 2 M e t h y l a t i o n o f benzoylecgonine ( t o c o c a i n e ) M e t h y l a t i n g r e a g e n t : Add 1 volume o f c o n c e n t r a t e d s u l p h u r i c a c i d s l o w l y and w i t h c o o l i n g , i n t o 2 volumes o f methanol. To t h e p u r i f i e d r e s i d u e o b t a i n e d f r o m t h e u r i n e sample ( 5 m l ) a f t e r e x t r a c t i o n w i t h 25 m l ethanol-chloroform (1:5),

i s added 0.6 m l o f t h e m e t h a n o l i c s u l p h u r i c a c i d , t h e m i x t u r e i s

v o r t e x e d t o ensure complete d i s s o l u t i o n , and k e p t a t 85OC f o r t e n m i n u t e s . A f t e r c o o l i n g , t h e r e a c t i o n p r o d u c t ( c o c a i n e ) i s e x t r a c t e d w i t h d i e t h y l e t h e r . The d i e t h y l e t h e r i s e v a p o r i z e d ,

1 m l w a t e r and s u f f i c i e n t s o l i d sodium b i c a r b o n a t e f o r minimal n e u t r a l i z a t i o n added. The r e a c t i o n p r o d u c t ( c o c a i n e ) i s e x t r a c t e d w i t h 0.2 m l o f c h l o r o f o r m , c o n t a i n i n g t h e i n t e r n a l standard (butylanthraquinone). Five 19

graphic analysis

pl

o f t h e o r g a n i c l a y e r a r e i n j e c t e d f o r gas chromato-

.

3 Reduction o f c o c a i n e and/or benzoylecgonine w i t h LiA1H4 t o 2-hydroxymethyl t r o p i n e and sub-

sequent 0 - a c y l a t i o n w i t h h e p t a f 1u o r o b u t y r i c a n h y d r i d e o r p e n t a f l u o r o p r o p i o n i c a n h y d r i d e . To 5.0 m l o f an aqueous s o l u t i o n c o n t a i n i n g c o c a i n e i s added 1 m l o f s a t u r a t e d sodium t e t r a b o r a t e s o l u t i o n and 2.0 m l o f cyclohexane. The d r u g i s e x t r a c t e d i n t o t h e cyclohexane phase and t h i s t r a n s f e r r e d t o a t e s t t u b e . 50

pl

o f a saturated LiAIH4 s o l u t i o n i n d i e t h y l

e t h e r a r e added. A f t e r 3 m i n u t e s 50 p1 d i s t i l l e d w a t e r a r e added t o t h e cyclohexane, and t h e m i x t u r e shaken. Next, 50

pl

o f heptafluorobutyric anhydride ( o r a l t e r n a t i v e l y pentafluoro-

p r o p i o n i c a n h y d r i d e ) a r e added t o t h e cyclohexane phase and a l l o w e d t o s t a n d f o r 3-5 m i n u t e s

References p. 8 4

TABLE 9.9 (continued) a t room temperature. 2.0 m l o f the cyclohexane s o l u t i o n are washed i n 6 m l saturated sodium tetraborate solution

-

and the cyclohexane phase t r a n s f e r r e d t o a clean tube, from which an

a l i q u o t can be taken f o r gas chromatographic-electron capture a n a l y s i s

8

.

4 Acylation o f ecgonine and benzoylecgonine w i t h hexafluoroi sopropanol - h e p t a f l u o r o b u t y r i c anhydride (1:Z). To 0.05-1.0

ug o f ecgonine and/or benzoylecgonine i s added 100 u l o f a m i x t u r e o f hexa-

fluoroisopropanol-heptafluorobutyric anhydride (1:Z). A f t e r heating f o r 30 min a t 75OC t h e excess o f reagents i s removed by evaporation. Then 1 m l o f cyclohexane i s added and 1 ~1 of 20 the sample gas chromatographed

.

5 Methylation o f benzoylecgonine w i t h diazomethane. The sample o f u r i n e (1-5 m l ) i s f i r s t e x t r a c t e d w i t h d i e t h y l ether ( 3 x 5 m l ) t o remove cocaine, then w i t h chloroform t o e x t r a c t benzoylecgonine. The chloroform e x t r a c t i s concent r a t e d t o 100 v l and t r e a t e d w i t h ethereal diazomethane (0.5 m l ) . Excess reagent i s removed

(4OoC) a f t e r 5 minutes the residue suspended i n saturated sodium bicarbonate s o l u t i o n (1 m l

.

from which the cocaine i s e x t r a c t e d w i t h d i e t h y l e t h e r ( 2 x 1 m l ) . The d i e t h y l e t h e r e x t r a c t i s concentrated (50 u l

and 1-2 p1 analyzed by GLC1'.

9.2 REFERENCES

1 C.E. 2 H.A.

Turner, C.Y. Ma and M.A. Elsohly, B u l l . Narc., 31 (1979) 71. Lloyd, H.M. Fales, P.F. Highet, W.J.A. VandenHeuvel and W.C. Wildman, J . dm. Chem. SOC., 82 (1960) 3791. 3 E. Brochmann-Hanssen and A. Baerheim Svendsen. J . Pharm. S c i . , 5 1 (1962) 1095. 4 K.D. Parker, C.R. Fontan and P.L. Kirk, Anal. Chem., 35 (1963) 356. 5 E. Brochmann-Hanssen and C.R. Fontan. J . Chromatogr., 19 (1965) 394. 6 S. Koontz, D. Besemer, N. Mackey and R. P h i l l i p s , J Chromatogr., 85 (1973) 75. 7 H. Kolb and P.W. Patt, drzneim.-Forsch., 15 (1965) 924. 8 J.W. Blake, R.S. Ray, J.S. Noonan and P.W. Murdick, Anal. Chem., 46 (1974) 288. 9 J.M. Moore, J. Chromatogr., 101 (1974) 215. 10 N.C. J a i n and P.L. Kirk, Microchem. J . , 12 (1967) 229. 11 E. Brochmann-Hanssen and R.C. Fontan, J . Chromatogr., 20 (1965) 296. 12 R.H. Hammer, J.L. Templeton and H.L. Panzik, J . Pharm. S c i . , 63 (1974) 1963. 13 F.P. S c a r i n g e l l i , J . d s s o c . off. A n a l . Chem., 46 (1963) 643. 14 A. Cavallaro, G. E l l i and G. Bandi, B o l l . ~ a b .Chim. Prov., 22 (1971) 813. 15 J.M. Moore, J . d s s o c . off. Anal. Chem., 56 (1973) 1199. 16 Anonym. J . d s s o c . off. Anal. Chem., 6 1 (1978) 473. 17 F. F i s h and W.D.C. Wilson, J . Chromatogr., 40 (1969) 164. 18 F. Fish and W.D.C. Wilson, J. Pharm. Pharmacol. suppl., 1969, 135 S. 19 J.E. Wallace, H.E. Hamilton, O.E. King, O.J. Bason, H.A. Schwertner and S.C. H a r r i s , Anal. Chem., 48 (1976) 34. 20 J.I. Javaid. H. Oekirmenjian. E.G. Brunngraber and J.M. Davis, J . C h r o m t o g r . , 110 (1975) 141. 21 J.I. Javaid, H. Dekirmenjian, J.M. Davis and C.R. Schuster, J . Chromatogr., 152 (1978) 105. 22 D.J. Berry and J. Grove, J . Chromatogr., 6 1 (1971) 111. 23 S.J. Mule, J. Chmmatogr. S c i . , 12 (1974) 245. 24 F. Medzihradsky and P. Dahlstrom, Pharmacal. Res. Commun.. 7 (1975) 55. 25 B. Dvorchik, S.H. M i l l e r and W.P. Graham, J . Chromatogr., 135 (1977) 141. 26 M.L. Bastos and O.B. Hoffman, J . Chromatogr. S c i . , 12 (1974) 269. 27 P . I . J a t l o v and D.N. Bailey, Clin. Chem.IWinston-Salem, N . C . J . 21 (1975) 1918. 28 M.J. Kogan, K.G. Verebey, A.C DePace, R.B. Resnick and S.J. MulB, Anal. Chem., 49 (1977 1965. 29 D.L. von Minden and N.A. D'Amato, Anal. Chem., 49 (1977) 1974. 30 N.C. Jain, D.M. Chinn, R.D. Budd, T.S. Sneath and W.J. Leung, J . Forensic S c i . , 22 (197

86

31 S.P. J i n d a l and P. Vestergaard, J. Pham. sci.. 67 (1978) 811. 32 J.C. Roberson, Anal. Chem.. 50 (1978) 2145. 33 J.J. Ambre, Tsuen-Ih Ruo. G.L. Smith, D. Backes and C.M. Smith, J . Anal. T o x f c o l . , 6 (1982) 26. 34 J.E. Graas and E . Watson, J. Anal. T o x i c o l . , 2 (1978) 80. 35 C.C. C l a r k , J. ASSOC. Off. Anal. Chem., 61 (1978) 683. 36 A . S . Christophersen and K . E . Rasmussen, J . Chromatogr., 174 (1979) 454. 37 L.L. P l o t c z y k , J . Chromatogr., 240 (1982) 349.

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11.3 QUINOLINE ALKALOIDS

Chapter 10 CINCHONA

ALKALO I DS

.......................................................... ..................................................................

10.1 Cinchona a l k a l o i d s 10.2 References

67 91

10.1 CINCHONA ALKALOIDS Q u i n i n e was gas chroratograohed by Lloyd e t a 1 . l i n 1960, when t h e y demonstrated t h a t high molecular weight alka'loids could be separated a t r e l a t i v e l y low temperatures on a 6 f e e t by 4 mm I . D .

packed column w i t h a 2-3 % SE-30 coated s o l i d support. This s t a t i o n a r y phase

and o t h e r non-polar s t a t i o n a r y phases have been used i n a number o f i n v e s t i g a t i o n s , mainly w i t h the purpose o f i n v e s t i g a t i n g the gas chromatographic behaviour o f a l k a l o i d s i n general 4 (Brochmann-Hanssen and Baerheim Svendsen', Smith e t a l . 3 , Sarsunovli and Hrivnak ), and i n 7 pharmacological o r t o x i c o l o g i c a l a n a l y s i s (Parker e t a ~ Kazyak ~ , and Knoblock6, S t r e e t , 9 Moffat e t a1.*, Midha and Charette ). However, w i t h non-polar s t a t i o n a r y phases no separat i o n o f the diastereoisomers quinine and quinidine, as w e l l as cinchonine and cinchonidine, can be achieved. Brochmann-Hanssen and Fontanlo'll

i n t h e i r systematic studies on gas chruma-

tography o f a l k a l o i d s w i t h s t a t i o n a r y phases o f various p o l a r i t y , found t h a t the diastereoisomers quinine and quinidine, cinchonine and cinchonidine showed s i g n i f i c a n t r e t e n t i o n time differences on p o l a r s t a t i o n a r y phases. I n s p i t e o f these i n v e s t i g a t i o n s , which showed new gas chromatographic p o s s i b i l i t i e s , most workers continued using non-polar s t a t i o n a r y phases i n t h e i r analyses. A combination o f t h i n l a y e r chromatography and gas chromatography on a non-polar s t a t i o n a r y phase was used by Sarsunova

4 t o achieve a separation o f cinchonidine and quinidine, which could n o t

and Hrivndk

be separated by t h i n - l a y e r chromatography as quinine and cinchonine. Smith e t a l . 3 a p p l i e d gas chromatography o f cinchona a l k a l o i d s as t r i m e t h y l s i l y l d e r i v a t i v e s t o d e t e c t a l k a l o i d a l i m p u r i t i e s i n pharmaceutical quinine and q u i n i d i n e using an OV-225 3 % column. They were n o t able t o separate the diastereoisomers, b u t they found t h a t the dihydroanalogs were present i n a l l 75 samples investigated, and the desmethoxyanalogs (cinchonine and cinchonidine) i n about h a l f o f the samples. The r e l a t i v e r e t e n t i o n times o f the a l k a l o i d s i n v e s t i c a t e d , as t h e i r t r i m e t h y l s i l y l d e r i v a t i v e s , are given i n Table 10.1. Midha and Charette 9 c a r r i e d o u t q u a n t i t a t i v e determinations o f q u i n i d i n e i n plasma and whole blood. Cinchonidine was added t o the plasma sample t o be analyzed as an i n t e r n a l standard. The alkaloidswere e x t r a c t e d w i t h benzene a t pH 12.0. The residue from the e x t r a c t was mixed w i t h 25 p1 o f t r i m e t h y l a n i l i n i u m hydroxide i n methanol, and a l i q u o t s (1-2 p l ) were i n j e c t e d i n t o the gas chromatograph i n which the i n j e c t i o n p o r t was h e l d a t 35OoC. The methyl d e r i v a t i v e s o f q u i n i d i n e and the i n t e r n a l standard gave w e l l separated symmetrical peaks. Det e c t i o n by flame i o n i z a t i o n gave a l i n e a r response over the range 0.2-12.0

pg q u i n i d i n e h l

plasma. The l i m i t of d e t e c t a b i l i t y was 0.05 pg/ml and the method was adequate f o r f o l l o w i n g blood p r o f i l e s o f 200 mg q u i n i d i n e sulphate doses i n humans. The recovery o f q u i n i d i n e

Refereneel p. 91

88

TABLE 10.1 RELATIVE RETENTION TIMES OF TRIMETHYLSILYL DERIVATIVES OF crNcHom ALKALOIDS3 Glass column 6.1 m x 3 m packed w i t h 3 I OV-225 on Gas Chrom

Q, temperature 225OC.

tRf o r

q u i n i d i n e approximately 21 min. A1 k a l o i d

R e l a t i v e r e t e n t i o n time

0.55 0.55 0.50 0.55 0.87 0.95 0.93 0.93 1.00 1.00 0.83 1.52 13.4

Cinchonidine Cinchonine D i hydroci nchonidi ne D i hydroci nchoni ne Epiquinidine Epiquinine D i hydroquini dine Di hydroqui n i ne Q u i n id i ne Quinine Quin i none Quinotoxine ( q u i n i c i n e ) Thioglycerol adduct o f q u i n i d i n e

TABLE 10.2

RECOVERY OF QUINIDINE AND CINCHONIDINE FROM PLASMA DETERMINED BY GLC ASSAY’ Microgram added t o 1 m l plasma

n

Mean microgram recovered

Mean percent recovery

5 5

1.98 7.53

100.71 95.84

Standard d e v i a t i o n of percent recovery 3.48 1.55

7

Mean 98.27 ? 3.61 % 1.72

81.63

2.84

~

Quinidine

1.96 7.86

Cinchonidine 2.11

TABLE 10.3 ESTIMATION OF QUINIDINE ADDED TO PLASMA BY GLC ASSAY’ Quinidine added ug

0.20 ~

0.39 0.79 1.96 3.93 7.86 11.78

n

Mean peak height

7 4 4

0.092 0.157 0.238 0.609 1.087 2.522 3.430

4 4 4

4

Standard d e v i a t i o n 0.009 0.002 0.007 0.031 0.044 0.086 0.198

*) w a n Cv = 4.61 %, y = mx where m = 0.295

f

0.008;

cv,

%

*)

9.68 1.38 2.83 5.12 4.09 3.43 5.76

r = 1.

and cinchonidine from plasma determined by t h e gas chromatographic method i s given i n Table 10.2 and the estimation o f q u i n i d i n e added t o plasma i n Table 10.3. I n a paper published l a t e r , Midha e t a1.l‘

described a comparison between a s p e c t r o f l u o r i -

m e t r i c method and the gas chromatographic technique published by Midha and Charette’.

and

they s t a t e d t h a t the recovery o f q u i n i d i n e (98 % f 4 %) and t h e c a l i b r a t i o n curve from plasma by GLC were e s s e n t i a l l y i d e n t i c a l t o the ones reported e a r l i e r9 For a f a s t determination o f

.

89

q u i n i d i n e i n plasma the authors found the GLC method preferable, as r e s u l t s can be a v a i l a b l e i n 40 minutes. Valentine e t al.13 applied, i n p r i n c i p l e , the method o f Midha and Charette’

f o r a quanti-

t a t i v e assay of quinidines ( q u i n i d i n e and hydroquinidine) i n plasma, using methylene c h l o r i d e instead o f benzene f o r the e x t r a c t i o n o f a small volume o f plasma a f t e r b a s i f i c a t i o n . To the e x t r a c t was added the i n t e r n a l standard, cinchonine; evaporation t o dryness and r e c o n s t i t u t i n g i n a methanolic s o l u t i o n o f t r i m e t h y l a n i l i n i u m hydroxide followed. An a l i q u o t o f t h i s s o l u t i o n was analyzed by GLC v i a on-column methylation reaction. Evaluation o f the method over the range 0.5

-

10 pg/ml i n human plasma gave a p r e c i s i o n and accuracy o v e r a l l o f

f

4.5 %

(RSD and RE). The plasma o f several p a t i e n t s were analyzed by the GLC method as w e l l as by a f l u o r i m e t r i c method f o r the l e v e l s o f quinidine. Results from the two methods were comparable. Moulin and Kinsun14 developed a simple, s e n s i t i v e and accurate method f o r q u i n i d i n e evalua t i o n i n serums, using chloroquine as an i n t e r n a l standard. One m l serum was made acid, ext r a c t e d w i t h hexane t o remove l i p i d s , made a l k a l i n e and again extracted, t h i s time w i t h d i e t h y l ether:methanol

(95:5) t o i s o l a t e quinidine. From using an O V - 1 3 % column on Gas Chrom

Q and a n i t r o g e n detector. determinations down t o 0.5 vg/ml could be done, i . e . s u f f i c i e n t f o r therapeutic q u i n i d i n e blood l e v e l determinations (1-5 pg/ml). For a simultaneous q u a n t i t a t i o n o f quinidine, procainamide and N-acetylprocainamide i n serum Kessler e t al.15 used a packed column, 1.83 m long by 2 mn I . D . w i t h 3 % OV-17 on Gas Chrom Q i n combination w i t h temperature programming from 23OoC t o 28OoC. The d i p r o p y l derivat i v e o f procainamide served as an i n t e r n a l standard. Good q u a n t i t a t i v e r e s u l t s were achieved w i t h a nitrogen-phosphorus detector. Although i t has been demonstrated t h a t quinine and q u i n i d i n e o n l y can be separated on packed columns w i t h p o l a r s t a t i o n a r y phaseslO.ll, phase

-

i n combination w i t h mass spectrometry

-

Furner e t a1.16 used a non-polar s t a t i o n a r y t o achieve a gas chromatographic d i f f e r e n t i -

a t i o n of the two isomers. When the mass spectrometer was operated i n the seleced i o n monit o r i n g (SIM) mode, 5 ng o r less o f quinine was detectable. That i s , quinine and q u i n i d i n e could be d i f f e r e n t i a t e d based upon selected ions. The p o s s i b i l i t y f o r developing a h i q h l y s e n s i t i v e q u a n t i t a t i v e assay was discussed. I n a study on high temperature q u a n t i t a t i v e glass c a p i l l a r y gas chromatography o f quinine/ quinidine, Verzele e t al.17 found t h a t untreated s o f t glass gave b e t t e r r e s u l t s than boros i l i c a t e glass. Some occasional t a i l i n g could be removed by the a n a l y s i s o f q u i n i n e / q u i n i d i n e by sodium c h l o r i d e dendrite deposition. With OV-1,

OV-17,

OV-225, Superox-4, RSL-802 and

RSL-903 good peak shapes were obtained. The r e s o l u t i o n o f quinine and q u i n i d i n e was zero on OV-1,

b u t improved w i t h increasing p o l a r i t y o f the s t a t i o n a r y phases, as already s t a t e d

and i t was complete on RSL-903 (a h i g h l y p o l a r polyby Brochmann-Hanssen and Fontan ’OS1’, aromatic sulfone). the most p o l a r phase of the s e r i e s . A 30 m by 0.3 mm 1.0. sodium d e n d r i t e column coated w i t h 0.15 p m l a y e r o f RSL-903 was used. A moving needle i n j e c t o r proved t o be the best choice i n order t o obtain accurate q u a n t i t a t i v e r e s u l t s . T y p i c a l gas chromatograms are shown i n Figure 2.2 and 2.3 (Chapter 2, C a p i l l a r y columns). The isothermal a n a l y s i s was a p p l i e d t o the assay o f quinine i n s o f t drinks, and o f quinine and q u i n i d i n e i n pharmaceutical preparations, w i t h good r e s u l t s . By m u l t i p l e a n a l y s i s the standard d e v i a t i o n f o r quinine i n s o f t d r i n k s was found t o be 1.97 % and f o r q u i n i n d q u i n i d i ne i n pharmaceutical preparations 1.07 % and 0.90 %, r e s p e c t i v e l y . Plotzcyk”

References p. 91

s t a t e d t h a t fused s i l i c a c a p i l l a r y columns w i t h non-extractable s t a t i o n a r y

90

phases and c o l d on-column i n j e c t i o n have given new p o s s i b i l i t i e s i n the gas chromatography o f a l k a l o i d s . The inertness and h i g h temperature s t a b i l i t y o f comnercially availabe fused s i l i c a columns have e l i m i n a t e d the need f o r d e r i v a t i z a t i o n o f many compounds, w h i l e p r o v i d i n g enhanced s e n s i t i v i t y . With siloxane deactivated cross-linked and gum phase SE-54 fused s i l i c a columns e x c e l l e n t gas chromatographic r e s u l t s were obtained f o r a number o f a l k a l o i d s , i . a . quinine. To improve p o l a r s o l u t e peaks a b i n a r y s o l v e n t o f 4 % methanol i n toluene was used w i t h c o l d on-column i n j e c t i o n . The thermal l a b i l i t y o f the underivatized a l k a l o i d was minimized i n s p l i t l e s s sampling by operating a t the lowest i n l e t temperature possible. Plotczyk found t h a t c o l d on-column i n j e c t i o n y i e l d e d a l i n e a r response from 1 t o 100 ng o f drug w i t h r e p r o d u c i b i l i t i e s o f 0.1-2 % a t t h e 10 ng l e v e l .

18

TABLE 10.4 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY OF crNcHoNA ALKALOIDS

%

Temperature

Comp. Prep.

Ref.

Column

S o l i d support mesh

Stat.phase

glass, 6 f t x 4 mn I.D. glass, 6 f t x 3 mm 1.0.

CW 80-100 GP ABS 100-140 t PEG

SE-30 SE-30

2-3 222OC 1.15 225OC

a l k . s. alk. s.

glass, 6.1 m x 3 n

GQ

OV-225

3

225OC

alk.s.id., qnt. imp. qn.qnd.der.

glass, 6 f t x 4 mm I.D.

Ana ABS 100-120

SE-30

1

s . s . , 5 f t x 118 i n 0.0. CW AW 60-80

QF-1 SE-30

3 5

glass, 80 cm x 1.5 mn I.D. GP AW 80-100 s . s . , 6 f t x 1/8 i n I.D. CW AW 100-120

OV-17 SE-30

2

225OC 25OoC 24OoC 23OoC 27OoC 23OoC 235OC 27OoC

OV-17 SE-30 OV-17 SE-30 XE-60 EGSSY HI-EFF 8B

5 2

glass, glass. s.s., glass,

1m 2 rn 1.2 m x 3 rmn 0.0. 3 f t x 3 mm I . D .

glass, 3 f t x 3 mm 1.0. glass, 1.83 m x 2

M

I.D.

CG AWS 80-100

CW AWS GP AWS 80-100

GQ S 80-100 GP AW 80-100

CW HP 80-100

NGS PVP + NGS OV-17

s . s . , 2 m x 2.17 mn GQ 100-120 glass. 1.83 m x 2 mm 1.0. GQ 100-120

ov-1 OV-17

glass S , 1.2 m x 2 mn I.D.

SP-2250

Sup. 100-200

glass cap. w i t h sodium dendrite 30 m x 0.3 mn f . s i l . cap. 25 m x 0.32 mm I.D.

RSL-903 SE-54

1 1 1 1 1 3 3

27OoC 225OC 220°c 23OoC 23OoC 24OoC 23OoC 23OoC

a1 k.tox.

6

a1 k. t o x .

5

. .

a1 k qnt

4

a1 k.

7

alk.

8 9

qnd.der.qnt.pl. a l k . s.

10

a l k . s.

11

225-28OoC p. r . qnd.hqnd . . der. qnt.pl. 27OoC qnd.qnt.bl.

:$$y:c

pr. qnd.qnt.pl.

220-2700c pr' qn.qnd.GC-MS 300C/rnin

0.15 prn 18OoC 75-260°C p r . 100C/mi n

1 2

alk. s. a l k . s.

,IJ 14 15 16 17

18

91

TABLE 10.5 CINCHONA

ALKALOIDS

-

LIST OF ABBREVIATIONS

ABS = a c i d , base washed, s i l a n i z e d AW = a c i d washed alk = alkaloid Ana = Anakrom b l = blood cap = c a p i l l a r y CG = Chromosorb G CW = Chromosorb W der = d e r i v a t i v e d i f f = differentiation f . s i l = fused s i l i c a ft = feet GP = Gas Chrom P GQ Gas Chrom Q HP = h i g h performance hqnd = h y d r o q u i n i d i n e

i d = identification I.D. = i n s i d e diameter imp = i m p u r i t y O.D. = o u t s i d e d i a m e t e r p l = plasma pm = p l a n t m a t e r i a l p r = (temperature) p r o g r a m i n g prep = pharmaceutical preparation PVP = p o l y v i n y l p y r r o l i d o n e qn = q u i n i n e qnd = q u i n i d i n e qnt = quantitative S = silanized s = separation s.s = s t a i n l e s s s t e e l Sup = S u p e l c o p o r t tox = toxicology

TABLE 10.6 SILYLATION OF CINCHONA ALKALOIDS

1. W i t h N-Methyl-14-trimethyl s i l y l - t r i f l u o r o a c e t a m i d e Add t o 0 . 1 mg a l k a l o i d i n a 0.3 m l c o n i a l v i a l 0 . 1 m l N-methyl-N-trimethylsilyl-trifluoroacetamide o r bistrimethylsilyl-trifluoroacetamide, c o v e r w i t h a T e f l o n - l i n e d septum secured 3 w i t h a screw cap, and h e a t a t 6OoC f o r 45 minutes. I n j e c t 3 p1 o f t h e sample .

10.2 REFERENCES L l o y d , H.M. Fales, P.F. H i g h e t , W.J.A. VandenHeuvel and W.C. Wildman, J. Am. C h e m . 82 (1960) 2791. E. Brochmann-Hanssen and A . Baerheim Svendsen, J . Pharm. S c i . , 5 1 (1962) 1095. E. Smith, S. Barkan, 8. Ross, M. M a i e n t h a l and J . Levine, J . Pharm. S c i . , 62 (1973) 1151. M. Sarsihova and J . HrivnBk, Pharmazie, 29 (1974) 608. K.O. Parker, C.R. Fontan and P.L. K i r k , Anai. C h e m . , 35 (1963) 356. L. Kazyak and E. Knoblock, A n a l . C h e m . , 35 (1963) 1448. H.V. S t r e e t , J . C h r o m a t o g r . , 29 (1967) 68. A.C. Moffat, A.H. Stead and K.W. Smalldon, J . C h r o m a t o g r . , 90 (1974) 19. K.K. Midha and C. C h a r e t t e , J . Pharm. S c i . , 63 (1974) 1244. E. Brochmann-Hanssen and C.R. Fontan, J . Chromatoqr., 19 (1965) 196. E. Brochmann-Hanssen and C.R. Fontan, J . C h r o m a t o q r . , 20 (1965) 394. K.K. Midha, I . J . McGilveray, C. C h a r e t t e and M.L. Rove, can. J . Pharm. S c i . , 12 1977) 41. J.L. V a l e n t i n e , P. D r i s c o l l , E.L. Hamburg and E.D. Thompson, J . Pharm. S c i . , 65 1976) 96. M.A. M o u l i n and H. Kinsun, C l i n . C h i m . d c t a , 75 (1977) 491. K.M. K e s s l e r , P. Ho-Tung, B. S t e e l e , J . S i l v e r , A. P i c k o f f , S. Narayanan and R.J Myerburg, C l i n . Chem. ( W i n s t o n - S a l e m , N . C . ) , 28 (1982) 1187. R.L. Furner, G.B. Brown and J.W. S c o t t , J . A n a l . roxicol., 5 (1981) 275. M. Verzele, G. Redant, P. Quereshi and P. Sandar, J . C h r o m a t o g r . , 199 (1980) 105 L.L. P l o t c z y k , J . C h r o m a t o g r . , 240 (1982) 349.

1 H.A.

SOC.,

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

This Page Intentionally Left Blank

93

Chapter 11

........................................................ ..................................................................

11.1 A m m y c h i d a l k a l o i d s 11.2 References

93 95

11.1 ACRONYCHIA ALKALOIDS Gainer and A r n e t t

1

described a method f o r the q u a n t i t a t i v e determination o f the antitumor

a l k a l o i d acronine. The a l k a l o i d was e x t r a c t e d from various comnonly used pharmaceutical exc i p i e n t s w i t h chloroform. The recovery was good, as can be seen frm Table 11.1. Cholesterol was used as an i n t e r n a l standard and a s h o r t packed column w i t h OV-17 as s t a t i o n a r y phase.

A chromatogram i s given i n Figure 11.1 and the a n a l y s i s o f acronine i n f i n i s h e d capsules i n Table 11.2.

CH, Acronine FIGURE 11.1

GAS CHROMATOGRAM OF ACRONINE/CHOLESTEROL~ on a packed OV-17 column, 0.61 m by 3 mm I.D., w i t h D i a t o p o r t S and 25OoC

1

1 = Cholesterol 2

lLL k-i-rr-

0 2 4 6 min

References p. 95

2 = Acronine

Reproduced from J . P h a r m . S c i . , 58 (1969) 1548, w i t h permission o f the c o p y r i g h t owner

94

TABLE 11.1

RECOVERY OF ACRONINE FROM MIXES WITH VARIOUS EXCIPIENTS’ Excipients

Acronine recovered,

Recovery

mglo

%

1000.4 508.3 500.6 521.0 499.8 503.8 495.5 426.1

Talc Stearic acid tlagnesi um s t e a r a t e S i l i c a gel Tartaric acid Ascorbic a c i d Oipotassium phosphate

100.4 101.7 100.1 104.2 100.0 100.8 99.1 85.2

TABLE 11.2 ACRONINE

IN FINISHED

CAPSULES~

Content Wt., mg

170 290 300 300 300 380

Excipient

Acronine mglcapsule 24.9 23.0 23.7 24.8 25.3 25.5

Microcrystalline cellulose S i l i c a gel Mg s t e a r a t e and s t a r c h S t e a r i c a c i d and s t a r c h Starch Talc

Fong and Farnsworth

2

n

5 5 4 5 5 5

Precision (RSD) % t 1.63 2 1.17 ? 2.71 2 1.16 f 1.74 t 1.59

used gas chromatography on two packed columns (10 % UC-98 and 3.5 %

SE-54) f o r t h e s e p a r a t i o n o f n i n e a l k a l o i d s o f Acronychia b a u e r i (Bauerella a u s t r a l i a n a ) . The r e t e n t i o n t i m e s r e l a t i v e t o a c r o n y c i d i n e on t h e UC-98 column and t o m e l i c o p i c i n e , on t h e SE-54 column a r e l i s t e d i n T a b l e 11.3. TABLE 11.3 RELATIVE

RETENTION

TIMES

OF ACRONYCHIA ALKALOIDS~

Alkaloid

S t a t i o n a r y phase 10 % UC-98

Alkaloid B Normel i c o p i n e Normel ic o p i d i n e Melicopidine Acronyci d i n e Me1 ic o p i c i n e Normelicopicine Melicopine Acronycine Acronycidine time (min) Melicopicine time (min)

0.60 ~.~~ 0.60 0.61 0.63 1.00 2.06 2.38 3.75 4.36

3.5 % SE-54 2.31 2.05

13.0 21.5

95

TABLE 11.4 EXPERIMENTAL CONDITIONS USE0 FOR GAS CHROMATOGRAPHY OF dcmwcmd ALKALOIDS Col umn

Sol id support

Stat.phase

%

Temperature

Canp.Prep.

Ref

mesh glass S, 0.61 m x 3 n 1.0. s . s . , 6 f t x 1/4 in O.D.

Dia S 80-100 GQ 100-120

OV-17 UC-98 SE-54

10 3.5

25OoC

acr.qnt.prep. 1

26OoC 23OoC

alk.pm.

Abbreviations; Dia = Diatoport, S = silanized, S . S . = s t a i n l e s s s t e e l , GQ = Gas Chrom Q, acr. = acronine, qnt = quantitative, prep = pharmaceutical preparation, pm = plant material

11.2 REFERENCES 1 F.E. Gainer and W.A. Arnett, J. Pbarm. sci., 58 (1969) 1548. 2 H.S.S. Fong and N.R. Farnsworth, Lloydia, 32 (1970) 110.

2

This Page Intentionally Left Blank

97

11.4.

PHENYLETHYLAMINE AN0 ISOQUINOLINE ALKALOIDS

Chapter 12 CACTUS ALKALOIDS 12.1. Cactus a1 k a l o i d s 12.2. References

...........................................................

97 10 1

.................................................................

12.1

CACTUS ALKALOIDS

An i n v e s t i g a t i o n o f the a l k a l o i d s o f Anhalonium l e w i n i i and some r e l a t e d compounds was c a r r i e d out by Kapadia and Rao1 using gas chromatography t o study the s t r u c t u r e - r e t e n t i o n time r e l a t i o n s h i p . A packed 1 % SE-52 column was used and the r e s u l t s are summarized i n Table 12.1. TABLE 12.1 GAS CHROMATOGRAPHY OF ANHALONILM ALKALOIDS AND RELATED BASES' Column 12 f e e t long, I . D . 4 mn (glass); support Gas Chrom P 100-140 mesh, s t a t i o n a r y phase and 18 a r e nonanhalonium bases. SE-52 1 %; oven temperature 18OoC. Compounds 6,8.10,12,14,17 N = n a t u r a l product from Anhalonium l e w i n i i . S = prepared s y n t h e t i c a l l y . Compd.

I Tetrahydroisoquinolines

1 2 3 4

5 6 7 8 9 10 11 12

13 14 15 16

17 18

Anhal ami ne Anhal i d i n e Anhalinine Anhalonidine Anhalonine Carnegine Lophophorine N-Methyl anhal i n i ne 0-Methyl anha 1on i d i ne 0-Methyl pel1o t i n e Pellotine Sa 1sol id i ne

Substi tuents

21 H H H

E113 CH3 CH:

H c"113 CH3 CH:

R2

R3

R4

H

OCH OCH~ OCH3 OCH~

OH OH OCH3 OH -0 H -0 OCH

r3 H H CH CH3 CH: H CH CH: H

o-ci,

OCH

O-d,

OCH OCH~ OCH3 OCH~ OCH;

OCH~ OCH: OH H

I 1 8-Phenylethylamines

R'

R"

N-Acetylmescal i n e Homoveratryl amine Mescaline N-Me t hy 1mes ca 1 ine

04-CH3 H H CH3

OCH3 H OCH OCH:

II I D i hydroi soquinolines

R

S c h i f f base A S c h i f f base B

H OCH3

Relemneer p. 101

tR(min)

Source

Mol. Wt.

N

208

S S N N

223 223 223 221 221 235 237 237 251 237 207

4.82 4.44 4.16 4.41 5.20 2.63 4.50 3.09 3.56 3.00 3.94 2.96

N S

237 181 211 225

7.00 1.44 3.38 2.90

S S

205 235

3.28 4.26

s

N S

s

S N S

S S

98

The authors concluded t h a t :

1. N-Monomethylation of primary and secondary amines, 0-methylation, o r C-monomethylation o f the bases studied,

r e s u l t e d i n a decrease i n r e t e n t i o n time. Although t h e r e was an increase

i n molecular weight corresponding t o a methylene group, the observed decrease i n r e t e n t i o n times o f the phehol e t h e r and the a l k y l a t e d amine could be r a t i o n a l i z e d as being due t o the corresponding decrease i n p o l a r i t y o f the d e r i v a t i v e s , as compared t o t h e i r parent compound. The decrease i n r e t e n t i o n o f C-methyl d e r i v a t i v e , as compared t o i t s lower homolog, may be hypothesized as due t o an i n t e r f e r e n c e o f the methyl group i n p e r m i t t i n g the molecule t o be adsorbed on the surface o f the chromatographic column. The methyl group might s t e r i c a l l y h i n der the i n t e r a c t i o n o f the p o l a r phenolic hydroxyl and/or amine groups w i t h the adsorbent. 2. I n t r o d u c t i o n o f hydroxyl o r methoxyl group, o r an unsaturation i n i s o q u i n o l i n e and methoxy l group i n 8-phenylethylamines, r e s u l t e d i n an increase i n the r e t e n t i o n time. It could again be conjectured t h a t the increase i n p o l a r i t y o f the d e r i v a t i v e s caused the increase i n adsorption on t h e l i q u i d phase. 3. I n tetrahydroisoquinolines, i n t r o d u c t i o n o f an hydroxyl group e f f e c t e d greater increase i n r e t e n t i o n than d i d a methoxyl group.

4. Replacement o f two methoxyl groups w i t h a methylenedioxy group i n t e t r a h y d r o i s o q u i n o l i n e s produced a noticeable increase i n r e t e n t i o n time. The a l k a l o i d s o f Lophophora williamsii (Lem. ex

SD)

Coult, belona t o the phenylethylamine

and the tetrahydroisoquinoline groups. Lundstram and Agurell 2 c a r r i e d o u t an i n v e s t i e a t i o n o f them by means o f gas chromatography on packed columns w i t h s t a t i o n a r y phases o f various p o l a r i t y . An SE-30 column was found t o resolve the low molecular weight (> 200) phenylethylamines well, whereas an XE-60 column was very useful f o r the i d e n t i f i c a t i o n o f the t e t r a h y d r o i s o q u i n o l i n e a l k a l o i d s . By means o f semi-preparative columns, a b e t t e r r e s o l u t i o n o f the a l k a l o i d mixture was obtained than by means o f the a n a l y t i c a l columns. I n Table 12.2 t h e gas chromatographic data f o r the peyote a l k a l o i d s and some r e l a t e d compounds are l i s t e d .

A t r a c e a l k a l o i d , 3,4-dimethoxyphenylethylamine was detected i n peyote by means of combined gas chromatographylmass spectroscopy. A chromatogram o f the phenol i c and the non-pheno l i c a l k a l o i d s i n peyote i s given i n Figure 12.1

For a screening of a s e r i e s o f c a c t i belonging mainly t o C e r e u s , E c h i n o p s i s , H e l i a n t h o c e r e u s and T r i c h o c e r e u s f o r the presence o r absence o f a l k a l o i d s , Agurell

3 adopted gas chro-

matography combined w i t h mass sepctroscopy, due t o the great s p e c i f i c i t y and s e n s i t i v i t y thereby obtained. The f o l l o w i n g a l k a l o i d s were found:

N-Methyl-3,4-dimethoxyphenylethylamine

Mescaline

3,4-Dimethoxyphenyl e t h y l amine

Horden ine

3-Methoxytyrami ne

Tyrami ne

N-Methyltyramine

Macromer ine

3,5-Dimethoxy-4-hydroxyphenyl e t h y l ami ne 3,4-Dimethoxy-5-hydroxyphenylethyl amine

Anhaloni dine

Candi c i ne Trichocereine

Retention times o f the reference compounds on two packed columns, one w i t h 5 % SE-30 and one w i t h 5 % XE-60, both operated a t 15OoC, are given i n Figure 12.2.

99

TABLE 12.2 GLC DATA FOR PEYOTE ALKALOIDS

AND RELATED COMPOUNDS ON PACKED COLUMNS OF VARIOUS

A,B and C: column 6 f t x 1/8 i n O.D.;

D and E: column 6 f t x 1/4 i n O.D.;

POLARITY'

A = 5 % SE-30 on

Gas Chrom P, 15OoC; 8 = 7 % F 60 t 2 % Z on Gas Chrom P, 17OoC; C = 5 % XE-60 on Chromosorb

W. 150%;

D = 5 % SE-30 on Chromosorb W, 190%;

E = 5 % XE-60 on Chromosorb

W,

184OC. ~

No

Alkaloid

Mol.Wt.

A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

4-Methoxyphenyl e t h y l ami ne Tvramine N-Methyl tyramine Hordenine Anhaline 3,4-Di methoxyphenyl e t h y l amine Mescaline N-Methylmescaline 0-Methylanhalidine Anhalinine 0-Methylanhalonidine Anhalidine Anhalamine Anhalonidine Pellotine Anhalonine Lophophorine Trichocereine

Retention t i m e (min) 8 C D

E

151 137 151 165

1.5 1.8 2.2 2.4

2.1

0.9 4.6 4.4 3.4

1.0 1.2 1.3 1.4

0.7 3.5 3.3 2.8

181 211 225 237 223 237 223 209 223 237 221 235 237

3.5 6.8 8.0 10.5 11.3 11.6 12.0 12.4 13.0 13.1 14.2 14.5 8.5

5.4 11.1 11.3 11.7 17.5 14.5

2.8 6.6 6.2 4.8 7.8 6.1 8.4 13.6 10.8 7.6 9.5 6.7 5.6

1.8 3.3 3.2 5.2 5.6 5.3 5.6 5.8 6.1 6.0 6.8 6.2 4.0

2.6 5.2 5.0 4.4 6.8 5.5 6.9 10.5 8.2 6.5 8.0 6.3 4.0

22.1 18.3 11.7

~~~

FIGURE 12.1 SEPARATION OF PHENOLIC (LEFT) AND NON-PHENOLIC (RIGHT) ALKALOIDS OF PEYOTE ON A 5 % PACKED XE-60 COLUMN AT 190°C2. Names o f the a l k a l o i d s a r e i n Table 12.3

References p. 101

100

FIGURE 12.2

RETENTION TIMES OF REFERENCE COMPOUNDS ON TWO PACKED COLUMNS3 Both columns 5 f t x 1/8 i n 0.0. ; 5 % SE-30 on Gas Chrom P and 5 % XE-60 on Chromosorb W, both a t a column temperature o f 15OoC. No. and names o f compounds as i n Table 12.3.

5 % X E - 6 0 150' 3L

15 LI/

39

21

0

2

L

6

8

10

12

14

37

18 19 2223 2728 3536

min

5% SE - 3 0 150' 3 4 32

0

2

L

6

8

10

12

1L

16

18

20

min

TABLE 12.3 CACTUS ALKALOIDS AN0 COMPOUNDS RELATED TO KNOWN CACTUS ALKALOIDS' 1 Tyramine

2 N-Methyl tyramine 3 Hordenine 4 N-Methyl-4-methoxyphenyl e t h y l ami ne 5 Dopamine 6 3-Methoxy-4-hydroxyphenyl e t h y l ami ne

7 3,4-Dimethoxyphenylethylamine 8 N-Methyl-3.4-dimethoxyphenylethylamine 9 N,N-Dimethyl-3,4-dimethoxyphenylethylamine 10 Macraerine 11 3,4-Oimethoxy-5-hydroxyphenyl e t h y l amine 12 3,5-Dimethoxy-4-hydroxyphenyl e t h y l ami ne 13 Mescaline 14 N-Methylmescal i n e 15 Trichocereine 16 Norcarnegine 17 Carnegine 18 Anhalamine 19 Anhalidine 20 Anhalinine 21 Anhalonidine 22 P e l l o t i n e 23 Anhalonine 24 Lophophorine 25 Peyophorine

26 27 28 29 30 31 32 33 34 35

0-Methyl anhaloni d i ne Lophocerine Pilocereine Phenylethylamine Octopanine Oxedrine 8-0-Methyloxedrine 0-Methyltyramine

4-Methoxy-3-hydroxyphenyl e t h y l ami ne

N-Methyl-4-hydroxy-3-methoxyphenylethylamine 36 N ,N-Dimethyl-4-hydroxy-3-methoxyphenyle t h y l ami ne 37 cis-l-Methyl-4-hydroxy-6,7-dimethoxytetrahydroisoquinol i n e 38 trans-1-Methyl -4-hydroxy-6 ,7-dimethoxytetrahydroisoquinol i n e 39 cis-1.2-Dimethyl-4-hydroxy-6.7-dimethoxyt e t r a h y d r o i soquinol i n e 40 trans-l,2-Dimethyl-4-hydroxy-6,7-dimethoxytetrahydroi soquinol i n e 41 0-Methylanhal i d i ne 42 0,N-Dimethylanhaloni dine

101 Ooetsch e t al.4 used gas chromatography f o r the separation o f the non-phenolic a l k a l o i d s and f o r the q u a n t i t a t i v e determination o f mescaline i n pereskia, P e r e s k i o p s i s and I s i a y a species. A packed 1.5 X OV-101 on Chromosorb G column and a column temperature o f 150°C was used f o r the analysis. The amount o f mescaline i n the plasma o f r a b b i t s a f t e r intravenous i n j e c t i o n was determined by Van Peteghem e t a?. Mescaline was converted t o the t r i f l u o r o a c e t y l d e r i v a t i v e and 2 t r i f l u o r o a c e t y l - H2-mescaline was used as an i n t e r n a l standard. The method t h a t was developed combined the s p e c i f i c i t y of gas chromatographic r e t e n t i o n times and mass s p e c t r a l frapmentation p a t t e r n w i t h the s e n s i t i v i t y

o f the mass fragmentographic detection. Plasma samples o f 0.5 m l were used. 200 ng o f the i n t e r n a l standard were added t o the sample, the pH

adjusted t o pH 10 and the e x t r a c t i o n performed w i t h benzene. The benzene e x t r a c t was evaporated t o dryness, the residue dissolved i n 0.2 m l e t h y l acetate and 0.2 m l o f t r i f l u o r o a c e t i c a c i d anhydride. A f t e r heating f o r 30 minutes a t 6OoC, t h e s o l v e n t was evaporated and the residue dissolved i n 10-20

pl

o f methanol, and 1-2 ~1 t n j e c t e d f o r gas chromatoqraohic analy-

s i s . The fragnentographic detection allowed d e t e c t i o n down t o 5 ng/ml and the r e l a t i v e standard d e v i a t i o n was 5

%.

TABLE 12.4 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY OF CACTUS ALKALOIDS Stat.phase

%

Temperature

Comp.Prep.

glass, 12 f t x 4 mn I . D . GP 100-140

SE-52

1

18OoC

Anh. a1k.

glass, 6 f t x 1/4 i n O.D.GP 100-120 CW AWS 80-100 GP AWS 100-120

SE-30 XE-60 F 60

5 5

15OoC 150°c

peyot a 1k .

2

c a c t .a1 k.

3

cact.al k. msc.qnt.pl.

5

Column

S o l i d support

t z

glass, 5 f t x 1/8 i n

s . s . . 2.5 m x 3.2 mn glass S , 1.5 m x 6.3

N II

.

Ref

1

17OoC

GP AWS CW AWS

SE-30 XE-60

CG 100-120 Var 100-120

ov-101

5 5 1.5 150°C

QF-1

2.5 195OC

Abbreviations: a l k = a l k a l o i d , Anh = Anhalonium, AWS = a c i d washed, silanized, comp = compound, CW = Chromosorb W, GP = Gas Chrom P, I.D. = i n s i d e diameter, msc = mescaline, O.D. = outside diameter. peyot = peyote, p l = plasma, prep = preparation, q n t = q u a n t i t a t i v e , S = silanized, S . S . = s t a i n l e s s s t e e l , Var =

4

c a c t = cactus, i n = inch, pharmaceutical Varaport.

12.3 REFERENCES

1 G.J. Kapadia and G.S. Rao, J . Pharm. s c i . , 54 (1965) 1817. 2 J. Lundstrom and S. Agurell, J . C h r o m a t o g r . , 36 (1968) 105. 3 S . Agurell, L l o y d i a , 32 (1969) 206. 4 P. W. Doetsch, J.M. Cassidy and J.L. McLaughlin, J . C h r o m a t o g r . , 168 (1980) 79. 5 G. van Peteghem, A. Heyndrickx and W. van Zele, J . Pharm. S c i . , 69 (1980) 118.

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103

Chapter 13 EPHEDRA ALKALOIDS

.........................................................

13.1. Ephedra a l k a l o i d s 13.1.1. Ephedrine i n p l a n t m a t e r i a l 13.1.2. Ephedrine i n pharmaceutical p k p a r a t i o n s 13.1.3. Ephedrine i n b i o l o g i c a l f l u i d s 13.2. References

103 105 105 106 109

....................................... .......................... .................................... ................................................................

13.1 EPXEDRA ALKALOIDS Ephedrine and i s t isomer pseudoephedrine are a l k a l o i d s i n Ephedra s p e c i e s . Brochmann-Hanssen and Baerheim Svendsen 1 reported the f i r s t gas chrmatopraphic separation o f these

two

a l k a l o i d s i n a study on the separation and i d e n t i f i c a t i o n o f 11 sympathomimetic amines on a 1.15 % SE-30 packed column a t 104OC. However, ephedrine and pseudoephedrine could n o t be separated as such, b u t were separated as t h e i r oxazolidine d e r i v a t i v e s a f t e r treatment w i t h acetone. A t y p i c a l chromatogram i s given i n Tale 13.1. FIGURE 13.1 GAS CHROMATOGRAM OF EPHEORINE (1) AND PSEUOOEPHEORINE ( 2 ) l a f t e r 3 hours a t room temperature i n acetone s o l u t i o n . Column: 1.15 % SE-30 on Gas Chmm P a t 104OC.

1 = Ephedrine 2 = Pseudoephedrine Reproduced from J . Pharm. S c i . , 5 1 (1962) 938, w i t h the permission o f the c o p y r i g h t owner.

Parker e t a1

.'

a p p l i e d gas chromatography f o r the separation and i d e n t i f i c a t i o n o f some

sympathomimetic amines. They used a packed column o f 5 % Carbowax 20 H on f i r e b r i c k t r e a t e d w i t h 5 % potassium hydroxide, a t a column temperature o f 17OoC o r 190°C.

Ephedrine and pseudo-

ephedrine were n o t separated, however. I n a couple o f papers, Beckett and

References p. 109

described the gas chromatographic separation

104

o f the o p t i c a l isomers o f "ephedrines" and "pseudoephedrines" as t h e i r N - t r i f l u o r o a c e t y l - L propyl d e r i v a t i v e s . For (+)ephedrine and (-)ephedrine a usable r e s o l u t i o n was obtained (res o l u t i o n f a c t o r 0.78), b u t f o r (+)pseudoephedrine and (-)pseudoephedrine the r e s o l u t i o n fact o r was poor ( r e s o l u t i o n f a c t o r 0.45).

5 succeeded i n separating the 4 diastereoisomeric ephedrines as t h e i r N-acetyl-0-trimethylsilyl d e r i v a t i v e s . For the separation o f enantiomers, conversion i n t o N-(R)-a-phenylbutyryl-0-trimethylsilyl d e r i v a t i v e was e f f e c t i v e f o r ephedrine and pseudoG i l b e r t and Brooks

ephedrine, but n o t f o r t h a t o f nor-ephedrine. The r e t e n t i o n i n d i c e s o f diastereomeric ephedr i n e s as t h e i r N-acetyl-0-trimethylsilyl e t h e r d e r i v a t i v e s are given i n Table 13.1 and the r e t e n t i o n i n d i c i e s o f ephedrines as diastereomeric N- (R)-a-phenyl b u t y r y l - 0 - t r i m e t h y l s i l y l ether d e r i v a t i v e s a r e i n Table 13.2. A gas chromatogram o f the separation o f nor-pseudoephedr i n e , ephedrine and pseudoephedrine as t h e i r N-acetyl-0-trimethylsilyl given i n Figure 13.1.

ether derivatives, i s

FIGURE 13.2 GAS CHROMATOGRAM OF EPHEDRINES5 on a 5 m by 3 m I.D. packed glass column w i t h 1 % OV-17 on Gas Chrom Q a t 17OoC; 1 = Norpseudoephedrine, 2 = Ephedrine, 3 = Pseudoephedrine as t h e i r N-acetyl-0-trimethylsilylether derivatives.

1

23

TABLE 13.1 RETENTION INDICES DF DIASTEREOMERIC EPHEDRINE? as N-acetyl-0-trimethylsilyl e t h e r d e r i v a t i v e s Compound (-)-Ephedrine (-)-Pseudoephedrine +) -Nor-ephedri ne -)-Nor-pseudoephedrine

I

Configuration 1R,2S 1R,2R 1S,2R 1R,2R

Retention index, 5 m 1 % OV-17, 17OoC 1925 1945 1865 1870

106

TABLE 13.2 RETENTION I N D I C E S OF EPHEORINES' as diastereomeri c N- (R)-a-phenyl b u t y r y l - 0 - t r i m e t h y l s i l y l e t h e r d e r i v a t i v e s ~

Retention i n d i c e s Parent compound (+)-Ephedrine (-)-Ephedrine (+)-Pseudoephedri ne * ( - ) -Pseudoephedrine ( t ) -Nor-ephedri ne ( - ) -Nor-ephedri ne * (+) -Nor-pseudoephedrine ( - ) -Nor-pseudoephedri ne

Configuration

*

2300 2310 2290 2325 2280 2280 2240 2260

1s,2s 1R,2R

* 13.2.1.

1 % O V - 1 (190OC)

1S,2R 1R,2S 15,2s 1R,2R l S , 2R 1R,2S

1 % OV-17 (210OC) 2555 2570 2570 2600

2550 2550 2520 2545

Data obtained from (*) sample

Ephedrine i n p l a n t m a t e r i a l

Yamasaki e t a1 .6 a p p l i e d gas chromatography t o t h e separation and q u a n t i t a t i v e a n a l y s i s o f the a l k a l o i d s i n some Ephedra species c o l l e c t e d around the Himalayas. A s a t i s f a c t o r y sepa r a t i o n o f the a l k a l o i d s and q u a n t i t a t i v e determination o f L-ephedrine and D-pseudoephedrine 1

was achieved using oxazolidine formation w i t h acetone 13.2.2.

.

Ephedrine i n pharmaceutical preparations

I n order t o determine ephedrine i n aerosols, Lawless e t a l . 7 e x p i r e d the aerosol through a piece o f s t a i n l e s s - s t e e l tubing i n t o a 10 m l volumetric f l a s k c o n t a i n i n g acetone. The v o l umetric f l a s k was brought t o volume and an a l i q u o t i n j e c t e d f o r t h e gas chromatographic analys i s on a 1.15 % SE-30 packed column a t 171OC. Over 99.5 % o f the amount o f ephedrine present i n the aerosol could be determined by t h i s method. For the determination o f ephedrine i n t a b l e t s a l s o containing phenobarbital and theophyll i n e , E l e f a n t e t a1.'

converted ephedrine t o benzaldehyde by periodate oxydation p r i o r t o

gas chromatography. Using benzyl alcohol as an i n t e r n a l standard and a packed column o f 3 % HI-EFF 8 BP on Gas Chrom Q and temperature programming from 80°C t o 13OoC, good r e s u l t s were obtained. The standard d e v i a t i o n f o r 10 i n d i v i d u a l t a b l e t s declared t o contain 24 mg ephedrine was found t o be 1.1 %. The same method was a l s o a p p l i e d by Vuorinen and Halmenkoski9. For t a b l e t s c o n t a i n i n g 25 mg and 22 mg ephedrine hydrochloride, a recovery o f 99.98 % and 99.96 X was found, w i t h a standard d e v i a t i o n o f i 1.25 % and i 0.81 %. r e s p e c t i v e l y . An 5 % Carbwax 4000 packed column on Chromosorb

W was used.

Iconomou e t a1.l'

determined the ephedrine content i n cough syrup and e x t r a c t e d i t w i t h

chloroform a f t e r b a s i f i c a t i o n o f the syrup w i t h ammonia. The chloroformextract was evaporated and the residue dissolved i n a chloroform s o l u t i o n c o n t a i n i n g the i n t e r n a l standard, diphenyl. Chromatograms o f ephedrine-diphenyl s o l u t i o n s (0.1 11g/v1 diphenyl) on a 2 m l o n g packed 2 % Versamide 900 on Chromosorb W column w i t h 5 % KOH a t 175OC are given i Figure 13.3. The " J o i n t Comnittee" o f the Pharmaceutical Society and the Society f o r A n a l y t i c a l Chemist r y " recomnended a gas chromatographic method f o r the determination o f ephedrine i n t a b l e t s , e l i x i r and nasal drops, using phenmetrazine as an i n t e r n a l standard. Ephedrine was e x t r a c t e d

Rcfersncw p. 109

106

from aqueous s o l u t i o n s o f the preparations by means o f d i e t h y l ether a f t e r b a s i f i c a t i o n w i t h sodium hydroxide. The d i e t h y l e t h e r e x t r a c t was concentrated and used f o r the assay on a 2 % Carbowax 6000 packed column on Chromosorb G impregnated w i t h 5 X sodium hydroxide. C o e f f i c i ents o f v a r i a t i o n s w i t h i n l a b o r a t o r i e s were n o t greater than 3 % f o r t a b l e t s c o n t a i n i n g 30 mq ephedrine. FIGURE 13.3

OF SOLUTIONS OF EPHEDRINE~' 1 = Diphenyl ( i n t e r n a l standard) (0.1 pg/ul) and 2 = Ephedrine: A = 0.5 pg/ul, B = 0.75 pg/v1, C = 1.0 pg/ul, 0 = 1.25 pg/pl, E = 1.50 pg/ul. A packed column w i t h 2 % Versamide 900 on Chromosorb W t 5 % KOH a t 175OC.

CHROMATOGRAMS

A

B

C

D

E

1i4J 5

13.2.3.

10

5

10 mi5n

10

5

1

10

5

0

Ephedrine i n b i o l o g i c a l f l u i d s

Urine

Beckett and Wilkinson"

developed a method f o r the i d e n t i f i c a t i o n and e s t i m a t i o n o f ephed-

r i n e and i t s congeners i n u r i n e . Urine samples o f 1-5 m l were a c i d i f i e d and e x t r a c t e d w i t h d i e t h y l e t h e r t o remove n e u t r a l and a c i d i c compounds. The u r i n e was then made a l k a l i n e and the amines were e x t r a c t e d w i t h d i e t h y l ether. This e x t r a c t was used a f t e r concentration f o r gas chromatographic analysis. Because ephedrine and pseudoephedrine could n o t be separated as such, t h e i r acetone d e r i v a t i v e s were prepared'.

The r e t e n t i o n times o f some ephedrines

congeners are given i n Table 13.3. Welling e t a l . I 3 used gas chromatography f o r the determination o f ephedrine i n u r i n e . Samples o f 5 m l u r i n e were a c i d i f i e d and e x t r a c t e d w i t h d i e t h y l ether; the d i e t h y l e t h e r was discarded. A f t e r b a s i f i c a t i o n w i t h sodium hydroxide the sample was e x t r a c t e d again w i t h

107 d i e t h y l ether. This e x t r a c t was used f o r the gas chromatographic analysis; 4-aminoacetophenone was used as an i n t e r n a l standard. The i n t e r n a l standard was added t o the u r i n e sample p r i o r t o the a l k a l i n e e x t r a c t i o n . A t y p i c a l chromatoyam i s given i n Fipure 13.4. FIGURE 13.4

GAS CHROMATOGRAM OF EPHEDRINE FROM URINE EXTRACT13 on a 3 % O V - 1 packed column on Gas Chrom Q, 1.83 m b y 4 mn I.D., a t 14OoC. A = e x t r a c t o f blank u r i n e spiked w i t h ephedrine sulphate (12 ug/ml) and 4-aminoacetophenone ( 5 pg/ml); B and C = gas chromatograms o f a 0.5-1.0 h r and a 6-9 h r u r i n e sample o f a subject having received 25 mg o f ephedrine sulphate. 1 = ephedrine, 2 = 4-aminoacetophenone, 3 = norephedrine.

C

B

1 1

2

3 2

L 6 8

min

TABLE 13.3

RETENTION

TIMES OF SOME EPHEDRINE

CONGENERS~'

on a 2 % PEG 6000 t 5 % KOH packed column on Chromosorb G, 1 m by 1/8 i n c h O.D.,

Compound I n t e r n a l marker (2,6-dimethyl phenoxy)ethylamine Methylephedrine Pseudoephedrine Ephedrine Nor-pseudoephedrine Nor-ephedri ne

a t 165OC

Ret e n t ion time (min ) Base Acetone d e r i v a t i v e 4.9 6.8 8.2 8.2 10.5 11.3

8.0 n o t formed 3.6 4.0 4.2 4.0

Plasma

Pickup and Paterson14 developed a method f o r the determination o f ephedrine i n plasma. To plasma samples o f 3 m l the i n t e r n a l standard (phendimetrazine) and a l k a l i were added p r i o r t o e x t r a c t i o n w i t h d i e t h y l ether. The d i e t h y l ether e x t r a c t was concentrated t o about 20 u1

Referencer p. 109

108

and 4 ul i n j e c t e d f o r the gas chromatographic assay on a 8 % Carbowax 20 M on Chromosorb W column a t 180OC. Accuracy and p r e c i s i o n o f the method was good. With the assay o f twelve plasma samples containing 25 ng ephedrine base per m l . the c o e f f i c i e n t o f v a r i a t i o n was 9.7 %. TABLE 13.4 EXPERIMENTAL CONDITIONS USE0 FOR GAS CHROMATOGRAPHY OF EPHEDRA ALKALOIDS

Col urnn

S o l i d support

Stat.Dhase

glass, 6-8 f t x 3 mn 1.0. GP A M 100-140 CW A#S 60-80 glass, 4 f t x 5 mn 1.0. F i b 100-120

SE-30 SE-30 Cab 20M t KOH s . s . , 2 m x 1/8 i n 0.0. CG AWS 100-120 SE-30 glass S, 3 m X 3 mn 1.0. GQ OV-1 glass S, 5 m x 3 mm 1.0. OV-17 S . S . , 6-8 f t x 3 1.0. CP 100-140 SE-30 glass, 6 f t x 0.25 i n 0.0. GQ 100-120 HI-EFF 8B

s . s . , 10 f t x 1,8' i n 1.0. CW 80-100 glass, 2 m x 2.5 mm 1.0. GP S 80-100 CW 80-100 t

1 m x 4 mn 1.0.

S.S.,

5.5..

CG Ab!S 80-100 t

1 m x 118 i n 0.0.

CG AWS 80-100 t

1.83 m x 4 mn 1.0. glass, 2 m x 1/4 i n 0.0.

GQ 100-120 CW 80-100 t

Cab 4000 SE-30 Ve r KOH Cab 6000 KOH PEG 6000 KOH OV-1 Cab KOH 2o

%

Temperature

1.15 104OC 1.15 104OC 170-1900c

3 170°C 1 1 1.15 17loC 3 80=130°C p r 6"CImi n 5 14OoC 3 145OC 175OC

'5

; " ; 3

ComD.PreD.

Ref.

ep.psep.s.

1

ep.psep.s.

"eDS". , - " DSe . DS" . 3.4 d i a s t .enant.eps. 5 ep.qnt.aeros. 7 ep.qnt.bnzld. 8 eD.ant.bz1d. . . ep.qnt.syr.

9 10

1500c

ep.qnt.prep.

11

1650c

ep.cg.qnt.ur. ep.qnt.ur. ep.qnt.pl.

12 13 14

14OoC l8OoC

TABLE 13.5 EPHEDRA

ALKALOIDS

-

LIST OF ABBREVIATIONS

aeros = aerosol AWS = a c i d washed, s i l a n i z e d b z l d = benzaldehyde Cab = Carbowax cg = congener CG = Chromosorb G comp = compound CW = Chromosorb W d i a s t = diastereomeric enant = enantiomeric ep ephedrine "eps" = "ephedrines" Fib = firebrick ft = f e e t

GP = Gas Chrom P GQ = Gas Chrom Q 1.0. = i n s i d e diameter i n = inch O.D. = o u t s i d e diameter psep = pseudoephedrine "pseps" = "pseudoephedrines" p l = plasma prep = pharmaceutical preparation qnt = quantitative s = separation S = silanized S.S. = stainless steel s y r = syrup u r = urine

109

13.2 REFERENCES 1 E. Brochmann-Hanssen and A. Baerheim Svendsen. J . Pharm. s c i . , 51 (1962) 938. 2 K.D. Parker, C.R. Fontan and P.L. K i r k , Anal. C h e m . , 34 (1962) 1345. 3 A.H. Beckett and B. Testa, J . Pham. P h a m c o l . , 25 (1973) 382. 4 A.H. Beckett and B. Testa, J. Chromatogr., 69 (1972) 285. 5 M.T. G i l b e r t and Ch.J.W. Brooks, B i o m e d . Mass. S p e c t r o m . , 4 (1977) 226. 6 K. Yamasaki, K. F u j i a , M Sakamoto, M. Yoshida and 0. Tanaka, Chem. Phann. ~ u l l . .22 (1974) 2898; c . A . , 82 (1975) 103221 v. 7 G.B. Lawless, J.J. Sciarra and A.J. Monte-Bovi, J . Phann. S c i . , 54 (1965) 273. 8 M. Elefant, L. Chafetz and J.M. Talmage, J . Pham. S c i . . 56 11967) 1181. 9 L. Vuorinen and J. Halmenkoski, Farm. A i k a k . , 8 1 (1972) 185. 10 N. Iconomou. J. Biichi. R. Jaspersen-Schib and H.-P. Jaspersen. Pharm. Acta Helv.. 42 (1967) 334. 11 J o i n t Comnittee, Pharm. SOC. and SOC. Anal. Chem., Analyst, 100 (1975) 136. 12 A.H. Beckett and G.R. Wilkinson, ,7. Pharm. Ph-col., 17 (1965) s u p p l . , 104 S. 13 P.Welling, K.P. Lee, J.A. Patel. J.E. Walker and J.G. Wagner, J . Phann. S c i . , 60 (1971) 1629. 14 M.E. Pickup and J.W. Paterson, J.Pharm. Pharmacol.. 26 (1974) 561.

This Page Intentionally Left Blank

111

Chapter 14 OPIUY ALKALOIDS

........................................................... .................................................... ........................... ........;. .................... .................................. .............................. ........................... ............ ...................... ...................................... ................................................. ................................................ .................................................. ................................................................

14.1. Opium a l k a l o i d s 14.1.1. Packed columns 14.1.1.1. Separation and i d e n t i f i c a t i o n 14.1.1.1.1. Derivatization 14.1.1.1.2. Combination o f GLC w i t h o t h e r a n a l y t i c a l techniques 14.1.1.2. Q u a n t i t a t i v e determination 14.1.1.2.1. Morphine i n opium 14.1.1.2.2. Morphine i n b i o l o g i c a l m a t e r i a l s 14.1.1.2.3. Non phenolic a l k a l o i d s 14.1.1.2.4. Heroin 14.1.2. C a p i l l a r y columns 14.1.2.1. Morphine 14.1.2.2. Heroin 14.2. References

111 111 112 114 116 117 117 120 125 129 136 136 137 144

14.1. OPIUM ALKALOIDS 14.1.1.

Packed columns

The opium a l k a l o i d s have engaged the i n t e r e s t o f s c i e n t i s t s s i n c e the i s o l a t i o n o f morphine by Serturner i n 1806. The i s o l a t i o n , c h a r a c t e r i z a t i o n and q u a n t i f i c a t i o n o f these a l k a l o i d s have been a continuing challenge. Gas chromatography o f opium a l k a l o i d s has been performed i . a . f o r the analysis o f the a l k a l o i d s present i n t h e crude drug i t s e l f , especiall y f o r the q u a n t i t a t i v e determination o f morphine, as w e l l as f o r the a n a l y s i s o f opium a l k a l o i d s , mainly morphine

-

and heroin

-

i n b i o l o g i c a l materials. Most studies have so f a r

been c a r r i e d o u t w i t h packed columns, o n l y a l i m i t e d number w i t h c a p i l l a r y columns. Gas chromatography o f opium a1 kaloids, metabolites and congeners has mostly been c a r r i e d o u t on non-polar o r s l i g h t l y p o l a r s t a t i o n a r y phases, such as SE-30, SE-52, OV-17, OV-1, QF-1, and XE-60.

I n some cases, however, more p o l a r s t a t i o n a r y phases have been employed

because o f t h e i r g r e a t e r s e l e c t i v i t y (PEG 20 M, DEGS, EGSS-Y, NGS and HI-EFF 86). However, the r e t e n t i o n times o f the a1 k a l o i d s are s i g n i f i c a n t l y increased on very p o l a r s t a t i o n a r y phases, which f o r h i g h molecular weight a l k a l o i d s may l e a d t o very high, and f o r p r a c t i c a l use o f t e n undesirably high, r e t e n t i o n times, even when working w i t h columns w i t h a low conc e n t r a t i o n o f s t a t i o n a r y phase.

To avoid adsorption due t o " a c t i v e s i t e s " on t h e s o l i d support when i t i s coated w i t h a low percentage o f s t a t i o n a r y l i q u i d , an acid/base washed and s i l a n i z e d support i s mostly used. Brochmann-Hanssen and Furya' a l s o coated the s o l i d support w i t h polyethylene g l c o l 4000 and

nonylphenoxyethyleneoxyethanol, 0.05 X o f each, before coating with 2 X SE-30. S t r e e t2 used a d e a c t i v a t i o n procedure f o r s o l i d support, whereby a water saturated s o l u t i o n o f the s t a t i o n a r y phase (SE-52) i n toluene was used, followed by heating o f the product i n an oxygen-free n i t r o g e n stream a t 37OoC f o r 18 hours. Later S t r e e t e t a l . 3 showed t h a t a c y l a t i o n o f the s o l i d support (diatomaceous e a r t h ) w i t h benzoylchloride i n p y r i d i n e p r i o r t o c o a t i n g w i t h a s i l i c o n e s t a t i o n a r y phase and followed by heating i n an atmosphere o f nitrogen, l e d t o gas chromatographic columns w i t h a marked reduction i n adsorption compounds such as morphine.

Referencesp. 144

-

even f o r unmodified p o l a r

112

14.1.1.1.

Separation and i d e n t i f i c a t i o n

I n t h e i r f i r s t paper on gas chromatography o f a l k a l o i d s , Lloyd e t a1.4 chromatographed the main opium a l k a l o i d s on a SE-30 column. Eddy e t a l . 5 made use o f the same s t a t i o n a r y phase i n studies on the r e l a t i o n s h i p between the r e l a t i v e amount o f the main opium a l k a l o i d s and the o r i g i n o f opium. They i n v e s t i g a t e d a number o f a u t h e n t i c UN opium samples on a 1 % SE-30 column on Gas Chrom P w i t h temperature p r o g r a m i n g and concluded t h a t gas chromatograp h i c analysis immediately establishes a c h a r a c t e r i s t i c f i n g e r p r i n t o f the i n d i v i d u a l a l k a l o i d sample and thus promises t o a i d i n t h e c o r r e l a t i o n between source country and sample. Yamaguchi e t a1 .6 gas chromatographed more than 40 opium a l k a l o i d s and r e l a t e d compounds t o determine whether o r not a c o r r e l a t i o n e x i s t e d between the r e t e n t i o n t i m e and the chemical s t r u c t u r e (Table 14.1). TABLE 14.1 RELATIVE RETENTION TIMES OF COOEINE AND RELATED ALKALOIDS~ Column: 6 f t long by 8 mm, 1 % SE-30 on Gas Chrom P (100-140 mesh) a t 185OC. Codeine, tR 4.71 min 2

16 7

Compound

D i hydrudesoxycodeine D

Characteristics

t~ [ r e l )

Desoxycodeine E Dihydrodesoxycodeine 0 Isocodeine Tetrahydrodesoxycodei ne Pseudocodei 14-Hydroxydinone hydrodesoxycodeine

0.64 0.66 0.74 0.77 0.80 0.84

A

D i hydrodesoxythebainone A1 lopseudocodeine 9 14-Hydroxydesoxycodeine E 10 14-Acetoxydihydrodesoxycodeine D 11 D i hydrocodeine 12 Neopine 13 Codeine methyl e t h e r 14 Codeine 15 Pseudocodei ne 16 Dihydro-6a-thebainol methyl e t h e r 17 D i hydroi socodeine 18 Oionine (ethyl morphine) 19 Norcodei ne 20 14-Hydroxytetrahydrodesoxycodeine 21 D i hydrocodei none 22 Morphine 23 Dihydro-66-thebainol methyl e t h e r 24 D i hydrothebainone methyl e t h e r 25 14-Hydruxydi hydrodesoxycodeine C

0.86 0.91 0.91 0.93 0.96 0.97 0.98 1.00 1.03 1.07 1.07 1.09 1.09 1.13 1.15 1.17 1.20 1.22 1.30

Obining the e t h e r r i n g C6-0, C4 desoxy

1 2 3 4 5 6 7

a

7.8

C6B-OH t$e&ng the e t h e r r i n g , C4 -OH t 6 ,J10i8=0

ring,C4-OCH3.

C6a-OH

C66-DH C -0C H C a-OH i7’8: Cia-06,5i-H6 O$&ffing the e t h e r r i n g ,

c6-o

, C -OH, O$&ffing3the Opening the Opening the C146-OH

A

C4-OH,

CllB-OH

C a-OH etfier r i n g , C4-OCH C B-OH e t h e r r i n g , C4-OCH3: C6=0 e t h e r r i n g , A ~ , ~?,-Oh,,

113 TABLE 14.1 (continued) Compound 26 27 28 29 30 31 32 33 34 35

14-Hydroxydi hydrocodeine 14-Hydroxycodei ne 8.14-0i hydroxydi hydrocode inone 14-Hydroxycodei none 14-Hydroxydi hydrocodei none 14-Acetoxycodeinone 14-Hydroxydi hydroi socodeine Thebai ne 8-Methoxy-14-hydroxydi hydrocodeinone Oihydrothebainone

36 37 38

14-Acetoxydi hydrocodeinone O i hydrothebai none N-ProDal avl - 14-hvdroxvdi - - hvdronorcodeinoneSinomenine methyl e t h e r

39

.+

Characteristics

t~ ( r e l ) 1.35 1.35 1.37 1.38 1.39 1.41 1.43 1.48 1.48 1.52

!c l 4

6a-OH, C B-OH C aidH, C p-OH C~B-OH, C ~ ~ ~ - O H A C8=0, C 8 I O H

~$8: c ; ~ ~ - o H ~ ~

C6=0, C I3-OAc C6B-OH, C1413-h8 C -0CH c 38-OH t6*ti:A~+iCH6, Olening’the e z h e r l t i n g , A8,14, C -OH, C6-OCH c4=0, c 13-OA~ OBening’Phe e t h e r r i n g , C4-OH, C6=0 A7

1.52 1.61

C =O, C 13-OH, N-CH CPCH Afitipode? opening t i e e t h e r r i n g , A C -0CH , C -0, C7-OCH3 C4 e-04, NgH Afitipode? opening the e t h e r r i n g , C -OH, C -0, C -0CH Ojening t i e e t i l e r r?ng, C ~ - O H , c6=0, C b-OH Aieipode, opening the e t h e r r i n g , C4-OH, C6=0, C7-OCH3, A7,8

1.69 1.87

40 14-Hydroxydi hydronorcodei none 41 D i hydros inomeni ne

1.91 2.00

42

14-Hydroxydi hydrothebai none

2.45

43

Sinomenine

2.83

C74:

For the a p p l i c a t i o n o f gas chromatography t o t o x i c o l o g i c a l analysis. Parker e t a l . ’ studied a s e r i e s o f a l k a l o i d s i n c l u d i n g the main opium a l k a l o i d s and some r e l a t e d compounds, using a SE-30 column; so a l s o d i d Kazyak and Knoblock

8 . I n a study on the systematic a p p l i -

c a t i o n o f gas chromatography t o t o x i c o l o g i c a l analysis, J a i n and K i r k

9

u t i l i z e d the very

p o l a r HI-EFF-86 ( 1 %) as s t a t i o n a r y phase f o r a number o f opium a l k a l o i d s and r e l a t e d

CM-

pounds: Codeine, dihydrocodeine, d i hydrohydroxycodeinone, heroin, morphine, noscapine, papaverine and thebaine. For the i d e n t i f i c a t i o n o f a number o f opium a l k a l o i d s (morphine, codeine, thebaine, noscapine, papaverine, cryptopine, narceine) and heroin, 3-O-acetylmorphine, and acetylcodeine, V i a l a e t a1.l’ p o l a r i t i e s (SE-30 3

I,

6-0-acetylmorphine

made use o f t h r e e gas chromatographic columns o f d i f f e r e n t

O V - 1 2 % and OV-1 2 % plus Igepal 0.2 %).They obtained r e l i a b l e sep-

a r a t i o n and i d e n t i f i c a t i o n i n t h i s way, which was based on the d i f f e r e n c e i n the r e t e n t i o n times i n the three columns. To achieve a b e s t possible i d e n t i f i c a t i o n based on r e t e n t i o n times, M o f f a t e t a1.l’

cal-

c u l a t e d the r e t e n t i o n indices f o r a number o f basic drugs, i n c l u d i n g opium a l k a l o i d s and congeners, on e i g h t st.ationary phases o f various p o l a r i t y . They concluded t h a t l o w - p o l a r i t y columns, such as SE-30 o r OV-17, should be p r e f e r r e d f o r the i d e n t i f i c a t i o n o f b a s i c drugs w i t h gas chromatography. Later Moffat”

c a l c u l a t e d the r e t e n t i o n i n d i c e s f o r a s e r i e s o f opium a l k a l o i d s and con-

geners on a SE-30 column (Table 14.2). Liras13 used an OV-17 column f o r i n v e s t i g a t i o n s on the metabolism o f morphine and codeine by drthrobacter species, and gave the gas chromatographic parameters o f e i g h t o x i d i z e d compounds, found as metabolites (14-hydroxymorphine,

14-hydroxymorphinone,

morphinone, dihydromorphinone, codeinone, 14-hydroxycodeine, 14-hydroxydi hydrocodeinone).

References p. 144

14-hydroxydi hydro-

14-hydroxycodeionone and

114 TABLE 14.2 RETENTION INDICES FOR SOME OPIUM ALKALOIDS AND CONGENERS ON SE-3012

RI Codeine Cot a r n i ne Desoxymorphi ne Heroin D i hydrocodeine Dihydrocodeinone Dihydromorphine Ethyl morphi ne D i hydrmorphinone

14.1.1.1.1.

RI

2385 1780 2275 2615 2365 2425 2440 2415 2490

Methyl d i hydromorphine Morphine Nalorphine Noscapine Oxycodone Oxymorphone Papaverine Phol codi ne Theba ine

2375 2435 2570 3100 2425 2520 2808 2380 2525

DERIVATIZATION

To o b t a i n a b e t t e r gas chromatographic i d e n t i f i c a t i o n o f a l k a l o i d s c o n t a i n i n g phenolic

or a l c o h o l i c hydroxyl groups, Anders and Mannering14 introduced on-column d e r i v a t i z a t i o n o f the f r e e base ( i . a . morphine) w i t h a c e t i c anhydride and/or p r o p i o n i c anhydride. M r p h i n e r e acted completely w i t h a c e t i c anhydride g i v i n g two peaks. one o f heroin and one, possibly, o f 6-O-monoacetylmorphine, and w i t h propionic anhydride t o give o n l y one peak, probably o f d i propionylmorphine. The usefulness o f the on-column d e r i v a t i z a t i o n technique depends upon the r e l a t i o n s h i p between the r e t e n t i o n times o f the d e r i v a t i v e s formed. The same technique was employed by M u d 5 f o r a number o f opium a l k a l o i d s and r e l a t e d compounds, as w i l l be seen from Table 14.3. TABLE 14.3 RETENTION DATA OF SOME OPIUM ALKALOIDS AND CONGENERS15 Column: 6 f t by 3 mm 1.0..

SE-30 on Gas Chrm S (80-100 mesh) a t 215OC

Free* Morphine Normorphine Codeine Norcodeine Heroin Nalorphine Methyl dihydromorphinone D i hydromorphi none E t h y l m r p h i ne D i hydro-14-hydroxymorphinone O i hydromorphi ne D i hydrocodeine

6.94 6.94 6.0 6.0 11.34 9.84

7.50 7.31 6.71 8.81 6.94 6.94

Oihydro-14-hydroxycodeine8.62 6-0-monoacetylmorphine

*

8.62

Retention time (min) k i t h 5 u l a c e t i c anhydride With 5 111 p r o p i o n i c anhydride tR[rel ** 1.16 1.16 1.00 1.00 1.89 1.64 1.25 1.22 1.12

8.06 23.81 6.0 16.12

9.84 6.71

11.44 9.28

12.37 6.71

18.94 11.68

1.47 1.16 1.16 1.44 1.44

12.56 8.06

15.94 10.31

15.62 10.31 6.94

26.63 16.31 10.87

tR f o r f r e e untreated compound,

11.34 29.34 8.40 22.50

10.50 31.70 6.0 19.78

15.94

14.81

11.34 11.62

26.34 12.56

6.94 8.62 11.34 tR (re,)

18.37 57.37 10.54 34.87

11.34

10.12

**

35.62

8.62 14.67

t o f r e e codeine

Brochmann-Hanssen and Baerheim Svendsen16 observed t h a t phenolic a l k a l o i d s , such as morphine and apomorphine, were o f t e n d i f f i c u l t t o gas chromatograph because o f adsorptive e f f e c t s . Morphine generally gave o n l y about one-half o f the peak h i g h t which one would expect on t h e

116 b a s i s o f t h e amount a p p l i e d . Apomorphine w i t h two p h e n o l i c h y d r o x y l groups a l s o produced badl y t a i l i n g peaks. It was t h e r e f o r e recormended t h a t p h e n o l i c a l k a l o i d s s h o u l d be c o n v e r t e d t o t h e i r t r i m e t h y l s i l y l e t h e r s , which g i v e symmetrical peaks w i t h o u t any t a i l i n g o n t h e c h r o matogram t h a t a r e w e l l s u i t e d even f o r q u a n t i t a t i v e d e t e r m i n a t i o n s . H e x a m e t h y l d i l s i l a z a n e was u t i l i z e d as s i l y l a t i n g reagent. F i s h and Wilson17 used N,O-bis(trimethylsily1)acetamide

for

t h e same purpose i n s t u d i e s on morphine i n u r i n e . To be a b l e t o s e p a r a t e and i d e n t i f y picogram q u a n t i t i e s o f morphine and c o d e i n e a f t e r ext r a c t i o n from b i o l o g i c a l f l u i d s ,

Kogan and Chedekel18 adopted t h e method o f D a h l s t r o m and

Paalzow19, u s i n g p e n t a f l u o r o p r o p i o n i c a n h y d r i d e and o f f - c o l u m n r e a c t i o n w i t h an e l e c t r o n 63 . c a p t u r e ( N i ) d e t e c t o r ; an OV-22 column was used. Kaneshina e t a1.20 d e s c r i b e d t h e gas c h r o matographic d e t e c t i o n of morphine and codeine i n opium poppies as f r e e bases, a c e t y l - and t r i f l u o r o a c e t y l - as w e l l as t r i m e t h y l s i l y l d e r i v a t i v e s . F o r i n v e s t i g a t i o n s on morphine, i t s m e t a b o l i t e s and conaeners (codeine, morphine-N-oxide, norcodeine, n o m o r p h i n e and pseudomorphine) ,Yeh2’

a p p l i e d nas c h r o m a t o y a p h y o f t h e t r i m e t h y l -

s i l y l - , a c e t y l and t r i f l u o r o a c e t y l d e r i v a t i v e s ( T a b l e 1 4 . 4 ) . TABLE 14.4 RETENTION

TIMES

(MIN)

OF MORPHINE

Compound

SE-30 3 % 3 f t 205OC

Trimethyl s i l y l -codeine -morphine -morphine N-oxide -morphine N-methyl i o d i d e * - n o r c o d e i ne -normorphine *+ -pseudomorphi ne Acetyl -codeine -morphine -morphine N-oxide -morphine N-methyl i o d i d e -norcodeine -normorphine T r i f l uoroacetyl -codeine -morphine -morphine N-oxide -norcodei ne *** -normorphine -pseudomorphine

AND TIS 215OC 7 6.0 6.0 4.0 + 6.2 6.3 8.5

CONGENERS”

OV-17 3 % 3 f t 205OC 215OC

-

6.0

-

5.2 4.4 6.0 7.5 7.1 53

45.4 34.5 34.5

41.3

13.5 24 23.2 19 13.5 19 (25OOC)

8.0 7.8 8.0 19.7 27.5 3 2.8 4.5

7.7 6.7 6.7 4.7 t 8.2 10.3 a. 3

OV-17 3 % 6 f t 2OO0C 23OoC 25OoC

7.7 5.1 7.0 13.0 8.7

4 3.5

13.4 10.5 10.5 12.0

6.5 5.7 5.7 5.8

19 12.5

8.0 6.5

15 20

SE-30 3 % on V a r a p o r t 100-120 mesh, OV-17 3 % on Gas Chrom 0 60-80 mesh,* Morohine N-methyl i o d i d e gave two peaks which may a r i s e from thermal decomposition, ** T r i m e t h y l s i l y l - o s e u d o morphine d i d n o t emerge from t h e column a t 290OC a f t e r 1 h r . , *** The r e t e n t i o n t i m e f o r t r i fluoroacetyl-pseudomorphine was o b t a i n e d a t a column temperature o f 29OOC On-column d e t i v a t i z a t i o n w i t h trifluoroacetylimidazole and heptafluorobutyrylimidazole f o r q u a n t i t a t i v e d e t e r m i n a t i o n o f morphine and codeine was used by Brugaard and Rasmussen2? Docosane was u t i l i z e d as an i n t e r n a l standard. The r e l a t i v e s t a n d a r d d e v i a t i o n o b t a i n e d f r o m t h e r e p r o d u c i b i l i t y t e s t s v a r i e d f o r morphine and codeine between 1.1 % and 2.8 %. The h e p t a -

References p. 144

116

fluoroacetyl d e r i v a t i v e s gave b e t t e r r e p r o d u c i b i l i t y than the corresponding t r i f l u o r o a c e t y l d e r i v a t i v e s : 1.6 X f o r morphine and 1.1 % f o r codeine r e s p e c t i v e l y , compared w i t h 1.9 % and 2.8 % respectively, The authors found f u r t h e r t h a t "clean chromatogram" w i t h o u t i n t e r f e r i n g peaks could be achieved by u s i n g f r e s h l y d i s t i l l e d reagents s t o r e d i n t i g h t l y capped dark b o t t 1es I n analysis o f morphine and codeine i n b i o l o g i c a l m a t e r i a l o f t e n o n l y micro amounts have t o be determined. Christophersen and Rasmussenz3 developed an on-column d e r i v a t i z a t i o n procedure f o r such determinations by p e r f l u o r o a c y l a t i o n o f both compounds i n combination with e l e c t r o n capture detection. Heptafluorobutyrylimidazole was used as d e r i v a t i z a t i o n reagent. Excess o f reagent must be removed p r i o r t o e n t e r i n g t h e e l e c t r o n capture detector, otherwise i t w i l l depress the standing c u r r e n t and detector response long enough t o i n t e r f e r e w i t h t h e q u a n t i t a t i o n o f the peaks o f i n t e r e s t . The removal o f excess o f reagent was obtained w i t h a precolumn venting system. The d e r i v a t i z a t i o n r e a c t i o n was found t o be complete w i t h t h e model substances used: codeine, ethylmorphine and morphine. The minimum detectable amount o f codeine was found t o be about 100 pg and t h a t o f morphine about 20 pg. A packed glass column w i t h 3 % SE-30 on Supelcoport a t 2OO0C was used. A number o f various d e r i v a t i z a t i o n reactions have been used, as shown i n Table 14.25. The

.

d e r i v a t i z a t i o n has been c a r r i e d o u t as off-column o r on-column d e r i v a t i z a t i o n . mostly i n o r der t o g i v e morphine and r e l a t e d a l k a l o i d s w i t h phenolic o r a l c o h o l i c hydroxyl groups b e t t e r gas chromatographic properties. E s p e c i a l l y f o r q u a n t i t a t i v e determination o f morphine i n opium o r b i o l o g i c a l m a t e r i a l such d e r i v a t i z a t i o n was necessary. S t r e e t e t al.3 found, however, t h a t the problem o f adsorption o f a phenolic a l k a l o i d , such as morphine, because o f " a c t i v e s i t e s " on the s o l i d support could be solved by d e a c t i v a t i o n o f the s o l i d support by treatment o f i t (diatomaceous e a r t h ) w i t h benzoyl c h l o r i d e i n p y r i d i n e before c o a t i n g i t w i t h the s t a t i o n a r y phase.

14.1.1.1.2.

COMBINATION OF GLC WITH OTHER ANALYTICAL TECHNIQUES

A combination o f gas Chromatography w i t h o t h e r a n a l y t i c a l techniques has o f t e n been used t o ascertain the presence o f opium a l k a l o i d s i n b i o l o g i c a l f l u i d s . J a i n e t a l . 2 4 detected

morphine and codeine i n u r i n e e x t r a c t s by GLC and confirmed t h e i r presence by TLC. Sine e t screened u r i n e samples f o r morphine by TLC and each specimen was then examined by gas chromatography. Mulez6 u t i l i z e d GLC detection o f drugs o f abuse as an adjunct confirmatory t e s t f o l l o w i n g r o u t i n e TLC o f u r i n e extracts; Watanabe e t a1.27 made use o f GLC-MS f o r t h e detection o f morphine and heroin i n urine. A s i m i l a r technique was employed by Weber and Ma28 f o r a r a p i d i d e n t i f i c a t i o n o f morphine and codeine i n opium. SmithZg developed a method f o r the i d e n t i f i c a t i o n o f suspected opium samples based on computerized GC-MS o f three o f the main opium a l k a l o i d s , morphine, codeine and papaverine. The separation o f the t r e e a l k a l o i d s was obtained on a 3 % OV-101 column on Chromosorb W HP.

117

14.1.1.2.

puanti t a t i v e determination

14.1.1.2.1.

MORPHINE I N OPIUM

The observation made by Brochmann-Hanssen and Baerheim Svendsen"

t h a t morphine, when gas

chromatographed as f r e e base, was t o some e x t e n t adsorbed on the chromatographic column, was confirmed by Schmerzler e t al.30 and by M a r t i n and Swinehart31. Brochmann-Hanssen and Baerheim Svendsen3' used tetraphenylethylene o r laudanosine as i n t e r n a l standards i n

t h e i r assay

o f morphine i n opium, the tetraphenylethylene e l u t i n g p r i o r to, and the laudanosine e l u t i n g a f t e r the morphine t r i m e t h y l s i l y l ether. The most c r u c i a l step i n the procedure was the ext r a c t i o n o f the t o t a l a l k a l o i d s and the separation o f the phenolic and the non-phenolic a l kaloids. This was achieved by means o f a s t r o n g l y a c i d i c c a t i o n exchange r e s i n ( e x t r a c t i o n and p u r i f i c a t i o n ) and a s t r o n g l y basic anion exchange r e s i n (separation o f phenolic and nonphenolic a l k a l o i d s ) . Using standard curves f o r morphine w i t h both i n t e r n a l standards, the morphine content i n a number o f opium samples was analyzed and the gas chromatographic values compared w i t h the r e s u l t s obtained, using a m o d i f i e d Mannich method ( o f f i c i a l i n the Pharmacopoea Nordica, 1 s t E d i t i o n ) . TABLE 14.5

MORPHINE CONTENT I N OPIUM SAMPLES (%)32 Opium sample

Gas chromatography* Value determined Calc. t o anhydr. base

U.S.P. UN 2A UN 15 UN 386

11.4 14.0 16.5 18.5

14.9 17.5 19.7

UN 137A UN 25A

15.1 18.3

16.1 19.5

UN UN UN UN UN UN

13.8 11.0 12.8 15.5 12.4 12.2

14.6 11.7 13.6 16.3 13.1 13.2

E529 E531 265 E612 E627 E631

Other methods 10.5** 13.5*** 16.1**** 17.0*** 20.3**** 13.8*** 18.1*** 19.1**** 13.8*** 12.0*** 13.1*** 15.5*** 11.0*** 12.0***

* Average values based on two o r more determinations. ** Opium assay U.S.P. X V I . *** Modified Mannich method, Pharmacopoea Nordica Ed.1; sample w i t h o u t drying. **** United Nations Secretariat; c a l c u l a t e d t o anhydrous base. M a r t i n and Swinehart31 found t h a t morphine base gave a constant value o n l y a f t e r approximately 24 chromatographic analyses o f 25 p1 o f a 0.1 % s o l u t i o n o f morphine and codeine, thus i n d i c a t i n g t h a t some adsorption took place on t h e column. A f t e r s u f f i c i e n t chromatograms, the adsorption s i t e s became saturated w i t h morphine and the sorption-desorption processes became equalised. For t h e q u a n t i t a t i v e determination o f morphine and codeine i n opium, t h e trim e t h y l s i l y l ethers o f the a l k a l o i d s were chromatographed. using squalene as an i n t e r n a l standard. The values obtained f o r morphine i n a number o f authentic opium samples are given i n Table 14.6, together w i t h the morphine values obtained w i t h the method o f the A u s t r i a n Pharmacopoeia, 9 t h E d i t i o n . P a r i s e t a1.33 estimated the morphine content i n opium poppies a f t e r s i l y l a t i o n . Notwithstanding the observations mentioned above concerning adsorption o f morphine when

Rehrences p. 144

118 TABLE 14.6 PERCENTAGE ANHYDREOUS MORPHINE I N OPIUM3' determined b y GLC as t r i m e t h y l s i l y l e t h e r and w i t h t h e method o f t h e A u s t r i a n Pharmacopoeia, 9th Edition Opium sample UN UN UN UN UN

Aus t r i an Pharmacopoeia

GLC-TMS

M-1 M-2 M-3 M-4 M-5

9.9 11.5 10.0 11.0 5.9

9.34 12.45 11.23 12.26 5.6

gas chromatographed as f r e e base, N i e ~ n i n e nd~e s~c r i b e s a method f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f morphine and t h e o t h e r main a l k a l o i d s o f opium w i t h o u t d e r i v a t i z a t i o n on a 3.5 % SE-30 column. The r e s u l t s a r e seen i n Table 14.7. TABLE 14.7 CONTENT OF ALKALOIDS I N OPIUM, OPIUM EXTRACT AND OPIUM TINCTURE34 determined w i t h o u t d e r i v a t i z a t i o n on a 3.5 % SE-30 column Sample

Test

Morphine Ph. N o r d i c a

Opium

1 2 3 4 5 6 Mean S t d .dev. Coeff. o f var. %

Opi um Opium e x t r a c t Opium t i n c t u r e

9.8 19.9 0.93

Codeine

Papaverine

Noscapine

Thebaine

Gas chromatoqraphy 11.8 11.2 11.0 11.6 11.0 11.4

0.82 0.84 0.75 0.86 0.80 0.78

1.17 1.20 i.24 1.16 1.09 1.19

4.37 4.80 4.36 4.50 4.36 4.47

11.3 0.33 2.9

0.81 0.04 4.9

1.18 0.05 4.4

4.48 0.17 3.8

0.87

21.0 1.08

1.72 0.16

1.43 0.18

4.5 0.32

1.4 0.13

0.83 0.90

8 e ~ h t e ul t~i l~i z e d a s t r o n g p o l a r s t a t i o n a r y phase (HI-EFF 88) 0.75 % on Chrumosorb G HP f o r t h e same purpose and succeeded i n d e t e r m i n i n g t h e main opium a l k a l o i d s i n opium e x t r a c t s w i t h o u t any d e r i v a t i z a t i o n . The r e l a t i v e s t a n d a r d d e v i a t i o n f o r t h e i n d i v i d u a l a l k a l o i d s l a y between

0.6 % and i 8 . 8 %, and f o r morphine a t

f

2.3 %.An i n t e r n a l s t a n d a r d was used f o r

t h e e v a l u a t i o n . On a packed column w i t h a 1 : l m i x t u r e o f OV-17 and SE-30 ( 3 % on V a r a p o r t 30 ~~ codeine, morphine, thebaine, p a p a v e r i n e and 5 % on Chromosorb W ASW), F ~ r m a n e cdetermined and noscapine s i m u l t a n e o u s l y and q u a n t i t a t i v e l y w i t h o u t d e r i v a t i z a t i o n and w i t h a s t a n d a r d d e v i a t i o n f o r t h e i n d i v i d u a l a l k a l o i d s between 0.05 and 0.18 %.

A s i m p l e and r a p i d method f o r gas chromatographic assay o f t h e morphine and c o d e i n e cont e n t i n opium was proposed by Nakamura and N ~ g u c h ui s~i n~g N , O - b i s ( t r i m e t h y l s i l y 1 )acetamide b o t h as a s o l v e n t f o r t h e opium sample t o be a n a l y z e d and as d e r i v a t i z a t i o n r e a g e n t f o r morphine and codeine f o r t h e gas chromatographic a n a l y s i s . Amount o f 2-4 mg opium was i n t r o d u c e d i n a 3 m l glass-stopped tube, 200 ~1 o f 40 % N,O-bis(trimethylsily1)acetamide was added and s o l u t i o n was e f f e c t e d by mechanical m i x i n g o n a V o r t e x v i b r a t o r . The 200 u l o f i n t e r n a l s t a n dard s o l u t i o n ( 2 mg n a l o r p h i n e p e r m l p y r i d i n e ) was added t o t h e sample s o l u t i o n and mixed. One p1 o f t h e r e s u l t i n g s o l u t i o n was i n j e c t e d f o r q u a n t i t a t i v e gas chromatographic a n a l y s i s

119

on a 3 % OV-17 column and a 3.8 % UCW-98 column. Typical chromatograms are given i n Fioure 14.1. FIGURE 14.1 CHROMATOGRAMS

OF OPIUM EXTRACT 37

A: 3 % OV-17 column, 8: 3.8 % UCW-98 column. 1 = codeine, 2 = morphine, 3 = nalorphine ( i n t e r n a l standard) as t r i m e t h y l s i l y l d e r i v a t i v e s .

A

j 3

I

1

,

2

A comparison o f opium assay values i s given i n Table 14.8. TABLE 14.8

COMPARISON OF OPIUM ASSAY VALUESJ7 Method

Percent morphine Opium UN-25 A Opium UN-38 G

Present GLC method GLC method o f Brochmann-Hanssen and Baerheim Svendsen 1963 Isotope d i l u t i o n method o f Brochmann-Hanssen 1972 TLC method o f Mary and Brochmann-Hanssen 1963 Mannich method (Baerheim Svendsen 1959)

17.92

-

17.50 17.51 17.00

18.92 17.83 18.4-18.5 17.85 18.10

I n a r e p o r t published by the J o i n t Committee o f the Pharmaceutical Society and the Anal y t i c a l D i v i s i o n o f the Chemical Society on Pharmaceutical Analysis3'

a gas chromatographic

method was proposed f o r the q u a n t i t a t i v e determination o f morphine i n opium and o p i a t e prepa r a t i o n s t o replace the assay l a i d down i n the B r i t i s h Pharmacopoeia. Several s t a t i o n a r y phases were t r i e d and a mixture o f 4:6 o f OV-25 and OV-225 was preferred. Since no complete recovery o f morphine from the gas chromatographic column could n o t be obtained, when chromatographed as f r e e base, s i l y l a t i o n o f morphine was recomnended. Nalorphine was used as an i n t e r n a l standard, as proposed by Wallace e t a1.51

Rqltmncn p. 144

, since

it i s structurally identical t o

120 morphine i n t h e v i c i n i t y o f t h e hydroxyl group and r e a c t s s i m i l a r l y t o morphine by t h e s i l y l a t i o n r e a c t i o n . The values obtained i n e i g h t l a b o r a t o r i e s f o r t h e morphine and codeine cont e n t i n f i v e United Nations raw opium samples a r e l i s t e d i n Table 14.9. TABLE 14.9 MORPHINE AND CODEINE CONTENT I N UNITED NATIONS RAW OPIUM SAMPLES38 Column: 1.5 in by 4.0 mn I.D.,

glass, 1 % o f a 4:6 m i x t u r e o f OV-225 and OV-25 on D i a t o m i t e

CQ o r Gas Chrom Q (100-120 mesh) a t 200-210°C.

Laboratorv

UN-M-1

UN-M-2

UN-M-3

UN-!1-4

M0rph.X Cod.% Morph.% Cod.% Morph.% Cod.%

~

_

_

UN-M-5 _

_ _____

Morph.% Cod.% Morph.% Cod.%

1

11.57 11.59

4.34 4.25

2

11.36 11.27

3.73 4.26

14.23 14.63

2.24 2.60

13.82 14.22

1.28 1.35

12.98 13.05

3

11.21 11.29

3.92 4.04

15.12 14.74

2.32 2.30

13.65 13.47

1.20 1.19

4

11.0 11.45

4.10 3.99

14.50 14.11

2.36 2.36

14.39 14.20

5

11.69 11.50

4.29 4.27

14.30 14.46

2.55 2.51

6

12.01 11.80

3.49 3.45

14.75 14.71

7

10.17 10.42

3.80 3.75

15.64

8

11.72 11.84

3.83 4.02

Mean

7.04 7.19

3.16 3.23

3.51 3.50

6.93 7.09

2.86 2.97

13.34 13.32

3.63 3.62

7.04 7.09

2.65 2.83

1.36 1.34

13.17 13.46

3.54 3.43

7.16 7.23

2.89 3.20

13.86 13.85

1.41 1.42

12.76 12.64

3.56 3.52

6.89 6.91

2.83 2.73

2.56 2.59

14.05 14.00

1.45 1.47

13.28 13.10

3.55 3.54

7.23 7.21

2.92 2.86

2.54

14.01 14.07

1.37 1.35

12.64 12.53

4.11 4.03

6.58 6.36

2.77 2.68

11.37

3.97

14.65

2.45

13.97

1.35

13.02

3.62

7.00

2.90

Stand.dev.

0.49

0.28

0.43

0.13

0.25

0.09

0.31

0.21

0.25

0.18

Coeff.var. %

4.35

7.07

2.95

5.4

1.82

6.57

2.41

5.88

3.63

6.36

Two a l i q u o t s from a s i n g l e weighing o f each opium sample were chromatographed by each 1a bora t o ry

.

An e l e g a n t and r a p i d method f o r q u a n t i t a t i v e d e t e r m i n a t i o n o f morphine i n opium e x t r a c t s was published by R a s m u ~ s e nusing ~ ~ t r i m e t h y l s i l y l i m i d a z o l e f o r on-column s i l y l a t i o n o f morphine and n-tetracosane (C24) as an i n t e r n a l standard. The gas chromatography was c a r r i e d o u t on a 3 % Dexsil 300 column. A n a l y s i s o f a standard opium e x t r a c t w i t h 14.9 m / m l o f morphine gave an average content o f 15.0 mg/ml and a c o e f f i c i e n t o f v a r i a t i o n o f 1.4 %.

14.1.1.2.2.

MORPHINE I N BIOIOGIW

MATERIALS

For t h e determination o f morphine i n opium, Ikekawa e t a1.40 developed a r a p i d method whereby morphine and morphine glucuronide were i s o l a t e d from t h e u r i n e sample (50 m l ) by ads o r p t i o n on a charcoal column (1 g i n a column o f 1 cm I.D.),

washed w i t h water t o remove i m -

p u r i t i e s , and e l u t e d w i t h g l a c i a l a c e t i c a c i d (20 m l ) . A f t e r evaporation t o dryness i n vacuo, t h e morphine glucuronide was hydrolyzed w i t h h y d r o c h l o r i c a c i d , and t h e pH a d j u s t e d t o 2.5 w i t h sodium hydroxide and e x t r a c t e d w i t h ch1oroform:isopropanol

( 9 : l ) . The water l a y e r was

121 adjusted t o pH 9.0 and e x t r a c t e d w i t h the same solvent. The d r y residue o f the s o l u t i o n obt a i n e d was dissolved i n N,O-bis(trimethylsily1)acetamide

(0.1-0.2 m l ) and a f t e r 10 minutes

a t roan temperature 10 u1 was i n j e c t e d f o r gas chromatographic analysis. Nalorphine was used as an i n t e r n a l standard

-

and was added t o t h e u r i n e sample before any treatment. The o v e r a l l

recovery was about 60 %.5-10 ug morphine i n 1000 m l u r i n e was the l i m i t o f d e t e c t a b i l i t y w i t h t h i s method. Packed columns (1.8 m by 4 mm 1.0.) w i t h 1.5 % OV-1, SE-30,

1.5 % OV-17,

1.5 %

1.5 % QF-1 o r 1.5 % XE-60 on Shimalite W 80-100 mesh were used f o r the gas chromato-

graphy. Payte e t a1.41 s t a t e d t h a t the s e n s i t i v i t y o f d e t e c t i o n o f morphine i n u r i n e could be i n creased by a c i d h y d r o l y s i s o f the glucuronide metabolite. L a t e r Yeh e t a1.42 showed t h a t 50 %

o f the morphine found i n u r i n e was as morphine.3-glucuronide. Truhaut e t a1.43 i n v e s t i g a t e d the detection o f f r e e morphine i n the u r i n e o f druo addicts and used a mixture o f ch1orofonn:ethyl acetate:ethanol ( 3 : l : l )

f o r the e x t r a c t i o n , a f t e r ad-

justment o f the pH t o 8-8.5 w i t h bicarbonate. Morphine was d e r i v a t i z e d w i t h N,O-bis(trimethy1sily1)acetamide and the gas chromatography was c a r r i e d o u t on a 3 % Dexsil column u s i n g tetracosane (CZ4) as an i n t e r n a l standard. 5 p g i n 10 m l u r i n e could be determined by t h i s method.

A r a p i d and simple method f o r t o t a l morphine i n u r i n e was developed by F r y e t a1.44 using 14C morphine t o c o r r e c t f o r e x t r a c t i v e losses. Morphine conjugates were hydrolyzed by p-glucuronidase and morphine e x t r a c t e d w i t h ether. Nalorphine was used as an i n t e r n a l standard and both compounds (morphine and nalorphine) were converted i n t o t h e i r t r i m e t h y l s i l y l e t h e r s using N,O-bis(trimethylsily1)acetamide as reagent. By adding 14C morphine t o t h e u r i n e sample t o be e x t r a c t e d and analyzed no attempt was made t o e x t r a c t the morphine q u a n t i t a t i v e l y , since thefinal

r e s u l t could be corrected f o r recovery by the use o f 14C-morphine. The c o e f f i -

c i e n t o f v a r i a t i o n over a period o f s i x months was 5.6

%.A

90 cm l o n g packed column w i t h

Chromosorb G coated w i t h a mixture o f 0.35 % JXR and 0.35 % CDMS was used a t 21OoC. The mass fragmentographic technique w i t h s t a b l e isotopes as i n t e r n a l standards shows h i g h s e n s i t i v i t y ( i n the picogram range), s p e c i f i c i t y due t o the focusing on s p e c i f i c mass f r a 5 ments, and good r e p r o d u c i b i l i t y i n q u a n t i t a t i v e work because the i n t e r n a l s t a b l e isotope standard makes i t possible t o c o r r e c t f o r losses i n the preparative phase o f the a n a l y s i s . Ebbighausen e t a l . 4 5 developed a method f o r the assay o f morphine and codeine i n u r i n e using deuterium labeled morphine and codeine. Codeine was d e r i v a t i z e d w i t h h e p t a f l u o r o b u t y r i c a c i d and morphine w i t h t r i f l u o r o a c e t i c a c i d before gas chromatography on a packed column w i t h 1 %

OV-17 on Gas Chrom Q. The LKB 9000 combination o f gas chromatograph-mass spectrometer equipped w i t h a m u l t i p l e - i o n detector/peak matcher was used. With a simple e x t r a c t i o n procedure of 1 m l u r i n e a f t e r h y d r o l y s i s a p r a c t i c a l l i m i t o f d e t e c t a b i l i t y was found t o be approximately 500 pg. Clarke and F ~ l t developed z ~ ~ a s i m i l a r method f o r the analysis o f morphine i n u r i n e w i t h N-C2H3-morphine as i n t e r n a l standard. The i n t e r n a l standard was added t o 10 m l urine. the u r i n e b u f f e r e d t o pH 8.5 and e x t r a c t e d w i t h ch1orofon:isopropanol ( 4 : l ) . The e x t r a c t i o n residue was t r i m e t h y l s i l y l a t e d by adding 25

pl

of N,O-bis(trimethylsily1)acetamide

and heat-

i n g a t 6OoC f o r about 1 h. About .? 111 was analyzed on a 3 % OV-17 column a t 23OoC coupled d i r e c t t o a Finnegan 1015 quadrupole mass spectrometer equipped w i t h a chemical i o n i z a t i o n source, which was operated a t an i o n i z i n c energy o f 100 eV, an i o n r e p e l l e r voltage o f 0 V and a filament emission o f 300 PA. The mass spectrometer was i n t e r f a c e d w i t h a System Indus-

Referencesp. 144

122 t r i e s 250 computer system. Morphine concentrations as low as 5 ng/ml could be measured by s e l e c t i n g i o n monitoring. Wilkinson and Way47 developed a method f o r q u a n t i t a t i v e determination o f morphine i n p l a s ma and celebrospinal f l u i d (0.1-1.0

m l ) . Morphine was e x t r a c t e d w i t h e t h y l acetate contain-

i n g 10 % isopropanol, back e x t r a c t e d w i t h h y d r o c h l o r i c a c i d and the residue o f t h i s s o l u t i o n t r e a t e d w i t h N.0-bis(trimethylsily1 )acetamide c o n t a i n i n g 1 % t r i m e t h y l c h l o r o s i l a n e (25 u l ) . The amount o f morphine was determined

using tetraphenylethylene as i n t e r n a l standard on a

packed column w i t h 3 % OV-10 as s t a t i o n a r y phase a t 215OC. L i m i t o f s e n s i t i v i t y o f the method was found t o be about 25 ng per sample. To be able t o determine morphine a t sub-microgram l e v e l s i n urine, b i l e , whole blood and t i s s u e homogenates w i t h a h i g h degree o f s p e c i f i c i t y , Wallace e t al.48 converted morphine t o i t s d i a c e t y l d e r i v a t i v e p r i o r t o Gas chromatography. Since morphine i s excreted as t h e glucuronide conjugate, a c i d h y d r o l y s i s o f the u r i n e sample was recommended t o enhance t h e s e n s i t i v i t y o f detection. The recovery o f morphine a f t e r i n v i t r o a d d i t i o n t o u r i n e and homogenized l i v e r i s summerized i n Table 14.10. TABLE 14.10

RECOVERY OF MORPHINE AFTER I N VITRO ADDITION TO URINE AND HOHOGENIZEO LIVER48 Amount added ug/ml

Recovery, mean t standard d e v i a t i o n (ug/ml) Urine

2.5 5.0 10.0

2.44 4.75 9.36 47.02

50.0 Average recovery

L 0.03 t 0.13 t 0.33 i

95.1 %

2.05

Liver 2.0 i 0.12 3.76 i 0.13 7.40 i 0.20 (not determined) 76.4 %

The average recovery f o r morphine as determined by the gas chromatographic technique and calculated from the mean value f o r each concentration. G a r r e t t and Giirkan4' compared a number o f s e n s i t i v e methods f o r the determination o f morphine i n b i o l o g i c a l f l u i d s and found no s i g n i f i c a n t d i f f e r e n c e s among: a method based on 1 i q u i d s c i n t i l l a t i o n , a radioisotope method and e l e c t r o n capture gas chromatography. The morphine was determined by GLC a t 215OC on a 3 m long column as t h e pentafluoropropionate der i v a t i v e w i t h nalorphine pentafluoropropionate as an i n t e r n a l standard. The estimated standard d e v i a t i o n ( i n percent o f concentration) o f an assay from 0.5 m l o f plasma ranaed from

1 % a t 2500 ng/ml t o 9.2 % a t 5 ng/ml.

To be able t o measure morphine l e v e l s i n b r a i n i n connection w i t h studies on the concent r a t i o n o f morphine and a n a l g e t i c a c t i v i t y , Hipps e t al.50 u t i l i z e d t h e m u l t i p l e i o n detect i o n method (mass fragmentography) using a computer-control l e d gas chromatopraph-mass spectrometer (OP-12

-

LKB-9000 GC-MS). The morphine was converted t o i t s t r i f l u o r o a c e t y l d e r i v a t i v e

immediately before the analysis and the gas chromatography was c a r r i e d o u t on a 50 cm l o n g by 6 mm 0.0. glass column packed w i t h 3 % SE-30 on Gas Chrom Q a t 21OoC. Deuterium l a b e l e d 2 t r i f l u o r o a c e t y l m o r p h i n e (N-methyl- H3-morphine) was used as an i n t e r n a l standard. The authors stressed t h a t the power o f the m u l t i p l e i o n detection method i s p a r t l y due t o the s e n s i t i v i t y a t t a i n a b l e using the mass spectrometer as a gas chromatographic detector, b u t , more s i g n i f i c a n t l y , t o the f a c t t h a t fragment ions i n the mass spectra o f substances can be used as chemically s e l e c t i v e detectors.

123 I n v e s t i g a t o r s have d e r i v a t i z e d morphine w i t h a v a r i e t y o f r e a g e n t s , a c h i e v i n g b o t h sup e r i o r gas chromatographic c h a r a c t e r i s t i c s and enhanced s e n s i t i v i t y . I n many cases pre-chromat o g r a p h i c d e r i v a t i z a t i o n o f morphine may be advantageous, u t i l i z i n g r e a g e n t s such as t r i f l u o r o a c e t i c a n h y d r i d e t o g i v e t r i f l u o r o a c e t y l morphine, which can be d e t e c t e d i n a q u a n t i t y o f l e s s t h a n 0.1 nanogram, when u s i n g a c y l i n d r i c a l r o d 63Ni e l e c t r o n c a p t u r e detector5’.

Since

t h e d e r i v a t i z a t i o n i s q u a n t i t a t i v e and t h e gas chromatograph response i s l i n e a r , w i t h t h e c o n c e n t r a t i o n o v e r a wide range

-

a l s o f o r n a l o r p h i n e , used as an i n t e r n a l s t a n d a r d

- quanti-

t a t i v e d e t e r m i n a t i o n s o f morphine can be c a r r i e d o u t i n b i o l o g i c a l f l u i d s . The method o f Wallace e t a l ?

o f f e r s p o s s i b i l i t i e s o f d e t e r m i n i n g plasma o r serum l e v e l s down t o 25 ng/ml

o f morphine i n a 2 m l specimen w i t h a c c e p t a b l e p r e c i s i o n , a l t h o u g h c o n c e n t r a t i o n s o f 5 ng/ml a r e d e t e c t e d by t h i s procedure. Nalorphine, s t r u c t u r a l l y i d e n t i c a l t o morphine i n t h e v i c i n i t y o f t h e h y d r o x y l group, r e a c t s s i m i l a r l y t o morphine and t h u s s e r v e s as a c o n t r o l b o t h f o r t h e d e r i v a t i z a t i o n r e a c t i o n and f o r v a r i a t i o n s i n t h e chromatoaraphic technology. Nakamura and Nay5* determined morphine and codeine i n cadaverous body f l u i d s and t i s s u e samples a f t e r e x t r a c t i o n w i t h o r g a n i c s o l v e n t and d e r i v a t i z a t i o n w i t h N . 0 - b i s ( t r i m e t h y l s i l y 1 ) acetamide, u s i n g n a l o r p h i n e as an i n t e r n a l s t a n d a r d and OV-17 3 % and UCW-98 3.8 % a s s t a t i o n a r y phases. Because o f t h e h i g h e r s t a b i l i t y o f p e n t a f l u o r o p r o p i o n y l morphine compared w i t h t r i f l u o r o a c e t y l morphine and i t s h i g h e r e l e c t r o n c a p t u r e p r o p e r t i e s , D a h l s t r o m and Paalzow19 p r e f e r r e d t h i s d e r i v a t i v e f o r a method f o r q u a n t i t a t i v e d e t e r m i n a t i o n o f morphine i n b i o l o c l i c a l samples, u s i n g e l e c t r o n c a p t u r e ( t r i t i u m f o i l ) d e t e c t o r . N a l o r p h i n e was used as an i n t e r n a l s t a n d a r d . The s e n s i t i v i t y l i m i t o f t h e d e t e c t o r was about 5 pg o f morphine and t h e c o r r e s p o n d i n g l i m i t when e x t r a c t e d f r o m plasma a b o u t 500 pg m l - l .

I n t h e b r a i n , 100 pg morphine c o u l d be d e t e r -

mined i n a 30 mg b r a i n t i s s u e sample. Analyses o f n i n e d i f f e r e n t plasma samples c o n t a i n i n g 0.750 ng m l - l morphine, showed a v a r i a t i o n c o e f f i c i e n t o f 5.3

i

1.2 %.The s e n s i t i v i t y i s

t h u s i n t h e same range as r e p o r t e d f o r t h e immunological t e c h n i q u e . Furthermore, gas chromatography has t h e advantage o f combining r a p i d i t y and s p e c i f i c i t y w i t h h i g h s e n s i t i v i t y and s e l e c t i v i t y o f the e l e c t r o n capture detector. O e r i v a t i z a t i o n o f codeine and morphine w i t h p e n t a f l u o r o p r o p i o n i c a n h y d r i d e u s i n 9 e l e c t r o n c a p t u r e d e t e c t i o n , as d e s c r i b e d b y D a h l s t r o m and Paalzow”

was used b y D a h l s t r o m e t a l . 53

f o r simultaneous d e t e r m i n a t i o n o f codeine and morphine i n plasma and b r a i n samples. The a l k a l o i d s were e x t r a c t e d from 0.05-1.0 m l samples a f t e r a d d i t i o n o f t h e i n t e r n a l s t a n d a r d s and c a r b o n a t e b u f f e r t o a d j u s t t h e pH t o 8.9, w i t h ( 9 : l ) . Back e x t r a c t i o n w i t h a c i d and r e - e x t r a c t i o n w i t h to1uene:butanol gave to1uene:butanol ( 3 - e t h y l m o r p h i n e and N-ethyl-normorphine)

a d r y r e s i d u e o f t h e a l k a l o i d s , which was t r e a t e d w i t h p e n t a f l u o r o p r o p i o n i c a n h y d r i d e f o r 30 min a t 65OC. Excess o f r e a g e n t was removed and t h e sample d i s s o l v e d i n e t h y l a c e t a t e f o r t h e gas chromatographic a n a l y s i s on a 3 % OV-17 column on Gas Chrom Q 100-120 mesh a t 215OC. The s e n s i t i v i t y o f t h e method ( 0 . 7 5 ng o f morphine and 7.5 ng o f codeine i n a sample) makes i t u s e f u l f o r p h a r m a c o k i n e t i c i n v e s t i g a t i o n s . The r e p r o d u c i b i l i t y was good. P e n t a f l u o r b e n z y l a t i o n o f morphine and r e l a t e d p h e n o l i c a l k a l o i d s b y e x t r a c t i v e a1 k y l a t i o n was used b y Cole e t a1.54 f o r t h e d e t e r m i n a t i o n o f morphine i n plasma b y gas chromatography and mass fragmentography u s i n g morphine-d3 as i n t e r n a l standard. The e x t r a c t i v e a l k y l a t i o n a f f o r d s a method o f i s o l a t i n g p o l a r compounds such as morphine w i t h s i m u l t a n e o u s d e r i v a t i z a t i o n . To 1 n i l o f plasma c o n t a i n i n g unknown amounts o f morphine. t h e i n t e r n a l s t a n d a r d was added, as w e l l as t r i f l u o r o a c e t i c a n h y d r i d e , p e n t a f l u o r o b e n z y l bromide, a l k a l i and e t h y l a c e t -

References p. 144

124

ate as s o l v e n t t o remove the r e a c t i o n products a f t e r t h e i r formation. By back e x t r a c t i o n , f i r s t w i t h a c i d and water, then w i t h e t h y l acetate a f t &

b a s i f i c a t i o n , the GC-MS was c a r r i e d

o u t using a packed column w i t h 3 % OV-17 on Diatomite C a t 265OC i n an MS 30 gas chromatograph mass spectrometer (AEI). When using pentafluorobenzyl bromide d e r i v a t i v e s no i m p u r i t i e s w i t h l o n g gas chromatographic r e t e n t i o n times were encountered, such as when usina the d i tri f l u o r o a c e t i c anhydride d e r i v a t i v e s . Anders and Mannering14 introduced on-column d e r i v a t i z a t i o n o f morphine w i t h p r o p i o n i c anhydride and obtained o n l y one peak. They believed t h a t i t was dipropionylmorphine. Von Meyer e t al.55 made use o f the same d e r i v a t i z a t i o n r e a c t i o n f o r the q u a n t i t a t i v e determination o f morphine i n blood, but they found t h a t the monopropionyl d e r i v a t i v e s o f morphine and n a l o r phine (used as an i n t e r n a l standard) were found i n t h e p r o p o r t i o n 99:1,

as compared w i t h the

R

dipropionyl d e r i v a t i v e s . The e x t r a c t i o n o f blood was performed by means o f E x t r e l u t w i t h

(1:l) and the gas chromatography was c a r r i e d o u t on a 3 % OV-1 on Chromosorb W HP packed column a t 23OoC. Good r e s u l t s were obtained i n the range o f

petroleum ether 40°C:isoamylalcohol 300-600 ng/ml blood.

Cimbura and K ~ v e applied s ~ ~ an o t h e r e x t r a c t i o n procedure f o r morphine from blood samples: adsorption on an XAD-2 r e s i n and subsequent e l u t i o n w i t h an organic solvent mixture. Morphine was d e r i v a t i z e d w i t h a c e t i c anhydride p r i o r t o gas chromatoaraphy, which was c a r r i e d o u t on a 3 % OV-17 on Chromosorb W HP packed column a t 24OoC. An o v e r a l l recovery o f 70-80 % was achieved and an o v e r a l l p r e c i s i o n o f the method w i t h a c o e f f i c i e n t o f v a r i a t i o n o f 4.2

%.The

detection l i m i t was 0.05 mg/l using a NP-detector. Saady e t a l .57 p r e f e r r e d a c y l a t i o n o f morphine w i t h N-methyl-bis-trifluoroacetamide f o r i t s determination i n blood and serum. E x t r a c t i o n was performed a t pH 9.9 w i t h to1uene:hexane: isoamylalcohol (78:20:2),

and the e x t r a c t i o n e f f i c i e n c y o f the method was found t o be between

55 and 60 %. The use of nalorphine as an i n t e r n a l standard, coupled w i t h the s e n s i t i v i t y o f the GC-MS,

d i d n o t necessitate f u r t h e r e x t r a c t i o n . Q u a n t i t a t i v e determinations were c a r r i e d

o u t from less than 0.2 mg/l t o a t l e a s t 8 mg/l f o r morphine and codeine w i t h good r e s u l t s . The problem of adsorption o f a phenolic a l k a l o i d such as morphine during gas chromatography because o f " a c t i v e s i t e s " on the s o l i d support (diatomaceous e a r t h ) has mostly been avoided by converting morphine t o d e r i v a t i v e s such as a c e t y l , propionyl, f l u o r o a c e t y l , f l u o r o propionyl and s i l y l d e r i v a t i v e s . S t r e e t e t a l .3, however, developed a technique f o r preparat i o n o f low a c t i v i t y packed columns f o r gas chromatography o f p o l a r compounds, i.a. morphine, i n unmodified form i n nanogram amounts using a n i t r o g e n - s p e c i f i c detector. The diatomaceous e a r t h (Chromosorb G) was washed w i t h a c i d and water, d r i e d and acylated by treatment w i t h benzoyl c h l o r i d e i n p y r i d i n e 48 h. A f t e r washing w i t h acetone and drying, the support was coated w i t h SE-52 and d r i e d under n i t r o g e n i n a Pyrex tube

(I.D. 4 cm) and

then heated a t 41OoC - a l s o under nitrogen. Pyrex columns ( 6 f t by 3 mm 0.0.) were cleaned w i t h concentrated hydrochloric acid, washed w i t h water and acetone and dried. Then they were f i l l e d w i t h a m i x t u r e o f benzoyl c h l o r i d e and p y r i d i n e (2:3) and kept f o r three days, emptied, rinsed w i t h toluene and d r i e d a t 100°C. Two v a r i a t i o n s o f d e a c t i v a t i o n were performed; (A) A 5

I

s o l u t i o n o f SE-52 i n methylene c h l o r i d e was drawn by suction i n t o three wind-

ings o f the column and slowly moved through i t t o the o t h e r end. This procedure was repeated and, a f t e r pouring out the excess l i q u i d , the column was d r i e d f i r s t a t 100°C and f i n a l l y a t 35OoC under nitrogen f o r 10 h. The glass column was then ready f o r f i l l i n g .

126

( 6 ) The glass column was f i l l e d w i t h coated support m a t e r i a l and heated a t 4OO0C under n i t r o g e n f o r 1 h, then t h i s powder was poured out. The column was r e f i l l e d w i t h coated supp o r t and heated under n i t r o g e n a t 35OoC i n the gas chromatograph u n t i l a s t a b l e baseline was obtained. The gas chromatography was c a r r i e d o u t a t a column temperature o f 250°C f o r morphine base. When decreasing amounts o f morphine were.chromatographed on a commercial O V - 1 column and on the proposed deactivated column, and graphs showing the r e l a t i o n s h i p between peak h i g h t and amounts o f morphine were constructed,

i t was found t h a t the comnercial OV-1 column had a c u t -

o f f p o i n t a t 0.4 pg morphine, whereas the proposed deactivated column had a c u t - o f f p o i n t a t 0.8 ng. Hence, the proposed column showed an improvement i n d e t e c t i o n l i m i t o f the o r d e r o f 1000-fold. Coating o f the glass surface as w e l l as the diatomaceous e a r t h (Procedure A ) produced r e s u l t s f o r morphine t h a t showed l e s s adsorption r e l a t i v e t o a CZ4 hydrocarbon than when d i a tomaceous e a r t h o n l y was treated. With the n i t r o g e n - s p e c i f i c d e t e c t o r t h e r e was, however, only a s l i g h t d i f f e r e n c e between the methods A and B over a range o f about 25-100 ng o f morphine. The proposed column showed good thermal s t a b i l i t y over a t l e a s t 18 months under r o u t i n e conditions o f a busy t o x i c o l o g y l a b o r a t o r y . 14.1.1.2.3.

NON-PHENOLIC ALKALOIDS

The p r i n c i p a l non-phenol i c a l k a l o i d s were determined by Baerheim Svendsen and BrochmannH a n ~ s e non~ ~ a SE-30 column, 4 %.Although the s o l i d support, Gas Chrom P, was a c i d and base washed, s i l a n i z e d and precoated w i t h 0.1 % o f polyethylene g l y c o l 9000 before c o a t i n g w i t h SE-30, appreciable amounts o f the a l k a l o i d s were adsorbed on the column, the adsorption i n creasing i n magnitude w i t h an increase i n the r e t e n t i o n time o f the a l k a l o i d . Although t h e chromatographic peaks showed s a t i s f a c t o r y symnetry, t h i s i s i n i t s e l f no guarantee against loss o f sample through adsorption. For any one a l k a l o i d the amount adsorbed appeared t o be f a i r l y constant and reasonable independent o f the sample size. Consequently, the percentage o f the i n j e c t e d sample l o s t by adsorption decreased as the sample s i z e increased. Thus i t was not possible t o obtain reproducible values f o r the c o r r e c t i o n f a c t o r s . The same d i f f i c u l t i e s were experiences w i t h the unknown s o l u t i o n t o be analyzed. Under such circumstances a quantit a t i v e determination was subject t o e r r o r s o f considerable magnitude. Nevertheless, a select i o n o f authentic opium samples were analyzed, usin9 the average c o r r e c t i o n f a c t o r s , given i n Table 14.11. The a n a l y t i c a l r e s u l t s a r e given i n Table 14.12. By means o f i o n - p a i r e x t r a c t i o n w i t h d i e t h y l h e x y l phosphate and gas chromatooraphy on an

OV-17 column Guttman e t al?'

determined papaverine i n plasma a t l e v e l s as low as 0.01 ug/ml.

To reduce any p o t e n t i a l adsorption o f papaverine by the gas chromatopraphic column, t h r e e i n j e c t i o n s o f a concentrated s o l u t i o n o f papaverine and the i n t e r n a l standard, dibucaine, i n d i e t h y l ether were made p r i o r t o any s e r i e s o f analyses. N i e ~ n i n e nand ~ ~ B e ~ h t e do l ~ n~o t mention any loss o f a l k a l o i d s due t o adsorption on the chromatographic column o f the p r i n c i p a l non-phenolic a l k a l o i d s i n opium. They used respecti v e l y SE-30 (3.5 % ) and HI-EFF 8B (0.75 % ) as s t a t i o n a r y phases. Q u a n t i t a t i v e determinations o f codeine i n pharmaceutical preparations, e.g. i n mixtures w i t h o t h e r compounds, were c a r r i e d o u t by Wesselman6' on s i l i c o n e cum (Linde W-98), b y Stev-

References p. 144

126 ens61 on OV-17 and by Dechene e t a1.62 on Dow Corning F l u i d No.200.

N i e ~ n i n e ndetermined ~~

codeine phosphate i n pharmaceutical preparations a f t e r e x t r a c t i o n o f the f r e e base, u t i l i z i n g a UCC-W 982 10 % column. TABLE 14.11 CALCULATION OF CORRECTION FACTORS BASED ON GLC OF ALKALOID MIXTURES OF KNOWN CONCENTRATIONS5' (1) Concentration of a l k a l o i d i n s o l u t i o n (mg/ml), ( 2 ) Peak area ( a r b i t r a r y u n i t s ) , ( 3 ) Corr e c t i o n f a c t o r based on strychnine, (4) Average value o f c o r r e c t i o n f a c t o r . Codeine a

b

6.0 444 0.40

1.5 2.25 117 83 0.45 0.43 0.427

(3) (4)

Thebaine a

c

b

Papaverine c

1.0 2.25 4.0 103 128 372 0.34 0.28 0.30 0.307

a

b

Strychnine c

a

3.5 2.5 1.5 215 73 73 0.57 0.60 0.62 0.597

5.0 175 1.00

b

Noscapi ne c

a

5.0 5.0 88 150 1.00 1.00 1.00

6.75 235 1.01

b

c

7.5 7.5 147 230 0.90 0.92 0.963

TABLE 14.12 GLC DETERMINATION

OF CODEINE,

THEBAINE,

PAPAVERINE

AND NOSCAPINE

IN O P I U M ~ ~

The values i n parentheses obtained w i t h o t h e r methods. Sample

UN UN UN UN UN UN

2A 15 25A 27A 37C 386

Codeine % 1.97 1.21 1.38 2.45 5.82 1.76

(2.07) (1.33) (1.50) (2.35) (4.07) (1.52)

Thebaine % 1.22 1.38 0.66 3.02 2.73 1.15

(2.08) (1.75) (1.15) (3.21) (3.23) (1.28)

Papaverine % 1.03 1.80 2.18 1.00 1.13 2.57

(1.44) (1.80) (3.34) (1.35) (1.40) (3.19)

Noscapine % 6.52 5.80 6.69 6.19 7.05 6.29

(6.67) (6.18) (6.66) (6.28) (6.68) (6.47)

Wesselman and K ~ c determined h ~ ~ codeine phosphate together w i t h metapyri lene fumarate and ephedrine hydrochloride i n syrup a f t e r e x t r a c t i o n w i t h chloroform o f the b a s i f i e d syrup and a d d i t i o n o f the i n t e r n a l standard, amobarbital. The gas chromatography was c a r r i e d o u t by temperature programming from 145 t o 225OC a t a heating r a t e o f 10°C/min on a 3.8 % Linde W-98 s i l i c o n e gum on Diatoport S column. The r e l a t i v e standard d e v i a t i o n f o r the t h r e e components were

f

3.76 % (ephedrine), i 2.38 % (metapyrilene) and

*

2.02 % (codeine).

Schmerzler e t al.30 found t h a t codeine and i t s metabolite norcodeine could n o t be separated on a SE-30 column, and peaks observed represented any c o n t r i b u t i o n from e i t h e r compound. However, under m i l d a c e t y l a t i o n conditions there i s a great d i f f e r e n c e i n r e a c t i o n r a t e between codeine and norcodeine. This was used t o remove norcodeine before i n j e c t i o n on SE-30 columns. Norcodeine may be estimated by d i f f e r e n c e i f an i n j e c t i o n before a c e t y l a t i o n i s made f i r s t . However, on an XE-60 column the two a l k a l o i d s were resolved as f r e e bases and acetylat i o n was unnecessary. On an OV-17 column Serfontein e t a1.65 determined codeine e x t r a c t e d from serum by microphase e x t r a c t i o n , whereas Brunson and Nash66 made measurements o f codeine and norcodeine i n plasma, whereby norcodeine was d e r i v a t i z e d w i t h e t h y l chloroformate. For the gas chromatographic determination o f codeine i n plasma Zweidinger e t a l .67 u t i l i z e d n - b u t y l c h l o r i d e f o r the e x t r a c t i o n o f codeine from plasma i n s t e a d o f chloroform, which has been mostly used, because i t does n o t r e a d i l y g i v e emulsions w i t h plasma such as chloroform. On an OV-17 column

or an XE-60 column q u a n t i t a t i v e determinations i n low nanogram q u a n t i t i e s

(50 ng/ml) were c a r r i e d out, b u t the l i m i t o f d e t e c t i o n was as low as 5 ng/ml. Dihydrocodein-

127

one was used as an internal standard. In a paper on a comprehensive gas chromatography procedure f o r measurement of drugs in biological material Shipe and Savory68 also studied morphine and codeine. The drugs were extracted from 1 ml of serum or urine a f t e r addition of buffer, and nas chromatographed on a 3 % OV-17 column by temperature programing. There has been an increased i n t e r e s t in Papaver bracteatum Lindl. because of the relatively high content of thebaine in i t s latex, capsules, leaves and mots, and a number of analytical methods f o r i t s quantitative determination have been developed, among them oas chromatographic methods. The extraction of the alkaloid from the plant material and i t s purification without losses prior t o gas chromatography, seems t o be a major problem. Ping Chen and DorenbosC9 and Vincent e t al.70 used extraction with acetic acid (3-5 %), removal of i . a . pigments with diethyl ether or hexane, and isolation of the alkaloid a f t e r adjustment of the pH t o 9-9.5 by extraction with diethyl ether or chloroform - followed by gas chromatography. The gas chromatography has mostly been done on an OV-17 packed c o l u m n 6 g ~ 7 1 y 7 2 * 7 3 y 7 4 ~ 7 5 9 7 6 ~ 77y78. The method developed by Fairbairn and H e l l i ~ e l gave l ~ ~ a satisfactory reproducibility with a wide range of thebaine content (0.1-36 %) in latex, capsule, roots and leaves. The coefficient of variation f o r single assays ranged from 0.63 t o 3.3 %.The accuracy was almost 100 % with recovery experiments and examination of the marks l e f t a f t e r the assay process. No decomposition of thebaine seemed t o take place, since an almost 100 % recovery was obtained using added thebaine or thebaine alone. Fen-Fen Wu and Dobberstein7’ found t h a t the compound eluted from the gas chmmatocraphic column and recorded as a thebaine peak on the chromatogram, did not contain any thebaine a s analyzed by TLC, i f a column temperature of 27OoC was used in the gas chromatopraphy. However, when gas chromatographed a t 225OC, a single thebaine s p o t was observed with TLC. A t t h i s low temperature severe t a i l i n g o f the thebaine peak was observed, making accurate quantification difficult. Kippers e t a1 .71 stated t h a t more reproducible quantitative results were obtained when thebaine base was gas chromatographed, than with thebaine hydrochloride. Vincent e t a l . 7 0 observed t h a t a small amount of thebaine was always adsorbed during GLC ( < 1 %)(newOV-17 2 % on Gas Chrom Q columns). The columns were therefore saturated with an alkaloid standard containing 0.2-0.4 pg thebaine by injection. By carefully standardizing and checking each step of the procedure for loss of thebaine, the method was assumed t o be suitable f o r i n t r a - and inter-laboratory analyses. For most gas chromatographic analyses cholesterol acetate was u t i l i z e d as an internal standard.

For the d e t e n i n a t i o n of papaverine in the blood of r a t s and dogs, Mussini and Marzo7’ developed a method based on the extraction of the alkaloid with diethyl ether from 2 ml blood, a f t e r basification with sodium hydroxide, back extraction with hydrochloric acid and extraction with diethyl ether ( a f t e r adjustment of the pH to l o ) . To the dried residue a solution of the internal standard (penfluridol) was added and the gas chromatographic analysis carried o u t on a packed column with 3 % OV-17 on Gas Chrom Q a t 29OoC, and a 63Ni electron-capture detector. The s e n s i t i v i t y of the method was 10 ng/ml blood. De Graeve e t a1.80 described two methods for the papaverine determination in blood samples for phamacokinetic studies, one utilizing a packed column with OV-1 1 % and interfaced with a LKB 9000 S mass spectrometer equipped with a multiple ion detector f o r mass fragmentography,

References p. 144

128

and the o t h e r using a c a p i l l a r y solumn w i t h SE-30 as s t a t i o n a r y phase and flame i o n i z a t i o n detection. Owing t o i t s h i g h s p e c i f i c i t y , t h e mass fragmentographic method was g r e a t l y sup e r i o r t o c a p i l l a r y gas chromatography, which was sometimes subject t o i n t e r f e r e n c e by s o l vent i m p u r i t i e s . Under selected conditions, a p r e c i s i o n o f about 2 % was ontained by the mass fragmentography technique, the r e p r o d u c i b i l i t y o f the o v e r a l l method was 10 % and the l i m i t o f detection i n blood about 5 ng/ml. To omit unconventional instrumentation t h a t was n o t s u i t a b l e f o r r o u t i n e analysis, as proposed by De Graeve e t a1.80,

B e l l i a e t a1.81 developed a q u i t e simple method t o determine

papaverine i n blood samples, using conventional flame i o n i z a t i o n o r a nitroqen-phosphorus detector. The i n t e r n a l standard, strychnine, was added t o the sample p r i o r t o e x t r a c t i o n , which was c a r r i e d o u t w i t h toluene a f t e r b a s i f i c a t i o n , back e x t r a c t i o n w i t h a c e t i c a c i d and e x t r a c t i o n o f the l i b e r a t e d base w i t h d i e t h y l ether. The gas chromatography was done on a packed column w i t h 2 % OV-101 a t 275OC. To minimize the adsorption e f f e c t s , the column was s i l a n i z e d by in situ i n j e c t i o n and by i n j e c t i o n o f a concentrated s o l u t i o n o f papaverine and the i n t e r n a l standard p r i o r t o r o u t i n e analysis. Precision and accuracy o f the method i s shown i n Table 14.13. TABLE 14.13

PRECISION

AND ACCURACY OF PAPAVERINE

ANALYSIS

IN

BLOOD^^

Each r e s u l t i s the mean o f f o u r determinations. SD = standard deviation, RSD = r e l a t i v e SD Nanogram added

20.0 40.0 80.0 160.0 100.0

250.0 500.0 1000.0

Gazdag and Nyiredy"

Nanogram found 19.8 40.0 79.5 159.5 100.3 250.0 502.5 995.5

SD

2.5 2.7 3.9 4.0 10.5 26.9 20.6 56.9

RSD ( % ) 12.7 6.7 4.9 2.6 10.5 10.8 4.1 5.7

Detector

NPD NPD NPD NPD FID FID FID FTD

a p p l i e d gas chromatography t o determine ethylmorphine hydrochloride

(20 mg) i n t a b l e t s a l s o containing phenacetin (300 mg) and aminophenazone (300 mg). On a SE30 packed column the compounds were separated and could be determined, the ethylmorphine hydrochloride had a r e l a t i v e standard d e v i a t i o n o f i 2.7 %. I n i n v e s t i g a t i o n s on various drugs o f abuse i n human b i o l o g i c a l f l u i d s ( u r i n e and plasma) and tissues, t h e r e has a l s o been a need f o r the determination o f n a r c o t i c antagonists. Digreg o r i o and OtBrien8' made use o f gas chromatography on an OV-17 3 % column f o r such i n v e s t i gations a f t e r e x t r a c t i o n o f the compounds from a l k a l i n i z e d u r i n e w i t h chloroform and conversion i n t o the corresponding t r i m e t h y l s i l y l ethers. The recoveries o f each compound v a r i e d from 62 t o 78 % f o r naloxone. depending on concentration, t o 97 t o 100 % f o r naltrexone. Smith and S t ~ c k l i n s k i ~ ~ studied '*~ the metabolites o f apomorphine (apocodeine, isoapocodeine, norapocodeine) i n u r i n e and faeces o f rats, and worked o u t a method f o r the d e t e r mination o f apomorphine as t r i m e t h y l s i l y l e t h e r and the metabolites as f r e e bases on an OV17 3 % column. Average recovery o f apocodeine and isoapocodeine was between 85 and 90 % f o r 500-1000 ug. Because o f the l a c k o f s e l e c t i v i t y o f t h i s method, Baaske e t a1.86 developed a method

using d e r i v a t i z a t i o n o f apomorphine w i t h h e p t a f l u o r o b u t y r i c anhydride. N-n-propyl -norapanorphine was u t i l i z e d as an i n t e r n a l standard. Amounts o f 1 t o 10 pg apmorphine per m l plasma could be determined. Q u a n t i t a t i v e r e l a t i v e recoveries w i t h a r e l a t i v e standard d e v i a t i o n o f 4.6 % were achieved by the method, which a l s o permits a n a l y s i s o f apomorphine i n the presence o f i t s two monomethyl e t h e r d e r i v a t i v e s , apocodeine and isoapocodeine. The l a t t e r compounds were chromatographically resolved as t h e i r heptafluorobutyrate d e r i v a t i v e s . 14.1.1.2.4.

HEROIN

Brochmann-Hanssen and Baerheim Svendsen16 found t h a t heroin was e l u t e d as a sharp peak when gas chromatographed alone on a SE-30 column. However, i n mixtures w i t h codeine, morphine o r o t h e r a l c o h o l i c o r phenolic substances, reactions t a k i n g place i n the i n j e c t o r pave r i s e t o several new esters, n o t present i n the o r i g i n a l s o l u t i o n . Both 3-0-acetyl-

and 6-0-acetyl-

morphine gave s i n g l e peak chromatograms i n the absence o f glass wool i n the column entrance. An apparent c a t a l y t i c e f f e c t o f glass wool r e s u l t e d i n peaks corresponding t o morphine, monoacetylmorphine and heroin. Curry and PattersonE7 combined i n f r a r e d spectroscopy, t h i n - l a y e r and g a s - l i q u i d chromatography f o r the analysis o f i l l i c i t heroin samples, whereby c o n o n l y found a l k a l o i d s , such as morphine and the monoacetylmorphines were taken i n t o account. However, no problems concerning the gas chromatography o f heroin i n mixtures w i t h morphine, as mentioned by Brochmann-Hanssen and Baerheim Svendsen16 were mentioned. A packed column w i t h cyclohexane succinate 3 % and dibenzyl phthalate as an i n t e r n a l standard were used. For q u a n t i t a t i v e determination o f h e r o i n i n i l l i c i t samples i n t h e presence o f a l k a l o i d s such as morphine and monoacetylmorphine GroomsE8 s i l y l i z e d the sample p r i o r t o the !as

chro-

matography, u t i l i z i n g cinchonine as an i n t e r n a l standard. De Zan and FasanelloE9 determined heroin hydrochloride i n i l l i c i t preparations by d i r e c t l y i n j e c t i n g a sample s o l u t i o n containi n g an i n t e r n a l standard ( c h o l e s t e r o l ) w i t h o u t p r i o r e x t r a c t i o n , whereby the most comnon adu l t e r a n t i n heroin s t r e e t dosage preparations, quinine hydrochloride, could be determined on an 3 X OV-1 column. The same s t a t i o n a r y phase was employed by Moore and Benag'

f o r the analy-

s i s o f heroin i n n a r c o t i c paraphernalia. Shaler and Jerpegl

combined gas chromatography and i n f r a r e d spectroscopy f o r the i d e n t i -

f i c a t i o n o f heroin and commonly used d i l u e n t s , such as quinine and procaine i n i l l i c i t seizures. The q u a n t i t a t i v e determination o f heroin was c a r r i e d o u t gas chromatographically using cholesterol TMS as an i n t e r n a l standard. To be able t o compare heroin samples s o l d i n i l l i c i t channels through the determination o f i m p u r i t i e s present, such as 6-0-monoacetylmorphine, acetylcodeine, morphine and codeine, Sobol and Sperlingg2 developed a method f o r q u a n t i t a t i v e determination o f h e r o i n and the compounds mentioned. Two chromatograms were run o f each sample: one a f t e r d e r i v a t i z a t i o n w i t h N,O-bis(trimethylsily1 ) t r i f l u o r o a c e t a m i d e and one by d i r e c t gas Chromatography o f the sample

-

i n both cases on a packed column w i t h 3 % OV-25 on Gas Chrom Q. Typical chromatograms are

given i n Figure 14.2. The analyses were l i m i t e d t o s p e c i f i c e x h i b i t s where i n t e l l i g e n c e had i n d i c a t e d a probable connection between two o r more cases and the l a b o r a t o r y examination was c a r r i e d o u t t o prove o r disprove t h i s connection. A constant r e l a t i o n s h i p o f the r e l a t i v e concentrations o f the by-products were found i n samples from a comnon source (No. 1771-1832 i n Table 14.14) whereas the spectrum d i f f e r e d i n a sample from another source (No. 1833 i n Table 14.14).

Reference8 p. 144

130 FIGURE 14.2 GAS CHROMATOGRAMS OF HEROIN SAMPLE"

Column: OV-25 3 % on Gas Chrom Q, D e r i v a t i z e d heroin a t 24OoC. Underivatized h e r o i n a t 265OC

Der iv a t ized heroin

1= 2 = 3 = 4 = 5 =

Underivatized h e r o i n

Morphine TMS Codeine TMS 6-0-Monoacetylmorphine TMS Acetylcodeine Heroin

1 = Codeine 2 = Morphine 3 = Acetylcodeine 4 = 6-0-Monoacetylmrphine 5 = Heroin

TABLE 14.14 RATIO OF BY-PRODUCTS I N HEROIN SAMPLES" Sample No.

Morphine

1771 1772 1774

6-0-MonoacetylMorphine 1.70 1.81 1.89

Codeine AcetylCodeine 0.13 0.09 0.06 0.12

1833

0.03

2.40

0.30

Heroin hydrochloride

2.69 2.85 2.88 2.97 3.25 3.01

95.5 95.2

95.0 94.7 95.2

3.35

93.2

9.4. .. 7.

Van der Slooten and Van der Helmg3 developed a s i m i l a r method t o be able t o draw conclusions about the common o r i g i n o r c m o n trade r o u t e o f d i f f e r e n t heroin samples by determining the r a t i o heroin:6-0-monoacetylmorphine:morphine.

Other c o n s t i t u e n t s o f the samples

such as amphetamines, quinine, methadone, cocaine and strychnine were determined i n an analogous way. Because o f the great v a r i e t y o f substances which may be present, t h r e e d i f f e r e n t packed columns (SE-30 3 %, QF-1 3 % and Apiezon L 10 % + KOH 2.5 I ) were used f o r the i d e n t i -

f ica t ion. I n a study o f procedures f o r the i d e n t i f i c a t i o n o f heroin Clarkg4 a l s o used gas chromatography and separated heroin and 17 chemically c l o s e l y r e l a t e d compounds on packed columns o f O V - 1 and OV-17 a t 25OoC. The r e t e n t i o n times r e l a t i v e t o heroin are piven i n Table 14.15.

131

TABLE 14.15 RETENTION TIMES OF HEROIN AND RELATED COMPOUNDSg4 Glass columns 6 f t by 4

mm I.D. a t 25OoC ~~

3 % ov-1

Compound Morphine D i hydromorphi none D i hydromorphine Codeine D i hydrocodeine Ethylmorphine Monoacetyl morphi ne Monoacetyl d i hydromorphi ne Acetyl codeine Acetyl e t h y l morphi ne Propionyl codeine Diacetylmorphi ne (heroin) Pseudoheroin ( a c e t y l d i hydromorphinoneenol acetate Propi onyl e t h y l morphi ne Diacetyl d i hydromorphine Monoacetyl monopropionyl d i hydromorphi ne D i propionylmorphi ne Dipropionyl dihydromorphine

0.80

~~

3 % OV-17

0.65 0.80 0.79 0.77 0.83 0.94 1.00 (3.95**)

0.55 0.70 0.52 0.49 0.63 0.47 0.68 0.61 0.64 0.68 0.81 1.00 (4.50**)

1.00 0.99 0.93 1.12 1.51 1.34

1.13 0.85 0.88 1.09 1.55 1.32

- *

0.80 0.61

- *

*) Retention time could n o t be determined because o f excessive peak symmetry, **) Retention time o f diacetylmorphine (heroin) i n minutes I n order t o i d e n t i f y and determine 3-0-acetylmorphine i n i l l i c i t h e r o i n samples Moore and Kleing5 applied gas chromatography. 3-0-acetylmorphine r e s u l t s from an incomplete e s t e r i f i c a t i o n o f morphine w i t h a c e t i c anhydride and the amount may be o f value f o r f o r e n s i c purposes. Because o f the very small amounts present i n heroin, the compound was d e r i v a t i t e d w i t h heptaf l u o r o b u t y r i c anhydride and gas chromatographed w i t h a 6 3 N i e l e c t r o n capture d e t e c t o r on a 3 % OV-17 on Gas Chrom Q packed column a t 23OoC usin? chlorpromazine as an i n t e r n a l standard. The h e p t a f l u o r o b u t y r i c anhydride d e r i v a t i v e s were e x t r a c t e d q u a n t i t a t i v e l y from the r e a c t i o n mixture w i t h l i g h t petroleum and were s t a b l e f o r several hours i n t h i s solvent. However, i t was recommended t h a t upon formation, the a n a l y s i s should be completed w i t h o u t delay. The a n a l y s i s was c a r r i e d o u t w i t h 1-10 mg heroin samples and the amount o f 3-0-acetylmorphine varied from 0.1 t o 2 %, acetylcodeine from 3 t o 15 %, morphine and codeine from 0.01 t o 0.5 %. I n a review on the f o r e n s i c i d e n t i f i c a t i o n o f heroin Manura e t al.96 t e s t e d GLC and anal y z e d heroin and 56 morphine d e r i v a t i v e s on f o u r GC columns using 3 % O V - l ,

3 % OV-17,

3 %

OV-25 and 6 % Dexsil as s t a t i o n a r y phases a t column temperatures between 24OoC and 280°C. The compounds analyzed and t h e i r r e t e n t i o n times r e l a t i v e t o heroin are given i n Table 14.16. With q u a n t i t a t i v e gas chromatography Han-Yong Lim and Sui-Tse Chowg7 analyzed more than a thousand i l l i c i t samples o f heroin and found t h a t a l l o f them contained monoacetylmorphine, acetylcodeine and the d i l u e n t c a f f e i n e . The heroin content o f most samples was w i t h i n the range 30-50 %

-

i n some cases even over 70 %

-

w i t h corresponing low values f o r c a f f e i n e . The

GLC was c a r r i e d o u t on 1 m long packed columns w i t h 3 % OV-17 on Gas Chrom Q a t 235OC using codeine as i n t e r n a l standard - w i t h o u t d e r i v a t i z a t i o n . The standard d e v i a t i o n o f samples i n the range o f 10.0 t o 55.0 mg, c o n t a i n i n g about 36 % heroin, was f o r h e r o i n 0.52, and the coe f f i c i e n t o f v a r i a t i o n 1.4

%.

A r a p i d and s e n s i t i v e method t o determine morphine, codeine and 6-0-monoacetylmorphine i n

References p. 144

132

TABLE 14.16 RETENTION TIMES OF MORPHINE DERIVATIVES RELATED TO HEROIN ON FOUR GLC COLUMNSg6 1 = 3 % OV-1 on Chromosorb W, 80-100 mesh, glass column 1.2 m by 6.35 mn O.D. a t 25OoC 2 = 3 % OV-17 on Chromosorb W, 80-100 mesh, glass column 1.2 m by 6.35 rmn O.D. a t 280oC 3 = 3 % OV-25 on Gas Chrom Q, 80-100 mesh, stainless s t e e l column 1.8 m by 3.18 mn 0.0. a t 24OoC 4 = 6 % Dessil 400 on Gas Chrom Q, 80-100 mesh, s t a i n l e s s steel column 1.8 m by 3.18 m 0.0. a t 240 C Compound Column

Morphine D i hydromorphi ne 3-0-Acetylmorphi ne 3-0-Acetyl d i hydromorphine 6-0-Acetylmorphi ne 6-0-Acetyl d i hydromorphi ne Heroin Diacetyl dihydromorphine 3-0-Propi onylmorphi ne 3-0-Propionyl d i hydromorphine 6-0-Propionylmorphine 6-0-Propionyl d i hydromorphi ne Dipropionylmorphi ne O i propi onyl d i hydromorphine 3-0-Acetyl -6-0-propionylmorphi ne 3-0-Acetyl -6-0-propionyl d i hydromorphine 3-0-Propionyl -6-0-acetylmorphine 3-0-Propionyl -6-0-acetyl d i hydromorphine 3-0-Butionylmorphi ne 3-0-Butionyl d i hydromorphine 6-0-Butionylmorphine 6-0-Butionyl d i hydromorphi ne D i butionylmorphine D i butionyl d i hydromorphine 3-0-Butionyl -6-0-acetyl morphine 3-0-Buti onyl-6-0-acetyl d i hydromorphi ne 3-0-Acetyl-6-0-butionylmorphi ne 3-0-Acetyl-6-0-butionyldihydromorphine 3-0-Butionyl-6-0-propionylmorphine 3-0-Buti onyl -6-0-propi onyl d i hydromorphine 3-0-Propi onyl -6-0-butionyl morphine 3-0-Propionyl -6-0-butionyl d i hydromorphine Codeine D i hydrocodeine Acetylcodeine D i hydroacetyl codeine Propionyl codeine Propi onyl d i hydrocodei ne Buti onyl codei ne Butionyl d i hydrocodeine Ethylmorphi ne D i hydroethylmorphi ne Acetylethylmorphi ne Acetyl d i hydroethyl morphi ne Propionyl ethylmorphine Propionyl d i hydroethyl morphine Butionyl ethylmorphine Butionyl d i hydroethylmorphi ne Oxymorphone D i hydromorphi none Oxycodone D i hydrocodei none Racemorphan

0.56 0.60

0.60 0.73 0.71 0.79 0.73 1.00 0.90 0.88 0.94

OI96

0.59 0.74 0.69 0.74 0.68 1.00 0.91 0.86 0.83 ..-. 0.88 0.80 1.48 1.35 1.23 1.07 1.30 1.09 1.09 1.07 1.09 0.95 2.21 1.85 1.56 1.33 1.48 1.31 1.89 1.60 1.85 1.58 0.51 0.49 0.69 0.60 0.83 0.75 1.04 0.86

-

0.40 0.67 0.63 0.67 0.64 1.00 0.86 0.84 0.79 .. . . 0.82 0.74 1.54 1.26 1.23 1.04 1.23 1.05 1.03 0.98 1.02 0.88 2.36 2.28 1.59 1.30 1.51 1.24 1.88 1.54 1.86 1.51 0.44 0.42 0.63 0.53 0.77 0.63 0.96 0.74 0.55 0.46 0.54 0.43 0.73 0.64 0.65 0.54 0.86 0.78 0.75 0.64 1.10 0.98 0.89 0.75

0.98 1.63 1.48 1.31 1.08 1.38 1.13 1.17 1.19 1.17 1.14 2.58 2.25 1.75 1.50 1.58 1.33 2.17 1.74 2.13 1.70 0.57 0.55 0.77 0.70 1.02 0.93 1.30 1.05 0.64 0.61 0.84 0.75 1.07 0.95 1.32 1.14 0.86 1.11 0.80 ~.~~ 0.83 . . ~ . 0.66 0.65 0.61 0.39 0.28 0.22

-

0.36 0.80 0.82 0.85 0.86 1.00 0.86 1.05 0.96 .... 1.00 0.97 1.66 1.40 1.29 1.09 1.29 1.12 1.38 1.23 1.27

i.io

2.78 2.64 1.70 1.45 1.66 1.36 2.12 1.77 2.12 1.71 0.63 0.61 0.67 0.59 0.85 0.74 1.10 0.90 0.69 0.65 0.72 0.62 0.92 0.76 1.17 0.94

0.69 0.30

133

TABLE 14.16 (continued) Compound

Column 2 3

1 0.30 0.52 0.55 0.77

Racemethorphan Leva1 lorphan Apomorphi ne Theba i n e

4

0.21 0.40

0.14 0.31

0.19 0.44

0.82

0.84

0.73

-

i i l i c i t heroin was developed by !looreg8. A f t e r d e r i v a t i z a t i o n w i t h h e p t a f l u o r o b u t y r i c anhyd r i d e the f l u o r i n a t e d d e r i v a t i v e s were extracted f r o m an a c e t o n i t r i l - s o d i u m bicarbonate s o l u t i o n i n t o l i g h t petroleum i n a one-step e x t r a c t i o n procedure and gas chromatographed on a 3 % OV-17 column a t 200-240°C. heroin

Morphine, codeine and 6-0-acetylmrphine were q u a n t i t a t e d i n

samples a t l e v e l s as low as 0.001 %, 0.01 % and 0.001 % r e s p e c t i v e l y . The gas chroma-

tographic column was conditioned a t 175OC f o r 1 h by i n j e c t i n ? 5 x 5 u1 o f S i l y l - 8 (Pierce) and 5 x 5 111 o f l i g h t petroleum c o n t a i n i n g 6-0-acetylmorphine (HFB) a t a concentration o f 5 mg/ml. The temperature was then increased t o 285OC and maintained f o r 48 h. Minimum detectable q u a n t i t y was ca. 20 pg f o r morphine, 80 pg f o r codeine and 100 pg f o r 6-0-acetylmorphine. The content o f morphine and 6-0-acetylmorphine i n i l l i c i t heroin samples was determined by Machata and Vycudilikg9 by e x t r a c t i v e p r o p i o n y l a t i o n i n aqueous s o l u t i o n (disodium hydrogenphosphate b u f f e r ) w i t h e t h y l acetate as e x t r a c t i v e solvent. Only the phenolic 3-OH-group o f morphine reacts t o 3-0-propionylmorphine; 6-0-propionylmrphine cannot be prepared by t h i s method. On a 2.5 % OV-1 o r SE-52 column these compounds were well separated, as shown i n Table 14.17. The standard d e v i a t i o n and v a r i a t i o n o f the q u a n t i t a t i v e determinations using a m i t r i p t y l i n e as an i n t e r n a l standard are given i n Table 14.18. TABLE 14.17 RETENTION INDEX (KOVATS) OF HEROIN AND RELATED COMPOUNDS I N ILLICIT SAMPLES” On OV-1 and SE-52 a t 25OoC O V - 1 column

Ca f f e i ne A m i t r i p t y l i n e ( i n t e r n a l standard) Codeine Thebai ne 6-Acetyl codeine 3-Propionylmorphi ne Heroin

6-Acetyl-3-propionylmorphine Papaveri ne

1818 2240 2429 2522 2535 2609 2638 2722 2808

SE-52 column 1990 2288 2501 2629 2616 2701 2741 2828 2936

TABLE 14.18 STATISTICAL DATA FOR QUANTITATIVE DETERMINATIONS OF HEROIN AND RELATED COMPOUNDSg9 % Standard d e v i a t i o n V a r i a t i o n (95 X ) n = 10 6.9 15.6 Codeine 5.1 11.6 6-Acetyl codeine 6.0 13.6 3-Propionylmorphine 5.5 12.4 Heroin 6-Acetyl -3-propi onyl morphi ne 7.6 17.2

Rslslsmca p. 144

134

Van Vendeloo e t al.loo developed a gas chromatographic method f o r f i n p e r p r i n t analysis of i l l i c i t heroin samples, capable of d e t e c t i n g the main components: acetylcodeine, c a f f e i n e . codeine, heroin, 6-0-monoacetylmorphine, morphine and quinine i n one r u n i n 25 min. Heroin. morphine, codeine and c a f f e i n e could be q u a n t i f i e d d i r e c t l y , 6-0-monoacetylmorphine and acetylcodeine were n o t f u l l y separated. Q u a n t i t a t i o n o f the l a t t e r two r e q u i r e d a c e t y l a t i o n o f 6-0-monoacetylmorphine t o heroin. A packed column o f 1 % O V - 1 on Chromosorb G HP was used and the analysis c a r r i e d o u t a t a column temperature o f 275'C w i t h a m i t r i p t y l i n e as an i n t e r 99 nal standard, as

.

Gough and Baker"' studied a number o f conventional and m o d i f i e d s t a t i o n a r y phases i n o r der t o f i n d the best one f o r q u a n t i t a t i v e gas chromatography of heroin and s t r u c t u r a l l y r e l a t e d compounds. S i l a n i z e d OV-210 was found t o be the most s u i t a b l e f o r the separation o f heroin, codeine, acetylcodeine, morphine and 6-0-monoacetylmorphine. I t gave the best reprod u c i b i l i t y o f r e t e n t i o n times and l e s s losses o f the compounds by adsorption. For q u a n t i t a t i v e analysis 2.8 m by 4 mm 1.0. glass columns and Diatomite CLQ, 80-100 mesh, as s o l i d supp o r t were used a t a column temperature o f 225OC. Despite the f a c t t h a t some o f the compounds, p a r t i c u l a r l y morphine, s u f f e r e d adsorption losses d u r i n g gas chromatography, these losses were reproducible, and s a t i s f a c t o r y q u a n t i t a t i v e data could be obtained, as shown i n Table 14.19. TABLE 14.19 PERCENTAGE COMPOSITION

Sample 1 2 3 4 5 6 7 8 9 10 11 12 13

Caffeine Mean 3.7 4.0 18.7 19.0 27.9 14.8 15.4 15.3 19.0 4.5 4.7 18.0 17.5

OF ILLICIT

Codeine

HEROIN SAMPLES DETERMINED

Morphine

True Mean True Mean True 4.5 6.9 6.9 4.5 13.8 13.7 19.5 8.1 7.5 20.9 17.5 16.0 28.0 27.9 27.4 15.8 47.5 48.4 15.6 48.7 47.6 15.7 48.2 48.2 19.1 58.9 58.4 4.7 4.2 4.9 9.3 9.7 4.8 9.4 9.9 13.9 14.7 18.6 4.5 4.8 9.6 9.7 18.1 9.0 9.4 9.3 9.4

Acetylcod.

-

Acetylmor.

Heroin

Amount i n j .

Mean True Mean True Mean True Mean True

-

-

B Y GLC"'

-

-35.8 19.0 22.0 14.7 7.3 14.8 22.0

36.8 18.5 22.5 14.6 7.5 14.5 21.2

~~

11.0 17.1 16.8 16.5

10.2 17.3 16.6 17.7

37.7

-

78.4 65.1 56.4 47.0 44.2

35.8

-

17.5

-

9.7 15.1 14.7 14.3

-

57.4 48.3 37.7 27.4

56.4 48.0 37.7 27.6

17.5 -

9.9 16.4 15.4 14.8

78.4 64.5 56.4 45.4 44.6

2.68 2.68 2.48 2.31 1.17 3.10 3.07 3.16 2.63 2.64 2.56 2.60 2.67

2.65 2.69 2.47 2.29 1.22 3.03 3.08 3.05 2.51 2.57 2.52 2.58 2.65

Caffeine, Codeine, Morphine, Acetylcodeine, Avetylmorphine and Heroin i n %, Amount i n j e c t e d i n ug

The use o f a stable-isotope l a b e l l e d molecule i s probably the c l o s e s t approach t o an i d e a l i n t e r n a l standard, because o f the n e a r l y i d e n t i c a l physical and chemical p r o p e r t i e s between the molecules. Under most gas chromatographic systems, the compound and i t deuterated analogue w i l l co-chromatograph, w i t h the d i s t i n c t i o n t h a t the molecular i o n i n the f r a p 102 mentography p a t t e r n w i l l d i f f e r i n IMSS by s u b s t i t u t e d deuterons. However, Jerpe e t a l . noticed a small d i f f e r e n c e i n gas chromatographic r e t e n t i o n times between the i n t e r n a l standard o f deuterated heroin and heroin. The a d d i t i o n o f three protons p e r a c e t y l group and i n crease i n mass o f 6 p a r t s p e r 369 e v i d e n t l y changes t h e p a r t i t i o n c h a r a c t e r i s t i c s o f deuter-

136

ated heroin enough t o account f o r t h i s s l i g h t separation ( o f f o u r t o s i x seconds on a 3 % OV-17 column, 4 ft long). The i n t e n s i t y o f each s p e c i f i c i o n fragment i s recorded on a sepa r a t e channel o f a mu1 t i p l e recorder. S p e c i a l l y d i l u t e d s o l u t i o n concentrations a r e graphed t o determine l i n e a r i t y of response w i t h i n the magnitude o f 1 t o 100 ng o f the sample i n j e c t ed on the column. Q u a n t i t a t i o n i s achieved by comparing the r e l a t i v e peak areas. Shui-Tse Chowlo' s t a t e d t h a t the o n l y physical method o f f e r i n g the necessary i d e n t i f i c a t i o n s e l e c t i v i t y w i t h q u a n t i t a t i v e c a p a b i l i t y f o r the gas chranatographic a n a l y s i s o f heroin i n i l l i c i t samples i s selected i o n monitoring (SIM) mass spectrometry. Deuterated heroin and the i o n s m / e 369 and 327 f o r heroin and 375 and 331 o f deuterated h e r o i n were used as i n t e r nal standards and the ions m / e 369 and 327 were q u a n t i f i e d and c a l c u l a t e d as heroin. The c a l i b r a t i o n curves f o r both m / e 375/369 and m / e 331/327 were l i n e a r w i t h i n the concentrations studied. The gas chromatographic a n a l y s i s was q a r r i e d o u t on a 1.8 m by 6.35 mn O.D.

glass

column packed w i t h 3 % OV-17 on Chromosorb W HP, 100,120 mesh, a t a column temperature o f 270OC. For GC-MS 1.8 m by 6.35 mn 0.0. glass columns were used, packed w i t h 3 % OV-1 on Chromosorb W HP, 100-120 mesh, and a column temperature o f 25OoC. The r e s u l t s obtained are sumnarized i n Table 14.20. TABLE 14.20 HEROIN PERCENTAGES DETERMINED BY GLC AND S I M ~ O ~ A l l r e s u l t s were the average o f three i n j e c t i o n s

SIM

Sample m / e 375f369

2 3 4 5 Mean Standard d e v i a t i o n Coefficient o f variation, %

39.2 39.7 39.8 40.3 39.6 0.42 1.06

m / e 331/327

40.5 41.1 40.8 41.3 40.7 0.47 1.17

GLC 38.9 40.3 40.0 39.7 39.6 0.54 1.36

HPLC 39.3 39.5 38.7 38.6 38.9 0.39 1.01

I n a study on heroin metabolism E l l i o t e t a1 .Io4 separated codeine, morphine, norcodeine, normorphine, 6-O-acetylmorphine,

3-0-acetylmorphine as t r i m e t h y l s i l y l d e r i v a t i v e s and h e r o i n

on packed columns w i t h SE-30 and QF-1, and assayed t h e amounts o f morphine, 6-0-acetylmorphine and heroin i n ,blood and u r i n e samples, using methyl arachidate as an i n t e r n a l standard. Whereas detection l i m i t s f o r heroin w i t h conventional FID are r e p o r t e d as 0.05 mg/ml i n blood from i l l i c i t preparations, a n i t r o g e n s p e c i f i c d e t e c t o r can q u a n t i t a t e l e v e l s o f 100

-

ng/ml and d e t e c t i o n l i m i t s can be as low as 20 ng/ml according t o Smith and Colelo5. They determined heroin and i t s metabolite 6-0-acetylmorphine i n blood a f t e r e x t r a c t i o n and derivat i z a t i o n t o i t s t r i f l u o r o a c e t a t e . t o prevent adsorption during the gas chromatopraphic analys i s when the f r e e hydroxyl group was present. Ethylmorphine, d e r i v a t i z e d i n the same way was used as an i n t e r n a l standard. To be able t o determine heroin and i t s m e t a b o l i t e 6-O-acetylmorphine,

morphine and nor-

morphine i n human u r i n e , Yeh and McQuinnlo6 made use o f a f r a c t i o n a t e d e x t r a c t i o n : Heroin was e x t r a c t e d w i t h chloroform a t pH 4.5, 6-0-acetylmorphine and morphine w i t h ethylene d i c h l o r i d e containing 30 % isopropanol a t pH 8.5 and normorphine a t pH 10.4 w i t h the same s o l vent. The e x t r a c t was d e r i v a t i z e d w i t h t r i m e t h y l s i l y l i m i d a z o l e and chromatographed a t 23OoC

Referencesp. 144

136

for the determination of 6-0-acetylmorphine and morphine. and a t 220°C f o r normorphine and morphine. The overall recoveries f o r 6-0-acetylmorphine and morphine in concentrations of 2-15 pg/5ml of urine were 68-70 % and 60-62 % respectively. Heroin was determined by temperature programming from 200 t o 25OoC using cholestane a s an internal standard. This substance was also used for the determination of morphine and normorphine, and f o r 6-0-acetylmorphine and heroin, whereas tetraphenylethylene was used as an internal standard f o r the determination of morphine and 6-0-acetylmorphine. The metabolites of heroin in urine following intravenous administration of a single 10 mg dose were studied by Yeh e t a l . 4 2 using TLC and column chromatography w i t h the isolation of the various compounds and GLC or GC-MS of the s i l y l , trifluoroacetyl, acetyl or propionyl derivatives f o r the identification. Free morphine, 6-O-acetylmorphine, f r e e normorphine, morphine 3-glucuronide, morphine 6-glucuronide, 6-0-acetylmorphine glucuronide and normorphine glucuronide were found as metabolites, morphine 3-glucuronide beina the major metabolite ( a b o u t 50 % of the administered dose of heroin). A 3 f t glass column with 3 % OV-17 on Gas Chrom 0 and a 5 f t stainless steel column with 3 % SE-30 on Varaport were used by the gas chromatographic analysis. The retention times of some of the heroin metabolites are given in Table 14.21. TABLE 14.21 RETENTION TIMES OF SOME HEROIN METABOLITES4’ Tri f l uroracetyl derivative 3 % OV-17 21ooc 6-0-Acetyl morphi ne Codeine Morphine Norcodeine Normorphi ne

6.9 min 5.8 3.8 9.1 6.2 -

-

Trimethyl si 1yl derivative 7 % nv-17 22ooc 23OoC

7 . 1 min 7.4 min 8.8

4.3

-

-

14.1.2. Capillary columns 14.1.2.1. Morphine Capillary gas chromatography of morphine and codeine was described by Christophersen and R a s m u ~ s e nin~ ~connection ~ with t h e i r studies on flash heater derivatization o f drugs f o r gas chromatographic analysis. Derivatization was found necessary f o r relative polar compounds such as morphine and codeine because of the often undesirable adsorption observed with glass capillary columns. Trimethylsilylation was performed with N,O-bis-(trimethylsilyl )acetamide and ethylmorphine was used as an internal standard f o r the quantitative determinations on a 20 m by 0.35 mn 1.0. glass capillary, wall coated w i t h SE-30. Calibration graphs for concentrations of 1-10 pg/ml in ethylacetate were constructed. The data obtained from the reproducibility t e s t showed t h a t a t 5.0 vg/ml the relative standard deviation was 1.5 % for codeine and 4.3 % f o r morphine. Huhtikangas e t a1.1°8 used also glass capillary columns f o r quantitative determination of morphine in plasma levels. A relative simple method was developed based on extractive alkylation of morphine with pentafluorobenzyl bromide. A glass capillary, 18 m by 0.35 mn I.D. coated with OV-225, and nalorphine as internal standard were used. The detection limit with FID

137

was 2-5 ng, w i t h ECO about 5 pg. Edlundlo9 described a method f o r simultaneous determination o f morphine, 6-0-acetylmorphine and codeine i n human plasma o r blood. The samples were b u f f e r e d t o pH 9 and e x t r a c t e d on s i l i c a columns, cleaned by e x t r a c t i o n and f i n a l l y acylated w i t h p e n t a f l u o r o p r o p i o n i c anhydride. The d e r i v a t i v e s formed were separated on a glass c a p i l l a r y column (25 m b y 0.36 mm

I.O.,

coated w i t h OV-1) a t 22OOC. The f a l l i n g needle i n j e c t i o n and e l e c t r o n capture d e t e c t i o n

were used. Although degradation o f the solutes was observed, the degradation observed was very reproducible, so t h a t q u a n t i t a t i v e analyses c o u l d be c a r r i e d o u t i n s p i t e o f the degradation. The author recomnended r e g u l a r c o n t r o l and c a l i b r a t i o n i n o r d e r t o o b t a i n r e l i a b l e resul t s . Because o f adsorption and degradation problems when using glass c a p i l l a r y columns f o r the analysis o f morphine and codeine, Plotczykl"

p r e f e r r e d fused s i l i c a columns t o t h e a n a l y s i s

o f underivatized drugs, i.a. codeine. He used c o l d on-column i n j e c t i o n and c r o s s - l i n k e d polysiloxane deactivated columns and obtained l i n e a r q u a n t i t a t i o n from 1 t o 100 ng w i t h p r e c i sions o f 0.1-2 % f o r some o f the drugs analyzed. 14.1.2.2.

Heroin

A number o f heroin seizures i n Wunsterland (G.F.R.) glass c a p i l l a r y gas chromatography. With a 12

ton X 350 and temperature p r o g r a m i n g (200-250°C) 6-O-acetylmorphine,

were analyzed by Bohn e t a1.l"

with

m long column by 0.3 mn 1.0. coated w i t h T r i a good separation was obtained f o r heroin,

morphine, acetyl codeine, c a f f e i n e and the i n t e r n a l standard, dotriacon-

tan. The heroin samples contained heroin (19.1-58.2

%), 6-0-acetylmorphine (1.0-7.3

%),

acetylcodeine (2.4-3.8 %), c a f f e i n e (26.5-64.3 % ) and strychnine (0-1.26 % ) . Glass c a p i l l a r y gas chromatography was used by Christophersen and R a s m ~ s s e na l~s ~o ~f o r heroin

.

Whereas morphine, codeine, mono-acetylmorphine and ethylmorphine were d e r i v a t i z e d

w i t h N,O-bis(trimethylsily1)acetamide

w i t h f l a s h heater d e r i v a t i z a t i o n , h e r o i n was n o t d e r i -

vatized, b u t i t was w e l l separated from the o t h e r compounds mentioned, as w e l l as c a f f e i n e and strychnine, which were found i n the heroin samples analyzed. I n a paper on p r o f i l i n g heroin samples by c a p i l l a r y chromatography f o r f o r e n s i c a p p l i c a t i o n Neumann and Gloger'" SE-54, and FIO o r

used glass c a p i l l a r y columns (25 m by 0.2 mn 1.0.) coated w i t h

NFIO. The t r a c e i m p u r i t i e s o f the heroin samples were e x t r a c t e d w i t h t o l u -

ene and d e r i v a t i z e d w i t h N-~4ethyl-N-trimethylsilyltrifluoroacetamide o r N,O-bis(trimethy1sily1)acetamide. Remarkable d i f f e r e n c e s were observed f o r samples from various o r i g i n , i.a. Turkey, Malaysia and Lebanon. Compounds such as a c e t y l thebaol

, diacetylnorcodeine,

4-0-dcetyl-

3,6-dimethoxy-5- (2-N-methyl acetami do) - e t h y l phenantrene and meconin were detected i n v a r y i n g amounts i n the d i f f e r e n t heroin samples. The " f i n g e r p r i n t i n g ' c a p i l l a r y gas chromatography o f i l l i c i t heroin samples seems t o have d i s t i n c t advantage over most o f the methods used so f a r f o r the f o r e n s i c comparison o f such samples. The i n t r o d u c t i o n o f fused s i l i c a c a p i l l a r y columns opened new ways i n the f o r e n s i c analys i s o f heroin samples. Demedts e t al.l13madeuse o f a CP-Si1 5 fused s i l i c a column p e m n e n t l y deactivated w i t h a polysiloxane a t h i g h temperature and a NP-detector and c a r r i e d o u t determinations of h e r o i n i n the nanogram range w i t h o u t d e r i v a t i z a t i o n . Down t o 250 pg h e r o i n was detectable. Standard d e v i a t i o n and c o e f f i c i e n t s o f v a r i a t i o n were comparable t o p r e v i ously used packed columns. Acetylcodeine and 6-monoacetylmorphine, which could n o t be separated on the CP-Si1 5 column, were separated on a CP-Si1 8 column. The columns were 25 m by

References p. 144

1S8 0.32 mn o r 0.22 mn I.D.

Temperature programming was used: 2OO0C f o r 2 min, then 5'C/min

to

26OoC ( h e l d f o r 1 min), then 10°C/min (held f o r 1 min). Helium was used as c a r r i e r gas and diacetylnalorphine as i n t e r n a l standard. The r e p r o d u c i b i l i t y data o f the method a r e shown i n Table 14.22. TABLE 14.22 REPRODUCIBILITY DATE FOR DETERMINATION OF HEROIN113 Within-run data and day-to-day p r e c i s i o n and % heroin found Simulated heroin samples t 40 % t 70 % Sample N r . 1 2 3 4 5

Within-run

Day- to-day

6 7

8 9 10 Average Standard d e v i a t i o n Variation %

I l l i c i t heroin sample

34.17 34.30 33.82

67.27 65.00 63.71

47.04 46.17 47.70 46.42 46.20

32.64 32.94 34.31 35.46 34.45

64.81 63.93 64.02 63.17 64.12

45.92 46.66 47.85 47.06 47.12

34.15 0.86 2.52 %

64.52 1.18 1.84 %

46.81 0.65 1.39 %

TABLE 14.23 EXPERIMENTAL CONDITIONS USE0 FOR GAS CHROMATOGRAPHY OF OPIUM ALKALOIDS Stat.phase

%

Temperature

Comp.Prep.

GA ABS t PEG SE-30 t NPE s . s . , 6 f t x 2 mn I.D. C# 100-120 SE-30 glass, 6 f t x 3 mm CG 100-120 SE-52 acyl glass, 6 f t x 4 mn I . D . cw 80-100 SE-30 glass, 6 f t x 4 mn I . D . GP 100-140 SE-30 6ftx8mn GP 100-140 SE-30 s . s . . 5 ft x 0.093 i n CW AH 60-80 SE-30 glass, 6 f t x 4 mn I . D . Ana ABS 100-120 SE-30 glass, 3 f t x 3 mm O.D. GP S 100-120 HI-EFF 8B s . s . , 1.8 m x 3 mn CG AlJS 30-100 SE-30 s . s . , 1.5 in x 3 mn OV-1 Aer 30 80-100 Aer 30 80-100 OV-1 s . s . , 1.0 m x 3 nun t Igepal 2m CG Ah' 80-100 SE-30 l m CG AW 80-100 OV-17 glass, 1.5-2 m x 2-4 mm CW. CG, Cel. SE-30 I.D. Oiat. SUO. OV-17 glass, 3 f t x 6 mn r - 01-1953 -- ----. - . GS S 80-100 SE-30 glass, 6 f t x 7 mn I.D. glass, 6 f t x 3 mm I.D. GS ABS 80-100 SE-30 glass, 6 f t x 3 mm 1.0. GP ABS PEG 100- SE-30 140

2

207OC

a l k . s.

1

235-270°C 250°C

a l k . s. tox. mo.qnt.ng.fb.

2 3

204OC 150-250°C p r 185OC 210oC 27OoC 2000C 2500C 22OoC 25OoC 210-28OoC p r 200-260°C p r

a1k.s. alk.qnt.O a1k.s. alk.s.tox. alk.s.tox. alk.s.tox.

4 5 6 7

Column

S o l i d support mesh

glass, 6 f t x 3 mn I.D.

.

2-3 1 1 5 1 1 3 3 0.2

205OC

2-3 3

1.15

8 9

alk.0.s.her. mam

10

a1k.s. R I

11

a1k.s. 21oOc 215OC 215OC 175OC 225OC

Ref.

RI

m0.co.der.s. a1 k.ocd. a1 k.ocd. a1k.s.

12

13 14 15 16

139 TABLE 14.23 (continued) Col umn

S o l i d support

Stat.phase

%

glass, 1 m x 3 mn 1.0. glass, 6 f t x 2 mn 1.0.

CJ! AWS 100-120 Sup. 80-100

OV-17 Ov-22

2.9 205'C 3 215OC

glass S, 5 f t x 2 mm I.D.GQ 100-120

OV-17

3

glass, 3 f t glass, 3 f t glass, 6 f t

Var. 100-120 GQ 60-80 GQ 60-80

SE-30 OV- 17

3

OV-17

3

glass 2 m x 2 nun I.D.

Sup. 80-100

SE-30

glass, 4 f t x 4 mn I . D . glass. 1.5 m x 2.3 nun 1.0. glass, 6 f t x 2 nun 1.0.

215OC 205OC 215OC

2050c 2150c

SE-30

5

100-120 HP AWS 80-100 100-120 80-100 HP 80-100 AW 80-100

ov-1 OV-101 SE-30

Dia.S 80-100 SE-30 CG HP AWS 80-100 HI-EFF 8B

CW AWS 80-100 Var.30 80-100

GQ 80-100 glass, 6 f t x 2 m G@ 100-120 glass, 6 f t x 2 m glass, 1.5 m x 4 mm 1.0. GQ

glass, 2 m x 2 mm 1.0.

SUP. 100-120

glass, 1.8 m x 4 mm I . D .

Shimalite AWS 80-100

2oooc

,ltl

Dr 250-2800c

OV-17 3 24OoC 3.8 23OoC

tJJ

alk.fb.ant.0.

36

mo.co.VS qnt. 37 0. 38

mo.TMS qnt.

39

1.5 3 3 3

195% 21Oo-22OoC 23OoC 25OoC

her.met.TFA, 42 TMS qnt. mo.TPIS qnt.ur. 43

ov1 -. -

23OoC

s . s . , 5 f t x 3 ~~XII O.D. 6 ft glass S, 3 m glass, 0.5 m x 6 mn 0.0.

GQ 100-120 CW HP 80-100

ov-1

3 3

215OC 235OC 2150C 210°C

References p. 144

34 35

mo.TKS qnt.O.

3

OV-17

GQ

SE-30

Sul. AFS 80-100 GQ 100-120 CW HP 80-100 GQ 100-120

OV-17 OV-17 UCW-98 OV-17

3 3 3.8 3

220°c 24OoC 240% 215OC

S

OV-17

2

245OC

OV-1

3

240'C

CW HP 100-120

qnt. 33

210°c

OV-17

Diat.C

30 31 32

25OoC

GQ 100-120

glass S, 1.8 m x 2 mn I . 0. glass S , 213 cm x 4 mm I.D. glass, 4 f t x 2 m I.D.

24 26 28 29

1

glass, 2 m x 2 mn I . D .

I.D.

LJ

3

0 * 3 5 21ooc 0.35 1 228OC

nun

13

. . n

glass, 6 f t x 2 mm 1.0.

glass, 3 f t x 4 glass

,,

LL

3.5 200°C 24OoC alk.fb.qnt.0. 0.75 150-235oC alk.fb.qnt.0.

SE-30 OV- 17 UClJ-98 OV-225 2t3 t OV-25 Dex.300

mo.co.T!IS

21

POP.

OV- 17 SE-30 QF-1 XE-60 GQ 60-80 OV- 17 Var. 100-120 SE-30 CW 80-100 Oex. CG AWS 100-120 JXR t CDMS GQ 100-120 OV-17

glass, 3 ft x 2 mm S.S., 5 f t x 2 mm s.s.. 1.5 m x 2 mn 1.0. glass, 0.9 m x 4 mn I . D .

mo.co.met.ox.

Aer. 100-120

Poly-A 103 OV-25 SE-30

m

.

SE-30 SE-30

glass, 1.8 m x 2 m I.D. GQ glass, 1.8 m x 2 mn I . D . CW glass, 6 f t x 6 nun O.D. GQ GW glass, 6 f t x 2 mn I.D. GW glass, 6 f t x 2 nun I.D. glass S, 6 f t x 4 mn 1.D.GP

1.0.

Ref

Dia.S 60-80 GP ABS PEG. 60-80

SE-30

0.65 m x 3

Como.Prep.

mo.MS ant.ur. 17 mo. co nl' p. PFP 18 ecd. mo. PFP qnt. b i . 13 ecd.

2OOoC 23OoC 250'C 3 210°C 220°C mo.co.TFA,HFA ocd. qnt. 3 2oooc mo.co.etmo.HFA q n t . ocd. 3 23OoC mo.co.qnt.ur. 3 2400C 3 205OC alk.ab.ur. 3 24OoC mo.co.id.O.HS 3 25OoC id.0.N-tlS 5 24OoC co.met.qnt.ser. ur. 3.8 225OC mo.co.TMS qnt. mo.TMS qnt.O. 4 183OC

glass, 1.3 m x 3 mn 1.0. Sup. 80-100

glass 5, 6 f t x 6 mm glass, 4 f t x 4 mn I . D .

Temperature

mo.THS qnt.ur. 44 deu.mo HFB. C O . TFA. ant.MF 46 deu.mo. qnt. u r . MF mo. TMS qnt.bi.47 mo.qnt. der.bi.48 mo. PFA. ~ 1 . 49 deu .mo. TFA q n t 5o br. mo.TFA qnt.ur. 5 1 mo.co. TMS qnt.52 pmt. mo.co.PFP qnt. 53 pl.bi. 265OC mo.PFB,TFA qnt.54 01. MF mo.der.qnt.bi. 55

.

,

~ ~

140 TABLE 14.23 (continued) Column

Stat.phase

X

Temperature

Comp.Prep.

OV-17

3

24OoC

mo. der. qnt. b l .56

ov-101 SE-30

3 4

2O5-24O0C p r mo.der.qnt.bl.57 18OoC npa. qnt.O. 58

OV-17

1

275OC

Dia.S 80-100

Li.W.-98

CW AWS

S o l i d support

CW HP 100-120 glass, 1.2 m x 3 mn I.D. 90 cm x 2 mn I.0.Sup. 100-120 glass, 4 f t x 4 mn 1.0. GP ABS 60-80 PEG glass, 1.8 m x 2 mn GQ S 80-100

Ref

pa.qnt.pl.

59

3.8 140-275OC p r

co.qnt.prep.

60

OV- 17

3

24OoC

co.qnt.prep.

61

CW AW

SE-30

10

195-260°C p r

co.qnt.prep.

62

CW AWS 80-100 Dia. 80-100 Sup. 100-120

UCC-W 382 L i .W.-98 OV-17

10 24OoC 3.8 145-255OC p r 3 250°C

63 64 65

Ana.A 90-100

XE-60

2

23OoC

co.qnt.prep. co.qnt.prep. co.norco.qnt. bi. co.qnt.pl.

GQ 100-120

OV-17

3

100-240°C p r mo.co.qnt.bi.

68

GQ 100-120 CP 100-120

OV-17 SE-30

3 10

26OoC 29OoC

th.qnt.P.b. th.qnt.p.b.

69 70

CG AWS 100-120 GQ 100-120

OV-17 OV-17

2 270-290°C p r 3.8 26OoC

a1k.qnt.p.b. th.qnt. P.b.

71 72

OV-17 OV-17 OV-17

2 3 2.5 2

27OoC 27OoC 27OoC 27OoC

th.qnt.m. th.qnt. P.b. th.qnt. ~ . b . th.qnt. p.b.

73 74 75 76

OV-17 OV-17 OV-17 OV-17 SE-30

3 2 3 3

26OoC

77 th.qnt. P.b. th.qnt. ~ . b . 78 pa.qnt.bl. 79

ov-1

1.0. glass, 0.91 m x 6.4 mm 0. D. glass, 1.0 m x 6.4 mn 0.0. s . s . , 6 f t x 3.1 mn 1.0. s.s., 6 f t x 3 mm glass, 0.9 m x 6.4 mn glass, 1.0 m x 3 m 1.0. glass S . 1.8 m x 2.5 n

I. 0. glass, 3 ft x 2 mn

I.D.

s.s,

7 ft x 3 n glass, 1.5 m x 4 mn 1.0. glass, 6 f t x 3 mn 1.0. glass, 4 f t x 3.5 mn 1.u. glass. 5 f t x 4 mn 1.0. glass glass, 6 f t x 3 mm 0.0. glass S , 1.5 m x 4 mn

...

CW GQ GQ GQ

AWS 80-100 100-120 100-120 100-120

GQ GQ GQ GQ

100-120 100-120 100-120 AWS 100-120

OV-17

I.D.

glass, glass, glass, glass, glass

1.5 m x 4

n

1 in x 3 . n I.D. 2 m x 3 mn 1.0. cap. 30 m x 0.25 mn 1.0. glass, 2 m x 3 mn 1.0. glass, 1.2 m x 3 IIIII 1.0. glass, 1.2 m x 2 mn

29OoC 27OoC 225OC

GP 100-120 GQ 80-100

OV-17

1 3

225% 265OC

CW HP 100-120

OV-101

2

275OC

CW 60-80

SE-30

2

23OoC

CW HP 80-100 glass, 6 ft x 3 mn 1.0. GQ 100-120

OV-17 OV-17

3

3

27OoC 26OoC

glass, 6 f t x 3 mn GQ 100-120 1.0. glass S. 76 un x 4 mn CW HP 100-120 1.0. glass, 5 f t x 4 mn I.D. CW AW 80-100 S.S.. 6 ft x 3 mn O.D. CW AUS 60-80 6 ft x 2 mn I.D. CW HP 80-100 glass, 6 f t x 4 mn CW HP 80-100 5.5, 6 f t x 3 mn WHP 100-120 glass, 6 f t x 3 mn GQ 100-120

ov-17

3

22OoC 26OoC

OV-17

3

190°c

CHDS SE-30

3 5 3 2 3 3

250°C 200% 235OC 275OC 2600C 240°C

3 3

235OC 235OC

67

pa.qnt.bl. MF

8o

pa.qnt.bl.

81

I.D. glass, 2 m x 32. n 1.0.

glass, 1.2 m x 3 mn I.O.CW glass, 2.1 m x 3 mn 1.D.GQ glass, 0.9 m x 3 mn I.D.CW

HP 80-100 AWS 80-100

OV-1 OV-1 OV-17 OV-25 SE-30 QF- 1 Apiezon t KOH

15.:

15OoC

et.mo.qnt. 82 prep. a n t .up. 83 a p w . met. qnt 84 ser. apano.TMS qnt.85 ur. apomo.qnt.pl. 86

.

her.cg.qnt. her.qn t , her. qn t her.qnt. her.gnt.id.IR mo. co acco. mam.TMS qnt.

.

.

her.qnt

.

87 88 89 90 91 92 93

141

TABLE 14.23 (continued) Column glass, 6 ft x 4 mn I.D.

S o l i d support

glass, 6 f t x 4 mn I . D . glass, 3 f t x 2 mn I.D. glass, 1.2 m x 6.35 mn - O.D. S.S.. 1.8 m x 3.18 mn 0.0. qlass, 1 m x 6.35 mn O.D. glass, 1.8 m x 4 mn I.D. glass, 2 m x 2 mn I.D.

GQ 100-120 GQ 80-100 CW 80-100

-

GQ 80-100

Stat.phase ov-1 OV-17 OV-17 OV- 17 ov-1 OV-17 OV-25 Dex.400 DV-17

X 3 3 3 3 3 3 3 6 3

Temperature Comp.Prep. Ref 25D°C her.cg. mam. 94 250°C HFB qnt. 23OoC mam.HFB.qnt. 95 180% D r her. ECD. MS 2500C ' 28OoC 56 mo.der. s. 96 24OoC 24OoC 235OC her.cg.qnt. 97

GQ 100-120

DV-17

3

200-220°c 220-240°C

CG AUS 80-100

SE-52

GQ 80-100

-

nv-1 -.

:::

glass, 1.8 m x 2 nun I.D. CG HP 80-100 OV-1 glass, 2.8 m x 4 mn I . D . Diat.CLQ 80-100 OV-210 S

1 3

24D-2800c pr 235OC 225OC

glass, 4 f t x 6 mn glass, 1.8 m x 6.35 mm O.D. s . s . , 5.3 f t x 2 mn I.D. s . s . , 9 f t x 2 mn I.D. glass, 213 cm x 4 mn I.D. qlass, 3 f t x 2 mn

OV-1 OV-1 OV-17 SE-30 QF-1 OV-17

1 3 3 3 3

23OoC 25OoC 27OoC 218OC 218OC 250°C

OV-17

3

CW HP 80-100 CW HP 100-120 CW HP 100-120 CW HP 100-120 Diat.C AW 100120 GQ 60-80

2

20O-25O0C p r 23OoC 22ooc

glass cap. 20 m x 0.35 nnn 1.13.

SE-30

5O-25O0C p r

glass cap. 18 m x 0.35 mn I.D. glass cap. 25 m x 0.36 mn I.D.

OV-225 ov-1

50-230°C 22D°C

f . s i l cap. 25 m x 0.32 mn I.D. glass cap. 12 m x 0.3 mn I.D.

SE-54 Tr X305

8D-28OoC p r 200-250% p r

glass cap. 25 in x 0.2 mn I.D. f . s i l cap. 25 in x 0.32 mn I.D.

SE-54 CP-Si1 5

150-3OOoC o r 200-3OO0C p r

.

co. mo.mam HFB qnt. mo.mam.prop. der. qnt.her. f p r i n t . her. co.mo.acco. mam.her.qnt. her.ant.HF her .qnt. S IH her.met FIS qnt.ur. her.mam.TFA. ant.bl. ND her. mo.mam.Tt1S mo. nonno. TMS qnt.ur. mo.co.TMS qnt. ocd. mo.PFB q n t . p l . mo co mam PFP. qnt.bi. co. mam.acco.ca. s t r . her. qnt. orof.her. her.tox.qnt.

. . .

ND

TABLE 14.24 OPIUM ALKALOIDS

-

LIST OF ABBREVIATIONS

ABW = acid, base washed ab = abuse acco = acetylcodeine alk = alkaloid Ana = Anakrom angt = n a r c o t i c antagonist apmo = apmorphine AW = a c i d washed b i = biological material b l = blood b r = brain CA = Chromosorb A ca = c a f f e i n e cap = c a p i l l a r y Cel = C e l i t e CG = Chromosorb G cg = congener co = codeine CHDS = cyclohexane dimethanol succinate

CG = Chromosorb G CW = Chromosorb W der = derivative deu = deuterium Dex = Dexsil Dia S = D i a t o p o r t S D i a t = Diatomite ECD = e l e c t r o n capture d e t e c t o r etmo = ethylmorphine f b = f r e e base f p r i n t = finger p r i n t f . s i l = fused s i l i c a ft = f e e t GA = Gas Chrm A GP = Gas Chrom P GQ = Gas Chrom Q GS = Gas Chrom S her = heroin HFA = heptafluoroacetyl

90 9g 100

,,,, I"'

102 103 104 105

106 107 108 110 111 112 113

142

TABLE 14.24 (continued) HFB = heptafluorobutyryl HP = high performance I.D. = inside diameter Igepal = nonyl phenoxy polyoxy ethylene ethanol in inch IR = infra red Li = Linde mam = monoacetylmorphine met = metabolite MF = mass fragmentography MS = mass spectroscopy ND = nitrogen detector NGS = neopentyl glycol succinate nlp = nalorphine norco = norcodeine normo = nonorphine nos = noscapine npa = non phenolic alkaloids NPE = nonyl phenoxyethylene oxyethanol 0 = opium ocd = on-column derivatization OD = outside diameter ox = oxydation product

pa = papaverine P.b. = Papaver bracteaturn PEG = polyethylene glycol PFB = pentafluorobenzyl PFP = pentafluoropropionyl pl = plasma pin = plant material pmt = post mortem tissue POP = POPPY pr = (temperature) proFraminn prof = profilino prop = propionyl qnt = quantitative RI = retention index S = silanized s = separation ser = serum SIM = selective ion monitorinp s.s = stainless steel str = strychnine Sup = Supelcoport TFA = trifluoroacetyl th = thebaine TMS = trimethylsilyl tox = toxicology TrX305 = Triton X 305 ur = urine Var = Varaport

TABLE 14.25 DERIVATIZATION OF OPIUM ALKALOIDS SILYLATION

1. With hexamethyldisilazane Morphine (10-25 mg) is dissolved in 2 ml of dry pyridine followed by 1 ml of hexamethyldisilazane. The vial is stopped with a polyethylene stopper and set aside at room temperature for 16-18 hours. The solution is gas chromatographed directly32

.

2.

With hexamethyldisilazane t trimethylchlorosilane

Morphine (1 mg) is dissolved in a solution of 0.75 ml of pyridine, 0.25 nl of trimethylchlorosilane and 0.25 ml of hexamethyldisilazane. A 0.25 ul sample is injected for qas chromatography31. 3. With N,O-bis( trimethylsilyl )acetamide 50 ~1 of a stock solution of morphine, codeine and/or nalorphine (0.1 mp/ml) is evaporated in a vial, the residue taken up in 100 p1 of a 40 % solution of N,O-bis(trimethylsily1) acetamide in pyridine and 1 ul is injected into the pas chromatonraph52.

Morphine and/or monoacetylmorphine is solved in 1.0 ml of pyridine and 0.5 ml of N,O-bis(trimethylsily1)acetamide in a vial. The vial is capped and the sample and reaaents are mixed by tapping about 15 second. The sample is allowed to stand 5 minutes before injection of 2 u1 88

.

143 TABLE 14.25 (continued) One m l o f N,O-bis(trimethylsily1)acetamide

i s added t o 0.07-3.4

mg o f aoomorphine i n a

Microflex tube f i t t e d w i t h T e f l o n - l i n e d cap. A f t e r shaking g e n t l y f o r 20 minutes 1 p1 i s used f o r gas ~ h r o m a t o g r a p h y ~ ~ . 4.

With N,O-bi s ( t r i m e t h y l s i l y l ) t r i fluoroacetamide t 10 % t r i m e t h y l c h l o r o s i l a n e Add t o morphine, codeine, notmorphine. norcodeine, morphine-N-oxyde and/or morphine-N-

methyl iodide, placed i n an a c y l a t i o n tube 0.05 m l a c e t o n i t r i l e and 0.05 m l o f N,O-bis(trim e t h y l s i l y l ) t r i f l u o r o a c e t a m i d e c o n t a i n i n g 10 % t r i m e t h y l c h l o r o s i l a n e . Cap the tube and shake i n a Vortex mixer f o r about 10 seconds. Heat a t 60-70°C f o r 30 minutes and i n j e c t 1 p l i n t o 21

the gas chromatograph 5.

.

With t rime thy1s i1y l i m i dazo 1e Morphine and/or n o n o r p h i n e (0.5-1 mg) i s mixed i n a sealed a c y l a t i o n tube w i t h 50 p1 o f

25 % t r i m e t h y l s i l y l i m i d a z o l e i n p y r i d i n e and heated i n an o i l bath a t 90-95OC f o r 1 hour. One 111 i s i n j e c t e d f o r gas chromatographic a n a l y s i s 106

.

ON COLUMN SILYLATION

1. With t r i m e t h y l si l y l imidazole One 111 of a morphine s o l u t i o n i n e t h y l a c e t a t e i s drawn i n t o a Hamilton s y r i n o e 750 N followed by 2

pl

o f trimethylsilylimidazole

-

and i n j e c t e d i n t o t h e ?as chromatograph 39

.

ACYLATI ON

1.

With a c e t i c anhydride Morphine (normorphine) (0.5-1 mg) i s dissolved i n 0.2 m l o f a c e t i c anhydride and 0.1 m l

o f p y r i d i n e i n a sealed a c y l a t i o n tube and heated a t 60-70°C f o r

4

hour. The excess of a c e t i c

anhydride i s removed by evaporation and the residue dissolved i n 50 p1 o f e t h y l acetate. One 106 . 111 o f the s o l u t i o n is i n j e c t e d i n t o the gas chromatograph 2.

With t r i f l u o r o a c e t i c anhydride Morphine (25 ng-5 mg) i s solved i n 0.1 m l e t h y l acetate and 0.1 m l t r i f l u o r o a c e t i c an-

hydride i n a tube. The tube i s t i g h t l y capped and shaken i n a Vortex mixer. Then i t i s placed i n a water bath o f 5OoC f o r 20 minutes, a f t e r which the content i s evaporated t o dryness under a gentle stream o f f i l t e r e d a i r a t 5OoC. The residue i s solved i n 0.1 m l e t h y l acetate and 1-5 111 used f o r gas chromatographic analysis 5 1. 3.

With pentafluoropropionic anhydride Morphine ( 1 ng-1 pg) i s solved i n 50

pl

d r y e t h y l acetate and 100 111 pentafluoropropion-

i c anhydride i s added. The t i g h t l y stoppered tube i s placed i n an oven a t 6OoC f o r 30 minutes. Excess o f reagent i s removed by a g e n t l e stream o f d r y n i t r o g e n and the residue d i s s o l v e d i n 50 p1 d r y e t h y l acetate. O f t h i s s o l u t i o n 1-2 t ~ 1i s i n j e c t e d f o r pas chromatopraphic analy19 sis .

A s o l u t i o n c o n t a i n i n g 1.0 ng t o 1.0 pg f r e e base o f e i t h e r morphine o r codeine i s evaporated t o dryness i n a 15 m l conical t e s t tube which has p r e v i o u s l y been t r e a t e d w i t h a 5 % ( v / v ) s o l u t i o n o f D r i - F i l m SC-87 i n toluene (Pierce Chemical Co.).

References p. 144

To t h e tube 100 111 o f

144

TABLE 14.25 (continued) glass d i s t i l l e d benzene and 100 u1 o f pentafluoropropionic anhydride i s added. The tube i s capped and then allowed t o r e a c t f o r 25 minutes a t

7OoC. A f t e r r e a c t i o n the s o l v e n t w i t h

the reagent i s evaporated t o dryness a t room temperature under a stream o f d r y nitropen. The sample i s taken up i n e t h y l acetate and 1 u l , containing 5-1000 pq per 111 i s i n j e c t e d i n t o the gas chromatograph

18

.

ON COLUMN ACYLATION 1. With a c e t i c anhydride The i n j e c t i o n o f the sample o f morphine (0.5 % s o l u t i o n i n methanol, e t h y l acetate o r tetrahydrofuran) (6 ~ 1 i )s followed w i t h i n 5 second o f t h a t o f 5

pl

o f a c e t i c anhydride

14

.

2. With propionic anhydride The same technique as described above but using 5 u l o f p r o p i o n i c 3. With trifluoroacetylimidazoleor heptafluorobutyrylimidazole One p 1 o f a s o l u t i o n o f morphine o r codeine (0.1-2.5

my/ml) i n e t h y l acetate i s drawn i n -

t o a Hamiltion 701-N syringe followed by 2 u l o f the a c y l a t i n g reaaent. The m i x t u r e i s i n j e c t e d i n t o the gas chromatograph

22

.

14.2 REFERENCES 1 E. Brochmann-Hanssen and T. Furya, J . Pham. S c i . , 53 (1965) 1549. 2 H.V. Street, J . Chromatogr., 37 (1968) 162. 3 H.V. Street, W. V y c u d i l i k and G. Machata, J . Chromatogr., 168 (1979) 117. 4 H.A. Lloyd, H.M. Fales, P.F. Hiphet. J.W.A. VandenHeuvel and W.C. Wildman, J . ~ m .Chem. SOC., 82 (1960) 3791. 5 N.B. Eddy, H.M. Fales, E. Haahti, P.F. Highet, E.C. Horning. E.L. May and 1I.C. blildman, U . N . Document ST/SOA/SER.K/114, 1961. 6 S. Yamaguchi, I . Seki. 5. Okuda and K. Tsuda, Chem.Pharm. B u l l , 10 (1962) 775. 7 K.D. Parker, C.R. Fontan and P.L. Kirk, m a ] . Chem., 35 (1963) 356. 8 L. Kazyak and E. Knoblock, Anal. Chem., 35 (1963) 1448. 9 N.C. J a i n and P.L. K i r k , Microchem. J . , 12 (1967) 229. 10 A. Viala. F. Gonezo. J. C a t a l i n and J.-P. Cano, J . E u r . T o x i c o l . , 1971, 375. 11 A.C. Moffat, A.H. Stead and K.E. Smalldon, J . Chromatogr., 90 (1974) 19. 12 A.C. k f f a t , J . Chromatogr., 113 (1975) 69. 13 P. LiraS, J . Chromatogr., 106 (1975) 238. 14 M.W. Anders and G.J. flannering, A n a l . Chem., 34 (1962) 730. 15 S.J. Mule, Anal. Chem., 36 (1964) 1907. 16 E. Brochmann-Hanssen and A. Baerheim Svendsen, J . Pharm. S c i . , 5 1 (1962) 1095. 17 F. Fish and W.D.C. Wilson, J . Chromatogr., 40 (1969) 164. 18 M.J. Kogan and M.A. Chedekel. J . Pham. Phamacol., 28 (1976) 261. 19 B. Dahlstram and L. Paalzow, J . Pharm. Pharmaml., 27 (1975) 172. 20 H. Kaneshina. Y. Kinoshita, 14. Mori, T. Yamagishi, S . Honma and H. tlitubashi, Shoyakugaku Zasshi, 28 (1974) 127; C.A. , 83 (1975) 152416 S . 21 S.Y. Yeh, J . Pharm. S c i . , 62 (1973) 1827. 22 G. Brugaard and K.E. Rasmussen, J . Chromatogr., 147 (1978) 476. 23 A.S. Christophersen and K.E. Rasmussen, J . Chromatogr., 168 (1979) 216. 24 N.C. Jain, Th.C. Sneath, R.D. Budd and W.J. Leung, C l i n . Chem. (Winston-Salem, N.C.), 21 (1975) 1486. 25 H.E. Sine, N.P. Kubasik and T.A. Rejent, C l i n . Biochem., 7 (1974) 102. 26 S . J . Mule, J. Chromatogr., 55 (1971) 255. 27 K. Watanabe. Y. Makino. H. Yanai, H. Hirobe and T. Okamoto, Yakugaky Zasshi, 93 (1973) 695; C . A . , 79 (1973) 49592 q. 28 J.M. Weber and T.A. Ma, Microchirn. A C t d , 1975, 401. 29 R.H. Smith, J. Forensci Sci., 13 (1973) 327.

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E. Schmerzler, W. Yu, M.I. Hewitt and I . J . Greenblatt, J . Pharm. sci., 55 (1966) 155. G.E. Martin and J.S. Swinehart, Anal. Chem., 38 (1966) 1789. E. Brochmann-Hanssen and A. Baerheim Svendsen, J. P h a m . sci., 52 (1963) 1134. M. Paris, J.-P. Gramnd and R.-R. Paris, Ann. P h a m . F r . , 32 (1974) 97. E. Nieminen, Farm. d i k a k . , 80 (1971) 342. A. Bechtel, C h r o m a t o y r a p h i d , 5 (1972) 404. D. Furmanec, J. C h r o m a t o y r . . 89 (1974) 76. G.R. Nakamura and T.T. Noguchi, J. F o r e n s i c sci., 14 (1974) 347. J o i n t Comnittee P h a n . SOC. and Chem. SOC., A n a l y s t , 103 (1978) 268. K.E. Rasmussen, J . C h r o m a t o y r . , 120 (1976) 491. N. Ikekawa, K. Takayama, E. Hosoya and T. Oka, A n a l . B i o c h e m . , 28 (1969) 156. J.T. Payte, J.E. Wallace and K. Blum, C u r r . T h e r . R e s . , 13 (1971) 412. S . Y . Yeh, R . L . McQuinn and C.W. Gorodetzky, J. Pharm. sci., 66 (1977) 201. R. Truhaut, A. Esmailzadeh, J . Lebbe, J.-P. Lafare and Nyyen Phu Lich, Ann. B i o l . C l i n . ,

32 (1974) 429. 44 D.E. Fry, P.D. # i l l s and R.G. TWyCrOSs, Clin. C h i m . d c t a , 5 1 (1974) 183. 45 W.0.R. Ebbighausen, J . Mowat, P. Vesteyaard and N.S. Kline, Adv. B i o c h e m . P s y c h o p h a m a c o l . , 7 (1973) 135. 46 P.A. Clarke and R . L . Foltz, C l i n . Chem. (Winston-Salem), N.C.). 4 (1974) 465. 47 G.R. Wilkinson and E.L. Way, B i o c h e m . P h a r m a c o l . , 18 (1969) 1435. 48 J.E. Wallace, J.D. Biggs and K. Blum, C l i n . C h i m . d c t d , 36 (1972) 85. 49 E.R. Garrett and T. Giirkan, J. P h a r m . S c i . . 67 (1978) 1512. 50 P.P. Hipps, M.R. Eveland, E.R. llayer, W.R. Sherman and T.J. Cicero, J. P h a r m a c o l . E x p . T h e r . , 196 (1976) 642. 51 J.E. Wallace, H.E. Hamilton, K. Blum and C. Petty. A n a l . Chem., 46 (1974) 2107. 52 G.R. Nakamura and E.L. Way, A n a l . C h e m . , 47 (1975) 775. 53 B. Dahlstrcm, L. Paalzow and P.O. Edlund, d c t a P h a r m a c o l . T o x i c o l . , 4 1 (1977) 273. 54 W.J. Cole, J . Parkhous and Y.Y. Yousef, J . C h r o m a t o q r . , 136 (1977) 409. 55 L. von Meyer, G. Kauert and G. Drasch, B e i t r . G e r i c h t l . M e d . , 39 (1981) 113. 56 G. Cimbura and E. Koves, J . A n a l . T o x i c o l . , 5 (1981) 296. 57 J . J . Saady, N . Narasimhachari and R.V. Blanke, J. A n a l . T o x i c o l . , 6 (1982) 235. 58 A. Baerheim Svendsen and E. Brochmann-Hanssen, U.N. D o c u m e n t s S T / S O A / S E R . K / I Q ~ , 1965. 59 D . E . Guttman, H.B. Kostenbauer, G.R. Nilkinson and P.H. Dub6, J. Pharm. S c i . , 63 (1974) 1625. 60 H.J. Wesselman, J . Pharm. S c i . , 57 (1968) 1412. 61 M. Stevens, J . P h a r m . S c i . , 64 (1971) 1686. 62 E . B . Oechene, L.H. Booth and M.J. Cauphey, J . P h a r m . P h a r m a c o l . , 21 (1969) 678. 63 E . Nieminen, ~ u l l .N a r c . , 23 (1971) 23. 64 H.J. Wesselman and W.L. Koch, J . P h a r m . S c i . , 57 (1968) 845. 65 W.J. Serfontein, D. Botha and L. L k V i l l i e r s , J . Pharm. P h a r m a c o l . , 27 (1975) 937. 66 M.K. Brunson and J.F. Nash, C l i n . Chem. ( W i n s t o n - S a l e m , N . c . ) , 2 1 (1975) 1956. 67 H.A. Zweidinger, F.11. bieinberq and R.W. Handy, J . Pharm. S c i . , 65 (1976) 427. 58 J.R. Shipe and J . Savory, dnn. C l i n . L a b . S c i . , 5 (1975) 57. 69 Pin; Chen and N.J. Dorenbos, u . N . D o c u m e n t s S T / S O d / S E R . J / 5 , 1973. 70 P.G.Vincent and W.A. Gentner, U . N . D o c u m e n t s S T / S O d / S E R . J / 9 , 1974. 71 F.J.E.M. Kippers, R . J.J.C. Lousberp, C.A.L. 8ercht and C.A. Salemink, U . N . Documents s T / SOA/SER. J/8, 1974. 72 M.J. Duffy, P.A. Ackert and C.F. Hiskey, U.N. D o c u m e n t s S T / S O d / S E R . J / l S , 1975. 73 J.W. Fairbairn and K. Helliwell, J. Pharm. P h a r m a c o l . , 27 (1975) 217. 74 C.B. Coffman, C . E . Bare and W.A. Gentner, B u l l . N a r c . , 27 (1975) 41. 75 Fen-Fen Wu and R . H . Oobberstein, J . C h r o m a t o q r . , 140 (1977) 65. 76 P.G. Vincent, C.E. Bare and W.A. Gentner, J . P h a r m . sci., 66 (1977) 1716. 77 G . Siniscalco Gigliano, B o l l . C h i m . Farm., 117 (1978) 678. 78 P.G. Vincent, W.A. Gentner, F.J.E.M. Kippers and C.A. Salemink, J . P h a r m . sci., 68 (1979) 87. 79 E. Mussine and A. Ilarzo, E X P . B i o l . , 10 (1972) 331. 80 J . De Graeve, J.van Cantfort and J . Gielen, J. C h r o m a t o q r . , 133 (1977) 153. 8 1 V. Bellia, J . Jacob and H.T. Smith, J . C h r o m a t o q r . , 161 (1978) 231. 82 M. Gazdag and Sz. Nyiredy, P h a r m a z i e , 32 (1977) 724. 83 G.J. Digregorio and C. O'Brien, J . C h r o m a t o y r . , 101 (1974) 424. 84 R . V . Smith and A.W. Stocklinski, J . C h r o m a t o y r . , 77 (1973) 419. 85 R . V . Smith and A.W. Stocklinski, A n a l . C h e m . , 47 (1975) 1321. 86 D.M. Baaske, J.E. Keiser and R . V . Smith, j . C h r o m a t o q r . , 140 (1977) 57. 87 A.S. Curry and A . Patterson. J . P h a r m . P h a r m a c o l . , 22 (1970) 198. 88 J . Grooms, J. d s s o c . Off. A n a l . C h e m . , 51 (1968) 1010. 89 P. DeZan and J . Fasanello, J . C h r o m a t o g r . S c i . , 10 (1972) 333.

146

90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108

J.M. Moore and F.E. Bena, mal. Chem., 44 (1972) 385. R.C. Shaler and J.H. Jerpe, J . Forensic sci.. 16 (1972) 668. S.P. Sobol and A.R. Sperling, Forensic S c i . Symp.(G. Davies, E d i t . ) , 1974, 170. E.P.J. van der Slooten and H.J. van der Helm. Forensic S c i . , 6 (1975) 83. C.C. Clark, J . Forensic S c i . , 22 (1977) 418. J.M. Moore and M. Klein. J . Chromatogr., 154 (1978) 76. J.J. Manura, Jew-Ming Chao and R. Saferstein, j . Forensic s c i . . 23 (1978) 44. Han-Yong Lim and Sui-Tse Chow, J . Forensic S c i . , 23 (1978) 319. J.M. Moore, J . Chromatogr., 147 (1978) 327. G. Machata and W. Vycudilik, J . rsnai. T o x i c o l . , 4 (1980) 318. F. van Vendeloo, J.P. Franke and R.A. de Zeeuw, Phann. Weekbl. S c i . E d . , 2 (1980) 129. T.A. Gough and P.B. Baker, J . Chromatogr. S c i . , 19 (1981) 227. J.H. Jerpe, F.E. Bena and W. Morris, J . Forensic S c i . , 19 (1975) 557. Shui-Tse Chow, J. Forensic S c i . , 27 (1982) 32. H.W. E l l i o t , K.D. Parker, J.A. Wright and N. Nomof, C l i n . Phannacol. Ther., 12 (1971) 803. D.A. Smith and J.W. Cole, J. Chromatogr., 105 (1975) 377. S.Y. Yeh and R.L. McQuinn, J. Pharm. S c i . , 64 (1975) 1237. A . S . ChristoDhersen and K.E. Rasmussen. J . Chromatoor.. 454. . , - 174 (1979) > , A. Huhtikangas, K. Wickstrom and T. Vartiainen, Prog.Clin.Pharm.111 (H. Turakka and E . v . d. K l e i j n , E d i t . ) 1981, 89. 109 P.O. Edlund, J . Chromatogr., 206 (1981) 117. 110 L.L. Plotcryk, J. Chromatogr., 240 (1982) 349. 111 G. Bohn, E. Schulte and W. Audick, A r c h . Kriminol.. 160 (1977)’ 27. 112 H. Neumann and H. GloSer, Chromatographid, 16 (1982) 261: 113 P. Oemedts, t1.Van den Heede, J. Van der Verren and A. Heyndrickx, J . A n a l . T o x i c o l . , 6 (1982) 30.

147

Chapter 15 APORPHINE ALKALOIDS 15.1 Aporphine a l k a l o i d s ........................................................ 15.2 References .................................................................

147 150

15.1 APORPHINE ALKALOIDS Arndt e t a1 . l gas chromatographed 27 aporphine d e r i v a t i v e s , n a t u r a l l y occurrinv a l k a l o i d s and s y n t h e t i c d e r i v a t i v e s , in order t o study the c o r r e l a t i o n between molecular weight and/ or other s t r u c t u r a l f e a t u r e s and retention times. A packed 0.3 % SE-30 on g l a s s micro beads column and two column temperatures, 235OC and 25OoC, were used f o r the i n v e s t i n a t i o n s . A number of the phenolic alkaloids could not be e l u t e d from the column and they were converted t o their ethyl e s t e r s . Even a f t e r e s t e r i f i c a t i o n some of them, boldine, corytuberine and hernandine f a i l e d t o nive an observable response. Other aporphine d e r i v a t i v e s behaved in a simil a r way. The r e s u l t s in Table 15.1 show t h a t t h e r e i s no l i n e a r r e l a t i o n s h i p between the r e l a t i v e retention times and molecular weight o f the compound investigated.

TABLE 15.1 RELATIVE RETENTION TIMES OF APORPHINE DERIVATIVES VS. 2-CHLOROAPORPHINE' 2-Chloroaporphine has a retention time of 3.7 and 1.9 m i n a t 235OC and 25OoC, respectively; glass column 2 m by 5 mm I . O . , 0.3 % SE-30 on micro c l a s s beads.

2

Substituents Rinq 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

3-CF3 3-F 3-CH3 1-CH3 1-6 =H 1-C1 3-C1 2-c1 3-CF -5,6-di-OCH3 3-OCd 2 - 0 ~ ~ ~ 5,6-d?-OCH3 3,4-di-OCH 3-OCH -4-0?2H5 5,6-d?-OCH3 2-OC2H5

References p. 1 5 0

Nitrogen Me Me Me Me Me Me Me Me Me Me Me Me Me Me H

M e

R RT 235OC 25OoC 0.49 0.52 0.73 0.78 0.80 0.89 0.99 1.00 1.02 1.20 1.21 1.26 1.29 1.30 1.35 1.38

Molecular weight 303 253 249 249 235 269.5 269.5 269.5 363 265 265 295 295 309 28 1 279

148

TABLE 15.1 (continued)

17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Substi tuents Ring 5,6-di-OCH 3.4-di-OC 3,4.6-tri ?O?H3-5-OC2H5 3.4.6-tri-OCH3 3-OCH -4-OH 3,4-b&ZO 3-OCH -4-OC2H5-5 ,6-CH202 3,4-d?-OCH -5.6-CH202 2,3,5,6-te&a-OCH3 5,6-di-OCH 2-OC2H5-3,$,6-tri-OCH3 _ _

4

Nitrogen Ct

Me Me Me Me He

RRT 235OC 25OoC 1.44 1.52 2.04 2.36 2.44

2.50

Me Me Me Me Me

2.54 2.78 4.37 4.52 30

Molecular weight 309 323 369 325 ~~

281 285 353 339 355 323 397

Cashaw et a1.2 carried out a comparative study on the gas chromatographic properties of the TMS derivatives of some naturally occurring aporphines and tetrahydroprotoberberines (THPB) and the TWS derivatives o f their demethylated products on OV-1 and OV-17. A better separation of the silyl derivatives o f the aporphine and the THPB alkaloids was achieved on the OV-1 column. A direct correlation of the number o f TMS groups or molecular weight with the elution pattern of the aporphines was not apparent. Boldine, with two TRS Croups, had a greater retention time than silylated 1,2,9,10- and 1,2,10,11-tetrahydroxyaporphine on both the OV-1 and OV-17 columns. Corydine, containing one nS group, was eluted before the two tetrahydroxyaporphines on OV-1, but its retention time was greater than that of the tetrahydroxyisomers, 1,2,9,10- and 1,2,10.11-tetrahydroxyaporphine, on OV-17. The two tetrahydroxyisomers had similar retention times on OV-17, but the 1,2,9,10-isomer was eluted first on ov-1. In contrast to the derivatized aporphines, the elution pattern of the silylated tetrahydroprotoberberines had a direct correlation with the number of silyl groups (or molecular weight) on OV-1 and an inverse correlation on OV-17. Three tetrahydroprotoberberines, viz. scoulerine, i socorypalmine, and stylopine, which yield 2,3,9,10- tetrahydroxyberbine on demethylation are eluted in increasing order of their molecular weiaht on OV-1 and in decreasing order o f their molecular weight on OV-17, as is seen in Table 15.2. Similarly, coreximine is eluted before its demethylated product, 2,3,10,11-tetrahydroberbine, on OV-1. This pattern is reversed on OV-17. The two isomeric tetrahydroxyberbines, 2,3,9,10- and 2,3,10,11-tetrahydroxyberbine, are separated on OV-1 but have similar retention times on OV-17. The two naturally occurring isomeric alkaloids, coreximine and scoulerine, which are derived from reticuline in plants, are separated on OV-17 but have similar retention times on OV-1 at 26OoC. The separation of four derivatized aporphines and four tetrahydroberbines on OV-1 is shown in Figure 15.1. Although both classes of alkaloids have similar molecular weights (Table 15.2) the aporphines are all eluted before the tetrahydroberberines on a non-polar phase (OV-1), indicating that differences in the retention times o f these alkaloids may be due to the geometry of the molecules. A highly specific and sensitive method for the determination of berberine in urine was developed by 14iyazaki et a1 . 3 using chemical ionization fragmentography and ( 2H3)berberine chloride as an internal standard. The berberine was reduced with sodium borohydride in methanol to give tetrahydroberberine, which was submitted to GC-HS analysis. Berberine concen-

149

TABLE 15.2 RETENTION TIMES OF SILYLATED DERIVATIVES OF APORPHINE AND TETRAHYDROPROTOBERBERINE

ALUALOIDS~ Glass column: 6 ft by 4 m, 3 % OV-1 o r OV-17 on Gas Chrom Q 100-120 mesh at 26D°C Compound

Mol .wt. Number TMS groups

Aporphines Corydine (l-hydroxy-2,10,11-trimethoxyaporphine Boldine (l,lO-dimethoxy-2,9-dihydroxyaporphine 1,2,9,1O-Tetrahydroxyaporphine l,Z,lO,ll-Tetra hydroxyaporphi ne Tetrahydroprotoberberi nes Stylopine (2,3,9,10-bis (methylenedioxy) -berbi ne Isocorypalmine (Z-hydroxy-3,9,10-trimethoxyberbine Scoulerine (2,9-dihydroxy-3,10-dimethoxyberbine) Coreximine (2,ll-di hydroxy-3,lO-dimethoxyberbi ne ) 2,3,9,10-Tetrahydroxyberbine 2,3,10,1l-Tetrahydroxyberbi ne

OV-1 t,(min)

OV-17 t,(min)

413

1

4.9

7.2

471 587 587

2

4 4

8.6 7.9 6.6

10.8 6.8 6.8

323

0

9.2

25.0

413

1

11.2

22.9

471

2

13.6

19.1

471 587 587

2

13.8 17.8 16.6

20.5 18.0 18.2

4 4

FIGURE 15.1

CHROMATOGRAPHIC SEPARATION OF SILYLATEO APORPHINES A N D TETRAHYOROPROTOBERBERINES~ Glass column: 6 ft by 4 m, 3 % OV-1 on Gas Chrom Q 100-120 mesh at 260OC 1 = Corydine, 2 = 1 , Z , 10,ll-Tetrahydroxyaporphine, 3 = 1,2,9,lO-Tetrahydroxyapornhine, 4 = Boldine, 5 = Isocorypalmine, 6 = Coreximine, 7 = 2,3,10,ll-Tetrahydroxyberbine, 8 = 2.3,9,10-Tetrahydroxyberbi ne

1

5 1

7

I 2

0 2

L 6 8 1 0 1 2 1L 16 1 8 2 0 2 2 2 L

min

References p. 160

150 t r a t i o n s down t o 1 ng/ml u r i n e c o u l d be determined. Samples o f 200 m l u r i n e were used f o r t h e a n a l y s i s . A f t e r a d d i t i o n o f t h e i n t e r n a l s t a n d a r d (100 p q ) t h e m i x t u r e was l y o p h i l i z e d and t h e r e s i d u e e x t r a c t e d w i t h e t h a n o l . The s o l u t i o n was chromatographed o v e r XAD-2, and t h e e l u a t e , o b t a i n e d w i t h 1 M h y d r o c h l o r i c acid-methanol, evaporated t o dryness

-

and t h e r e d u c -

t i o n c a r r i e d out. The f r e e base was l i b e r a t e d w i t h potassium h y d r o x i d e and e x t r a c t e d w i t h c h l o r o f o r m , t h e c h l o r o f o r m s o l u t i o n c o n c e n t r a t e d and analyzed. Recovery o f b e r b e r i n e added t o u r i n e was 95.7 t 0.5 %. Because N-n-propylnorapomorphine i s r e p o r t e d t o be s e v e r a l times more p o t e n t t h a n apomorphine i n several b i o l o g i c a l systems, Green e t a1.4 developed a GC-HS method f o r t h e d e t e c t i o n , i d e n t i f i c a t i o n and q u a n t i f i c a t i o n o f aporphines. The 0 - t r i f l u o r o a c e t y l d e r i v a t i v e s o f a s e r i e s o f N-methyl- and N-propylaporphines as w e l l as n o r a p o r p h i n e s were p r e p a r e d and examined. The r e t e n t i o n d a t a a r e g i v e n i n Table 15.3. TABLE 15.3 RETENTION DATA OF APORPHINE DERIVATIVES' The values a r e i n methylene u n i t s . Column 183 cm b y 2 mm, 1 X OV-17, Compound Apomorphine Apocodeine Isoapocodeine Bulbocapnine Z , l O , l l - T r i hydroxy-N-methylaporphine 2 , l l - D i hydroxy- 10-methoxy-N-methyl aporphi ne 2,lO-Di methoxy- 11-hydroxy-N-methyl a porph ine

N-n-Propylnorapomorphine N-n-Propyl norapocodei ne 10-Hydroxy-N-n-propyl noraporphine 11-Hydroxy-N-n-propylnoraporphi ne

temp.program.

f r o m 200°C

Trimethyl s i l y l derivatives

Trifluoroacetyl d e r i va ti ves

27.56 28.50 27.79 31.60 29.12 30.24 30.88 28.67 29.52 29.08 27.07

23.78 26.62 25.56 31.00 24.33

26-83 29.33 25.04 27.67 25.80 24.33

TABLE 15.4 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY OF APORPHINE ALKALOIDS Column glass, 2 m x 5 nnn 1.0. glass, 6 f t x 4 mm

S o l i d support mesh

Stat.phase

m i c r o Class beads SE-30 GQ 100-120 ov-1 OV-17 glass, 1 m x 2 nnn I . D . GQ Dex. 300 g l a s s , 163 cm x 2 mm I . D . S u p e l c o p o r t 100 OV-17

%

Temperature

0 . 3 235OC 25OoC 3 26OoC 3 26OoC 1 23OoC 1 f r o m 200°C p r

Comp. Prep.

Ref.

a1k.s.

1

a1k.der.s.

2

ber.qnt.ur. a1k.der.s.

3 4

A b b r e v i a t i o n s : Dex.300 = D e x s i l 300, p r . = ( t e m p e r a t u r e ) proorammino, a l k . = a l k a l o i d , s. = s e p a r a t i o n , d e r . = d e r i v a t i v e , b e r . = b e r b e r i n e , q n t . = q u a n t i t a t i v e , u r = u r i n e

15.2 REFERENCES 1 R.R. A r n d t , W.H. Baarschers, B. Oouolas, E.C. Shoop and J.A. Ueisbach, Chem. I n d . , 1963, 1163. 2 J.L. Cashaw, K.D. McMurtey, L.R. Meyerson and V.E. Davis, A n a l . B i o c h e m . , 74 (1976) 343. 3 H. Miyazaki, E. S h i r a i , M. I s h i b a s h i and K. N i i z i m a , J. C h r o m a t o g r . , 152 (1978) 79. 4 J.F. Green, G.N. Jham, J.L. Neumeyer and P. Vouras, J. Pharm. Sci., 69 (1980) 936.

161

Chapter 16 ISDQUINDLINE RELATED ALKALOIDS

16.1 A m a r y l l i d a c e a e a l k a l o i d s ................................................... 16.2 Erythrina a l k a l o i d s ........................................................ 16.3 References .................................................................

151 153 154

16.1 AMARYLLIDACEAE ALKALOIDS M i l l i n g t o n e t a1 .l demonstrated t h e v a l u e o f u s i n g ?as chrornatoaraphy-mass s p e c t r o m e t r y f o r t h e a n a l y s i s o f a crude m i x t u r e o f A m a r y l l i d a c e a e a l k a l o i d s i s o l a t e d f r o m Crinum g i a u c m . Because o f t h e low v o l a t i l i t y o f most A m a r y l l i d a c e a e a l k a l o i d s t h e y a r e u n s u i t a b l e f o r d i r e c t a n a l y s i s b y gas chromatography-mass s p e c t r o m e t r y . They were t h e r e f o r e c o n v e r t e d t o t h e i r t r i m e t h y l s i l y l d e r i v a t i v e s , h a v i n g t h e h i g h v o l a t i l i t y and s t a b i l i t y r e q u i r e d f o r ?as chrumatography and e x h i b i t i n g mass s p e c t r a which p r o v i d e u s e f u l s t r u c t u r a l i n f o r m a t i o n . The GC-MS d a t a o b t a i n e d w i t h t h e TMS d e r i v a t i v e s o f some A m a r y l l i d a c e a e a l k a l o i d s a r e l i s t e d i n T a b l e

16.1. The s t r u c t u r e o f some h y d r o x y l i c a l k a l o i d s and t h e i r RIS e s t e r s a r e shown i n F i c u r e 16.1 TABLE 16.1 GC-MS DATA OF THE TMS DERIVATIVES OF SOME AMARYLLIDACEAE ALKALOIDS'

Compound

'R

irpl\

TMS-Ambell i n e ( 0 )

1.00

T M S - C r i nami ne

0.83

T M S - C r i we1 1 ine

0.87

Bis-TMS-Deacetyl bowdensine

1.28

Bis-TMS-Lycorine (C)

0.82

Bis-TMS-Criglaucine ( F )

1.31

Bis-TMS-Criglaucidine ( 0 ' )

1.05

U n i d e n t i f i e d (A)

0.64

U n i d e n t i f i e d (B)

0.73

U n i d e n t i f i e d (E)

1.20

References p. 154

P r i n c i p a l m / e values ( r e l . i n t e n s i t i e s )

403(100), 254(19), 373(82), 211(78), 403(26), 185(16), 463(49), 232(15), 431(9), 227(100), 463(59), 217(40), 461(16), 181(24), 343(54), 226(14), 373(100), 256(30), 403(100), 254(20),

388(17), 241(69), 342(15), 181(100), 373(39), 181(56), 347(25), 231(35), 340(8), 226(62), 254(20), 204(13), 319(10), 103(12), 288(28), 201(18),

372(21), 231(37), 388(17), 242(19),

373(19), 212(23), 252(28), 132(43), 243(29), 162(18), 305(14), 228(20),

315(8), 75(9), 232(49), 156(33), 301(27), 75(13), 287(16), 198(28), 358( 19), 218(18), 373(26), 241(65),

372(65), 211(64), 240(21), 73(40), 212(19). 73(100), 292(16), 204(19), 250(9), 73(47), 231(17), 147(20), 211(19), 73(100), 254(29), 171(16), 284(18), 204(18), 372(60), 211(64),

282(56), 73(65), 224(30), 211(72), 70(34), 244(12), 73(100), 228(17), 218(23), 73(100), i99(9), 70(13), 227(24), 73(ioo), 282(21), 73(58), 282(56), 73(91),

162

FIGURE 16.1 ALKALOIDS FROM cRr"IM

GLAUCUM'

I = Ambelline, I1 = c r i w e l l i n e . 111 = crinamine, I V = l y c o r i n e , V = deacetylbowdensine; R =

H. T r i m e t h y l s i l y l derivatives, R

= TMS.

OCH,

Ia R:H I b R=TMS

OR

0%

ma R=H

0

IUb R = T M S

Ro-Q N I

OCH,

Pa

R=H

P b R=TMS

FIGURE 16.2

GAS CHROMATOGRAM OF CRINUM

GLUCI/IY

ALKALOIDS AFTER TRIMETHYLSILYLATION'

Column: 3 % O V - 1 on Gas Chrom Q, temperature programming 215-250°C;

0

2

4

6

8

10

12

1L

16

18

min

f o r names see Table 16.1.

163

16.2 ERYTHRINA ALKALOIDS To be able t o c a r r y o u t a comprehensive i n v e s t i g a t i o n of the a l k a l o i d composition o f a l a r g e and diverse c o l l e c t i o n o f E r y t h r i n a species f l i l l i n n t o n e t a1.‘

a p p l i e d gas chromato-

graphy-mass spectrometry as a t o o l f o r e f f e c t i n g complete c h a r a c t e r i z a t i o n o f the a1 k a l o i d s i n p l a n t species o r genus. o f i d e n t i f y i n g new a l k a l o i d s and a s s i y i n g s t r u c t u r e s t o them, and o f employing the a l k a l o i d a l composition e s t a b l i s h e d f o r i n d i v i d u a l species f o r a chemotaxonomic guide w i t h i n the p l a n t genus. Because most o f the a l k a l o i d s encountered contained one o r more hydroxyl groups, i t was necessary t o improve t h e i r v o l a t i l i t y f o r the GC-11s analysis. This was achieved by conversion t o t h e i r t r i m e t h y l s i l y l d e r i v a t i v e s . The pas chromatography was c a r r i e d o u t w i t h a packed column o f 3 % OV-17 on Gas Chrom Q. I n o r d e r t o i d e n t i f y the gas chromatographically separated compounds the pas chromatoyaph was i n t e r f a c e d w i t h a low-resolution mass spectrometer. The a l k a l o i d s studied are shown i n Figure 16.3. FIGURE 16.3

2

ERYTHRINA ALKALOIDS

CH30

3 1

4 a-erythroidine

5 6 7 8 9

2

(erysopine) R2.= H, R 3 = CH3 (erysonine) R 1 = R3 = H, R2 = CH3 (erysoline) R2 = R 3 . z H, R, = CH3 (erysodine) R1 = H, R2 = R 3 = CH3 (erysovine) R2 = H, R1 = R3 = CH3 (erythravine) R3 H, R1 = R2 = CH3 (erysotrine) R1 = R 2 = R 3 = CH3 (erythraline) R 1 + R 2 = CH2, R 3 = CH3

R1 =

10

11 ( e r y s o t i n e ) R1 = H, R2 = CH3, X = H,OH 12 (erysosalvine) R2 = H, R1 = CH3, X 0,OH 13 ( e r y t h r a t i d i n e ) H,OH R1 = R2= CH3, X 14 ( e r y t h r a t i n e ) R 1 t R2 = CHz, X = H,OH 15 (dihydroerysodine) R1 = H, R2.= CH3, X = H2 16 ( e r y s o f l o r i n o n e ) R 1 = R2 = H, X = 0 17 (erysotinone) R1 = H, R2 = CH3, X 0 18 (erysosal vinone) R2 = H. R1 = CH3, X 0 19

(erythratinone) R1 t R2 = CH2,

0-e r y t h r o id ine

X = 0

TABLE 16.2 RELATIVE RETENTION TIMES OF ERYTHRINA ALKALOIDS (FREE BASES AND Tf1S-DERIVATIVES)2 Column: glass, 6 f t by 2 m I . D . ,

3 % OV-17 on Gas Chrom Q, temperature proaramning 225-25OoC

A1 k a l o i d p-Erythroidine Erysotrine Erythraline Erysol i n e Erysonine Erysopine Erythravine

References p. 154

R e l a t i v e r e t e n t i o n time Free base P I -S - _d_e r i v-a t i v e 1.39 1.17 1.21

0.43

0.95 0.89 0.95 1.07

154

TABLE 16.2 (continued) A1 k a l o i d

Re1a ti ve r et e n t ion ti me Free base TNS-derivative

Erysodine Erysovine D i hydroerysodine Erysoti ne Erysosalvine Erythratidine Erysoflorinone Erysotinone Erysosal vinone

1.00 1.07 0.93 1.18 1.25 1.34 1.58 1.66 1.47

TABLE 16.3 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY OF ISOQUINOLINE RELATED ALKALOIDS Column

S o l i d support mesh

glass, 6 f t x 2 mn I.D. GQ 100-120 glass, 6 f t x 2 mn I.D. GQ

Stat.phase

ov-1 OV-17

%

Temperature

3 3

215-250°C pr. 225-25OoC pr.

Comp.Prep.

Ref.

a1k.der.s. a1k.der.s.

1 2

16.3 REFERENCES 1 D.S.

M i l l i n g t o n , D.E.

1972, 277. 2 D.S. M i l l i n g t o n . D.H.

Games and A.H.

Jackson, Proc.Int.Symp.Gaschromatogr.Masspectrom.,

Steinman and K.L. Rinehart, Jr., J. Am. Chem.

SOC.,

96 (1974) 1909.

166

11.5 INDOLE ALKALOIDS

Chapter 17

TERPENOID INDOLE ALKALOIDS AND SIMPLE INDOLE ALKALOIDS

....................................... .................................................. ........................................................ ......................................... ........................................................... ........................................................ ................................................... .................................................... .............................................. ............................................. .................................................. .................................................................

17.1. Tryptamine and B-carboline a l k a l o i d s 17.2. Heteroyohimbine a l k a l o i d s 17.3. R a u w o l f i a a1 kal oids 17.3.1. Reserpine and rescinnamine 17.3.2. Ajmal i n e 17.4. Strychnos a l k a l o i d s 17.5. Various i n d o l e a1 kaloids 17.5.1. Vinca a1 kaloids 17.5.2. Physostigma a l k a l o i d s 17.5.3. dspidosperma a l k a l o i d s 17.5.4. uncaria a1 kaloids 17.6. References

155 158 160 160 161 162 165 165 166 166 168 172

17.1. TRYPTAMINE AND 8-CARBOLINE ALKALOIDS I n a cas c h r m a t o y a p h i c study on b i o l o F i c a l l y important amines, Fales and Pisano

1

inves-

t i g a t e d some i n d o l e bases, such as tryptamine and serotonine. When the i n d o l e bases were qas chromatographed on a 0.75 % SE-30 column on Gas Chrom P, extensive t a i l i n a was observed, probably due t o adsorptive i n t e r a c t i o n s between the comDound and the support. However, when a t h i c k e r l a y e r o f SE-30 was used ( 4 %) the compounds showed well-formed peaks w i t h o u t t a i l ing. Holmstedt e t a1.2 found t h a t terminal primary amines condense r e a d i l y w i t h acetone, theref o r e a phenolic amine (serotonine) gives the corresponding t r i m e t h y l s i l y l ether-acetone condensation product when s i l y l a t i o n i s c a r r i e d o u t i n acetone s o l u t i o n . Primary amines may be e n t i r e l y l o s t on a NGS column, b u t the acetone condensation products have a normal behaviour under these conditions. The r e l a t i v e r e t e n t i o n times f o r i n d o l e bases r e l a t e d t o tryptamine are summarized i n Table 17.1. TABLE 17.1

RELATIVE

RETENTION

TIIES

FOR INDOLE

BASES RELATED TO TRYPTAIINE~

Glass columns 6 f t by 4 mn w i t h 7 % F-60 plus 1 % EGSS-Z a t 182OC, and 10 % NGS a t 216OC; Antracene time 7.0 min and 6.6 min r e s p e c t i v e l y - and 4.9 min on NGS a t 227OC Compound Anthracene N,N-Dimethyl tryptamine N,N-Diethyl tryptamine

F-60-Z,

182OC

1.00 1.05 1.71 4-Trimethylsilyloxy-N,N-dimethyltryptamine 2-89 5-Trimethyl s i l y l oxy-N,N-dimethyl tryptamine 3.19 3.70 6-Trimethyl silyloxy-N,N-dimethyltryptamine 7-Trimethylsilyloxy-N,N-dimethyltryptamine 2.23 5-Trimethyl s i lyloxy-N ,N-diethyl tryptamille 5.10 Try ptami ne 1.00 Acetone condensation product o f tryptamine 1.86 5-Methoxytryptamine 2.74 Acetone condensation product o f 5-methoxytryptamine 4.50

References p. 172

NGS, 216OC 1.00 1.68 2.14 3.21 3.74 1.72 3.96 3.26 9.50

158 TABLE 17.1 (continued) Compound

F-60-Z,

182OC

5-Methoxy.-N,N-dimethyltryptamine 2.69 Serotonin 3.10 Acetone condensation product o f serotonin a. 74 T r i m e t h y l s i l y l e t h e r o f acetone condensations product o f derotonin 5.29 ~~

NGS, 216OC 5.10

NGS, 227OC 4-Hydroxy-N,N-dimethyl tryptamine 5-Hydroxy-N ,N-dimethyl tryptami ne 6-Hydroxy-N,N-dimethyl tryptamine 7-Hydroxy-N, N-dimethyl t r y p t a m i ne 5-Hydroxy-N,N-diethyl tryptamine Holmstedt e t a1.'

3.46 5.77 5.92 4.41 8.11

7.22 15.0 15.9 11.9

a p p l i e d gas chromatography t o solve the problem o f the s t r u c t u r e o f the

c h i e f i n d o l e bases o f e p e n d , a South American s n u f f reported t o produce h a l l u c i n a t i o n s . With the gas chromatographic method mentioned above the f o l l o w i n g compounds were found: Tryptamine, N,N-dimethyl tryptamine, 5-hydroxy-tryptamine,

4-hydroxy-N,N-dimethyl t ryptami ne,

5-hydroxy-N,N-dimethyltryptamine, 6-hydroxy-N,N-dimethyltryptamine and 7-hydroxy-N,N-dimethyltryptamine. Agurell e t a1.3 i n v e s t i g a t e d by means o f gas chromatography c e r t a i n species o f v i r o l a and other South American p l a n t s t h a t have been used as sources o f i n t o x i c a t i n p s n u f f s by c e r t a i n South American I n d i a n t r i b e s . The hallucinogen 5-methoxy-N,N-dimethyltryptamine and a number o f o t h e r indoles were found. One I n d i a n s n u f f proved t o be unusually r i c h i n a l k a l o i d s (11 %). Considerable d i f f e r e n c e s i n the a l k a l o i d composition o f d i f f e r e n t p a r t s o f s i n g l e p l a n t s were encountered, N,N-dimethyltryptamine N,N-dimethyltryptamine

being the major component i n the leaves and 5-methoxy-

i n the bark o f v i r o l a t h e i d o r a . O f the other species o f v i r o l a inves-

tigated, v. rufula, contained s u b s t a n t i a l amounts o f tryptamines, whereas v. m u l t i n e r v i a and venosa were almost devoid o f a l k a l o i d s . v. c a l o p h y l l a contained h i g h amounts o f a l k a l o i d s

v.

o n l y i n the leaves. Two new 8-carbolines o f a type c a r r y i n g the s u b s t i t u e n t s i n the 6-posit i o n o f the 8-carboline nucleus were found i n v. t h e i d o r a , v. rufula, and Anadenanthera ( P i p t a d e n i a ) p e r e g r i n a . From spectrometric and o t h e r data t h e i r s t r u c t u r e s were shown t o be

2-methyl-6-methoxy-l,2,3,4-tetrahydro-~-carboline and 1,2-dimethyl-6-methoxy-l,2,3,4-tetrahydro-pcarboline. The a l k a l o i d s found are l i s t e d i n Table 17.2 and t h e i r chemical s t r u c tures i n Figure 17.1. Typical gas chromatograms are given i n F i y r e 17.2 and 17.3. The a l k a l o i d bases were e x t r a c t e d w i t h chloroform a f t e r b a s i f i c a t i o n o f the methanolic e x t r a c t , the s a l t s back e x t r a c t e d i n t o aqueous h y d r o c h l o r i c a c i d and the bases again e x t r a c t e d f r a n the aqueous s o l u t i o n w i t h chloroform a f t e r a d d i t i o n o f sodium carbonate. The ?as chromatooraphic separation was performed on packed columns o f various p o l a r i t i e s and the i d e n t i f i c a t i o n achieved by mass spectrometry. TABLE 17.2 ALKALOIDS FOUND I N v m o u AND OTHER SOUTH AMERICAN PLANTS3 N,N-Dimethyl t r y p tami ne N-Methyl tryptami ne Tryptami ne 5-Methoxy-N,N-dimethyl tryptamine 5-Methoxy-N-methyl tryptami ne 5-Methoxvt rvotami ne

5-Hydroxy-N,N-dimethyltryptamine (bufotenine) 5-Hydroxy-N-methyl tryptami ne 5-Hydroxytryptamine (serotonin)

2-Nethyl-1,2,3.4-tetrahydro-pcarbol i n e 2-Methyl -6-methoxy-l.2,3,4 ,-tetrahydro-p-carbol i n e 1.2-Dimethvl-6-methoxv-l.2.3.4. -tetrahvdro-8-carbol ine

157

FIGURE 17.1 CHEMICAL STRUCTURES OF ALKALOIDS FOUND IN v m o u AND OTHER SOUTH AMERICAN PLANTS3 l a Tryptamine, l b N-methyl tryptamine. l c N.N-dimethyl tryptamine, 2a 5-methoxytryptaminer 2b 5-methoxy-N-methyl tryptamine, 2c 5-methoxy-N,N-dimethyl tryptamine, 3a 5-hydroxytryptamine (serotonin), 3b 5-hydroxy-N-methyl tryptamine, 3c 5-hydroxy-N,N-dimethyl trvptamine (bufotenine), 4a 2-methyl tetrahydro-8-carbol ine, 4b 2-methyl -6-methoxytetrahydro-p-carboline, 5 1,2-dimethyl-6-methoxytetrahydro-p-carboline, 6a tetrahydrohamine, 6b 2-methyl t e t r a h y d r o h a n i n e .

, Rl W

NN

\

R

2

FIGURE 17.2 GAS CHROMATOGRAMS OF ALKALOID FRACTIONS OF SOUTH AMERICAN SNUFFS3 Column: 1.3 m by 3.2 mn I.D. packed glass column; 7 % F-60 t 2 % EGSS-2 on Gas Chrom P a t 193OC. A: E x t r a c t o f E D h from Rio Cauaburi; B: E x t r a c t o f Nyakwsna from Rio Tototobi. Upper chromatogram h i g h magnification, lower chromatogram low magnification. A: 1 = DMT, 2 = M T , 3 = MTHC, 4 = 5-MeO-OMT. 5 = 6-MeO-THC. 6: 1 = DMT, 2 = MMT, 3 = 5-MeO-DMT, 4 = 5-Me0MMT. 5 = 6-MeO-THC.

References p. 172

10

20

30

LO

50

60

10

10

30

40

50

60

10

I0

30

LO

50

60

I0

20

30

LO

50

60

168

FIGURE 17.3 ALKALOIDS I N FLOWERING SHOOTS, LEAVES, BARK AND ROOTS OF VIROLA

THEIODORA~

Glass column 1.8 m by 3.2 mm w i t h 7 % F-60 t 2 % EGSS-Z on Gas C h r a 1 = DMT, 2 = WT, 3 = 5-MeO-DMT, 4 = 5-MeO-MMT.

io

ia

io

eaves

a;

P a t 193OC

i,

ia

3ot

1

1

min

Audette e t a1 .4 performed a gas chromatooraphic screenin!

o f Phalaris species f o r

a l k a l o i d s , mainly o f the tryptamine type. Samples of 20 g d r i e d p l a n t m a t e r i a l were e x t r a c t ed w i t h ethanol i n a Soxhlet apparatus, the ethanol was evaporated and the residue dissolved i n d i l u t e s u l p h u r i c acid. A f t e r a d d i t i o n o f excess o f ammonium hydroxide, the a l k a l o i d bases were extracted w i t h chloroform, and a f t e r concentration the gas chronatocraphi c a n a l y s i s was c a r r i e d out on packed columns o f d i f f e r e n t p o l a r i t i e s , usin? Teflon tubings. A f t e r c o n d i t i o n i n g o f the columns they were s i l a n i z e d w i t h 10 u l o f S i l y l 8 (Pierce Chem. Co.). The retent i o n times o f the a l k a l o i d s on the various columns used are piven i n Table 17.3. 17.2. HETEROYOHIMBINE ALKALOIDS I n a gas chromatographic study o f the e f f e c t o f methoxy s u b s t i t u t i o n and c o n f i g u r a t i o n of heteroyohimbine a l k a l o i d s on the r e t e n t i o n times on a 1 % SE-30 packed column, Beckett 5 and huma-Badu showed t h a t they were i n the order pseudo < a110 < normal. The i n t r o d u c t i o n of one methoxy group i n t o the i n d o l e nucleus doubled the r e t e n t i o n time, w h i l e two methoxy groups increased i t by a f a c t o r of four.

I n Table 17.4 the r e t e n t i o n times and the c o n f i g u r -

ations of closed r i n g E heteroyohimbine a l k a l o i d s a r e l i s t e d .

169

TABLE 17.3 RETENTION TIMES OF TRYPTAMINE ALKALOIDS I N Pmmrs SPECIES4 Teflon tubing 6 f t by 1/8 i n c h I . D . packed w i t h Amine 200, CHMIS and DEGS on Gas Chrom G (Amine 200 and CHDMS) and Chromosorb W (DEGS), a t temperatures o f 165oC, 195OC and 18OoC, respectively. Compound

Column Amine 200 CHDMs

Gramine Hordenine Dimethyl t r y p tamine 5-tlethyl tryptamine Tryptamine N-Methyl tryptamine N,N-Oimethyl-5-methoxytryptamine 5-Methoxyt ryptami ne 5-Methoxy-N-methyl tryptamine Bufotenine

DEGS

0.51 0.58 3.03 5.41 4.28 4.00 9.16 14.41 12.42 42.16

1.31 1.04 12.08 20.09 18.35 15.40 36.05 66,04 50.04

0.52 1.08 3.29 8.13 6.58 5.09 10.50 24.48 18.07 74.00

TABLE 17.4 RETENTION TIMES AND CONFIGURATIONS OF CLOSED R I N G E HETEROYOHItlBINE ALKALOIDS5 Glass column 1 m, packed w i t h SE-30 1 % on Gas Chrom P, a t 215OC Configuration R

Alkaloid Ajmalicine Tetraphyll i n e Raumi t o r i n e Rauvanine

Normal Normal Normal Normal

H 11-OMe 10-OMe 10,ll-di-OMe

Rauni ti c i ne Tetra hydroal ston ine Ra un it i d i ne Aricine Reserpinine Iso-reserpi 1 i n e

A110 A110 A110 A110 A110 A1 l o

H

Akuammi gine Iso-reserpi nine Iso-rauni t i d i n e Reserpi l i n e

Epiallo Epiallo Epiallo Epiallo

H 11-OMe 11-OMe 10,ll-di-Otle

Iso-ajmal i c i n e Mitrajavine Epirauvanine

Pseudo H Pseudo 9-One Pseudo 10 ,ll-di-OMe

Configuration o f C-19-Me a

a B P P a P a a

H 11-OMe 10-OMe 11-OMe 1 0 , l l - d i -0Me

a

a a P a

Retention time (min)

pKa

10.5 22.5 22.7 40.1

6.31 6.33

7.1 8.9 16.3 19.7 20.3 34.0

6.24 5.83 6.20 5.75 6.01 6.07

7.1 15.6 17.9 26.3

6.49 6.42 6.20

5.3 3.9 23.6

a a P

The s t r u c t u r e o f heteroyohimbine a l k a l o i d s w i t h open r i n g E and closed r i n g E. I n the a l k a l o i d s w i t h open r i n g E (1) R = H o r OCH3 and R' = -C2H5 o r -CH=CH2, w i t h closed r i n g

i n the alkaloids

E (2) R

Me 0 O c

0 OC I

= H, mono

H

o r di-OCH3 groups.

I

References p. 172

R'

., Me

II

160

17.3.1.

Reserpine and rescinnamine

S e t t i m j e t a l e 6 described a gas chromatopraphic method f o r the e s t i m a t i o n o f reserpine and rescinnamine i n v o l v i n g a l k a l i n e h y d r o l y s i s o f the a l k a l o i d s and subsequent e s t e r i f i c a t i o n o f the acids formed by means o f diazomethane. Reserpine pave q u a n t i t a t i v e l y 3.4.5-trimethoxybenz o i c a c i d methylester, whereas the trans-3,4,5-trimethoxycinnamic a c i d methylester. which should be expected f o r rescinnamine, was p a r t l y isomerized t o the cis-trimethoxycinnamic a c i d methylester o r formed an adduct w i t h a molecule o f methanol, y i e l d i n g 3-methoxy-3-(3,4.5-trimethoxyphenyl) propionic a c i d methylester.

A gas chromatogram o f the compounds are given i n Figure 17.4 FIGURE 17.4 GAS CHRO:IATOGRAM

OF HYDROLYSIS

PRODUCTS OF RESERPINE

AND RESCINNAIWE

AFTER ESTERIFICATION~

1 = methyl-3,4.5-trimethoxybenzoate, 2 = methyl-3-methoxy-3-(3,4,5-trimethoxyphenyl)propionate, 3 = cis-methyl-3,4.5-trimethoxycinnamate, 4 = trans-methyl-3,4,5-trimethoxycinnainate, 5 = the i n t e r n a l standard (methyl stearate). Column: plass, 2.40 m x 2 mn I.D., 15 % Apiezon L on Chromosorb W a t 230 C.

0

I

I

1

5

10

15

I

20

T

25

min

Pantarotto e t a1

.

developed a !as

chromatonraphic-mass spectronraphic method f o r the

i d e n t i f i c a t i o n and q u a n t i t a t i v e determination o f reserpine i n nanonram amounts i n r a t b r a i n . The e x t r a c t i o n o f homogenized b r a i n t i s s u e was performed w i t h acetone c o n t a i n i n g 03-reserpine (as i n t e r n a l standard). A f t e r concentration o f t h i s e x t r a c t i t was used f o r nas chromatographic analysis on an OV-1 1 % packed column on Gas Chrom Q a t 285OC. No column adsorption, decomposition o r C-3-epimerization was observed d u r i n g the analysis. A comparison o f the data obtained by means o f gas chromatography-mass fragmentoyaphy w i t h those obtained by r a d i o i s o t o p i c assay gave s a t i s f a c t o r y agreement. A gas chromatogram o f the a l k a l o i d s i s Civen i n Figure 17.5.

161

FIGURE 17.5 GAS CHROFATOGRAH OF OESERPIOINE ( l ) , RESERPINE ( 2 ) AND RESCINNAMINE ( 3)7 on a glass column, 40 cm by 4 mn I . D .

17.3.2. Forni

packed w i t h 1 % O V - 1 on Gas Chrom Q a t 285OC

Ajmal i n e

8

described a method f o r q u a n t i t a t i v e determination o f ajmaline i n bark and r o o t

samples o f Rauwolfia vomitoria. Because o f the presence o f many products i n the raw rnethanolic e x t r a c t o f the crude drug, an e x t r a c t i o n o f the a c i d i f i e d e x t r a c t was performed w i t h c h l o r o form p r i o r t o e x t r a c t i o n o f the a l k a l o i d s w i t h the same solvent a f t e r adjustment o f the pH t o 8.5. Because o f the p o l a r i t y o f the a l k a l o i d s , they were s i l a n i z e d before gas chromatography on a 3 % OV-17 on Chromosorb U column. A gas chromatogram showing the good separation o f ajmaline from other a l k a l o i d s w i t h s i m i l a r s t r u c t u r e s and arbutin, which was used as an i n t e r n a l standard, i s found i n Figure 17.6.

. Ri’R,

Rafumncem p. 172

Ajmaline

R=R3=OH R1=R2=R5=H R4= C2H5

Isoajmaline

R=R2=OH R1=R3=R4=H R5= C2H5

Sandwichine R1=R3=OH R =R2=R5=H R4-- C2H5 T e t r a p h y l l i c i n e R=OH R3=H

162 FIGURE 17.6

GAS CHROMATOGRAM OF AJMALINE AND SOME RELATED ALKALOIDS~ on a glass column, 2 m by 3 mn I.D. packed w i t h 3 % OV-17 on Chromosorb W a t 27OoC 1 = A r b u t i n ( i n t e r n a l standard), 2 = sandwichine. 3 = i s o a j m l i n e , 4 = ajmaline, 5 = t e t r a phyl 1 ic ine

3

I

,

.

,

,

0

5

10

15 min

20

25

30

Ten analyses o f the same batch o f crude drug were c a r r i e d out. The r e s u l t s obtained were 2.41, 2.55,

2.52,

2.22,

2.41,

2.43, 2.36, 2.44 and 2.45 % o f ajmaline w i t h a mean o f 2.43 %,

a standard d e v i a t i o n o f 0.098 % and a c o e f f i c i e n t o f v a r i a t i o n o f 4.05 % 17.4. STRYCHNOS ALKALOIDS O f the Strychnos alkaloids, mainly strychnine and brucine have been examined by cas chromatography. The gas chromatography has mostly been c a r r i e d o u t on non-polar o r s l i c h t l y pol a r s t a t i o n a r y phases (SE-30, SE-52, XE-60, O V - l and OV-17), b u t a l s o more p o l a r l i q u i d s 9 have been employed (NGS, EGSS-Y, HI-EFF 8B). As s t a t e d by Brochmann-Hanssen and Fontan pol a r s t a t i o n a r y phases are more s e l e c t i v e than non-polar phases, b u t w i t h increasin? p o l a r i t y o f the s t a t i o n a r y phase the r e t e n t i o n times o f the a l k a l o i d s are s i a n i f i c a l l y increased. I f brucine and l e s s p o l a r bases l i k e vomicine and novacine are gas chranatographed on very POl a r s t a t i o n a r y phases, t h e i r r e t e n t i o n times w i l l be very long. Such s t a t i o n a r y phases are, therefore, n o t s u i t a b l e f o r r a p i d analysis. Non-polar o r s l i a h t l y p o l a r phases should be pref e r r e d f o r t h i s type o f a l k a l o i d s . Strychnine and brucine i n a l k a l o i d a l mixtures i s o l a t e d frm seeds o f Strychnos nux vomica 10

L. were separated on a SE-30 1.15 % column by Brochmann-Hanssen and Baerheim Svendsen whereas Bisset and Fouche”

and Bisset, D e j e s t r e t and Fouche”

,

p r e f e r r e d the s l i p h t l y p o l a r

SE-52 f o r the examination o f a l k a l o i d a l mixtures from s. nux vomica and s. i c a j a . The more p o l a r a l k a l o i d s , such as d i a b o l i n e and r e t u l i n e , were e l u t e d w i t h s h o r t r e t e n t i o n times a t 23O-28O0C. w h i l e the l e s s p o l a r bases, l i k e vomicine and novacine, had much longer r e t e n t i o n

163 times. A t y p i c a l gas chromatogram o f some

Strychnos

a l k a l o i d s i s Given i n F i y r e 17.7, the

r e l a t i v e r e t e n t i o n times of the same a l k a l o i d s are presented i n Table 17.5 and t h e i r chemic a l formulas i n Figure 17.8. FIGURE 17.7

GAS CHROMATOGRAM OF SOME

ALKALOIDS~~

STRYCHNOS

on a s t a i n l e s s s t e e l column, 6 f t by 1/8 inch packed w i t h 5 % SE-52 on Aeropak 30 1 = Pseudostrychnine s u b s i d i a r y peak (?), 2 = strychnine, alon? w i t h peak 1 may i n d i c a t e t h a t pseudostrychnine i s present as w e l l , 3 = a-colubrine, 4 = p-colubrine, 5 = i c a j i n e , 6 = brucine, 7 = vomicine, 8 = novacine

TABLE 17.5

RELATIVE

RETENTION

TIMES

OF TERTIARY

STRYcHNos

ALKALOIDS'~

gas chromatographic conditions, see Figure 17.8 Column temperature Spermos t r y c h n i ne Retul i n e Diabol i n e Strychnospemi ne Strychnine * Holstiine a-Col u b r i n e 8-Colubrine Icajine Brucine ** Vomi c in e Nova c i n e

250°C

Rstry

Rstry

Rbru

0.45

0.44 0.49 0.69 0.75 1.00 0.94 1.71 1.82 1.96 2.61 3.62 5.48

0.17 0.19 0.26 0.29 0.38 0.36 0.66 0.70 0.78 1.00 1.41 2.18

0.50 0.76 0.82 1.00 1.03

* Actual r e t e n t i o n times: 23OoS, 11.0; ** Actual r e t e n t i o n times: 250 C, 15.2

Refereneea p. 172

2800c

23OoC

Rstry

Rbru

0.69

0.31

1.00

0.45

1.59 1.66 1.88 2.24 3.10 4.24

0.71 0.74 0.84 1.00 1.39 2.12

250°C, 5.55; 28OoC, 2.07 min. and 28OoC, 4.55 min.

164

FIGURE 17.8 TERTIARY

SmycHNoS

ALKALOI D S ~

R

n

I

‘I R=H Spermost r y c h n i n e R=OCH3 Strychnospetmine

I1 Retul i n e Ac

I11 Diaboline

IV

m

Hol s t i i n e V

R=R1=H Pseudostrychnine R=R1=OCH3 Pseudobrucine

VI R=R1=H Strychnine R=H, R1=OCH3 a-Colubrine R=OCH3, R1=H 8-Colubrine R=R1=OCH3 Brucine

P V II

R=R1=R2=H I c a j i n e R=Rl=H.

R2=0H V m i c i n e

R=R1=OCH3. R2=H Novacine

Using gas chromatography o f the N-oxides o f the pseudo s e r i e s o f the a l k a l o i d s , decompos i t i o n o f the a l k a l o i d s was observed i n a l l cases, g i v i n g strychnine as t h e main peak on the chromatograms. The authors presumed t h a t t h i s decomposition mipht be due t o t h e use o f a s t a i n l e s s s t e e l i n j e c t o r and column instead o f working i n an a l l - g l a s s system. Sondack and Koch13 c a r r i e d o u t q u a n t i t a t i v e determinations o f strychnine and brucine i n phatmaceutical preparations using OV-1 and a glass column. Papaverine was used as an i n t e r nal standard. The a l k a l o i d a l bases were e x t r a c t e d w i t h chloroform a f t e r adding sodium hydroxide t o the preparation. Hanks e t a l . 1 4 u t i l i z e d 1.3.5-triphenyl

benzene as an i n t e r n a l stan-

dard f o r determinations o f strychnine down t o 2 ug i n g r a i n b a i t s on an SE-30 5 % column. Since p l a n t s may take up strychnine through t h e r o o t s when used as b a i t s above and below

165

ground t o c o n t r o l rodents, M i l l e r e t a1.15 developed a gas chromatographic method f o r determining strychnine residues i n a l f a l f a . Samples o f 50 g a l f a l f a were e x t r a c t e d w i t h e t h y l acetate a f t e r b a s i f i c a t i o n w i t h sodium carbonate. the a l k a l o i d back e x t r a c t e d i n t o 0.1 N s u l phuric a c i d and e x t r a c t e d w i t h dichloromethane a f t e r b a s i f i c a t i o n aoain. A f t e r concentration the amount o f strychnine was determined on a packed column w i t h 1.5 % OV-17 as s t a t i o n a r y phase. The average recovery o f strychnine from spiked a l f a l f a (0.05 ppm) was 88 i 11 X (8 determinations). For the determination o f strychnine i n b i o l o g i c a l materials, Platonow e t a1.16 e x t r a c t e d the a l k a l o i d from the b i o l o g i c a l m a t e r i a l w i t h chloroform as t r i c h l o r o a c e t a t e . This s t r y c h nine s a l t i s more soluble i n chloroform than i n water. The chloroform s o l u t i o n o f t h e s a l t was i n j e c t e d i n t o the gas chromatograph f o r analysis. Tissue concentrations o f s t r y c h n i n e as low as 0.01 ppm could be detected. The recovery o f s t r y c h n i n e added t o l i v e r was approximately,90

%.

17.5. VARIOUS INDOLE ALKALOIDS 17.5.1.

Vinca a l k a l o i d s

The pharmacokinetics o f vincamine a f t e r intravenous and o r a l a p p l i c a t i o n t o the dog was i n v e s t i g a t e d w i t h gas chromatooraphy by Laufen e t al.17. w i t h dich1oromethane:ethanol

Samples o f plasma were e x t r a c t e d

( 9 9 : l ) a f t e r a d d i t i o n o f quinine as i n t e r n a l standard and so-

dium hydroxide t o pH 12.5. The residue obtained a f t e r evaporation o f the solvents was t r e a t ed w i t h N-methyl-N-trimethylsilylfluoroacetamide and the r e a c t i o n product taken up i hexane. Gas chromatography was performed on a 1 m long packed column w i t h 1 X OV-1 o r a 25 m lonp glass c a p i l l a r y column w i t h OV-101. Good q u a n t i t a t i v e r e s u l t s were obtained. Detection l i m i t was 0.5 ng/ml and the recovery o f the e x t r a c t i o n procedure about 85 %. Hoppen e t a1.18 used a packed SE-30 column f o r the assay o f vincamine i n plasma. The mole c u l a r i o n (m/e 426) and the parent peak ( m / e 367) o f the t r i m e t h y l s i l y l d e r i v a t i v e s were assayed simultaneously by selected i o n monitoring.Down t o 30 pc/ml plasma could be assayed. The method i s s u i t a b l e f o r pharmacokinetic studies. Plasma samples o f EDTA-anticoagulated blood (1-2 m l ) were e x t r a c t e d w i t h hexane a f t e r a d d i t i o n o f tris(hydroxymethy1)aminomethane. The aqueous phase was frozen o u t and the hexane phase decanted i n t o a c o n i c a l glass tube. The solvent was evaporated and N-methyl-N-trimethylsilyltrifluoroacetamide

was added. The

r e a c t i o n mixture was used f o r the gas chromatographic analysis on an 1 % SE-30 packed column on Chromosorb W HP a t 210OC.

A number o f c l o s e l y r e l a t e d Vinca a l k a l o i d s

-

homologues o f vincaminic and apovincaninic acids Chrom Q column by Gazdag e t al.”.

such as stereo and s t r u c t u r a l isomers, e s t e r

-

were separated on a packed OV-101 on Gas

Oerivatization ( s i l y l a t i o n with N.0-bis(trimethylsily1)

t r i f l u o r o a c e t a m i d e ) was needed f o r those a l k a l o i d s c o n t a i n i n n f r e e hydroxy croups and/or a carboxylic a c i d group (vincaminic and apovincaminic a c i d s ) . I n Table 17.6 the r e t e n t i o n times and e l u t i o n temperatures o f the Vinca a l k a l o i d s i n v e s t i g a t e d

- as

such and as d e r i v a t i v e s

are given. Polgar and Vereczkey2O applied gas chromatography w i t h a glass c a p i l l a r y column f o r the determination o f apovincaminic acid, the main m e t a b o l i t e o f apovincaminic a c i d e t h y l e s t e r (vinpocetine) i n human plasma. Apovincaminic a c i d was recovered from plasma by a d d i t i o n o f tetrabutylammonium hydroxide and e x t r a c t i o n w i t h chloroform. I t was transformed i n t o i t s

Relemncsr p. 172

-

166

TABLE 17.6 RETENTION TIMES AND ELUTION TEMPERATURES OF VINCA ALKALOIDS" on a glass column, 1 m by 3.2 mn I.D. packed w i t h 3 % OV-101 on Gas Chrom Q, w i t h temperat u r e p r o g r a m i n g f r o m 200°C t o 330OC, 5OC/min. tR(min)

Compound cis-Vi ncamenine

cis-Vincanol cis-Isovincanol cis-Vi ncamone cis-Vi ncaminic a c i d cis-Epivincamine trans-Epi vincamine cis-Epivincaminic a c i d e t h y l e s t e r trans-Vincamine trans-Apovincaminic a c i d e t h y l e s t e r cis-Vi ncamine cis-Vincaminic a c i d e t h y l e s t e r cis-Apovi ncami ne trans-Apovi ncamine cis-Apovincaminic a c i d e t h y l e s t e r cis-Apovi ncamini c a c i d cis-11-Bromo-vi ncami ne cis-10-Bromo-vincamine cis-Apovi ncamini c a c i d phenylester

4.77 4.77 4.77 6.56 6.96 7.20 7.20 7.44 7.67 7.78 7.87 7.08 8.30 8.30 8.88 8.93 10.98 11.97 14.92

E l u t i o n temperature 223.9 223.9 223.9 232.8 234.13 236.0 236.0 237.2 238.4 238.9 239.4 239.4 241.5 241.5 244.4 244.7 254.9 259.9 274.6

methylester w i t h diazomethane and gas chromatographed on an Sp 2100 glass c a p i l l a r y column, 10 m long by 0.25 nun I . D . a t 22OoC u s i n g 9-brom-apovincaminic a c i d as an i n t e r n a l standard. U i t h a NP-detector the detection l i m i t was 2 ng/ml plasma. 17.5.2.

Physostigma

alkaloids

Physostigmine s a l i c y l a t e i n 0.5 % aqueous s o l u t i o n was determined by Teare and Borst"

by

freeze-drying o f samples o f 0.2 m l , and conversion o f the compound i n t o i t s t r i m e t h y l s i l y l d e r i v a t i v e by d i s s o l v i n g the residue i n 5 ~l o f dry p y r i d i n e and 10 ~l o f N,O-bis(trimethy1sily1)acetamide. The s o l u t i o n was allowed t o stand f o r 1 h before i n j e c t i n g 1.2 111 i n t o the gas chromatograph. A 3.8 % SE-30 on D i a t o p o r t S column and a temperature o f 145OC was used f o r the analysis. The physostigmine THS and the s a l i c y l i c a c i d TVS d e r i v a t i v e s were e l u t e d separately. Routine a n a l y s i s over several days gave a p r e c i s i o n o f t 11.5 %. 17.5.3.

dspidosperma

a1 k a l o i ds

The usefulness o f the a p p l i c a t i o n o f a d i r e c t l y coupled gas chromatograph-mass spectromet e r f o r the a n a l y s i s o f complex a l k a l o i d mixtures was demonstrated by Thomas e t a1." i n an i n v e s t i g a t i o n o f the a l k a l o i d s i n dspidosperma neblinae. A m i x t u r e o f the minor a l k a l o i d s was f i r s t chromatographed on alumina columns and the f r a c t i o n s obtained were gas chromatopraphed on a packed column, 1 % SE-30 on Gas Chrom Q, by temperature p r o g r a m i n g (150-250OC). The exc e l l e n t r e s o l u t i o n o f the pas chromatograph and the mass spectra obtained l e d t o the i d e n t i f i c a t i o n o f 11 a l k a l o i d s . The a l k a l o i d s and the percentages i n which they were found i n the e x t r a c t a r e given i n Table 17.7. A gas chromatogram o f some a l k a l o i d s i s given i n Figure 17. 9.

167

TABLE 17.7 ALKALOIDS OF A S P I W S P E M NEBLINAE

22

% o f extract Aspi dospermi dine

1,2-Dehydroaspidospermidine Deacetyl p y r i f o l i d i n e

1.2-Dehydrodeacetylpyrifol i d i n e Demethoxyaspi dospermi ne Aspi dospermine Demethyl aspi dospermi ne Pyrifolidine Aspi docarpine Eburnamoni ne Neb1i n i n e

peak no.

0.1

3.3 0.9 1.3 4.7 26.3 18.7

1 2

3 4

0.05

0.8

5

FIGURE 17.9

GAS CHROMATOGRAI! OF ALKALOIDS I N A S P I ~ P E R M ANEBLINAE~' on a glass column, 5 f t by 1/0 inch packed w i t h 1 % SE-30 on Gas Chrom Q, w i t h temperature programming from 150°C t o 250OC. For numbering o f peaks, see Table 17.6.

3

During a d e t a i l e d i n v e s t i a a t i o n o f the a l k a l o i d s o f voacanga africana Stapf, by Thomas and B i e n ~ a na ~number ~ o f a l k a l o i d s were i s o l a t e d by means o f ?as chromatography. They were i d e n t i f i e d by comparison o f t h e i r mass spectra w i t h a v a i l a b l e data. However, o n l y few data o f the gas chromatographic experimental conditions were given i n the paper.

Referencei p. 172

168

17.5.4.

uncaria a l k a l o i d s

P h i l l i p s o n and H e m i n g ~ a yapplied ~~ a combination o f t h i n - l a y e r chromatonraphy, g a s - l i q u i d chromatography, u l t r a v i o l e t spectroscopy and mass spectrometry techniques f o r t h e a1 k a l o i d screening o f herbarium samples o f the genus Uncaria (Rubiaceae). Some s i x t y a l k a l o i d s were distinguished by the screening procedure, and they represented heteroyohimbine, oxindole, roxburghine, simple p c a r b o l i n e , pyridine-indole-quinolizidine and nambirtannine types. Gas chromatograms o f some a l k a l o i d s included i n t h e screening are shown i n F i y r e 17.10 and the r e t e n t i o n times of some o f the

Uncaria

a l k a l o i d s are l i s t e d i n Table 17.8.

FIGURE 17.10 GAS CHROMATOGRAM OF SOME uNcARrA ALKALOIDSL4

on a glass column, 2 f t by 1/4 i n c h I.D.. For numbering o f peaks, see Table 17.7.

packed w i t h 5 % SE-52 on Varaport 30, a t 240OC.

9

?

6

6

io

is

b

O ; min

i

io

11

9

b

1;

;O min

TABLE 17.8 RETENTION TIMES

OF

U

N

~

ALKALOIDSE4 A

For experimental conditions, see Figure 17.10.

A1 k a l o i d

tR(min)

No.

Alkaloid

tR(min)

No.

Pent a c y c l i c heteroyohimbines

Ajmalicine Isoajmal 'c 'ne Mitrajavfnd Tetrahydroa 1st o n i ne Akuamjgine 4-R akuamnigine N-oxide 19-epf -Ajmalicine 3-Iso-19-epi-ajmal i c i n e

20.6 19.5 17.5 14.3 14.3 19.5 12.7

6

Rauni t i cine

15.5

Tetracyclic heteroyohimbines

3

D i hydrocorynanthei ne Gambirine Speci oqyni ne H ir s u t ine Hirsuteine

18.8

5

37.7 12.2 11.8

2

169

CHEMICAL STRUCTURES OF uticdmd ALKALOIDSE4

I Pentacyclic heteroyohimbine ( R = H, OH o r OMe) 11 T e t r a c y c l i c heteroyohimbine ( R = H. OH o r OMe. R ' = E t o r v i n y l ) 111 Pentacyclic oxindole (R = H, OH o r M e ) I V T e t r a c y c l i c oxindole (R = H, OH o r OMe, R ' = E t o r v i n y l ) 111 and I V can e x i s t as A o r B isomers

I - I V can e x i s t as isomers defined as

i n which the lactam carbonyl can l i e

C-3 H C-20 H all0

a

a

below (A) o r above ( B ) the plane of

epial l o

B

a

the C-D r i n g s

normal

a

B

pseudo

B

B

C-3 H

C-19 H

V Roxburghine C

a

a

D

B

a

E

B

B

V I Hannane Hanine

(R =

V I I Harmaline V I I I Angustine ( R = CH=CH2, R ' = H) Angustoline (R = CH(OH)Me, R'= H) Angustidine ( R = H, R ' = Me)

I X Gambirtannine (R = H2) Oxogambirtannine (R = 0 )

X Dihydrogambirtannine

H)

(R = OMe)

R

R

Me MeOOC I

m

3 /

R

m

Refereneesp. 172

\ N R'

x

Ix MeOOC

MeOOC

170 TABLE 17.8 (continued) A1 k a l o i d

tR(min)

No.

T e t r a c y c l i c heteroyohimbines (continued)

Mitraciliatine Corynantheidine M i t ragynine Isocorynantheidi ne Specioci 1i a t i n e

Alkaloid

I socorynoxei ne Rotundifol i n e Rhynchoci 1i n e Rhynchophyll i n e Rhynchophyll i n e N-ox ide Corynoxei ne Isorotundifol ine C i 1iaphyl 1 i n e Corynoxi ne Corynoxine B SpecSofol i n e

Pentacyclic oxindoles

14.0 14.0 23.2 14.0 14.0 12.4 12.4 12.4 12.4 12.4 12.4 12.4 12.4 13.5 13.5

T e t r a c y c l i c oxindoles

Isorhynchophyll i n e an t i - Isorhyncho phyl 1ineN-oxide

10.9 10.9

No.

T e t r a c y c l i c oxindoles (continued)

22.3 16.9 33.2 16.7 32.1

Isomitraphylline Isomitraphyll i n e N-oxide Javaphylline Mitraphylline M i t r a p h y l l i n e N-oxide Isopteropodine Isopteropodine N-oxide Pteropodi ne Pteropodine N-oxide Speciophyl l i n e Speciophyll i n e N-oxide Uncarine F Uncarine F-oxide Uncarine A Uncarine 8

tR(min)

10.3 16.0 17.9 10.9 10.9 10.3 16.0 17.9 10.2 10.2 15.5

Roxburphine C Roxburghine D Roxburghine E Dimeric i n d o l e a l k a l o i d Hannane Harmine Harmaline

0.6 1.5 1.4

Angust ine Angustidine Angustol i n e Gambi rtannine D i hydroaambi r t a n n i ne Oxogambi rtannine

20.8

TABLE 17.9 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY OF TERPENOID INDOLE ALKALOIDS AND SIMPLE INDOLE ALKALOIDS Column

S o l i d support mesh

glass S, 6 f t

GP AWS 80-100

glass, 6 f t x 4 mn glass, 1.8 m x 3.2 mn

-

SE-30 SE-30 GP S 80-100 F-60 t EGSS-2 NGS GP AWS 100-120 F-60

CW AGlS 80100 CG AWS 60-80 CG 60-80 l m GP AWS 60-80 2.4 m x 2 mn I.D. CW A# 80-100 40 cm x 4 imn I.D. GQ 100-120 2 m x 3 mn I.D. CW HP 100-120 3 f t x 0.07 i n I.D. GP AWS 80-100 I.D.

%

Temperature

SE-30 OV-17 DEGS

0.75 16OoC 4 205OC 182OC 10 216OC 7 193OC 5 1 18OoC

Amine 220 CHMlS SE-30 Apiezon L ov-1 OV-17 SE-30 XE-60 EFSS-Y HI-EFF 8B

0.5 0.4 1 15 1 3 1 1 1 1

t EGSS-Z

2.25 m x 3.2 mn

Teflon, 6 f t x 1/8 i n

glass, glass, glass, glass,

Stat.phase

Comp. Prep.

ind.b. ind.b.p.

.

a1k id .MS. alk.id.

165OC 195OC 215OC 23OoC 285OC 27OoC 225OC 220°c 23OoC 24OoC

a1k.s. rs.rc.qnt. rs. on t. br. aj.qnt.pm. a1k.s.

Ref.

171 TABLE 17.9 (continued) ~~

Column

S o l i d support mesh

Stat.phase

Temperature

%

Comp. Prep.

GP ABS PEG 100- SE-30 140 Aer. 100-120 SE-52 s . s . , 2 f t x 1/8 i n OV-1 glass, 0.9 m x 6.4 nnn O.D. GQ 80-100

1.15 225OC

alk.pm.

5 3

230-280°C 280°C

glass, 6 f t x 6 mm cw s glass, 1.2 m x 2.0 m I . D . GQ 100-120

SE-30 OV-17

5 1.5

27OoC 275OC

glass, 1 m x 2 mm I . D . glass cap., 25 m glass, 150 cm x 2 mm

GQ 80-100

ov-1

1

CQ HP 100-120

ov-101 SE-30

1

235OC 150-250°C p r 210°c

alk.pm. str.bru.qnt. prep. str.qnt.bts. str.qnt.res. Pm. vi.R!S. qnt. p!. v i .TMS.qnt. P' . ~i~a1k.s.

glass, 6 f t x 3 mm 1.0.

glass S, 1 m x 3.2 mn I . D . GQ 80-100 glass cap.,

10 m x 0.25 mn 1.0.

200-3300c pr 50C/min 22ooc

OV-101 sp.2100

AVA.me.qnt.

Ref. 10

11 13 14 15

17 18 19

2o

-1

PI.

alass. 4 f t x 3 mn I . D . glass; 5 f t x 1 / ~ in glass, 0.5 in x 1/4 i n - 2 f t x 1/4 i n I.D.

Dia S 80-100 GQ 100-120 Var.80-100

-

SE-30 SE-30 SE-52 SE-52

3.8 1 5 5

145OC . 150-250°C p r 230OC 24OoC

Dhv.TMS.ant. . ds.alk.s.' un.alk.id.MS

TABLE 17.10 TERPENOID INDOLE ALKALOIDS AND SIMPLE INDOLE ALKALOIDS ABS = acid, base washed Aer = Aeroport a j = ajmaline alk = alkaloid Amine 220 = 1-hydroxy-2-heptadecenyl imidazoline A s = Aspidosperma AVA = apovincaminic a c i d AW = a c i d washed b r = brain b r u = brucine bts = baits cap = c a p i l l a r y CG = Chromosorb W CHDMS = cyclohexane dimethanol succinate CW = Chromosorb W DEGS = diethyleneglycol succinate Dia S = D i a t o p o r t S GP = Gas Chrom P GQ = Gas Chrom Q HP = h i g h performance ID = i n s i d e diameter

References D. 172

-

LIST OF ABBREVIATIONS

i d = identification i n = inch i n d . b = i n d o l e bases me = methyl e s t e r MS = mass spectrometry 0.0. = outside diameter phy = physostigmine p l = plasma pm = p l a n t m a t e r i a l prep = pharmaceutical preparation qnt = quantitative r c = rescinnamine r e s = residue r s = reserpine S = silanized S.S. = stainless steel s t r = strychnine TMS = t r i m e t h y l s i l y l Un = lhlcaria Var = Varaport v i = vincamine Vi = V i n c a

21 22 24

172

17.6 REFERENCES

1 H.M. Fales and J.J. Pisano. Anal. Biochem., 3 (1962) 337. 2 B. Holmstedt, W.J.A. VandenHeuvel, W.L. Gardiner and E.C. Horning, Anal. Biochem., 8 (1964) 151. 3 S. Agurell, B. Holmstedt, J.-E. Lindgren and R.E. Schultes, Acta Chem. scand., 23 (1969) 903. 4 R.C.S. Audette, J. Bolan, HJ!. Vijayanagar, R. B i l o u s and K. Clark, J . Chromatogr., 43 (1969) 295. 5 A.H. Beckett and 0. Ihruma-Badu, J . Pharm. Pharmacol., Suppl. 1968, 74 S. 6 G. Settimj, L. D i Simone and M.R. Del Giudice, J . Chromatogr.. 116 (1976) 263. 7 G. Pantarotto, G. Belvedere, A. F r i a e r i o , T. Mennini and L. Manara, E u r . J . Drugmetabol. Pharmkin., 1976, 25. 8 G.P. Forni, J. Chromatoqr., 176 (1979) 129. 9 E. Brochrnann-Hanssen and C.R. FOntan,'J. Chromatogr., 19 (1965) 296. 10 E. Brochmann-Hanssen and A. Baerheim Svendsen, J . Pharm. S c i . , 51 (1962) 1095. 11 N.G. B i s s e t and P. Fouchg, J . Chromatogr.. 37 (1968) 172. 12 N.G. Bisset, M. k j e s t r e t and P. Fouch6, d ~ Pharm. . Franc., 27 (1969) 147. 13 D.L. Sondack and W. Koch, J . Pharm. S c i . , 62 (1973) 101. 14 A.R. Hanks, B.S. Engdahl and B.M. Colvin, J . d s s o c . off. Anal. Chem., 58 (1975) 961. 15 G. M i l l e r , J. Warren, K. Gohre and L. Hanks, J . d s s o c . off. Anal. C h e m . , 65 (1982) 901. 16 N. Platonow, H.S. Funnel1 and W.T. Oliver, J . Forensic S c i . , 15 (1970) 443. 17 H. Laufen, W. Juhran, W. F l e i s s i g , R. Gdtz, F. Scharf and G. Bartsch. drzneim.-Forsch., 27 (1977) 1255. 18 H.-0. Hoppen, R. Heuer and G. Seidel. Biomed. Mass spectrom., 5 (1978) 133. 19 M. Gazdag, K. M i h t i l y t i and G. Szepesi, Fresenius' z. Anal. Chem., 309 (1981) 105. 20 M. Polgar and L. Vereczkey. J . Chromatogr., 241 (1982) 29. 21 F.H. Teare and S. I. Borst, Y. Pharm. Pharmacol., 21 (1969) 277. 22 D.W. Thomas, H.K. Schnoes and K. Biemann, Experientia. 25 (1969) 678. 23 D.W. Thomas and K. Biemann, L l o y d i a , 31 (1968) 1. 24 J.D. P h i l l i p s o n and S.R. Hemingway, J . Chromatogr., 105 (1975) 163.

173

Chapter 18 ERGOT ALKALOIDS

............................................ ............................................................. ..................................................................

18.1 Lysergic a c i d diethylamide (LSD) 18.2 Ergot a l k a l o i d s 18.3 References 18.1 LYSERGIC A C I D DIETHYLACIIDE

-

173 176 184

LSO

The e a r l i e s t i n v e s t i g a t i o n s on gas chromatography o f Ervot a l k a l o i d s were performed w i t h l y s e r g i c a c i d diethylamide, LSD. i n order t o develop methods f o r i t s d e t e c t i o n and i d e n t i f i c a t i o n i n t r a c e amounts i n n a r c o t i c seizures; i . a . i n sunar cubes impregnated w i t h the psychomimetic drug. Due t o the low v o t a t i l i t y o f the compound, Radecka and Nioam' hydrogenated i t p r i o r t o gas chromatography t o increase i t s s t a b i l i t y . LSD was e x t r a c t e d f r o n the suaar

cubes as a f r e e base w i t h dichloromethane. hydrogenated w i t h Adam's c a t a l y s t and gas chromatographed on 0.2 % SE-30 on micro glass beads. The hydroTenated LSD was e l u t e d i n 3.6 min a t a column temperature o f 24OOC. By means o f a stream s p l i t t e r . eluates were c o l l e c t e d and analyzed by t h i n l a y e r chromatooraphy f o r confirmation o f i d e n t i t y . Convincing t h i n - l a y e r chromatograms were obtained. Because o f the low s e n s i t i v i t y and n o n - r e p r o d u c i b i l i t y o f the method developed by Radecka and Nigam'

-

50 pg f a i l e d t o give convincing detectable r e s u l t s

-

Katz e t a1.'

developed a

method f o r d i r e c t gas chromatography o f LSD i n submicrogram amounts using a packed column o f SE-30. 0.3 %, on glass micro beads. Symmetrical peaks were obtained t h a t were w e l l s u i t e d f o r q u a n t i t a t i v e work. The detector response was l i n e a r a t low concentrations and 0.5 pg o f LSD could e a s i l y be detected and q u a n t i f i e d . Lerner3 developed a GLC method f o r detection o f LSD d i r e c t l y on a 2 % SE-52 column a t

250°C i n an a l l - g l a s s system, which minimize decomposition. However, f o r preater s e n s i t i v i t y and s t a b i l i t y i n gas chromatography, the author p r e f e r r e d t o use t h e t r i m e t h y l s i l y l derivat i v e o f LSD. N,O-bis(trimethylsily1)acetamide

was u t i l i z e d as s i l y l a t i o n reanent and LSD was

solved i n dimethylformamide by the reaction, which was performed a t 55OC f o r 30 minutes. The t r i m e t h y l s i l y l d e r i v a t i v e o f LSD was gas chromtoqraphed a t 245OC

-

a l s o on a 2 % SE-52 c o l -

umn. LSD was e x t r a c t e d from sugar cubes w i t h chloroform-methanol ( 9 : l ) .

The e x t r a c t i o n was

performed from aqeous s o l u t i o n a f t e r a d d i t i o n o f bicarbonate. Figure 18.1 shows a gas chromatogram o f 30 p g LSD and Figure 18.2 one obtained w i t h 0.2 ufl o f the t r i m e t h y l s i l y l d e r i v a t i v e o f LSD. B a i l e y e t a l . 4 synthesized a s e r i e s of dialkylamides o f l y s e r g i c and i s o l y s e r g i c a c i d i n order t o d i s t i n g u i s h LSD from these compounds. The f o l l o w i n g compounds were made:

I Lysergic a c i d dimethylamide I 1 I s o l y s e r g i c a c i d dimethylamide 111 IV V VI VII VIII IX X

References p. 184

Lyseraic a c i d diethylamide I s o l y s e r g i c a c i d diethylamide Lysercic a c i d methylpropylamide I s o l y s e r p i c a c i d methylpropylamide Lyserpic a c i d ethylpropylamide I s o l y s e r a i c a c i d ethylpropylamide Lyserqic a c i d dipropylamide I s o l y s e r g i c a c i d dipropylamide

114 FIGURE 18.1 GAS CHROMATOGRAM OF 3 0 ug LSD3

on a stainless steel column. 1 . 5 m by 3 mn I.D., 0 . 2 % SE-30 a t 27OoC

packed with glass micro beads, coated with

FIGURE 18.2 GAS CHROMATOGRAM OF 0.2 ug TRIMETHYLSILYL DERIVATIVE OF LSD Gas chromatographic conditions. see Figure 18.1.

176

The compounds were gas chromatographed on a 3 % SE-30 column as pure compounds a t 225oC and as s i l y l a t e d compounds a t 25OoC, as shown i n Table 18.1. TABLE 18.1 RETENTION TIMES (MINUTES) OF LYSERGIC AND ISOLYSERGIC A C I D AMIOES4 on a 3 f t long glass column, packed w i t h 3 % SE-30 on Chromosorb \I Compound *)

3 % SE-30 225'

1/11 III/IV V/VI VII/VIII 1x;x

3 % SE-30 250' s i l y l a t e d compound

20.0

11.1

24.7 23.9 32.3 35.6

13.6 15.2

** 1

19.4

*) For names o f compounds see t e x t , **) t h i s compound was completely decmposed As can be seen i t i s easy t o d i t i n g u i s h LSD from i t s homoloyes and from the isomeric pair

V

and

VI

by using SE-30. Retention times increase w i t h molecular weight. and the i s o -

meric p a i r s are n o t separated under these conditions. Some decomposition o f the amides and t h e i r s i l y l a t e d d e r i v a t i v e s took place i n the gas chromatographic process. Lerner and K a t s i a f i c a s

5 studied the separation o f a number o f h a l l u c i n o g e n i c drugs: N,N-

dimethyltryptamine, mescaline, psilocybine, ibogaine and l y s e r q i c a c i d diethylamide by gas chromatography on a 2 % SE-52 column. Only ibooaine nave a good, sharp mas chromatographic peak, and one microgram could be detected. The o t h e r compounds nave e i t h e r assymetrical peaks o r r e l a t i v e l y poor s e n s i t i v i t y . They were, therefore, converted i n t o t r i m e t h y l s i l y l derivat i v e s using dimethylformamide, N.0-bis(trimethylsily1)acetamide s i l a n e (1:4:1)

and 4,5,6-trimethylchloro-

as solvent-reaction m i x t u r e and heatinn f o r one hour a t 7OoC. When ibonaine

was present i n a complex mixture, l e s s than 50 % was u s u a l l y converted t o the t r i m e t h y l s i l y l d e r i v a t i v e , b u t converted and unconverted ibogaine appeared as an e a s i l y recoonizable double peak on the gas chromatogram. The r e s u l t s are presented i n Table 18.2. TABLE 18.2

GAS CHROMATOGRAPHY OF LSD AND OTHER HALLUCINOGENIC DRUGS5 on a 2 % SE-52 column Compound Dimethyl tryptamine Dimethyl tryptamine (TMS d e r i v a t i v e ) D i methvl t rvotami - . ne (TMS d e r i va ti ve) Mescaljne Mescaline (TMS d e r i v a t i v e ) Mescaline (TMS d e r i v a t i v e ) Psilocybine Psi 1ocybi ne (TMS d e r i va t i ve ) Psi 1ocybi ne (TMS d e r i va t i ve ) Ibogaine Ibogaine (THS d e r i v a t i v e ) Ibogaine Ibogaine (TMS d e r i v a t i v e ) Lysergic a c i d dietylamide Lysergic a c i d diethylamide TMS der.) Lysergic a c i d diethylamide ITMS der.) ~

References p. 184

mperature OC Temperature oort Column I n j e c t i oin n p 140 140 Proqraned i40 140 Programmed 180 180 Programed 220

220 Progamned Progamed

250 245 Programed

190 190 260 190 190

260 235 235 260 280 280 260 260 280 260 260

tR (mi n ) 8.0 10.0 12.0 5.4 32.8 19.7 4.6 21.8 30.6 11.7 12.7 39.3 39.9 14.6 11.8 47.6

176

6 To characterize LSD i n i l l i c i t preparations, Jane and klheals applied on-column s i l y l a t i o n o f LSD. N.0-bis(trimethylsily1)acetamide was found t o g i v e b e t t e r r e s u l t s than N-methyl-N-

trimethylsilyl-trifluoroacetamide as s i l y l a t i n g reagent. the l a t t e r r e s u l t e d i n lower y i e l d s o f LSD and l e d t o greater v a r i a t i o n i n the peak heights o f r e p l i c a t e d i n j e c t i o n s . The use o f glass columns was found t o be e s s e n t i a l i f on-column decomposition was t o be avoided. The oncolumn s i l y l a t i o n procedure was found t o work very w e l l , and l i n e a r , reproducible graphs could be obtained f o r s o l u t i o n s containing from 0.1 t o 5 p g o f LSD. Sperling7 i s o l a t e d LSD from d i f f e r e n t LSD t a b l e t s by column chromatography, converted i t i n t o i t s t r i m e t h y l s i l y l d e r i v a t i v e by N,O-bis(trimethylsily1)trifluoroacetamide

and heating

f o r one hour a t 8OoC. He was able t o separate LSD-TMS from the N-methyl-N-propyl-isaer TMS. The LSD f r e e base has a r e t e n t i o n time t h a t i s o n l y s l i g h t l y longer than t h a t o f the TWS der i v a t i v e and so, i f the s i l y l a t i o n i s incomplete, LSD w i l l appear as a shoulder on the main peak on the 0.25 % OV-17 column, using micro glass beads as s o l i d support. Such columns do n o t have a very l o n g l i f e time because o f bleeding o f f o f the s t a t i o n a r y phase a t the temperat u r e used. 18.2 ERGOT ALKALOIDS The Ergot a l k a l o i d s may be d i v i d e d i n t o two groups. One group comprises a s e r i e s o f simp l e r e r g o l i n e derivatives, t h e c l a v i n e a l k a l o i d s , the o t h e r group may be considered as amides o f l y s e r g i c acid. Whereas t h e f i r s t group o f a l k a l o i d s has low molecular weights (mol.wt. 238-260, fumigaclavine A 229) the amide type o f a l k a l o i d s have such h i c h molecular weiahts t h a t only the simpler amides such as l y s e r g i c a c i d amide, l y s e r g i c a c i d diethylamide (mol. w t . 323) and ergometrine pass a gas chromatographic column.

Agurell and Ohlsson8 gas chromatographed a number o f c l a v i n e a l k a l o i d s on t h r e e d i f f e r e n t

A l l c l a v i n e a l k a l o i d s could be chromatos t a t i o n a r y phases, JxR 3 X, SE-30 5 % and XE-60 5 %. graphed w i t h s a t i s f a c t o r y r e s u l t s on JxR and SE-30 columns and most s a t i s f a c t o r i l y on XE-60. The a l k a l o i d s containing hydroxyl groups showed l i m i t e d t a i l i n p . Attempts t o overcome t h i s by formation o f s u i t a b l e d e r i v a t i v e s ( t r i m e t h y l s i l y l , h e p t a f l u o r o b u t y r y l o r t r i f l u o r o a c e t y l y i e l d e d no adequate r e s u l t s w i t h e x t r a c t e d a l k a l o i d mixtures. None o f the columns were able t o separate stereoisomers. I n Figure 18.3 the separation o f the c l a v i n e a l k a l o i d s i n c l u s i v e LSD i s given on the columns u t i l i z e d . O f the l y s e r g i c a c i d d e r i v a t i v e s o n l y LSD, l y s e r p i c

- the l a s t one a f t e r p y r o l y s i s i n the i n - could be chromatographed on SE-30 and JxR columns a t compara-

a c i d amide and l y s e r g i c a c i d methylcarbinolamide j e c t o r t o l y s e r g i c a c i d amide

t i v e l y h i g h temperatures. Thus, l y s e r g i c a c i d amide had a r e t e n t i o n time o f 14.7 minutes on 9 3 % JxR a t 24OoC. Sondack developed a method f o r the ?as chromatographic determination o f ergometrine as t r i m e t h y l s i l y l d e r i v a t i v e . As s i l y l a t i n g reaqent, a m i x t u r e o f 75 u1 o f N-tri-

methylsilyldiethylamine. 150 u l o f d r y p y r i d i n e and 100 p1 o f N-trimethylsilylimidazole was used. Brucine was u t i l i z e d as an i n t e r n a l standard and q u a n t i t a t i v e estimations c a r r i e d o u t f o r ergometrine maleate i n t a b l e t s and injectables. c o n t a i n i n g 0.2 mg per t a b l e t o r m i l l i l i t e r . A residual standard d e v i a t i o n o f f 1 X f o r i n j e c t a b l e s and f 3 % f o r t a b l e t s was found, and a r e l a t i v e e r r o r o f less than 1 % f o r i n j e c t a b l e s . Denraded samples a l s o showed on the chromatograms peaks o f d e r i v a t i z e d ergometrinine and d e r i v a t i z e d lumierganetrine. A method f o r the gas chromatographic determination o f agroclavine was developed by Barrow and Quigley". The t r i f l u o r o a c e t y l d e r i v a t i v e and the t r i m e t h y l s i l y l d e r i v a t i v e were chroma-

FIGURE 18.3

RETENTION TIMES OF CLAVINE A L K A L O I D S ~ on 3 % JxR at 24OoC. 5 % SE-30 at 265OC and 5 % XE-60 at 225OC (6 ft by 3 n plass columns). 1 = agroclavine, 2 = elymoclavine, 3 = chanoclavine-I, 4 = chanoclavine-11, 5 = isochanoclavine-I, 6 = setoclavine, 7 = isosetoclavine, 8 = penniclavine, 9 = isopenniclavine. 10 = adihydrolysergol, ll = festuclavine, 12 = pyroclavine. 13 = costaclavine, 14 = fumioaclavine B, 15 = fumigaclavine A , 16 = lysergene. 17 = lyseroine, 18 = lyseroole, 19 = isolyserpole, 20 = cycloclavine, 21 = LSD.

3% J x R I Gas- Chrom 0 . 2LO' 6 3 7 L15

5JL

13 11 1

I

I

0

I

19 2

I

L

2

6

10 min

8

5 % SE-30lGas Chrom P.265'

21

5 % XE-60IGas-Chrom 11 1312

1

0

1

1

2

5

171

1

P.225O 3

15 16 14

1

L

'

I

6

'

l

8

I

1

lOmin

tographed. Derivatization led to increase of the thermostability and change in retention times, permitting a better GLC identification. Mhen using 1-3 % of SE-30, an extensive tailing o f TMS-agroclavine took place, presumably by partial adsorption to active sites in the solid support. With a higher percentage of stationary phase, no tendency to tailina was observed. The OV-phases showed very long retention times and offered no advantage over the SEphases. The TMS-agroclavine gave one single peak on the gas chromatograms, whereas the TFAagroclavine cave two peaks of derivatized agroclavine and sometimes, also, a peak of underivatized agroclavine. The authors were not able to reproduce a sinole product or reproducibly obtain a consistent mixture of products with trifluoroacetic anhydride. Because of the low volatility and thermal instability at hiph temperatures o f the dihydroergotoxine alkaloids, Szepesi and Gazdag" developed a gas chromatopraphic method for the separation and identification of these a1 kaloi ds (dihydroergocristine, di hydroergokryptine

References p. 184

178

and dihydroergocornine) based on q u a n t i t a t i v e decomposition o f the a l k a l o i d s catalyzed by the metal surface o f the i n j e c t i o n p o r t and d i f f e r e n t m i g r a t i o n r a t e s o f the peptide moieties from the various a l k a l o i d s during the decomposition. The f r e e bases were i n j e c t e d f o r pas chromatography

- and,

depending on the temperature o f the i n j e c t i o n p o r t , the chromatooram

varied, as can be seen i n Figure 18.4. FIGURE 18.4

GAS CHROMATOGRAM OF DIHYDROERGOTOXINE

ALKALOIDS~~

on a s t a i n l e s s s t e e l column, 1 m long, packed w i t h 2 % Dexsil 300; temperature p r o o r a m i n o 180-28OOC a t 5oC/min; i n j e c t i o n p o r t temperature (A) 195OC. ( 8 ) 210°C, (C) 235OC, ( 0 ) 290bC 1 = dihydroergocornine, 2 = dihydroergokryptine, 3 = d i h y d r o e r g o c r i s t i n e , x = unknown. mV

5

io

min

TABLE 18.3 RETENTION TIMES OF ERGOTOXINE AND DIHYDROERGOTOXINE ALKALOIDS” on the columns as described i n Figure 18.4; temperature: 2OO0C f o r 18 min, then proprammed from 200°C t o 3OO0C a t 20°C/min; temperature i n j e c t i o n p o r t : 2350C. Compound

tR(min) Column temp. OC

Compound

tR(min)

Column temp.0C

Ergocorn ine Ergocorni n i ne D i hydroergocornine D i hydroergocornini ne

9.01

225

Ergocri s t i n e Ergocri s t i n i n e D i hydroergocri s t i n e D i hydroergocri s t i n i ne

14.6

253

Ergokrypti ne Ergokrypti nine D i hydroe r go krypt ine D i hydroergokrypti n i ne

10.03

230

Phenyl butazone

11.92

239

179

De Zeeuw e t a1.l’

developed a method f o r the i d e n t i f i c a t i o n o f ergotamine present i n

p u t r i f i e d blood samples. The samples were hydrolyzed w i t h h y d r o c h l o r i c acid, t h e pH adjusted t o 9.5 and e x t r a c t i o n performed w i t h chloroform. Gas chromatooraphy o f the r e s i d u e obtained i n t h i s way on a SE-30 column gave one major peak. By computerized GC-tIS, the compound r e sponsible f o r the peak was i d e n t i f i e d as a c y c l i c dipeptide, phenylalanine-proline lactam.

A minor peak was i d e n t i f i e d as i t s pyruvoyl precursor. I n p u t r i f i e d blood the L-phenylalanineL - p r o l i n e lactam dominated q u a n t i t a t i v e l y , b u t a l s o small amounts o f L-phenylalanine-D-prol i n e lactam were found. I n a hydrolysate o f ergotamine i n water and non-decomposed blood, Lphenylalanine-D-pro1 i n e lactam was the major component, whereas L-phenylalanine-Lbprol i n e lactam was present i n minor amounts together w i t h i t s pyruvoyl precursor. The presence i n blood samples o f these compounds seems t o be s p e c i f i c f o r erpotamine, dihydroergotamine and t h e i r respective - i n i n e stereoisomers. I n a l a t e r p u b l i c a t i o n , Van Mansvelt e t a1.13 s t a t e d t h a t the degradation o f ercotamine took place o n l y t o a l i m i t e d extent d u r i n g the a c i d h y d r o l y s i s and t h a t i t occurred mainly i n the i n j e c t i o n p o r t o f the gas chromatograph a t the h i g h temperatures used. They s t u d i e d the a p p l i c a b i l i t y o f t h i s thermal decomposition o f s i x ergot-peptide a l k a l o i d s : ergotamine, ergosine, e r g o c r i s t i n e , ergokryptine, eroocornine and eroostine i n connection with t h e i r i d e n t i f i c a t i o n w i t h gas chromatography. The gas chromatopraphy was performed i n an a l l - g l a s s system and the i n j e c t i o n p o r t temperature was 30OoC. The decomposition pathways o f erpotamine are given i n Figure 18.5 and a gas chromatogram o f the degradation products i n Figure 18.6. FIGURE 18.5 DECOHPOSITION PATHWAYS OF ERGOTAtIINEl3

1 = Ergotamine. LSA i n d i c a t e s the l y s e r g i c a c i d moiety. The dashed l i n e i n d i c a t e s t h a t the cleavage takes place between the a-nitrogen atom and the a-carbon atom o f the amino a c i d i n volved, namely a-hydroxyalanine. This r e s u l t s i n a pyruvoyl precursor o f phenylalanine-prol i n e lactam which can have s t r u c t u r e 2 o r 3. S t r u c t u r e 3 i s t h a t o f pyroerclotamine, a r e f e r ence sample o f which showed the same GC and MS behaviour as the above pyruvoyl precursor. However, t h i s does n o t preclude s t r u c t u r e 2 (which i s more s t a b l e ) f o r t h i s precursor, b u t f o r which no reference sample was a v a i l a b l e . S t r u c t u r e 4 i s the phenylalanine-praline lactam, which i s obtained i n two forms, namely L-phe-D-pro lactam and L-phe-L-pro lactam.

/

180 FIGURE 18.6 GAS CHROMATOGRAM OF ERGOTAMINE BASE13 on a 1.8 m by 2 mn glass column. packed w i t h 3 I SE-30 on Chromosorb W HP a t 225OC. 1 = L-phe-1-pro lactam. 2 = L phe-D Dro lactam, 3 = pyruvoyl precursor o f L-phe-0-pro lactam

The o t h e r Ergot-peptide a1 kaloids: ergosine, ergostine, e r g o c r i s t i n e , ergokryptine and ergocornine, showed s i l i m a r decomposition p a t t e r n s i n the i n j e c t i o n p o r t , i n t h a t they a l l gave c y c l i c lactams c o n t a i n i n g two amino acids and a precursor o t these lactams, which, besides these two amino acids, contained a deaminated t h i r d hydroxyamino acid. T h i s was confirmed by GC-MS.

Table 18.4 summarizes the various degradation products, t h e i r r e t e n t i o n

times and r e t e n t i o n indices on SE-30 and t h e i r quasi-molecular ions i n C I - H S . F i y r e 18.7 shows a gas chromatogram o f a mixture o f the s i x Ergot-peptide a l k a l o i d s , i n j e c t e d as f r e e bases i n ethanol: each i n d i v i d u a l a l k a l o i d can be i d e n t i f i e d by the presence o f two o r t h r e e c h a r a c t e r i s t i c degradation products. Plomp e t a l .14 i n v e s t i g a t e d f u r t h e r the thermal decomposition o f the dihydroergotoxine a l k a l o i d s f o r q u a n t i t a t i v e purposes and they found t h a t the r e a c t i o n i s n o t catalyzed by a metal surface as s t a t e d by Szepesi and Gazdagll. They used an a l l - g l a s s system and a select i v e and s e n s i t i v e n i t r o g e n detector and obtained a s e n s i t i v i t y o f about 1-10 ng f o r the various dihydroergotoxine a l k a l o i d s . Using a combination o f GC and chemical i o n i z a t i o n MSS

(CI)

fragmentography, a 1 0 - f o l d increase i n s e n s i t i v i t y , compared w i t h the GC method used,

was obtained. By means o f e l e c t r o n impact ( E I ) and chemical i o n i z a t i o n mass spectrometry, the mechanism o f the thermal decomposition o f the dihydroerpotoxine a l k a l o i d s was elucidated. The r e l a t i v e r e t e n t i o n times o f the decomposition products o f the dihydroernotoxine a1 k a l o i d s are given i n Table 16.5. The i n f l u e n c e o f the i n j e c t i o n p o r t temperature on the decomposition o f the a l k a l o i d s i s i l l u s t r a t e d i n Figure 18.9. The r e p r o d u c i b i l i t y o f the method i s good, as can be seen from Table 18.6.

181

TABLE 18.4 GLC DEGRADATION PRODUCTS. RETENTION TItlES. RETENTION INDICES AND QUASI-HOLECULAR IONS OF SOME ERGOT PEPTIOE ALKALOIOS13

A1 k a l o i d

Amino acids i n peptide Degradation products Peak moiety detectable by GLC *)

Ergotamine

a-hydroxy-Ala

Ergosi ne

a-hydroxy-Ala

Ergostine

a-hydroxy-aami n obu tyr ic acid

Ergocristine

a-hydroxy-Val

Ergokryptine

a-hydroxy-Val

Ergocornine

a-hydroxy-Val

tR

Phe Pro L-Phe-L-Pro lactam 1 6.33 L-Phe-D-Pro 1 actam 2 5.76 pyruvoyl-Phe-Pro lactam **) 3 7.00 Leu Pro Leu-Pro lactam 4 1.93 pyruvoyl-Leu-Pro 5 2.81 lactam **) Phe P r o L-Phe-L-Pro lactam 1 6.33 L-Phe-0-Pro lactam 2 5.76 a-ketobutyryl -Phe6 9.20 Pro lactam **) Phe Pro L-Phe-L-Pro lactam 1 6.33 L-Phe-0-Pro lactam 2 5.76 a - k e t o i s o v a l e r y l -Phe7 10.58 Pro lactam **) Leu Pro Leu-Pro lactam 4 1.93 a - k e t o i s o v a l e r y l -LeuPro lactam 8 4.28 Val Pro Val-Pro lactam 9 1.51 a-ketoisovaleryl-valPro lactam 10 3.55

Ret. index

HH' (CI-MS)

2300 2275

245 245

2340 1900

315 211

2075 2300 2275

281 245 245

2435 2300 2275

329 245 245

2480 1900

343 211

2175 1810

309 197

2100

295

*) Peak number i n Figure 18.7, **) The exact s t r u c t u r e o f t h i s component i s unknown a t the present time. As i n d i c a t e d i n Figure 18.5, pyruvuoyl-Phe-Pro lactam may be present as an a,B-diketo s t r u c t u r e ( 2 ) o r as a s t r u c t u r e w i t h a dioxane r i n g (3). This a l s o a p p l i e s t o the other deaminated t r i p e p t i d e lactam components i n t h i s t a b l e . FIGURE 18.7 GAS CHROMATOGRAM OF S I X ERGOT-PEPTIDE ALKALOIDSI3 on SE-30 (see Figure 18.6). For peak numbers, see Table 18.4

References p. 184

182 FIGURE 18.8 STRUCTURES OF DIHYDROERGOTOXINE ALKALOIDS AND THEIR THERMAL DECOMPOSITION PRODUCTS14

DLAA +

nome

I

-R

R peptide moiety

TABLE 18.5 RELATIVE RETENTION TIMES OF DECOMPOSITION PRODUCTS OF OIHYDROERGOTOXINE ALKALOIDS14 on a glass column, '1.25 m x 3.8 n I.D., temperature programming

packed w i t h 3 % SE-30 on Supelcoport, a t 20O-27O0C,

Compound I n t e r n a l standard O i hydroergocorni ne D i hydroergokrypti ne D i h y d r o e r g o c r i s t i ne Dihydrolysergic a c i d amide

Re1a t i ve r e t e nt i on time 1-00 ( t R = 12.67 min) 0.46 0.57 1.42 2.42

A p p l i c a t i o n o f the method t o the determination o f the dihydroergotoxine a l k a l o i d s i n commercial samples and pharmaceutical preparations were a l l c a r r i e d o u t w i t h the f r e e base o f the a l k a l o i d s since a n a l y s i s o f t h e i r s a l t s cannot be performed w i t h o u t excessive decomposit i o n . The mean dihydroergotoxine methane sulphonate content o f f i v e commercial samples was 95,2 t 2.9 %, i n c l u d i n g a mean dihydroergocornine content o f 30.9 f 0.8 %, a mean dihydroergokryptine content of 32.8

f

2.1 % and a mean d i h y d r o e r g o c r i s t i n e content o f 31.5

f

3.5 %.

Almost the same data were observed f o r d i hydroergotoxine t a b l e t s obtained from two manufacturers.

183 FIGURE 18.9

GAS CHROMATOGRAM OF DIHYDROERGOTOXINE ALKALOIDS14 GLC conditions, see Table 18.5. 1 = Dihydroergocornine, 2 = dihydroergokryptine, 3 = dihydroergocristine. 4 = i n t e r n a l standard.

A

B

C

I, 1! io- to

i

1

20

10 20

min

TABLE 18.6 RESULTS OF REPRODUCIBILITY STUDIES14 Standard d e v i a t i o n (%)

Concentration o f compound (ugh11

D i hydroergocornine

Dihydroergokryptine

Dihydroergocristine

11 6 2

16 6 4 3 3

0.10

0.20 0.50 1.00 3.00

1 2

TABLE 18.7 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY OF ERGOT ALKALOIDS Column

S o l i d support mesh

Stat.phase

s . s . , 1.5 m x 3 mn I.D. glass, 1.8 m x 3 mm glass

MGB MGB

SE-30 SE-30 SE-52

glass, 3 f t

CW 80-100

SE-30

References p. 184

%

Temperature

Comp. Prep.

Ref.

0.2 0.3 2

27OoC 28OoC 245OC

1 2

3

225OC 25OoC

LSD hy.id. LSD i d . LSD i d . LSD M S i d . ly.am. 1y am. TMS

.

4

184

TABLE 18.7 (continued) Col umn

S o l i d support mesh

Stat.phase SE-52

glass, 1.8 m x 3 rn 1.0. CW AlJS 80-100 glass, 6 . f t x 2 mn I.D. TMGB 100-120 glass, 6 f t x 3 mn GQ AWS 100-120 GP AWS 100-120 GP AWS 100-120 glass, 1.2 m x 6.4 mn O.D. GQ 80-100 glass, 5 f t x 3 rmn CW AWS 80-100

s.s.,

1 m x 3.2 mm

GQ 80-100

glass, 1.8 m x 2 mn 1.0. CG HP 80-100 glass S, 1.8 m x 2 n I.D.

CG HP 80-100

glass, 1.25 m x 3.8 mn 1.0. Sup. 80-100

OV-17 OV-17 JxR SE-30 XE-60 ov-1 SE-30 SE-52 OV-17 DeX.

% 2' 2 1.5 0.25 3 5 5 1 10 10

SE-30 SP 22'50 SE-30

2 3 3 3

SE-30

3

Temperature

Comp.Prep.

25OoC 245OC 27OoC 258OC 24OoC 265OC 225OC 26OoC 210Oc 1980c pr 220OC

hal.LSO.id. hal.LSD.TMS.id. LS0.ocd.id. LS0.isom.TMS.

Ref.

6 7

clav.alk.der. 5.

ernm.TMS.ant.

9

anr.TFA.qnt. ay.TMS. qn t .

180-280°C pp rr dh.ergt. 200-300°C decp. 2250C ertm.decD.id. 225°C tox. 225OC erg.pept.alk. decp.id. 200-270°C p r e r p t . a l k . q n t . decp.

11 12

,, I J

14

TABLE 18.8 ERGOT ALKALOIDS

-

LIST OF ABBREVIATIONS

agr = agroclavine alk = alkaloid AW = a c i d washed clav = clavine CG = Chromosorb G CW = Chromosorb W decp = decomposition product der = d e r i v a t i v e Dex = Dexsil 300 dh.ergt = dihydroergotoxine erg.pept = e r g o t peptide e r g t = ergotoxine ergm = ergometrine ertm = ergotamine GP = Gas Chrom P GQ = Gas Chrom Q ha1 = hallucinogenic drugs HP = h i p h performance hy = hydrogenated

1.0. = i n s i d e diameter i d = identification i s m = isomer LSD = l y s e r g i c a c i d diethylamide ly.am = l y s e r g i c a c i d amide MGB = micro glass beads ocd = on-column d e r i v a t i z a t i o n 0.0. = outside diameter p r = (temperature) pray-amin? s = separation S = silanized s.s = stainless steel Sup = Supelcoport TFA = t r i f l uoroacetyl TMGB = t e x t u r e d micro c l a s s beads TMS = t r i m e t h y l s i l y l tox = toxicology

18.3 REFERENCES 1 C. Radecka and I.C. tiigam, J . Pharm. S c i . , 55 (1966) 861. 2 N.A. Katz, G. Tadjer and !J.A. A u f r i c h t . J . C h r o m a t o g r . , 21 (1967) 545. 3 M. Lerner, m i l . Narc., 19 (1967) 39. 4 K. Bailey, 0. Verner and 0. Legault, J. dssoc. off. Anal. C h e m . , 56 (1973) 88. 5 M. Lerner and M.D. Katsiaficas. B u l l . arc., 21 (1969) 47. 6 J. Jane and B.B. Wheals, J . C h r o m a t o q r . , 84 (1973) 181. 7 A.R. Sperling, J . P h a r m . S c i . , 12 (1974) 265. 8 S. Agurell and A. Ohlsson, J. C h r o m a t o q r . , 6 1 (1971) 339. 9 D. Sondack, J . Pharm. S c i . , 63 (1974) 584. 10 K.D. Barrow and F.R. Quigley, J . C h r o m a t o q r . , 105 (1975) 393. 11 G. Szepesi and M. Gazdag, J . C h r o m a t o q r . , 122 (1976) 479. 12 R.A. De Zeeuw, F.J.W. Van Hansvelt and J.E. Grevin?, J. Forensic S c i . , 22 (1977) 550. 13 F.J.W. Van Mansfelt, J.E. Greving and R.A. De Zeeuw, J . C h r o m a t o q r . , 151 (1978) 113. 14 T.A. Plomp, J.G. L e f r i n k and R.A.A. Maes, J . C h r o m a t o q r . , 151 (1978) 121.

Refsmncea p. 184

185

11.6 STEROIDAL ALKALOIDS (GLYCOALKALDIDS) Chapter 19 SOLANUN

ALKALOIDS

.......................................................... .................................................................

19.1. SO~ZIWI a1 k a l o i d s 19.2. References

185 186

19.1. SOLANUM ALKALOIDS To be able t o gas chromatograph the h i g h molecular weiqht p l y c o a l k a l o i d s present i n potatoes (solanine, m.w.

869, demissine, m.w.

1034) Herb e t al.'

p e m e t h y l a t e d the glyco-

a l k a l o i d s t o improve t h e i r v o l a t i l i t y f o r gas chromatographic analysis. Pennethylated derivat i v e s o f glycoalkaloids were p r e f e r r e d t o t r i m e t h y l s i l y l d e r i v a t i v e s because o f the greater v o l a t i l i t y and lower molecular weights o f the former. The a l k a l o i d s were e x t r a c t e d f r o m samples o f 20 g ground fresh potatoes w i t h chloroform-methanol (1:2).

A f t e r a d d i t i o n o f an

aqueous s o l u t i o n o f 0.8 % sodium sulphate. the a l k a l o i d s were found i n the methanol l a y e r . This was separated from the r e s t and evaporated t o dryness. The p l y c o a l k a l o i d s were e x t r a c t e d w i t h absolute methanol and the pennethylation performad w i t h methyl sulphate. sodium hydride and methyl iodide. The permethylated compounds were e x t r a c t e d w i t h benzene and the oas chromatography performed on a packed 3 % Dexsil 300 column and an 3 % DV-1 column. With the OV-1 column i t was found t h a t the glycoalkaloids could be e l u t e d a t temperatures 25-30'

lower

than on the Dexsil 300 column, w i t h equivalent separation. FIGURE 19.1 CHROMATOGRAM OF PERRETHYLATED GLYCDALKALDIDS~

on a 90 cm by 2 m glass column, packed w i t h 3 % Dexsil 300 on Supelcoport; temperature prog r a m i n g 275-35DoC. 1 = solanidine, 2 = B-chaconine, 3 = a-chaconine, 4 = a-solanine.

1

F2O/min

200

ReferencesD. 186

+1Olrnin

275

1

I

350 O C

186 Because o f the r e l a t i v e h i g h column temperature t h a t had t o be used (Oexsil 300: 275-35OoC by temperature programing; OV-1:

33OoC isothermal) bleeding took place and a l s o d e t e r i o r a -

t i o n o f the column. This r e s u l t e d i n poor separation and the formation o f a r t e f a c t s a f t e r some days o r weeks o f use o f the column. A chromatogram o f some permethylated p l y c o a l k a l o i d s i s found i n Figure 19.1 and the r e l a t i v e r e t e n t i o n and the r e t e n t i o n temperatures i n Table 19.1. TABLE 19.1 RELATIVE RETENTION AND RETENTION TEMPERATURES OF PERMETHYLATED GLYCOALWILOIDS' on a 3 % O V - 1 packed column on Gas Chrom Q, 120 cm by 2 m, a t 33OoC. Retention r e l a t i v e t o a-solanine, time o f e l u t i o n 12 min. A l k a l o i d (permethylated)

Re1a t i ve r e t e n t i o n

Solani d i ne b-Chaconine a-Chaconi ne a-Solanine Demi ssine Tomatine

Retention temperature ,OC 223 303 324 327 330 330

0.35 0.87 1.00 2.84 4.23

TABLE 19.2 EXPERIMENTAL CONDITIONS USE0 FOR GAS CHROMATOGRAPHY OF STEROIDAL ALKALOIDS (GLYCOALKALOIOS) Column

S o l i d support mesh

glass, 90 cm x 2 mn glass, 120 cm x 2 nun

Sup. 100-120 GQ 100-120

Statephase

%

Temperature

3

275-3500c pr 33OoC

Oex.

ov-1

Comp.Prep.

prm.alk.s.

Abbreviations: Sup = Supelcoport, GQ = Gas Chrom Q, Dex = Oexsil 300, p r = (temperature) programming, prm = permethylated, a l k = a l k a l o i d , s = separation 19.2

REFERENCES

1 S.F. Herb, Th.J. F i t z p a t r i c k and S.F. Osman, J. A g r i c . Food Chem., 23 (1975) 520.

Ref.

1

187

11.7 XANTHINE ALKALOIDS

Chapter 20 XANTHINE ALKALOIDS

. . .. . . . .. . . .. . . . . . . . . . . . . .. . . .. ................................................................... . . ... . . ... . . . . . . . . .................................................

20.1. Xanthine a l k a l o i d s . . . . . . .. . . . . . . .. . .. .. , .. .. 20.2. C a f f e i n e 20.2.1. C a f f e i n e i n c o f f e e , t e a and beverages . . . . . . .. . . .. 20.2.2. C a f f e i n e i n plasma 20.2.3. C a f f e i n e i n p h a r m a c e u t i c a l p r e p a r a t i o n s .. . .. 20.3. T h e o p h y l l i n e . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 20.3.1. T h e o p h y l l i n e i n p h a r m a c e u t i c a l p r e p a r a t i o n s .. . . . . . . . .. 20.3.2. T h e o p h y l l i n e i n b i o l o g i c a l f l u i d s 20.3.2.1. D e t e r m i n a t i o n o f t h e o p h y l l i n e as such . . . .. . . . . . . . 20.3.2.2. Determination o f theophylline a f t e r d e r i v a t i z a t i o n A. On packed column I. Propylation . , .. .. . . .. ...... . I I. M e t h y l a t i o n . . ... .. . . .... . ... 111. B u t y l a t i o n I V . Pentylation V. P e n t a f l u o r o b e n z y l a t i o n . .. .... .. .. .. B. On c a p i l l a r y columns I. Ethylation 20.4. References

. . .. . . . .. . .. . . . . . . . . ..

187 190 190 19 1 194 199 200 200 20 1 202 202 202 20 3 204 206 207 208 208 2 10

.. . . . . ... . .. . .... . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. .................... .............. ... . . .. . ....... ...................................... . . . . . .. ... . . .. . . . . ... . . ... . . . . . . . . .. . . . . . . .. . .

......................................... ........................................ . . . . . . . . . . . .. . .. .................................. ......................................... .................................................................

20.1.

XANTHINE ALKALOIDS

Several a u t h o r s have i n c l u d e d one o r more n a t u r a l l y o c c u r r i n g x a n t h i n e d e r i v a t i v e s ( c a f f e i n e , theobromine and t h e o p h y l l i n e ) i n t h e i r gas chromatographic s e p a r a t i o n o f a l k a l o i d s . L l o y d e t a 1 . l used a packed column w i t h SE-30 on Chromosorb !I, Baerheim Svendsen‘,

so d i d Brochmann-Hanssen and

P a r k e r e t a l . 3 and Kazyak and Knoblock4. K o l b and P a t t 5 i n t r o d u c e d more

p o l a r s t a t i o n a r y phases ( n e o p e n t y l g l y c o l sebacate). Brochmann-Hanssen and Fontan6”

applied

v a r i o u s p o l a r s t a t i o n a r y phases (EGSS-Y and HI-EFF-8B) and such phases on p o l y v i n y l p y r r o l i d one t r a t e d s u p p o r t . J a i n and K i r k

8 used HI-EFF-8B f o r t h e s e p a r a t i o n o f c a f f e i n e and t h e o -

bromine. The f i r s t s y s t e m a t i c i n v e s t i g a t i o n on t h e aas chromatographic s e p a r a t i o n o f x a n t h i n e de9 r i v a t i v e s was p u b l i s h e d by Reisch and I4alker . The n a t u r a l l y o c c u r r i n n x a n t h i n e s , c a f f e i n e , theobromine and t h e o p h y l l i n e , as w e l l as a number o f d e r i v a t i v e s , were gas chromatographed on an 1.5 % packed SE-30 column on Chromosorb

W. I n Table 20.1 t h e compounds a r e g i v e n and

and i n F i g u r e 2 0 . 1 t y p i c a l gas chromatograms. Kamei and A t s u s h i ”

chromatographed x a n t h i n e d e r i v a t i v e s on packed columns w i t h SE-30,

SE-52, QF-I, OV-1, 0V-17 and XE-60 as s t a t i o n a r y phases. C a f f e i n e , theobromine, t h e o p h y l l i n e , o x y e t h y l t h e o p h y l l i n e , o x y p r o p y l t h e o p h y l l i n e and d y p h y l l i n e were i n v e s t i g a t e d . Hass s p e c t r o m e t r y was c a r r i e d o u t w i t h an o n - l i n e H i t a c h i 002-type h i g h r e s o l u t i o n mass system. The r e l a t i v e r e t e n t i o n times o f t h e compounds i n v e s t i g a t e d a r e g i v e n i n T a b l e 20.2. The TMS- and T F A - d e r i v a t i v e s o f t h e m e t h y l a t e d x a n t h i n e s had good ?as chromatographic p r o p e r t i e s . T y p i c a l chromatograms a r e g i v e n i n F i g u r e 20.2. I n t h e i r p u b l i c a t i o n on t h e gas chromatography o f a l k a l o i d s on c a p i l l a r y columns, l l a s s i n g i l l and Hodokins”

References p. 210

a l s o chromatographed c a f f e i n e . A 100 f e e t l o n g c a p i l l a r y c o a t e d w i t h

188

TABLE 20.1 XANTHINE DERIVATIVES GAS CHROMATOGRAPHED BY REISCH AND WALKER'

1 = Caffeine 2 = Theobromine 3 = 4 = 5 = 6 = 7= 8= 9=

Theophyl 1i n e 7-(2'-Hydroxyethyl )-theophyll i n e 1-( 2' -Hydroxypropyl ):theobrunine

7-(N-methyl-N-hydroxyethyl)-3-amino-2-hydroxypropyl)-theophyll ine 7-(2,3-Dihydroxypropyl)-theophylline 1-Ally1 theobromine 7-Propin-(2' )-yltheophyll i n e

10 = l-Propin-(2')-yltheobromine 11 = 1-n-Hexyl theobromine 12 = 8-Chl orotheophyl 1i n e FIGURE 20.1 GAS CHROMATOGRAMS OF XANTHINE DERIVATIVES LISTED IN TABLE 20.1' on a 6 f t by 1/4 i n c h packed column, w i t h 1.5 % SE-30 on Chromosorb W. (1) i n diethylamin s o l u t i o n and tem erature p r o g r a m i n g 200-3OO0C, ( 2 ) i n aqueous s o l u t i o n and temperature programning 200-300gC and (3) i n chloroform solution. 7 min isothermal, then temperature prog r a m i n g t o 25OoC. For peak numbers, see Table 20.1.

2

I

i i 1 Z i b +

I

1

Qmin

1

1

2

I

I

'

l

l

I

1

1

2

3

I 5

6

7

8

9

1

3

1

I

5

I

10 I l m i n

1

6

1

7

I

0

9

I

I

m

i

n

189

TABLE 20.2 RELATIVE

RETENTION TIMES OF METHYLATED XANTHINES~O on s t a i n l e s s s t e e l columns, 1 m by 3 mn, packed w i t h various s t a t i o n a r y phases on Chrmosorb W. a t d i f f e r e n t temneratures

Compound

1.5 % SE-30 3 190°c Caffeine 0.43 Theobromine 0.61 Theophyl 1ine 1.02 Oxypropyl theophyl - 1.00 l i n e (4.1 min) Oxyethyltheophyl- 1.15 1i n e Dyphyll i n e 3.29

% SE-30 1.5 % SE-52 3 20oOc 21OoC 0.42 0.45 0.82 0.61 1.00 0.95 1.OO 1.00 (3.3 min) (4.4 min) 1.03 1.16 3.82

2.75

% QF-1 2 % OV-1 2 % OV-17 3 21OoC 205OC 24OoC 0.46 0.49 0.45 0.77 0.77 0.54 0.85 0.80 0.81 1.00 1.00 1.00 (3.9 min)(3.5 min)(6.3 min) 1.18 1.29 1.19 2.51

3.23

2.49

% XE-30 23OoC 0.32 0.57 1.19 1.00 (3.7 min) 1.38 3.78

FIGURE 20.2

GAS CHROMATOGRAMS OF ( 1 ) TFA AND (2) TMS DERIVATIVES OF METHYLATED XANTHINES~O on a s t e e l column, t r e a t e d with MDS, 1 m by 3 mn, packed w i t h 3 % XE-60 on Chromosorb W, 220 C. 1 = oxypropyltheophylline, 2 = oxyethyltheophylline. 3 = dyphylline, 4 i n t e r n a l standard ( d i o c t y l p h t h a l a t e )

stainless

1

I‘ -

2

-

0

2

i

6

8 min

2

i

6

8 mir

QF-1, a 200 f e e t long one coated w i t h SE-30 and a 100 f e e t l o n g one coated w i t h Apiezon L

were used w i t h temperature p r o g r a m i n g up t o 25OOC. On the Apiezon L column c a f f e i n e was n o t eluted. Dohn e t a1.12 used a glass c a p i l l a r y coated w i t h T r i t o n X 305 f o r a study on i l l i c i t heroin samples, which r e g u l a r l y contained c a f f e i n e . Caffeine was e l u t e d as a sharp peak. Christophersen and R a s m ~ s s e n ’ included ~ c a f f e i n e i n a study on glass c a p i l l a r y gas chromatography of n a r c o t i c drugs, using SE-30 as s t a t i o n a r y phase and temperature programming up t o 25OoC. Polysiloxane deactivated c a p i l l a r i e s glass and fused s i l i c a - coated w i t h Carbowax 20 M

-

Rshnnca p. 210

190

14

or CP-Sil 5 ( a dimethylpolysiloxane phase prepared from SE-30) were used by Schepers e t a l . for the analysis of drugs including alkaloids such as caffeine and theophylline. Because of the extensive use of theophylline f o r the treatment of bronchial asthma and other cardiorespiratory disorders, many investigations have been carried out by means of gas chromatography t o determine theophylline in serum, plasma and saliva. In many cases theophylline has been gas chromatographed as such, in some other cases a f t e r derivatization. Methyl derivatives15916y17 'I8, n-butyl deri vati ves19~20y21y22*23~24.25,26, propyl derivatives27 ,

pentyl derivatives28y29a n d pentafluorobenzyl derivatives30y31 have been made i n order t o obtain better gas chromatographic properties of theophylline and thus b e t t e r detection possib i l i t i e s . A number of stationary phases have been applied, from non-polar ones (SE-30) t o very polar ones (HI-EFF 88). OV-17 i s the stationary phase t h a t has mostly been used. 20.2. CAFFEINE 20.2.1. Caffeine in coffee, tea and beverages A great number of methods have been developed f o r the determination of caffeine in coffee, gravimetric, spectrophotometric, iodometric and colourometric methods. Vitzthum3' applied gas chromatography f o r such determinations. He developed a method for the determination of caffeine in decaffeinated coffee.

e.g.

Because gas chromatography of caffeine on packed columns of 2.5 % SE-30 on Chromosorb gave t a i l i n g , Vitzthum3* preferred t o use a packed column of 0.2 % SE-30 on micro-glass beads After extraction of the coffee-extract with 96 % ethanol under reflux, the ethanolic extract was gas chromatographed directly, using pyrene as an internal standard. A typical chromatogram i s shown in Figure 20.3. FIGURE 20.3 CHROMATOGRAM OF CAFFEINE I N DECAFFEINATED SOLUBLE COFFEE3'

on a 2 m long packed column of 0.2 % SE-30 on micro-glass beads a t 210OC. 1

=

caffeine, 2 =

pyrene (internal standard).

I I

0

1

20

min

191 Good agreement was found between the r e s u l t s obtained by means o f gas chromatography and by UV-spectrophotometry.

However, the gas chromatographic method i s more s u i t a b l e f o r r a p i d

determinations. Newton33 developed a gas chromatographic method f o r the determination o f c a f f e i n e i n i n s t a n t tea, using the e x t r a c t i o n and clean-up procedure o f Yeransian e t a1.34, p r i o r t o the gas chromatographic analysis. This was c a r r i e d o u t on a packed column o f 10 % DC-200 on Gas Chrom Q a t 190°C using a thermoionic KC1 detector. This detector i s s e n s i t i v e t o as l i t t l e as 1 ng caffeine. Comparative studies showed t h a t the r e s u l t s obtained w i t h the gas chromatographic method and w i t h the UV-spectroscopic method o f Yeransian e t a1.34were i n good agreement. Fogden and U r r ~ determined ~ ~ caffeine i n c o l a d r i n k s a f t e r a thorough e x t r a c t i o n o f the c a f f e i n e from a l k a l i n i z e d c o l a (amnonia) w i t h dichloromethane, concentration o f the s o l u t i o n , and gas chromatography on a packed column of‘Versamide 930 2 % on Phasesep

N

using procaine

as an i n t e r n a l standard. The method gives a recovery o f 95 t o 98 % o f the c a f f e i n e added t o cola preparations. S c h i l l i n g and G8136 described methods f o r a l l types o f marketed c o f f e e products. Coffee was e x t r a c t e d w i t h chloroform and the chloroform s o l u t i o n was gas chromatographed d i r e c t l y using pyrene as an i n t e r n a l standard. A packed column o f 10 % SE-30 on Varaport was used f o r the analysis. Sharp peaks were obtained, very s u i t a b l e f o r q u a n t i t a t i v e determinations.

A r a p i d method t o assay c a f f e i n e i n c o n e r c i a l c o f f e e samples was developed by Vitzthum e t a l . 3 7 . Aqueous e x t r a c t s were automatically gas chromatographed d i r e c t l y , using 5-aminoq u i n o l i n e as an i n t e r n a l standard. A packed column o f Carbowax 20 M on Chromosorb G HP was used. The analysis was c a r r i e d o u t a t 220°C using a n i t r o g e n s e n s i t i v e flame i o n i z a t i o n det e c t o r . For coffee, a sample o f 150 mg was ground, mixed w i t h magnesium oxide and b o i l e d f o r

10 n i n . w i t h 50 m l o f water. A f t e r f i l t r a t i o n the f i l t e r was washed w i t h 10 m l h o t water three times. A s o l u t i o n o f the i n t e r n a l standard was added and the t o t a l volume brought t o 100.0 m l . 2 p l o f the s o l u t i o n was used f o r the gas chromatographic a n a l y s i s . The main advantage o f the method i s the e x t r a c t i o n w i t h water, which i s r a p i d and complete. B a n d i ~ ndescribed ~~ a method f o r the assay o f c a f f e i n e and quinine i n beverages. The a l k a l o i d s were e x t r a c t e d from the a l k a l i n i z e d d r i n k (NaOH) w i t h chloroform c o n t a i n i n g the i n t e r n a l standard (pyrene); the solvent was evaporated and the r e s i d u e d i s s o l v e d i n pyreneA l c o h o l i c bever-

f r e e chloroform. An a l i q u o t was used f o r the gas chromatographic analysis.

ages were d i l u t e d w i t h water before e x t r a c t i o n w i t h chloroform ( c o n t a i n i n g pyrene). I n the range of 50-200 mg c a f f e i n e per l i t r e of non-alcoholic beverages, an accuracy o f f 3.6 % was obtained and f o r a l c o h o l i c beverages (coffee l i q u e u r ) i t was between

-

2.4 and t 3.6

%.De-

t e c t i o n l i m i t was 4 mg c a f f e i n e per l i t r e . 20.2.2.

Caffeine i n plasma

To overcome the major drawbacks o f previous methods f o r the determination o f c a f f e i n e i n body f l u i d s ( d i f f i c u l t i s o l a t i o n from i n t e r f e r i n g m a t e r i a l s , a s u b s t a n t i a l blank e r r o r and low s e n s i t i v i t y ) Grab and Reinstein3’ developed a gas chromatographic method f o r such determinations. The method i n v o l v e d e x t r a c t i o n o f c a f f e i n e from plasma samples ( 2 m l ) w i t h c h l o r o form a f t e r the aqueous phase was adjusted t o pH 11.5-12.0.

The chloroform e x t r a c t was evapor-

ated t o dryness and redissolved i n carbon disulphide. Hexobarbital was used as an i n t e r n a l

References p. 210

192

standard, and the gas chromatographic a n a l y s i s c a r r i e d o u t on a packed column o f 3 % OV-17 on s i l a n i z e d Chromosorb W AW a t 20OoC. Caffeine was determined a t a concentration o f 0.25 ug per m l . Recovery o f c a f f e i n e added t o plasma samples (0.5-1.5

pg/ml) was 98-102 %.A t y p i c a l

chromatogram i s shown i n Figure 20.4. FIGURE 20.4 CHROMATOGRAM OF CAFFEINE I N PLASMA3’ on a 3 % OV-17 column, 6 f e e t by 1/8 i n c h O.D.. barbital, 3 = caffeine.

a t 200°C; 1 = carbon disulphide, 2 = hexo-

_j

. I

I

I

1 2 3 1 5 6 7

min

Reproduced from ~ . P h a m . S c i . , 57 (1968) 1703. w i t h permission o f the c o p y r i g h t owner.

Merriman e t al.40 improved the method o f Grab and Reinstein3’.

They developed a gas chro-

matographic-mass spectrometric micromethod. As l i t t l e as 20 ng o f c a f f e i n e can be measured, and, t h e r e f o r e accurate estimates o f c a f f e i n e concentrations i n 100 p l o r l e s s o f b i o l o g i c a l samples can b: made. The method i s rapid, s p e c i f i c and s e n s i t i v e . A packed column o f 3 % k x s i l 300 on Chromosorb Q was used a t 21OoC, and g l u t e t h i m i d e was used as an i n t e r n a l standard. Blood samples (0.1 m l ) were mixed w i t h 0.2 m l o f 0.9 % Na C1 s o l u t i o n and 0.5 m l o f g l u t e thimide i n chloroform s o l u t i o n . The m i x t u r e was mixed f o r 60 seconds and the phases separated by low-speed c e n t r i f u g i n g . A l l the c a f f e i n e and g l u t e t h i m i d e p a r t i t i o n e d i n t o the c h l o r o form phase. The chloroform phase was evaporated t o dryness, dissolved i n 0.5 m l o f acetone and gas chromatographed. The c a f f e i n e concentrations r e l a t i v e t o the g l u t e t h i m i d e concentrat i o n s were determined by monitoring the molecular i o n o f c a f f e i n e ( m / e 194) t o the M-28 i o n o f glutethimide (m/e 189). The increased s e n s i t i v i t y and s e l e c t i v i t y o f the a l k a l i flame detector f o r nitrogen-cont a i n i n g compounds, l e d t o the development o f an assay f o r c a f f e i n e i n plasma, b y Cohen e t ala4’. Amounts down t o 0.25 pg/ml plasma can be determined.

193

Plasma samples o f 1.0 m l were a l k a l i n i z e d (NaOH), the i n t e r n a l standard (mepivacaine) added and the mixture e x t r a c t e d w i t h chloroform. The chloroform s o l u t i o n was evaporated and the residue redissolved i n methanol (25 u l ) . 1.5 p1 o f the methanol s o l u t i o n was i n j e c t e d f o r the gas chromatographic analysis on a packed column o f 3 % OV-17 on Chromosorb P. A t y p i cal chromatogram i s given i n Figure 20.5. Peak h e i g h t r a t i o measurements produced l i n e a r range. Absolute s e n s i t i v i t y from a 1.0 m l plasma standard curves i n the 0.25-10.0 sample was 0.1 Ug/ml. The r e l a t i v e d e v i a t i o n o f a 2.0 pg/ml pooled plasma standard curve (done repeatedly over several months) was 5.2 %. FIGURE 20.5 CHROMATOGRAM OF HUMAN PLASMA SAMPLES41 on a 3 % OV-17 packed column on Chromosorb p, 1.82 m by 2.5 mn I.D., a t 21OoC. A = pooled plasma blank, B = plasma from a normal volunteer c o n t a i n i n g 5 ug o f c a f f e i n e (1) and 12 ug o f mepivacaine/ml ( 2 ) .

1

B

A

2

\

\ \

Reproduced from J.Pharm.Sci., 67 (1978) 1093. w i t h permission o f t h e c o p v r i a h t owner.

I n a comparative study o f micromethods f o r the determination o f c a f f e i n e i n small plasma samples (10-50 u l ) a gas chromatographic and a r a d i o a c t i v e l a b e l l i n g method were used by Milon and Antonioli4’. The c a f f e i n e was e x t r a c t e d i n t o chloroform and the gas chromatography c a r r i e d o u t on a packed column using 3 % SE-30 on Chromosorb W, and (1-CH314C) c a f f e i n e as an i t i t e r n a l standard. Because the c a f f e i n e peak had an assymetrical form, f o r m i c a c i d vapour was added t o the c a r r i e r gas, as proposed by W e l t ~ n ~The ~ . r e p r o d u c i b i l i t y o f t h e gas chromatographic method f o r the range 1-20 m g / l i t r e o f caffeine, added t o plasma, was found t o be b e t t e r than 5 %. Bradbrook e t al.44 described a method f o r the assay o f c a f f e i n e i n 0.5 m l samples o f plasma. The sample was made a l k a l i n e w i t h NaOH and a f t e r a d d i t i o n o f the i n t e r n a l standard (phenacetin) the e x t r a c t i o n was achieved w i t h 3 m l o f e t h y l acetate by means o f v o r t e x m i x i n g followed by c e n t r i f u g i n g . The residue o f the e t h y l acetate s o l u t i o n was dissolved i n 20 u l

R.femncan p. 110

194 methanol and 1-3 p1 used f o r the gas chromatographic a n a l y s i s on packed columns o f Apiezon

M 10 % o r Poly S-179 3 % a t 225OC. Comparison o f a t h i n l a y e r chromatographic method w i t h the gas chromatographic method showed t h a t the gas chranatographic method r e q u i r e d more comp l e x sample preparation, b u t had g r e a t e r s e n s i t i v i t y than the t h i n l a y e r chromatographic method. However, both methods were s u i t a b l e f o r use i n studies o f c a f f e i n e i n pharmacokinetics.

20.2.3. Caffeine i n pharmaceutical preparations Caffeine i s o f t e n present i n pharmaceutical preparations i n combination w i t h other drugs, such as a c e t y l s a l i c y l i c acid, phenacetin,antipyrin,

etc. Because c l a s s i c a l a n a l y t i c a l tech-

niques (e.g. spectrophotometric and c o l o u r i m e t r i c methods) can be q u i t e time consuming and leave much t o be desired i n accuracy and p r e c i s i o n , gas chromatography has been q u i t e extens i v e l y applied f o r the analysis o f such multicomponent preparations. I n 1963 Hoffman and M i t c h e l l 4 5 described a method f o r determining i n a s i n g l e run the act i v e ingredients i n APC-tablets ( = A c e t y l s a l i c y l i c acid, Phenacetin, Caffeine t a b l e t s ) . Due t o the high r a t i o o f phenacetin t o c a f f e i n e (162 mg : 32 mg) and the small d i f f e r e n c e s i n t h e i r r e t e n t i o n times, i t was n o t very s a t i s f a c t o r y f o r the determination o f c a f f e i n e . However, the means o f several analyses y i e l d e d r e s u l t s close t o e i t h e r the l a b e l e d claim o r the composition by synthesis. The r e s u l t s o f a s e r i e s o f analyses o f s y n t h e t i c APC mixtures are l i s t e d i n Table 20.3. TABLE 20.3

PRECISION AND ACCURACY ANALYSIS

OF A

SYNTHETIC APC MIXTURE45

on a packed column o f DC 200, 2 % on Haloport F, 6 f e e t by 4 mn I.D. ming from 75OC t o 200OC

Component

Synthetic mixture

Tablet A

Acetylsalic* acid Acetophenetidin Caffeine

Acetylsalic. acid Acetophenetidin Caffeine

Tablet B A c e t y l s a l i c . acid Acetophenetidin Caffeine

ComDosi t i o n by synthes. % by w t . Run 1

53.52 38.34 7.65

Run 2

and temperature program-

Av. Deviation f ran Run 3 Run 4 Mean Mean %

53.31 55.06 54.33 54.75 54.36 38.36 37.12 37.74 37.96 37.79 8.31

7.81

7.92

7.28

7.83

Run 4

Run 5

Av. E r r o r 4;

0.6 1.0

t

-

-

1.6 1.4

*

3.6

t

2.3

k

Tablet Assays

tion on

Run 1

m9 226.8

mg mg 223.53 216.14 221.56 235.10 221.17 222.10 223.14

162.0

168.35 172.61 167.92 162.66 166.29 166.92 167.45

32.4 226.8 162.0 32.4

mg

32.4

Run 2 mg

mg

mg

Run 6

Mean

mg

31.72 31.34 33.70

32.10 32.28

224.64 217.04 226.36 227.70 225.23 161.78 170.21 161.48 160.00 162.16

224.19 163.11

34.75

32.4

Run 3

33.87 33.28 33.45 33.78

Two m i c r o l i t e r i n j e c t i o n s o f chloroformic s o l u t i o n s were used throughout.

33.85

195 Haefelfinger e t al.46 developed a s i m i l a r method f o r the determination o f phenacetin, i s o p r o p y l a n t i p y r i n , c a f f e i n e and persedon. A powdered t a b l e t was brought i n t o a 200 m l columetr i c f l a s k , Sedormid ( = a l l y l - i s o p r o p y l a c e t y l urea) was added as an i n t e r n a l standard, and acetone t o 200 m l . 1 p1 o f the s o l u t i o n was gas chromatographed. A comparative study o f a t h i n - l a y e r chromatographic method and an i o n exchange r e s i n separation, i n combination w i t h UV-determination,

and the gas chromatographic method, showed t h a t t h e l a t t e r was the more

elegant and rapid. A packed column o f !O % SE-30 on C e l i t e was used f o r the gas chromatography, which operated a t 215OC t o assay c a f f e i n e i n the multicomponent preparations mentioned. Dechene e t a l . 4 7 determined a c e t y l s a l i c y l i c acid, phenacetin, c a f f e i n e and codeine i n tabl e t s . A c e t y l s a l i c y l i c a c i d and phenacetin were separated and determined on one packed column o f DS 200 on Haloport by temperature programing, 100-180°C, whereas c a f f e i n e and codeine were separated on a packed column o f SE-30 (10 %) on Chromosorb W AW by temperature programming, 195-260°C. Codeine and c a f f e i n e were e x t r a c t e d from the powdered t a b l e t m a t e r i a l w i t h chloroform a f t e r the a d d i t i o n o f a l k a l i , and the chloroform s o l u t i o n used f o r the gas chromatographic assay. Results obtained w i t h the method are l i s t e d i n Table 20.4. TABLE 20.4 RESULTS OF GAS CHROMATOGRAPHIC ANALYSES OF APC TABLETS47 ~

~~

~

Ingredient assay Average o f 10 assays High value Low value Standard d e v i a t i o n

Acetyl sal ic. acid 98.30 103.30 93.76 i 2.77

Recovery ( % ) Phenacetin Caffeine 100.77 106.20 95.46 k 3.26

100.0 105.04 97.92 i 1.16

Codeine phosphate 102.88 109.60 94.06 f 4.83

The s e n s i t i v e thermoionic KC1 detector used by Newton33 f o r the determination o f very low concentrations o f c a f f e i n e i n i n s t a n t tea, was a l s o used by Alber and Overton4* f o r the determination o f c a f f e i n e i n t a b l e t s c o n t a i n i n g salicylamide and acetaminophen.

The t a b l e t

powder was e x t r a c t e d w i t h acetone a f t e r a d d i t i o n o f the i n t e r n a l standards (amobarbital and c y c l i z i n e ) and the s o l u t i o n used f o r the gas chromatographic analysis. This was c a r r i e d o u t on a packed column o f OV-17 3 % on Gas Chrom Q a t 165OC. The s i g n a l s from the KC1 thermoi o n i c d e t e c t o r were a m p l i f i e d and fed d i r e c t l y i n t o t h e a n a l o g - t o - d i g i t a l converter o f a POP 12 A LINC system computer. The r e s u l t s obtained by means o f the computer were compared

t o manual peak height measurements f o r 6 commercial preparations i n j e c t e d i n duplicate, and t o r e s u l t s obtained from a UV-procedure. A t y p i c a l chromatogram i s given i n Figure 20.6 and the r e s u l t s obtained a r e l i s t e d i n Table 20.5. The gas chromatographic method i s p a r t i c u l a r l y w e l l s u i t e d f o r m u l t i p l e analyses, which can be a p p l i e d t o automated techniques and i n d i v i d u a l t a b l e t a n a l y s i s . Because drug assay i n suppositories f o l l o w i n g the c l a s s i c a l techniques ( e . g . spectrophotom e t r i c and c o l o u r i m e t r i c methods) i s cumbersome and n o t very accurate o r precise due t o i n terference o f the e x c i p i e n t s and o t h e r additives, Cometti e t a l .49 a p p l i e d gas chromatography

for such assays. The suppositories were dissolved i n ethanol o r chloroform, c o n t a i n i n g the i n t e r n a l standards t o be used and depending on the drugs present. The gas chromatography was c a r r i e d o u t on d i f f e r e n t packed columns. depending on the drugs t o tie determined. Typical

References p. 210

196

gas chrcinatograms o f suppositories containing c a f f e i n e and some o t h e r drugs are given i n Figure 20.7. Analytical r e s u l t s o f some suppositories are l i s t e d i n Table 20.6. FIGURE 20.6 GAS CHROMATOGRAM OF SALICYLAMIDE (1). AMOBARBITAL (2). ACETAMINOPHEN (3), CAFFEINE ( 4 ) AND CYCLIZINE (5)48

on a 3 % OV-17 packed column on Gas Chrom Q a t 165OC

TABLE 20.5 COMPARISON OF GLC, GLC-COMPUTER, AND UV ASSAY RESULTS OF SOME FORMULATIONS4' Manual peak ht. Declared mg/tablet

mg/tablet

Computer peak h t .

X

UC spectrophotometry

X

% of declared

mg/tablet

o f declared

mg/tablet

o f declared 99.88 99.76

ComDound Salicylamide Acetaminophen Caffeine

250 250 18.0

249 241 18.8

99.6 96.4 104

251 243 18.6

100 91.2 103

249.7 249.4

Salicylamide Acetaminophen Caffeine Salicylamide Acetaminophen Caffeine Salicylamide Acetaminophen Caffeine

200 120 30 180 180 30 230 150 30

204 124 32.5 175 182 31.9

102 103 108

200 124 32.4

100 103 108

204.3 120.0

102.2 100.0

97.2 101 106

176 180 31.6

97.8 100 105

189 185 30.9

105 103 103

221 158 28.4

98.1 105 94.7

224 157 27.3

97.4 105 91.0

223.2 150.7 25.1

97.1 100.5 83.6

-

-

197

FIGURE 20.7 CHROMATOGRAMS OF SUPPOSITORIES49 L e f t : 1 = diphenylamine ( i n t e r n a l standard), 2 = lidocaine, 3 = chlorpheniramine, 4 = d-propoxyphene, 5 = c a f f e i n e on a packed column o f 2 % Carbowax 20 M and 2 % SE-30 on Gas Chrom P, impregnated w i t h 5 X KOH, a t 195OC. Right: 1 = phenacetin, 2 = caffeine, 3 = aminophenazone, 4 = chlorpheniramine and 5 = chlorothioxantone ( i n t e r n a l standard) on a packed column o f 10 X UCC W98 on Diatoport S. a t 205OC Reproduced from J. Pharm. S c i . , 60 (1971) 1074. w i t h permission o f the copyright owner.

1

L

I

6

0Cmz.:

attenuation

m2. a i 0 2 . i ~

attenuation

TABLE 20.6 ANALYSIS OF SIMULATED AND COMMERCIAL SUPPOSITORY PREPARATIONS4’ Simulated preparation 1 Aminophenazone 300 d-Propoxyphene hydrochloride 60 Caffeine 50 Chlorpheniramine maleate 3 L i docai ne 20 Phenacetin 300 Aminophenazone 400 60 Ca f f e i ne Chlorpheniramine maleate 4 30 Fenalamide Sodi urn benzoate 60

2 299.8 60.4 50.5 3.04 20.2

3 6 6

4 1.01 0.67 1.62 2.58 2.40

298.8 399.4 59.9 3.99 29.9 59.6

6

0.97 1.25 1.18 2.72 1.45 1.32

5 -0.07 +0.67 +1.00 t1.33 +1.00 -0.40 -0.15 -0.17 -0.25 -0.33 -0.67

Comnercial suppository 2 304.2 61.3 51.9 3.08 20.6

6 29 25

4 2.38 2.56 3.33 5.16 2.98

5 t1.40 t2.17 +3.80 t2.67 t3.00

299.3 397.7 59.5 3.94 29.6 59.0

20

2.20 2.61 3.53 5.90 3.46 6.18

-0.23 -0.58 -0.83 -1.50 -1.33 -1.67

1 = weighed o r declared amount i n mg, 2 = found amount, average i n mg, 3 = number o f repl i c a t e analyses, 4 = r e l a t i v e standard d e v i a t i o n ( p = 0.05), 5 = r e l a t i v e e r r o r , 6 = number o f l o t s examined.

Rofemoea p. 210

198 '

I n order t o r u n a s e r i e s o f analyses o f drugs i n pharmaceutical preparations, F r i c k e

50

made use o f simple e x t r a c t i o n s . Tablets c o n t a i n i n g c a f f e i n e , phenacetin and a s p i r i n were brought i n t o a volumetric f l a s k , t o which chloroform and a small amount o f g l a c i a l a c e t i c a c i d were added. A f t e r shaking f o r 1 h chloroform was added and an a l i q u o t was t r a n s f e r r e d t o a C e l i t e column t h a t was t r e a t e d w i t h 1 N NaHC03 s o l u t i o n . A f t e r e l u t i o n w i t h chloroform, the e l u a t e was evaporated t o dryness, and the residue dissolved i n methanol t o about 0.3 mg/

m l o f c a f f e i n e . The gas chromatography was c a r r i e d o u t on a packed column using Dexsil 300 10 % o r 17 % on Chromosorb W HP a t 22OoC o r 222OC r e s p e c t i v e l y . I n a paper on the gas chromatographic assay o f phenacetin, caffeine, a n t i p y r i n and d i methylaminoantipyrin i n pharmaceutical preparations, Oesch and Sahli51 e x t r a c t e d the components by means o f chloroform. A f t e r evaporation o f the solvent the residue was r e d i s s o l v e d i n chloroform and gas chromatographed on a packed column o f 2.5 % SE-30 on Chromosorb W HP a t 18OoC A t y p i c a l chromtogram i s given i n Figuse 20.8. FIGURE 20.8 CHROMATOGRAM OF PHENACETIN ( l ) , CAFFEINE ( 2 ) , ANTIPYRIN (3) AND DIMETHYLAMINOANTIPYRIN ( 4 ) 5 1 on a packed column o f 2.5 % SE-30 on Chromosorb W HP. 2 m lone, a t 18OoC

De Vos and Jonkhoff5' described a gas chromatographic method t o determine c a f f e i n e i n seve r a l pharmaceutical preparations, e.g. t a b l e t s and suppositories. The t a b l e t and suppository assay was c a r r i e d o u t by d i s s o l v i n g o r suspending the preparation i n chloroform, t o which the i n t e r n a l standard, lidocaine, had been added. The gas chromatographic separation was o b t a i n ed on a 3 % OV-1 packed column a t 16OoC. Caffeine could be determined w i t h a standard deviat i o n o f about 2 %.

199 20.3.

THEOPHVLLINE

Because theobromine and t h e o p h y l l i n e have very low v o l a t i l i t y and low s o l u b i l i t y i n most solvents, and are d i f f i c u l t t o gas chromatograph w i t h o u t considerable adsorption losses, Brochmann-Hanssen and Oke16 p r e f e r r e d t o convert both compounds i n t o c a f f e i n e on flash-heater methylation by means o f t r i m e t h y l a n i l i n i u m hydroxide. Caffeine can be r e a d i l y gas chromatographed. To a screw-cap containing the substance t o be gas chromatographed, t r i m e t h y l a n i l i n ium hydroxide was added i n approximately 100 % excess. The s o l u t i o n was d i l u t e d w i t h methano l when necessary, and 1 p1 containing 0.2 t o 1.0 pg o f the compound was i n j e c t e d i n t o the f l a s h heater w i t h a m i c r o syringe. The temperature o f the f l a s h heater was 275OC and the column temperature 137OC. A packed column o f 3 % SE-30 on Gas Chrom Q was used. I n order t o o b t a i n b e t t e r gas chromatographic p r o p e r t i e s f o r the q u i t e p o l a r xanthine deintroduced f l a s h heater b u t y l a t i o n w i t h tetra-n-butylamnonium r i v a t i v e s , Kowblansky e t a l . l9 hydroxide, using the comnercially availabe t i t r a n t , a 25 % methanol s o l u t i o n (about 1 M ) .

7 p l o f the reagent were added t o 1 m l methanolic xanthine s o l u t i o n c o n t a i n i n g n o t more than about 1.5 p equiv. o f N-H groups, a t l e a s t a 4 : 1 molar r a t i o o f a l k y l a t i n g agent t o t e s t compound. 1 p l o f the s o l u t i o n was i n j e c t e d i n t o t h e gas chromatograph, using a minimum i n j e c t o r block temperature o f 27OoC. On a packed column o f 3 % OV-17 on Gas Chrom Q and a c o l umn temperature o f 22OoC, very good separation o f xanthines was obtained, as can be seen i n the Figure 20.9. FIGURE 20.9 CHROMATOGRAMS OF XANTHINES FOLLOWING FLASH HEATER N-BUTYLATIONl' on a packed 3 % OV-17 column on Gas Chrom Q; i n j e c t o r block temperature 27OoC, column temperature 220°C; 1 = caffeine, 2 = theophylline, 3 = theobromine, 4 = 1-methylxanthine, 5 = xanthine.

1

\I

c

I 5 I

200

20.3.1.

Theophylline i n pharmaceutical preparations

E l e f a n t e t a1.53 were the f i r s t t o use gas chromatography f o r the determination o f theop h y l l i n e i n t a b l e t s t h a t a l s o contained other components (phenobarbital and ephedrine hydrochloride). One fine-ground t a b l e t c o n t a i n i n g 130 mg t h e o p h y l l i n e was e x t r a c t e d w i t h a c h l o r o fonn-methanol ( 1 : 1) s o l u t i o n o f the i n t e r n a l standard (4,4'-methylene-bis-(N,N-dimethylanil i n e ) (0.6 mg/ml) and a f t e r c e n t r i f u g i n g , the c l e a r s o l u t i o n was i n j e c t e d i n t o t h e gas chromatograph on a packed column w i t h HI-EFF 88 3 % on Gas Chrom Q a t a column temperature o f 25OoC. Results o f the assay are given i n Table 20.7. TABLE 20.7

ASSAY OF INDIVIDUAL TABLETS CONTAINING THEOPHYLLINE, EPHEDRINE AND PHENOBARBITAL53 on a 3 % packed column o f HI-EFF 8B on Gas Chrom Q a t 250°C Tablet

Ephedrine HC1

Phenobarbital

Theophylline

1 2 3

4 5 6 7 8 9 10 Average R e l a t i v e standard d e v i a t i o n

23.8 23.6 23.6 23.4 23.8

7.6 7.8 7.8 7.7 7.8

127.0 130.0 126.0 126.0 126.0

23.7

7.7 1.0 %

127.0 0.9 %

1.1 %

Schul t z and P a ~ e e n b a m p e ndetermined ~~ theophyll i n e i n a suspension c o n t a i n i n g ephedrine and phenobarbital, using e x t r a c t i o n , f i r s t o f ephedrine a t pH 11 w i t h chloroform, and then theophylline and phenobarbital a t pH 4.8 w i t h the same solvent. Hexobarbital was used as an i n t e r n a l standard f o r the t h e o p h y l l i n e and phenobarbital determination. A recovery o f 99.4 % and a c o e f f i c i e n t o f v a r i a t i o n o f

f

0.3 % was found f o r t h e o p h y l l i n e i n a comnercial suspen-

sion. 20.3.2.

Theophylline i n b i o l o g i c a l f l u i d s

Theophylline i s c u r r e n t l y being used f o r the treatment o f bronchial asthma and other card i o r e s p i r a t o r y disorders. Because t h e r e i s good evidence t h a t both the t h e r a p e u t i c response and the t o x i c s i d e - e f f e c t s are r e l a t e d t o the concentration o f the t h e o p h y l l i n e i n the p l a s ma, r a t h e r than t o i t s dosage, accurate a n a l y t i c a l methods a r e needed t o make i t p o s s i b l e t o c o n t r o l treatment and reduce the r i s k o f dangerous t o x i c symptoms.

Gas chromatographic methods have been developed t o solve t h i s problem. Theophylline has been gas chromatographed and determined as such, b u t i n most cases d e r i v a t i z a t i o n has been c a r r i e d o u t t o o b t a i n b e t t e r gas chromatographic p r o p e r t i e s and b e t t e r d e t e c t i o n p o s s i b i l i t i e s . For q u a n t i t a t i v e work several substances have been used as i n t e r n a l standard, mainly b a r b i t u r a t e s (alphenal, amobarbital, aprobarbital, heptabarbital, hexabarbital, t h i o b a r b i t a b u t a l s o other substances have been used (fluoranthene, codeine, theobromine, medazepam, pramoxine hydrochloride, cyheptamine, probenecid p r o p y l e s t e r ) . Recently 3-isobutyl-1-methyl

201

xanthine has been introduced as internal standard. In derivatization reactions t h i s compund and theophylline are quite similar, as also in e x t r a c t a b i l i t y , s t a b i l i t y and chromatographic properties. 20.3.2.1. Determination of theophylline a s such

A highly specific and reasonably sensitive method t o determine theophylline in human serum was developed by Chrzanowski e t al.55. Samples of 3 ml serum were acidified with 0.1 N HC1 and extracted with chloroform-isopropanol (95 : 5). After evaporation of the solvent, the residue was dissolved in 50 ul of a n internal standard solution containing 5 mg of Pramoxine hydrochloride in 50 ml chloroform-isopropanol mixture. About 1.3 p1 was injected onto the column of 3 % OV-17 on Chromosorb W a t a t column temperature of 24OOC. Determination of theophylline in serum containing 4.9 ug/ml to 10.1 ug/ml could be carried out with a standard deviation of 0.4 ug/ml t o 0.3 ug/ml, giving coefficients of variation of 8.0 % and 3.3 % respec t i vel y . In a following paper, Chrzanowski e t al.56 stated t h a t abnormal theophylline values obtained for blood serum samples that were kept in glass tubes sealed with butyl rubber stoppers, were caused by a substance which leached from the stoppers. Wesley-Hadzi ja57 worked o u t a simple method for the determination of theophylline in serum samples. To samples of 1 ml serum was added 0.5 ml 0.1 N HC1 and the extraction was carried o u t with diethyl ether-dichloromethane ( 7 : 4 ) , the organic solution evaporated to dryness, and the residue dissolved in 50 pl methanol containing the internal standard (500 ug/ml of codeine). An aliquot of 2-5 ul was gas chromatographed on a packed column of 3 % OV-17 on Gas Chrom Q a t a column temperature of 245OC. The reproducibility of the procedure was good. Because most gas chromatographic methods for the determination of theophylline in biological fluids involve tedious extraction procedures and/or derivatization prior t o the gas chromatographic analysis, Sheehan and Haythorn” developed a rapid method for such determinations. Theophylline was extracted from acidified blood with chloroform a f t e r the addition of the internal standard, cyheptamide, and i t was gas chromatographed d i r e c t l y on a packed column of 3 % OV-1 on Chromosorb W using temperature programming from 160°C t o 28OoC. Concentrations down t o 2 pg/ml blood could be determined, with recoveries ranging from 90 t o 110 %. A typical chromatogram i s given in Figure 20.10. An interesting technique f o r gas chromatographic determination of theophylline in dried whole blood was developed by Albani and Toseland5’. About 35 mg whole blood was applied on f i l t e r paper cards by capillary blood sampling techniques. 3 ul of the internal standard solution (heptabarbital) were added t o the spot. Three t o six spots were punched o u t and were extracted with 500 p1 of phosphate buffer pH 4.5 and 5 ml of d i s t i l l e d diethyl ether. After evaporation of the diethyl e t h e r , the residue was dissolved in 15 pl of methanol. 2 pl were injected in the gas chromatograph, which was equipped with a nitrogen specific detector and a packed column of 0.5 % HI-EFF 8B on Chromosorb W HP. Column temperature was 24OoC. Analysis of ten samples, with identical concentration, on ten sucessive days, revealed a day-to-day variance o f f 2.9 %; the precision of the method, as studied by analysis of ten different samples with identical concentrations, revealed a coefficient of variation of 5.2 % (range 6.7 % t o 3.8 % ) in five subsequent investigations.

Refemnces p. 210

FIGURE 20.10

CHROMATOGRAM OF AN EXTRACT FROM 2 ML BLOOO CONTAINING 40 pg

OF THEOPHYLLINE5'

on a 3 % packed column o f OV-1 on Chromosorb W and temperature p r o g r a m i n g from 160 t o 280°C 1 = theophylline, 2 = cyheptamide ( i n t e r n a l standard).

2

I

I

I

1

2

L

6

8

I

10

I

12

min

Chambers6' described a r a p i d method f o r the determination o f underivatized t h e o p h y l l i n e i n plasma. To a 500 p1 sample o f plasma was added the i n t e r n a l standard, heptabarbital, then the sample was a c i d i f i e d and e x t r a c t e d w i t h chloroform. The residue a f t e r evaporation o f the chloroform was dissolved i n 10 ~1 acetone and 4 p1 was gas chromatographed on a 3 % p o l y (cyclohexyldimethanol) succinate on Diatomite column a t 225OC. A chromatogram o f an e x t r a c t o f the plasma i s given i n Figure 20.11. Both Albani and Toseland5'

and Chambers"

made use o f an organic-nitrogen s p e c i f i c ( a l k a l i -

flame i o n i z a t i o n ) detector. 20.3.2.2

Determination of theophylline after derivatization

A. Packed columns I . Propylation

Shah and RiegelmanZ7 e x t r a c t e d t h e o p h y l l i n e from plasma and/or s a l i v a samples (1 m l ) w i t h a mixture o f d i e t h y l ether, dichloromethane and isopropanol (6 : 4 : 1). Theophylline was r e moved from the organic s o l u t i o n by means o f aqueous sodium hydroxide, t h e a l k a l i n e s o l u t i o n was a c i d i f i e d w i t h phosphoric a c i d (pH 5) and t h e o p h y l l i n e was re-extracted w i t h the organic mixture mentioned. The i n t e r n a l standard ( t h i o b a r b i t a l o r fluoranthene) was added, t h e s o l vent evaporated, and the residue dissolved i n 25

pl

o f tetrapropylamnonium hydroxide and Gas

chromatographed on a packed column o f 3 I OV-17 on Gas Chrom Q a t 190°C.

I n the i n j e c t i o n

heater (kept a t 265OC) theophylline i s q u a n t i t a t i v e l y converted t o i t s propyl d e r i v a t i v e , which gives a symmetrical peak d i s t i n c t from any o t h e r xanthines, o r b a r b i t u r a t e s .

203 FIGURE 20.11 CHROMATOGRAM OF AN EXTRACT OF PLASMA CONTAINING

THEOPHYLLINE~'

on a 3 % poly(cyclohexyldimethano1) succinate on Diatomite packed column a t 225OC; 1 = heptab a r b i t a l ( i n t e r n a l standard, 160 p m l e / l ; 2 = theophylline, 95 pmole/l

I 0 I 0 12 t6 20 min

Zoidema e t a1.61 used Propyl-8 (= dimethylformamide-dipropylacetal)

f o r propylation o f

theophylline. Fluoranthene was used as an i n t e r n a l standard f o r the determination o f theop h y l l i n e i n b i o l o g i c a l f l u i d s . E x t r a c t i o n was obtained w i t h chloroform-isopropanol (95 : 5) a f t e r a c i d i f y i n g the sample ( 1 m l ) w i t h hydrochloric a c i d . A f t e r evaporation o f the s o l v e n t the residue was dissolved i n 0.5 m l o f a methanolic s o l u t i o n o f the i n t e r n a l standard. The methanol was evaporated and the residue dissolved i n 30 p1 Propyl-8 and gas chromatographed on a packed column o f 3 % OV-17 on Gas Chrom

Q

a t a column temperature o f 22OoC

11. Methylation I n a determination o f purine and pyrimidine base r a t i o s i n n u c l e i c acids and oligonucleot i d e s , Mac Gee15 methylated them i n the f l a s h heater o f the gas chromatograph by thermal decomposition o f t h e i r tetramethylamnonium s a l t s . Theophylline and theobromine (50 m o l e s ) were dissolved i n 0.5 m l o f 1 M tetramethylamnonium hydroxide i n ethanol. While the f r e e purines are n o t very s o l u b l e i n ethanol, t h e i r tetramethylammonium s a l t s are q u i t e soluble. The gas chromatography was performed on a 6 f e e t by 4 mn s t a i n l e s s s t e e l column packed w i t h

80-100 mesh glass beads coated w i t h 0.5 % Carbowax 20

M. Column temperature was 143OC and

f l a s h heater temperature 36OoC. With complete methylation, theophyll i n e and theobromine yielded caffeine. Because the peak shape o f t h e o p h y l l i n e can be poor i n gas chromatography, as a l s o can be the s e n s i t i v i t y o f t h e method, Dusci e t a l . 1 7 p r e f e r r e d t o convert t h e o p h y l l i n e w i t h on-column methylation by means o f t r i m e t h y l a n i l i n i u m hydroxide t o c a f f e i n e . In t h a t manner they

204

developed a method f o r the assay o f theophylline i n plasma t h a t was s e n s i t i v e down t o 0.1 mg% theophylline. A s i m i l a r e x t r a c t i o n and p u r i f i c a t i o n procedure was proposed by Shah and Riegelman27. A packed column o f 3 % OV-225 on Chromosorb W was used and the column temperature was 235OC. Recovery was f o r amounts o f 6-48 ug theophylline added t o plasma samples (2 m l ) 88 f 8 %. I n t e r n a l standard was medazepam. used on-column methylation, but trimethylamonium hydroxide was used as Kinsun e t a l . l8 the methylating agent. A s i n g l e e x t r a c t i o n o f serun samples (50 p l ) with chloroform-diethyl e t h e r (6 : 4) was c a r r i e d out and theophylline could be determined by t h e method down t o 0.5 ug/ml. A nitrogen s e l e c t i v e detector was used, w i t h heptabarbital as i n t e r n a l standard. The gas chromatographic column was a packed one w i t h 3 % OV-1 on Gas Chrom Q operating a t 21OoC. 111. B u t y l a t i o n For the determination o f theophylline and probenecid i n b i o l o g i c a l material, Arbin and Edlund" used dimethylformamide di-n-butyl acetal as the b u t y l a t i n g agent. The samples (0.21.0 ml) were extracted w i t h chloroform over a c e l l u l o s e column treated w i t h phosphate b u f f e r pH 7. The eluate containing theophylline and probenecid was evaporated t o dryness and the residue was dissolved i n 60 u l i n t e r n a l standard s o l u t i o n (probenecid propylester). A f t e r the a d d i t i o n o f 40 111 o f the b u t y l a t i n g agent, t h e mixture was k e p t a t room temperature f o r f i v e minutes and then 1 p l was i n j e c t e d i n t o the gas chromatograph. A packed column o f 3 % OV-17 on Chromosorb W and temperature programing from 190°C t o 24OoC was used. A chromatogram i s given i n Figure 20.12. FIGURE 20.12 CHROMATOGRAM OF CAFFEINE (1). THE N-BUTYL DERIVATIVES OF THEOPHYLLINE (Z), THEOBROMINE ( 3 ) AND PROBENECID (5) AND PROBENECID PROPYLESTER ( 4 ) ( = INTERNAL STANOARD)~~ on a 3 % OV-17 packed column on Chromosorb W w i t h temperature programming, 19O-24O0C

1

1

1

.

,

2

.

.

.

L

,

.

6

,

.

,

8

.

,

.

,

.

,

.

,

10 12 1L 16 18 min

206

The e x t r a c t i o n procedure and the d e r i v a t i z a t i o n give the method high s e l e c t i v i t y . Quantit a t i v e determinations down t o 2 ug theophylline and 4 ug probenecid can be done. The addit i o n o f 5 ug theophylline t o a sample gave an absolute recovery o f 91 ? 5 %. Johnson e t a l . ' l made use of the b u t y l a t i o n o f theophylline i n connection w i t h i t s quantit a t i v e determination i n human serum and saliva. The b u t y l a t i o n described by Kowblansky e t a l . 19, whereby mixtures o f caffeine, theobromine and theophylline can e a s i l y be separated on a p o l a r phase such as SP 2250 and OV-17. requires q u i t e a high i n j e c t o r temperature (300OC) t o o b t a i n optimum a l k y l a t i o n . The b u t y l a t i o n described by Greely6', which i s an off-column but y l a t i o n by means o f b u t y l iodide. avoids the p y r o l y t i c production o f t r i a l k y l a m i n e t h a t OCcurs when a tetraalkylamnonium hydroxide i s i n j e c t e d , and t h i s r e a c t i o n i s t h e r e f o r e p r e f e r able f o r the analysis o f xanthines. Aprobarbital and alphenal were used as i n t e r n a l standards. A packed column w i t h 3 % SP 2250 on Supelcoport and temperature programming f r o m 16OoC t o 24OoC was used. P e r r i e r and LearZ4 converted theophylline t o i t s b u t y l d e r i v a t i v e by means o f t e t r a b u t y l amnonium hydroxide and on-column a l k y l a t i o n i n connection w i t h a r a p i d e x t r a c t i o n procedure. Samples o f 100 ~1 plasma were e x t r a c t e d w i t h chloroform-isopropanol (95 : 5 ) containing the i n t e r n a l standard, amobarbital. The solvent was evaporated and the residue dissolved i n 1 m l toluene. With the a i d o f 10 u1 o f an aqueous s o l u t i o n o f tetrabutylamnonium hydroxide, theop h y l l i n e and amobarbital were q u a n t i t a t i v e l y e x t r a c t e d from the toluene, and by i n j e c t i o n of an a l i q u o t of t h i s s o l u t i o n i n t o the gas chromatograph, a l k y l a t i o n took place i n t h e i n j e c t o r , and q u a n t i t a t i o n o f theophylline i n concentrations i n 100 ~1 plasma was possible w i t h a flame i o n i z a t i o n detector, using a packed column w i t h 3 % OV-17 on Chromosorb G a t 25OOC. Least e t al.23 reduced the sample size t o 20 u1 serum, plasma. o r saliva, i n a microscale procedure. The e x t r a c t i o n was c a r r i e d o u t w i t h a chloroform-isopropanol s o l u t i o n o f the i n t e r n a l standard, 3-isobutyl-1-methylxanthine. A f t e r a1 k y l a t i o n w i t h tetramethylamnonium hydroxide and pentyliodide, gas chromatography was c a r r i e d o u t on a packed 3 % OV-17 on Gas Chrom Q column, by temperature programing from 18OoC t o 260°C, and using a n i t r o g e n sensit i v e detector. With the sample volume used, the background i n t e r f e r e n c e i s equivalent t o about 0.1 m g / l i t r e , and 0.5 mg theophylline per l i t r e can e a s i l y be measured. Between-run precision was 2.8 % by theophylline concentrations o f 14.8 m g / l i t r e . To improve the theophylline analysis, B a i l e y e t a1." used 3-isobutyl-1-methylxanthine as an i n t e r n a l standard. This substance had already been used by Dechtiaruk e t a1.28, Schwertner e t al.31 and Least e t al.23. Extraction was perfotmed w i t h serum samples o f 2 m l w i t h a chloroform-isopropanol s o l u t i o n (95 : 5 ) o f the i n t e r n a l standard. A f t e r p u r i f i c a t i o n v i a e x t r a c t i o n i n t o aqueous sodium hydroxide, and back e x t r a c t i o n i n t o chloroform-isopropanol, the sample was d e r i v a t i z e d w i t h n-butyl-8-reagent. No interference was encountered from normal serum constituents o r methylxanthines. Recovery was 85 %, and p r e c i s i o n and accuracy were good. An 10 % SE-30 on Chromosorb W column a t 200°C was used. Pranskevich e t a1.25 p r e f e r r e d a packed column o f 3 % SP 2250 DB as s t a t i o n a r y phase because the b u t y l d e r i v a t i v e , which i s a weak base, gas chromatographs markedly b e t t e r on t h i s phase than on the same percentage loadings of o t h e r brands o f OV-17. A f t e r an i n i t i a l chloroform e x t r a c t i o n o f 1.0 m l serum sample, a toluene wash and back e x t r a c t i o n i n t o 0.5 M ammonium hydroxide, d e r i v a t i z a t i o n was accomplished by t h e a d d i t i o n o f 15 u l o f an 8 : 1 mixture o f N,N-dimethylacetamide (400 111) and 2.4 % tetramethylammonium hydroxide i n methanol (50 u l ) t o the d r i e d e x t r a c t . Butyliodide (15 u l ) was then added and the gas chromatographic assay

Referencesp. 210

206

c a r r i e d out. The s t a t i o n a r y phase SP 2250 DB was i d e a l f o r the a n a l y s i s since an a l k a l i n e tetramethylanonium hydroxide s o l u t i o n was used. Typical gas chromatograms are given i n F i g u r e 20.13. FIGURE 20.13

GAS CHROMATOGRAMS OF THEOPHYLLINEE5 Column: Glass, 90 cm by 2 mm I.D., packed w i t h 3 % SP 2250-DB on Supelcoport, temp. 200°C. (A) standard-theophylline 20 pg/ml and i n t e r n a l standard ( h e p t a b a r b i t a l ) 10 ug/ml; ( 9 ) cont r o l serum-theophylline 10 uq/ml and i n t e r n a l standard 10 u g h 1 ; patient-serum specimen (C) from p a t i e n t r e c e i v i n g theophylline and i n t e r n a l standard 10 pg/ml; (D) blank-serum and i n t e r n a l standard 10 ug/ml. (1) = theophylline, ( 2 ) = i n t e r n a l standard.

rn

1 2 3

7

-

m

0 1 2 3 0 1 2 3 0 1 2 3

min

A seven minutes determination o f t h e o p h y l l i n e i n l e s s than 100 p1 o f serum was described 50 u l methanol and 2OOu1 o f a mixture o f d i e t h y l ether, dithloromethane and isopropanol ( 6 : 4 : 1) A f t e r shaking, 1.6 u l o f the e x t r a c t plus 0.4 u l o f a m i x t u r e o f b u t y l i o d i d e , methanol and tetrabutylamnonium hydroxide, was i n j e c t e d i n t o the gas chromatograph using a 3 % OV-17, 0.1 % therephthalic a c i d on Chromosorb 750 a t a column temperature o f 23OoC. As i n t e r n a l stanby Vinet and ZizianZ6. To 100 u1 o f serum was added 50 p1 phosphate b u f f e r pH 4.2,

dard 3-isobutyl-1-methylxanthine was used, w i t h a s e n s i t i v e n i t r o g e n detector. The r e s u l t s c o r r e l a t e d w e l l w i t h those obtained by u l t r a v i o l e t spectrometry.

I V . Pentylation The method described by Johnson e t al.'l,

whereby off-column b u t y l a t i o n by means o f b u t y l

i o d i d e was used f o r the b u t y l a t i o n , was improved by Dechtiaruk e t a1."

by using 3 - i s o b u t y l -

1-methylxanthine as i n t e r n a l standard and p e n t y l i o d i d e as a l k y l a t i n g agent. Pentyl i o d i d e gave optimal r e s o l u t i o n between the d e r i v a t i v e s o f the compounds o f i n t e r e s t . Figure 20.14 shows the separation of the pentyl d e r i v a t i v e s of theophylline, theobromine, phenobarbital and the i n t e r n a l standard. To o b t a i n greater s e n s i t i v i t y and greater s e l e c t i v i t y i n the a n a l y s i s o f serum theophyl-

207

FIGURE 20.14 PENTYL DERIVATIVES OF THEOPHYLLINE (1) 9 THEOBROMINE (2),

3-ISOBUTYL-1-METHYLXANTHINE

(3)

AND PHENOBARBITAL ( 4)28 on a packed column of 3 % OV-17 on Gas Chrom Q, w i t h temperature programing, 190-240°C

10

-

987-

65L-

321-

I

i

0-

l i n e , Lowry e t al."

1

i

i, min

used a n i t r o g e n s e n s i t i v e d e t e c t o r and 3-isobutyl-1-methylxanthine as

an i n t e r n a l standard. The method involved a s i n g l e e x t r a c t i o n o f 50 v l serum, and the der i v a t i z a t i o n o f t h e o p h y l l i n e and t h e i n t e r n a l standard t o t h e i r p e n t y l d e r i v a t i v e s was performed by d i ssol v i n g the compounds i n N,N-dimethyl acetami de, and adding t r i m e t h y l a m o n i um hydroxide and propyliodide. A packed column w i t h 3 % OV-17 on Gas Chrom Q was used a t a c o l umn temperature o f 24OoC. Using 50 u l o f serum, concentrations o f 1 ug/ml i n serum could e a s i l y be measured. Precision o f the method was 3.4 % f o r therapeutic doses o f t h e o p h y l l i n e .

V. Pentafl uorobenzylation To be able t o determine t h e o p h y l l i n e i n the ng range, Arbin and Edlund3O improved t h e i r method2' by d e r i v a t i z i n g theophylline and the i n t e r n a l standard theobromine using pentafluorobenzyl bromide, i n connection w i t h an e l e c t r o n capture detector. To 100 p 1 samples o f plasma c o n t a i n i n g 50-1000 ng theophylline, 100 u l o f a s o l u t i o n o f the i n t e r n a l standard were added. The e x t r a c t i o n was performed by column e x t r a c t i o n ( c e l l u l o s e ) using d i c h l o r o methane as solvent. A f t e r back e x t r a c t i o n i n t o an a l k a l i n e aqueous phase (NaOH), theophyll i n e and theobromine were a1 k y l a t e d w i t h pentafluorobenzyl bromide by an e x t r a c t i v e a l k y l a t i o n technique. The d e r i v a t i v e s were e x t r a c t e d w i t h cyclohexane and, a f t e r concentration t o 3 m l , 2 u1 were i n j e c t e d f o r the gas chromatographic assay. Standard d e v i a t i o n o f the method was 2.5 % a t a concentration l e v e l o f 200 ng per sample, and 8 % a t 20 ng per sample. The sample amounts were 100 u l plasma and 250 mg r a t b r a i n tissue, and the s e n s i t i v i t y l i m i t

Refereneea p. 210

208

about 5 ng per sample. A packed column w i t h 3 % XE-60 on Gas Chrom Q a t 220' was used f o r the gas chromatographic assay. Schwertner e t a1 .31 introduced a s a l t i n g - o u t procedure, i n combination w i t h s i n g l e e x t r a c t i o n using chloroform-isopropanol

(95 : 5). 100 111 plasma samples could be e f f e c t i v e l y ex-

t r a c t e d w i t h 2 m l chloroform-isopropanol. Theophylline was d e r i v a t i z e d w i t h pentafluorobenz y l chloride, and 3-isobutyl-1-methylxanthine was used as an i n t e r n a l standard. This standard i s s i m i l a r t o t h e o p h y l l i n e i n e x t r a c t a b i l i t y , d e r i v a t i z a t i o n rates, s t a b i l i t y and chromatographic properties. Accurate measurements o f plasma concentrations ( + 0.22 pg/ml) could be obtained w i t h l i t t l e o r no i n t e r f e r e n c e from theophylline metabolites and o t h e r coextracta b l e material. A packed column w i t h 3 % OV-17 on Gas Chrom Q and temperature p r o g r a m i n g from 15OoC t o 25OoC was used i n combination w i t h an e l e c t r o n capture detector. B.Capi 1l a r y columns

I . Ethylation Glass c a p i l l a r y gas chromatography was a p p l i e d by Floberg e t al.63 f o r the simultaneous determination o f theophylline and c a f f e i n e i n small volumes (50 p1) o f plasma from premature i n f a n t s , a f t e r the samples' e x t r a c t i o n as i o n - p a i r s w i t h tetramethylamnonium i n t o d i c h l o r o methane and a c y l a t i o n w i t h e t h y l i o d i d e

-

followed by GC-MS.

The gas chromatography was per-

formed on a 25 m OV-225 glass c a p i l l a r y column and temperature p r o g r a m i n g (17O-21O0C

-

10°C

per min) using t r i d e u t e r o t h e o p h y l l i n e and hexadeuterocaffeine as i n t e r n a l standards. The compounds could a l s o be separated as pentafluorobenzyl d e r i v a t i v e s on a 25 m OV-17 glass c a p i l l a r y . The p r e c i s i o n o f the method was 3.9 % f o r t h e o p h y l l i n e and 3.2 % f o r c a f f e i n e (0.6 ug per m l plasma, n = 10) and the d e t e c t i o n l i m i t was 20 and 40 ng/ml plasma f o r t h e o p h y l l i n e and caffeine, respectively.

TABLE 20.8. EXPERIMENTAL CONDITIONS USED FOR Column glass, 6 f t x 4 m glass, 6 f t x 3 mn S . S . , 5 f t x 2.4 glass, 6 f t x 4 mn

GAS CHROMATOGRAPHY OF XANTHINE ALKALOIDS

S o l i d support mesh

I.D. I.D. I.D. I.D.

Stat.phase

CW 80-100 GP 100-140 C AW 60-80 Ana ABS 100-120

glass, 1 m x 2 mn I.D. CW S 80-100 glass. 3 f t x 0.07 i n I.D.GP AS 80-100

GQ S 100-120 GP 80-100

glass, 3 f t x 1/8 i n O.D. 6 f t x 1/4 i n s.s.S, 1 m x 3 mn I.D.

GP S 100-120 CW 60-80 CW AWS 80-100

+

%

SE-30 2-3 SE-30 1.15 SE-30 5 SE-30 1 QF-1 3 NPGS 1 SE-30 1 XE-60 1 EGSS-Y 1 HI-EFF 8B1 NGS 1 PVP NGS HIlEFF 8Bl SE-30 1.5 SE-30 1.5 SE-30 3 SE-52 1.5 QF-1 3 OV-17 2 XE-60 3

;

Temperature

Comp.Prep.

Ref.

204OC 175OC 210°C 150-180°C 2OO0C 2oooc 175OC

a1 k. s. a1k.s. alk.s.id.tox. alk.s.id.tox.

1 2 3

a1k.s.

5

2200c 23OoC 23O-24O0C 23OoC

a1k.s.

6

7 2oooc 220-230°C alk.s.id.tox. 8 200-300°C p r . xths.s. 9 1900C 2OOoC 210°C xths . , t p TMS. TFA. 210°C der.s.id.MS 24OoC 23OoC

.

209

TABLE 20.8 (continued) ~

Col umn

~~

S o l i d support mesh

Stat.phase

100 f t x 0.01 i n I.D. 200 f t x 0.01 i n 1.0. 100 f t x 0.01 i n I . D . glass cap., 12 m x 0.3 mn I . D . glass cap., 20 m x 0.35 mn I . D . glass cap., 12 m x 0.49 mn 1.0. f . s i l . cap., 12 m x 0.22 mn 1.0. s . s . , 5 f t x 1/8 i n 0.0. GQ 100-120 glass S . 6 f t x 4 mn 1.0. CW HP 100-120 s . s . . 2 m x 2.37 n GQ 100-120 GQ glass S , 180 cm x 3 mn 1.0. CW 80-100 sup. 100-120 glass, 122 cm x 2 mn 1.0. S.S.

-

cap.,

%

QF- 1 SE-30 Apiezon L T r i t o n X 305 SE-30 CP-Si1 5 CP-Si1 5 SE-30 3 OV-225 3 ov- 1 3 OV-17 3 OV-17 3 SP 2250 3

Temperature IOO-ZOO~C pr. 100-250°C p r . 100-250°C p r . 20O-25O0C pr. 50-25OOC pr. 137OC 235OC 21ooc 220°c 19O-24O0C pr. 160-240°C pr.

Comp.Prep.

Ref.

ca.

11

ca. her.smp1. ca.

12 13

drg.ca.s.id.

14

tb.tp.Me.der. tp.Me.der. to.Me.der. - r - - - - xths.Bu.der. tp.Bu. der. t p . Bu .der. qnt,

16 17 18 -~ 19 20

21

-1

glass 5, 5 f t x 0.25 i n 0.D.CW HP 100-120

SE-30

10

glass, 9 1 cm x 2 mn I . D . glass, 1.8 m x 2 nun I . D .

GQ 100-120 CG 100-120

OV-17 OV-17

3

180-260' 250°C

glass, 90 cm x 2 m I.D.

Sup.100-120

SP 225006

3

2oooc

OV-17

3 0.1

glass, 1.8 m x 2 mn I . D .

C 750 100-120

+

3

3

240°C

t p .Pe .der

XE-60

3

220°c

to.PFbz.der. qnt.bi. tp.PFbz.qnt.pl ca.qnt.coex. ca.qnt.tea ca .qnt.col a ca .qnt. co. ca.qnt.co. ca .qnt. d r . ca. qnt. p l . ca.qnt.bl. ca .qnt. p l ca.qnt.pl.

+

glass, 2 m x 4 mm I.D. 0ia.S 80-100 glass S , 6 f t x 4 mm 1.0. CW HP 80-100 - 8 f t x 4 ~n 1.0. CW HP 80-100 glass, 2 m CW HP 80-100 glass, 150 cm x 3 mm 1.0. CW HP 80-100 glass, 6 f t x 0.25 i n O.O.GQ 100-120 glass, 1.8 m x 4 mm 1.0. GQ 100-120 glass S , 6 f t x 1/8 i n 1.0. CW HP 100-120 glass, 6 f t x 1/4 i n 0.0. GQ 100-120 glass, 1.83 m x 2 mm 1.0. CW HP 80-100 CW HP 80-100 glass, 4 f t x 2 mn 1.0. Oiat CLQ glass, 1 m x 4 mn 1.0. GQ 100-120 2mx3mn

References p. 210

OV-17 3 Cab 20M 0.2 DC 200 10 Ver.930 2 SE-30 10 Cab 20M 2 OV-17 2.5 OV-17 3 Dex 300 3 OV-17 3 3 SE-30 Apiezon M 10 Poly S-179 3 DC 200 2 SE-30 10 SE-30 10 OV-17 3 Cab 20M 2 SE-30 2 UCC W98 10 Dex 300 10 Oex 300 17 SE-30 2.5 OV-1 3 HI-EFF 88 3 OV-17 3 OV-17 3 OV-17 3 OV-1 3 HI-EFF 86 0.5 PCHDS 3 OV-17 3

22 23

24 25

PI.

OV-17

glass, 6 ft x 2 mn 1.0.

glass, 1 m x 4 mn I . D . GQ 100-120 2 m MGB slass. 6 f t x 4 mn 1.0. GQ 80-100 glass; 5 f t x 1/4 i n Phs.N 60-85 AWS glass, 5 ft x 3 mn I.D. Var.30 70-80 s . s . , 80 cm x 1/8 i n CG HP AWS 100-120 glass, 90 cm x 2 mn 1.0. CG AWS 80-100 s . s . , 6 f t x 1/8 i n 0.0. CW HP AWS 100-120 glass, 5 f t x 0.25 i n 0.O.CQ 80-100 glass, 182 cm x 2.5 mn I . D . CP 60-80 glass, 180 cm x 2 mm 1.0. CW AWS 80-100 glass, 1.5 m x 4 mm 1.0. Srb 60-80 glass, 2.4 m x 4 mn 1.0. GQ S 60-80 s . s . , 6 f t x 4 mn I.D. Ha1.F 60-80 s . s . , 1.8 m x 2 m Cel. 80-100 s . s . , 6 f t x 1/8 i n O.D. CW AW GQ 100-120 glass, 4 f t x 4 mn 1.0. GP + KOH 5 % glass, 2 m x 4 mn 1.0.

.

3

3

GQ 80-100

pr.

t p . Bu .der .qn t. ser. tp.qnt . p l tp.Bu.der.qnt. PI. t p . Bu .der .qnt.

190°c 190-240°C p r .

OV-17 OV-17

glass S, 180 cm x 3 mn I . D .

220°c

t p . Bu.der.qnt. P'. tp.Pr.qnt.bi. t p . Pe.der.qnt.

thra.

GQ glass, 1.83 m x 3.1 mm glass, 122 cm x 2 mn 1.0. GQ 100-120 GQ 100-120

IJI *

230°C

150-250°C pr. 210°c 190°c 2oooc 215OC 220°c 260°C 2oooc 210°c 210°c 19ooc 225OC 225OC 75-200°C p r . 2 15OC 195-260°C pr. 165OC

.:

. ca.qnt. p l .

26 27 28 29 30

. 31 32 33 35 36 37 38 39 40 41 42 44

ca. qn t. pre. ca.qnt.pre. ca. qnt. pre. ca.qnt.pre.

45 46 47 48

ca.qnt.sup.

49

ca.qnt.pre.

50

2oooc 205OC 2zooc 222oc 18OoC 16OoC 250°C 2oooc 240°C 245OC 180-280°C p r . 240°C 225OC 190-220°C

ca.qnt.pre. 51 ca.qnt.pre. 52 tp. tb. qnt 53 tp.qnt.pre. 54 tp.qnt.ser. 55,56 tp.qnt.ser. 57 tp.qnt.bl. 58 tp.qnt.bl. 59 tp.qnt.pl. 60 tp.Pr.der.qnt. 61 bi

.

.

210 TABLE 20.8 (continued) Col umn

S o l i d support

Stat.phase OV-225 OV-17

glass cap., 25 m

I

Temperature

Comp. Prep.

170-210°C p r . tp.Eth.der.qnt. 170-210°C pr. p l .

Ref. 63

TABLE 20.9 XANTHINE ALKALOIDS

-

LIST OF ABBREVIATIONS

ABS = acid, base washed, s i l a n i z e d alk = alkaloid Ana = Anakrom AW = a c i d washed b i = biological material b l = blood Bu = b u t y l C = Chromosorb ca = c a f f e i n e cap = c a p i l l a r y Cel = C e l i t e co = c o f f e e coex = coffee e x t r a c t CP =Chromosorb P CW = Chromosorb W Dia = Diatoport D i a t = Diatomite der = d e r i v a t i v e Dex = Dexsil d r = drink drg = drug Eth = e t h y l f . s i l = fused s i l i c a GP = Gas Chrom P Gq = Gas Chrom Q Hal = Haloport her = heroin HP = high performance 1.0. = i n s i d e diameter i d = identification i n = inch

Me = methyl MGB = micro glass beads NGS = neopentyl g l y c o l succinate NPGS = neopentyl g l y c o l sebacate 0.0. = outside diameter PCHDS = poly(cyclohexy1 dimethanol succinate) Pe = p e n t y l PFbz = pentafluorobenzyl Phs.N = Phasesep N p l = plasma p r = (temperature) programming P r = propyl pre = pharmaceutical preparation qnt = quantitative s = separation S = silanized ser = serum smpl = sample Srb = Supasorb S.S. = stainless steel sup = suppository Sup = Supelcoport t b = theobromine TFA = t r i f l u o r o a c e t y l thra = therephthalic acid TMS = t r i m e t h y l s i l y l tox = toxicology t p = theophylline Var = Varaport xths = xanthines

20.4 REFERENCES

1 H.A. Lloyd, H.M. Fales, P.F. Highet, W.J.A. VandenHeuvel and W.C. Wildman, J. Am. Chem. Soc., 82 (1960) 3791. 2 E. Brochmann-Hanssen and A. Baerheim Svendsen, J. Pharm. S c i . , 51 (1962) 1095. 3 K.D. Parker, C.R. Fontan and P.L. Kirk, Anal. Chem., 35 (1963) 356. 4 L. Kazyak and E. Knoblock, dnd.Chem., 35 (1963) 1448. 5 H. Kolb and P.W. Patt, Arzneim.-Forsch., 15 (1965) 924. 6 E. Brochmann-Hanssen and C.R. Fontan, J. Chromatogr.. 19 (1965) 296. 7 E. Brochmann-Hanssen and C.R. Fontan, J . Chromatogr., 20 (1965) 394. 8 N.C. J a i n and P.L. Kirk, Nicrochem. J., 12 (1967) 229. 9 J. Reisch and H. Walker, Pharmazie, 21 (1966) 467. 10 K. Kamei and M. Atsushi, Chem. Phann. B u l l . , 21 (1973) 122811 J.L. M a s s i n g i l l and J.E. Hodgkins. Anal. Chem., 37 (1965) 95212 G. Bohn, E. Schulte and W. Audick, Arch. Kriminol., 160 (1977) 27. 13 A.S. Christophersen and K.E. Rasmussen, J. Chromatogr., 174 (1979) 454. 14 P. Schepers, J . Wijsbeek. J.P. Franke and R.A. de Zeeuw, J . Forensic S c i . , 27 (1982) 49. 15 J. MacGee, Anal. Biochem., 14 (1966) 305. 16 E. Brochmann-Hanssen and T.O. Oke, J . Pharm. S c i . , 58 (1969) 370. 17 L.J. Dusci, P. Hackett and I.A. McDonald. J . Chromatogr., 104 (1975) 147.

211 18 H. Kinsun, M.A. Moulin, R. Venezia, D. Laloum and M.C. Bigot, C l i n . C h i m . A c t a , 84 (1978) 315. 19 M. Kowblansky, B.M. Scheinthal, G.D. Cravello and L. Chafetz, J . C h r o m a t o g r . , 76 (1973) 467. 20 A. Arbin and P.-0. Edlund, Acta P h a m . s u e c . , 11 (1974) 249. 21 G.F. Johnson, W.A. Dechtiaruk and H.M. Solomon, C l i n . Chem. ( W i n s t o n - S a l e m , N . c . ) , 2 1 11975) 144. , 22 D.G. Bailey, H.L. Davis and G.E. Johnson, J. Chromatogr.. 121 (1976) 263. 23 Ch.J. Least, G.F. Johnson and H.M. Solomon, C l i n . Chem. ( W i n s t o n - S a l e m , N . c . ) , 22 (1976) 765. 24 D. P e r r i e r and E. Lear, C l i n . Chem. ( W i n s t o n - S a l e m , N . c . ) , 22 (1976) 898. 25 C.A. Pranskevich, J . I . Swihart and 5.3. Thoma, J . A n a l . T o x i c o l . , 2 (1978) 3. 26 B. Vinet and L. ZiZian, C l i n . Chem. ( W i n s t o n - S a l e m , N . C . ) , 25 (1979) 156. 27 V.P. Shah and S. Riegelman, J . P h a r m . S c i . , 63 (1974) 1283. 28 W.A. Dechtiaruk, G.F. Johnson and H.M. Solomon, C l i n . Chem. ( W i n s t o n - S a l e m , N . c . ) , 2 1 (1975) 1038. 29 J.D. Lowry, L.J. Williamson and V.A. Raisys, J . C h r o m a t o q r . , 143 (1977) 83. 30 A. Arbin and P.-0. Edlund, Acta P h a r m . S u e c . , 12 (1975) 119. 31 H.A. Schwertner, Th.M. Ludden and J.E. Wallace, Anal. C h e m . , 48 (1976) 1975. 32 0.G. Vitzthum, T r o i s i e m e C o l l o q u e Internat. sur l a C h i m i e d e s C a f & 1 9 6 7 , E d i t . Assoc. Sci. I n t . Cafe ( P a r i s ) 1968, 216. 33 J.M Newton, J. ASSOC. Off. A n a l . C h e m . , 52 (1969) 653. 34 J.A. Yeransian, H. Kadim, E. Borker and A. Stefanucci, J. ~ s s o c . off. m a . Chem. 46 (1963) 315. 35 E. Fogden and S. Urry, J . ~ s s o c .P u b l . A n a l . , 11 (1973) 104. 36 P. S c h i l l i n g and S. G a l , Z. L e b e n s m . - U n t e r s . F o r s c h . , 153 (1973) 94. 37 O.G. Vitzthum, M. B a r t e l s and H. Kwasny, Z. L e b e n s m . - U n t e r s . F o r s c h . , 154 (1974) 135. 38 F. Bandion, M i t t . R e b e , W e i n , O b s t b a u , F r d c h t e n v e r w e r t u n q , 25 (1975) 107. 39 F.L. Grab and J.A. Reinstein, J . P h a r m . S c i . , 57 (1968) 1703. 40 R.L. Merriman, A. Swanson, M.W. Anders and N.E. Sladek, J C h r o m a t o q r . , 146 (1978) 85. 41 J.L. Cohen, Chi Cheng, J.P. Henry and Yuen-ling Chan, J . P h a r m . S c i . , 67 (1978) 1093. 42 H. Milon and J.A. A n t o n i o l i , J. C h r o m a t o g r . , 162 (1979) 223. 43 B. Welton, C h r o m a t o g r a p h i d , 3 (1970) 211. 44 I . D . Bradbrook, C.A. James, P.J. Morrison and H.J. Rogers, J . C h r o m a t o q r . , 163 (1979) 118. 45 A.J. Hoffman and H . I . M i t c h e l l , J . P h a r m . sci., 52 (1963) 305. 46 P. Haefelfinger, €3. Schmidli and H. R i t t e r , A r c h . P h a r m . ( w i n h e i m ) , 297 (1964) 641. 47 E.B. Dechene, L.H. Booth and M.J. Coughey, J . P h a r m . S c i . , 2 1 (1969) 678. 48 L.L. Alber and M.W. Overton, J. ~ s s o c . off. A n a l . C h e m . , 54 (1971) 620. 49 A. Cometti, G. Bagnasco and N. Maggi, J . P h a r m . S c i . , 60 (1971) 1074. 50 F.L. Fricke, J . ASSOC. Off. A n a l . C h e m . , 55 (1972) 1162. 51 M. Oesch and M. S a h l i , Pharm. Acta Helv., 49 (1974) 317. 52 D. de Vos and G. Jonkhoff, P h a r m . Weekbl., 113 (1978) 1282. 53 M. Elefant, L. Chafetz and J.M. Talmage, J . P h a r m . S c i . , 56 (1967) 1181. 54 H.W. Schultz and C. Paveenbampen, J . P h a r m . S c i . , 62 (1973) 1995. 55 F.A. Chrzanowski, P.J. Niebergall, J.G. N i k e l l y , E.T. Sugita and R.L. Schnaare, B i o c h e m . Med., 11 (1974) 26. 56 F.A. Chrzanowski, P.J. Niebergall, R. Maycock, J. Taubin and E.T. Sugita, J . P h a r m . S c i . , 65 (1976) 735. 57 B. Wesley-Hadzija, B r . J . C l i n . P h a r m a c o l . , 1 (1974) 515. 58 M. Sheehan and P. Haythorn, J . C h r o m a t o g r . , 117 (1976) 393. 59 M. Albani and P.A. Toseland, N e u r o p a d i a t r i e , 9 (1978) 97. 60 R.E. Chambers, J . C h r o m a t o g r . , 171 (1979) 473. 61 J. Zuidema, J.E.C.P.M. L i c h t , J. P r i n s and F.W.H.M. Merkus, P h a r m . Weekbl., 111 (1976) 570. 62 R.H. Greeley, J. C h r o m a t o g r . , 88 (1974) 229. 63 S. Floberg, 5. Lindstrom and G. Lonnerholm, J . C h r o m a t o g r . , 221 (1980) 166. I

-

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213

11.8 MISCELLANEOUS ALKALOIDS Chapter 21

21.1. Cephalotaxus a l k a l o i d s 21.2. References

..

.................................... ....................................

..............

213 2 15

21.1. CEPHRMTAXUS ALKALOIDS Many a1 k a l o i d s o f the genus Cephalotaxus have demonstrated antitumour a c t i v i t y . Spencer e t a1 .lapplied gas chromatography t o separate the components o f crude a1 k a l o i d mixtures o f Cephalotaxus species. By combined GC-MS,

the i d e n t i t y o f known a l k a l o i d s could be confirmed

and the presence o f unknown a l k a l o i d s demonstrated. Estimation o f the various a l k a l o i d s was obtained by using methyl l i g n o c e r a t e (C24:o) as an i n t e r n a l standard by gas Chromatography on a packed column o f 3 % Dexsil 300 on Gas Chrom Q and temperature programming from 180°C t o 285'C.

Gas chromatograms o f standards and.of a p l a n t e x t r a c t are found i n Figure 21.1.

the

chromatographic data i n Table 21.1 and the formulas o f the a l k a l o i d s i n Figure 21.2. The r e s u l t s obtained by the assay o f Cephalotaxus a l k a l o i d e x t r a c t s o f various species a r e l i s t e d i n Table 21.2. FIGURE 21.1 CHROMATOGRAMS OF STANDARDS (A) AND A PLANT EXTRACT ( B ) ' .

1 = Cephalotaxine ( I a ) , 2 = homoerythrina a l k a l o i d s (VIa and b and V I I a and b). 3 = cephalotaxinone a r t e f a c t , 4 = drupacine (11), 5 = 11-hydroxycephalotaxine (111), 6 = homoerythrina a l k a l o i d s ( V I I I a and b), 7 = i n t e r n a l standard (C ), 8 = caphalotaxinone ( I V ) , 9 = uni d e n t i f i e d a l k a l o i d , 10 = deoxyharringtonine (Ie)T419 = i s o h a r r i n g t o n i n e ( I d ) , 12 = h a r r i n g tonine ( I b ) , 13 = homoharringtonine ( I c ) .

.

I *

0

References p. 216

?

10

20

30

LO

50 min

214

TABLE 21.1

CHROMATOGRAPHIC DATA FOR C E P m o T m u s ALKALOIDS' Peak numbers refer to Figure 21.1. Peak No.

Compound

tR (Rel)

1 2 3 4 5

Cephalotaxine Homoerythrina alkaloids (VIa and b, VIIa and b and IX) Cephalotaxinone artefact Drupacine (11) Acetylcephalotaxine (If) 11-Hydroxycephalotaxine(I1 I)

6 7

Homoerythrina alkaloids (VIIIa and b) Internal standard (C24:o) Cephalotaxinone (IV) Unidentified a1 kaloid Deoxyharringtonine (Ie) Isoharringtonine (Id) Harringtonine (Ib) Homoharringtonine (Ic)

Desmethylcephalotaxinone

8

9 10 11 12 13

0.55 0.67 0.71 0.73 0.79 0.80 0.80 0.88 1.00 1.05 1.24 1.76 1.90 2.00 2.14

(V)

TABLE 21.2 GAS CHROMATOGRAPHIC ANALYSIS OF SELECTED CEPHdLOTdXUS ALKALOID EXTRACTS'

Plant part

Species

Ia harrinqtonia harringtonia harringtonia harringtonia fortunei f o r t unei wilsoniana griffithii

tr.

=

var. var. var. var.

drupacea harringtonia harringtonia harringtonia

Seed Root Leaf Whole plant Leaf Seed Seed Root-leaf

Percentage of total alkaloids Ib Ic Id Ie I1 111

33 39 32 45 64 52 29 40

8.3 6.6 2.4 3.7 0.3 4.4 6.1 0.9

1.2 3.9 7.5 19 4.1 13 8.5 20 0.1 0.3 tr. 0.9 0.2 0.4 1.5 11

0.7 2.4 1.3 1.5

35 3.7 5.3 3.7 - 6.8 0.4 7.0 - 0.7 4.3 9.0

VIIIa

and b 9.6 6.5 1.1 3.4 2.6 2.9 2.1 2.2 1.4 4.2 4.6 2.2 0.9 45 1.7 2.5

trace

TABLE 21.3 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY OF Column glass, 4 ft

Solid support mesh GQ 100-120

cmmLoTdxus

ALKALOIDS

Stat.phase %

Temperature Comp.Prep.

Dex. 300

180-285'C

3

pr. a1k.s.

Abbreviations: Dex. 300 = Dexsil 300, pr. = (temperature) programming, alk. = alkaloid, s = separation

Ref. 1

216 FIGURE 21.2 CEPHALOTAXUS

ALKALOIDS

1

Numbers r e f e r t o Figure 2 1 . 1 and Table 21.1.

OJ

no llDl

R.H

lib1 R I

ocn,

ocn,

1 y?COIC~I

-c-c-cn2-cn,-c-Icnltl I on

o cn2co,cn1 IICI

R=

1 I I

- c - c - i c n , ~ l - c - IcnIIl I on

I on

IIVI

on I

lid1 R

s

o nc-colcul II I -c-c-cn2-cn,-cn I

-Icnlll

on 11.1

1111

R

o cnIcolcnl II I -c-c-cnl-cnlI on

cn--lcnII1

R

0 IVI

~.-c-cn,

21.2 REFERENCES 1 G.F. Spencer, R.D. Plattmer and R.G. Powell,

J . Cbromatogr.,

120 (1976) 335.

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217

Chapter 22 IMIDAZOLE ALKALOIDS 22.1 Pilocarpine 22.2 References

................................................................. ..................................................................

217 218

22.1 PILOCARPINE

1 P i locarpine was gas chromatographed by Bruchmann-Hanssen and Baerheim Svendsen on a packed column o f 1.15 % SE-30 a t 175OC. Brochmann-Hanssen and F ~ n t a n " ~chromatographed p i l o carpine on packed columns of various p o l a r i t y , e . q . XE-60, EGSS-Y, HI-EFF 8 B. as w e l l as 4 NGS and PVP + NGS. M a s s i n g i l l and Hodgkins used packed columns o f various p o l a r i t i e s and they separated p i l o c a r p i n e and i s o p i l o c a r p i n e on 1%JXR and 0.5 % Epon 1001 Resin w i t h r e t e n t i o n times o f 9.08 min and 8.75 min, r e s p e c t i v e l y , on JXR, and 11.50 min and 11.67 min, respectively. on Epon 1001 Resin. Bayne e t a l ? developed a method f o r the assay o f sub-nanograms o f p i l o c a r p i n e i n b i o l o g i cal f l u i d s . Because a s i g n i f i c a n t t a i l i n g was observed when p i l o c a r p i n e and i s o p i l o c a r p i n e were gas chromatographed, as w e l l as a tendency f o r p i l o c a r p i n e t o epimerize t h e r m a l l y t o isopilocarpine, the authors acylated t h e imidazole r i n g w i t h h e p t a f l u o r o b u t y r i c anhydride, using t r i e t h y l a m i n e as a c a t a l y s t . The p i l o c a r p i n e base was e x t r a c t e d w i t h dichloromethane from an aqueous s o l u t i o n a f t e r adjustment o f the pH t o 9. I t was subjected t o d e r i v a t i z a t i o n and a clean-up procedure, and gas chromatographed on a packed 3 % OV-17 on Chromosorb W c o l umn a t 190°C. The method described i s s p e c i f i c f o r p i l o c a r p i n e , w i t h the i s o p i l o c a r p i n e der i v a t i v e e l u t i n g p r i o r t o the p i l o c a r p i n e d e r i v a t i v e . The l i m i t o f s e n s i t i v i t y was 25-50 pg of p i l o c a r p i n e using EC-detection. Methazolamide was used as an i n t e r n a l standard after derivatization

-

t o dimethylmethazolamide.

-

also

Typical gas chromatograms are found i n Fig-

ure 22.1. TABLE 22.1 EXPERIMENTAL CONDITIONS USED FOR GAS CHROMATOGRAPHY OF PILOCARPINE Column

S o l i d support mesh

glass, 6 f t x 3 nun 1.0. glass, 3 f t x 2 mn I . D .

GP 100-140 GP AWS 80-100

glass, 3 f t x 2 mn copper, 6 f t x 1/8 copper, 2 f t x 1/8 glass S , 1.8 m x 2

Rafertnces D. 218

Stat.phase

SE-30 SE-30 XE-60 EGSS-Y HI-EFF 88 1.0. GP AWS 80-100 NGS GP 80-100 PVP + NGS i n O.D. GP 100-120 JXR Epon i n 0.0. Dia S 80-100 OV-17 m I.D. CW 100-120

%

1.15 1 1 1 1 1

1 1 0.5 3

Temperature

Comp. Prep.

Ref.

175OC 175OC 220°c 23OoC 23OoC 23OoC

a1k.s.

1

a1k.s.

2

a1k.s.

3

2oooc 100-300°C pr. 100-250°C p r . alk's' 190% p i 1. der. q n t .b i .

4

5

218

FIGURE 22.1 CHROMATOGRAMS OF P ILOCARP INE DER IVAT I VE5 prepared from aqueous p i l o c a r p i n e n i t r a t e s o l u t i o n c a r r i e d through e x t r a c t i o n , d e r i v a t i z a t i o n and clean-up procedure. A = i n t e r n a l standard, methazolamide d e r i v a t i v e ; B = p i l o c a r p i ne d e r i v a t i v e ; I = p i l o c a r p i n e base equivalent t o 50 pg; I 1 = p i l o c a r p i n e base e q u i v a l e n t t o 1 ng. Reproduced from J . P h a r m . sci., 65 (1976) 1724 w i t h permission o f the copyright owner.

1

I

0 1 2 3 1 5 6 7 8

0 1 2 3 1 5 6 7 8

min

TABLE 22.2 PILOCARPINE

-

LIST OF ABBREVIATIONS

alk = alkaloid AWS = a c i d washed, s i l a n i z e d b i = biological f l u i d CW = Chromosorb W Dia = Diaport Epon = Epon 1001 Resin GP = Gas Chrom P

1.0. = i n s i d e diameter O.D. = outside diameter

pi1 PVP qnt s = S =

= pilocarpine = p o l y v i n y l p y r r o l idone = quantitative separation silanized

22.2 REFERENCES 1 E. Brochmann-Hanssen and A. Baerheim Svendsen, J . P h a r m . sci., 5 1 (1962) 1095. 2 E. Brochmann-Hanssen and C.R. Fontan, J . C h r o m a t o g r . , 19 (1965) 296. 3 E. Brochmann-Hanssen and C.R. Fontan, J . C h r o m a t o g r . , 20 (1965) 394. 4 J.L. M a s s i n g i l l J r . and J.E. Hodgkins, A n a l . C h e m . , 37 1965 952. 5 W.F. Bayne, L.-C. Chu and F.T. Tao, J . P h a r m . Sci., 65 119761 1724.

H IGH-PER FORMANCE LlQUID CHROMATOGRAPHY

This Page Intentionally Left Blank

221

CONTENTS

.

I GENERAL PAR7 Chapter 1. General aspects o f HPLC o f a l k a l o i d s Chapter 2

. HPLC analysis

o f various a l k a l o i d s

...............................

.................................

223 235

.

I 1 SPECIAL PART Chapter 3 . P y r r o l i d i n e . p y r r o l izidine. p y r i d i n e . p i p e r i d i n e and q u i n o l i z i d i n e

a1 k a l o i d s .......................................................... . Tropane a l k a l o i d s : 4.1 Tropine a l k a l o i d s ........................... 4.2 Pseudotropine a l k a l o i d s ..................... Chapter 5 . Q u i n o l i n e a l k a l o i d s : Cinchona a l k a l o i d s ............................ Chapter 6 . Phenylethylamines and i s o q u i n o l i n e a l k a l o i d s ....................... Chapter 7 . Opium a l k a l o i d s .................................................... Chapter 8 . Terpenoid i n d o l e a l k a l o i d s and simple i n d o l e a l k a l o i d s ............. Chapter 9 . Ergot a l k a l o i d s .................................................... Chapter 10. S t e r o i d a l a l k a l o i d s ................................................ Chapter 11. Xanthine a l k a l o i d s ................................................. Chapter12 . Diterpene a l k a l o i d s ................................................ Chapter 4

................................... ................................................ Chapter 15 . Quaternary ammonium compounds ...................................... Appendix ...................................................................... Subject index ................................................................. Compound index ................................................................

241 249 260 269 287 297 331 357 381 387 415

Chapter 13 . Colchicine and r e l a t e d a l k a l o i d s

417

Chapter14 . Imidazole a l k a l o i d s

421 425 432 437 443

222

LIST OF ABBREVIATIONS USED FOR DESCRIPTION OF MOBILE PHASES ACN

Acetoni t r i l e

AcOH

Acetic a c i d

AmOH*

Amy1 alcohol

BuOH*

Butanol

BU*O

Dibutyl ether

OEA

Diethylamine

DMFA

Dimethy1 fonnami de

EtOAc

Ethyl acetate

EtOH

Ethanol

EteO

D i e t h y l ether

IsoprOH

Isopropanol

(IsoPr)*O Me2C0

D i isopropyl e t h e r Acetone

MeEtCO

Methyl e t h y l ketone

MeOH

Methanol

PrOH

Propanol

THF

Tetrahydrofuran

TrEA

Triethylamine

* I n the Tables the p r e f i x 1it e r a t u r e .

!,

or

5

i s added i f i t was mentioned i n t h e o r i g i n a l

1. GENERAL PART

This Page Intentionally Left Blank

223

Chapter 1 GENERAL ASPECTS OF HPLC OF ALKALOIDS

1.1. Ion-exchange HPLC ................................................................ 1.2. Reversed-phase HPLC 1.3. I o n - p a i r HPLC.. 1.4. S t r a i g h t - p h a s e HPLC 1.5. D e t e c t i o n methods 1.6. Sample p r e p a r a t i o n References

.............................................................. .................................................................. .............................................................. ................................................................ ............................................................... ............................................................................

223 224 227 228 230 231 232

F o r a s u c c e s s f u l a n a l y s i s o f a l k a l o i d s by means o f HPLC a s e r i e s o f f a c t o r s has t o be considered.They a r e o f t h i s book

-

-

b e s i d e s those o f an i n s t r u m e n t a l n a t u r e , which a r e beyond t h e scope

t h e s t a t i o n a r y phase, t h e m o b i l e phase, t h e method o f d e t e c t i o n and t h e sam-

p l e p r e p a r a t i o n . T h e y w i l l be d i s c u s s e d b r i e f l y i n t h i s c h a p t e r . So f a r HPLC a n a l y s i s o f a l k a l o i d s has been performed by means o f ion-exchange,

reversed-

phase, i o n - p a i r and s t r a i g h t - p h a s e chromatography. A l s o , some g e n e r a l aspects o f t h e a l k a l o i d a n a l y s i s by means o f these types o f chromatography w i l l be d e a l t w i t h i n t h i s c h a p t e r .

1.1. ION-EXCHANGE HPLC Although t h e i o n i c p r o p e r t i e s o f a l k a l o i d s would make them s u i t a b l e o b j e c t s f o r i o n - e x change chromatography, t h i s t e c h n i q u e has o n l y found l i m i t e d a p p l i c a t i o n s i n t h e a n a l y s i s o f a1 k a l o i ds. The s t a t i o n a r y phases employed i n t h e HPLC a r e u s u a l l y c h e m i c a l l y bonded ion-exchange groups ( a l k y l s u l f o n i c groups) on s i l i c a g e l . The a n a l y s i s o f some opium a l k a l o i d s on such a s t a t i o n a r y phase has been s t u d i e d b y Knox and Jurand112 (see Chapter 7 ) . R e t e n t i o n o f t h e a l k a l o i d s c o u l d be a l t e r e d by changes i n t h e m o b i l e phase, i . e .

t h e i o n i c s t r e n g t h , t h e na-

t u r e o f t h e c o u n t e r i o n , t h e pH and t h e a d d i t i o n o f an o r g a n i c s o l v e n t t o t h e m o b i l e phase. T w i t c h e t t e t a1.3 e v a l u a t e d ion-exchange chromatography f o r t h e a n a l y s i s o f a w i d e var i e t y o f drugs ( s e e Chapter 7 ) . They found t h a t r e t e n t i o n i s n o t o n l y due t o ion-exchange mechanisms, b u t p a r t i t i o n chromatographic mechanisms a l s o p l a y e d a r o l e . Whereas Knox and Jurand”*

o n l y used l o w percentages o f o r g a n i c s o l v e n t s i n t h e m o b i l e phase, T w i t c h e t t e t

a l . r e p o r t e d t h a t a t l e a s t 40% a c e t o n i t r i l e o r methanol s h o u l d be used t o o b t a i n good e f ficiency.McMurtrey

e t a1 . 4 r e p o r t e d t h e a n a l y s i s o f some i s o q u i n o l i n e a l k a l o i d s b y means o f

ion-exchange chromatography (see Chapter 6 ) . They a l s o used l o w p e r c e n t a g e s o f o r g a n i c mod i f i e r s i n t h e m o b i l e phase, t h e n a t u r e o f t h i s m o d i f i e r had o n l y l i t t l e i n f l u e n c e on t h e s e l e c t i v i t y o f t h e s e p a r a t i o n . Walton and M ~ r g i a ~ r’ e~p o’ r~t e d t h e s e p a r a t i o n o f v a r i o u s a l k a l o i d s on cation-exchange r e s i n s l o a d e d w i t h m e t a l i o n s capable o f f o r m i n g complexes

w i t h amnonia (Cutt,Nitt,Zntt,Agt). T h i s t y p e o f chromatographic t e c h n i q u e has n o t f o u n d any f u r t h e r a p p l i c a t i o n s i n t h e a n a l y s i s o f a l k a l o i d s . I n t h e appendix t h e p r o p e r t i e s o f some commercially a v a i l a b l e ion-exchange s t a t i o n a r y phases a r e summarized.

224

1.2. REVERSED-PHASE HPLC

-

Reversed-phase chromatoqraphy has most widely been used i n HPLC alkaloid analysis Part i c u l a r l y on microparticulate s i l i c a qel with chemically bonded alkyl qroups, i . e . octyl and octadecyl qroups. However, in several cases i t has been found t h a t reversed-phase materials were unsuitable f o r the analysis of basic compound^^'^. So Baker e t a1.l' reported highly variable column efficiency ( p l a t e number varying from 100 t o 2500, see Tables 2.2 and 2.3) f o r various basic compounds, when using an octadecyl column. The t a i l i n g and variable p l a t e numbers are probably caused by f r e e silanol groups i n the stationary phase. The number of silanol groups i n s i l i c a gel i s about 6 per nm 2 For hydrocarbons d i r e c t l y bonded t o s i l i c a , a n average of about 4 silanol groups i s bonded. For the smallest group trimethylsilyl - t h i s may even be 5, whereas f o r the bulky octadecyl group only 2 s i l a n o l groups a r e bonded per nm2 11'12. Unger e t a1 ,13 came t o an even lower coverage of the s i lano1 groups (see Table 1.1). The remaining silanol groups a r e shielded by the hydrocarbon group. According t o Karch e t a1.14 the optimum coverage and shielding of the s i l a n o l groups i s obtained w i t h C4-alkyl chains. The number of f r e e silanol groups i n long chain alkyl bonded packing material can be reduced by endcapping, i . e . the bonded phase i s f u r t h e r treated with a small monofunctional s i l a n e , such as t r i m e t h y l ~ i l a n e ' ~ . The best r e s u l t s in the analysis of alkaloids will generally be obtained with a s t a t i o nary phase with the highest possible coverage of the silanol groups. The amount of adsorption power ( f r e e silanol groups) l e f t in a reversed-phase material, can be t e s t e d by using the column i n the adsorption mode with a non-polar mobile phase, such as heptane. and a sol u t e such as methanol or acetone. I f no adsorption i s present, a symmetrical peak with a k' (capacity factor) equal t o 0 i s expected13. The mobile phases employed i n reversed-phase chromatography usually consist of water t o which an organic solvent - miscible w i t h water (mostly methanol o r a c e t o n i t r i l e ) - i s added. Generally an increased polarity of the mobile phase gives a decreased solvent strength, i . e . an increased k ' . For alkaloids - analyzed by means of water methanol mixtures - systematical studies have been made16. Karch e t al.14 presented an eluotropic s e r i e s f o r some common solvents used in reversed phase HPLC (see Table 1.2). The series i s based on the retention of the solvents mentioned on an alkyl bonded stationary phase u s i n g water as eluent.The higher the value f o r the sol-

.

-

TABLE 1.1 SURFACE 5QNCENTRATION OF SOME CHEMICALLY BONDED ALKYL GROUPS IN REVERSED-PHASE SILICA GEL PACKINGS

.

Bonded organic functional group

Surface concentration (prnole/m2)

Trimethylsi lyl Dimethylphenyl s i lyl Triphenylsi lyl n-Butyl di methyl s i lyl n-Butyl di phenyls i lyl n-Octyl di methyl s i l y l n-Hexadecyldimethylsi l y l silanol arouDs S i l i c a ael

4.5 2.6 1.5 3.6 1.8 3.8 3.4 8.0

-

226

TABLE 1.2 AELATIVE RETENTIOfiS14RELATIVE TO METHANOL) ON REVERSED PHASES WITH WATER AS ELUENT (ELUOTPOPIC SEPI E S I Compound

S t a t i o n a r y phase C8 1.0 2.7 3.2 3.3 8.4

Met ha no 1 Acetic a c i d Ethanol Acetoni t r i l e Isopropanol

5 8 1.0 3.1 3.1 8.3

Compound

S t a t i o n a r y phase

Oimethyl formami de Acetone n-Propanol Oioxane

vent. t h e more i t w i l l reduce t h e r e t e n t i o n o f a sample

-

C8

c18

9.4

7.6

9.3

8.8 10.1 11.7

10.8 13.5

i f t h e s o l v e n t i s used i n a m i x -

t u r e w i t h w a t e r as e l u e n t . B a k a l y a r e t a1 .17 found t h a t t h e s e l e c t i v i t y o f e l u e n t s c o n t a i n i n g m i x t u r e s o f w a t e r w i t h methanol, a c e t o n i t r i l e o r t e t r a h y d r o f u r a n v a r i e d f o r d i f f e r e n t f u n c t i o n a l groups.The s e l e c t i v i t y c o u l d be v a r i e d by u s i n g t e r n a r y s o l v e n t s . The s e l e c t i v i t y o f a reversed-phase s e p a r a t i o n on a l k y l bonded phases c o u l d b e s t be v a r i e d by a l t e r i n g t h e m o b i l e phase; t h e n a t u r e o f t h e a l k y l group had o n l y l i m i t e d i n f l u e n c e on t h e s e l e c t i v i t y .

I n o r d e r t o reduce t a i l i n g on reversed-phase m a t e r i a l s , b a s i c m o b i l e phases can be used, However, t h e s t a b i l i t y o f t h e c h e m i c a l l y bonded groups above pH 8.5 i s 1 i m i t e d . T h e s t a b i l i t y of some reversed-phase m a t e r i a l s f o r v a r i o u s amines was s t u d i e d b y W e h r l i e t a1.18.

When

u s i n g i n o r g a n i c bases, t h e columns had a l i f e t i m e o f o n l y a few days. S i l i c a g e l d i s s o l v e d much f a s t e r than t h e reversed-phase m a t e r i a l s . Using p r i m a r y . secondary and t e r t i a r y amines o r ammonia i n t h e m o b i l e phase, a n e g l i g i b l e d e c o m p o s i t i o n o f t h e reversed-phase m a t e r i a l s was found. Sodium h y d r o x i d e and q u a t e r n a r y amines d i s s o l v e d t h e s t a t i o n a r y phases, p a r t i c u l a r l y t h e s i l i c a g e l , l e a v i n g t h e hydrocarbon groups on t h e remnants o f t h e s u p p o r t . The a t t a c k o f t h e s i l i c a g e l l a t t i c e decreased i n t h e s e r i e s p r i m a r y , secondary, t e r t i a r y amine and i n t h e s e r i e s m e t h y l , e t h y l , p r o p y l . The w a t e r s o l u b l e t r i e t h y l a m i n e was f o u n d t o be most s u i t e d as b a s i c m o d i f i e r f o r reversed-phase chromatography. For c h e m i c a l l y bonded s t a t i o n a r y phases, column l i f e can be improved by u s i n g a p r e - i n j e c t i o n s i l i c a g e l guard column19 (see b e l o w ) . To overcome t h e problem o f t a i l i n g i n reversed-phase HPLC, s a l t s can a l s o be added t o t h e m o b i l e phase

-

s o - c a l l e d i o n - s u p p r e s s i o n . Ammonium carbonate, sodium a c e t a t e and sodium

phosphate have been used f o r t h i s purpose i n t h e a n a l y s i s o f a l k a l o i d s . Also, i o n - p a i r i n g has p r o v e d t o be s u c c e s s f u l i n a l k a l o i d a n a l y s i s , I t a l l o w s a n a l y s i s o f a l k a l o i d s u n d e r aci d i c c o n d i t i o n s , t h u s a v o i d i n g t h e problem o f c h e m i s o r p t i o n o f t h e b a s i c compounds on t h e a c i d i c s i l a n o l groups. A d d i t i o n of l o n g a l k y l c h a i n amines i n low c o n c e n t r a t i o n s t o t h e m o b i l e phase has been d e s c r i b e d as a method t o improve peak performance i n t h e a n a l y s i s o f b a s i c

compound^^^-^^.

Low c o n c e n t r a t i o n s o f t e t r a m e t h y l ammonium i n t h e m o b i l e phase have a l s o been r e p o r t e d t o improve peakshape i n reversed-phase HPLC28'29.

The b e n e f i c i a l e f f e c t o f t h e a d d i t i o n o f

amines t o t h e m o b i l e phase was e x p l a i n e d b y t h e masking o f f r e e s i l a n o l groups b y t h e amines. 23.25,26,27 Several a u t h o r s compared v a r i o u s amines f o r e f f e c t on t h e column performance G i l l e t a1.27 found t h a t an i n c r e a s e i n c h a i n l e n g t h o f t h e amine a d d i t i v e r e s u l t s i n a

s i g n i f i c a n t improvement o f peakshapes o f b a s i c compounds. The i n t r o d u c t i o n o f h y d r o x y l

References p. 232

226

groups i n the amine considerably reduced the b e n e f i c i a l e f f e c t o f the amine a d d i t i v e . Also, the geometry o f the amine was an important f a c t o r . O f the isomers, t r i e t h y l a m i n e and hexylamine, the l a t t e r gave b e t t e r r e s u l t s than the former, i n d i c a t i n g t h a t t h e primary amine i n t e r a c t s more s t r o n g l y with the a c t i v e s i l a n o l groups than does the former. I n t r o d u c t i o n of one o r two f u r t h e r methyl groups on the n i t r o g e n i n hexylamine d i d n o t a f f e c t the i n t e r a c t i o n o f the amine w i t h the s i l a n o l groups. B i j e t a1.25 found t h a t the long chain amines even a t very low concentrations ( 1 mM) were much more e f f i c i e n t i n masking s i l a n o l groups than, f o r example, the more bulky t r i e t h y l a m i n e . The authors described a method t o measure the s i l a n o l masking e f f e c t and found hexadecyltrimethyl amnonium bromide t o be t h e most acti ve compound.

Comparison of various a l k y l chain lengths o f the chemically bonded groups has l e d t o t h e conclusion t h a t s e l e c t i v i t y i s n o t changed, b u t t h a t k ' increases w i t h increased a l k y l chain length12 ~ 1 3 ~ 1 4 Goldberg3' compared several types o f octadecyl columns f o r t h e i r r e t e n t i o n o f n e u t r a l

,

a c i d i c and basic compounds. Vastly d i f f e r i n g chromatographic p r o p e r t i e s were found. A more extended study o f the d i v e r s i t y o f octadecyl bonded phases was made by Engelhardt e t a l . 31

.

The s u i t a b i l i t y o f the various m a t e r i a l s f o r the analysis o f basic compounds i s a l s o taken i n t o consideration. An important p o i n t made by these authors i s the pH o f the s i l i c a gel i n aqueous suspension.This pH may range from 3.8

-

9.932. Reversed-phase m a t e r i a l s made from

such basic s i l i c a g e l s showed b e t t e r peakshapes f o r basic compounds than d i d m a t e r i a l prepared from a weakly a c i d i c s i l i c a gel. Besides s t a t i o n a r y phases containing chemically bonded a l k y l chains, such phases w i t h cyano-alkyl and alkyl-amino chains have been used i n the a n a l y s i s o f a l k a l o i d s . However. so f a r , no extensive comparative s t u d i e s have been performed on the usefulness o f the various reversed-phase m a t e r i a l s f o r the analysis o f basic compounds. Several studies on the mechanism of r e t e n t i o n i n reversed-phase chromatography have been .

c a r r i e d o u t and d i f f e r e n t theories have been developed t o e x p l a i n the r e t e n t i o n mechanism (ref.33-37).Reviews

on the various aspects o f reversed-phase HPLC have been given by a num-

ber o f authors 12.15,36,38.39,40,41 An advantage o f reversed-phase chromatography i n the a n a l y s i s o f a1 k a l o i d s i n b i o l o g i c a l f l u i d s i s t h a t an analysis can be c a r r i e d o u t d i r e c t l y w i t h o u t any laborious sample clean-up procedure. However, the use o f a precolumn t o avoid a too r a p i d d e t e r i o r a t i o n o f the HPLC column i s advisable (see Chapter 11). When using aqueous s a l t s o l u t i o n s i n reversed-phase chromatography, one has t o be aware o f the r i s k o f corrosion o f s t a i n l e s s s t e e l columns (see Table 1.3)38. TABLE 1.3 TYPICAL FOR STAINLESS STEEL CORROSIVE SOLUTIONS38.

W i l l corrode a t a l l concentrations

W i l l corrode a t 10% i n water

A1 umini um f l u o r i d e Ammonium chloride, amnonium f l u o r i d e Amy1 chloride, benzoyl c h l o r i d e Aqua r e g i a (HC1 + HN03) Chloroacetic a c i d HF, HCl. HBr and B r 2 Pb. L i Ma halides K C l , Kh,-NaCl, NaBr

Aluminium c h l o r i d e , aluminium n i t r a t e Ammoni um d i phosphate Ammonium p e r c h l o r a t e Boric acid Potass iurn c h l ora t e Sodium bicarbonate. sodium carbonate

.

227

SCHEME 1.1 GENERAL OUTLINES OF A REVERSED-PHASE HPLC SYSTEM FOR THE SEPARATION OF BASIC COMPOUNDS Stationary phase

Mobile phase

Octadecyl ( o c t y l ) bonded phase w i t h low percentage o f f r e e s i l a n o l groups

ION-SUPPRESSION MODE : methanol (acetoni tri l e ) water c o n t a i n i n g ca. 0.01 0.1 M phosphate b u f f e r . 7). ammonium carbonate o r sodium acetate (pH 4

-

-

-

-

ION-PAIR MODE : methanol ( a c e t o n i t r i l e ) water containing ca. 0.005 M a l k y l s u l f o n a t e and 1%a c i d 4. ( a c e t i c acid), pH 2

-

Besides chemically bonded phases on s i l i c a gel supports m i c r o p a r t i c u l a t e macroporous polymer r e s i n s a l s o have been used i n the analysis o f a l k a l o i d s (see Chapters 7 and 8). A d i s advantage o f macroporous polymer r e s i n s i s t h a t they are n o t as r i g i d as the reversed-phase materials based on s i l i c a g e l . I n a d d i t i o n they may s h r i n k o r swell s l i g h t l y

- depending on

the composition o f the mobile phase. On the o t h e r hand they are more s t a b l e than chemically bonded phases on s i l i c a g e l . They can be used i n the e n t i r e pH range

- and a column l i f e o f

more than two years w i t h o u t loss i n e f f i c i e n c y has been reported42s43'44. Robinson e t a l . 44 presented an e l u o t r o p i c s e r i e s o f solvents f o r such r e s i n s .

A sumnary o f the p r o p e r t i e s o f some o f the c o m n l y used reversed phase s t a t i o n a r y phases i s given i n the appendix. The general o u t l i n e s f o r a reversed-phase HPLC-system f o r b a s i c compounds i s given i n Scheme 1.1. 1.3. ION-PAIR HPLC The i o n i c p r o p e r t i e s o f a l k a l o i d s under a c i d i c conditions make them s u i t a b l e f o r i o n - p a i r chromatography. I n i o n - p a i r chromatography the a l k a l o i d a l c a t i o n i s b a s i c a l l y combined w i t h an anion t o y i e l d an i o n - p a i r , which can a c t as a n e u t r a l molecule. The i o n - p a i r i s p a r t i tioned between a mobile phase and a s t a t i o n a r y phase during the chromatographic process. I o n - p a i r chromatography can be p r a c t i s e d i n the normal o r i n the reversed-phase mode. The theory o f the r e t e n t i o n mechanisms i n reversed-phase i o n - p a i r chromatography has been the subject o f several c o n t r o v e r s i a l p u b l i c a t i o n s . Models have been described i n which the i o n - p a i r i t s e l f i s adsorbed on the s t a t i o n a r y phase o r i n which the p a i r i n g i o n i s adsorbed on the s t a t i o n a r y phase, thus a c t i n g as a s o r t o f ion-exchange s t a t i o n a r y p h a ~ e ~ ~ -Terms ~'. such as soap ~ h r o m a t o g r a p h '47 y ~ ~ and dynamic ion-exchange chromatography4' have been used i n t h i s connection. A more general theory f o r i o n - p a i r chromatography has been given by Bidlingmeyer e t a l . 51'52

and Knox and H a r t ~ i c k ~I t~ explains . r e t e n t i o n by adsorption o f the

p a i r i n g ions (hetaerons) on the s t a t i o n a r y phase, r e s u l t i n g i n a charged surface. The higher the concentration o f the hetaeron on the surface, the stronger the r e t e n t i o n o f opposite charged ions. Knox and H a r t ~ i c kfound ~ ~ a l i n e a r r e l a t i o n between surface concentration o f the hetaerons and k ' o f opposite charged s o l u t e ions. Solute ions w i t h the same charge as the hetaeron showed decreasing k ' upon increasing hetaeron surface concentration, e v e n t u a l l y leading t o negative k'-values. By comparison o f hetaerons o f d i f f e r e n t chain lengths i t was observed t h a t the k ' d i d n o t depend on chain length

- when

the surface concentrations o f the

hetaerons were compared. From loading a reversed-phase s t a t i o n a r y phase w i t h the hetaerons i t was n o t i c e d t h a t the

Referencesp. 232

228

longer chain ions had l a r g e r breaktrough volumes. thus r e q u i r i n g longer e q u i l i b r a t i o n tim e ~ In ~ ~order . t o reduce the e q u i l i b r a t i o n time, h i g h concentrations o f hetaeron could be used t o l o a d the column; subsequently the concentration o f the hetaeron was reduced t o the desired l e v e l , u n t i l e q u i l i b r a t i o n occurred. The strongly’adsorbed hetaerons were d i f f i c u l t t o wash o u t o f t h e column, e.g. 53 tadecyl s t a t i o n a r y phase

dodecyl-sulfate could n o t be removed completely from an oc-

.

Straight-phase i o n - p a i r HPLC has been a p p l i e d f o r the analysis o f alkylamines (see Chapt e r 15) using a s t a t i o n a r y phase loaded w i t h an aqueous p i c r a t e o r naphtalenesulfonate sol u t i o n . These counter-ions a l s o improve the d e t e c t i o n l i m i t o f poorly UV-absorbing compounds. Also inorganic counter-ions. such as bromide and perchlorate, have been used i n s t r a i g h t phase i o n - p a i r chromatography (see Chapter 15). Reversed-phase i o n - p a i r chromatography has a l s o been used f o r the a n a l y s i s o f a l k y l amines (see Chapter 15). Most c o n o n l y a l k y l s u l f o n a t e s and a l k y l s u l f a t e s have been used as counter-ions. The i n f l u e n c e o f the a l k y l - c h a i n l e n g t h o f the counter-ion on the r e t e n t i o n has been i n ~ e s t i g a t e d ~ ~ - ~ ~equal . F o rconcentrations o f i t i n the mobile phase the r e t e n t i o n generally increases w i t h increased a l k y l - c h a i n l e n g t h o f the hetaeron (see Chapter 2 and 7). A d d i t i o n o f amines t o the mobile phase seems t o improve peakshape and t o r e g u l a t e the

A comparison o f some reversed-phase s t a t i o n a r y phases used i n i o n p a i r HPLC has been p e r f ~ r m e d ~ (see ~ ’ ~ Chapter ~ ’ ~ ~ 2 and 7 ) . Lurie56s57’58 found t h a t the r e t e n t i o n o f c l o s e l y r e l a t e d compounds could be best improved by changing the water content o f the mobile phase o r the a l k y l - c h a i n length o f the p a i r i n g ion. Bidlingmeyer51’52 optimized a separation by using mixed p a i r i n g - i o n s . Reviews on i o n - p a i r chromatography have been The general o u t l i n e s of a reversed-phase i o n - p a i r HPLC-system are summarized i n Scheme 1.1. 1.4. STRAIGHT-PHASE HPLC Due t o the extensive experience gained i n analysis o f a l k a l o i d s by TLC on s i l i c a gel p l a tes and the i n i t i a l poor peak performance o f basic compounds i n reversed-phase HPLC, the number o f a p p l i c a t i o n s o f s i l i c a gel as s t a t i o n a r y phase i n the HPLC of a l k a l o i d s i s r e l a t i v e l y high, as compared w i t h o t h e r groups o f compounds. Also, because o f the weakly a c i d i c s i l a n o l groups present i n s i l i c a gel, b a s i c m o d i f i e r s , such as ammonia, diethylamine and triethylamine, have o f t e n been added t o the s o l v e n t systems used i n TLC-analysis o f a l k a l o i d s t o avoid t a i l i n g caused by chemisorption (see Fig. 8.9).

However, under basic conditions the s t a b i l i t y o f s i l i c a gel i s poor18. Depending on

the nature o f the organic solvents used i n combination w i t h the basic m o d i f i e r , the s i l i c a gel i s dissolved a t various rates. This l i m i t s the a p p l i c a b i l i t y o f s i l i c a gel as s t a t i o n a r y phase i n the HPLC analysis o f a l k a l o i d s . For c e r t a i n solvent systems the column l i f e can be extremely s h o r t Atwood e t a1.l’

- even

some days.

studied the d i s s o l u t i o n o f s i l i c a gel i n the mobile phase by measuring

the concentration o f s i l i c a gel i n the column e f f l u e n t by atomic absorption. For uncoated s i l i c a gel and water as mobile phase a t a f l o w r a t e o f 1 ml/min the concentration o f s i l i c a gel i n the column e f f l u e n t was 38 ug/ml. For s i l i c a g e l w i t h chemically bonded octadecyl groups, no measurable amounts o f s i l i c a gel were found i n the e f f l u e n t under the same experimental conditions ( d e t e c t i o n l i m i t 0.3 pg/ml). The same was t r u e f o r uncoated s i l i c a

229

gel when methanol was used as mobile phase. However, by using a p r e - i n j e c t i o n guard column

-

which saturates t h e mobile phase w i t h s i l i c a gel

tonitrile

- water

-

a column c o u l d be used w i t h ace-

(2:3) containing ammonia (pH 10.7) as mobile phase w i t h o u t t h e r a p i d l o s s

o f e f f i c i e n c y t h a t was observed f o r an a n a l y t i c a l column w i t h o u t a guard column. The amount o f s i l i c a gel i n the column e f f l u e n t , when using a guard column, was about 100 pg/ml. Also, reversed-phase m a t e r i a l s could be protected i n the same way against d i s s o l u t i o n . Engelhardt and MUller3* reported on the differences i n the physical p r o p e r t i e s , such as s p e c i f i c surface area, s p e c i f i c pore volume and average pore diameter

-

and on the d i f f e r e n t

amounts o f s t a t i o n a r y and mobile phase per u n i t column volume f o r various comnercially avail a b l e s i l i c a gels. If the r e t e n t i o n f o r various s o l u t e s were normalized f o r these f a c t o r s , d i s t i n c t s e l e c t i v i t i e s were s t i l l noticed. This could be explained by d i f f e r e n c e s i n the surface pH o f the s i l i c a s . I r r e g u l a r ones were u s u a l l y n e u t r a l o r weakly a c i d i c , whereas the spherical ones were e i t h e r a c i d i c (pH ca.4) o r basic (pH ca.9)

(see Table 1.4).To o b t a i n the

required and optimum s e l e c t i v i t y , the pH o f s i l i c a gel can e a s i l y be adjusted. For b a s i c compounds more symmetrical peak shapes were obtained on s i l i c a w i t h a basic character. The a c t i v i t y o f t h e s i l i c a gel i s i n HPLC o f a l k a l o i d s of l i m i t e d value, since t h e mobile phases mostly used contain a r e l a t i v e l y high percentage o f p o l a r m o d i f i e r s , such as alcohols and/or bases. Deactivation o f the s i l i c a gel by water present i n the samples o r i n the mob i l e phase w i l l be swamped by the e f f e c t o f the m o d i f i e r s mentioned. I n straight-phase chromatography a change o f the mobile phase w i l l r e q u i r e longer e q u i l i b r a t i o n time than i n reversed-phase chromatography w i t h chemically bonded phases. A more d e t a i l e d discussion on 61-64 the p r o p e r t i e s o f s i l i c a gel i s given i n the l i t e r a t u r e

.

Aluminium oxide, which f r e q u e n t l y has been used i n TLC analysis o f a l k a l o i d s , has o n l y found l i m i t e d a p p l i c a t i o n i n HPLC. Concerning the mobile phases used i n straight-phase HPLC, the reader i s a l s o r e f e r r e d t o the discussion on mobile phases i n Volume 23A o f t h i s s e r i e s : Chromatography o f A l k a l o i d s Part A: Thin-layer chromatography. The general o u t l i n e s o f a straight-phase HPLC-system f o r basic compounds i s given i n Scheme 1.2. TABLE 1.4 pH VALUES OF DIFFERENT SILICA GELS MEASURED I N A' l % ( w / w ) AQUEOUS SUSPENSION3'. S i l i c a gel Zorbax 8PSil Lichrospher S i l o 0 Nucleosi 1 100-7 H 80-10 (home-made) Lichrosorb S i 100 Porasi 1 P a r t i s i l 10

Rcfesnncea p. 232

pH 3.9 5.3 5.7 6.5 7.0 7.2 7.5

Regular(R) o r i r r e g u l a r ( I)

R R R I

I I I

S i l i c a gel Lichrosorb Si60 P o l y g o s i l 60-5 Spherosil XOA 400 Hypersi 1 Lichrospher Silo00 Spherisorb S l o w Lichrospher Si500

pH 7.8 8.0 8.1 9.0 9.2 9.5 9.9

Regular(R) o r irregular(1)

I I R R R R R

230 SCHEME 1.2

GENERAL OUTLINES OF A STRAIGHT-PHASE HPLC SYSTEM FOR THE SEPARATION OF BASIC COMPOUNDS Stationary phase

Mobile phase

S i l i c a gel

Dichloromethane, Chloroform, Ether ( d i e t h y l - .isopropyl-), Tetrahydrofuran o r Ethyl acetate

-

Methanol or Isopropanol

-

Ammonia, Diethylamine o r Triethylamine (ca. 1%o f the mobile phase)

1.5. DETECTION METHODS I n the HPLC analysis o f a l k a l o i d s , UV d e t e c t i o n has mostly been used.Detectors w i t h f i x e d wavelength (254 and/or 280 nm) have been widely employed.These detectors are f a i r l y sensitive

-

a l l o w i n g detection i n the ng range o f a l k a l o i d s . Multiwavelength UV detectors can be

used t o achieve a more s e l e c t i v e and/or more s e n s i t i v e detection. However, d e t e c t i o n i n the shorter wavelength region o f UV l i m i t s the number o f solvents t h a t can be used i n the mobile phase. O f the p o l a r solvents, alcohols can be used down t o about 205 nm and a c e t o n i t r i l e and water down t o a l i t t l e below 200 nm. O f the non-polar solvents, alkanes can be used t o about

200 nm, w h i l e moderately p o l a r ethers have a UV c u t - o f f p o i n t a t about 220 nm. Due t o the great d i f f e r e n c e s i n the e x t i n c t i o n c o e f f i c i e n t s o f a l k a l o i d s - f o r example 1% E:Zm = 6 f o r a t r o p i n e and Elcm = 888 f o r serpentine a t 254 nm - equal amounts o f a l k a l o i d s may give q u i t e d i f f e r e n t peak areas. A chromatogram recorded by means of an UV detector may therefore be misleading i n a d i r e c t comparison o f the amounts o f d i f f e r e n t a l k a l o i d s present based on peak areas (see a l s o Table 7.7). The d i f f e r e n c e i n UV absorption o f a compound, measured a t two d i f f e r e n t wavelengths by means o f a dual wavelength detector, has been used f o r i d e n t i f i c a t i o n purposes i n HPLC o f a l k a l o i d s too. The absorption r a t i o o f a compound measured a t two d i f f e r e n t wavelengths i s c h a r a c t e r i s t i c f o r a compound. Baker e t a1.l'

( r a t i o 254:280.

Table 2.2 and 2.3) and L u r i e

e t al.65 ( r a t i o 220:254, Table 7.6) applied t h i s method t o the analysis o f a s e r i e s o f drugs. The absorption r a t i o mentioned can a l s o be used t o c o n t r o l the p u r i t y o f a compound e l u t e d from a HPLC column and recorded as one peak. I f a peak represents more than one compound, the absorption r a t i o w i l l d i f f e r from t h a t o f a pure compound66

.

Fluorescence d e t e c t i o n i s h i g h l y s p e c i f i c and o f t e n extremely sensitive.However, i s l i m i t e d t o fluorescent a l k a l o i d s

-

i t s use

eventually t o a l k a l o i d s made f l u o r e s c e n t by d e r i v a t i -

zation. Various chemical reactions can be used f o r t h i s purpose, such as o x i d a t i o n t o g i v e fluorescent o x i d a t i o n products, coupling w i t h f l u o r e s c e n t groups, i.e. dansyl groups, and i o n - p a i r i n g w i t h fluorescent ions, i.e. p i c r a t e , 8-naphtalenesulfonate, 9,lO-dimethoxyanthracenesulfonate. Pre- and post-column d e r i v a t i z a t i o n techniques have been described (see Chapt e r 4). Reviews on pre- and post-column reactions have been g i ~ e n ~ ~ - ~ ' .

A h i g h l y s p e c i f i c d e t e c t i o n method was introduced by Westwood e t Using a CD ( c i r c u l a r dichroism) spectrophotometer as detector, CD a c t i v e compounds could be detected select i v e l y . This was demonstrated f o r some Amaryllidaceae a l k a l o i d s ( s e e Chapter 6). Also, electrochemical d e t e c t i o n has been applied i n a few cases t o a l k a l o i d s (see Chapters 7, 8 and 11). An advantage o f the method i s t h a t i t enables a s e l e c t i v e a t t e n u a t i o n o f i n t e r f e r i n g compounds. The s e n s i t i v i t y i s a t l e a s t comparable w i t h UV detection74.

231

The most selective method of detection i s probably the coupling of HPLC and MS. Eckers e t al.75 used i t in connection with the analysis of alkaloids. A detailed discussion of the various detection methods in HPLC i s given by Scott76. 1.6. SAMPLE PREPARATION Sample preparation i s an important step in HPLC. I t influences n o t only the s e n s i t i v i t y and the selectivity of the analysis, b u t also the column l i f e and the analysis time. The general problems in alkaloid sample preparation have been dealt with in Volume 23A of t h i s series. Some special problems connected with the analysis of alkaloids in body fluids have also been discussed there (Chapter 12), while other special problems have been dealt with in connection with the analysis of xanthine derivatives in biological fluids (Chapter 11 of t h i s volume). I t i s obvious t h a t the cleaner the sample. the longer the column l i f e . On the other hand extensive clean-up procedures are time consuming. A compromise can be the use of precolumns t o protect the analytical column against contamination caused by impurities present in the sample. Precolumns can be prepared from less expensive material t h a n the analytical columns, i .e. pellicular packings or large diameter p a r t i c l e s , which can be easily dry-packed. However, microparticulate stationary phases have also been used. As HPLC analysis of alkaloids often deals with the determination of low concentrations of alkaloids in a complex matrix, some factors concerning the sample preparation which influence the detection limit are worth consideration. Karger e t a1.77 pointed o u t t h a t one o f the most important ways t o improve the s e n s i t i v i t y i s t o inject large sample volumes. When the alkaloids t o be analyzed are dissolved in the same solvent as i s used as the mobile phase, as much as several hundreds of pl can be injected without loss of column efficiency. When a solvent i s used from which the solute i s strongly retained on the analytical column. a concentration of the alkaloids t o be analyzed can be achieved on the t o p of the column - avoiding laborious extraction techniques. Afterwards elution can take place with a suitable mobile phase. Such a procedure can be used t o enrich trace components in urine samples for instance, and i t has been demonstrated f o r the analysis of dihydroergocri~tine~'. I n this study i t was found that f o r equal amounts of alkaloid sample, an increase of the volume injected from 50 p1 t o 7 ml only s l i g h t l y increased the peakwidth. Even as large injection volumes as 165 ml gave reasonable reproducible separations, allowing analysis in the lower ppb range. By using a specially devised step-gradient elution system, a further improvement of the method was possible. allowing injection volumes as large as one liter7'. A selective detection method can reduce the interference of other compounds. On column "preconcentration techniques" have extensively been applied in several and t h e i r fundamentals have been discussed theoretically82 . Van Vliet e t al.83 performed an enrichment of trace components by pumping the sample solution through a precolumn via a valve t o the drain: the precolumn was then - via the same valve connected t o the analytical column and a stepwise gradient elution was applied. The precolumn was designed t o allow rapid sampling from large volumes and t o cause only negligible band spreading. For the components tested (phtalates) the best results were obtained by means of a 2 x 4.6 nun ID precolumn packed with 5 pm particles of an octadecyl bonded stationary phase. Trace enrichment from samples up t o 1 1 were possible. Band broadening observed for the described system was not more than can be observed f o r conventional 10 20 ul injections.

-

-

References p. 232

232 Even a t the high f l o w rates used f o r pumping t h e sample s o l u t i o n through the precolumn, a complete recovery o f the samples was found. Reviews o f various preconcentration methods have been given by K i rklandE4 and Freia5.Ki r k l a n d e t a1.86 i n v e s t i g a t e d peak broadening caused by t h e i n j e c t i o n mode, column design, det e c t o r s and guard columns. REFERENCES

1 J.H. Knox and J. Jurand, J. Chromatogr., 82 (1973) 398. 2 J.H. Knox and J. Jurand. J. Chromatogr., 87 (1973) 95. 3 P.J. Twitchett, A.E.P. Gorvin and A.C. Moffat, J. Chromatogr., 121 (1976 359. 4 K.D. McMurtrey, J.L. Cashaw and V.E. Davis, J. L i q . Chromatogr., 3 (19801 663. 5 H.F. Walton, J. Chromatogr. I 102 (1974) 57. 6 E. Murgia and H.F. Walton, J . Chromatogr., 104 (1975) 417. 7 E.O. Murgia, D i s s . A b s t r . I n t . B , 36 (1976) 3911. 8 P.J. T w i t c h e t t and A.C. Moffat, J. Chromatogr., 111 (1975) 149. 9 6.6. Wheals, HPLC i n C l i n i c a l Chemistry,Ed. P.F. Dixon, Academic P r e s s , London,l976,p 211. 10 J.K. Baker. R.E.Skelton and Ch.Y. Ma, J . Chromatogr., 168 (1979) 417. 11 R.K. G i l p i n and M.F. Burke, A n a l . Chem., 45 (1973) 1383. 12 6.8. COX, J. Chromatogr. S c i . , 15 (1977) 385. 13 K.K. Unger, N. Becker and P. Roumeliotis, J . C h r o m a t o g r . , 125 (1976) 115. 14 K. Karch, I. Sebastian, I . Halasz and H. Engelhardt, J. Chromatogr., 122 (1976) 171. 15 R.E. Majors, J. Chromatogr. S c i . , 18 (1980) 488. 16 E. Soczewinski and 1. Dzido. J . L i q . Chromatogr., 2 (1979) 511. 17 S.R. Bakalyar, R. McIlwrick and E. Roggendorf, J. Chromatogr., 142 (1977) 353. 18 A. Wehrli, J.C. Hildenbrand, H.P. K e l l e r , R. Stampfli and R.W. F r e i , J. Chromatogr.,149 (1978) 199. 19 J.G. Atwood, G.J. Schmidt and W. Slavin, J. Chromatogr., 171 (1979) 109. 20 I.M. Johansson, K.G. Wahlund and G. S c h i l l , J. Chromatogr., 149 (1978) 281. 21 K.G. Wahlund and A. Sokolowski, J. Chromatogr., 151 (1978) 299. 22 W.R. Melander, J. Stoveken and C. Horvath, J . Chromatogr., 185 (1979) 111. 23 0. Westerlund and E. Erixson, J. Chromatogr., 185 (1979) 593. 24 A. Sokolowski and K.G. Wahlund, J . Chromatogr., 185 (1979) 299. 25 K.E. B i j , C. Horvath, W.R. Melander and A. Nahum, J. Chromatogr., 203 (1981) 65. 26 C.T. Hung, R.B. Taylor and N. Paterson, J. Chromatogr.. 240 (1982) 61. 27 R. G i l l , S.P. Alexander and A.C. Moffat, J. Chromatogr., 247 (1982) 39. 28 F.P.B. van der Maeden, P.T. van Rens, F.A. Buytenhuys and E. Buurman, ~.Chromatogr., 142 (1977) 715. 29 M.G.M. de Ruyter, R. Cronelly and N. Castagnoli, J. Chromatogr., 183 (1980) 193. 30 A.P. Goldberg, A n a l . Chem.. 54 (1982) 432. 31 H. Engelhardt. B. Dreyer and H. Schmidt, Chromatographid , 16 (1982) 11. 32 H. Engelhardt and H. Mdller, J. Chromatogr., 218 (1981) 395. 33 C. Horvath, W. Melander and I . Molnar, J. Chromatogr., 125 (1976) 129. 34 C . Horvath, W. Melander and I . Molnar. A n a l . Chem.. 49 (1977) 142. 35 C. Horvath and W. Melander, J . Chromatogr. S c i . . 15 (1977) 393. 36 H. C o l i n and G. Guiochon. J. Chromatogr., 141 (1977) 289. 37 C. Horvath and W. Melander, rnt. L a b . , (1978) 11. 38 R.E. Majors, J. ASS. off. A n a l . Chem., 60 (1977) 186. 39 N.H.C. Cooke and K. Olsen, J . Chromatogr. s c i . , 18 (1981) 512. 40 H. Engelhardt and G. Ahr, Chromatographia. 14 (1981) 227. 41 E. Grushka,Editor, Bonded S t a t i o n a r y Phases i n Chromatography, Ann Arbor S c i e n c e P u b l i s h e r s i n c . , Ann Arbor Michigan, 1974. 42 R. Matsuda, 1. Yamamiya, M. Tatzuzawa. A. Ejima and N. Takai, J . Chromatogr., 173 (1979) 75. 43 K. Aramaki. T. Hanai and H.F. Walton, A n a l . Chem., 52 (1980) 1963. 44 J.L. Robinson, W.J. Robinson, M.A. Marshall, A.D. Barnes, K.J. Johnson and D.S. Salas, J. Chromatogr., 189 (1980) 145. 45 C. Horvath, W. Melander, I . Molnar and P. Molnar, Anal. Chem., 49 (1977) 2295. 46 J.H. Knox and G.R. Laird, J. Chromatogr., 122 (1976) 17. 47 J.H. Knox and J . Jurand, J . Chromatogr., 125 (1976) 39. 48 P.T. Kissinger, A n a l . Chem., 49 (1977) 883. 49 C.P Temey-Groen, S. Heemstra and J.C. Kraak, J . Chromatogr., 161 (1978) 69. 50 R.P.W. Scott and P.J. Kucera, J. Chromatogr., 175 (1979) 51. 51 B.A. Bidlingmeyer, S.N. Deming, W.P. Price, B. Sachok and M. Petrusek, J. Chromatogr.

233 186 (1979) 419. B.A. Bidlingmeyer, J. C h r o m a t o g r . S c i . , 18 (1980) 525. J.H. Knox and R.A. Hartwick, J ; C h r o m a t o g r . ; 204'(1981) 3. R.G. Achari and J.T. Jacob, J . L i q . C h r o m a t o g r . , 3 (1980) 81. E.J. Kubiak and J.W. Munson, J. Pharm. S c i . , 69 (1980) 152. I.S. L u r i e and S.M. Demchuk, J . Liq. C h r o m a t o g r . , 4 (1981) 337. I.S. L u r i e and S.M. Demchuk, J. L i q . C h r o m a t o g r . , 4 (1981) 357. I.S. Lurie, J. L i p . C h r o m a t o g r . , 4 (1981) 399. E. Tomlinson, T.M. Jefferies and C.M. Riley, J. C h r o m a t o g r . , 159 (1978) 315. M.T.W. Hearn, i n A d v a n c e s i n C h r o m a t o g r a p h y , vol. 1 8 , E d i t e d b y J . G i d d i n g s , E. G r u s h k a , J . C a z e s a n d R.P. B r o w n , Marcel D e k k e r Inc., New Y o r k , 1980. p. 59. 6 1 L.R. Snyder, P r i n c i p l e s of a d s o r p t i o n c h r o m a t o g r a p h y , Marcel D e k k e r I n c . , New Y o r k , 1963. 62 K.K. Unger, P o r o u s S i l i c a , J . C h r o m a t o g r . L i b r a r y , vol. 1 6 , E l s e v i e r , A m s t e r d a m , 1979. 63 S.R. Abbott, J . C h r o m a t o g r . S c i . . 18 (1980) 540. 64 R. Ohlacht and I . Halasz, C h r o m a t o g r a p h i a 14 (1981) 216. 65 I.S. Lurie. S.M. Sottolano and S. Blasof, J. F o r e n s i c S c i . , 27 (1982) 519. 66 R. Yost, J. Stoveken and W. MacLean, J. C h r o m a t o g r . , 134 (1977) 73. 67 R.W. F r e i and W. Santi, 2. A n a l . C h e m . , 277 (1975) 303. 68 M.S.F. ROSS, J . C h r o m a t o g r . , 141 (1977) 197. 69 R.W. F r e i , A n a l . Proc., 16 (1979) 289. 70 R.W. F r e i and A.H.M.T. Scholten, J . C h r o m a t o g r . S c i . , 17 (1979) 152. 7 1 R.W. F r e i , J. C h r o m a t o g r . . 165 (1979) 75. 72 A.F. F e l l , A n a l . Proc., 17 (1980) 512. 73 S.A. Westwood, D.E. Games and L. Sheen, J. C h r o m a t o g r . , 204 (1981) 103. 74 J. Frank, C h i m i a , 35 (1981) 24. 75 C. Eckers, D.E. Games, E. Lewis. K.R.N. Rao, M. R o s s i t e r and N.C.A. Weerasinghe, i n A . Q u a y l e ( E d i t o r ) , A d v a n c e s i n M a s s S p e c t r o m e t r y , vol. 8 , H e y d e n , L o n d o n , 1980, p.1396. 76 R. P.W. Scott, L i q u i d C h r o m a t o g r a p h y Detectors, J . C h r o m a t o g r . L i b r a r y , vol. 1 1 , E l s e v i e r , Ams t e r d a m , 19 77. 77 B.L. Karger. M. M a r t i n and G. Guiochon, A n a l . C h e m . , 46 (1974) 1640. 78 P. Schauwecker, R.W. F r e i and F. E r n i , J . C h r o m a t o g r . , 136 (1977) 63. 79 F. E r n i , R.W. F r e i and W. Lindner. J. C h r o m a t o g r . , 125 (1976) 265. 80 J.N. L i t t l e and G.J. F a l l i c k , J . C h r o m a t o g r . , 112 (1975) 389. 81 K. K r u n e n and R.W. F r e i , J . C h r o m a t o g r . , 132 (1977) 429. 82 J.F.K. Huber and R.R. Becker, J . C h r o m a t o g r . , 142 (1977) 765. 83 H.P.M. van V l i e t , Th.C. Bootsman, R.W. F r e i and U.A.Th. Brinkman, J . C h r o m a t o g r . , 185 (1979) 483. 84 J.J. Kirkland. A n a l y s t , 99 (1974) 859. 85 R.W. F r e i , A n a l . Proc., 17 (1980) 519. 86 J.J. Kirkland, W.W. Yau. H.J. Stoklosa and C.H. D i l k s , J . C h r o m a t o g r . S c i . . 15 (1977) 303. 52 53 54 55 56 57 58 59 60

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236

Chapter 2 HPLC ANALYSIS OF VARIOUS ALKALOIDS

........................................... ... .... .......... ................................................... .................. ................................................... ........... ................................................... ......................... ...................................................

2.1. Ion-exchange HPLC.. 2.2. Reversed-phase HPLC.. 2.3. Ion-pair HPLC 2.4. Straight-phase HPLC References

235 235 236 236 240

A number o f papers have been pub ished on the HPLC analysis o f a l k a l o i d s i n o r d e r t o s t u dy the p o s s i b i l i t i e s f o r such analy ?s i n general. The papers comprise a wide v a r i e t y o f a l kaloids and HPLC systems f o r t h e i r separation. The data published i n these papers may be useful i n connection w i t h many separation and o t h e r HPLC problems i n the a n a l y s i s o f alkal o i d s . Therefore some o f the data o f general i n t e r e s t are sumnarized i n the present chapter. However, i n other chapters o f t h i s book, HPLC systems t h a t may be u s e f u l f o r a l k a l o i d anal y s i s i n general, have a l s o been d e a l t w i t h (see Table 2.1). 2.1.

ION-EXCHANGE HPLC

Walton and c o - ~ o r k e r s ~ ’reported ~’~ the analysis o f various a l k a l o i d s by means o f ligand-exchange chromatography. As s t a t i o n a r y phases, ion-exchange m a t e r i a l s loaded w i t h metal-ions t h a t are capable o f g i v i n g a m n i a complexes (Cu++,Ni++,Zn++

and Ag’)

were used.

A cation-exchange s t a t i o n a r y phase f o r the separation o f various drugs. i n c l u d i n g several a l k a l o i d s , has been t e ~ t e d ” ~ .The method was found t o be s u i t a b l e f o r b a s i c compounds. An aqueous amnonium phosphate b u f f e r c o n t a i n i n g a t l e a s t 40% methanol, o r b e t t e r , a c e t o n i t r i l e . should be used as mobile phase. V a r i a t i o n i n the s o l v e n t r a t i o . i o n i c s t r e n g t h and pH could be used t o vary the r e t e n t i o n o f the a l k a l o i d s . 2.2.

REVERSED-PHASE HPLC

4 Twi t c h e t t and M o f f a t tested a m i c r o p a r t i c u l a t e octadecyl s t a t i o n a r y phase f o r t h e analys i s o f some drugs, i n c l u d i n g several a l k a l o i d s . For the systems t e s t e d (methanol

-

aqueous

ammonium phosphate b u f f e r s , pH varying from 3 t o 9) severe t a i l i n g was observed f o r basic drugs, making these systems less s u i t a b l e f o r the a n a l y s i s o f such compounds. The analysis o f a series o f basic drugs by means o f reversed-phase HPLC was reported i n connection w i t h the i d e n t i f i c a t i o n o f these compounds by dual-wavelength detection” Table 2.2). The i n f l u e n c e o f the methanol

-

on reversed-phase packings has been studied

(see

water r a t i o on the r e t e n t i o n o f some a l k a l o i d s 14

.

Aramaki e t a1.15 studied the chromatographic behaviour o f some a l k a l o i d s on a macroporous styrene-divinylbenzene copolymer (see Fig.8.6 and Table 8.4). The i n f l u e n c e o f changes i n the mobile phase on the r e t e n t i o n was studied.

Robrenca p. 240

236 TABLE 2.1 ANALYSIS OF VARIOUS ALKALOIDS, DEALT WITH I N OTHER CHAPTERS. ~

Ref.

Ref, i n o t h e r chapter

4 5 7 8 9 12 13

21 22 27 30 38 56 60

2.3.

ION-PAIR HPLC Lurie9’18’19s20

Chapter

Fig.

Table

Ref.

7.16

7.8, 7.9

15

7 7 7 7 7 7

7.11

7

17 18 19 20 22

7.3

Ref. i n o t h e r chapter

Chapter

43 91 98 99 100 121

8 7 7 7 7 7

Fig. 8.6

Table 8.4 7.11

7.6

7.14

reported the analysis o f a s e r i e s o f drugs o f f o r e n s i c i n t e r e s t by means

o f reversed-phase i o n - p a i r HPLC (Chapter 7, Fig.7.11 and Table 7.3). Also Achari and Jacob 16 studied t h i s method i n more d e t a i l . D i f f e r e n t reversed-phase packings were t e s t e d and the i n f l u e n c e o f the mobile phase composition on the r e t e n t i o n was studied. It was concluded t h a t a l k y l bonded phases produced longer r e t e n t i o n than d i d phenyl o r cyclohexyl bonded phases. The higher the organic content o f the s t a t i o n a r y phase the longer the r e t e n t i o n . Retention

i s a l s o increased by increasing the water content o f the mobile phase and by increased chain length o f the a l k y l s u l f o n a t e counter-ion. 2.4. STRAIGHT-PHASE HPLC Verpoorte and Baerheim Svendsenl reported the analysis o f a s e r i e s o f a l k a l o i d s on microp a r t i c u l a t e s i l i c a gel using d i e t h y l e t h e r o r chloroform w i t h various percentages o f methanol as mobile phase (Table 2.4). The optimum wavelength o f d e t e c t i o n f o r f i x e d wavelength detect o r s o f 254 and 280 nm f o r the a l k a l o i d s i n v e s t i g a t e d were a l s o reported. 5 Jane found h i g h l y p o l a r mobile phases, c o n s i s t i n g o f methanol - aqueous ammonium n i t r a t e solution

-

amonia i n combination w i t h s i l i c a gel columns s u i t a b l e f o r the a n a l y s i s o f drugs

w i t h a wide range o f p o l a r i t y (Chapter 7. Fig. 7.16 and Table 7.8 and 7.9). A s i m i l a r system was employed by Baker e t a1.12 (Table 2.3). Pharmaceutical preparations containing a l k a l o i d s have been analyzed on s i l i c a gel w i t h the - methanol ammonia10 (Table 2.5). Oiethyl e t h e r saturated w i t h

mobile phase dichloromethane

-

water t o which a small amount o f diethylamine was added has a l s o been used f o r the same purpose13 (Table 2.6).

I n connection w i t h the t e s t i n g o f a new detector, a separation o f some

a l k a l o i d s on s i l i c a gel has been reported

.

11

Flanagan e t a l . * l reported the analysis o f basic drugs on s i l i c a gel using non-aqueous i o n i c eluents: methanol

-

d i e t h y l e t h e r mixtures containing 0.02

-

0.1% p e r c h l o r i c a c i d o r

perchlorate s a l t s . Retention and s e l e c t i v i t y i n such systems could be adjusted by the pH, i o n i c strength and solvent r a t i o . Also, bonded phases could be used i n combination w i t h such solvent systems. Basic compounds were r e t a i n e d o n l y when ionized, the r e t e n t i o n o f such compounds could therefore be p r e d i c t e d by t h e i r pKa values. However, a t low pH. r e t e n t i o n was decreased again; the best r e s u l t s are t h e r e f o r e obtained a t intermediate pH-values. Quaternary amnonium compounds gave t a i l i n g i n t h i s type o f solvent system. The authors suggested t h a t r e t e n t i o n i s caused by i o n i z e d s i l a n o l groups (ion-exchange mechanisms) b u t a l s o t h a t i o n - p a i r formation could p l a y a r o l e .

FF a

TABLE 2.2

P

P

IDENTIFICATION OF SOME DRUGS BY HPLC WITH DUAL WAVELENGTH UV-DETECTION12 Column,uBondapak C18(300x3.9 mn I D ) , m o b i l e phase 0.025 M sodium dihydrogen phosphate i n methanol f l o w r a t e 2 ml/min.

-

water (2:3),

N

0

Drug

relative absorption r e t e n t i o n time r a t i o A254/A280

Barbituric acid 0.21a Sul f a n i 1ami de 0.23 Phenylephri ne 0.27 Theobromi ne 0.28 Pa racetamol 0.30 A c e t y l s a l i c y l i c a c i d 0.31 Hydroxyamphetarni ne 0.32 Phenylpropanolamine 0.34 Theophyll ine 0.34 Ilimenhydrinate 0.38 Oxymorphone 0.41 Ephedrine 0.41 Mescaline 0.43 Caffeine 0.48 Ectylurea 0.50 Procaine 0.51 Amphetamine 0.52 Salicylamide 0.53 N i kethamide 0.58 Phenacernide 0.59 Oxycodone 0.60 Morphine 0.62 Dichloralphenazone 0.66 Mephenoxal one 0.67 Methocarbamol 0.72 Dimethyltryptarnine 0.75 Methylamphetamine 0.75 Tetrahydrozoline 0.77 P h e n m t r a z i ne 0.80 O i hydrocodei ne 0.81 O i hydromorphi none 0.84 Nicotine 0.85 Mephentermi ne 0.88

19.1 5.25 1.73 2.25 8.23 0.32 0.94 16.0 3.24 3.67 2.80 17.1 5.41 2.15 17.9 3.0 20.4 0.83 16.5 33.5 2.1 1.67 6.0 1.79 1.43 1.89 6.50 9.46 13.3 1.08 2.0

14.4 7.67

A254C

Nd

Drug

r e 1a t i ve absorption r e t e n t i o n time r a t i o A

A254

254'A280

2.9 2.7 0.049 0.23 1.8 0.029 0.043 0.0088 0.46 0.16 0.046 0.0083 1.3 0.013 0.61 0.019 0.094 0.15 0.0055 0.0026 0.011 0.026 0.0035 0.0035 0.0021 0.0054 0.0021 0.0043 0.0070 0.056 0.002

;The column v o i d volume was s l i g h t l y l e s s than 0.21 R e t e n t i o n time 6.8 min

4520 2680 3190 3750 3750 3750 1420 4660 5320 7580 1150 1600 2330 260 1250 630 2680 1480 110 230 950 240 240 190 750 230 360 670 270 460 990 450

Codeine Bromural Nal oxone Phenacetinb Heroin Lobe1 ine Mephenesi n Naphazoline F1uorescei n Methylphenidate Nylidrine D i hydrocodei none Ethylmorphi ne Levorphanol Chlordiazepoxide Pentazoci ne Oiphenylhydantoin G l u t e t h i m i de Phencyclidine Leva1 l o r p h a n Phenaglycodol Ooxyl ami ne F1 urazepam Thi opental Oxymethazoline Methaqualone Phenazoci ne Oxazepam Thi amyl a1 Methohexi t a l Papaveri ne Diazepam

0.90 0.95 0.96 1.00 1.11 1.11 1.11 1.11

1.15 1.46 1.59 1.66 1.69 1.78 2.02 2.03 2.07 2.12 2.16 2.35 2.90 2.95 3.21 3.55 3.62 3.94 4.05 4.05 4.58 4.80 7.06 9.56

2.47 14.4 2.21 8.42 1.75 8.52 3.23 1.14 2.12 17.0 1.76 1.94 2.18 0.35 3.69 0.46 16.4 12.0 19.3 0.44 16.5 11.4 3.76 0.33 1.00 2.51 0.72 6.16 0.31 9.0 3.0 6.04

940 0.0059 710 0.0077 600 0.49 2620 1190 0.0028 1920 0.0092 880 0.019 500 0.21 2220 0.0018 320 0.0025 480 0.0029 400 0.0057 660 0.0006 370 0.045 1180 0.0009 360 0.0091 630 0.0039 1350 250 0.0007 650 0.0026 860 0.0093 340 0.016 690 0.013 3150 0.0014 650 0.049 920 0.0011 780 0.061 1510 0.016 2370 0.0015 2600 0.029 2440 0.10 2650

:Absorbance o f a 10-111 i n j e c t i o n o f a 1.0 mg/ml s o l u t i o n o f t h e drug Number o f t h e o r e t i c a l p l a t e s

238

TCBLE 2.3 IDENTIFICATION OF SOVE DRUGS BY HPLC WITH DUAL WAVELENGTH UV-DETECTION1’ Column pPorasil ( 3 0 0 ~ 3 . 9mn I D ) , mobile phase methanol - 2 11 ammonia n i t r a t e (27:2:1), f l o w r a t e 2.0 ml/rin.A254/A280 = absorption r a t i o UV. Druq Noscapine Phenacetin Nal oxone Papaver ine Benzphetamine Piminodine Cocaine Phenazoci ne Procaine Nylidrin Leva1 lorphan tlethyl phenidate Pentazocine Phendimetrazine Et h inamat e Phenmetrazine fieperi dine Quinine Promethazi ne Diphenhydramine Methapyri lene Phenyl propanolamine Heroin Pethadone Phencyclidine T h i o r i daz ine Amphetamine Oxymorphone Ooxylamine Ethylmorphi ne Hydroxyamphetami ne Propyl hexedrine Oxycodone Codeine Porphine Dimethyl tryptami ne Methamphetamine Ephedrine Phenyl e r h r i n e D i hydrocodei none Ethohertazine Mescaline Xylometazol i n e t!ephenterami ne D i hydrocodeine Oxymetazol i n e Tetrahydrozol i n e D i hydromorphi none Strychnine Dextromethorphan Naphazoline Levorphanol

r e l a t i v e r e t e n t i o n t i m e absorption r a t i o A254/A280 0.53a 0.53 n.56 0.56 0.58 0.58 0.61 0.61 0.61 0.61 0.64 0.67 0.67 0.67 0.70 0.72 0.75 0.75 0.76 0.77 0.77 0.78 0.80 0.83 0.83 0.83

o .a6

0.86 0.89 0.92

0.e2 0.92 0.92

1.oo

0.61 0.84

0.82 1.06

2.10 3.02 0 -86 0.24 0.44 0.75 0.12 9.50 0.16 8.00

1.oo

31.0 30.7 0.62 2.21 90.0 1i n 9 65.0 0.64 1.57 22.0

2.08 60.0

1.17 17.7 1.15 0.38 4.0 1.13 1.09 0.77 31.0

1.20

52.0 0.50

1.28 1.31 1.31 1.33 1.36 1.36 1.36 1.42 1.43 1.54 1.56 1.61 1.64

0.93 27.4 2.93 8.67 36.3 0.53 0.31 16.1 .~ 1.09 3.22 0.14 0.49 ~~

0.12

The column void volume was s l i g h t l y l e s s than 0.53 Morphine was used as standard, r e t e n t i o n time 3.5 min Absorbance o f a 10-ul i n j e c t i o n o f a 1.0 mg/ml s o l u t i o n Number o f t h e o r e t i c a l p l a t e s

111 amnonium

-I

A254C

NU

0.035 0.051 0.025 0.070 0.069 0.16

0.0081 0.048 01013 0 A026 0.0062 0.0049

0.010

0.0020 0.040 0.0030 0.0042 0.14 0.059 0.13

710 1,700 710 1,600 750

0.0021

1.800 0.072

1,140 1,900

0.20

840

0.0039 0.0044 0.0017

940

0.020 0.011 0.0005 0.0029

0.88

1.00b 1.09 1.19 1.22

-

0.016 0.0034 0.0034 0.0045

0.0085 0.0022 0.0034 0.0038

0.0065 0.012 0.038 0.0091 0.0045

0.0022 0.062 0.0013

110 2.330 2,440 1.080 290 100 450 470 3,190 1,862 1,050 466 1,170 1,510 2,140 2,290 1.370 1;290 1,370 1.460 970 370 1,750 1,210

1.210

239

TABLE 2.4 RETENTION TIMES OF SOHE ALKALOIOS' Column, Perckosorb SI 60. 5 M (300x2 n ID), d e t e c t i o n UV 254 nm.

RT = r e t e n t i o n time; FR = f l o w - r a t e .

A1 k a l o i d

Solvent system Chl oroform-methanol

9:l 8:2 7:3 8:2 7:3 6:4 -----RT FR RT FP. RT FR RT FR RT FP RT FP Quinine Q u in i d i ne C i nchoni ne C i nc honi d i ne Atropine Scopolamine Cocaine Strychnine Brucine rbrphine Codeine Thebai ne Heroin Narceine Noscapine Papaverine Emetine Cephael i n e Serpentine A1 s t o n i n e Reseroine Yohimbine Raubasine Tetrahydroalstonine

-

6.4 6.5 10.6 9.0

5.9 6.2 11.0 9.0

0.75 0.75 0.75 0.75

1.5 0.81 2.1 0.81 3.9 0.81 3.9 0.81 3.5 0.81 2.7 0.81 2.7 0.81 1.7 0.81 7.5 0.81 1.1 0.81 1.0 0.81 1.1 @.El 1.1 0.81 1.1 0.81 1.1 0.81 5.9 0.81

1.5 3.9 4.7 4.6 3.3 2.9 3.0 1.7 5.7 1.1 1.2 1.2 8.6

0.75 0.75 0.75 0.75 n.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75

0.135 2.7 1.43 2.2 1.34 0.85 2.9 1.43 2.4 1.34 0.85 5.1 1.43 4.2 1.34 0.65 3.7 1.43 3.0 1.34 9.4 1.38 7.4 1.20 1.7 1.50 1.2 1.38 1.3 1.20 3.2 1.50 2.0 1.38 2.9 1.20 9.4 1.43 7.3 1.34 17.2 1.43 13.1 1.34 4.0 1.50 4.3 1.43 4.3 1.34 3.4 1.50 3.6 1.43 3.4 1.34 2.7 1.50 3.7 1.A3 3.7 1.34 2.1 1.50 2.2 1.43 2.2 1.34 8.5 1.34 0.6 1.50 0.6 1.43 0.7 1.3A 0.8 1.50 0.8 1.43 0.8 1.34 0.6 1.50 0.7 I.A3 0.8 1.34 5.1 1.34

2.2 3.7 2.2 2.1

1.1 1.7 1.1 1.1

0.75 0.75 0.75 0.75

2.9 4.6 2.9 2.5

2.0 3.4 6.9 6.8 8.6 4.6 4.1 2.3

0.~1 0.01 0.81 0.81

0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81

-

0.38 0.38 0.38 0.38

1.0 1.5 1.0 1.0

0.81 0.81 0.01 0.81

3.9 3.7 5.9 4.5

-

-

-

0.33 0.33 0.33 0.33

3.0 3.8 2.9 2.7

0.33 0.33 0.33 0.33

2.2 2.8 2.? 2.2

0.40 0.40 0.40 0.40

TABLE 2.5 RETENTION DATA OF VARIOUS

-

dichloromethane Column, P a r t i s i l , 10 wn (250~4.6mm ID), mobile phase methanol (3:l) c o n t a i n i n g 1% 29% ammonia, f l o w r a t e 0.7 ml/min, d e t e c t i o n UV 254 nm. k' Theophyll i n e Sodi urn Sul facetamide 6-Hydroxydopamine Ethaveri ne Papaverine Tropicamide Caffeine Theobromine Scopolamine Pyr ilami ne Epinephrine Phenylpropanolamine Quinidine Quin j ne Code 1 ne

0.08" 0.16 0.54 0.92 0.92 0.92 0.92 0.96 1.00 1.33 1.50 1.66 1.71 1.71 1.88

~

*

r e t e n t i o n time 2.59 min.

References p. 240

Tailing Factor ( % )

50.0 25.0 25.0 25.0 66.7 25.0 29.4 47.0 58.8 60.0 64.0 50.0 55.6 40.0

k' Brompheniramine Chlorpheniramine Hydroquinidine Amphetamine Phenyl ephr ine Ephedrine Strychnine Dextromethorphan h t a z o l ine Atropine Homatropi ne Naphazoline Xylometazol i n e Oxymetazol i n e

1.92 1.92 2.50 2.66 2.66 3.25 3.33 3.58 4.00 4.13 4.42 9.58 13.58 15.25

Tai 1ing Factor ( % )

50.0 55.6 50.0 58.8 35.7 19.2 25.0 25.0 18.8 20.9 17.9 24.0 53.3

240

TABLE 2.6 SEPARATION OF SOME ALKALOIDS AND

DRUGS (SEE ALSO FIG.7.14)13

Column, P a r t i s i l 5 um (250~4.6mn I D ) . mobile phase d i e t h y l e t h e r 95% saturated w i t h water t 0.4% diethylamine, f l o w r a t e 2 ml/min, d e t e c t i o n UV 254 nm. ~~

A1 k a l o i d Aconitine N-methyl ephedri ne Ephedrine Emetine Cephael ine Ethylmorphi ne Codeine Papaverine Noscapine Narceine Scopol ami ne Atropine Homatropine Quinine Strychnine Caffeine Phenytoi ne Sulfani 1amide Phenobarbi t a l

k'

1.51 0.58 2.89 2.36 3.95 4.29 5.58 2.15 0.35 0.58 4.11 8.93 9.55 4.34 6.32 5.62 6.73 8.82 14.9

REFERENCES

8 9 10 11

R. Verpoorte and A. Baerheim Svendsen, J . Chromatoyr., 100 (1974) 227. H.F. WaltOn. J. Chromatoyr., 102 (1974) 57. E. Murgia and H.F. Walton, J . Chromatogr.. 104 (1975) 417. P.J. T w i t c h e t t and A.C. Moffat. J. Chromatoyr.. 111 (1975) 149. I. Jane, J . Chromatoyr., 111 (1975) 227. E.O. Murgia, D i s s . Abstr. I n t . B , 36 (1976) 3911. P.J. Twitchett, A.E.P. Gorvin, A.C. Moffat, P.L. Williams and A.T. S u l l i v a n , i n HPLC i n C l i n i c a l Chemistry, E d i t o r P . F . Dixon, Academic Press, London, (1976), p. 201. P.J. Twitchett, A.E.P. Gorvin and A.C. Moffat, J. Chromatoyr., 120 (1976) 359. I. Lurie, J. A S S O C . off. Anal. Chem., 60 (1977) 1035. R.G. Achari and E.E. Theimer, J . Chromatoyr. S c i . , 15 (1977) 320. Y. Hashimoto, M. Moriyasu, E. Kato, M. Endo, N. Miyamoto and H. Uchida, Mikrochim. d c t a

12 13 14 15 16 17 18 19 20 21 22

J.K. Baker, R.E. Skel t o n and Ch.Y. Ma, J . Chromatoyr., 168 (1979) 417. R. Gimet and A. F i l l o u x , J . Chromatoyr.. 177 (1979) 333. E. Soczewinski and T. Dzido. J . L i q . Chromatoyr., 2 (1979) 511. K. Aramaki, T. Hanai and H.F. Walton, A n a l . Chem., 52 (1980) 1963. R.G. Achari and J.T. Jacob, J . L i q . Chromatoyr., 3 (1980) 81. J.D. Wittwer, Forensic sci. I n t . , 18 (1981) 215. I . S . L u r i e and S.M. Oemchuk, J . L i q . Chromatogr.. 4 (1981) 337. I . S . L u r i e and S.M. Demchuk, J . L i q . Chromatoyr., 4 (1981) 357. I . S . Lurie, J . L i q . Chromatoyr., 4 (1981) 399. R.J. Flanagan, G.C.A. Storey, R.K. Bhamra and I . Jane, J . Chromatoyr., 247 (1982) 1 5 . I.S. Lurie, S.M. Sottolano and S . Blasof, J. F o r e n s i c S c i . , 27 (1982) 519.

1 2 3 4 5 6 7

2 (1978) 159.

II. SPECIAL PART

This Page Intentionally Left Blank

241

CHAPTER 3 PYRROLIDINE, PYRROLIZIDINE, PYRIDINE, PIPERIDINE AN0 QUINOLIZIDINE ALKALOIDS 3.1. Reversed-phase HPLC.. 3.2. S t r a i g h t phase HPLC 3.3. Detection References

.............................................................

241 242 242 242

............................................................... ......................................................................... .............................................................................

N i c o t i n e i s an a l k a l o i d which i s comnonly found i n u r i n e and, therefore, n i c o t i n e has been included i n several studies concerning the analysis o f drugs and drugs o f abuse i n u r i n e samples (Table 3.1). Analysis o f tobacco a l k a l o i d s i n general has been performed by HPLC; a l s o i n i n v e s t i g a t i o n s on p y r r o l i z i d i n e a l k a l o i d s , many o f which possess hepatoxic, carcinogenic, teratogenic and mutagenic properties, has HPLC been s u c c e s s f u l l y applied. 3.1. REVERSED-PHASE HPLC P i e t r z y k e t a1.'

s t u d i e d t h e e f f e c t o f s o l u t e i o n i z a t i o n on the chromatographic reten-

t i o n . As s t a t i o n a r y phase the polystyrene-divinylbenzene copolymer XAD2 was used. One o f the model compounds was n i c o t i n e , f o r which the r e t e n t i o n a t various pH o f the mobile phase was determined. Hanks e t a l . w i t h methanol

-

2?

analyzed n i c o t i n e - c o n t a i n i n g p e s t i c i d e s by means o f an octadecyl column

0.05 M amnonium hydrogen phosphate (pH 7.5) (3:2) as mobile phase.

Q u a n t i t a t i v e analysis o f the a l k a l o i d s i n tobacco was reported by Piade and Hoffmanne3. Two octadecyl columns were coupled i n s e r i e s and t h e a l k a l o i d s separated w i t h a g r a d i e n t o f an increasing amount o f a c e t o n i t r i l e i n a t r i e t h y l a m i n e

-

phosphoric a c i d b u f f e r (Fig.3.1).

The i n f l u e n c e o f the pH on the separation o f the a l k a l o i d s was studied; optimum r e s u l t s were obtained a t pH 7.56. The r e s u l t s were compared w i t h a GLC and a spectrophotometric method. Analysis o f the major tobacco a l k a l o i d s has been performed on an octadecyl column using a triethylamine

- phosphoric

a c i d b u f f e r (pH 7.25) t o which 40% methanol was added (Fig.3.2)

(24). The method was compared w i t h a GLC method. Several e x t r a c t i o n solvents were compared as t o t h e i r effectiveness. An 0.025 M phosphate b u f f e r (pH 7.8) was found t o be s u i t a b l e . I n a series of papers, Segall and co-workers described various HPLC methods f o r the anal y s i s o f p y r r o l i z i d i n e a l k a l o i d s i n p l a n t m a t e r i a l . A cyano-type column i n combination w i t h tetrahydrofuran

-

0.01 M amnonium carbonate was used (Fig.3.3)

f i c a t i o n o f some Senecio a l k a l o i d s 11'12913a20.

f o r t h e i s o l a t i o n and i d e n t i -

A g r a d i e n t o f 13% tetrahydrofuran, i n c r e a s i n g

t o 26%, gave good separations. S i m i l a r r e s u l t s could be obtained w i t h an i s o c r a t i c system containing 16% tetrahydrofuran. An octadecyl column was a l s o employed and methanol

-

potassium dihydrogen phosphate b u f f e r

(pH 6.3) as mobile phase. The advantage o f t h i s system over the above mentioned one was t h a t i t allowed detection a t a s h o r t e r wavelength (218 nm). i.e.

a l k a l o i d s (Fig.3.4)16.

a t the absorption maxima o f the

I t could a l s o be applied t o preparative17 and semipreparative'l

sepa-

r a t i o n s . Huizing and Malingr@l5 p u r i f i e d and separated p y r r o l i z i d i n e a l k a l o i d s on a p o l y s t y r ene-divinylbenzene r e s i n (XAD2) using a c i d i c methanol

R.tmncap. 242

- water mixtures.

An increased percen-

242

tage o f methanol suppressed the t a i l i n g o f the peaks. An a n a l y t i c a l separation o f p y r r o l i z i d i n e a l k a l o i d s on a styrene-divinylbenzene r e s i n has been reported by Ramsdell and BuhlerZ5. Such a s t a t i o n a r y phase can be used a t r e l a t i v e l y h i g h pH (Fig.3.5). The analysis o f p y r r o l i z i d i n e a l k a l o i d s i n R a d i x s y m p h y t i was reported by T i t t e l e t a l .

(18.26). Analysis. o f the N-oxides could be performed on an octadecyl column (Fig.3.6). The reduced a l k a l o i d s could be separated on an alkylamino column. Because o f i n t e r f e r i n g peaks, the l a t t e r method was n o t s u i t a b l e f o r the analysis o f p l a n t m a t e r i a l . The N-oxides could a l s o be separated on an alkylamino s t a t i o n a r y phase using a c e t o n i t r i l e mobile phase (Fig.3.7)

26

.

-

water (98:2) as

3.2. STRAIGHT-PHASE HPLC 8

Watson

analyzed n i c o t i n e and c o t i n i n e e x t r a c t e d from u r i n e on a s i l i c a gel column using

e t h y l acetate

-

isopropanol

r a t e d the same a l k a l o i d s resin

-

-

- ammonia

(80:3:0.4)

as mobile phase. Maskarinec e t a1.l'

sepa-

a f t e r i s o l a t i o n from b i o l o g i c a l f l u i d s by adsorption on a XAD

on a s i l i c a gel column w i t h dioxane

-

isopropanol

-

2

ammonia ( 8 0 : 3 : 0 . 4 ) .

3.3. DETECTION For the s e n s i t i v e detection o f p y r r o l i z i d i n e a l k a l o i d s i t i s necessary t o use solvent systems t h a t enable detection a t the absorption maxima o f these a l k a l o i d s , below 220 nm16'18 Baker e t a l . 1 4 reported the i d e n t i f i c a t i o n o f various drugs by means o f t h e i r r e l a t i v e r e t e n t i o n times i n combination w i t h the absorbance r a t i o , c a l c u l a t e d from the peak heights observed w i t h 254 and 280 nm UV-detection. REFERENCES

1 H.F. Walton. J. C h r o m a t o g r . . 20 (1967) 57. 2 E. Murgia and H.F. Walton, J . C h r o m a t o g r . , 104 1975) 417. 3 P.J. Twitchett and A.C. Moffat, J . C h r o m a t o g r . , 111 (1975) 149. 4 I. Jane, J. C h r o m a t o g r . . 111 (1975) 227. 5 E. Murgia, Diss. Abstr. I n t . B . , 36 (1976) 3911 6 P.J. Twitchett. A.E.P. Gorvin and A.C. Moffat, . C h r o m a t o g r . , 120 (1976) 359. 7 B.B. Wheals, J. C h r o m a t o g r . , 122 (1976) 85. 8 I . D . Watson, J. C h r o m a t o g r . , 143 (1977) 203. 9 D.J. Pietrzvk. E.P. K r o e f f and T.D. Rotsch. And . Chem., 50 (1978) 497. 10 M.P. Maska6nec. R.W. Harvey and J.E. Caton, J . A n a l . Toxicol.. 2 (1978) 124. 11 C.W. Q u a l l s Jr. and H.J. Segall, J . C h r o m a t o g r . , 150 (1978) 202. 12 H.J. Segall and R.J. Molyneux, Res. Commun. i n Chem. P h a r m . and T o x . , 19 (1978) 545. 13 H.J. Segall, T o x i c o l . L e t t . , 1 (1978) 278. 14 J.K. Baker, R.E. Skelton and Ch.Y. Ma, J . C h r o m a t o g r . , 168 (1979) 417. 15 H.J. Huizing and T.M. Malingre, J . C h r o m a t o g r . , 176 (1979) 274. 16 H.J. Segall, J. L i q . C h r o m a t o g r . , 2 (1979) 429. 17 H.J. Segall, J. L i q . C h r o m a t o g r . . 2 (1979) 1319. 18 G. T i t t e l . H. Hinz and H. Wagner, P l a n t a M e d . , 37 (1979) 1. 19 N. Ota and Y. Mino. S h o y a k u g a k u Z a s s h i , 33 (1979) 140. CA, 92 (1980) 169114a. 20 H.J. Segall and T.P. Krick, T o x i c o l . L e t t . , 4 (1979) 193. 21 G.P. Dimnena, T.P. K r i c k and H.J. Segall, J . C h r o m a t o g r . , 192 (1980) 474. 22 A.R. Hanks, L.R. Schronk and T.C. Arnst, J. L i q . Chromatogr., 3 (1980) 1087. 23 J.J. Piade and 0. Hoffmann, J . Liq. C h r o m a t o g r . , 3 (1980) 1505. 24 J. Saunders and D.E. Blume, J. C h r o m a t o g r . , 205 (1981) 147. 25 H.S. Ramsdell and D.R. Buhler, J . C h r o m a t o g r . . 210 (1981) 154. 26 H. Wagner, U. Neidhardt and G. T i t t e l , P l a n t a Med.. 41 (1981) 232.

243 5

25

'hbuffi

pH 256 1(

I' 01 C

n 01 L

-.x L

0

U 01

>

3

1

0

I

I

I

I

1

1

,

10 20 30 40 50 60 70 80 90 min

23 F i g . 3.1. HPLC s e p a r a t i o n o f Tobacco a l k a l o i d s Pre-column Bondapak C18, f o l l o w e d by two columns L i c h r o s o r b RP18 (250x2 mm 10) i n s e r i e s , m o b i l e phase l i n e a r g r a d i e n t o f 0-25% a c e t o n i t r i l e (90 m i n ) i n 0.07 M t r i e t h y l a m i n e (pH 7.56 w i t h p h o s p h o r i c a c i d ) , f l o w r a t e 1.5 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, n o r n i c o t i n e ; 2, anabasine; 3, c o t i n i n e ; 4, N ' - f o r m y l - n o r n i c o t i n e ; 5, anatabine; 6, n i c o t i n e ; 7. mjosmine; 8, 2 , 3 ' - d i p y r i d y l . (Reproduced w i t h p e r m i s s i o n f r o m r e f . 2 3 , by c o u r t e s y o f Marcel Dekker, I n c . )

3

2

I

,

0

5

,

,

,

,

10

15 min

20

25

F i g . 3.2. HPLC s e p a r a t i o n o f major Tobacco a l k a l o i d s z 4 Column UBondapak C18 (300x4 m I D ) , m o b i l e phase methanol - w a t e r ( 2 : 3 ) b u f f e r e d w i t h 0.2% phosphoric a c i d t o which t r i e t h y l a m i n e i s added u n t i l pH 7.25, f l o w r a t e 0.5 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, n o r n i c o t i n e ; 2, anabasine; 3, anatabine; 4, n i c o t i n e . 11 F i g . 3.3. HPLC s e p a r a t i o n o f some Senecio a l k a l o i d s Column PBondapak CN (300x4 mm I D ) , m o b i l e phase t e t r a h y d r o f u r a n - 0.001 M aimonium c a r b o n a t e (pH 7.8)(16:84), f l o w r a t e 1.8 m l / m i n , d e t e c t i o n UV 235 nm. Peaks: 1, unknown; 2, r e t r o r s i n e ; 3, s e n i c i p h y l l i n e ; 4, senecionine.

References p. 242

244

1

I

I

12

I

I

8 min

I

I

1

L

r

0

16 Fig, 3.4. HPLC separation of some senecio alkaloids Column UBondapak C18 (300x4 mn ID), mobile phase methanol - potassium dihydrogen phosphate (pH 6.3)(55:45), flow rate 1.2 ml/min. detection UV 225 nm. Peaks: 1, retrorsine; 2, seneciphylline; 3, senecionine. (Reproduced with permission from ref. 16, by courtesy of Marcel Dekker. Inc.) 25 Fig. 3.5. HPLC separation of Senecio jacobaea alkaloids Column PRP-1 (styrene-divinylbenzene resin)(l50x4.1 mn ID). mobile phase acetonitrile - 0.1 M anonia, 20 min. linear gradient from (1:9) to (3:7), flow rate 1 ml/min, detection UV 220 nm. Peaks: 1, jacoline; 2, jacozine; 3, jacobine; 4, jaconine; 5, seneciphylline; 6, senecionine.

1

Fig. 3.6. HPLC separation of some symphytum alkaloids 18 Column MN-Nucleosil C18 10 pm (300x4 mm ID), mobile phase methanol water (45:55). flow rate 2 ml/min, detection UV 220 nm. Peaks: 1, echimidine N-oxide; 2, symphytine N-oxide. (Reproduced with permission from ref. 18, by courtesy of Hippokrates Verlag)

-

2

L 6 8 10 12 min

246

0 7

0

al (II

I

C 0

6.0 m l l m i n

L

E C

0 0 N

>

3 c L

0

+-

r

UI

I

II

23 L

1

I

I

10

5 1

I

I

20

30

LO

min F i g . 3.7. HPLC s e p a r a t i o n of some Symphytum a l k a l o i d s 26 Column UBondapak NH (300x4 mm I D ) , m o b i l e phase a c e t o n i t r i l e - w a t e r (92:8), f l o w r a t e g r a d i e n t from 2 m l / k n t o 6 m l / m i n i n 18 min, d e t e c t i o n UV 200 nm. Peaks: 1, a l l a n t o i n ; 2, symphytine N-oxide; 3, e c h i m i d i n e N-oxide; 4 , a c e t y l - l y c o p s a m i n e N-oxide; 5, lycopsamine N-oxide. (Reproduced w i t h p e r m i s s i o n f r o m r e f . 23, by c o u r t e s y o f H i p p o k r a t e s V e r l a g )

TABLE

3.1

PYRROLIDINE, PY RROLI Z I O I N E , PY R I D I N E . PIPERIDINE AND QU INOLIZIDI NE ALKALOIDS I N THE CONTEXT OF HPLC ANALYIS OF DRUGS OF ABUSE (CHAPTER 7 ) A1 k a l o i d s

Ref.

Nicotine Nicotine Nicotine Nicotine N i c o t i n e , lobe1 ine

4 6 7 14

References p. 242

3

R e f . i n Chapter 7 21 22 30 32 56

TABLE 3.2 HPLC ANALYSIS OF VARIOUS COMPOUNDS INCLUDING PYRROLIOINE, PYRROLIZIOINE, PYRIDINE, PIPERIDINE AND QUINOLIZIDINE ALKALOIDS Alkaloid

Aims

Stationary phase

Ni cotine,quini ne, strychnine,opium and tropane alkaloids

Separation on ionexchange resins (ligand-exchange LC)

hydrolyzed Porqqel PT. loaded with Cu 470~6.3 Bio-Rad PC20,loaded with Cu++ 470~6.3

Ref.

Column dim. Mobile phase LxID mn 0.06M NH OH 0.2M NH40H 0.05M NH40H 0.03M HO:,,

in in in in

33% 33% 33% 33%

EtOH EtOH EtOH EtOH

1,2.5

TABLE 3.3 HPLC ANALYSIS TOBACCO ALKALOIDS A1 kaloi d

Aims

Stationary phase

Column dim. Mobile phase LxID m

Nicotine ,coti ni ne Nicotine

Analysis in urine Effect solute ionization on retention

Micropak SI 10 XA02, 45-65 urn

250x2 250x2

Nicotine ,nornicotine, cotini ne Nicotine

Analysis in biological fluids

Zorbax-Sil

250~4.6

Analysis in pesticides

UBondapak C18 or 300x4 Lichrosorb RP18, 10 urn 2 5 0 ~ 4 . 1

Nicotine ,norni coti ne, anabasine,anatabine, cotinine,N'-formylnornicotine,myosmine, 2,3' -dipyri dyl

Analysis in tobacco (Fig.3.1)

Lichrosorb RP18

Ni cotine,norni cotine, anabasine,anatabi ne

Analysis in tobacco and fresh plant material (Fig.3.1)

EtOAc-isoprOH-NH40H(80:3:0.4)

Ref. 8

0.01M phosphate buffers of various pH, ionic strength made up to 0.1M

with NaCl in ACN-H20(1:9) Dioxane-isoprOH-NH40H(80:3:0.4)

9 10

MeOH-0.05M

(NH4)2P04 (pH 7.5)(3:2) 22

250x2 (two in series)

A 0.07M TrEA (pH 7.56 with H3P04)

300x4

0.2% H PO (pH 7.25 with TrEA) in MeOH-HzO($: 3)

B ACN

linear gradient 0-25% B in A in 90 min

23 UBondapak C18

24

TABLE 3.4

?

HPLC ANALYSIS PYRROLIDINE, PYRROLIZIDINE. PYRIDINE, PIPERIOINE AN0 QUINOLIZIDINE ALKALOIDS

N N

Alkaloid

Aims

Retrorsine,seneciphylline,

Analysis alkaloids in species

senecionine,ridelline,ja-

Senecio

Stationary phase

Column dim. Mobile phase LxIO m

p8ondapak CN

300x4

MN Nucleosil C18 10 Lichrosorb NH2

THF - 0.01M (NH ) CO (pH 7.8) (16:84) or lineir'gr$dient 13-26% 11,12 THF in 30 min 13,20 MeOH - H 0 in various ratios, 260x13, MeOH gradientg - H30(1:1)+ and pH HCl(pH 3.5) 610x20, 15 150x10 MeOH-O.OiM KH PO (pH 6.3)(1:1), 300~3.9 (55:45),(3:2)' 16 2 columns MeOH-0.005M KH2P04(pH 6.3)(3:2) 17 CH C1 sat. with 1% aq.(NH4)2C03300x4 is6pr8H (20: 1) MeOH-H20(45:55) 18 300x4 no detai 1s ACN-aq. H3P04(pH 2.0)

,Bondapak C18

300~7.8

MeOH-O.01M KH2P04( 7:33)

PRP-1

150x4. I

ACN-O.1M NH OH(1:3) or linear gradient (1?9) t o (3:7)

300x4

ACN-H20(92:8)

cobine,jacoline,jaconine

Echimi di ne ,echinati ne, symphytine

Purification and separation alkaloids from Boraginaceae

XAO 50-100 742250 urn

Retrorsi ne, ride1 1 i ne ,senecionine,seneciphylline, senkerki ne Retrorsine ,senecioni ne, seneciphylline Echimidine,symphytine and their N-oxides

Identification in species (Fig.3.4)

pBondapak C18

Senecio

Preparative LC of s e n e c i o alkaloids Analysis in Symphytum plant material (Fig .3.6)

Matrine,oxymatrine,sophoAnalysis in Sophorae radix ranol ,sophocarpine N-oxide, amagyri ne Echiumine,intermedine,jaIsolation by semipreparative cobine,jacoline,jaconine, HPLC from plant material jacozi ne,lycopsami ne ,senecionine,seneciphylline, sincamidine Jacobine,jacoline,jaconine, Separation on styrene-diviretrorsine,senecionine,senylbenzene resin (Fig.3.5) niciphyll ine Echimidine-,lycopsamine-, Analysis in Symphytum plant acetyl lycopsamine- ,symmaterial (Fig.3.7) phytine-N-oxide

Ref.

pm

or

500/C18 column MN Nucleosil NH2 10 ,m

19

,Bondapak NH

2

This Page Intentionally Left Blank

249

Chapter 4 TROPANE ALKALOIDS 4.1. Tropine a l k a l o i d s 4.1.1. Ion-exchange HPLC.. 4.1.2. Reversed-phase HPLC.. 4.1.3. I o n - p a i r HPLC.. 4.1.4. Straight-phase HPLC.. 4.1.4. Detection 4.2. Pseudotropine a l k a l o i d s 4.2.1. Ion-exchange HPLC.. 4.2.2. Reversed-phase HPLC.. 4.2.3. I o n - p a i r HPLC.. 4.2.4. Straight-phase HPLC 4.2.5. Detection.. References

........................................................ ...................................................... ............................................................ ...................................................... .................................................................. ........................................................ ...................................................... ............................................................ ........................................................ ................................................................ ........................................................................

4.1.

249 249 250 252 252 260 260 26 1 261 262 262

TROPIYE ALKALOIDS*

Most o f the work performed so f a r on t r o p i n e a l k a l o i d s concerns the a n a l y s i s o f such a l k a l o i d s i n pharmaceutical preparations (Table 4.5).

Special methods have been i n v e s t i g a t e d

i n order t o lower the d e t e c t i o n l i m i t o f such alkaloids13’16’28’33’35’37’41’43’45. on the a p p l i c a t i o n o f d e r i v a t i z a t i o n techniques i n LC given by F r e i and S a n t i ”

Reviews and F r e i 39 ,

include some examples o f t r o p i n e a l k a l o i d s . The a n a l y s i s o f atropine and i t s degradation products has a l s o been i n v e ~ t i g a t e d ~ ~ * ~ ~ ’ ~ ~ * ~ ~ . 4.1.1.

ION-EXCHANGE HPLC

Ligand-zxchange chromatography o f a l k a l o i d s by means o f ion-exchange m a t e r i a l s loaded w i t h metal ions was described by Walton and M ~ r g i a ~ ’ ~ ’ ’The ~ ~ technique . i s discussed i n Chapter 7. Instead o f a HPLC system using a d i o l c ~ l u m n ~ Huen ~ ’ ~ and ~ , T h e ~ e n i nfound ~ ~ a micropartic u l a t e s u l f o n i c a c i d cation-exchange m a t e r i a l t o be b e t t e r f o r a s e l e c t i v e separation o f t r o pane a l k a l o i d s p r i o r t o post-column f l u o r i m e t r i c i o n - p a i r d e r i v a t i z a t i o n (Fig.4.1).

Atropine

and scopolamine could be separated from i t s major decomposition products: apoatropine, t r o p i c acid, t r o p i n e and scopoline. 4.1.2.

REVERSED-PHASE HPLC

Honigberg e t a1 .15 described the HPLC analysis o f antispasmodic mixtures, c o n t a i n i n g i . a . atropine and scopolamine. The r e t e n t i o n behaviour was studied by changing three o p e r a t i n g parameters, i . e . the s t a t i o n a r y phase, the methanol

-

water r a t i o , and the pH o f the mobile

phase. A p e l l i c u l a r octadecyl and a phenyl column were used. I t was found t h a t more b a s i c mob i l e phases

-

containing ammonium carbonate - l e d t o t a i l i n g and m u l t i p l e peaks f o r a t r o p i n e

and scopolamine. Generally b e t t e r r e s u l t s were obtained on a phenyl column. Lund and Hansen3’ studied the separation o f a t r o p i n e and some o f i t s decomposition products using m i c r o p a r t i c u l a t e medium p o l a r i t y reversed-phase m a t e r i a l s as s t a t i o n a r y phase, i . e . s t a t i o n a r y phases containing chemically bonded cyano o r alkylamino groups. Atropine and apoatro*Because atropine and 1-hyoscyamine behave s i m i l a r i n a l l HPLC systems, the term a t r o p i n e w i l l be used i n the t e x t and i n a l l tables and f i g u r e s t o describe both a l k a l o i d s . Refcrenea p. 262

250

p i n e c o u l d be s e p a r a t e d on a cyano column, whereas t r o p i c a c i d and a t r o p i c a c i d were s e p a r a t e d o n a n a l k y l a m i n o column. C o u p l i n g o f a cyano and an a l k y l a m i n o column i n s e r i e s enabled t h e sep a r a t i o n o f a l l t e s t compounds ( T a b l e 4.1).

A mixed bed column (cyano : a l k y l a m i n o 2 : l ) was

found t o a l l o w f a s t s e p a r a t i o n ; however, a t r o p i n e c o u l d n o t be determined q u a n t i t a t i v e l y because i t was e l u t e d c l o s e t o t h e s o l v e n t f r o n t . The problem c o u l d be s o l v e d by i n c r e a s i n g t h e l e n g t h o f t h e cyano column t o 15 cm. Column s w i t c h i n g was used t o improve t h e d e t e c t i o n l i m i t o f a p o a t r o p i n e and s t i l l a l l o w a q u a n t i t a t i v e d e t e r m i n a t i o n o f a t r o p i n e ( F i g . 4 . 2 ) . G f e l l e r e t a1 . 3 5 * 4 1 used a m i c r o p a r t i c u l a t e c h e m i c a l l y bonded d i o l s t a t i o n a r y phase i n comb i n a t i o n w i t h an e x c l u s i v e l y aqueous m o b i l e phase. I t a l l o w e d post-column i o n - p a i r d e r i v a t i z a t i o n o f t h e separated a l k a l o i d s . By changing t h e pH and t h e i o n i c s t r e n g t h o f t h e b u f f e r , t h e c a p a c i t y f a c t o r s o f t h e a l k a l o i d s c o u l d be v a r i e d . A t r o p i n e was i n c l u d e d i n a s e r i e s o f a l k a l o i d s w h i c h were s e p a r a t e d on a macroporous s t y r e n e - d i v i n y l b e n z e n e copolymer47 (Chapter 8. Table 8 . 4 ) . 4.1.3.

ION-PAIR HPLC

F o r most o f t h e HPLC analyses o f t r o p i n e a l k a l o i d s , i o n - p a i r chromatography has been a p p l i e d . 26 analyzed methylscopolamine u s i n g sodium d e c y l s u l f a t e as i o n - p a i r i n g r e a g e n t . W a l t e r s

Burdo"

used a s i m i l a r method f o r t h e d e t e r m i n a t i o n o f a t r o p i n e and scopolamine i n t a b l e t s . Octanesulf o n i c a c i d (0.01 M) s e r v e d as t h e p a i r i n g - i o n i n an a c e t a t e b u f f e r o f pH 3.5 c o n t a i n i n g 34% a c e t o n i t r i l e . The a n a l y s i s was performed on an o c t a d e c y l column. F o r q u a l i t a t i v e work a decrease o f t h e percentage o f a c e t o n i t r i l e t o 28 was p r e f e r e d ( T a b l e 4 . 2 ) . Brown e t a l . 2 7 ' 3 0 used h e p t a n e s u l f o n i c a c i d (0.01 M) as p a i r i n g - i o n and an aqueous m o b i l e phase o f pH 3.40 cont a i n i n g 35% a c e t o n i t r i l e i n t h e a n a l y s i s o f a n t i c h o l i n e r g i c drugsz7, and a t r o p i n e 3 ' on m i c r o p a r t i c u l a t e o c t a d e c y l columns. Hartmann3* c o n t i n u e d t h e work s t a r t e d by Burdo22. mentioned above, and used sodium d e c y l s u l f a t e and d i o c t y l s u l f o s u c c i n a t e as i o n - p a i r i n g r e a g e n t s i n t h e a n a l y s i s o f methylscopolamine i n neomycine c o n t a i n i n g v e t e r i n a r y p r e p a r a t i ons ( F i g .4.3). A c h a r i and Jacob5'

s t u d i e d s e v e r a l parameters t h a t have an i n f l u e n c e on t h e r e t e n t i o n o f

b a s i c drugs i n i o n - p a i r HPLC, i . a .

a t r o p i n e and scopolamine. Some g e n e r a l c o n c l u s i o n s were

drawn concerning t h e n a t u r e o f t h e p a i r i n g - i o n , t h e t y p e o f c h e m i c a l l y bonded s t a t i o n a r y phase and t h e c o m p o s i t i o n o f t h e m o b i l e phase (Chapter 2 ) . F o r t h e a n a l y s i s o f a t r o p i n e and i t s m a j o r a c i d i c decomposition products, K r e i l g a r d Z 5 used 0.01 M tetrabutylammonium s u l p h a t e as p a i r i n g - i o n i n a m o b i l e phase o f a c e t o n i t r i l e

-

0.05 M

a c e t a t e b u f f e r (pH 5 . 5 ) ( 1 : 4 ) ( T a b l e 4 . 3 ) . S a n t i e t a l . 1 3 t e s t e d v a r i o u s s t a t i o n a r y phases f o r s t r a i g h t - p h a s e i o n - p a i r p a r t i t i o n chromatography. P i c r i c a c i d served as precolumn p a i r i n g - i o n f o r t h e s e p a r a t i o n o f a t r o p i n e , scopolamine and ergotamine because o f i t s s t r o n g UV-absorption, which enabled a 100-300 t i m e s i m provement o f t h e d e t e c t i o n l i m i t o f t h e p o o r l y UV-absorbing t r o p i n e a l k a l o i d s (see d e t e c t i o n ) . Good r e s u l t s were o b t a i n e d w i t h m i c r o p a r t i c u l a t e K i e s e l g u h r , b u t t h e r e p r o d u c i b i l i t y o f t h e column performance was d i f f i c u l t because o f v a r i a t i o n i n t h e q u a l i t y o f t h e K i e s e l g u h r . M i c r o p a r t i c u l a t e s i l i c a g e l w i t h a pore s i z e o f 100 o r 1000 the pairing-ion,

8 was

found t o be b e t t e r as s u p p o r t f o r

b u t low f l o w r a t e s had t o be used ( 0 . 2 m l / m i n ) ( F i g . 4 . 4 ) .

S i l i c a gel w i t h a

s m a l l e r pore s i z e gave p o o r r e s u l t s , due t o t h e mechanical i n s t a b i l i t y o f t h e system a t t h e r a t h e r h i g h l i n e a r v e l o c i t i e s used.

261 TABLE 4 . 1 RETENTION DATA AND DETECTION LIMITS FOR ATROPINE AND I T S DEGRADATION PRODUCTS3' Compound

k'

Atropine Apoatropine Tropic acid Atropic acid d e l ladonnine

0.57 2.0 2.4 3.0 3.5

minimum d e t e c t a b l e amount ( n g ) 5 40 2

Compound

k'

6-Be1 l a d o n n i n e 6-Isatropic acid Scopolamine Homatropi ne

5.9 3.0 0.36 0.44

Column N u c l e o s i l 5 CN ( 5 0 ~ 4 . 6 nnn I D ) and N u c l e o s i l 5 NH ( 5 0 ~ 4 . 6mn I D ) connected i n s e r i e s , m o b i l e phase 0.05 M sodium a c e t a t e b u f f e r (pH 5) - methino1 (3:1), d e t e c t i o n UV 254 nm. TABLE 4.2 SEPARATION

OF SOME TROPANE ALKALOIDS'~

Compound

k'

Compound

Tropic acid Scopol ami ne Homatropine Scopolamine N-oxi de Methylscopolamine

0.65 2.70 2.76 2.88 3.24

Methyl a t r o p i ne Atropine Hyoscyami ne Cocaine Benztropine

~

~~

~~

~

k' 3.63 4.00 4.00 11.6 not eluted

~~

Column UBondapak C18 ( 3 0 0 ~ 3 . 9 mm I D ) , m o b i l e phase 28% a c e t o n i t r i l e i n 0 . 0 1 M aqueous octanes u l f o n i c a c i d a d j u s t e d t o pH 3.5, f l o w r a t e 1 ml/min, d e t e c t i o n UV 230 nm. TABLE 4.3 SEPARATION OF SOME TROPANE ALKALOIDSz5 Compound

k'

Compound

k'

Atropine Belladonnine Tropic acid Apoatropine

0.2 0.3 1.5 2.5

Atropic acid 8-Isatropic acid 4-Methylbenzoic a c i d ( i n t e r n a l standard)

4.5 6.2 6.5

Column L i c h r o s o r b RP8, 5 p m ( 1 0 0 ~ 4 . 6 mm I D ) , m o b i l e phase 0.01 M tetrabutylammonium s u l p h a t e i n 0.05 M a c e t a t e b u f f e r - a c e t o n i t r i l e ( 4 : l ) a t pH 5.5, f l o w r a t e 1 ml/min, d e t e c t i o n UV 254 nm

.

Huen e t a1.28 found t h e optimum pH f o r i o n - p a i r s e p a r a t i o n s w i t h p i c r i c a c i d t o be 5-6 i n t h e i r i n v e s t i g a t i o n s , when t h e e f f e c t o f t h e v a r i a t i o n o f p i c r i c a c i d c o n c e n t r a t i o n and temp e r a t u r e was s t u d i e d . G f e l l e r e t a1 . 3 3 3 3 7 d e s c r i b e d a u t o m a t i z a t i o n o f precolumn d e r i v a t i z a t i o n f o r t h e systems mentioned above. Post-column d e r i v a t i z a t i o n w i t h i o n - p a i r i n g t e c h n i q u e has a l s o been used t o improve t h e de43

t e c t i b i l i t y o f t r o p i n e a l k a l o i d s ( s e e d e t e c t i o n ) . The t e c h n i q u e was used by Lawrence e t a l . after

s e p a r a t i o n o f t h e a l k a l o i d s on a m i c r o p a r t i c u l a t e s i l i c a g e l column w i t h a m o b i l e phase

o f c h l o r o f o r m and methanol, c o n t a i n i n g t h e weakly i o n - p a i r i n g b u t y r i c a c i d . C r ~ m m e ni n~v~e s t i g a t e d t h e r e t e n t i o n o f o r g a n i c compounds on s i l i c a g e l u s i n g aqueous mob i l e phases. A r e t e n t i o n model was presented, based o n t h e d i s t r i b u t i o n o f i o n - p a i r s . A p p l i c a t i o n s t o t h e s e p a r a t i o n o f some t r o p i n e a l k a l o i d s a r e shown i n Fig.4.5.

References p. 262

4.1.4.

STMIGHT-PHASE HPLC

The f i r s t HPLC separation of t r o p i n e a l k a l o i d s was performed on a s i l i c a gel column by means o f tetrahydrofuran

- 28% ammonia (100:l)’. Verpoorte and Baerheim Svendsen” separated some - methanol - diethylamine (9O:lO:l) on m i c r o p a r t i c u l a t e

tropine alkaloids with diethyl ether s i l i c a gel (Fig.4.6).

Rather l a r g e amounts o f a l k a l o i d s had t o be i n j e c t e d due t o the poor

chromophore. which caused severe t a i l i n g on p e l l i c u l a r s i l i c a gel columns. Chloroform cont a i n i n g mobile phases d i d n o t p e r m i t useful separations o f the a l k a l o i d s because o f l a r g e d i f ferences i n capacity f a c t o r s o f a t r o p i n e and scopolamine. Also, by means o f a mobile phase cons i s t i n g o f d i e t h y l ether and diethylamine. separations were achieved4’

( Chapter 7,Fig.7.14).

-

dichloromethane ( 3 : l ) . t o which 1%o f 29% ammonia was added. as mobile phase and a m i c r o p a r t i c u l a t e s i l i c a gel column. Atropine, homatropine and Achari and TheimerL4 used methanol

scopolamine showed d i f f e r e n t capacity f a c t o r s . Straight-phase separations by means o f p o l a r mobile phases were used by Jane

12

(Chapter 7.

Table 7.8). Aigner e t a1.18 separated some drugs, i n c l u d i n g tropane a l k a l o i d s . on s i l i c a gel columns impregnated w i t h s i l v e r iodide. By using gradient e l u t i o n , multicomponent mixtures o f drugs could be separated. 4.1.5.

DETECTION

A major problem i n the analysis o f t r o p i n e a l k a l o i d s by HPLC i s t h e i r poor chromophore. The decomposition products t r o p i n e and scopoline completely lack any chromophore. UV d e t e c t i o n a t 254 nm has. according t o Stutz and Sass

1 a d e t e c t i o n l i m i t o f about 1 pg, and R I d e t e c t i o n o f

about 50 ug. WaltersL6 found a wavelength o f 230 nm t o be t h e optimum compromise between great e r a b s o r p t i v i t y o f the a l k a l o i d s and increasing background absorbance o f the mobile phase (detection l i m i t 0.5 ug). Brown and S1eeman3’ reported a d e t e c t i o n l i m i t o f 200 ng f o r a t r o pine a t 254 nm. Because o f the low absorbance o f t r o p i n e a l k a l o i d s , Santi e t al.13’28

developed an i o n - p a i r

separation using the strong UV absorbing p i c r i c a c i d as p a i r i n g - i o n . Compared t o a reversed-phase separation followed by d e t e c t i o n a t 210 nm, a f i f t y - f o l d enhancement o f the d e t e c t i o n l i m i t was observed using the p i c r i c a c i d i o n - p a i r technique and d e t e c t i o n a t 254 nm. The det e c t i o n l i m i t f o r a t r o p i n e was 5 ng and f o r scopolamine 50 ng. The non UV absorbing scopoline could be detected a t a l e v e l as low as 2.5 ng. By measuring a t the UV maximum o f p i c r i c a c i d

-

345 nm - interference o f the s i g n a l s o f non-pairing compounds was suppressed. G f e l l e r e t a1 .33*37 developed the technique t o an automated pre-column d e r i v a t i z a t i o n method. G f e l l e r e t a1 .35941 a l s o developed an automatic post-column d e r i v a t i z a t i o n method. A f l u o rescent pai r i n g - i o n

-

9,lO-dimethoxyanthracene-2-sul fonate (DAS)

-

was used t o improve the

detection l i m i t o f atropine. The best r e s u l t s were obtained w i t h an e x c l u s i v e l y aqueous mobile phase, t o which the reagent was added as an aqueous s o l u t i o n . Subsequently the i o n - p a i r was extracted w i t h chloroform o r dichloromethane. An increase i n the percentage o f methanol i n the mobile phase reduced the s e n s i t i v i t y . The method was found t o be 200 times more s e n s i t i v e than UV detection a t 208 nm; the minimum detectable amount was 200 pg. Peak broadening caused by

the post-column r e a c t o r was about 40%. To meet the requirement o f a s e l e c t i v e separation w i t h a mobile phase containing only small amounts o f organic solvents, Huen and T h e ~ e n i nprefer~~ red ion-exchange separation (Fig.4.2).

263

Lawrence e t a1 .43 described a simpler post-column ion-pair derivatization technique. whereby the alkaloids were separated by means of an organic mobile phase on s i l i c a gel. The column eluate and the aqueous OAS solution were mixed and the two imniscible phases separated. About 50% of the organic phase was led t o the fluorimetric detector. Various parameters influencing the bandwidth were investigated, i.a. the influence of methanol in the mobile phase. An increasing methanol content deteriorated the signal t o noise r a t i o , and the most useful range was 0-15% methanol. The detection l i m i t f o r atropine was found t o be 40 ng. Hashimoto e t al.36 described a capacitance-conductance detector f o r HPLC; i t had a detection l i m i t of 5 p g f o r scopolamine.

References p. 262

264

NH?+CN NHZ

-

1

'

20

min

"

'

I

10

'

'

-

~

I

0

-

0

5

10

15 m\

Fig. 4.1. HPLC analysis a t r i E i n e w i t h post-column f l u o r i m e t r i c i o n - p a i r d e r i v a t i z a t i o n t o improve the detection l i m i t Column Nucleosil 10 SA (300x4 mm I D ) , mobile phase methanol 0.2 M aqueous diammonium hydrogen phosphate (pH 3)(1:9), f l o w r a t e 2 ml/min, column temperature 85 C. d e t e c t i o n UV 254 nm (1). f l u o r i m e t r i c d e t e c t i o n a f t e r post-column d e r i v a t i z a t i o n w i t h Na-DAS ( e x c i t a t i o n 383 nm, emission 446 nm) (2). Peaks: 1, scopolamine; 2, atropine; 3, apoatropine. (Reproduced w i t h permission from r e f . 45) 32 Fig. 4.2. HPLC analysis atropine and i t s degradation products usinq column s w i t c h i n g Columns Nucleosil 5NH ( 5 0 ~ 4 . 6mn 10) and Nucleosil 5CN(100x4.6 mn I D ) , mobile phase methanol - 0.05 M sodium azetate b u f f e r (pH 5)(1:3), d e t e c t i o n UV 254 nm.(see a l s o Table 4 . 1 ) . Peaks: 1, t r o p i c acid; 2, a t r o p i c acid; 3, atropine; 4, apoatropine.

-

i

1

A

i2

1'6

min

38 Fig. 4.3. HPLC analysis methylscopolamine and some added i m p u r i t i e s Column UBondapak C18 ( 3 0 0 ~ 3 . 9mn I D ) , mobile phase 0.01 M d i o c t y l s u l f o s u c c i n a t e sodium s a l t water (55:45). pH adjusted t o 3.5 w i t h g l a c i a l and 0.01 M anonium n i t r a t e i n 95% ethanol a c e t i c acid, f l o w r a t e 1 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, t r o p i c acid; 2, a t r o p i c acid; 3, DOSS a r t i f a c t ; 4, scopolamine; 5, methylscopolamine; 6, aposcopolamine; 7. methylaposcopolamine. (Reproduced w i t h permission from r e f . 38, by courtesy o f Journal Association of o f f i c i a l a n a l y t i c a l chemists).

-

255

2

i1

0.001A

i -

5

I

min 10 8 6 L 2 Fig. 4.4. HPLC analysis t r o p i n e a l k a l o i d s 28

Column S i l i c a gel S i l o 0 5 p m (100x3 m I D ) impregnated w i t h 0.06 M p i c r i c a c i d (pH 6). mobile phase chloroform saturated w i t h the s t a t i o n a r y phase 0.06 M p i c r i c acid, f l o w r a t e 0 . 2 ml/min, detection UV 345 nm. Peaks: 1. dodecylbenzene ( t o ) ; 2, apoatropine; 3, ergotaminine; 4 , a t r o pine; 5, ergotamine; 6, scopolamine. 44 Fig. 4.5. HPLC analysis a n t i c h o l i n e r g i c s Column Lichrospher S i l o 0 10 pm (200x4 nm ID), mobile phase 0.1 M sodium phosphate b u f f e r pH 2 . 2 containing 1.9% n-amylalcohol, l i n e a r v e l o c i t y 1 . 6 nm/sec., d e t e c t i o n UV 254 nm. Peaks: 1. scopolamine; 2 , afropine; 3, benactyzine; 4, adiphenine; 5, methylatropine; 6, oxypyrronium; 7, oxyphenonium.

h

Rafaence# p. 262

F i g . 4 . 6 . HPLC analysis o f some t r o p i n e a l k a l o i d s 1 9 Column P a r t i s i l 5 pm ( 3 0 0 ~ 4 . 6mn I D ) , mobile phase d i methanol diethylamine (90:10:1), f l o w ethyl ether r a t e 2.29 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, scopolamine; 2. apoatropine; 3, atropine.

-

-

N

8:

TABLE 4.4 HPLC ANALYSIS OF VARIOUS COMPOUNDS INCLUDING TROPINE ALKALOIDS ~~

~

Aims

Stationary Phase

Column Dim. LxID mn Mobile Phase

A,S,22 other alkaloids

Analysis alkaloids

Merckosorb Si60. 5pm

300x2

CHC13-HeOH( 9: 1) .(8:2) ,( 7:3) Et20-MeOH(8:2),(7:3),(6:4) 5

A.opium alkaloids, quinine.cinchonine.strychnine, n i c o t i ne, coc

Separation on ionexchange resins (ligand-exchange LC)

Hydrolyzed Portgel PT loaded w i t h Cu Bio-Rad2$C20 .loaded w i t h Cu

470~6.3

0.06M NH OH i n 33% EtOH 0.2M NH 8H i n 33% EtOH 0.05M NA OH i n 33% EtOH 0.03M NHdOH i n 33% EtOH

A,benztropine. various alkaloids

Analysis drugs o f abuse(Tab1e 7.8)

P a r t i s i l 6 um

250~4.6

Detection w i t h conductance detector

S i l i c a gel 10

A1 k a l o i d *

Other Compounds

Various drugs o f forensic interest

S,Cinchona a1 k a l o i ds .bruc i ne.strychnine, emeti ne ,reserp i ne,yohimbine. caffeine

470~6.3

6.10, 17

MeDH-2M NH OH-1M NH N03(27:2:1) MeOH-O.2M AH4N03()3 !: 12 CHC13-MeOH-hexane(7:3: 10)

Vm

36

A.S,codeine, papaveri ne .qui nine,caffeine, ephedrine

Various drugs

Retention behaviour basic drugs i n ionp a i r HPLC

pBondapak C18 VBondapak Phenyl pBondapak CH VBondagel Chromegabond C8 Chromegabond C6Hl,

300x4

0.005M heptanesulfonic acid i n H O-MeOH-AcOH(50:49:1) (PH a.0,

A,codeine.morphine,dihydrocodeine,quinine, q u i n i d i ne,caffeine ,theophyl1ine ,ajmal ine

Various basic drugs

Separation o f basic drugs on s i l i c a gel w i t h non-aqueous i o n i c eluents

Spherisorb S5W S i l i c a

250~4.9

MeOH-hexane(85:15)containing 0.10% HC104

*

Ref.

For abbreviations see footnote Table 4.5

50

66

k?

H

i P N

TABLE 4.5 HPLC ANALYSIS TROPINE ALKALOIDS I N PHARMACEUTICAL PREPARATIONS

(0 ~~

A1 k a l o i d *

Other Compounds

A.H .S .apoA ,t r o p HMe,codeine,dihydrocodeinone,ephedrine, strychnine A,S .ergotami ne

Aims

S t a t i o n a r y Phase

Column Dim. LxID mn Mobile Phase

Ref.

Separation

Sil-X

1000~4.5

1

Various analgesics. Determination i n C o r a s i l C18 1220~2.3 a n t i h i s t a m i n i c s and cough-cold a n t i t u s s i ves mixtures Separation w i t h i o n p a i r LC S p h e r o s i l XOB.5-1@m 1 0 0 ~ 2 . 8 loaded w i t h 0.03M p i c r i c a c i d and b u f f e r DH 5 S i l i c a g e l 100.5pm 100~2.8 loaded w i t h 0.06M p i c r i c a c i d and b u f f e r PH 5 Chlordiazepoxide, propanthel i n e . i s o propamid,clidinium, Determination o f C o r a s i l C18 o r 1220~2.3 phenobarbital ,pro- antispasmodic Corasi 1 Phenyl chlorperazine mixtures

THF-28% NH40H( 1OO:l) ACN-1%NH40Ac(3: 2) (pH 7.4)

7

CHCl s a t . w i t h 0.05M p i c r i c a c i d 3 i n pH 5 b u f f e r CHCl s a t . w i t h 0.06M p i c r i c a c i d 3 i n pH 6 b u f f e r

13.16

MeOH-l% aq.(NH ) H P04(6:4) (pH 5.85),(1:17(~4 5.50). (2:3)(pH 5.50). MeOH-1% aq. (NH )H PO -1%aq. (NH ) HPO ( 3 : 1 : f ) ( i H 4.20). (2:f : (pa 7. go), (4:3: 3) (pH 7.60) MeOH-0.5% aq. (NH ) CO (3:2) 8.74) (pH 8.65),(1:1)(pa (2:3)(pH 8.80) 15

3)

A.S,ergotamine.er-

B u t a l b i t a l .pheno-

Separation on s i l -

Lichrosorb Silo0

gotami n i ne , c a f f e i ne

barbital

v e r impregnated s i l i c a gel

5,,m,imp. with 1.09% AgI

A. S ,apoA

Separation

P a r t i s i l 5um

n o t given

CHCl -DEA(99.99:0.01)

A C H h -hexane(l:l) B CHCl 3-MeOH-DEA(90: 1 O : O . 5)

300~4.6

l i n e a r 3 g r a d i e n t 16-92% B i n A (1.5-2.5 min.)

18

Et20-MeOH -OEA(90: 10: 1 )

19 N

3

N 01 W

A,H,S,quini ne,qui n i dine.dihydroquinidine ,xanthi nes .strychnine.ephedrine,codeine ,papaverine

Various drugs

A.apoA.trop ac.atrop ac .B ,8-i s a t r o p i c a c

.

Analysis i n pharmaceuticals

P a r t i s i l 10pm

250~4.6

CH C1 -MeOH(1:3)with NH~OH~

1%29%

24

Separation a t r o p i n e L i c h r o s o r b RP8.5pm and degradation' products (Table 4.3)

100~4.6

0.01M tetrabutylamnonium i n 0.05M aq. a c e t k e buffer-ACN (4:l)(pH 5.5)

25

A.H ,S AMe .SMe, trap ac,S N-ox,coc,benztropine

L i d o c a i ne

Determination i n tablets(Tab1e 4.2)

UBondapak C18

300~3.9

0.01M 1 - o c t a n e s u l f o n i c a c i d (pH 3.5)-ACN(66:34),(72:28)

A

Various drugs

Separation a n t i chol in e r g i c drugs

pBondapak C18

300~3.9

0.01M h e p t a n e s u l f o n i c a c i d -ACN(65:35)

A.S,B ,apoA. scop ,ergotami ne ,di hydroergotami ne . c a f f e i ne

Barbiturates, pizotifene

Separation w i t h i o n - p a i r HPLC ( F i g . 4.4)

L i c h r o s o r b Si100.5pm 150x3 loaded w i t h 0.06M p i c r i c acid(pH=6)

CHCl s a t . w i t h 0.06M p i c r i c acid3(pH 6 )

Sepa r at ion

pBondapak C18

300~3.9

0.01M h e p t a n e s u l f o n i c a c i d (pH 3.40)-ACN( 65 :35)

50x4.6

eOH-O.025M NaOAc b u f f e r (PH 5)(1:3)

A,trop

ac

A,H .S .a-B, 8-B .aPoA, t r o p ac,atrop ac. 8 - i s a t r o p i c ac

26

Determination a t r o - N u c l e o s i l 5CN and p i n e and i t s degra- N u c l e o s i l 5NH2 i n d a t i o n products series (Table 4.1 ,Fig.4.2) Various drugs

Post-column d e r i v a - L i c h r o s o r b DIOL.lOpm 250x4 t i z a t i o n (fluoroL i c h r o s o r b RP8,lOpm 1 0 0 ~ 4 . 6 rescent ion-pairs)

sk,S ,apoS .apoSMe.

Neomycin,benzphetami ne

Determination i n pBondapak C18 v e t e r i n a r y formulat i o n s (Fig.4.3)

A.H.S ,various a1 k a l o i d s

A.ergotami ne A,

S ,AMe

A S , apoA

S u l f a n i l a m i d e .phenytoine,phenobarbital Hydroxyatrazine Various drugs

Identification pharmaceuticals ( F i g . 7.14) Post-column d e r i v a t i z a t i on

P a r t i s i l PXS 5/25

28.33, 37

30

32

A,emtine,ephedrine, d i hydroergotami ne, bromocrypti ne t r o p ac,atrop ac

27

300~3.9

0.1M phosphate b u f f e r ( p H 3) MeOH-0.02M phosphate b u f f e r (PH 3)(3:2) 0.01M Na-decylsulfate,O.OlM NH NO i n MeOH-H 0(3:2)

35,41

0.~lM3Na-dioctyl~ulfosuccinate,O.OlM NH NO i n 95% EtOH-H20( 55:49) ($H 3.5)

38

250~4.6

E t 0 s a t . w i t h 50-100% H20 +0?05-0.8% DEA

60x3

0.1M b u t y r i c a c i d i n CHC13 -MeOH (9: 1)

200x4

0.1M Na-phosphate b u f f e r ( p H 2. .*A4 +1.3% AmOH MeOH-0.2M aq. (NH4)2HP04(1:9) 45

42 L i c h r o s o r b Si60,5pm

I o n - p a i r chromato- Lichrospher S i 100, 5pm graphy(Fi g .4.5) Post-column d e r i Nucleosi 1 l O S A v a t i z a t i o n ( F i 9.4.1)

300x4

43

? 3

HMe,codeine.morphine, noscapine,papaverine, thebai ne

p

SMe A,S,theophyl l i n e

I Q N N

Determination i n N u c l e o s i l 5C8 pharmaceutical preparations

120~4.6

ACN-O.01M phosphate b u f f e r (pH 5.0)(2:3)

Pyri1amine.phenirami ne

Analysis i n t a b l e t s P a r t i s i l 10 ODS

250~4.6

ACN-2.85mM ethylenediamine b u f f e r ( p H 7.44)(1:1)

Phenobarbital

A n a l y s i s i n pharma- Spherisorb 5pm c e u t i c a l preparations

250x4

MeOH-O.05M tetramethylamm n i u m phosphate b u f f e r ( p H 2.0) (21:lO) 68.69

* A b b r e v i a t i o n s used i n Tables 4.4 and 4.5 A Me apoA a t r o p ac

B COC

H HMe S SMe S N-OX apoS s cop trop t r o p ac

a t r o p i n e .hyoscyamine methyl a t r o p i ne apoatropi ne atropic acid belladonnine cocaine homatropine methylhomatropine scopolamine methyl scopol ami ne scopolamine N-oxide aposcopolamine scopoline tropine tropic acid

54 61

4.2.

PSEUDOTROPINE ALKALOIDS

Most of the i n v e s t i g a t i o n s on HPLC analysis o f cocaine and r e l a t e d compounds concern the abuse o f cocaine, A series o f methods has been described f o r the i d e n t i f i c a t i o n o f s t r e e t drugs

-

i n c l u d i n g cocaine (Table 4.11). HPLC systems used f o r the a n a l y s i s o f drugs o f abuse are Tables 7.6, 7.8 discussed i n the Chapter 7 4s9,ii I12,20,23.30~53,58959,60965 966 D67 (see also

A review on the analysis o f cocaine has been given64. Although HPLC should be a

and 7.11).

w e l l s u i t e d method f o r the analysis o f cocaine metabolites i n b i o l o g i c a l m a t e r i a l , o n l y two papers seem t o have been published on t h i s matter31y48. The separation o f the f o u r p o s s i b l e 49 diastereoisomers o f cocaine was achieved by Olieman e t a1.46 and Lewin e t a l . . 4.2.1.

ION-EXCHANGE HPLC

Ion-exchange chromatography has been used f o r the analysis o f s t r e e t drugs caine

-

i n a number o f i n v e s t i g a t i o n s 3 ~ g s 2 0 s * 1 Walton . e t a l .6’10’17

-

i n c l u d i n g co-

a p p l i e d ligand-exchange

chromatography f o r alkaloids, i . a . f o r cocaine. None o f the methods mentioned was designed e s p e c i a l l y f o r cocaine, b u t r a t h e r f o r s t r e e t drugs i n general. They are discussed i n more d e t a i l i n Chapter 7. 4.2.2.

REVERSED-PHASE HPLC

Reversed-phase HPLC was used by Jatlow e t a1.31 f o r the a n a l y s i s o f cocaine and i t s metab o l i t e s i n u r i n e (Fig.4.7).

An amount o f 0 . 1 pg/ml cocaine could be detected by using a micro-

p a r t i c u l a t e octadecyl column and an a c i d i c mobile phase c o n s i s t i n g of 0.25 M potassium dihydrogen phosphate (pH 2.7) containing 17% a c e t o n i t r i l e . For the analysis o f cocaine i n plasma, an octadecyl column has been employed i n combination w i t h the mobile phase methanol potassium phosphate b u f f e r (pH 6.6)(3:1),

-

0.05 M

using t e t r a c a i n e as i n t e r n a l standard48. Noggle and

Clark63 reported a method f o r the i d e n t i f i c a t i o n of

cis- and trans-cinnamoylcocaine

in illicit

cocaine. The a l k a l o i d s and some o f t h e i r degradation products were separated on an octadecyl type o f column, using methanol

-

aqueous phosphate b u f f e r (pH 3)(1:2) as mobile phase.

T r i n l e r and R e ~ l a n dused ~ ~ a chemically bonded d i p h e n y l s i l y l s t a t i o n a r y phase f o r the ident i f i c a t i o n o f cocaine and some l o c a l anaesthetics, comnonly used as adulterants i n cocaine samples. A mobile phase o f a c e t o n i t r i l e

-

water (85:15) containing 1%ammonium carbonate was

used. The same system was a l s o used f o r semi preparative work

-

t o allow further i d e n t i f i c a t i o n

o f the separated drugs by means o f I R spectroscopy. To study the h y d r o l y s i s o f cocaine i n u r i n e and plasma samples, F l e t c h e r and H a n ~ o c kused ~ ~ an octadecyl column and methanol

-

water con-

t a i n i n g phosphoric a c i d t o g i v e pH 3.8 as mobile phase. Above pH 7 a s i g n i f i c a n t h y d r o l y s i s o f cocaine t o benzoylecgonine took place.This may e x p l a i n the d i f f e r e n c e which has been reported i n the l i t e r a t u r e i n the estimations o f unchanged cocaine i n body f l u i d s . Jane e t a l .56 obtained u n s a t i s f a c t o r y r e s u l t s f o r analysis o f cocaine and r e l a t e d compounds when using s i l i c a gel o r octadecyl modified s i l i c a gel as s t a t i o n a r y phase. However, good separations and peak performance were obtained w i t h a chemically bonded d i m e t h y l s i l y l phase and methanol

-

aqueous 0.1 M amnonium n i t r a t e (2:3)(pH 4.3) as mobile phase (Table 4.6).

261 TABLE 4.6 HPLC RETENTION DATA OF COCAINE AND RELATED ( r e l a t i v e r e t e n t i o n times(RRT) were calculated w i t h respect t o c o c a i n e ( r e t e n t i o n time 2.7 min)) Compound

RRT

Compound

RRT

Procaine Chloroprocaine L i gnocai ne Pyrrocai ne Benzoylecgonine Dimethocai ne Octacai ne Propoxycaine Prilocaine Mepivacaine Orthocaine Cocaine Benzocaine Butani 1icai ne P i perocai ne cis-Cinnamoylcocaine Leucinocaine Proxymetacai ne Amylocaine

0.65 0.67 0.70 0.74 0.77 0.77 0.77 0.80 0.83 0.83 0.89 1.00 1.13 1.13 1.17 1.18 1.26 1.32 1.41

Butacaine trans-cinnamoylcocaine Amydri c a i ne Phenacaine Cinchocaine Cy c 1omethyca ine

1.47 1.49 1.69 1.72 3.00 3.00

Morphine Codeine 0-acetylmorphi ne Heroin Acetyl codeine

0.45 0.52 0.61 0.91 0.92

Ephedrine Caffeine Amphetamine Me thy1amp he t ami ne Cycl i z i ne D i p i panone

0.60 0.69 0.69 2.58 2.58

0.58

Column Lichrosorb RP2, 5 p m ( 1 5 0 ~ 4 . 6m ID), mobile phase methanol - 0.1 M ammonium n i t r a t e (2:3) adjusted t o pH 4.3 w i t h 2 M h y d r o c h l o r i c acid, f l o w r a t e 1.5 ml/min, d e t e c t i o n UV 279 nm. TABLE 4.7 RETENTION VOLUME OF COCAINE AND RELATED COMPOUNDSZ3 ( r e t e n t i o n volume (Rr) r e l a t i v e t o cocaine, see a l s o Table 7.3) Compound

Rr

Compound

Rr

Anti p y r i ne Procaine Benzocai ne Lidocaine

0.27 0.34 0.45 0.62

Cocai ne Methaqualone Mecl oqual one Tetracaine

l.OO(26.4 m l ) 1.11 1.31 2.60

Column pBondapak C18 (300x4 m I D ) , mobile phase methanol - a c e t i c a c i d - water (40:1:59) (pH 3.5) containing 0.005 M n-heptanesulfonic acid, f l o w r a t e 2 ml/min, d e t e c t i o n UV 254 nm. 4.2.3.

ION-PAIR HPLC

L u r i e and co-workers 23’58’59*60’67

used i o n - p a i r chromatography f o r the a n a l y s i s o f s t r e e t

drugs. The r e s u l t s obtained f o r cocaine and some l o c a l anaesthetics on an octadecyl column using heptanesulfonic a c i d as p a i r i n g - i o n i n a mobile phase o f methanol (40:1:59)

-

water

-

acetic acid

are given i n Table 4.7 (see a l s o Table 7.6). Olieman e t a l . 4 6 separated t h e f o u r

cocaine diastereoisomers on a m i c r o p a r t i c u l a t e octadecyl column by means o f a mobile phase o f tetrahydrofuran

-

water (1:4) containing 0.005 M _I-heptanesulfonic a c i d (Fig.4.8).

Lichrosorb

RP18 was found t o be less s u i t a b l e as s t a t i o n a r y phase than Nucleosil C18 because o f t a i l i n g and peak broadening. 4.2.4.

STRAIGHT-PHASE HPLC

Lewin e t a l .49 separated the f o u r isomeric cocaines by straight-phase chromatography

Referenca p. 262

262

(Fig.4.9).

HPLC was prefered over GLC because o f the decomposition o f some o f the a l k a l o i d s

during GLC. Some o t h e r straight-phase systems f o r drugs o f abuse are d e a l t w i t h i n Chapter 74.12 4.2.5,

DETECTION

The UV spectrum o f cocaine shows maxima a t 229. 274 and 281 nm ( i n ethanol). The l a t t e r two maxima have o n l y low i n t e n s i t y and are, therefore, n o t s u i t a b l e f o r a s e n s i t i v e detection.

A detection a t 230-235 nm has u s u a l l y been prefer re^?^'^^'^^'^^, thus l i m i t i n g the choice o f the mobile phase. Olieman e t al.46 found the d e t e c t i o n l i m i t o f cocaine a t 235 nm t o be 2.8 ng i n t h e i r system. Jatlow e t a1.31 detected cocaine both a t 200 nm and a t 235 nm. The former wavelength gave a 2.5 times more s e n s i t i v e d e t e c t i o n o f cocaine. The debenzoylated metabolites (ecgonine, norecgonine and methylecgonine) had i n s u f f i c i e n t UV absorption f o r UV d e t e c t i o n even a t 200 nm. Comparison o f peak h e i g h t absorbance a t the wavelength o f d e t e c t i o n could be

-

used f o r f u r t h e r v e r i f i c a t i o n o f the i d e n t i t y . Jane e t a l ? p r e f e r r e d UV d e t e c t i o n a t 279 nm, because i t allowed d e t e c t i o n o f r e l a t i v e l y small amounts o f t h e strong UV absorbing cinnamoylcocaine i n cocaine samples. Baker e t a1.40 used the r a t i o o f absorbance a t 254 and 280 nm t o characterize drugs o f f o rensic i n t e r e s t

-

and a l s o cocaine. S i m i l a r l y , L u r i e e t a1.66 used the 220/254 absorbance r a -

t i o (Table 7.6). Noggle and Clark63 applied t h i s method t o the i d e n t i f i c a t i o n o f trans-cinnamoylcocaine i n i l l i c i t cocaine samples (254/280 r a t i o ) .

-

cis- and

REFERENCES 1 M.H. Stutz and S.'Sass, Anal. Chem., 45 (1973) 2134. 2 C.Y. Wu. S. Siggia, T. Robinson and R.D. Waskiewicz. Anal. Chim. A c t a , 63 (1973) 393. 3 J.D. Wittwer, J . Forensic S c i . , 18 (1973) 138. 4 M.L. Chan, C. Whetsell and J.D. McChesney, J . Chromatogr. S c i . , 12 (1974) 512. 5 R. Verpoorte and A. Baerheim Svendsen, J . Chromatogr., 100 (1974) 227. 6 H.F. Walton. J . Chromatogr.. 102 (1974) 57. 7 I.L. Honigberg, J.T. Stewart and A.P. Smith, J . Pharm. S c i . , 63 (1974) 766. 8 V. Quercia, B. Tucci Bucci and A.R. La Tegola, F i t o t e r a p i a , 46 (1975) 3. 9 P.J. Twitchett. J . Chromatogr., 104 (1975) 205. 10 E. Murgia and H.F. Walton. J . Chromatogr., 104 (1975) 417. 11 P.J. T w i t c h e t t and A.C. Moffat, J . Chromatogr., 111 (1975) 149. 12 I . Jane, J . Chromatogr., 111 (1975) 227. 13 W. Santi. J.M. Huen and R.W. Frei. J. Chromatogr., 115 (1975) 423. 14 W.A. T r i n l e r and D.J. Reuland, J . Forensic sci. Soc., 15 (1975) 153. 15 I.L. Honigberg, J.T. Stewart, A.P. Smith, R.D. P l u n k e t t and E.L. Justice, J . Pharm. sci., 64 (1975) 1389. 16 R.W. F r e i and W. Santi, 2. Anal. Chem., 277 (1975) 303. 17 E.O. Murgia, D i s s . A b s t r . I n t . B , 36 (1976) 3911. 18 R. Aigner, H. S p i t z y and R.W. F r e i . J . Chromatogr. S c i . , 14 (1976) 381. 19 R. Verpoorte and A. Baerheim Svendsen, J . Chromatogr., 120 (1976) 203. 20 P.J. Twitchett. A.E.P. Gorvin and A.C. Moffat, J . Chromatogr., 120 (1976) 359. 21 M. Deki and K. Mizuki. Kanzei Chuo Bunsekishoho, 16 (1976) 23. CA 87 (1977) 16622m. 22 T.G. Burdo, 4 t h Ann. Fed. Anal. Chem. and S p e c t r . SOC. Meeting, Detroit,paper no 176 (1977). 23 I . S . Lurie, J . ASSOC. Off. Anal. Chem., 60 (1977) 1035. 24 R.G. Achari and E.E. Theimer, J . Chromatogr. Sci., 15 (1977) 320. 25 8. Kreilgard, Arch. Pharm. Chem., 6 (1978) 109. 26 M.J. Walters, J . ASSOC. Off. Anal. Chem., 6 1 (1978) 1428. 27 N.D. Brown. L.L. H a l l . H.K. Sleeman, B.P. Doctor and G.E. Demaree. J . Chromatogr., 148 (1978) 453. 28 J.M. Huen, R.W. Frei. W. Santi and J.P. Thevenin. J . Cbromatogr., 149 (1978) 359. 29 K. Sugden. G.B. Cox and C.R. Loscombe, J . Chromatogr., 149 (1978) 377.

263

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69

Brown and H.K. Sleeman, J . Chromatogr., 150 (1978) 225. P . I . Jatlow, C. Van Dyke, P. Barask and R. Byck, J . Chromatogr., 152 (1978) 115. U. Lund and S.H. Hansen, J . Chromatogr., 161 (1978) 371. J.C. G f e l l e r , J . Huen and J.P. Thevenin, J . Chromatogr.. 166 (1978) 133. W.A. T r i n l e r and D.J. Reuland, J . F o r e n s i c S c i . , 23 (1978) 37. J.C. G f e l l e r , G. Frey, J.M. Huen and J.P. Thevenin, HRC 8 cc., J . High R e s o l u t . Chromatoqr., Chromatogr. Commun., 1 (1978) 213. Y . Hashimoto, M. Moriyasu, E. Kato, M. Endo, M. Miyamoto and H. Uchida. Mikrochim. d c t a 2 (1978) 159. J.C. G f e l l e r , J.M. Huen and J.P. Thevenin, Chromatographia. 12 (1979) 368. P.A. Hartmann. J . ASSOC. off. A n a l . Chem...~ 62 (19791 1099. R.W. Frei, J . Chromatogr., 165 (1979) 75. J.K. Baker, B.E. Skelton and Ch.Y. Ma, J . Chromatoqr., 168 (1979) 417. J.C. G f e l l e r , G. Frey, J.M. Huen and J.P. Thevenin, J . Chromatogr., 172 (1979) . . 141. R. Gimet and A. F i l l o u x , J . Chromatogr., 177 (1979) 333. J.F. Lawrence, U.A.T. Brinkman and R.W. Frei. J . Chromatogr.. 185 (1979) 473. J. CrOIIBnen, J . Chromatogr., 186 (1979) 705. J.M. Huen and J.P. Thevenin, HRC & CC., J.High Resol. Chromatogr. ,Chromatogr.Commun., 2 ( 1 9 7 9 ) 1 5 4 . C. Olieman, L. Maat and H.C. Beyenan, Recl. T r a v . Chim. Pays-Bas, 98 (1979) 501. K. Aramaki, T. Hanai and H.F. Walton. Anal. Chem., 52 (1980) 1963. A.N. Masoud and D.M. Krupski, J . A n a l . T o x i c o l . , 4 (1980) 305. A.H. Lewin, S.R. Parker and F.I. C a r o l l , J . Chromatogr.. 193 (1980) 371. R.G. Achari and J.T. Jacob, J . L i g . Chromatogr., 3 (1980) 81. G.K. Poochikian and J.C. Cradock, J . Pharm. s c i . . 69 (1980) 637. A . I . Da Rocha, A . I . Reiz-Luz and F. Marx, d c t a dmazonica, 11 (1981) 661. J.D. Wittwer, F o r e n s i c s c i . I n t . . 18 (1981) 215. P. Majlat. P. Helboe and A.K. Kristensen, r n t . J . Pharm., 9 (1981) 245. S.M. Fletcher and V . S . Hancock. J . Chromatogr., 206 (1981) 193. I. Jane, A. Scott, R.W.L. Sharpe and P.C. White, J . Chromatogr., 214 (1981) 243. G. Hoogewijs. Y . Michotte, J. Lambrecht and D.L. Massart. J . Chromatogr.. 226 (1981) 42. I . S . L u r i e and S.M. Demchuk, J . L i q . Chromatogr., 4 (1981) 337. I . S . L u r i e and S.M. Demchuk, J. L i q . Chromatogr., 4 (1981) 357. I . S . Lurie, J . L i q . Chromatogr., 4 (1981) 399. D.R. Heidemann, J . Pharm. S c i . , 70 (1981) 820. V. Das Gupta. r n t . J . Pharm., 10 (1982) 249. F.T. Noggle and C.R. Clark. J. ASSOC. Off. A n a l . Chem., 65 (1982) 756. T.A. Gough and P.B. Baker. J . Chromatogr. s c i . , 20 (1982) 289. J.G. Umans, T.S.K. Chiu, R.A. Lipman, M.F. Schultz and S.U. Shin, J . Chromatogr., 233 (1982) 213. R.J. Flanagan, G.C.A. Storey, R.K. Bhamra and I. Jane, J . Chromatogr., 247 (1982) 15. I.S. Lurie, S.M. Sottolano and S. Blasof, J . F o r e n s i c S c i . , 27 (1982) 519. L.J. Pennington and W.F. Schmidt. J . Pharm. s c i . , 7 1 (1982) 951. W.F. Schmidt and L.J. Pennington. J . Pharm. S c i . , 71 (1982) 954.

N.D.

\

I

TABLE 4.8

N

a P

HPLC ANALYSIS COCAINE I N COMBINATION WITH OTHER COMPOUNDS Alkaloid

*

Aims

S t a t i o n a r y phase

Column Dim. Mobile phase LxID mn

Coc.opiun a1 kaloids,quinine, cinchonine,caffeine

Separation by means o f dynamic c o a t i n g HPLC

C o r a s i l I and II,dyn a m i c a l l y coated w i t h P o l y 6-300(2%)

1000x1

Heptane-EtOH(1:l) w i t h d i f f e r e n t percentage s a t u r a t i o n w i t h P o l y 6-300

2

Coc.23 o t h e r a l k a l o i d s

Analysis a l k a l o i d s

Merckosorb Si60.5pm

300x2

CHCl -MeOH( 9: 1) ,(8: 2) ,( 7: 3) Et203MeOH(8: 2), (7: 3), (6:4)

5

C0c.a tropine,opi um a1ka-

Separation on ion-exchange resins(1igand-exchange LC)

Hydrolyzed Porpqel PT, 470~6.3 loaded w i t h Cu Bio-Rad+$CZO,loaded w i t h Cu 470~6.3 L i c h r o s o r b Si100,5,,m 150x5

loids,nicotine.strychnine, q u i n i n e .cinchoni ne Coc,amyl o c a i ne .benzocai ne, b u t a c a ine

Separation b a s i c drugs on s i l i c a g e l w i t h aqueous s o l v e n t s

Coc.benzoy1ecgonine.morphi-

S t a b i l i t y COC and h e r o i n i n pharmaceutical dosage form

ne.heroin.0Ac-morphine, benzoic a c i d

pBondapak C1B

300x4

0.o6M 0.2M 0.05M 0.03M

Ref.

NH OH i n 33% EtOH NH40H i n 33% EtOH NH40H i n 33% EtOH NHfOH i n 33% EtOH

6.10. 17

MeOH-H 0(7:3) c o n t a i n i n g NH - f o r mate.Nfi NO o r Na-fonnate v a r i ~ u s ~ c o ~ c e n t r a t i oand n s pH 29

i4

ACN-0.015M

Na2HP04(pH 3.0)(1:3) 51

TABLE 4.9 HPLC ANALYSIS OF COCAINE AND RELATED ALKALOIDS Alkaloids* Coc.pseudococ.al allopseudococ

lococ.

Coc.pseudococ,allococ. allopseudococ

Aims

S t a t i o n a r y phase

Column Dim. Mobile phase

Separation o f d i a s t e r e o i s o mers ( F i g .4.8)

N u c l e o s i l C18,km

150~4.0

THF-H 0(1:4) c o n t a i n i n g 0.005M h e p t a g e s u l f o n i c a c i d and 2% AcOH

Separation o f d i a s t e r e o i s o mers ( F i g .4.9)

P a r t i s i l 10 PXS

250~4.6

Heptane-isoprOH-DEA(25:75:0.1)

Analysis i n Erytroxylum p l a n t material F o r a b b r e v i a t i o n s see f o o t n o t e Table 4.11

COC

Ref.

46 49

S i l i c a g e l ODS C18.8um 250x4

MeOH-0.05M phosphate b u f f e r ( p H 7 ) (32:68) 52

Coc.benzoylecgonine, e t h y l -p-ami nobenzoate

S t a b i l i t y COC i n phannaceutic a l p r e p a r a t i o n s a t v a r i o u s pH

Bondapak CN

300x4

MeOH-O.02M NH40Ac(3: 1) 62

N N

TABLE 4.10 HPLC ANALYSIS OF COCAINE AN0 RELATED ALKALOIDS I N BIOLOGICAL MATERIAL -

*

Aims

S t a t i o n a r y phase

Coc,morphine,codeine.caffeine, theophylline

Analysis u r i n e

BOP(no f u r t h e r d e t a i 1s)

COC .benzoyl ecgonine.norcoc,benzoylnorecgonine

Determination i n u r i n e ( F i g .4.7)

P a r t i s i l 10 ODS

Various l o c a l Coc,benzoylecgon i n e .A.S,morphi ne, anaesthetics and caffeine analgesics

Analysis

Coc ,benzoyl ecgonine

Alkaloids

Other comounds

Column Dim. M o b i l e phase LxID mn

Ref.

Heptane-prOH(9:l) 8 250~4.6

ACN-0.25M KH2P04(pH 2.7)(17:83)

_..

JA

ODS-HC SIL-X-1

250~2.6

MeOH-O.05M phosphate b u f f e r ( p H 6.6)(3:1)

H y d r o l y s i s COC i n b i o l o gical fluids

H y p e r s i l 5 ODS.5pm

100~4.6

MeOH-H20(55:45).pH

Various drugs Coc,papaverine, yohimbi ne .heroi n, s t r y c h n i n e , c a f f e i ne

Analysis papaverine i n blood

Micropak CN-10

300x4

Coc.opium alka1o i ds . c a f f e i ne, a u i n i ne

Determination h e r o i n i n blood

L i c h r o s o r b S i 6 0 . 5 ~ m 300x4

Various drugs

COC

i n plasma

4a

*For a b b r e v i a t i o n s see f o o t n o t e Table 4.11

3.8 w i t h H PO 455 Hexane-CH C1 -ACN-propylamine (50: 25: 25?0. 57

I)

ACN-MeOH-(MeOH-NH OH(2:1))-(AcOH -MeOH( 1: 1)) (75: 25?0.040:0.216)

__

b5

TABLE 4.11

EJ

m

HPLC ANALYSIS OF COCAINE I N CONNECTION WITH THE ANALYSIS OF DRUGS OF ABUSE Ref.

A1 kaloids*

Other compounds

Aims

S t a t i o n a r y phase

Column Dim. M o b i l e phase LxIO mn

Coc,various opium a1 k a l o i d s . q u i n i nine.quinidine

Procaine.ani l e r id i ne .methapyrilene

A n a l y s i s drugs o f abuse

Zipax SAX

1ooox2.1

A 0.01M b o r i c a c i d b u f f e r pH 9.5 w i t h 1M NaOH 6 0.01M KH PO b u f f e r pH 6.0 w i t h 1M NaOH g r a d i e n t A+6(85:15) t o 6 , l i n e a r 50r 10% p e r min 3

Coc.various opium alkaloids ,quinine.LSD,rnescal ine

Procaine,benzoc a i ne ,various o t h e r drugs

Identification street drugs

C o r a s i l II,37-50pm,

500~2.3

Cyclohexane-MeOH-cyclohexylamine (98.3: 1.5: 0.2) ,(94.5: 4.5: 1) A S k e l l y 8-952 EtOH-dioxane-cyclohexylamine(991.3:50:25:13) 6 idem (686:100:200:14) l i n e a r g r a d i e n t from A t o 6

A1 0 .Woelm 618, 1823dpm

500~2.3

Cyclohexane-cycl ohexyl ami ne (98.8: 0.2)

Coc,various opium alkaloids,caffeine,s t r y c h n i ne, quinine,ephedrine

Procaine.1 ignocaine,barbiturat e ,paracetam01

Analysis i l l i c i t heroin p r e p a r a t i o n s ( F i g . 7.2)

Zipax SCX

12oox2.1

Coc,various drugs o f abuse(Tab1e 7.8)

Local anaesthetics.various o t h e r drugs

Separation drugs o f abuse

P a r t i s i l 6pm

250~4.6

Screening o f drugs o f abuse

Bondapak C18/Corasi1

610x2

Eva1u a t i o n ion-exchange ana reversed-pnase columns f o r t h e a n a l y s i s o f drugs

P a r t i s i l SCX 10 m

250~4.6

UBondapak C18

300x4

Identification

Zipax SCX

Coc,morphine.heroin.methadone

-

Coc .morphine ,n i cotine.ephedrine. c a f f e i n e , q u i n i ne, tubocurarine

Coc,various opium a1k a l o i d s

Various drugs

4 A 0.2M b o r i c a c i d buffer.pH 9.3 w i t h 40% NaOH B 0.2M b o r i c a c i d buffer-ACN-prOH (86:12:2),pH 9.8 w i t h 40% NaOH l i n e a r g r a d i e n t 0-100% B i n 6 min 9 MeOH-2M NH OH-1M NH NO (27:2:1) MeOH-0.2M 4NH4N03(3?2)3

12 ACN-H O(9:l) ,(65:35) ,( 1: 1 ) a1 1 contaqning 0.1%( N H ~ ) ~ c o ~

14

0.5.0.1.0.05.0.01M (NH )H PO b u f f e r s o f pH 3.5 o r 7,wi?h 6.28,40 o r 60% MeOH 0.025M NaH PO o r Na HPO b u f f e r s of pH 3,5,3 o$ 9, wi?h 0?20,40,60 o r 60% MeOH 11.20 0.2M NaOH+5% prOH,l% KN03 and 2% ACN(pH 9 )

21

R1 a I 69

P N

N

0.005M h e p t a n e s u l f o n i c a c i d i n MeOH-AcOH-H20(40:1:59)(pH 3.5)

Coc.various opium Local anaesthet i c s .various and e r g o t a1 kaloids,caffeine, o t h e r drugs theophylline, q u i n i n e . s t r y c h n i ne COC Local anaesthetics

I o n - p a i r chromatography f o r the separation o f drugs o f abuse(Tab1e 4.7)

I d e n t i f i c a t i o n s t r e e t drugs Bondapak PhenyllPo- 1 2 0 0 ~ 3 . 2 rasil 8

ACN-H 0(85:15) by we?ght

Coc.various a l k a loids(Tab1e 2.2 and 2.3)

Various drugs

I d e n t i f i c a t i o n by means o f dual wavelength d e t e c t i o n

Coc,various opium a1 k a l o i d s , c a f f e i ne.quinine,qui n i d i n e , s t r y c h n i ne

Local anaesthetics,hypnotics, analgesics

COC, benzoylecgonine,cinnamoylcoc,opium a l k a l o i ds . c a f f e i ne

Local anaesthetics

VBondapak C18

300x4

23 w i t h 0.1% (NH ) CO 334

UBondapak C18

300~3.9

0.25M NaH2P04 i n MeOH-H20(2:3).

PPorasi 1

300~3.9

Analysis h e r o i n seizures (Table 7.11)

pPorasi 1

300x4

MeOH-2M NH OH-1M NH NO (27:2:1) CH2C12-con?.NH40H( d o : $ ) Cyil ohexane- (CHCl 3-MeOH-NH40H (800:200:1))(3:1) conc. NH40H: 28, 14 o r 7%

A n a l y s i s i l l i c i t cocaine samples(Tab1e 4.6)

L i c h r o s o r b RP2.5pm

150~4.6

Coc,various o p i um Local anaesthea l k a l o i d s and t i cs ,amphetami other alkaloids nes ,barbi t u r a t e s

I o n - p a i r HPLC o f drugs o f forensic i n t e r e s t

pBondapak C18, 300~3.9 UBondapak Phenyl o r UBondapak CN

COC,transand c i s - c i nnamoyl coc, benzoylecgoni ne, benzoic a c i d

A n a l y s i s i l l i c i t cocaine samples

pBondapak C18

300~3.9

56 0.005M a l k y l s u l f o n a t e ( C ,C C ) i n MeOH-H O-A~OH(40:59:1),~’ 58.59, (30:69:1)?(20:79:1), pH 3.5 60 MeOH-phosphate b u f f e r ( p H 3.0)(1:2)

Analysis h e r o i n seizures (Table 7.6)

pBondapak C18 o r P a r t i s i l 10-ODs-3

300~3.9 250~4.6

ACN-H 0-H PO (12:87:1) c o n t a i n i n g 0 . 0 2 M 2 m e t ~ a n ~ s u l f o n iacid, c pH 2.2

Coc,tropacoc,A, o p i um a1 k a l o i ds , various alkaloids

COC

S

40

53 MeOH-O.1M NH NO (2:3),pH w i t h 2M HC1

4.3

63 Local anaesthet i c s ,various o t h e r drugs

*Abbreviations used i n Tables 4.8 A

DH 7 . 0

cocaine atropine scopolamine

-

67 4.11

268

31 Fig. 4.7. HPLC analysis cocaine and metabolites i n u r i n e Column P a r t i s i l 10-005 ( 2 5 0 ~ 4 . 6m ID), mobile phase 0.25 M potassium dihydrogen phosphate (pH 2.7) c o n t a i n i n g 17% aceton i t r i l e . f l o w gate 2 ml/min, d e t e c t i o n UV 200 and 235 nm, column temperature 40 C. Peaks: 1, benzoylecgonine; 2, cocaine; 3, benzoylecgonine 2 - e t h y l e s t e r ( i n t e r n a l standard).

‘I,, 1 0 8 6

2,L

L

3

1 L

0.005 A

I

I,

I J 1

,

1

0

2

L

1

6

1

8

1

10 min

1

1

1

1

12

14

16

18

1

20

r

16

12

I

8

L

0

min

F i g . 4.8. HPLC separation o f isomeric cocaines 49 isopropanol - diethylamine Column P a r t i s i l lOPXS ( 2 5 0 ~ 4 . 6mn I D ) , mobile phase heptane (75:25:0.1), flow r a t e increasing e x p o n e n t i a l l y (see dashed l i n e ) , d e t e c t i o n UV 230 nm. Peaks: 1, N,N-dibenzylbenzamide ( i n t e r n a l standard); 2, cocaine; 3, allococaine; 4, pseudococaine; 55 Zllopseudococaine. 46 Fig. 4.9. Separation o f isomeric cocaines water (1:4) conColumn Nucleosil C18 5 pm ( 1 5 0 ~ 4 . 0mn ID), mobile phase tetrahydrofuran a c i d and 2% a c e t i c acid, f l o w r a t e 1.0 ml/min, d e t e c t i o n t a i n i n g 0.005 M !-heptanesulfonic UV 235 nm. Peaks: 1, cocaine; 2, pseudococaine; 3, allococaine; 4, allopseudococaine. (Reproduced w i t h permission from r e f . 46, by courtesy o f Recueil des travaux chimiques des Pays-Bas)

-

-

269

Chapter 5 QUINOLI NE ALKALOIDS : CINCHONA ALKALOIDS

............................................................... ............................................................... 5.3. Ion-pair HPLC ..................................................................... 5.4. Straight-phase HPLC............................................................... 5.5. Detection ......................................................................... References ............................................................................. 5.1. Ion-exchange HPLC.. 5 . 2 . Reversed-phase HPLC

270 270 271 271 272 273

hPLC has been applied successfully i n the a n a l y s i s o f Cinchona a l k a l o i d s whereby the f o l l o w i n g problems have been d e a l t w i t h :

1. Separation o f Cinchona a l k a l o i d s i n general. 2. Analysis o f such a l k a l o i d s present i n a ) n a t u r a l l y o c c u r r i n g mixtures and i n p l a n t m a t e r i a l , b) pharmaceutical preparations, c ) drugs o f abuse, d) food and beverages, e ) biological fluids. Q u i n i d i n e has, as an antiarrhythmic drug. a narrow therapeutic range, and consequently i t s q u a n t i t a t i v e analysis i n blood, plasma and serum has been the subject o f a s e r i e s o f investigations(Tab1e 5 . 5 ) . Several authors compared HPLC methods w i t h o t h e r methods, such o r f l u o r i m e t r i c assays 18,21.22,24,25,27,28.35,36,38,46,56

as TLC31, GLC27’46,

The l a t t e r method i s the l e a s t s p e c i f i c one and i t u s u a l l y gives higher q u i n i d i n e values because of i n t e r f e r e n c e o f i m p u r i t i e s (dihydroquinidine) and metabolites.The same a p p l i e s f o r EMIT. Many o f the methods used a l s o determine d i h-v d r o ,a u i n i d i n e 1 4 ’ 1 8 ’ 2 1 ~ 2 5 ~ 2 7 ~ 2 8 ~ 2 g ~ 3 1 1

34g35136,38.46,58.61.65,66.67.74.

The content o f dihydroquinidine i n q u i n i d i n e can be as Many q u i n i d i n e

high as 20%. I t s determination i n q u i n i d i n e has been

metabolites, some o f which have a s i m i l a r a c t i v i t y t o q u i n i d i n e , have been determined, together w i t h quinidine. by the e x i s t i n g methods

18,21,23,24.34,35.36.38,46,49.58,61,65,66,

67*74. A possible i n t e r f e r e n c e o f o t h e r drugs i n t h e analysis o f a u i n i d i n e has been reported22~25y28131’35’46’48and p a r t i c u l a r l y on t h e i n t e r f e r e n c e o f quinine22’25’27y46’58~ 67’74.Weidner e t a l .35 developed a method t h a t separated q u i n i d i n e and quinine, using the l a t t e r compound as an i n t e r n a l standard. An eventual i n t e r f e r e n c e o f quinine i n q u i n i d i n e determinations i n b i o l o g i c a l f l u i d s due t o the use o f s o f t d r i n k s containing quinine i s u n l i k e l y 18 , Bonora e t al!6*49 et al?

described a method f o r the a n a l y s i s o f q u i n i d i n e i n urine; Rakhit

and Pate174 reported methods s u i t e d f o r t h e analysis o f q u i n i d i n e i n u r i n e .

L a g e r ~ t r 6 mi n~j ~e c t e d u r i n e d i r e c t l y on the column f o r the analysis o f q u i n i d i n e . Barrow e t a1.47 reported t h e analysis o f q u i n i n e and q u i n i d i n e and t h e i r r e s p e c t i v e metabolites i n r a t u r i n e . Flood e t a l . 4 5 determined simultaneously three a n t i a r r h y t m i c drugs i n plasma by means o f HPLC. As a common d i l u t a n t f o r drugs o f abuse, q u i n i n e i s o f t e n found and determined i n mixtures o f such drugs (Table 5.1).

The analysis o f quinine i n s o f t

d r i n k s have a l s o been accomplished by means o f HPLC (Table 5.6).

Ralerenea p. 273

270

Table 5 . 1 CINCHONA ALKALOIDS I N THE CONTEXT OF HPLC ANALYSIS OF DRUGS OF ABUSE(Chapter 7)

Alkaloids

*

Ref

Q19d

3

Q

4

GQ

a

Ref Chapter 7 6 11

10

QBQd

Q

QvQd 0

11 13 19 26

Ref

Ref Chapter 7

39 59

9 9,Pd

15 0 iE(Fig. 7.2) Q.9d 9 21 22(Table 7.8) 30 Q.9d 38(Table 7.3) 9 47

7

0

Alkaloids*

60 62,63,64 69 70 71 73

56 91(Table 7.11) 93 98,99,100 113 117(Fig.7.9) 118 121(Table 7.6)

5.1 ION-EXCHANGE HPCC

3 Wittwer described an ion-exchange separation method f o r some drugs o f abuse. Q u i n i n e and q u i n i d i n e were among the compounds investigated. Murgia and W a l t ~ n used ~ ’ ~ cation-ex~ changers loaded w i t h metal ions (Cutt.Zntt.Nit+)

t o separate a number o f a l k a l o i d s , i n c l u d i n g

cinchonine and quinine (ligand-exchange chromatography). T w i t c h e t t e t a1 .13 evaluated a m i c r o p a r t i c u l a t e cation-exchange column f o r the analysis o f a s e r i e s o f drugs, i n c l u d i n g q u i nine. A discussion o f the r e s u l t s i s given i n Chapter 7. 5.2 REVERSED-PHASE HPLC Johnston e t a1.5a described reversed-phase HPLC o f a s e r i e s o f Cinchona a l k a l o i d s , whereby a chemically bonded octadecyl s t a t i o n a r y phase was used and methanol- watera c e t i c a c i d (25:75:1) as mobile phase (Fig. 5.1). This mobile phase was introduced by Crouthamel e t a l .18D23’42 f o r the a n a l y s i s o f q u i n i d i n e i n plasma. Q u i n i d i n e , dihydroquinidine and 3-hydroxyquinidine could be separated. The solvents mentioned above- i n the 50 r a t i o 20:80:1- were used f o r the separation o f cinchonine and cinchonidine , Most o f the mobile phases employed i n the analysis o f q u i n i d i n e i n b i o l o g i c a l f l u i d s are a c i d i c . Drayer e t al.21956 and Leroyer e t al.58*67 separated q u i n i d i n e and i t s metabolites by using a c e t o n i t r i l e - a c e t i c acid-water i n combination w i t h an octadecyl column (Fig. 5.2).

Barrow e t a l . 4 7 used a s i m i l a r system, b u t p r e f e r r e d a gradient e l u t i o n

t o separate quinine and q u i n i d i n e from t h e i r respective metabolites. A c i d i c b u f f e r s have a l s o been used i n mixtures w i t h a ~ e t o n i t r i l e ~ (Fig. ~ ’ ~ 5.3). ~ ~ ~ ~ ’ ~ ~ Powers and Sadee2* p r e f e r r e d a chemically bonded a l k y l phenyl s t a t i o n a r y phase over an octadecyl phase. because o f the poor chromatographic behaviour o f the l a t t e r type. They used 0.75 M sodium acetate (pH 3 . 6 ) - a c e t o n i t r i l e (3:2) as mobile phase. Because o f the s h o r t l i f e time of a s t r a i g h t phase column

-

due t o t h e accumulation o f

polar substances from the u r i n e on the s i l i c a gel used. Bonora e t a l ?

preferred

a reversed-phase separation f o r the analysis o f q u i n i d i n e i n u r i n e . To be able t o separate q u i n i d i n e and i t s metabolites on an a l k y l phenyl column, a c e t o n i t r i l e plus a b u f f e r o f pH 4,5 was used as mobile phase. A small amount o f tetrahydrofuran added t o the mobile phase was found t o improve the peak shape and the separation (Fig.5.4). Reece and P e i k e r t46 used a l k y l phenyl columns f o r the same k i n d of a n a l y s i s (Fig.5.5). column temperature o f 5OoC improved the peak r e s o l u t i o n .

*

For abbreviations see footnote Table 5.6

b u t they found t h a t a

271 The chromatographic system r e p o r t e d b y Weidner e t a l .35 s e p a r a t e d q u i n i d i n e , d i h y d r o q u i n i d i n e and 3 - h y d r o x y q u i n i d i n e . Q u i n i n e was used as an i n t e r n a l s t a n d a r d , a l t h o u g h i t had t h e same r e t e n t i o n t i m e as d i h y d r o q u i n i d i n e , t h i s made i t necessary always t o c a r r y o u t two anal y s e s : one w i t h and one w i t h o u t t h e a d d i t i o n o f q u i n i n e . K l i n e e t a l . 3 4 used a c e t o n i t r i l e

-

methanol

-

1%aqueous ammonium c a r b o n a t e (65:31:4) and

an o c t a d e c y l column t o analyze q u i n i d i n e and d i h y d r o q u i n i d i n e ( F i g . 5 . 6 ) . LagerstrSm61 i n j e c t e d u r i n e d i r e c t l y on an o c t y l t y p e of column f o r t h e a n a l y s i s o f q u i n i d i n e i n u r i n e . The a c i d i c m o b i l e phase, 25% a c e t o n i t r i l e i n 0.095 M sodium p e r c h l o r a t e and 0.005 M p e r c h l o r i c a c i d , a l l o w e d f l u o r e s c e n t d e t e c t i o n , which s o l v e d t h e p r o b l e m o f s o l v e n t peak observed w i t h UV d e t e c t i o n . R a k h i t e t

d5 reported

the analysis o f quinidine i n

v a r i o u s b i o l o g i c a l m a t e r i a l s ( F i g . 5 . 7 ) u s i n g an o c t y l t y p e o f s t a t i o n a r y phase.

A cyanopropyl bonded phase has been used f o r t h e s e p a r a t i o n o f t h e f o u r m a j o r Cinchona a1 k a l o i d s ( q u i n i n e , q u i n i d i n e , c i n c h o n i d i n e , c i n c h o n i ne) ( F i g . 5 . 8 ) 7 2 . Aramaki e t a1 .44 performed a n a l y s i s o f b a s i c drugs on a macroporous s t y r e n e - d i v i n y l b e n z e n e co-polymer (see Chapter 8, Table 8 . 4 ) . 5.3 I O N - P A I R HPLC Persson and Lagerstrom14’29 analyzed a n t i a r r h y t h m i c drugs i n plasma by means o f i o n - p a i r p a r t i t i o n chromatography. Q u i n i d i n e and d i h y d r o q u i n i d i n e were s e p a r a t e d on a m i c r o p a r t i c u l a t e s i l i c a g e l column loaded by an i n - s i t u t e c h n i q u e w i t h t h e aqueous s t a t i o n a r y phase: 0.2 M p e r c h l o r i c a c i d - 0 . 8 M sodium p e r c h l o r a t e ( c a . 35% o f t h e t o t a l w e i g h t o f t h e s i l i c a

g e l used) ( F i g . 5 . 9 ) .

The m o b i l e phase, n - b u t a n o l

-

d i c h l o r o m e t h a n e - n-hexane (1:7:2),

was

s a t u r a t e d w i t h t h e s t a t i o n a r y phase. A precolumn was used t o improve t h e e q u i l i b r a t i o n o f t h e m o b i l e phase. n - B u t a n o l was found t o improve t h e peak symmetry; i t lowered, however, t h e s e l e c t i v i t y o f t h e s e p a r a t i o n o f r e l a t e d compounds. Dichloromethane determined t h e s e l e c t i v i t y o f t h e system and n-hexane was found t o be i n e r t and c o u l d be used t o decrease t h e pol a r i t y o f t h e m o b i l e phase and - t h u s

-

increase the r e t e n t i o n times.

W i t h t o l u e n e s u l f o n i c a c i d as p a i r i n g - i o n ,

Gaetani e t a1 .16 a n a l y z e d q u i n i d i n e i n b l o o d

on a s i l i c a g e l column. However, q u i n i d i n e was n o t s e p a r a t e d f r o m i t s d i h y d r o d e r i v a t i v e and also quinine i n t e r f e r e d w i t h the analysis. Lurie19’62’63’64*73

d e s c r i b e d t h e a n a l y s i s o f some drugs o f f o r e n s i c i n t e r e s t , e.g. q u i -

nine, and a p p l i e d reversed-phase i o n - p a i r chromatography (see Chapter 7 ) . I n a s t u d y o f i o n - p a i r chromatography w i t h c a t i o n i c s u r f a c t a n t s , which a u t h o r s - c o u l d be c a l l e d dynamic ion-exchange chromatography

-

-

according t o the

Terwey-Groen e t a1.30 p r e -

s e n t e d t h e a n a l y s i s o f q u i n i n e i n s o f t d r i n k s , w i t h an a n i o n i c s u r f a c t a n t ( d o d e c y l s u l f a t e ) as an example ( F i g . 5 . 1 0 ) . J e u r i n g e t a l . 4 3 a n a l y z e d q u i n i n e i n s o f t d r i n k s . By u s i n g d o d e c y l s u l f o n i c a c i d as m i r i n g - i o n , a reversed-phase a n a l y s i s under a c i d i c c o n d i t i o n s became p o s s i b l e . T h i s was necessa r y f o r t h e f l u o r i m e t r i c d e t e c t i o n o f q u i n i n e . The method a l l o w e d a d i r e c t a n a l y s i s o f q u i n i n e i n s o f t drinks without extraction. 5.4 STRAIGHT-PHASE HPLC Pound and Sears

References p. 213

6 d e s c r i b e d a HPLC procedure f o r a simultaneous a n a l y s i s o f q u i n i n e , q u i -

272

nidine, t h e i r dihydroderivatives, cinchonine and cinchonidine i n c m e r c i a l preparations. The q u a n t i t a t i v e determination

- w i t h a n t i p y r i n e as an i n t e r n a l standard - was performed on micro-

p a r t i c u l a t e s i l i c a gel w i t h tetrahydrofuran

-

0.2% amnonia as mobile phase (Fig.5.1).

When

q u i n i d i n e and quinine s u l f a t e were analyzed, the percentage o f amnonia was increased t o 0.4% and 0.5% respectively. Frischkorn and Frischkorn15 determined quinine i n s o f t d r i n k s by employing methanol

-

0.5% a n o n i a as mobile phase and using a s i l i c a g e l column. However,

quinine and q u i n i d i n e could n o t be separated. Achari e t

d7 described

a s i m i l a r HPLC method

f o r the analysis o f q u i n i d i n e i n human plasma (Fig.5.12). Kates e t al.31 used a solvent system c o n s i s t i n g o f 0.001 M trimethylamine hydrochloride and 0.001 M potassium hydroxide (pH 9 )

- methanol

(1:4) and a m i c r o p a r t i c u l a t e s i l i c a gel

column t o separate q u i n i d i n e and dihydroquinidine. Cinchonine was used as i n t e r n a l standard. Q u i n i d i n e and dihydroquinidine could a l s o be separated by a method developed by Achari and Theimer"

f o r the analysis o f a s e r i e s o f drugs. However, quinine and q u i n i d i n e were n o t se-

parated. Guentert e t a l .24s38 described a straight-phase separation o f q u i n i d i n e and i t s major metab o l i t e s , extracted from human plasma. On a m i c r o p a r t i c u l a t e s i l i c a gel column and mobile phase hexane

-

ethanol

-

ethanolamine (91.5:8.47:0.03)

primaquine as an i n t e r n a l standard (Fig.5.13).

q u a n t i t a t i v e analysis was performed using The a d d i t i o n o f ethanolamine t o the mobile phase

eliminated t a i l i n g and reduced r e t e n t i o n times. Ethanolamine was found t o be s u p e r i o r t o ammonia because i t provides a more s t a b l e s o l v e n t mixture, enabling automatic i n j e c t i o n . The percentage o f ethanol could be adjusted i n order t o a l t e r the r e t e n t i o n times. A l s o the v a r i a t i o n o f the ethanolamine concentration l e d t o changes i n the r e t e n t i o n times. However, long e q u i l i b r a t i o n times (1-2 h r s ) were needed t o achieve s t a b l e conditions. Peat and JennisonZ5 used a p o l a r mobile phase i n combination w i t h a m i c r o p a r t i c u l a t e s i l i c a gel column

- as

o r i g i n a l l y described by Jane"

-

f o r the a n a l y s i s o f q u i n i d i n e i n plasma

(Fig.5.14).

Quinidine and quinine were not, however, separated; cinchonidine was used as i n t e r n a l standard. A s i m i l a r method was used by Pershing e t a l . 66

.

A m i c r o p a r t i c u l a t e s i l i c a gel column and a mobile phase o f dichloromethane nol

-

p e r c h l o r i c a c i d (65:35:5.5:0.1)

-

hexane

-

metha-

was applied by Sved e t a1.28 t o determine the q u i n i d i n e

and dihydroquinidine content i n plasma. P e r c h l o r i c a c i d was added t o induce fluorescence, b u t i t also had some i n f l u e n c e on the r e t e n t i o n times. 3-Hydroxyquinidine was separated from q u i -

niciine and dihydroquinidine; however, 2'-quinidinone had t h e same r e t e n t i o n time as q u i n i d i n e . Bauer and Untz51 analyzed a s e r i e s o f Cinchona a l k a l o i d s by means o f straight-phase HPLC (Fig.5.15).

They found t h a t the a d d i t i o n of 2.65 m l o f water t o 1 l i t e r o f the mobile phase

(chloroform

-

isopropanol

-

diethylamine(940:57:1)), which corresponds t o about 75% s a t u r a t i o n ,

gave optimum separation, as regards r e s o l u t i o n versus time o f analysis. To o b t a i n t h e c o r r e c t percentage o f water i n the mobile phase, the water content present i n the m i x t u r e was deterined by the Karl Fischer method, and water was then added t o o b t a i n a f i n a l concentration of 2.65 m l / l . The non-aqueous i o n i c solvents, as described by Flanagan e t al.71 (see Chapter 2) had only l i m i t e d a p p l i c a b i l i t y f o r quinine because o f t a i l i n g a t low pH. Raising the pH o f the ammonium perchlorate s o l u t i o n i n methanol t o about 9.2 improved the peakshape. 5.5 DETECTION Due t o t h e i r strong fluorescence under a c i d i c conditions, Cinchona a l k a l o i d s have been detec-

213

ted by means o f fluorescence detectors16*17D21'28'34'35g43'46.49,56g61~65y66~67 (Fig.5.2, 5.5, 5.6 and 5.7). Usually e x c i t a t i o n wavelengths o f 330-360 nm have been used, measuring

emission a t 420-450 nm. However, some authors p r e f e r r e d 245 nm and 340 nm r e s p e c t i ~ e l y ~ ~ * ~ ~ * ~ K l i n e e t a l . 3 4 described a post-column a d d i t i o n o f 1 M s u l p h u r i c a c i d t o induce fluorescence of q u i n i d i n e and dihydroquinidine (Fig.5.6), b u t a c i d i c mobile phases have mostly been used t o a l l o w fluorescence d e t e c t i o n16 17 9 21328,35,43, 46 949 956 s58 I 6 1 65 967 According t o Weidner e t a1 .35, fluorescence d e t e c t i o n i s more s e n s i t i v e than UV d e t e c t i o n a t 254 nm; however, f o r r o u t i n e analysis o f q u i n i d i n e i n serum, the a d d i t i o n a l s e n s i t i v i t y was n o t found t o be necessary. Verpoorte and Baerheim Svendsen 5 p r e f e r r e d d e t e c t i o n a t 280 nm over d e t e c t i o n a t 254 nm. Peat and JennisonZ5 found about equal responses a t 254 and 280 nm; however, the l a t t e r wavelength was p r e f e r r e d f o r UV-detection because o f l e s s i n t e r f e r e n c e o f spurious components i n the analysis o f q u i n i d i n e i n plasma. Many authors have p r e f e r r e d d e t e c t i o n o f q u i n i d i n e a t i t s maximum a t 235 nm27.3 1,36,38,49,72 b u t Powers and Sadee"

p r e f e r r e d t o detect q u i n i d i n e a t i t s secondary maximum a t 330 nm. A l -

though t h i s meant a f i v e times less s e n s i t i v i t y than a d e t e c t i o n a t 235 nm. d e t e c t i o n a t 330 nm was n o t i n t e r f e r e d w i t h by other compounds. For t h i s reason, d e t e c t i o n a t 325 nm has a l s o 4a been employed

.

For the determination o f antiarrhythmic drugs i n serum, Flood e t

a p p l i e d a simulta-

neous detection a t 205 and 254 nm. Bauer and Untz51 preferred d e t e c t i o n a t 312 nm. because a t t h i s wavelength the 6-methoxy s u b s t i t u t e d a l k a l o i d s have the same e x t i n c t i o n c o e f f i c i e n t as the non-substituted a l k a l o i d s , enabling a d i r e c t comparison o f the peak areas o f the d i f f e r e n t alkaloids. I d e n t i f i c a t i o n of drugs nm was reported by Baker e t

-

i n c l u d i n g quinine Lurie e t

-

by using the absorbance r a t i o a t 254 and 280 used the 220:254 r a t i o f o r the same reasons.

Kral and S ~ n t a gemployed ~ ~ an electrochemical d e t e c t o r f o r the a n a l y s i s o f q u i n i n e i n beverages. The maximum o x i d a t i o n p o t e n t i a l was a t -1.55 V vs. SSCE, b e s t r e s u l t s were, however, obtained a t -1.4 V. A capacitance-conductance detector f o r HPLC was developed by Hashimoto e t a l . 3 3 and a p p l i e d t o the analysis o f a l k a l o i d s , e.g. some Cinchona a l k a l o i d s . Eckers e t a1.54 reported the a p p l i c a t i o n o f a coupled LC-MS system t o the a n a l y s i s o f Cinchona a1 kaloids.

REFERENCES 1 C.Y. Wu, S. Siggia, T. Robinson and R.D. Waskiewicz, dnal. Chim. d c t a , 63 (1973) 393. 2 C.Y. M U , Diss. A b s t r . Int. 6, 33 (1973) 4166. 3 J.D. Wittwer. J . Forensic S c i . , 18 (1973) 138. 4 M.L. Chan, C. Whetsell and J.D. McChesney. J . Chrom. S c i . , 12 (1974) 512. 5 R. Verpoorte and A. Baerheim Svendsen, J . Chromatogr., 100 (1974) 227. 6 N . J . Pound and R.W. Sears, Can. J. Pharm. S c i . , 10 (1975) 122. 7 P.J. Twitchett, Chem. B r . , 11 (1975) 443. 8 P.J. Twitchett. J. Chromatogr., 104 (1975) 205. 9 E. Murgia and H.F. Walton, J. Chromatogr., 104 (1975) 417. 10 P.J. T w i t c h e t t and A.C. Moffat, J . Chromatogr., 111 (1975) 149. 11 I . Jane, J . Chromatogr., 111 (1975) 227. 12 E.O. Murgia, Diss. dbstr. I n t . B. 36 (1976) 3911. 13 P.J. Twitchett, A.E.P. Gorvin and A.C. Moffat,J. Chromatogr., 120 (1976) 359. 14 B.A. Persson and P.O. Lagerstrom, J . Chromatogr., 122 (1976) 305. 15 C.G.B. Frischkorn and H.E. Frischkorn, 2. Lebensm.-Unters. Forsch., 162 (1976) 273. 16 E. Gaetani, C . Laureri and G. Vaona, dteneo Parmense, dcta N a t . , 13 (1977) 577. 17 K.A. Conrad, B.L. Molk and C.A. Chidsey, C i r c u l a t i o n , 55 (1977) 1. 18 W.G. Crouthamel, B. Kowarski and P.K. Narang, C l i n . Chem., 23 (1977) 2030.

'

214

I. L u r i e , J . d s s o c . Off. d n a l . C h e m . , 60 (1977) 1035. R.G. A c h a r i and E.E. Theimer, J . C h r o m a t o g r . S c i . , 15 (1977) 320. D.E. Drayer, K. R e s t i v o and M.M. Reidenberg. J. L a b . C l i n . M e d . , 90 1977) 816. J.L. Powers and W. Sadee, C l i n . Chem. , 24 (1978) 299. W.G. Crouthamel, B. Kowarski and P.K. Narang, C l i n . Chem., 24 (1978) 1853. T.W. G u e n t e r t and S . Riegelman, C l i n . C h e m . , 24 (1978) 2065. M.A. P e a t and T.A. Jennison. C l i n . C h e m . , 24 (1978) 2166. O.J. Reuland and W.A. T r i n l e r , F o r e n s i c S c i . , 11 (1978) 195. R.G. A c h a r i , J.L. B a l d r i d g e , T.R. K o z i o l and L. Yu, J. ‘ C h r o m a t o g r . S c i . , 16 (1978) 271. S. Sved, I.J.M. G i l v e r a y and N. Beaudoin. J. C h r o m a t o g r . , 145 (1978) 437. P.O. Lagerstrom and B.A. Persson, J . C h r o m a t o g r . 149 (1978) 331. C.P. Terwey-Groen, S . Heemstra and J.C. Kraak, J. C h r o m a t o g r . , 161 (1978) 69. R.E. Kates, D.W. McKennon and T.J. Comstock. J. P h a r m . S c i . , 67 (1978) 269. K.E. Rasmussen. F. Tbnnesen, B. N i e l s e n . B. Lunde and J. Rbe, Medd. N o r s k F a r m . S e l s k . , 40 (1978) 117. 33 Y. Hashimoto, M. Moriyasu, E. Kato, M. Endo, N. Miyamoto and H. Uchida, M i k r o c h i m . d c t a , 2 (1978) 159. 34 B.J. K l i n e , V.A. Turner and W.H. Barr, d n a l . C h e m . , 51 1979 449. 35 N. Weidner, J.H. Ladenson, L. Larson, G. K e s s l e r and J.L. Mcdonald, C l i n . C h i m . d c t a , 9 1 (1979) 7. 36 M.R. Bonora, T.W. G u e n t e r t , R.A. Upton and S. Riegelman, C l i n . C h i m . d c t a , 9 1 (1979) 277. 37 J.M. M i l l e r and E. Tucker, I n t . L a b . , (1979) 16. 38 T.W. Guentert, P.E. Coates, R.A. Upton, C.L. Combs and 5. Riegelman, J. C h r o m a t o g r . , 162 (1979) 59. 39 J.K. Baker, R.E. S k e l t o n and Ch.Y. Ma, J. C h r o m a t o g r . , 168 (1979) 417. 40 R. Gimet and A. F i l l o u x , J. C h r o m a t o g r . , 177 (1979) 333. 41 E. Soczewinski and T. Dzido, J. L i q . C h r o m a t o g r . , 2 (1979) 511. 42 P.K. Narang and W.G. Crouthamel, J. P h a r m . S c i . , 68 (1979 z. L e b e n s m . 4 n t e r . s . Forsch., 43 H.J. Jeuring, W. van Hoeven, P. van Doorninck and R. t e n 169 (1979) 281. 44 K. Aramaki, T. Hanai and H.F. Walton, d n a l . C h e m . , 52 (1980) 1963. 45 J.G. Flood, G. Bowers and R.B. McComb, C l i n . C h e m . , 26 (1980) 197. 46 P.A. Reece and M. P e i k e r t , J. C h r o m a t o g r . , 181 (1980) 207. 47 S.E. Barrow, A.A. T a y l o r , E.C. H o r n i n g and M.G. Horning, J. C h r o m a t o g r . , 181 (1980) 219. 48 J.T. Ahokas, C. Davies and P.J. Ravenscroft, J . C h r o m a t o g r . , 183 (1980) 65. 49 T.W. Guentert, A. R a k h i t , R.A. Upton and S. Riegelman, J . C h r o m a t o g r . , 183 (1980) 514 50 M.A. Johnston, W.J. Smith, J.M. Kennedy, A.R. Lea and O.M. H a i l e y , J. C h r o m a t o g r . , 184 (1980) 241. 51 M. Bauer and G. Untz, J . C h r o m a t o g r . , 192 (1980) 479. 52 J . Pao and J.A.F. De S i l v a , J. C h r o m a t o g r . , 221 (1980) 97. 53 R.G. A c h a r i and J.T. Jacob, J . L i q . C h r o m a t o g r . , 3 1980) 81. 54 C. Eckers, D.E. Games, E . Lewis, K.R. Nagaraja Rao, .!I R o s s i t e r and N.C.A. Weerasinghe, i n A d v a n c e s of Mass s p e c t r o m e t r y , v o l . 8 , H e y d e n , L o n d o n , p . 1396. 55 H.R. Ra, G. Kewitz, M. Wenk and F. F o l l a t h , Er. J . C l i n . Pharmacol.. 111 (1981) 312. 56 O.E. Drayer, B. Lorenzo and M.M. Reidenberg, C l i n . C h e m . , 27 (1981) 308. 57 Dextraze. J . Foreman. W.C. G r i f f i t h s and I . Diamond,clin. T o x i c o l . . 18 (1981) 291. . P.G. 58 R. Leroyer, C: J a r r e a u and M. Pays, F e u i l l . B i o l . , 22 (1981) 111. 59 J.D. Wittwer, F o r e n s i c S c i . r n t . , 18 (1981) 215. 60 P.B. Baker and T.A. Gough, J . C h r o m a t o g r . S c i . , 19 (1981) 483. 61 P.O. Lagerstrom, J . C h r o m a t o g r . , 225 (1981) 476. 62 I . S . L u r i e and S.M. Demchuk, J. L i q . C h r o m a t o g r . , 4 (1981) 337. 63 I.S. L u r i e and S.M. Demchuk, J. L i g . C h r o m a t o g r . , 4 (1981) 357. 64 I . S . L u r i e , J. L i q . C h r o m a t o g r . , 4 (1981) 399. 65 A. R a k h i t , M. K u n i t a n i , N.H.G. H o l f o r d and S. Riegelman, Clin. Chem. 28 1982) 1505. 66 L.K. Pershing, M.A. Peat and B.S. F i n k l e , J. d n a l . T o x i c o l . , 6 (1982 153 67 R. Leroyer, C . J a r r e a u and M. Pays, J. C h r o m a t o g r . , 228 (1982) 366. 68 R.L.G. N o r r i s , J.T. Ahokas and P.J. R a v e n c r o f t , J . C h r o m a t o g r . , 230 1982 433. 69 J.G. Umans, T.S.K. Chiu, R.A. Lipman, M.F. S c h u l t z , S.U. S h i n and C. . I n u r r i s i , J. C h r o m a t o g r . , 233 (1982) 213. 70 B.C. P e t t i t t and C.E. Damon. J . Chromatoqr., 242 (19821 189. 71 R.J. Flanagan, G.C.A. S t o r e y , R.K. Bhamra and I . Jane,’J. C h r o m a t o g r . , 247 (1982) 15. 72 A. Hobson-Frohock and W.T.E. Edwards, J . C h r o m a t o g r . , 249 (1982) 369. 73 I . S . L u r i e , S.M. S o t t o l a n o and S . B l a s o f , J. F o r e n s i c S c i . , 27 (1982) 519. 74 C.P. P a t e l , T h e r . Drug. Monit., 4 (1982) 213. 75 K. K r a l and G. Sontag, z. L e b e n s . - U n t e r s . F o r s c h . , 175 (1982) 22.

19 20 21 22 23 24 25 26 27 28 29 30 31 32

~

~~~

276

Fig. 5.1. Reversed-phase HPLC separation o f some Cinchona alkaloids5' a c e t i c a c i d (25:75:1). Column UBondapak C18 (300x4 mn I D ) , mobile phase methanol - water flow r a t e 1.5 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, theophylline ( i n t e r n a l standard); 2, cinchonine; 3, cinchonidine; 4, q u i n i d i n e ; 5, q u i n i d i n e and dihydroquinidine; 6, dihydroquinine.

-

1

2

0

i

i

i

io

iL

min

Fig. 5.2. HPLC analysis q u i n i d i n e and metabolites i n serumz1 Column ulondapak C18 (300x4 mn I D ) , mobile phase 2.5% aqueous a c e t i c a c i d - a c e t o n i t r i l e (88:12), flow r a t e 1.8 m l h i n , fluorescence detector ( e x c i t a t i o n 340 nm, emission 418 nm). Peaks: 1, 3-hydroxyquinidine; 2, cupreidine( =desmethylquinidine) ; 3, quinidine; 4, cinchon i d i n e ( i n t e r n a l standard); 5, dihydroquinidine; 6, Z'-quinidinone. (reproduced w i t h permission from r e f . 21, by courtesy o f the C.V. Mosby Company).

.ri.

I

1

I

0

10 min

References p. 273

20

metabolite^^^

F i g . 5.3. Separation of q u i n i d i n e and i t s Column P a r t i s i l PXS 5/25 OOS ( 2 5 0 ~ 4 . 6mn I D ) w i t h guard column, mobile phase a c e t o n i t r i l e - 1 M sodium dihydrogen phosphate 1 M sodium perchlorate - 85% phosphoric a c i d - water ( 1 l : Z : l : l : 85), f l o w r a t e 1.5 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, 3-hydroxyquinidine; 2, cupreidine (=desmethylquinidine); 3, q u i n i dine; 4 , quinine; 5, dihydroquinidine. (Reproduced w i t h permission from r e f . 74, by courtesy o f Raven Press)

276

Fig. 5.4 Analysis q u i n i d i n e and metabolites i n urine36 Column pBondapak Phenyl ( 3 0 0 ~ 3 . 9mn ID), mobile phase 0.05 M phosphate b u f f e r (pH 4.5) aceton i t r i l e - tetrahydrofuran (80:15:5), f l o w r a t e 1.16 ml/min, d e t e c t i o n UV 230 nm. Peaks: 1, L'-quinidinone; 2, 3-hydroxyquinidine; 3, oxprenolol ( i n t e r n a l standard); 4, q u i n i d i n e ; 5, d i hydroquinidine ; 6, q u i n i d i n e N-oxi de.

-

0

6

12

18 min

24

30

36

A

I

I

0

8

C

B

I

I

1 6 0

8

I

I

16

0

I

8

16

min 46 Fig. 5.5. HPLC assay q u i n i d i n e i n plasma acetoColumn pBondapak Phenyl (300x4 mn ID), mobile phase 0.0015 M aqueous phosphoric a c i d n i t r i l e (9:l). f l o w r a t e 2 ml/min, fluorescence d e t e c t i o n ( e x c i t a t i o n 320 nm. emission 418 nm) Chromatogram A: blank plasma; chromatogram B: plasma standard ( 10 pmole/l quinidine, 5 m o l e / 1 3-hydroxyquinidine); chromatogram C: plasma from p a t i e n t on chronic o r a l q u i n i d i n e therapy. Peaks: 1, p o l a r metabolites; 2, 3-hydroxyquinidine; 3. i n t e r n a l standard (cinchonidine); 4. u n i d e n t i f i e d metabol it e ; 5, quinidine; 6, d i hydroqui n i dine.

-

211

3

2

1

l

6

3

E

. ,>. ".,.

",

--y..,y"""

.,.

L(",,"y"'-

.,",,,..

",

,.,-

,,,c*y""II.~_l

Column U l t r a s p h e r e C8 ( 1 5 0 ~ 4 . 6 mn ID), m o b i l e phase a c e t o n i t r i l e - methanol - t e t r a h y d r o f u r a n 0.01 M aqueous t r i e t h y l a m i n e (pH 2.5 w i t h p h o s p h o r i c a c i d ) ( 5 : 5 : 3 : 8 7 ) , f l o w r a t e 1.0 ml/min. f l u o r e s c e n c e d e t e c t i o n ( e x c i t a t i o n 245 nm, e m i s s i o n 435 nm). Peaks: 1, quinidine-l0,ll-dihydrod i o l ; 2, 3 - h y d r o x y q u i n i d i n e ; 3, q u i n i d i n e N-oxide; 4, q u i n i d i n e ; 5, d i h y d r o q u i n i d i n e ; 6, 3-met h y l - 5 - t r i a z o l o p h t a l a z i ne ( i n t e r n a l s t a n d a r d ) ; 7, 2 ' - q u i n i d i none. (Reproduced w i t h p e r m i s s i o n from r e f . 65, by t h e c o u r t e s y o f C l i n i c a l Chemistry)

1 2

I

- - "

'

1

I

0

5

1

10

1

15

min

References p. 273

I

I

'

20

25

30

Column S p h e r i s o r b CN 5 um ( 2 5 0 ~ 4 . 6 n I D ) , m o b i l e phase a c e t o n i t r i l e - methanol - t e t r a h y d r o f u r a n 0.0068 M p h o s p h o r i c a c i d (pH 7.0 w i t h 1 M sodium h y d r o x i d e ) . f l o w r a t e 1.5 ml/min. . - t e m.e r a t u r e 5OoC. d e t e c t i o n ' U V 231 nm. Peaks: 1, q u i n i n e ; 2, q u i n i - . d i n e ; 3, c i n c h o n i d i n e ; 4 , c i n c h o n i n e .

270

Fig. 5.9. Analysis uinidine and dihydroquinidine i n plasma 4.29 Column Lichrosorb Silo0 10 pm coated with ca. 35% 0.2 M perchloric acid and 0 . 8 M sodium perchlorate (150~4.5 mm I D ) , mobile phase n-butanol dichloromethane - n-hexane (T:7:2)saturated with the s t a t i o n a ry phase, detection UV 254 nm. Peaks: 1. quinidine; 2, dihydroquinidi ne. F i g . 5.10. Analysis quinine i n lemon toni c30 Column Lichrosorb RP8 10 pm (150x3 mn I D ) , mobile phase 25% propanol and 0.01% sodium dodecylsulfate in 0.025 M sodium s u l f a t e (pH 6.4), detection UV 243 nm. Peak: 1, quinine. 10 pl injection of mixture of 1 ml tonic diluted with 2 ml eluent.

?

-

min5

l

I

3 2 1 0

L

i I

0

.

0

k ,

L

.

,

.

8min

,

l

2

.

,

L min

Fig 5.11. Separation o f some Cinchona alkaloids6 Colum Lichrosorb Si60 10 wm ( 2 5 0 ~ 1 . 8mn ID), mobile phase tetrahydrofuran - 0.2% concentrated ammonia, flow r a t e 1 ml/min, detection UV 254 nm. Peaks: 1, cinchonine and cinchonidine; 2 , quinidine; 3, quinine; 4, dihydroquinidine; 5, dihydroquinine. (Reproduced with permission from r e f . 6 , by the courtesy of the Canadian Pharmaceutical Association)

279

I

a

I

b

23

I

O

T

L

I

-

8 min

I

1

'

I

2

'

I

I

0

L

I

I

I

8

12

16

rnin

l

l

,

,

I

I

0

L

8

12

16

20

min

F i g . 5.12. A n a l y s i s q u i n i d i n e i n plasma 27 Column P a r t i s i l 10 m ( 2 5 0 ~ 4 . 6 mn I D ) . m o b i l e phase methanol - 0.75% c o n c e n t r a t e d ammonia, f l o w r a t e 1.1 ml/min, d e t e c t i o n UV 236 nm. Peaks: 1, q u i n i d i n e ; 2, d i h y d r o q u i n i d i n e ; 3, s t r y c h n i n e ( i n t e r n a l s t a n d a r d ) . (Reproduced w i t h p e r m i s s i o n f r o m r e f . 27, by c o u r t e s y o f J o u r n a l Chromatographic Science). F i g . 5.13A. S e p a r a t i o n o f q u i n i d i n e and m e t a b o l i t e s i n a t e s t Column Micropak S i l o ( 2 5 0 ~ 2 . 1mm ID), m o b i l e phase hexanes - e t h a n o l - ethanolamine (91.5: 8.47:0.03), f l o w r a t e 1 ml/min, d e t e c t i o n UV 235 nm. Peaks: 1, q u i n i d i n e ; 2, d i h y d r o q u i n i d i n e ; 3, 2 ' - q u i n i d i none; 4, p r i m a q u i ne; 5, q u i n i d i ne-N-oxi de ; 6, 3-hydroxyqui n i d i ne. F i g . 5.136. C o n d i t i o n s and peaknumbering as i n F i g . 5.13A, e x c e p t f o r t h e r a t i o o f t h e m o b i l e phase, which i s (92.97:7.0:0.03). A n a l y s i s i n plasma.

A

C

B

1

---

0

2

4

60

2

min

References p. 273

L O

2

L

F i g . 5.14. A n a l y s i s 2 2 u i n i d i n e and d i h y d r o q u i n i d i n e i n plasma Column P a r t i s i l 10 urn ( 2 5 0 ~ 4 . 6mm I D ) , mob i l e phase methanol - 1 M ammonium n i t r a t e 2 M ammonia (27:2:1), f l o w r a t e 1.2 ml/min, d e t e c t i o n UV 280 nm. Peaks: 1, Q u i n i d i n e ; 2, d i h y d r o q u i n i d i n e ; 3, c i n c h o n i d i n e . (reproduced w i t h p e r m i s s i o n from r e f . 25, by t h e c o u r t e s y o f C l i n i c a l C h e m i s t r y )

-

280

1

h I

30

iI

I

10

20

1

0

min 51 Fig. 5.15. Separation Cinchona a l k a l o i d s Column Lichrosorb Si60 5 vn ( 1 5 0 ~ 4 . 6mn I D ) , mobile phase chloroform - isopropanol - d i e t h y l f l o w r a t e 1.2 ml/min, d e t e c t i o n UV 312 nm. Peaks: 1, q u i n i d i amine - water (940:57:1:2.65), none; 2, epiquinidine; 3, epiquinine; 4 , q u i n i d i n e ; 5, cinchonine; 6, cinchonidine; 7, quinine; 8, d i hydroqui n i d i ne; 9, d i hydroqui n i ne.

P

TABLE 5.2

a B

HPLC ANALYSIS OF VARIOUS COMPOUNDS INCLUDING CINCHONA ALKALOIDS

a B ?

N

COLUMN DIM. LxID mn

AIMS

STATIONARY PHASE

Q,C,opium a l k a loids,cocaine, caffeine

Separation by means o f dynamic c o a t i n g HPLC

C o r a s i l I and 11, dynamically coated w i t h Poly 6-300 (2%)

Q,Qd .C .Cd ,20 o t h e r a1 k a l o i d s

Analysis alkaloids

Merckosorb Si60,5

Q,C ,opium-,

Separation on ion-exchange r e s i n s (1igand-exchange LC)

ALKALOIDS*

OTHER COMPOUNDS

w

tropane a l k a l o i d s s t r y c h n i ne,nicotine

urn

Heptane-EtOH( 1 0 t l ) w i t h d i f f e r e n t percentage o f 1,2 saturation with the stationar y phase Poly 6-300 CHCl3-!kOH(9:1) ,(8:2),(7:3) Et20-!kOH(8:2),(7:3).(6:4)

Hydrolyzed Por e l PT, loaded w i t h Cu Bio-Rad PC20,l oaded w i t h Cu2+

470~6.3 470~6.3

0.06M N b O H i n 33% EtOH 0.2M NH40H i n 33% EtOH 0.05M NH40H i n 33% EtOH 0.03M NH40H i n 33% EtOH

250x3

CHCl3-MeOH-NH40H( 81.5: 18:O. 5)

99

Q u a n t i t a t i v e a n a l y s i s opium a1 k a l o i d s

Spherisorb s i l i c a . 5 ~ m

Q,Qd,C,brucine, s t r y c h n i n e , emet i n e , reserpine, yohimbi ne ,scopo1ami ne

D e t e c t i o n w i t h conductance detector

S i l i c a g e l 10 urn

5

9,?2 32

CHCl3-MeOH-hexane (7:3: 10)

33 Santoni ne

E f f e c t s o l v e n t composition on L i c h r o s o r b RP2, 10 urn r e t e n t i on

120~3.5

MeOH-HzO (1:4), (2:3), (3:Z) (4:l) MeOH

Separation on s t y r e n e - d i v i n y l - H i t a c h i g e l 3010, benzene polymer (Table 8.4) 10 m

220~4.6

ACN-O.02M NH OH(3:2) ACN-O.02M t e q r a b u t y l amnoniumhydroxide (3:7), (3:2)

R e t e n t i o n behaviour b a s i c drugs i n i o n - p a i r HPLC

300x4

0.005M h e p t a n e s u l f o n i c a c i d i n H20-MeOH-AcOH(50:49:1) pH 4.0)

250~4.9

Me H-HC1 4- onc.NH40H(1000:3.55. 9.$)(pH 8.25 7i

41

Q,C , a t r o p i ne,

a c r i d i ne ,ephedrine,opium a l k a l o i d s ,reset-p i ne ,s t r y c h nine , y o h i mbi ne

44

Qd,atropine,scoVarious drugs polami n e , c a f f e i ne, codeine,papaverine, ephedrine

@Ayarjousa l 01 s

REF.

300x2

C,opium a l k a l o i d s

Cd ,codei ne,narceine.brucine, colchicine,aconitine,caffeine

lOO0xl

MOBILE PHASE

F l u r a z am var i o u s %ugz

Se a r a t i o n b a s i c d r u s w ' t h nol-aqueous i o n i c so9venI.s

*For a b b r e v i a t i o n s see f o o t n o t e Table 5.6

VBondapak C18 VBondapak Phenyl ,,Bondapak CN ,,Bondage1 Chromegabond C8 Chromegabond C 6 H l l Spherisorb S5W s i l i c a

53

5

TABLE 5.3

3 .l N m

HPLC ANALYSIS CINCHONA ALKALOIDS I N PLANT MATERIAL OR NATURALLY OCCURRING MIXTURES ALKALOIDS *

AIMS

Q ,Qd ,C ,Cd ,HQ,HQd

Separation and d e t e r m i n a t i o n L i c h r o s o r b Si60 10

Q ,Qd ,C ,Cd ,HQ,HQd

Separation and d e t e r m i n a t i o n pBondapak C18

STATIONARY PHASE

COLUMN D I M LxIO mm pm

REF.

250~1.8

THF-25% NH OH(99.8:0.2).(99.6:0.4), (99.5 :O

6

300x4

MeOH-H20-A~OH(25:75:1),(20:80:1)

50

150~4.6

CHCl3-isoPrOH-H20-DEA( 940:57 :2.65 :1)

Q,Qd ,C,Cd ,HQ,HQd, Separation ( F i g .5.15) e p i Q,epi Qd,qui n i d i none

L i c h r o s o r b Si60 5

Q,C,HQ

Coupling LC-MS

Partisil 5

pm

n o t given

Q ,Qd ,C ,Cd

Separation a l k a l o i d s from plant material o r c e l l cult u r e s ( F i g . 5.8)

Spherisorb CN

250~4.6

pm

MOBILE PHASE

.t)

51

CHC13-ACN-aq.NH4DH(75:24.5:0.5)

54

ACN-MeOH-THF-0.0068 M phosphate b u f f e r ( p H 7)(17:28.7:3.3:50)

72

TABLE 5.4 HPLC ANALYSIS CINCHONA ALKALOIDS I N PHARMACEUTICAL PREPARATIONS ALKALOIDS *

OTHER COMPOUNDS

AIMS

STATIONARY PHASE

Q,Qd ,C ,Cd ,HQ, HQd

A n t i p y r i ne

Determination i n pharmaceut i c a l p r e p a r a t i o n s (Fig.5.11)

L i c h r o s o r b Si60 1OVm 2 5 0 ~ 1 . 8

THF-25% NH DH(99.8:0.2), (99.6 :O .4)499.5 :O .5)

Q,Qd,HQd,xanthiVarious drugs nes ,strychnine, tropane a1 k a l o i d s , codeine,papaverine,ephedrine

A n a l y s i s o f pharmaceuticals

P a r t i s i l 10 i ~ n

250~4.6

CH Clp MeOH(1:3) w i t h 1% z 9 i NH&

Q,opium-,tropSulfanilamide, ane a l k a l o i d s , phenytoine,phenostrychnine,cafbarbital f e i n e ,aconi t i n e , emeti ne,cephaeline

I d e n t i f i c a t i o n i n pharmac e u t i c a l s (Fig.7.14)

P a r t i s i l PXS 5/25

250~4.6

Et20 s a t w i t h 50-100% H20+ 0.05-0.8% DEA

Qd ,HQd

P u r i t y c o n t r o l Qd, raw m a t e r i a l ,and pharmaceuticals

,,Bondapak C18

300~3.9

MeOH-H20-AcOH(25:71:4)

*

Theobromine

COLUMN DIM. LxIO m

MOBILE PHASE

REF.

6

20

40

For a b b r e v i a t i o n s see f o o t n o t e Table 5 . 6

42

Q,Qd,C,Cd,HQ,HQd

Theophylline

P u r i t y c o n t r o l ,Q and Od, raw m a t e r i a l s , and pharmaceut i c a l s fFia.5.1)

UBondapak C18

300x4

&OH-H O-AcOH(25:75:1), (20 :8b :1) 50

~

~~~

TABLE 5.5 HPLC ANALYSIS CINCHONA ALKALOIDS I N BIOLOGICAL MATERIAL ALKALOIDS

*

COLUMN DIM. MOBILE PHASE LxID mn

REF.

AIMS

STATIONARY PHASE

Determination i n plasma, using ion-pair p a r t i t i o n HPLC (Fig.5.9)

L i c h r o s o r b Si100, 10 rn coated w i t h 35% 0.2M HC1044.8M NaC104

150 o r 200~4.5

n-BuOH-CH2C12-n-hexane T1:7:2)sat. w i n t h e s t a t i o n a r y phase

Determination i n blood

L i c h r o s o r b Si60

250x2

0.02M t o 1 uenesul f o n i c a c i d - 0.05M(NHq)H2P04 (pH 3)+40% MeOH

16

Determination i n b l o o d

Phenyl /Corasi 1

1000x2.5

Gradient e l u t i o n A 0.05M H3P04 c o n t a i n i n g 0.5M (NH4)2SO4 B MeOH &-adient:100% A t o A:B (67: 33)

17

Qd,HQd ,theobromine, 30HQd

D e t e r m i n a t i o n i n serum

pBondapak C18

300x4

MeOH-H O-AcOH(25:71:4) (PH 3.6)

Qd s HQd ,Cd 3 3OHQd,Cud, 2'-quinidinone

Determination i n serum ( F i g .5.2)

pBondapak C18

300x4

ACN-2.5% aq. AcOH(12:88)

D e t e r m i n a t i o n i n serum

UBondapak Phenyl

300~3.9

ACN-0.75M NaOAc b u f f e r (pH 3.6) (2:3)

Determination i n plasma (Fig.5.13)

Mikropak S i 10

250~2.1

Hexanes-EtOH-ethanolamine

Determination i n plasma ( F i g . 5.14)

P a r t i s i l 10

250~4.6

MeOH-1M NHqN03-2M NH40H (27: 2 :1)

25

Determination i n plasma ( F i g .5.12)

P a r t i s i l 10

250~4.6

MeOH-conc. NH40H (99.25 :O .75)

27

Qd,Q,2' -qui n i d i none,HQd

OTHER COMPOUNDS

18,23

21.56 Various drugs

Qd,HQd,30HQd, 2 ' - q u i n i d i none Qd N-oxide , primaquine Qd ,Cd, HQd ,Q Qd,HQd,Z'-quinidinone,strychnine

14.29

22

(91.5:8.47:0.03), (92.97 :7 .O :0.03) 24,38

Various drugs

h)

*For a b b r e v i a t i o n s see f o o t n o t e Table 5.6

m

w

N P W

Qd,HQd.2 ' -quini dinone,30HQd

Qd .HQd.C

Determination i n plasma, using ion-pair extraction Determination i n plasma

Lichrosorb Si60,

250x3

CH~Cl2-hexane-MeOH-70% HC104 (60:35:5.5:0.1)

P a r t i s i l lOSCX

250~4.6

&OH-0 .OO 1M tr imet hyl ami ne and 0.001M KOH i n H20 (pH ca 9)(4:1) 31

Determination i n plasma (Fig. 5.6)

VBondapak C18

300~3.9

ACN-methanol-1% aq.(NH4) CO3 (65:31:4)

Determination i n serum

Micropak MCH-10

250~2.1

0.01M KH PO4 i n H20 containing 0.8f% H3P04 and 10% 35 MeOH

5wn

28

34

Qd sHQd 930HQd 9 2'-quinidinone, Qd N-oxide

Oxprenolol

Determination i n urine (Fig. 5.4)

PBondapak Phenyl

300~3.9

ACN-THF-O.05M phosphate buffer(pH 4.5)(15:5:80)

Q,xanthine a1 kaloids

Various drugs

I d e n t i f i c a t i o n i n serum

P a r t i s i l - 1 0 ODS

250~4.6

&OH-0.049M H3P04,0.049M KH2P04 b u f f e r (pH 2.15) (2:3)

Qd

Disopyramide, 1idocai ne p-chl orodisopyramide

Determination i n serum

fiondapak C18

300x4

ACN-O.03M KH2P04 b u f f e r (pH 4.45)(28:72)

D i sopyrami de ,

Determination i n plasma (Fig. 5.5)

aondapak Phenyl

300x4

ACN-0.0015M aq H3P04(1:9)

Separation and i s o l a t i o n Q and Qd metabolites from r a t urine

J3ondapak C18

300~3.9

Gradient e l u t i o n A H~O-ACOH (99:l) B H -ACN-AcOH(40:59:1) g r a g e n t A:B(9:1) t o (15:85) gradient A :B( 9 :1)t o ( 2 :8), solvent 8 contained 0.1% THF 47

36

45

procainamide, N-acetyl procainamide Q,Qd > HQ, HQd9 Cu 9 Cud ,30HQd ,OHQ, Q-10,lldihydrodiol Qd-10,lldihydrodiol,2'-quinidinone,2'-quininone Qd

Qd,HQd,30HQd, 2'-quinidinone, Qd N-oxide

37

46

300~7.8

D i sopyramide ,pmcainamide, tocai n i de, 1ignocai ne, p h l orodi sopyram i de Pronethanol

-

Determination i n plasma

Lichrosorb RP8, 10 p

250~4.6

ACN-O.05M phosphate b u f f e r (pH 3.0)(27:73)

Determination i n plasma and urine

,Bondapak

300~3.9

ACN-THF-0.05M phosphate b u f f e r (pH 4.75)(15: 5:BO)

48 Phenyl

49

P N 0

Qd,HQd ,7' -trifluoromethyldihydrocinchonidine

Determination i n plasma and urine

P a r t i s i l , 10 m,,

250~4.6

CH C12-MeOH-conc.NHqOH (95.5:4:0.5 1

Q.Qd .HQd ,30HQd Qd,Hod, 30HQd

Determination i n plasma

UBondapak C18

300~3.9

ACN-AcOH-H20(10:4:86)

D i r e c t a n a l y s i s o f drugs i n urine

L i c h r o s o r b RP8

150~4.5

ACN-0.095M NaC104,0.005M HC104(1:3)

Qd ,HQd s30HQds 2'-quinidinone, Qd N-oxide, Qd 10.11-dihydrodiol

Determination i n plasma, u r i n e and b i l e (Fig.5.7)

U1t r a s p h e r e C8

150~4.6

ACN-MeOH-THF-O.01M TrEA (5:5:3:87)(pH 2.5 w i t h H3P04)

Qd ,HQd 9 30HQd 9 2'-quinidinone. Cud, p r imaquine

Determination i n plasma

P a r t i s i l 10

250~4.5

MeOH-1M NH4N03-2M NH40H (28: 1:1)

Qd

52 58,67 61

65

66 Disopyramide,monoDetermi n a t i o n i n plasma d e a l k y l a t e d disopyramide D e t e r m i n a t i o n i n serum and u r i n e (Fig.5.3)

Q.Qd,HQd 9 30HQd,Cud

Spheri-5 RP8

n o t specified

ACN-O.05M NaH PO4 b u f f e r (pH 3.00)(2:3$ 68

P a r t i s i l PXS 5/25 ODS

250~4.6

ACN-1M NaH PO - l M NaClO 85% H1P0,-6,0411:2: 1:1: i 5 )

74

TABLE 5.6 HPLC ANALYSIS CINCHONA ALKALOIDS ALKALOIDS

AIMS

BEVERAGES STATIONARY PHASE

COLUMN DIM. LxID nm

MOBILE PHASE

REF.

Q

Determination i n s o f t d r i n k s and aq. s o l u t i o n s

S p h e r o s i l 5 wn

250x3

NeOH-25% NH40H(99.5:0.5)

Q

Analysis i n s o f t d r i n k (Fig.5.10)

L i c h r o s o r b RP8, 10 Lull

150x3

25% PrOH,O.Ol% Na-dodecylsulfate 0.025M Na2S04

Q

Analysis i n s o f t drinks

L i c h r o s o r b RP8, 10 Lull

250~4.6

0.005M Na-dodecyl s u l f o n a t e ACN-H20(3:1).HC1O4 added t o pH 3

43

N u c l e o s i l 5C18

250x4

MeOH- 1M NaNO 1M c i t r i c a c i d - 1M N a - c i t r a t e - &O (64:10:2:3:21)(pH 6 )

75

Q

*

*

IN FOOD AND

Analysis i n s o f t drinks, using electrochemical d e t e c t i o n

15

in 30

Abbreviations used: C=cinchonine, Cd=cinchonidine, Q=quinine, Qd=quinidine. H-=dihydroN

in m

This Page Intentionally Left Blank

281

Chapter 6 PHENYLETHYLAMINES AND ISOQUINOLINE ALKALOIDS

................................................................ .................................................................... ................................................................ ..........................................................................

6.1. Ion-exchange HPLC.. 6.2. Reversed-phase HPLC ................................................................ 6.3. I o n - p a i r HPLC.. 6.4. S t r a i g h t - p h a s e HPLC 6.5. D e t e c t i o n References ..............................................................................

287 287 288 288 290 291

The i s o q u i n o l i n e a l k a l o i d s form a l a r g e group o f compounds c o m p r i s i n g a v a r i e t y o f chemic a l s t r u c t u r e s . HPLC methods have so f a r been a p p l i e d o n l y f o r a few o f them, i . e . m e s c a l i n e

-

as a drug of abuse ( T a b l e 6.1)

-

emetine and c e p h a e l i n e , as w e l l as b e r b e r i n e . The opium

a l k a l o i d s have been d e a l t w i t h s e p a r a t e d l y ( s e e Chapter 7 ) . 6.1. ION-EXCHANGE HPLC Walton3 s t u d i e d t h e a n a l y s i s o f a l k a l o i d s on ion-exchange m a t e r i a l s l o a d e d w i t h m e t a l i o n s (see Chapter 7 ) . McMurtrey e t a1.26 r e p o r t e d on t h e a n a l y s i s o f some dopamine d e r i v e d i s o q u i n o l i n e a l k a l o i d s by means o f ion-exchange HPLC. Three d i f f e r e n t c o m m e r c i a l l y a v a i l a b l e c a t i o n exchange m a t e r i a l s were compared. The e f f e c t o f t h e i o n i c s t r e n g t h , pH and o r g a n i c m o d i f i e r s i n t h e m o b i l e phase were i n v e s t i g a t e d . I t was found t h a t n o t o n l y ion-exchange mechanisms were i n v o l v e d i n t h e r e t e n t i o n o f t h e a l k a l o i d s . The e f f e c t o f t h e pH o f t h e m o b i l e phase on t h e r e t e n t i o n w i s m i n i m a l ; however, above pH 5 . 5 an i n c r e a s e d t a i l i n g was observed. A d d i t i o n o f o r g a n i c m o d i f i e r s decreased t h e e l u t i o n t i m e i n t h e o r d e r n - b u t a n o l

-

acetonitrile

-

-

dioxane

-

isopropanol

e t h a n o l . Increased temperature reduced t h e r e t e n t i o n times o f t h e a l k a l o i d s

and i n c r e a s e d column e f f i c i e n c y . P a r t i s i l as s t a t i o n a r y phase d i f f e r e d i n s e v e r a l a s p e c t s f r o m t h e o t h e r two s t a t i o n a r y phases t e s t e d . Some r e p r e s e n t a t i v e s e p a r a t i o n s a r e p r e s e n t e d i n ( F i g . 6 . 1 and 6 . 2 ) . The a n a l y s i s o f b e r b e r i n e i n crude drugs by means o f p e l l i c u l a r cation-exchange columns have been r e p o r t e d by Akada e t a l . 6.2. REVERSED-PHASE HPLC Q u a n t i t a t i v e a n a l y s i s o f t h e q u a t e r n a r y a l k a l o i d d - t u b o c u r a r i n e i n c u r a r e has been p e r f o r 9 med on an o c t a d e c y l bonded s t a t i o n a r y phase . The i n f l u e n c e o f pH, b u f f e r c o n c e n t r a t i o n , and t h e n a t u r e o f t h e c a t i o n i n t h e m o b i l e phase were i n v e s t i g a t e d . Optimum pH was f o u n d t o be 4. As t h e c a t i o n i n t h e m o b i l e phase, tetramethylammonium gave b e t t e r s e p a r a t i o n t h a n p o t a s s i u m

o r ammonium. The c o n c e n t r a t i o n o f t h e b u f f e r d i d n o t a f f e c t t h e r e t e n t i o n o f t h e a l k a l o i d s ; however, a h i g h e r c o n c e n t r a t i o n gave improved peak shape. To reduce t h e t i m e o f a n a l y s i s a g r a d i e n t e l u t i o n was p r e f e r r e d ( F i g . 6 . 3 ) . Berberine

-

also a quaternary a l k a l o i d

-

has been a n a l y z e d on an o c t a d e c y l column u s i n g 11

a c e t o n i t r i l e - phosphate b u f f e r (pH 5.2) ( 3 : 2 ) as m o b i l e phase

.

S e p a r a t i o n o f A m a r y l l i d a c e a e a l k a l o i d s has been a c h i e v e d on an o c t y l column w i t h methanol w a t e r ( 3 : 2 ) c o n t a i n i n g t r a c e s o f a m n ~ n i a ~G~f e. l l e r e t a1."

References p. 291

d e s c r i b e d t h e a n a l y s i s o f some

-

288

TABLE 6.1 ISOQUINOLINE ALKALOIDS I N THE CONTEXT OF HPLC ANALYSIS OF DRUGS OF ABUSE (CHAPTER 7) A1 kaloids

Ref.

Ref. i n Chapter 7

~~

Mescal i n e Tubocurari ne Tubocurarine Mescaline Cephael ine Mescaline

1

11 21 30 56 61 98.99,lOO

4 7 19 23 34,35,36

-

-

i n c l u d i n g emetine on a chemically bonded d i o l s t a t i o n a r y phase. I t allowed the alkaloids d e s i r a b l e f o r a post-column f l u o r i m e t r i c i o n - p a i r use o f an e x c l u s i v e l y aqueous phase

-

reactor. Davisz4 analyzed glaucine and i t s m e t a b o l i t e dehydroglaucine i n m i c r o b i a l c u l t u r e s . For the q u a n t i t a t i v e analysis on a phenyl-type o f bonded phase, papaverine was used as i n t e r nal standard. The a c e t o n i t r i l e content o f the mobile phase ( a c e t o n i t r i l e potassium dihydrogenphosphate (2:4:5))

-

-

methanol

0.05 M

was found t o be c r i t i c a l f o r t h e r e s o l u t i o n o f t h e a l -

kaloids and f o r t h e peakshape. OeBros and Gissen17 analyzed tubocurarine on an octadeceyl column u s i n g a c e t o n i t r i l e

-

wa-

t e r (18:82) containing 0.2 M p e r c h l o r i c a c i d (pH 5.4) as mobile phase. 6.3.

ION-PAIR HPLC

Bannister e t al.21determined emetine i n plasma w i t h the a i d o f HPLC. A f t e r e x t r a c t i o n from the b i o l o g i c a l f l u i d w i t h dichloromethane the a l k a l o i d was o x i d i z e d w i t h mercuric acetate, y i e l d i n g a fluorescent product. Several a l k y l s u l f o n a t e s were t e s t e d as the p a i r i n g i o n f o r bath emetine and i t s o x i d a t i o n product. An increased a l k y l ’ chain l e n g t h was found t o improve the peak shape. For emetine a mobile phase c o n s i s t i n g o f 0.0025 M octanesulfonate and 0.5% a c e t i c a c i d i n methanol

- water (56:44)

was found s u i t a b l e , i n combination w i t h an octadecyl

column. For the more p o l a r o x i d a t i o n product the r a t i o methanol

- water was

changed t o 3:2.

Several i n v e s t i g a t o r s analyzed quaternary protoberberine a1 k a l o i d s by means o f reversed-phase i o n - p a i r HPLC28’30*39. Hashimoto e t a1 .30 reported the use o f micromanipulators t o t r a n s f e r the a l k a l o i d s o f a p l a n t c e l l t o t h e HPLC system. The a l k a l o i d s were subsequently separated on an o c t y l bonded s t a t i o n a r y phase w i t h t h e mobile phase a c e t o n i t r i l e furan

- 0.1 N t a r t a r i c a c i d - sodium dodecylsulfate (20:20:59.5:0.5).

-

tetrahydro-

A s i m i l a r solvent has

been employed f o r the analysis o f berberine. palmatine and c o p t i s i n e i n p l a n t m a t e r i a l , though 39 the tetrahydrofuran was changed f o r methanol and the r a t i o o f the s o l v e n t was d i f f e r e n t Meulemans e t a1.33 determined tubocurarine i n plasma w i t h the a i d o f an HPLC system con-

.

s i s t i n g o f an octadecyl column i n combination w i t h t h e mobile phase methanol

-

containing triethylamine, phosphoric a c i d and pentanesulfonic a c i d (pH 3.4).

Parkin3‘

water (2:3) used

tubocurarine as i n t e r n a l standard i n the analysis o f alcuronium i n plasma. The a l k a l o i d s were separated on an octadecyl bonded phase with the mobile phase methanol

0.25% a c e t i c a c i d and 0.005 6.4.

M

- water

(4:l) containing

dodecylsulfate.

STRAIGHT-PHASE HPLC Emetine and cephaeline can be analyzed on s i l i c a g e l w i t h chloroform

- methanol

as mobile

289

TABLE 6.2 LIQUID CHROMATOGRAPHIC DATA FOR CACTACEAE ALKALOIDS AND RELATED C0MPOUNDSl4. (3DOx4.5 n ID), Column Lichrosorb Si60, 10 vm ( 3 0 0 ~ 4 . 5mn ID) i n s e r i e s w i t h VPorasil. 8 mobile phase 51 a c e t o n i t r i l e conc. a n o n i a (96:4). f l o w r a t e 2.0 ml/min and 52 chloroform 1%conc. ammonia i n methanol ( 9 : l ) . f l o w r a t e 1.0 ml/min, d e t e c t i o n UV 254 nm.

-

-

A1 k a l o i d

Retention time (min) and k ' s1 52 k' tr k'

Phene t h y 1 amines

3-Hydroxy-4-methoxyphenethyl ami ne 4-hydroxy-3-methoxyphenethyl ami ne

23.2 14.4

6.48 3.64

Sal s o l ine (6-hydroxy-7methoxy-l-methyl-) 15.0 I s o s a l s o l i n e (7-hydroxy-6-methoxy-1-methyl-) 17.5 Arizonine (8-hydroxy-7-methoxy-1-methyl-) 11.7 0-Methylcorypal l i n e (N-methyl-6.7-di methoxy-) 5.2

3.69

T et r a h y d r o i s o q u i n o l i n e s

**

-*.

-

-**

2.66

- ** ** -

0.63

11.1

0.79

3.9 7.7 7.6 3.9

0.22 1.31 1.48 0.22

8.8 33.9 31.8 9.0

0.42 4.38 4.00 0.45

3.9

0.22

8.0

7.4

1.31

29.0

3.60

7.1

1.22

22.8

2.70

10.2 8.3 5.4

2.19 1.59 0.69

41.5 37.4 17.8

5.59 4.94 1.82

4.47

N-Methyl-7,8-dimetboxy-l.2,3.4-tetrahydroisoquinol i n g * N-methylsalsoline N-methyl isosal sol ine Hydrocotarnine (N-methyl-8-methoxy-6.7-methyl enedioxy-) N-Methyl-6-methoxy-7 ,&methyl ene@ oxy-1,Z ,3,4- t e t r a h y d r o i soqui no1 i n e P e l l o t i n e (1,2-dimethyl-8-hydroxy-6.7-dimethoxy-) Gigantine (1,2-dimethyl-5-hydroxy-6,7-dimethoxy-) 6,7-Dimethoxy-l,2,3,4-tetrahydroi soqui no1 ine S a l s o l i d i n e (6,7-dimethoxy-l-methyl-) Carnegine ( 1 ,2-dimethyl-6,7-dimethoxy-)

*:

not identified i n cacti tr >40 min

Fkferences p. 291

0.3

290

With t h e a d d i t i o n o f

o r diethylamine4'

t h i s s o l v e n t system has been used

f o r the analysis o f these a l k a l o i d s i n Ipecac p l a n t m a t e r i a l . F r e i e t a l . 5'6s18 reported the analysis o f some a l k a l o i d s i n c l u d i n g emetine and cephaeline as dansyl d e r i v a t i v e s on s i l i c a gel (Fig.7.15).

A series o f i s o q u i n o l i n e a l k a l o i d s and phenylethylamine d e r i v a t i v e s from Cactaceae has been analyzed on s i l i c a gel14 (Table 6.2). A d d i t i o n o f a l k a l i t o the mobile phase was found t o r e duce t a i l i n g . The system decsribed cou1.d a l s o be used f o r semipreparative purposes. Mescaline, as w e l l as a series o f other s t i m u l a n t drugs w i t h a phenylethylamine s t r u c t u r e . has been analyzed as ~-naphtoquinone-4-sulfonated e r i v a t i v e s on a s i l i c a gel column. I n the analysis o f urine, i n t e r f e r i n g peaks were n o t observed25. The anti-tumor a l k a l o i d t h a l i c a r p i n e and some r e l a t e d a l k a l o i d s could be separated on s i l i c a gel15 (Fig.6.4)

a f t e r e x t r a c t i o n from

urine.

6.5. DETECTION Isoquinoline a l k a l o i d s are u s u a l l y detected a t 254 and 280 nm w i t h s u f f i c i e n t s e n s i t i v i t y . However t o increase the s e n s i t i v i t y o f the analysis o f tubocurarine i n plasma, deBros and 17 Gissen preferred 204 nm above 280 nm, because o f the t e n f o l d enhancement o f the UV-absorption a t the former wavelength. To improve the s e n s i t i v i t y and the s e l e c t i v i t y o f the d e t e c t i o n o f some alkaloids, F r e i e t a1 .5'6s18 prepared dansyl d e r i v a t i v e s . Emetine and cephaeline coupled with, respectively, one and two dansyl groups enabled f l u o r i m e t r i c d e t e c t i o n (see Chapter 7 f o r r e a c t i o n conditions, and Fig.7.15). Post-column fluorescent i o n - p a i r formation has been used t o improve the s e n s i t i v i t y and 20 o f emetine (Chapter 4).

s e l e c t i v i t y o f the detection, i.a.,

A more than 50-fold increase i n s e n s i t i v i t y i n t h e a n a l y s i s o f emetine i n plasma was achieved by precolumn o x i d a t i o n o f the a l k a l o i d w i t h 1%mercuric acetate i n a c e t i c a c i d aqueous sodium hydroxide

-

-

35 %

96 % ethanol (73:27:900) a t room temperature and w i t h one hour

r e a c t i o n time, i n combination w i t h f l u o r i m e t r i c d e t e c t i o n ( e x c i t a t i o n 225 nm, emission 418 nm)21. The fluorescent protoberberine a l k a l o i d s have been detected f l u o r i m e t r i c a l l y using ex39 c i t a t i o n a t 350 nm and measuring emission a t 520 nm , Endo e t a1 .25 reported a precolumn d e r i v a t i z a t i o n method w i t h sodium 1?-naphtoquinone-4-~~1fonate f o r stimulant amines. Detection o f the coloured d e r i v a t i v e s a t 450 nm increased the s e n s i t i v i t y about 25-fold, when compared w i t h UV-detection o f the underivatized compounds.UV detection o f the coloured d e r i v a t i v e s a t 245 and 280 nm was a l s o more s e n s i t i v e

-

2.4 and 3.7-

f o l d increase i n s e n s i t i v i t y r e s p e c t i v e l y . However, i n t e r f e r i n g peaks made i t l e s s s u i t a b l e f o r the analysis o f u r i n e e x t r a c t s . The r a t i o o f UV-absorbance a t 245 and 280 nm has been used as a f u r t h e r c h a r a c t e r i s t i c feature i n combination w i t h the r e t e n t i o n times f o r the i d e n t i f i c a t i o n o f various drugs 19 cluding mescaline

.

-

in-

An electrochemical d e t e c t i o n method has been a p p l i e d t o the analysis o f some i s o q u i n o l i n e alkaloids16y26. McMurtrey e t a1.26 found the method about 1000 times more s e n s i t i v e than UV detection a t 280 nm. Selective detection o f Amaryllidaceae a l k a l o i d s i n the low ug range w i t h a c i r c u l a r d i chroism spectrophotometer has been described by Westwood e t a l .31. The method o f f e r s a h i g h degree o f s e l e c t i v i t y f o r o p t i c a l l y a c t i v e a l k a l o i d s w i t h s u i t a b l e chromophores, since the

291 wavelength o f d e t e c t i o n can be varied. CD-active compounds can be detected i n complex mixtures o f other compounds without i n t e r f e r e n c e . Coupling o f LC w i t h mass-spectrometry has been reported f o r the analysis o f Amaryllidaceae a l k a l o i d s

29

.

REFERENCES

1 M.L. Chan, C. Whetsell and J.D. McChesney, J . C h r o m a t o g r . s c i . , 12 (1974) 512. 2 R. Verpoorte and A. Baerheim Svendsen, J . C h r o m a t o g r . , 100 (1974) 227. 3 H.F. Walton, J. Chromatogr., 102 (1974) 57. 4 P.J. Twitchett and A.C. Moffat, J . C h r o m a t o g r . , 111 (1975) 149. 5 R.W. F r e i and W. Santi, z. A n a l . C h e m . , 277 (1975) 303. 6 R.W. Frei. W. Santi and M. Thomas, J. C h r o m a t o g r . , 116 (1976) 365. 7 P.J. Twitchett, A.E.P. Gorvin and A.C. Moffat. J . C h r o m a t o g r . . 120 (1976) 359. 8 K. Hostettmann, M.J. P e t t e i , I. Kubo and K. Nakanishi, Helv. Chim. dcta. 60 (1977) 670. 9 F.P.B. van der Maeden, P.T. van Rens and F.A. Buytenhuys. J . C h r o m a t o g r . . 142 (1977) 715. 10 Y . Akada and T. Tanase, Yakugaku Z a s s h i , 97 (1977) 940. 11 T. H a t t o r i , N. Kamiya, M. Inoue and M. Hayakawa, Yakugaku Z a s s h i . 97 (1977) 1305. 12 V. Quercia. B. Bucci. C. Tela, M. Terracciano and N. P i e r i n i , ~ 0 1 1 .Chim. Farm., 117(1978)545 13 Y. Akada and Y . Kato, Herba p o l . , 24 (1978) 199. 14 J. Strombom and J. Bruhn, J . C h r o m a t o g r . . 147 (1978) 513. 15 M. Smellie, M. Corder and J.P. Rosazza, J . C h r o m a t o g r . , 155 (1978) 439. 16 Y. Hashimoto, M. Moriyasu. E. Kato, N. Miyamoto and H. Uchida, M i k r o c h i m . dcta, 2 (1978) 159. 17 F. DeBros and A.J. Gissen. A n e s t h e s i o l o g y , 51 (1979) 5265. 18 R.W. Frei, J. C h r o m a t o g r . , 165 (1979) 75. 19 J.K. Baker, R.E. Skelton and Ch.Y. Ma, J. C h r o m a t o g r . , 168 (1979) 417. 20 J.C. G f e l l e r , G. Frey. J.M. Huen and J.P. Thevenin. J . C h r o m a t o g r . , 172 (1979) 141. 21 S.J. Bannister, J. Stevens. 0. Musson and L. Sternson, J . C h r o m a t o g r . , 176 (1979) 381. 22 R. Gimet and A. F i l l o u x . J . C h r o m a t o g r . , 177 (1979) 333. 23 M.W. White, J. C h r o m a t o g r . . 178 (1979) 229. 24 P.J. Davis. J. C h r o m a t o g r . , 193 (1980) 170. 25 M. Endo, H. Imamichi. M. Morayasu and Y. Hashimoto. J . C h r o m a t o g r . , 196 (1980) 334. 26 K.D. McMurtrey, J.L. Cashaw and V.E. Davis, J . L i q . C h r o m a t o g r . , 3 (1980) 663. 27 R.V. Smith, A.E. K l e i n and 0.0. Thompson, M i k r o c h i m . d c t a . (1980) 151. 28 Y. Akada, S. Kawano and Y. Tanase, Yakugaku Z a s s h i . 100 (1980) 766. CA, 93 (1980) 245588~. 29 C. Eckers, D.E. Games, E. Lewis, K.R.N. Rao, M. Rossiter and N.C.A. Weerasinghe. i n A. P u a y l e (Editor), A d v a n c e s i n Mass S p e c t r o m e t r y , V o l . 8 , H e y d e n , London, 1980. p.1396. 30 Y . Hashimoto, K. Kawanishi, H. Tomita, Y. Uhara and M. Moriyasu, A n a l . L e t t . , 14 (1981) 1525. 31 S.A. Westwood, D.E. Games and L. Sheen, J . C h r o m a t o g r . . 204 (1981) 103. 32 J.E. Parkin, J. C h r o m a t o g r . , 225 (1981) 240. 33 A. Meulemans. J. Mohler. 0. Henzel and Ph. Duvaldestin, J. C h r o m a t o g r . , 226 (1981) 255. 34 I.S. L u r i e and S.M. Demchuk. J. L i g . C h r o m a t o g r . , 4 (1981) 337. 35 I.S. L u r i e and S.M. Demchuk, J . L i q . C h r o m a t o g r . , 4 (1981) 357. 36 1.S. Lurie, J. L i q . C h r o m a t o g r . , 4 (1981) 399. 37 E. Merck, Darmstadt, R e a g e n z i e n , Flussigkeitschromatographie u n t e r Druck, p. 71-3. 38 G.C. Subba and F. Sandberg, d c t a Pharm S u e c . , 19 (1982) 293. 39 T. Misaki, K. Sagara. M. Ojima, S. Kakizawa, T. Dshima and H. Yoshizawa. Chem. Pharm. B u l l . , 30 (1982) 354. 40 N.P. Sahu and S.B. Mahato, J . C h r o m a t o g r . . 23 (1982) 525.

292

6

a C

0

0 N I

INJ

INJ.

f 0

,

L

.

6

1

0

.

,

.

,

10 min

.

12

,

,

.

14

1

.

16

r

I

10

0

4

Fig. 6.1. Separation o f some dopamine derived i s o q u i n o l i n e a l k a l o i d s26 Column Nucleosil SA lOpm(250x3.2m ID).mobile phase 0.5 M NH H PO t 5% n-butanol, f l o w r a t e 1.0 ml/min. detection UV 280 nm. Peaks: 1, dopamine; 2, SalSh?nof; 3, Tetrahydropapaveroline; 4, 2.3,10,1l-tetrahydroxyberbi ne; 5, 2.3,9,lO-tetrahydroxyberbine and 3 ' -0-methyl tetrahydropapaverol ine; 6, 7-0-methyltetrahydropapaverol ine; 7. 4'-O-methyl tetrahydropapaveroline; 8, 6-0-methyl tetrahydropapaveroline; 9, 2-0-methyl tetrahydroxyberbine; 10, 11-0-methyl t e t r a h y droxyberbine and 10-0-methyltetrahydroxyberbine. Fig. 6.2. Separation o f some dopamine derived i s o q u i n o l i n e a l k a l o i d s26 Column Vydac TP 401 SCX 10 p m ( 2 5 0 ~ 3 . 2mn ID), mobile phase 0.2 M NH H PO4, f l o w r a t e 0.5 ml/min, column temperature 51OC. d e t e c t i o n UV 280 nm. Peaks: 1, dopalhize; 2, s a l s o l i n o l ; 3, tetrahydropapaverol ine; 4. 2,3,10,11-tetrahydroxyberbine; 5, 3'-O-methyl tetrahydropapaverol i n e ; 6, 2.3,9,10-tetrahydroxyberbine; 7, 7-0-methyltetrahydropapaveroline; 8. 4'-O-methyltetrahydropapaverol ine; 9, 6-0-methyl tetrahydropapaverol ine; 10, 2-0-methyl tetrahydroxyberbine; 11, 11-0-methyl tetrahydroxyberbine; 12, 10-0-methyl tetrahydroxyberbine; 13, 3-0-methyl tetrahydroxyberbine. (Fig. 6.1 and 6.2 were reproduced w i t h permission from r e f . 26, by courtesy o f Marcel Dekker, I n c . ) . 1

Fig. 6.3. HPLC analysis o f curare sample' Precolumn Corasil C18 ( 5 0 ~ 4 . 6mn ID), column p8ondapak C18 (300x4 mn I D ) , mobile phase A 0.025 M tetramethylammonium phosphate i n methanol - water (1:3)(pH 4), 8 0.025 M t e t r a methylamnonium phosphate i n methanol water ( 9 : l l ) ( p H 4), l i n e a r g r a d i e n t A t 8 ( 9 : l ) t o (15:85) i n 30 min, f l o w r a t e 1 m l h i n , d e t e c t i o n UV 280 nm. Peaks: 1, d-tubocurarine; 2, chondrocurine; 3, curarine; 4, isochon&-odendrine; 5. curine.

-

l

0

.

l

,

,

10 20 min

.

,

30

293

13

,

I

0

, 10

I

, 20

I

, 30

,

, LO

volume (ml) Fig. 6.4. Separation o f some bisbenzylisoquinoline alkaloids15 Column UPorasil (300x4 mn ID), mobile phase cyclohexane - chloroform - diethylamine (25:150: 0.3), f lo w rate 2.3 ml/min. detection UV 254 nm. Peaks: 1, thalicarpine; 2, hernandalinol; 3, hernandaline; 4, dehydrothalicarpine.

Referencesp. 291

TABLE 6.3 HPLC ANALYSIS OF VARIOUS ALKALOIDS INCLUDING ISOQUINOLINE ALKALOIDS ALKALOIDS

AIMS

STATIONARY PHASE

Emetine.cephaeline, 22 o t h e r a l k a l o i d s

Analysis alkaloids

Merckosorb Si60. 5~

300x2

CHCl -MeOH(9:1),(8:2),(7:3) Et202MeOH(8: 2), ( 7 :3), (6:4)

2

Mescaline,opium a l k a l o i d s ,

Separation on ion-exchange r e s i n s ( 1 i g a n d exchange LC)

Hydrolyzed Porpqel T loaded w i t h Cu Bio-Rad PC20,loaded w i t h Cu++

470~6.3

0.06M 0.2 M 0.05M 0.03M

3

quinine.cinchonine,strychnine. n i c o t i ne,atropi ne,cocaine

Emetine,Cinchona a l k a l o i d s , D e t e c t i o n w i t h conducscopolarnine,brucine,strych- tance d e t e c t o r n i ne,reserpine ,yohimbi ne. caffeine

COLUMN DIM. L x I 0 (m)

470~6.3

NH OH NH40H NH40H NH:OH

in in in in

REF.

33% EtOH 33% EtOH 33% EtOH 33% EtOH

CHCl 3-MeOH-hexane (7: 3: 10)

S i l i c a g e l 10 fl

16

Dopamine,salsolinol,6-OMe-. Analysis doparnine d e r i v e d P a r t i s i l 10 SCX 7-0&,3'-OMe,4'-OMe-tetraisoquinoline alkaloids hydropapaverol ine,2.3,9,10(Fig.6.1.6.2) t e t r a h y d r o x y b e r b i ne.2.3,lO ,11-tetrahydroxyberbi ne and i t s 11-OMe d e r i v a t i v e s

Vydac TP 401 SCX, 10 N u c l e o s i l SA, 10

Emeti ne.cephae1 ine

L i c h r o s o r b Si60, 5 fl

Separation

MOBILE PHASE

pin

250~4.6 250~3.2 250~3.2

0.1M NH H PO 0.2M NH4H2P04 0.5M NHH :P : O+ :5%

200x2

CHC13-MeOH(85:15)

n-BuOH 26 37

TABLE 6.4 HPLC ANALYSIS ISOQUINOLINE ALKALOIDS I N PLANT MATERIAL ALKALOI OS

AIMS

STATIONARY PHASE

D ihydrochelerythrine, N-methylflindersine

P r e p a r a t i v e HPLC p l a n t extracts

S i l i c a gel

300x50

E t O-hexane(l:g) EtaAc-hexane( 1:4)

8

aondapak C18

300x4

0.025M tetramethylamnonium phosphate i n A. MeOH-H20(1:3) pH 4, B. MeO-H O ( 9 : l l ) pH 4, l i n e a r g r a d i e n t A+B(9:1) f.0(3:17)

9

Tubocurarine,chondrocurine. Analysis c u r a r e (Fig.6.3) c u r a r i n e , isochondodendrine, curine

COLUMN DIM. LxID(mn)

MOBILE PHASE

REF.

? 2 ;a 0

P P N

2

Analysis crude drugs

Zipax SCX

1000~2.1 A. ACN-O.1M NaClO (3:2) B. 0.2M H BO -0.062M NaC104 in H20(pH 8.5) AtB(7:3) 10,13 ACN-phosphate buffer (pH 5.2)(3:2) UBondapak C18 300x4 Analysis in C o p t i s speBerberi ne 11 cies Analysis in Boldus extract 00s-HC-Si1 250~2.6 MeOH-H20(1:19) 12 Boldine Analysis Cactus alkaloids Lichrosorb Si60. 10 urn 300~4.5 ACN-conc. NH OH 16 Cactus alkaloids and Partisil 8 in CHC13-1% con$. NH40H in MeOH(9:l) (Table 6.2) series 14 Analysis in microbial ,Bondapak Phenyl 300~3.9 ACN-MeOH-O.05M KH2P04(2:4:5) G1 aucine,dehydroglaucine, cultures 24 papaverine Lycorine.ambel1 ine,criglau- LC-MS of C r i n u m alkaloids Spherisorb 00s. 5 vm not given ACN-H20-NH40H(79.3:20:0.3) 29 cine Histochemical chromatogra- Lichrosorb RP8 not given ACN-THF-O.1N tartaric acid-Na dodecylBerberi ne ,palmatine PhY sulfate(20:20:59.5:0.5) 30 MeOH-H 0(3:2) containing a trace of NH40H 150x5 Detection Amaryllidaceae C8 bonded phase Ambelline 2 31 alkaloids with CD-detector Analysis Ipecacuanha uPorasi 1 300~4.5 CHC13-(5% conc. NH40H in MeOH)(95:5) 38 Emetine.cephae1 ine Analysis plant material TSK gel LS-410, 5 um 150x4 ACN-MeOH-O.1N tartaric acid-Na dodecylBerberi ne,palmati ne,copsulfate (40:10:49.5:0.5) 39 tisine Analysis Ipecacuanha uPorasi 1 300~3.9 CHClg-MeOH-DEA(90:10:0.2) 40 Emeti ne,cephael ine

Berberine

TABLE 6.5 HPLC ANALYSIS ISOQUINOLINE ALKALOIDS IN PHARMACEUTICAL PREPARATIONS AL KALOI DS

OTHER COMPOUNDS

Emetine,cephaeline, codeine.morphine,noscapine.ephedrine Ernetine,atropine.di- Pindolol ,guanhydroergotamine,bro- facin.ketotifen. mocryptine.ephedri ne pi zoti fen ,clemastine

AIMS

STATIONARY PHASE

Separation as dansylderivati ves (Fig.7.15)

Silica gel Silo0

Post-column derivatization with fluorescent pairing ion

COLUMN DIM. MOBILE PHASE LxID(mn1

REF.

250~2.8 (isopr) O-isoprOH-conc.NH40H, (48:2:0?3), (isopr) 0 sat. with conc.NHgOH-i soprOH(89: 1) 5,6,18 Lichrosorb DIOL. 10urn 250x4 0.1M phosphate buffer(pH 3 ) Lichrosorb RP8. lOpm 100~4.6 MeOH-0.02M phosphate buffer (PH 3)(3:2) 20

Emeti ne ,cephael ine, opium a l k a l o i d s , various o t h e r s

Sul f a n i lami de. phenytoi ne.phenobarbi t a l

I d e n t i f i c a t i o n pharmaceuticals(Fig.7.14)

P a r t i s i l PXS 5/25

2 5 0 ~ 4 . 6 E t 0 s a t . w i t h 50-100% H20+ 0.85-0.8% DEA

Berberine

Acrinol

Ana 1ys is p ha rmaceu ti ca 1s

Zorbax ODS

250

22 0.005M d o d e c y l s u l f a t e i n ACN-H20(95: 5)

28

TABLE 6.6 HPLC ANALYSIS ISOQUINOLINE ALKALOIDS I N BIOLOGICAL MATERIALS ALKALOIDS ~

~~

OTHER COMPOUNDS

AIMS

STATIONARY PHASE

COLUMN DIM. LxIDfmn)

MOBILE PHASE

REF

~

Thal icarpine,hernandaline.hernandalino1. dehydrothalicarpine

A n a l y s i s i n urine(Fig.6.4)

UPorasi 1

300x4

Tubocurarine.isochondodendrocuri ne Emet i ne

A n a l y s i s i n plasma

pBondapak C18

300x4

Naphtalene

A n a l y s i s i n plasma

pBondapak C18

Various ami nes, amphetamines

A n a l y s i s s t i m u l a n t s i n u r i n e , L i c h r o s o r b Si100, 10,m 250x2 p r e c o l umn d e r i v a t i z a t i on o r Wakogel LC 5H, 5 m ,

Mescaline

*

300x4

Cycl ohexane-CHC1 3-DEA (25:150:0.3) 15 ACN-H 0 (18: 82) c o n t a i n i n g 0.2 M ~ H C I O ~(PH 5.4) 17 0.0025M octanesulfonate, 0.5% AcOH i n MeOH-H20(56:44) 20 CHCl -EtOAc-EtOH-2-hexane (25:jO: 1:50) 25 MeOH-ACN-acetate b u f f e r ( p H 3.5) (36:9:55) c o n t a i n i n g 0.001M Na-dodecylsul f a t e 27

N-n-propyl norapomorphine ,apomorphi ne, b o l d i ne

Analysis i n plasma

$ondapak

Phenyl

300x4

Tubocurarine ,alcur o n i um

Determination a1 curonium i n plasma

$ondapak

C18

3 0 0 ~ 6 . 4 MeOH-H O(4:l) c o n t a i n i n g 0.25% AcOH a6d 0.005M Na-dodecylsulfate 32

Tubocurari ne

Determination i n plasma

Radial-Pak

C18, 10

100x5

MeOH-(TrEA(lOg/l)-pentanesulf o n i c a c i d ( 1 ml)-H3P04(2 m1)H20 ad 11)(2:3)

33

297

Chapter 7 OPIUM ALKALOIDS

............................................................... ............................................................. ..................................................................... ............................................................. ......................................................................... ...........................................................................

7.1. Ion-exchange HPLC.. 7.2. Reversed-phase HPLC.. 7.3. Ion-pair HPLC 7.4. Straight-phase HPLC.. 7.5. Detection References..

HPLC has been used t o t r y and solve a great number o f problems o f opium a l k a l o i d s .

297 298 301 305 308 310

concerning the a n a l y s i s

(1) The separation o f the various components of n a t u r a l l y occurring mixtures o f opium a1kal oids. (2) The separation o f opium a l k a l o i d s and other compounds present i n pharmaceutical preparations. ( 3 ) The separation and i d e n t i f i c a t i o n o f drugs o f abuse. I n Tables 7.13-7.18

t h e d i f f e r e n t HPLC techniques t h a t have been applied so f a r t o

solve t h e a n a l y t i c a l problems mentioned, are sumnarized. They a r e arranged according t o the aim o f the analysis. A review o f HPLC analysis o f drugs abuse i s given by Wheals 32 and Gough and Baker"'. 7.1. ION-EXCHANGE HPLC Depending on the a n a l y t i c a l problem involved, a number o f chromatographic systems have been used f o r t h e separation o f opium a l k a l o i d s and r e l a t e d compounds. T w i t c h e t t and Moffat"

and T w i t c h e t t e t a1.27s30 evaluated several HPLC systems f o r t h e a n a l y s i s

o f 30 drugs. Both m i c r o p a r t i c u l a t e reversed-phase (octadecylsilane) and cation-exchange

-

( s u l f o n i c acid) columns were tested, the f i r s t i n combination w i t h methanol aqueous water (2:3) o f phosphate b u f f e r mixtures o f varying pH, the l a t t e r w i t h methanol

-

varying pH and i o n i c Strength. It was concluded t h a t the reversed-phase columns had poor ', the cation-exchange columns e f f i c i e n c y f o r most o f the basic drugs t e ~ t e d ~ l * ~whereas the optimum conditions could be selected by

were found t o be s u i t a b l e f o r

using eluents containing a t l e a s t 40% methanol o r a c e t o n i t r i l e , and by changing t h e i o n i c strength. V a r i a t i o n o f the pH could influence the r e t e n t i o n and the column e f f i c i e n c y ; a pH o f over 7 reduced the column l i f e considerably. The necessity c f adding an organic solvent t o the mobile phase f o r the analysis o f a l k a l o i d s on ion-exchange columns has been o u t l i n e d by Knox and J ~ r a n d ~ They ' ~ . invest i g a t e d the separation o f some opium a l k a l o i d s and analgesics by using strong anion o r cation-exchange columns. Six major opium a l k a l o i d s were separated on a strong c a t i o n exchange column by means o f a b o r i c a c i d b u f f e r o f pH 9.5, t o which 4% a c e t o n i t r i l e and 1%n-propanol was added (Fig. 7.1). Twitchett" a p p l i e d the same method t o t h e a n a l y s i s 6 o f i l l i c i t heroin preparations (Fig. 7.2), whereas Wittwer used a g r a d i e n t e l u t i o n t o separate t h e major opium a l k a l o i d s on a strong anion-exchange resin. Walton and Murgia13'19'26 described the separation o f some opium a l k a l o i d s and o t h e r compounds by means o f 1igand-exchange chromatography. D i f f e r e n t cation-exchange r e s i n s loaded w i t h metals t h a t form ammonia complexes (Cu",

Referenced p. 310

Ni",

Zn++ and Ag')

were i n v e s t i -

298

gated. As mobile phase amnonia-ethanol mixtures were used. Strong basic solvents may cause h y d r o l y s i s o f some e s t e r a l k a l o i d s , reducing t h e a p p l i c a b i l i t y o f t h e method. Hays e t a1.7 used Zipax SCX i n p u r i t y analyses o f h e r o i n and morphine. The a l k a l o i d s were e l u t e d w i t h a g r a d i e n t o f sodium p e r c h l o r a t e (0.4-1.4

M) i n a 0.01 M phosphate

b u f f e r o f pH 6.8 c o n t a i n i n g 10% ethanol. Matantseva and co-workers87y101~103 separated t h e major opium a l k a l o i d s on a p e l l i c u l a r s u l f o n i c a c i d cation-exchange s t a t i o n a r y phase, using a pH 4.5 phosphate b u f f e r c o n t a i n i n g 30% a c e t o n i t r i l e as mobile phase. 7.2. REVERSED-PHASE HPLC I n one o f the f i r s t HPLC separations described f o r the a n a l y s i s o f

alkaloid^"^

a

dynamic c o a t i n g technique was used i n order t o apply t h e s t a t i o n a r y phase (Poly 6-300) on the s i l i c a gel support. Using various percentages o f the s t a t i o n a r y phase i n t h e mobile phase, t h e loading o f the support w i t h the s t a t i o n a r y phase could be v a r i e d . However, t h e a v a i l a b i l i t y o f chemically bonded s t a t i o n a r y phases has reduced the value o f a dynamic c o a t i n g procedure t o merely a t h e o r e t i c a l l y i n t e r e s t i n g one. Despite t h e f a c t t h a t several authors t o begin w i t h reported poor r e s u l t s f o r the a n a l y s i s o f a l k a l o i d s w i t h reversedphase columns 7*21s30s56,

an ever i n c r e a s i n g number of HPLC separations w i t h reversed-

phase chromatography i s being published. Honigberg e t a1 .14 compared some chemically bonded s t a t i o n a r y phases -on p e l l i c u l a r s i l i c a gel- i n t h e a n a l y s i s o f cough-cold mixtures, containing, i . a . , some a l k a l o i d s . The authors found t h a t t h e peak shape f o r a packing m a t e r i a l w i t h chemically bonded phenyl groups was more s y m t r i c a l than t h a t with chemic a l l y bonded octadecyl groups, when e q u i v a l e n t mobile phases were used. Knox and Pryde23 a p p l i e d s i l i c a gel w i t h chemically bonded s h o r t chain alkanes f o r the a n a l y s i s o f opium a l k a l o i d s . With 0.025 M a m n i a i n methanol

-

water ( l : l ) ,

a good separation o f t h e major

a l k a l o i d s was obtained. Various authors have discussed t h e f a c t o r s i n f l u e n c i n g t h e r e t e n t i o n behaviour o f a l k a l o i d s , i.e., opium a l k a l o i d s , i n reversed-phase chromatography. Honigberg e t a1 ,14 found t h a t f o r chemically bonded phenyl and octadecyl s t a t i o n a r y phases a decrease o f t h e a c e t o n i t r i l e content i n a mobile phase c o n s i s t i n g o f a c e t o n i t r i l e and aqueous 0.1% ammonium acetate s o l u t i o n r e s u l t e d i n increased r e t e n t i o n times and i n c r e a s i n g bandspreading on both types o f s t a t i o n a r y phases. The r e t e n t i o n times on octadecyl phases were u s u a l l y longer than those on phenyl phases, when e q u i v a l e n t mobile phases were applied. T w i t c h e t t e t a1.21s27 r e p o r t e d t h a t a t pH 3 t h e r e t e n t i o n time o f morphine on an octadecyl phase column f i r s t decreased w i t h an increase o f t h e methanol content i n t h e mobile phase and then increased. An increase o f t h e pH l e d t o an increase o f t h e r e t e n t i o n time o f basic compounds (40% methanol as mobile phase). Thus, knowledge about t h e pKa and t h e l i p i d s o l u b i l i t y o f a compound enables p r e d i c t i o n about i t s r e t e n t i o n behaviour on octadecyl columns. According t o Wu and W i t t i ~ kt h~e ~r e t e n t i o n o f opium a l k a l o i d s on octadecyl columns i s p r i m a r i l y governed by t h e percentage o f a c e t o n i t r i l e i n a mobile phase c o n s i s t i n g o f a c e t o n i t r i l e and an aqueous phosphate b u f f e r . An increase o f t h e acetoni t r i l e content leads t o a decrease o f t h e r e t e n t i o n times. The pH i n f l u e n c e s t h e r e t e n t i o n : increase o f t h e pH leads t o an increased r e t e n t i o n time. Although a b a s e l i n e separation c o u l d be

299

TABLE 7.1 RETENTION

VOLUME OF SOME OPIUM ALKALOIOS~O

-

Column, Hypersil ODS, 5 wn (100~4.6 mn ID), mobile phase methanol 0.01 H phosphate b u f f e r (pH 3) containing 0.1 M potassium bromide (1:7), f l o w r a t e 0.75 ml/min. Compound Morphi ne-3-gl ucuronide Normorphine D i hydromorphi ne Morphine Morphine-N-oxide Codei ne-N-oxide

Retention Volume ( m l ) 1.0 1.7 1.7 1.8 1.8 4.3

Compound D i hydrocodei ne Nalorphine Codeine Norcodeine 6-0-acetylmorphine

Retention Volume (ml ) 4.8 4.9 5.3 5.7 10.4

-

obtained w i t h 0.1 M sodium dihydrogen phosphate i n a c e t o n i t r i l e water (1:3) (pH 4.8) (Fig. 7.3). the authors found t h a t f o r q u a n t i t a t i v e analysis o f morphine an a c e t o n i t r i l e concentration o f 5% was more suitable. This HPLC system was a l s o used f o r the determina52 t i o n o f morphine i n poppy straw Poochikian and C r a d ~ c kused ~ ~ a c e t o n i t r i l e - aqueous phosphate b u f f e r s i n combination w i t h octadecyl columns f o r the analysis o f heroin and i t s h y d r o l y s i s products. The retent i o n times were found t o be shorter w i t h increasing percentages o f a c e t o n i t r i l e and w i t h

.

more s t r o n g l y buffered mobile phases. Also, the peak sharpness increased under such conditions. Optimum pH was between 5 and 8.7. The order o f e l u t i o n was n o t e f f e c t e d by changes i n pH, b u f f e r strength, o r percentage o f a c e t o n i t r i l e i n t h e m b i l e phase. As well as mobile phases consisting o f a c e t o n i t r i l e and aqueous b u f f e r s , phases i n which a c e t o n i t r i l e has been replaced by methanol have been used i n combination w i t h octadecyl s t a t i o n a r y phases (see Tables 7.13-7.18). The separation o f codeine and morphine and some 80 o f t h e i r metabolites i n such a system i s summarized i n Table 7.1 . TABLE 7.2 RETENTION TIMES OF SOME OPIUM ALKALOIDS78

Systems A-D: column, 300x4 mm 1.0. Nucleosil 10CN; systems E-G: column, 300x4 mn 1.0. Nucleosil 10C18. Mobile phases: A, 1%amonium acetate (pH 5.8)-acetonitrile-dioxane (80:10:10);B, 1%amnonium acetate (pH 5 . 8 ) - a c e t o n i t r i l e (80:ZO); C and E, 1%amnonium acetate (pH 5 . 8 ) - a c e t o n i t r i l e (70:30); D and G, 1%amnonium acetate (pH 5 . 8 ) - a c e t o n i t r i l e (60:40); F, 1%amnonium acetate (pH 5 . 8 ) - a c e t o n i t r i l e (65:35). A l l systems: flow-rate, 1.5 ml/min. A1 k a l o i d Morphine Codeine Cryptopine Thebaine Noscapine Papaverine

Rthrencea p. 310

Retention time (min) A B C D 3.8 4.2 4.0 4.1 4.8 4.5 5.1 5.4 5.6 9.6 7.2 8.1 6.7 10.8 8.0 9.2 5.9 12.3 14.1 9.3 5.6 15.7 18.2 9.3

E 2.4 3.3 6.5 9.8 42.6 20.8

F 2.3 3.0 4.7 7.5 23.4 11.3

G 2.2 2.9 4.1 6.7 15.7 8.0

300

-

Wu and Dobberstein41 used methanol 0.3% amnonium carbonate i n water ( 4 : l ) as mobile phase i n the q u a n t i t a t i v e analysis o f thebaine i n Papuuer bracteatwn. However, they reported t h a t t h e i r octadecyl column had a c t i v e s i t e s on the s i l i c a gel support on which thebaine was adsorbed. For q u a n t i t a t i v e determinations i t was necessary t o s a t u r a t e t h e a c t i v e s i t e s by i n j e c t i n g 20 pg thebaine once a day and then 5 pg thebaine several times

-

u n t i l successive i n j e c t i o n s gave thebaine peak areas which v a r i e d w i t h i n 2% o r l e s s from each other. Rasmussen e t a1 .51 described a straight-phase and a reversed-phase HPLC separation f o r t h e determination o f morphine i n organic and aqueous poppy e x t r a c t s respectively. The reversed-phase system consisted o f an octadecyl column and methanol - 0.05 M aqueous ammonium carbonate ( 1 : l ) as mobile phase. This system gave b e t t e r r e s u l t s f o r t h e analysis o f morphine than t h e system mentioned by Wu and W i t t i ~ k ~because ~ ~ ~ ‘o f fewer i n t e r f e r i n g peaks, The separation was found t o be g r e a t l y influenced by t h e s a l t concentration. The column performance was n o t a f f e c t e d by the high pH. The reversed-phase system had the advantage over t h e straight-phase system i n t h a t crude e x t r a c t s could be analyzed. The straight-phase system could only be applied f o r more p u r i f i e d e x t r a c t s . Nobuhara e t al.78 separated opium a l k a l o i d s on octadecyl and cyanoalkyl phases using mixtures o f a c e t o n i t r i l e and 1%aqueous ammonium acetate (pH 5.8) as mobile phase. Both types o f column enabled baseline separation o f t h e major opium a l k a l o i d s (Fig. 7.4 and 7.5, TABLE 7.2). However, the cyanoalkyl s t a t i o n a r y phase gave the best separation f o r quantitat i v e analysis. Since no previously reported method adequately separated h e r o i n from i t s major impurit i e s and adulterants, Baker and Goughg3 tested various columns and mobile phases. Best r e s u l t s were obtained w i t h an aminopropyl bonded phase (Fig. 7.6), t h e o n l y disadvantage being the rather strong r e t e n t i o n o f morphine. For the analysis o f a l k a l o i d s i n Papauer somnifermm p l a n t material, an aminopropyl s t a t i o n a r y phase has been used”’. and except f o r noscapine and papaverine a l l major opium a l k a l o i d s were separated ( F i g . 7.7). For the analysis o f acetylprocaine i n heroin samples, Bernauer and Fuchslo5 employed an octadecyl s t a t i o n a r y phase i n combination w i t h the mobile phase a c e t o n i t r i l e water (91.5:8.5) containing 8 mg/100 m l t r i s ( hydroxymethyl ) -aminomethane (Fig. 7.8). As noticed by other authors, P e t t i t t and Damon117 found t h a t a phenyl-bonded phase gave b e t t e r peak shapes f o r the a l k a l o i d s than an o c t y l o r octadecyl type o f s t a t i o n a r y phase. However the a d d i t i o n o f N,N-dimethyloctylamine t o the mobile phase was found t o be essential i n order t o g r e a t l y reduce t a i l i n g o f opium a l k a l o i d s (Fig. 7.9). Tatsuzawa e t a1 .36337s45s5g separated c o l d drugs and neuroleptics by using a styrenedivinylbenzene-methyl methacrylate copolymer as s t a t i o n a r y phase. The b e s t r e s u l t s were obtained w i t h methanol amonia ( 9 9 : l ) as mobile phase. The e f f e c t o f t h e pH and o f t h e composition o f t h e mobile phase on t h e separation were discussed. Aramaki e t a1.70 analyzed a series o f a l k a l o i d s on a macroporous styrene-divinylbenzene copolymer w i t h a l k a l i n e a c e t o n i t r i l e water mixtures as mobile phase (Fig. 7.10). The columns showed e x c e l l e n t

-

-

-

s t a b i l i t y , and a l s o under t h e strong basic conditions used f o r the a n a l y s i s o f t h e a1 kal o i ds

.

301 7.3.

ION-PAIR HPLC

Because o f the low effectiveness o f reversed-phase columns f o r b a s i c compounds reported by several authors

-

-

as

i o n - p a i r chromatography has o f t e n preferred.

L ~ r i analyzed e ~ ~ drugs o f f o r e n s i c i n t e r e s t on octadecyl columns w i t h 0.005 M l-heptanes u l f o n i c a c i d i n methanol - w a t e r

- acetic

a c i d (40:59:l)(pH 3.5) (Table 7.3). The system

enabled a simultaneous analysis o f a c i d i c , n e u t r a l and basic drugs. Among the compounds analyzed were the opium a l k a l o i d s ( F i g . 7.11). A s i m i l a r system was a p p l i e d f o r t h e analysis o f papaverine i n plasma 67

.

TABLE 7.3

REVERSED-PHASE ION-PAIR CHROMATOGRAPHY OF SOME DRUGS OF FORENSIC INTEREST38 Column UBondapak C18 (300x4 mm I.D.), mobile phase 0.005 M heptanesulfonic a c i d i n methanol water - a c e t i c a c i d (40:59:1), pH ca 3.5, f l o w r a t e 2 ml/min.

-

Retention volume ( R r ) o f compounds r e l a t i v e t o noscapine Compound

Rr

Morphine Codeine 0-ace ty 1morph ine Procaine Acetylprocaine Acetvl codei ne Hero; n

0.28 0.36 0 . 37 0.38

0.50 0.69 0.70

Compound Thebai ne Noscapi ne Q u i n i d ine Methapyri lene Papaverine Ouinine

Rr 0.73 1.00 (23.0 m l ) 1.23 1.24 1.40 1.44

01 ieman e t a140 found t h a t reversed-phase i o n - p a i r chromatography was the most s u i t a b l e technique t o analyze a s e r i e s o f morphinan d e r i v a t i v e s . Normal reversed-phase chromatography on octadecyl columns, as w e l l as straight-phase systems gave too much t a i l i n g

-

p a r t i c u l a r l y f o r a l k a l o i d s w i t h a h i g h r e t e n t i o n t i m e (Table 7.4). Smith e t a1 .53958962 used i o n - p a i r chromatography t o separate apomorphine and r e l a t e d a l k a l o i d s . A diphenylsilane column was used i n combination w i t h methanol 0.02 M aqueous potassium dihydrogen phosphate 0.001 M sodium dodecylsulfate (36:9:55).

-

-

acetonitrile

-

0.03 M c i t r i c a c i d (pH 3.25) c o n t a i n i n g

The system allowed b a s e l i n e separation o f apo-

morphine, apocodeine. isoapocodeine and the i n t e r n a l standard N-n-propylnorapomorphine o r boldine. Dodecylsulfate as counter-ion gave b e t t e r r e s u l t s than heptanesulfonic acid. Without the a d d i t i o n o f a p a i r i n g - i o n , t a i l i n g was observed. Soni and D ~ g a analyzed r ~ ~ opiates w i t h reversed-phase i o n - p a i r chromatography; u s i n g the i o n - p a i r chromatography no t a i l i n g f o r t h e a l k a l o i d s was observed on o c t a d e c y l s i l a n e columns. Tetrabutyl ammonium phosphate and 1-heptanesulfonic a c i d were used as p a i r i n g ions (Table 7.5). Kubiak and M u n ~ o nstudied ~~ several p a i r i n g - i o n s f o r the analysis o f morphine, codeine and ethylmorphine on o c t y l s i l a n e o r octadecylsilane columns. A d d i t i o n o f 0.01 M ammonium n i t r a t e t o the mobile phase s i g n i f i c a n t l y improved the peak shape and reduced the t a i l i n g . I n order t o improve the separation f u r t h e r , d i f f e r e n t p a i r i n g - i o n s were t e s t e d i n combinat i o n w i t h a mobile phase c o n s i s t i n g o f 0.01 M ammonium n i t r a t e i n a c e t o n i t r i l e (375:625).

- water

Increase i n t h e alkane chain length o f the p a i r i n g - i o n by up t o e i g h t carbons

gave a r e l a t i v e l y minor increase i n the capacity f a c t o r s o f the a l k a l o i d s . However, f u r t h e r

References p. 310

302

TABLE 7.4 RETENTION TIVES ( n i n ) OF MORPHINANS FOR DIFFERENT SOLVENT (CONTAINING 0.005 tl n-HEPTANESULFONIC ACID). Column, uBondapak C18, (300x4 n),f l o w r a t e 1.2 ml/min. Dashes i n d i c a t e no e l u t i o n w i t h i n a reasonable time (>30 min). Compound

Solvent system

Florphi ne Codeine Heroin D i hydromorphine O i hydrocodei ne O i hydrocodei none 1-methyl -dihydrocodei ne 1-methyl - d i hydrocodeinone 1-bromo-di hydrocodei none 1.7-dibromo-di hydrocodeinone D i hydrothebainone 0-Acetyl - d i hydrothebai none N-nor-di hydrothebai none N-formyl - d i hydrothebai none 1-methyl - d i hydrothebainone N-formyl-1-methyl - d i hydrothebai none 1-bromo-di hydrothebai none 1,7-di bromo-di hydrothebai none 2-hydroxy-dihydrothebainone thebaine oripavine

#ethanol-water

tlethanol-water

Acetoni t r i l e - w a t e r

(50:50)

(40:60)

(25:75)

4.0 4.6 7.3 4.1 4.5 5.1 5.4 5.9

5.6 7.5 19.4 6.0 7.7

4.5 6.1 16.2 4.9 5.7

8.8

8.0

9.6 12.7 18.7

7.6 11.5 17.6

9 .o 10.1 9.5 9.2,g.R 12.6 13.3,14.7 21.4

7.5 8.7 6.7 10.0 10.1 14.4 14 .o

5.7 17.9 8.7

5.6 17.5 7.5

8.0 14.5 5.2 5.5 5.1 5.1.5.3 6 .O 6.5,7.0

8.0 11.7 4.0 7.5 4.8

TABLE 7.5 REVERSED PHASE ION-PfiIR CHROVATOGRAPHY OF SOME OPIUM ALKALOIDS AND THEIR ABSORPTION RATIOS RECORDED WITH DETECTION AT 254 AND 280 nm64

Column liBondapak C18 (300x4 mn! I.D.), mobile phase S 1 0.01M t e t r a b u t y l ammonium phosphate (pH 7.5) - methanol (53:47), S2 0.01!1 1-heptane s u l f o n i c a c i d (pH 3 w i t h 1%acetic acid) - methanol (65:35) a f t e r 10 min changed t o (45:55), f l o w r a t e 2 ml/min, detection UV 254 and 280 nm simultaneous. Retention time (mi n ) Compound Acetylcodeine Mor ph ine Oxymorphone Noroxymorphone O i hydromorphi none Nal orphi ne Codeine 0-acetylmorphi ne Oxycodone D i hydrocodei none Heroin Papaverine

s1 2.5 5.0 5 .O 5.2 5.8 7.1 8.3 10.1 10.4 11.8 15.4 16.7

s2

...

7.0 5.0 3.8

... ...

12.5 9.0 10.5 retained

...

...

Ratio 254/280

s1 5.25 0.75 0.85

0.87 0.77 0.79 1.10 0.59 o .a5 0.76 0.24 10.1

s2

...

0.72 0.94 0.84

...

...

1.05 0.54 0.84

...

...

9.9

303 increase i n t h e chain l e n g t h l e d t o a considerable increase. Best r e s u l t s were obtained w i t h d i o c t y l sodium sulfosuccinate. This p a i r i n g - i o n circumvents t h e s o l u b i l i z a t i o n problem o f some of t h e long chain alkanes p a i r i n g - i o n s (14 and 16 carbon atoms), b u t gives s i m i l a r capacity f a c t o r s as the long chain alkane p a i r i n g - i o n s mentioned. Morphine, codeine and ethylmorphine could be separated w i t h 0.005 M d i o c t y l sodium s u l f o s u c c i n a t e i n t h e mobile phase. Eriksson e t a1 .63 used straight-phase i o n - p a i r chromatography f o r t h e a n a l y s i s o f apomorphine i n b i o l o g i c a l m a t e r i a l . Best r e s u l t s were obtained w i t h p e r c h l o r i c a c i d as p a i r i n g - i o n . Dichloromethane

-

methanol

-

1

M p e r c h l o r i c a c i d (956:40:4) was used as mobile

phase i n combination w i t h a s i l i c a gel column. L u r i e and O e m c h u ~ k ~and ~ ’ L~u~r i e l o 0 extended t h e i r e a r l i e r s t u d i e s on reversed-phase i o n - p a i r HPLC of forensic drugs t o t h e f a c t o r s i n f l u e n c i n g the separation. The e f f e c t o f the s t a t i o n a r y phase, the water

-

methanol r a t i o i n the mobile phase, and the a l k y l - c h a i n

l e n g t h o f the counter-ion, as w e l l as i t s concentration

on the r e t e n t i o n o f a s e r i e s o f

a c i d i c , neutral and basic drugs, was studied. For s o l i d supports c o n t a i n i n g chemically bonded octadecyl and phenyl s t a t i o n a r y phases i t was found t h a t increased chain-length o f the p a i r i n g i o n ( a l k y l s u l f o n i c a c i d ) l e d t o an increase o f the k ’ o f basic compounds. This e f f e c t was less pronounced on a cyano-type s t a t i o n a r y phase. A comparison o f d i f f e r e n t s t a t i o n a r y phases o f cyano-, phenyl- and octadecyl types showed t h a t t h e r e t e n t i o n i n creased f o r basic compounds, from the cyano-type v i a the phenyl-type t o t h e octadecyltype, using t h e same mobile phase. However, the smaller the a l k y l group o f the counter-ion, the l e s s pronounced was the e f f e c t . The concentration o f the counter-ion had o n l y a l i m i t e d e f f e c t on the r e t e n t i o n , whereas increased water content i n the mobile phase l e d t o longer r e t e n t i o n times.

To improve the separation o f c e r t a i n compounds, t h e s e l e c t i v i t y can be improved by i n creasing the percentage o f water i n t h e mobile phase by changing t h e counter-ion ( d i f f e r e n t a l k y l group) f o r solutes o f d i f f e r e n t pKa. S e l e c t i v i t y f o r basic compounds i s l i t t l e enhanced by d i f f e r e n t s t a t i o n a r y phases, and the same holds t r u e f o r a l t e r a t i o n s o f t h e concentration o f the counter-ion. Two systems were developed t h a t were s u i t a b l e f o r the a n a l y s i s o f drugs o f abuse 100 One consisted o f a mobile phase o f methanol

-

water

-

.

1%a c e t i c a c i d (40:59:1) c o n t a i n i n g

0.02 M methanesulfonic a c i d (pH 3 . 5 ) i n combination w i t h a m i c r o p a r t i c u l a t e octadecyl column. It was recommended f o r samples containing compounds such as b a r b i t u r a t e s , l o c a l anaesthetics, LSO and r e l a t e d a l k a l o i d s . The other system made use o f the same column and a mobile phase o f methanol - water - 1%a c e t i c a c i d (20:79:1) containing 0.02 s u l f o n i c a c i d (pH 3.5).

M methane-

I t was p a r t i c u l a r l y s u i t a b l e f o r the a n a l y s i s o f phenethylamines

and f o r the separation o f heroin and acetylcodeine. The increased counter-ion concentrat i o n i n these mobile phases compared t o previously used systems was applied t o reduce the 121

v a r i a t i o n i n r e t e n t i o n times o f bases f o r d i f f e r e n t samples. I n a subsequent study

L u r i e e t a l . reported the analysis o f i l l i c i t heroin samples using s i m i l a r HPLC-systems; however, a c e t o n i t r i l e was used instead o f methanol i n order t o a l l o w f a s t e r f l o w r a t e s , thus reducing analysis time. Instead o f a c e t i c a c i d the mobile phase was a c i d i f i e d w i t h orthophosphoric a c i d t o a l l o w d e t e c t i o n a t 220 nm. The r e s u l t s are sumnarized i n Table 7.6.

References p. 310

304

TABLE 7.6 RELATIVE RETENTION TIMES AN0 220:254 ABSORBANCE RATIOS AND BY-PRODUCTS121.

FOR HEROIN AND ITS ADULTERANTS

S1: Column pBondapak C18 (300~3.9 mm I.D.), 52: column, P a r t i s i l 10 ODS-3 ( 2 5 0 ~ 4 . 6 mn 1.0.) both w i t h mobile phase 0.02 M methanesulfonic a c i d i n a c e t o n i t r i l e water phosphoric a c i d (12:87:l)(pH=2.2 w i t h 2 M sodium hydroxide), f l o w r a t e 3.0 ml/min.

-

Compound

r e l a t i v e r e t e n t i o n times S1

L-Ascorbic a c i d I s o n i c o t i namide Morphine Ami nopyri ne Procaine Ephedrine Paracetamol Theophyl 1 ine Methapyril ene T r i pel ennamine Codeine Pyri 1ami ne Quin i d i ne Barbital Quinine 0-Acetylmorphi ne Caffeine Phentermine Lidocaine Quinine (second peak) Acetyl procai ne P r i 1ocai ne Sal icylamide Antipyrine Hyoscyamine Strychnine Benzocai ne Acetylsalicylic acid Sodium s a l i c y l a t e Tropacocaine Phenobarbital Benzoyl t r o p e i ne Acetylcodeine Thebai ne Phenacetin Meperi d i ne Heroin Cocaine Amylocaine Phencyclidine Noscapine Tetracaine Papaverine Tartaric acid O i phenhydramine Methadone Phenyl butazone

...

0.05 0.09 0.15 0.12 0.14 0.11 0.12 0.15 0.16 0.17 0.18 0.19 0.23

...

0.23 0.19 0.28 0.30

... ... ...

0.28 0.36 0.41 0.38 0.50 0.60

...

0.71 0.81 0.82

0.85 0.88 0.81

...

1.00 1.05

...

2.12 2.30 2.28 2.80

...

...

‘Elutes near s o l v e n t f r o n t . bExhibits no UV a t 254 nm. % t e n t i o n time greater than 1 h.

220:254 r a t i o (S2)

s2 0.05 0.05 0.08 12.1 0.12 0.9 4.1 0.12 0.12 3.5 0.12 0.6 0.13 1.4 2.0 0.13 0.13 2.4 0.16 8.9 0.17 3.2 0.7 0.19 0.21 62.0 0.22 0.7 0.22 14.7 2.2 0.23 0.25 5.1 0.30 16.6 0.30 0.7 0.30 0.6 0.31 3.5 0.31 6.0 0.36 1.4 0.39 21.7 0.41 0.56 0.58 4.9 0.58 10.7 0.75 7.3 0.76 7.7 0.85 11.0 0.87 8.1 0.88 11.2 4.6 0.92 ... 0.95 0.5 1.oo 25.5 l.OO(19 min) 24.0 1.11 6.0 1.37 5.9 2.15 8.5 2.36 10.8 2.38 10.3 3.17 0.4

...

...

... ... ...

...

... ... ... ...

-

305

Achari and Jacobse1 (see CHAPTER 2) have made extensive s t u d i e s o f t h e i o n - p a i r HPLC of b a s i c drugs. Lindherg e t a1 .94 a p p l i e d s t a t i s t i c a l o p t i m i z a t i o n methods t o t h e separat i o n o f some opium a l k a l o i d s , whereby f o u r f a c t o r s were varied: t h e e l u e n t strength, t h e pH and t h e concentration o f t h e phosphate b u f f e r as w e l l as the camphorsulfonic a c i d ( t h e p a i r i n g - i o n ) . Via a s e r i e s o f experiments the k ’ - v a l u e o f each a l k a l o i d as a f u n c t i o n o f t h e above mentioned v a r i a b l e s was determined, and by s t a t i s t i c a l means t h e optimum c o n d i t i o n s f o r a d e s i r e d separation could be selected. Reversed-phase i o n - p a i r separations have been a p p l i e d t o t h e a n a l y s i s o f morphine and i t s metabolites i n b i o l o g i c a l samples11o and t o t h e a n a l y s i s o f t h e degradation products 116 of and morphine

.

7.4. STRAIGHT-PHASE HPLC Opium a l k a l o i d s and drugs o f abuse have been w i d e l y analyzed by straight-phase chromatography. Except f o r two i n v e s t i g a t i o n s l Y l 2

-

i n t h e e a r l y stage o f development o f HPLC

-

a l l HPLC analyses o f opium a l k a l o i d s have been performed w i t h s i l i c a gel columns i n comb i n a t i o n w i t h b a s i c solvents, i n o r d e r t o reduce t a i l i n g due t o chemisorption o f t h e a l k a l o i d bases on t h e weakly a c i d i c s i l i c a g e l . Only once does aluminium oxide seem t o have been used f o r t h e separation o f drugs o f abuse”. system was used, i.e.

I n t h a t case, a b a s i c s o l v e n t

0.22% cyclohexylamine i n cyclohexane o r i n cyclohexane

-

methanol

(98.5: 1.5). Smith e t a1.8’10’17

analyzed t h e opium a l k a l o i d s by means o f p e l l i c u l a r s i l i c a g e l

columns i n combination w i t h a m o b i l e phase o f n-hexane

-

chloroform

-

methanol

-

diethyl-

amine i n various r a t i o s . Thebaine could be determined i n Papauer bracteaturn and P.orientaZe by means o f an i s o c r a t i c system8. By using a s p e c i a l l y designed g r a d i e n t system, t h e s i x major opium a l k a l o i d s could be determined i n opium. Brucine was used as an i n t e r n a l standard”’

17.

Vincent and Engelke54 c a r r i e d o u t an i s o c r a t i c HPLC separation o f t h e f i v e major opium a l k a l o i d s i n Papaver somnifemm and o f thebaine i n P. bracteaturn on m i c r o p a r t i c u l a t e s i l i c a gel f o r a q u a n t i t a t i v e determination o f t h e a l k a l o i d s . A mobile phase c o n s i s t i n g o f f o u r components: n-hexane

-

dichloromethane

-

ethanol

separated t h e f i v e major opium a l k a l o i d s (TABLE 7.7, dichloromethane

-

-

diethylamine (300:30:40:0.5)

F i g . 7.12).

By a l t e r i n g t h e r a t i o

ethanol, the r e t e n t i o n o f t h e a l k a l o i d s could be v a r i e d . Capsular t i s s u e

e x t r a c t s could be analyzed d i r e c t l y . Chan e t a l . l l preferred cyclohexylamine as t h e b a s i c m o d i f i e r i n t h e a n a l y s i s o f drugs of abuse on p e l l i c u l a r s i l i c a gel o r aluminium oxide columns. V o l a t i l e amines were l e s s s u i t e d , because the s o l v e n t composition changed with time, and secondary and t e r t i a r y amines absorbed U V - l i g h t a t 254 nm. Caude e t a1 .25,48 used ethylamine as t h e basic m o d i f i e r i n a s o l v e n t system o f e t h y l acetate

- methanol

-

water i n order t o analyze some opium a l k a l o i d s i n pharmaceutical

preparations on m i c r o p a r t i c u l a t e s i l i c a gel columns, whereas Achari and Theimer3’ used chloroform

-

methanol ( 3 : l ) c o n t a i n i n g 1%amnonia f o r the same k i n d o f analyses

on m i c r o p a r t i c u l a t e s i l i c a gel.

-

also

No d r a s t i c d e t e r i o r a t i o n o f the column e f f i c i e n c y was

observed when a m n i a was added t o t h e solvent; t h e column was s t i l l usable even a f t e r two years.

Reference p. 310

306 TABLE 7.7.

RETENTION OF SOME PAPAVER ALKALOIDS AND RELATIVE DETECTOR RESPONSES~ 54

-

dichloromethane Column, pPorasi1 (300x4 mn I.D.), mobile phase n-hexane diethylamine (300:20:20:0.5), f l o w r a t e 1.8 ml/min, d e t e c t i o n UV 254 nm. Alkaloid Morphine Codeine Salutaridine Orioavine Thebaine Laudanosine Isothebaine

Rt sec

;R

Area' 110~)

1700 860 780 680 374 370 330

1.000 0.506 0.459 0.400 0.220 0.218 0.194

70 43

Rt sec

Alkaloid Cryptopine Alpinigenine Papaverine Noscapine Narcei ne Protopine Gnoscopine

500 11 118 60 270

193 189 175 170 170 168 160

-

Rxz 0.114 0.111 0.103 0.100 0.100 0.099 0.094

-

ethanol

Areal 11031 360 120 16 150 7 330 130

'Area i n pv/sec f o r each a l k a l o i d a t a concentration o f 20 pg/ul. 2 Rx=retention time r e l a t i v e t o morphine

A s i m i l a r solvent system was a l s o used by Rasmussen e t a l q 5 1 f o r the a n a l y s i s o f morphine i n organic poppy e x t r a c t s (Fig. 7.13). The e x t r a c t s could be analyzed w i t h o u t any p u r i f i c a t i o n p r i o r t o HPLC analysis. Gimet and F i l l o u x 6 0 performed analyses on a l k a l o i d s i n c l u d i n g opium a1 kaloids

-

-

i n pharmaceutical preparations, and used a m i c r o p a r t i c u l a t e

s i l i c a qel column and d i e t h y l ether o r d i e t h y l ether saturated w i t h water as mobile phase. I n both cases 0.4% diethylamine was added t o the mobile phase (Fig. 7.14). An increase o f the percentage o f s a t u r a t i o n o f the d i e t h y l e t h e r w i t h water, as w e l l as o f the percentage o f diethylamine

l e d t o a decrease o f the r e t e n t i o n times. F r e i e t a1.29 separated

the dansyl d e r i v a t i v e s o f some a l k a l o i d s , i . a . , morphine, and some non-derivatized a l k a l o i d s (Fig. 7.15). To circumvent the problems connected w i t h the a n a l y s i s o f a wide range o f drugs w i t h o f t e n q u i t e d i f f e r e n t p o l a r i t i e s , Janez2 used m i c r o p a r t i c u l a t e s i l i c a gel columns and very p o l a r mobile phases, such as methanol and methanol

-

-

0.2 M amnonium n i t r a t e (3:2)

2 M amnonia (Tables 7.8,

-

1 M ammonium n i t r a t e (27:2:1)

and 7.9,

F i g . 7.16). No n o t i c e a b l e

loss o f column performance was observed f o r r o u t i n e use over several months w i t h the HPLC system described. Baker e t a1.56 used a s i m i l a r system and found t h a t the r e t e n t i o n times o f the drugs analyzed c o r r e l a t e d q u i t e w e l l w i t h those reported by Janez2 (Table 2.2,2.3). The usefulness o f such p o l a r solvent systems i n the a n a l y s i s o f opium a l k a l o i d s has also been demonstrated by Feher e t a1.68 (Table 7.10). Wittwergl

i n v e s t i g a t e d the i n f l u e n c e o f t h e v o l a t i l i z a t i o n o f amines i n the mobile

phase by t e s t i n g the same solvent system, containing amnonia i n various concentrations ( w i t h o u t changing the water content o f the mobile phase) i n combination w i t h a s i l i c a gel column. For the compounds tested, comnon adulterants

or i m p u r i t i e s o f i l l i c i t h e r o i n

samples, o n l y a few changes i n the e l u t i o n order were observed, p a r t i c u l a r l y f o r the e a r l y e l u t i n g compounds, and furthermore an increase o f r e t e n t i o n time was observed upon decreasing ammonia concentration (Table 7.11).

However, the r e l a t i v e r e t e n t i o n v a r i e d

l i t t l e f o r most t e s t compounds. The water content o f the mobile phase was found t o p l a y an important r o l e i n the s e l e c t i v i t y o f the system. Retention times were reduced consider a b l y on increase o f the water content of the mobile phase b u t some compounds were more a f f e c t e d than others. Therefore, the water content o f the mobile phase should be c o n t r o l l e d

307

TABLE 7.8 OF COMPOUNDS OF FORENSIC

RETENTION

RELATIVE

INTEREST

TO M O R F I N E ~ ~

Column P a r t i s i l 6 pm ( 2 5 0 ~ 4 . 6mn ID), mobile phase methanol - 2 M ammonia - 1 M ammonium n i t r a t e (27:2:1), f l o w r a t e 1 ml/min. d e t e c t i o n U V 278 nm (see a l s o Fig.7.16). Compound

Rrel

Compound

Amethocai ne Atropine Antazol i n e Benzocaine Benztropine Bromodi phenhydramine Butacaine Caffeine Chlordiazepoxi de Chl orpheni rami ne Chlorprmazi ne Cocaine D i ethazi ne Dextropoxyphene D i azepam D i hydromorphi ne Diphenhydramine Oxymorphone Ethopropazi ne Heroin Lignocai ne Meclophenoxate

0.58 2.35 1.21 0.45 2.88 0.65 0.58 0.52 0.48 1.02 0.67 0.51 0.64 0.49 0.45 1.57 0.64 0.68 0.61 0.69 0.46 0.57

Methadone Methapyri lene Methaqualone 6-Methyldihydromorphine

6-0-acetylisopropylmorphine 6-0-acetylmorphine Morphine Nalorphi ne Nicotine Ni trazepam Paracetamol Meperidi ne Phenacetin Phenbutrazate Phencycl idene Procaine Quinidine Quinine Salicylamide Strychnine Theophylline

Rrel 0.74 0.59 0.45 1.26 0.66 0.75 l.OO(8.9 m l ) 0.55 0.57 0.46 0.46 0.62 0.45 0.45 0.66 0.56 0.63 0.65 0.46 1.57 0.49

TABLE 7.9

OF THE OPIUM ALKALOIDS

RETENTION

RELATIVE

MORPHINE^^

TO

Conditions as i n Table 7.8 (see a l s o Fig.7.16) A1 k a l o i d

Rrel l.OO(0.9ml) 0.95 0.79

Morphine Codeine Thebai ne Papaverine Noscapine Narcei ne Cotarni ne Sihyaromorphi none

0.47 0.47 0.92 4.10 1.50

R

A1 k a l o i d

D i hydrocodeinone Ethylmorphi ne D i hydrocodei ne Oxycodone Protopine Laudanosine A c e t y l d i hydrocodeinone (thebacone)

1

1.32 0.87 1.44 0.60 0.61 0.68 0.74

TABLE 7.10

CAPACITY

FACTORS OF SOME OPIUM ALKALOIDS

IN STRAIGHT-PHASE

AND REVERSED-PHASE SEPARATIONS~~

S t r a i g h t phase HPLC-system: column, P a r t i s i l 5 ~m(250x4.5 mm I . D . ) , mobile phase methanol 2 M ammonia - 1 M ammonium n i t r a t e (30:2:1), f l o w r a t e 0.9 ml/min. Reversed phase HPLC-system: column, lOum S i l i c a RP18 ( 2 5 0 ~ 4 . 0nun I.D.), mobile phase, a c e t o n i t r i l e - 0.01 M anunonium carbonate (4:6), f l o w r a t e 2.5 ml/min. A1 k a l o i d Papaverine Noscapi ne Oxycodone Thebai ne Ethylmorphine Codeine

References p. 310

k' Straight-phase

0.0 0.04 0.54 0.85 1.12 1.21

k' Reversed-phase 5.07 17.0 5.27 15.6 4.64 2.97

Alkaloid

-

k' k' straight-phase reversed-phase

Morphine Dihydrocodeinone D i hydrocodei ne Dihydromorphinone Dihydromorphine

1.32 2.20 2.46 2.65 2.82

1.25 7.02 4.73 2.14 1.67

308

TABLE 7.11 EFFECT OF AMMONIA CONCENTRATION I N MOBILE PHASE ON THE RETENTION OF SOME COMPOUNDS RELATIVE TO HEROIN91

-

Column, UPorasil (300x4 mn I.D.), mobile phase cyclohexane (chloroform - methanol amnonia(800:200:1))(3:1) S1: 28% ammonia; S2: 14% ammonia; S3: 7% armnonia , f l o w r a t e 2.0 ml/min, d e t e c t i o n UV 254 nm. r e l a t i v e r e t e n t i o n vs. h e r o i n i n Solvent Systems Compound Methaqualone Diazepam Lidocai ne Noscapine Cocaine Papaverine Aminopyrine Benzocaine Meperidine Methapyri lene Methadone Caffeine Barbital Phenaceti n

S 1

0.38 0.40 0.41 0.44 0.46 0.48 0.54 0.60 0.63 0.64 0.68 0.72 0.75 0.75

S 2

S 3

0.35 0.37 0.34 0.39 0.37 0.44 0.51 0.54

0.33 0.35 0.33 0.36 0.38 0.41 0.49 0.52

0.62

0.62

0.64 0.69 0.68

0.66 0.75 0.66 0.61 0.68

0.65 0.71

Comoound Phenobarbital Tetracaine Acetylcodeine Antipyrine Heroin Procaine Acetylprocaine Codeine 6-0-acetylmorphine Quinidine Quinine Strychnine Morphine Retention time o f h e r o i n (sec)

s 1 0.78 0.79 0.82 0.90 1.00 1.32 1.56 1.98

2.06 2.21 2.48 2.86 5.72 323

5 2 0.67 0.76 0.81 0.86 1.00 1.28 1.54 1.90 2.05 2.39 2.66 3.00 5.35 353

s 3 0.63 0.77 0.81 0.82 1.00 1.34 1.60 1.94

2.11 2.44 2.67 3.03 5.56 377

thoroughly, and the water content o f methanol p a r t i c u l a r l y should be checked p r i o r t o i t s use. Hanseng5 separated opium a l k a l o i d s on s i l i c a gel w i t h both p o l a r and non-polar solvents. He suggested an ion-exchange type o f mechanism f o r the r e t e n t i o n o f t h e a l k a l o i d s i n the p o l a r solvent systems. The i n f l u e n c e o f the changes i n the s o l v e n t composition on the k ‘ o f various a l k a l o i d s i s shown i n F i g . 7.17. The i n f l u e n c e o f pH and t h e p o l a r i t y o f the amine i n the s o l v e n t system were tested. Increased r e t e n t i o n was observed on increase o f pH o r increased p o l a r i t y o f the amines. The more p o l a r amines gave r i s e t o some peak asymmetry. Umans e t a l . l 1 3 developed a separation on s i l i c a gel f o r t h e determination o f h e r o i n and i t s metabolites. The pH o f 7 o f the mobile phase: a c e t o n i t r i l e - methanol - (conc. ammonia - methanol (1:2)) - ( a c e t i c a c i d methanol(1:l)) (75:25:0.04:0.216) avoided problems o f column degradation and h y d r o l y s i s o f h e r o i n d u r i n g the analysis and t h i s mobile phase a l s o allowed the more s e n s i t i v e d e t e c t i o n a t 218 nm.

-

7.5. DETECTION Detection of the opium a l k a l o i d s i s u s u a l l y performed a t 254 nm and 280 nm. However, the i n t e n s i t y o f the absorption v a r i e s f o r each a l k a l ~ i d ~ ~ ’ ~ ~ ( T7.7). a b l eThe r a t i o between the absorbance a t 254 nm and 280 nm i s a reproducible constant, c h a r a c t e r i s t i c f o r each compound56. By using t h i s absorbance r a t i o i n combination with the r e l a t i v e r e t e n t i o n times i n three d i f f e r e n t HPLC systems. Baker e t al.(Table 2.2, 2.3)56 were able t o i d e n t i f y 95% o f a series o f f o r e n s i c drugs. The r e l a t i v e r e t e n t i o n times and absorption r a t i o s f o r 101 drugs were presented. A s i m i l a r method was used f o r t h e i d e n t i f i c a t i o n o f opiates by Soni and D ~ g a r(Table ~ ~ 7.5) and by L u r i e a t a1.lZ1; the l a t t e r authors, however, used

so9

the r a t i o o f absorbances a t 220 and 254 nm (Table 7.6). Although most i n v e s t i g a t o r s used 254 o r 280 nm as the wavelength o f detection, several preferred d e t e c t i o n a t ca. 220 nm92y969113'121

o r 235 nm102, because o f t h e

greater s e n s i t i v i t y . F e l l e t a1 .lo6 reported a rapid-scanning multichannel d e t e c t i o n method and i t s a p p l i c a t i o n t o the i d e n t i f i c a t i o n o f h e r o i n and r e l a t e d compounds. The d e t e c t o r was capable o f simultaneously recording a t three d i f f e r e n t wavelengths, w h i l e a u t o m a t i c a l l y c a p t u r i n g spectra on-1 i n e f o r storage and subsequent manipulation. The recorded absorption spectra could be t r a n s f e r r e d t o t h e i r second o r h i g h e r d e r i v a t i v e s t o improve the select i v i t y . Additional i n f o r m a t i o n could be obtained from absorbance r a t i o s , c a l c u l a t e d continuously throughout the chromatogram. The natural fluorescence o f the opium a l k a l o i d s has been used t o o b t a i n a s e l e c t i v e and s e n s i t i v e d e t e c t i o n method. I n t h i s way codeine has been detected ( t h e d e t e c t o r operating w i t h an e x c i t a t i o n wavelength o f 213 nm and a c u t - o f f f i l t e r f o r the emission 96 o f 320 nm)lo8 and morphine ( e x c i t a t i o n 290 nm, emission 340 nm)

.

Whealsz8, Ross42 and F r e i 5 5 have reviewed the p o s s i b i l i t i e s o f improving the d e t e c t i o n p r o p e r t i e s and s e l e c t i v i t y by means o f r e a c t i o n chromatography. Jane and Taylor"

used

the o x i d a t i o n o f morphine t o pseudomorphine f o r a s p e c i f i c and q u a n t i t a t i v e f l u o r i m e t r i c determination method f o r morphine i n u r i n e . Some morphine analogues a l s o produced fluorescent dimers, b u t they could be separated d u r i n g the HPLC. The o x i d a t i v e dimerizat i o n was performed on the top o f the HPLC column ( s i l i c a g e l ) by i n j e c t i o n o f a m i x t u r e o f the u r i n e e x t r a c t and 0.04 M potassium f e r r i c cyanide. By i n t r o d u c i n g dihydromorphine as a r e a c t i v e i n t e r n a l standard, the problem o f v a r i a b l e r e a c t i o n y i e l d s was overcome. Dihydromorphine undergoes the same o x i d a t i v e d i m e r i z a t i o n as morphine, b u t a m i x t u r e o f both a l k a l o i d s y i e l d s a mixed dimer. The three possible dimers were separated by HPLC and from the r e l a t i v e peak areas the morphine concentration could be determined. The r e t e n t i o n time of a s e r i e s of dimers i s given i n Table 7.12.

For a l a r g e number o f drugs i t was

reported t h a t no interference was observed when t h i s method was used. The absolute s e n s i t i v i t y determined by the d e t e c t i o n l i m i t o f the f l u o r i m e t e r was 4 ng. TABLE 7.12

RETENTION TIMES OF THE PEAKS PRODUCED BY OXIDATION, RELATIVE TO THAT OF THE DIHYDROMORPHINE DIMER (6.1 min)2O X

Worphi ne Normorphine Nalorphi ne 6-0-acetylmorphine 6-Methyldihydromorphine

X-X

X-dihydro Dihydrod i hydro

0.47 0.66 1.00 1.88 1.37 1.00 1.00 0.25 0.42 0.31 0.50 1.00 three peaks which are n o t resolved O i hydrohydroxymorphone 0.54 0.71 1.OO

X

X-X

X-di hydro D i hydrod i hydro

Oxymorphone retained Dihydromorphinone retained Pentazocine 0.38 0.57 Phenazocine 0.31 0.40 Paracetamol 0.32 0.45

-

1.oo 1.00

1.oo 1.oo 1.oo

Column, P a r t i s i l 7 pm, ( 2 5 0 ~ 4 . 6nun I.D.), mobile phase methanol 2 M anonia - 1 M anmonium n i t r a t e (3:2:1), f l o w r a t e 2 ml/min, detection, f l u o r i m e t e r ( e x c i t a t i o n 320 nm, e m i s s i o n 436 nm).

References p. 310

310 Nelson e t a l . l 1 4 used t h e o x i d a t i o n o f morphine t o pseudomorphine i n a post-column reactor. For the post-column d e r i v a t i z i n g reagent, a s o l u t i o n o f 50 mg potassium f e r r i c cyanide i n 250 m l o f 4 M a m n i a was used. Methanol 0.1 M aqueous potassium bromide

-

(12.5:87.5) was used as mobile phase f o r t h e separation o f t h e a l k a l o i d s on a octadecyl s t a t i o n a r y phase. The fluorescence was measured a t 432 nm a f t e r e x c i t a t i o n a t 323 nm. I n a d d i t i o n t o morphine, normorphine, dihydromorphine, nalorphine and 6-0-acetylmorphine gave fluorescent dimers. The r e s u l t s w i t h t h e post-column r e a c t i o n showed good agreement w i t h those obtained w i t h the method o f Jane and Taylor". F r e i e t a1.29 made the dansyl d e r i v a t i v e s o f some a l k a l o i d s i n order t o improve the detection l i m i t and t o save elaborated clean-up procedures i n the a n a l y s i s o f pharmaceut i c a l preparations. The dansyl d e r i v a t i v e s were prepared by adding an excess o f 0.1% dansylchloride i n acetone and 0.1 M sodium carbonate t o an aqueous s o l u t i o n o f the a l k a l o i d , heating a t 45' C f o r 30 minutes and subsequently e x t r a c t i n g t h e dansyl d e r i v a t i v e s w i t h benzene. The r e s u l t s o f an analysis o f a cough syrup containing codeine and noscapine i s given i n Fig. 7.15. I n c o n t r a s t t o these a l k a l o i d s , morphine does r e a c t w i t h d a n s y l c h l o r i d e t o y i e l d a mono-dansyl d e r i v a t i v e . B o l l e t e t a1.48 described a new electrochemical d e t e c t o r f o r HPLC t h a t was a p p l i e d i n the analysis o f noscapine i n pharmaceutical preparations using a straight-phase separation system. The s e n s i t i v i t y o f the d e t e c t i o n o f noscapine was compatible w i t h UV-detection, b u t i m p u r i t i e s could o n l y be detected by means o f t h e electrochemical detector. White 6 1 developed an electrochemical d e t e c t o r f o r the analysis o f morphine i n b i o l o g i c a l samples. Because o f i t s phenolic group, morphine was most susceptible t o electrochemical o x i d a t i o n and thus gave the l a r g e s t detector respons. Indoles and phenothiazines were o x i d i z e d l e s s r a p i d l y and consequently gave lower detector responses. Codeine gave no s i g n i f i c a n t response a t a1l.The method was q u i t e s p e c i f i c f o r morphine, the s e n s i t i v i t y was l e s s than 1 ng o f morphine using an e l e c t r o d e p o t e n t i a l o f +0.60V (vs. s i l v e r / s i l v e r c h l o r i d e reference electrode (SSCE)). Several authors reported the use o f commercially a v a i l a b l e electrochemical detectors f o r the a n a l y s i s o f morphine and some morphine antagonists, +O.8V7O. +O.725l1l, +O.79ll2 and +0.6-0.8V115 ( a l l vs. electrode p o t e n t i a l s o f +lV6', SSCE) were employed. Codeine i s n o t detected under such conditions. For the electrochemi89 cal detection of apomorphine an e l e c t r o d e p o t e n t i a l o f +0.5V (vs. SSCE) has been used . RLFERENCES 1 2 3 4 5

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312

75 R.R. Brodie, L.F. Chasseaud, L.M. Walmsley, H.H. Soegtrop and A. Darragh, J . Chromatogr. 182 (1980) 379. 76 S.R. Gautam, A. Nahum, J . Baechler and D.W.A. Bourne, J . Chromatogr., 182 (1980) 482. 77 R.G. Peterson, B.H. Rumach, J.8. S u l l i v a n and A. Makowski, J . Chromatogr., 188 (1980) 420. 78 Y. Nobuhara, S. Hirano. K. Namba and M. Hashimoto, J . Chromatogr., 190 (1980) 251 79 J.L. Love and L.K. Pannell, J . Forensic S c i . , 25 (1980) 320. 80 P.E. Nelson, S.M. F l e t c h e r and A.C. Moffat. J . Forensic S c i . SOC., 20 (1980) 195. 81 R.G. Achari and J.T. Jacob, J . Liq. Chromatogr., 3 (1980) 81. 82 D.N. Harbin and P.F. L o t t . J . Liq. Chromtogr., 3 (1980) 243. 83 U. Das Gupta, J . Pharm S c i . , 69 (1980) 110. 84 E.J. Kubiak and J.W. Munson. J . Pharm. Sci., 69 (1980) 152. 85 G.K. Poochikian and J.C. Cradock. J . Pharm. Sci., 69 (1980) 637. 86 C.Y. KO, F.C. Marziani and C.A. Janicki, J . Pharm. S c i . , 69 (1980) 1081. 87 E.F. Matantseva, P.P. Gladyshev, M.I. Gorgaev and G.A. Bektensva, Khim. Prir. Soedin, (1980) 730. CA 94 (1981) 127428s. 88 P.O. Roksvaag, J.B. Frederikson and T. Waaler, Phurm. Acta Hetu., 55 (1980) 198. 89 R.V. Smith and D.W. Humphrey, A n a l . L e t t . , 14 (B8)(1981) 601. 90'H.E. Harvey and R.M. Chell, Aust. J . Pharm. Sci., 10 (1981) 115. 91 J.D. Wittwer, Forensic S c i . I n t . , 18 (1981) 215. 92 P. M a j l a t , P. Helboe and A.K. Kristensen, I n t . J . Pharm., 9 (1981) 245. 93 P.B. Baker and T.A. Gough, J . Chromtogr. Sci., 19 (1981) 483. 94 W. Lindberg, E. Johansson and K. Johansson, J . Chromtogr., 211 (1981) 201. 95 S.H. Hansen, J . Chromatogr., 212 (1981) 229. 96 J.A. Glasel and R.F. Venn, J . Chromatogr., 213 (1981) 337. 97 G. Hoogewijs, Y. Michotte, J . Lambrecht and D.L. Massart, J . Chromatogr., 226 (1981) 423. 98 I.S. L u r i e and S.M. Oemchuk, J . Liq. Chromatogr., 4 (1981) 337. 99 I.S. L u r i e and S.M. Oemchuk, J . Liq. Chromatogr., 4 (1981) 357. 100 I.S. Lurie, J . Liq. Chromatogr., 4 (1981) 399. 101 E.F. Matantseva, P.P. Gladyshev and B.A. Yankowskii, Khim, Farm. Zh., 15 (1981) 117. 102 H. Cooper, A.C. Mehta and R.T. Calvert, Pharm. J . , 226 (1981) 682. CA 95 (1981)103248c 103 P.P. Gladyshev, E.F. Matantseva and M.I. Goryaev, Zh. A m Z . Khim., 36 (1981) 1130. 104 I.M. Beaumont, Anat. Proc. (London), 19 (1982) 128. 105 D. Bernhauer and E.F. Fuchs, Arch. KriminoZ., 169 (1982) 25. 106 A.F. F e l l , H.P. Scott, R. G i l l and A.C. Moffat, Chromatographia, 16 (1982) 69. 107 M.B. Escribano and J. B o a t e l l a Riera, Circ. F a r m . , 40 (1982) 89. CA 97 (1982) 50716111. 108 I . W . Tsina, M. Fass, J.A. Debban and S.8. Matin, CZin. C h e m . , 28 (1982) 1137. 109 T.A. Gough and P.B. Baker, J . Chromatogr. S c i . , 20 (1982) 289. 110 J.O. Svensson, A. Rane, J. Sawe and F.Sj6qvist. J . Chromatogr., 230 (1982) 427. 111 K. Ishikawa, J.L. Martinez and J.L. McCaugh, J . Chromatogr., 231 (1982) 255. 112 R.D. Todd, S.M. Muldoon and R.L. Watson, J . Chromatogr., 232 (1982) 101. 113 J.G. Umans, T.S.K. Chiu, R.A. Lipman, M.F. Schultz, S.U. Shin and C.E. I n t u r r i s i , J . Chromtogr., 233 (1982) 213. 114 P.E. Nelson, S.L. Nolan and K.R. Bedford, J . Chromtogr., 234 (1982) 407. 115 R.B. Raffa, J.J. O ' N e i l l and R.J. T a l l a r i d a , J . Chromatogr., 238 (1982) 515. 116 I. Beaumont and T. Deeks, J . Chromatogr., 238 (1982) 520. 117 B.C. P e t t i t t and C.E. Oamon, J . Chromatogr., 242 (1982) 189. 118 R.J. Flanagan, G.C.A. Storey, R.K. Bhamra and I . Jane, J . Chrornatogr., 247 (1982) 15. 119 L.W. Ooner and A.F. Hsu, J . Chrornatogr., 253 (1982) 120. 120 S.T. Chow, J. Forensic S c i . , 27 (1982) 32. 121 I.S. Lurie, S.M. Sottolano and S. Blasof, J. Forensic Sci., 27 (1982) 519. 122 G.W. Halstead, J . Pharm. S c i . , 71 (1982) 1108. 123 W.E. Warren and A.O'Adamo, J . Pharm. S c i . , 71 (1982) 1115. 124 B. Stuber and K.H. Mdller, Pharm Acta HeZu.. 57 (1982) 181. 125 I.M. Beaumont, Pharm. J . , 229 (1982) 39.

313

0

10

20 30 mtn

io

so

6

5

10

rnin

Fig. 7.1. Separation o f some opium a l k a l o i d s 4 Column Zipax SCX 37-44 um ( 1 2 0 0 ~ 2 . 1mm ID), mobile phase 0.2 M sodium hydroxide, b o r i c a c i d added t o pH 9.5, 0.2 M KNO w i t h 4% a c e t o n i t r i l e and 1%n-propanol. l i n e a r v e l o c i t y 0.4 cm/sec, detection UV 254 nd. Peaks: 1. morphine; 2, codeine; 3, papaverine; 4. thebaine; 5, cryptopine; 6, noscapine. 18 Fig. 7.2. Separation o f some c o n s t i t u e n t s o f i l l i c i t h e r o i n samples Column Zipax SCX 37-44 pm ( 1 0 0 0 ~ 2 . 1mn ID), mobile phase gradient e l u t i o n w i t h A: 0.2 M b o r i c a c i d adjusted t o pH 9.3 w i t h 40% sodium hydroxide and B: 0.2 M b o r i c a c i d - a c e t o n i t r i l e n-propanol (86:12:2) adjusted t o pH 9.8 w i t h 40% sodium hydroxide, l i n e a r g r a d i e n t from 0-100% B i n 6 min, flow r a t e 2 ml/min, d e t e c t i o n UV 270 nm. Peaks: 1, barbitone; 2, c a f f e i n e ; 3, morphine; 4, 0-acetylmorphine; 5, strychnine; 6. heroin; 7, quinine; 8, cocaine.

I 0

,

10

, 20

mn

Rcferenca p. 310

,

M

, 10

Fig. 7.3. Separation o f major opium a l k a l o i d s 3 5 Column VBondapak C18 (300x4 mm ID), mobile phase 0.1 M sodium dihydrogen phosphate i n a c e t o n i t r i l e - w a t e r ( l : 3 ) . pH4.8, f l o w r a t e 1.25 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, morphine; 2, codeine; 3, thebaine; 4, papaverine; 5, noscapine. (Reproduced w i t h permission from r e f . 35, by courtesy o f the American Chemical Society)

314

6 2

l J i

d 8

L

12

16

20

24

min

0

L

8

12

16

20 min

F i g . 7.4. S e p a r a t i o n o f some opium a l k a l o i d s .78 Column N u c l e o s i l 1OC18 (300x4 mm I D ) , m o b i l e phase 1%ammonium a c e t a t e (pH 5.8) - a c e t o n i t r i l e (65:35), f l o w r a t e 1.5 ml/min. Peaks: 1. morphine; 2, codeine; 3, c r y p t o p i n e ; 4, t h e b a i n e ; 6, noscapine. 5, papaverine; . . Fig.7.5. S e p a r a t i o n o f some opium a l k a l o i d s 78 Column N u c l e o s i l lOCN (300x4 mm ID), m o b i l e phase 1%ammonium a c e t a t e (pH 6 . 3 ) - a c e t o n i t r i l e - dioxane (79:16:5), f l o w r a t e 1.5 ml/min. Peaks: 1, n a r c e i n e ; 2, morphine; 3, codeine; 4, c r y p t o p i n e ; 5, thebaine; 6, noscapine; 7, p a p a v e r i n e .

I"

F i g . 7.6. S e p a r a t i o n of h e r o i n and some common a d u l t e r a n t s and c o n t a m i n a t i o n s 93 Column amino-propyl bonded s i l i c a (S5NH )(Phase-Sep) (250x4 mrn I D ) , m o b i l e phase a c e t o n i t r i l e - 0.005 M t e t r a b u t y l a m n o n i u m phosphate f85:15), f l o w r a t e 1 ml/min, d e t e c t i o n U V 284 nm. Peaks: 1, noscapine; 2, papaverine; 3, c a f f e i n e ; 4, h e r o i n ; 5, a c e t y l c o d e i n e ; 6, 6 - 0 - a c e t y l morphine; 7, codeine; 8, s t r y c h n i n e ; 9, morphine. (Reproduced w i t h p e r m i s s i o n f r o m r e f . 93, by t h e c o u r t e s y o f J o u r n a l Chromatographic Science)

315

Fig. 7 . 7 . Separation of some opium a l k a l o i d s 119 Column Zorbax NH ( 2 5 0 ~ 4 . 6mn I D ) , mobile phase a c e t o n i t r i l e - 0?025 M potassium dihydrogen phosphate ( 3 : 1 ) , flow r a t e 2.0 ml/min, detection UV 286 nm. Peaks: 1, papaverine; 2 , thebaine; 3, narceine; 4, codeine; 5, morphine; 6 , tyrosine ( i n t e r n a l standard).

1

2

0

6

L

min

B

9 L

ij

c

I

rnin

10

8

6

L

2

0

10

1

20

20 rnin

Fig. 7.8. Separation of heroin and some common a d u l t e r a n t s and contaminations 105 Column VBondapak C18 (300x4 nm ID), mobile phase a c e t o n i t r i l e - water (91.5:8.5) containing 8 mg tris(hydroxymethy1)aminomethane per 100 ml, flow r a t e 2 ml/min, detection U V 254 nm. Peaks: 1, ascorbic a c i d ; 2 , c a f f e i n e ; 3, papaverine; 4 , noscapine; 5, 6-0-acetylmorphine; 6 , heroin; 7 , morphine; 8, acetylcodeine; _ _9 -, acetylprocaine; 10 procaine. Fig. 7.9. Separation of opium alkaloids"' (70x2 mm I D ) , mobile Column VBondapak Phenyl (250x5 mm I D ) , guard column Corasil C18 37-50 Dhase A: a c e t o n i t r i l e - water (5:95) and B : a c e t o n i t r i l e - water (20:EO) both containing 1 ml/l N,N-dimethyloctylamine (pH 3.5'with sodium hydroxide), l i n e a r g r a d i e n t from A t o f i n 20 min, flow r a t e 1.0 ml/min, detection UV 275 nm. Peaks: 1, morphine; 2 , codeine; 3, thebaine; 4, quinine; 5, papaverine; 6 , noscapine. Chromatogram A poppy-straw concentrate, B standard mixture.

References p. 310

316

2

, 0

15

45

30

60

min

F i g . 7.10. Separation a l k a l o i d s i n opium e x t r a c t 70 Column H i t a c h i g e l 3010 10 urn ( 2 2 0 ~ 4 . 6mn I D ) . mobile phase a c e t o n i t r i l e - 0.019 M p n o n i a (48:52), f l o w r a t e 1 ml/min, temperature 65 C, d e t e c t i o n UV 254 nm. Peaks: 1, morphine; 2, codeine; 3, papaverine; 4: thebaine; 5, noscapine. (Reproduced w i t h permission from r e f . 70, by courtesy o f the American Chemical Society).

I r

I

0

5

10

15min

Fia. 7.11. SeDaration o f some oDium alkaloids3' Coiumn dondapak C18 (300x4 nun iO), mobile phase 0.005 M heptanesulfonic a c i d i n nethanol water - a c e t i c a c i d (40:59:1) (pH ca. 3.5). f l o w r a t e 2 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, morphine; 2, codeine; 3. thebaine; 4, noscapine; 5, papaverine. (Reproduced w i t h permission from r e f . 38, by courtesy o f Journal Association o f f i c i a l a n a l y t i c a l chemists)

A

Fig. 7.12 Separation o f major opium alkaloidss4 Column PPorasil (300x4 mm I D ) , mobile phase n-hexane - dichloromethane - ethaaiethylamine (300:30:40:0.5), flow nol r a t e 2.4 ml/min, d e t e c t i o n UV 285 nm. Peaks: 1, noscapine; 2, papaverine; 3. thebaine; 4 , codeine; 5, morphine. Chromatogram A P a p a v e r bracteatum e x t r a c t , B P a p a v e r somniferum e x t r a c t . (Reproduced w i t h permission from r e f . 54, by courtesy o f Journal Association o f o f f i c i a1 a n a l y t i c a l chemists)

-

I

I

1

I

0

7

1L

21

1

28 0

1

1

7 min

I

14

I

r

21

28

317

18

1

12.13

L

0

4

8

12

2Omin

16

r.

-20

5

10

15 20

min

Fig. 7.13. Separation a l k a l o i d s from poppy e x t r a c t 3 ' Column Spherisorb 5 m (250x3 mn ID), mobile phase methanol - chloroform - amnonia (18:81.5: 0.5), f l o w r a t e 0.6 ml/min, d e t e c t i o n UV 280 nm. Peaks: 1, morphine; 2, cinchonine ( i n t e r n a l standard). (Reproduced w i t h permission from r e f . 51, by courtesy o f Norsk Farmaceutisk Selskap). Fig. 7.14. Separation o f some a l k a l o i d s and drugs6' Column P a r t i s i l 5 urn ( 2 5 0 ~ 4 . 6mn ID), mobile phase d i e t h y l e t h e r 95% saturated w i t h water t 0.4% diethylamine, f l o w r a t e 2 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, noscapine; 2, aconitine; 3, papaverine; 4, emetine; 5, ephedrine; 6, cephaeline; 7, scopolamine; 8, ethylmorphine; 9. codeine; 10, phenytoine; 11 homatropine; 12, N-methylephedrine; 13, narceine; 14, quinine; 15, caffeine; 16, strychnine; 17, sulfanilamide; 18, atropine; 19, phenobarbital,

i

Fig. 7.15. Analysis o f a l k a l o i d s i n cough yrup a f t e r d e r i v a t i z a t i on w i t h dansyl -chl o r i de2$ Column Lichrosorb S i l o 0 10 um ( 2 5 0 ~ 2 . 8n I D ) , mobile phase d i i s o p r o p y l e t h e r isopropanol conc. amnonia (48:2:0.3), d e t e c t i o n UV 254 nm. Peaks: 1. Dns-ephedrine; 2. noscapine; 3. Dns-cephaeline; 4, Dns-emetine; 5, codeine; t , dodecylbenzene. Fluorescence d e t e c t i o n w i t R e x c i t a t i o n 360 nm and emission 500-510 nm.

-

->

,

1 6 U 7

6

Reference p. 310

,

,

5 4 min

,

,

,

1

3

2

1

0

-

A

1

B

3

min 1'5

I

10

15

I

10

5

Fig. 7.16. Straight-phase HPLC separation o f opium a l k a l o i d s L L Column P a r t i s i l 6 pm ( 2 5 0 ~ 4 . 6n ID), mobile phase methanol - 2 M a n o n i a - 1 M a n o n i u m n i t r a t e (27:2:1). f l o w r a t e 1 ml/min. d e t e c t i o n U; 278 nm. A. Opium sample, peaks: 1, noscapine; 2, thebaine; 3. codeine; 4. morphine. 8. Chinese Heroin" sample, peaks: 1, c a f f e i n e ; 2, heroin; 3, 0-acetylmorphine; 4, morphine; 5, strychnine.

c

: i,

Fig. 7.17. P l o t s o f the r i t y o f the mobile phase @= codeine; O= morphine; Column Lichrosorb Si60 5

6;

values o f opiates obtained on unmodified s i l i c a versus the pola-

v= pin

normorphine; o= noscapine; A = papaverine; (150~4.65 mn ID).

a=

thebaine.

TABLE 7.13

I

? Iu

HPLC ANALY IS OF VARIOUS COMPOUNDS INCLUDING OPIUM ALKALOIDS

*

ALKALOIDS

0

C.M. Na ,No, P ,T 18 o t h e r s

OTHER COMPOUNDS

.

AIMS

STATIONARY PHASE

COLUMN D I M . LxID (mm)

MOBILE PHASE

A n a l y s i s a1 k a l o i d s Merckosorb Si60 5 300x2 CHCl3-Me0H(9:l).(8:2),(7:3) (Table 2.4) Et20-MeOH( 8: 2), (7: 3), (6 :4) Separation on ion-exchange Hydrolyzed Porhgel PT 0.06M NH40H i n 33% E t O H r e s i n s ( 1igand-exchange LC) loaded w i t h Cu o r N i 2+ 4 7 0 ~ 6 . 3

C ,M ,No, P ,EtM,Me t h , c0c.A.S ,ni c

.

C .M ,No, P, E t M ,Meth Q, cinchonine.coc,A.S,nic

Separation on ion-exchange Hydrolyzed Por/qel PT r e s i n s (ligand-exchange LC) loaded w i t h Cu Bio-Rad PC20 loaded w i t h Cu2+

0.06M 4 7 0 ~ 6 . 3 0.2 M 0.05M 4 7 0 ~ 6 . 3 0.03M

C,Na,B,aconitine.caf, Santonine c o l c h i c i n e ,ci nchonidine

E f f e c t s o l v e n t composition L i c h r o s o r b RP2 10 um on r e t e n t i o n

1 2 0 ~ 3 . 5 MeOH-H20 (1:4),(2:3).(3:2),(4:1) MeOH

C,P,A,scop.Qd,caf

C,M,diHC,Mep,prop,A, ajmaline,caf,Tp,Q.Qd

Various drugs Retention behaviour b a s i c drugs i n i o n - p a i r HPLC

Various drugs

VBondapak C18 VBondapak Phenyl pBondapak CN ,,Bondage1 Chromegabond C8 Chromegabond C6Hll

Separation b a s i c d r u g s w i t h S y l o i d 74 s i l i c a non-aqueous i o n i c s o l v e n t s on s i l i c a g e l Spherisorb S5W s i l i c a

300x4

NH OH i n 33% E t O H NH40H i n 33% E t O H NH40H i n 33% E t O H NHO ;H i n 33% E t O H

REF.

12 13

19,26 66

0.005M h e p t a n e s u l f o n i c a c i d i n MeOH-H20-AcOH(49:50: 1 ) (pH 4.0)

81 2 5 0 ~ 4 . 9 MeOH o r MeOH-CHC1 (2:3) c o n t a i n i n g 1.42 m l H C l O and33.68 m l conc. NH OH p e r 1 PpH 9.2) 2 5 0 ~ 4 . 9 Me$H-hexane(85:15) c o n t a i n i n g 0.02, 0.05 o r 0.10% HC104 118

TABLE 7.14 HPLC ANALYSIS I N THE PURITY CONTROL OF OPIUM ALKALOIDS ~~

ALKALOIDS* C,M,H

,AcC ,30AcM ,60AcM

AIMS

STATIONARY PHASE

P u r i t y p r o f i l e s M, H, MHCl

Zipax ETH Zipax OOS Zipax SCX

*For a b b r e v i a t i o n s see f o o t n o t e Table 7.18

COLUMN DIM. LxIO(mm)

MOBILE PHASE

1000~2.2 MeOH-H 0(4:6) 1 0 0 0 ~ 2 . 2 pH 8.62phosphate b u f f e r 1000~2.2 0.4-1.4M NaClO i n 0.01M pH 6.8 phosphate b u f f a r c o n t a i n i g 10% EtOH ,gradient e l u t i o n

REF.

7

w 0 N

C,M.Eph

S t a b i l i t y control

londapak C18

300x4

M,H,30AcM,60AcM M.P.H,OAcM

Study o f the hydrolysis o f H S t a b i l i t y H i n CHC13-H20

aondapak C18 llBondapak C18

M. H, 60AcM

S t a b i l i t y H i n CHC13-H20 a t various pH

Hypersil ODS 10

300~3.9 ACN-0.015M KH2PO4 (pH 3.5)(3:7) 300x4 ACN-phosphate b u f f e r (pH 3.5) (85: 15) 100 0.01M Na-pentanesulfonate-ACNH3PO4(69.5:30.O:0.5)(pH 2.0)

w

0.1M KH2P04-MeOH(9:l) 0.1M KH2P04-EtOH(52.5:47.5)

31 57 102 104,125

TABLE 7.15 HPLC ANALYSIS OPIUM ALKALOIDS I N PLANT MATERIAL AND OPIUM

*

ALKALOIDS

AIMS

STATIONARY PHASE

C.M.No,P.T.crypt

Separation by means o f ion-exchange LC (Fig.7.1)

Zipax SCX 37-44

C.M.No.P.T.Losine,crypt

Determination M i n opium

Zipax SAX

T,isoT,orientalidine

Determination T i n poppy plants

Corasil I 1 37-50

C, M ,No, P ,TI crypt

Separation opium alkaloids

C. M. No, P ,T, cryp t .B

COLUMN DIM. L x ID (nm)

w

MOBILE PHASE

REF.

1000~2.1 (0.2M NaOH, b o r i c acid added t o pH 4 9.5, 0.2M KN03)-ACN-prOH(95:4:1) 1000~2.1 A. 0.01M b o r i c acid b u f f e r pH 9.5 w i t h 1M NaOH B. 0.01M KH2P04 b u f f e r pH 6.0 w i t h 1 M NaOH gradient A+B(85:15) t o B (5% o r 10% per min, l i n e a r )

6

pm

300~2.8 n-Hexane-CHCl3-MeOH-OEA(90O:75:25: 1)

8

Corasil I 1 37-50

pm

500~2.8 A. CHCl -MeOH-DEA(lDO:300:1) B. Hexaje special gradient system s t a r t w i t h 30 m l A i n 3 1 B 10

Simultaneous assay alkaloids i n opi um

Corasil I 1 37-50

urn

C.M.Na.No,P,T,cot,Losine.prot

Separation alkaloids i n opium (Table 7.8.7.9 and Fig.7.16)

P a r t i s i l 6 urn

C, M, No, PIT. crypt

Performance o f new short chain reversed phase packing

S i l i c a gel SAS 6

C.M.NosPsT C.M.No,P,T

Separation alkaloids from opium

Partisil 5

250~4.9 MeOH-2M NH40H-1M NH4N03(27:2:1)

23 32

Determination C. M and T i n opium (Fig.7.3)

VBondapak C18

300~4.0 0.1M NaH2P04 i n ACN-H20(5:95) 0.1M NaH PO i n ACN-H20(25:75) pH adjus?ed4to 2.0. 4.8 o r 7.1

35

n.

1000~2.8 as i n 10, gradient s t a r t s w i t h 15 ml A i n 1 1 B

17

250~4.6 MeOH-2M NH40H-1M NH4N03(27:2:1) 22 prn

125x5

0.025M NH40H i n MeOH-H20(1:1)

C.M.No,P.T C.M.T,i

Separation a l k a l o i d s from opium (Table 7.3 and Fig.7.11)

soT,orientalidine

VBondapak C18

Determination T i n P a p a v e r b r a e VBondapak C18

300x4 300x4

0.005M Heptanesulfonic a c i d i n MeOH-H,O-AcOH( 40:59: 1)(pH 3.5) -

38

MeOH-0.3% aq. (NH4)2C03(4:1)

41

teatum

C ,No ,P .T

Separation

M.cinchonine

Spherisorb ODS 10 pm Determination M i n aqueous and o r g a n i c poppy e x t r a c t s (Fig.7.13) Spherisorb s i l i c a 5 ,m

P o l y s t y r e n e beads

M

D e t e r m i n a t i o n M i n poppy straw

VBondapak C18

c ,M,

Determination o f a l k a l o i d s i n

VPorasil 5

no d e t a i l s MeOH-!-hexane-28% 250x3 250x3

2 columns

NH40H(97:2:1)

MeOH-0.05M aq.(NH ) CO (1:l) CHCl3-MeOH-NH40H($1?5:38:0.5) 0.1M NaH2P04 i n ACN-H20(6:94)

300x4 Na, No, P,T, isoT,prot.cryPt. Losi ne, o r i pavi ne, s a l u t a r 1 d i ne, a l p i nigenine,gnoscopine C.M.No,P.T C,M,No,P,T,EtM,A,Eph,RSP,S,C,Q, yohimbine,acridine

Urn

54

A n a l y s i s a l k a l o i d s i n opium Partisil 7 250~4.5 (Table 7.10) S i l i c a RP18 10 urn 250~4.0 Separation a l k a l o i d s on s t y r e n e - H i t a c h i g e l 3010 10 urn 2 2 0 ~ 4 . 6 d i v i n y l -benzene polymer (Figs. 7.10 and 8.6)

C.M,Na,No,P,T,crypt

Determination a l k a l o i d s i n opium N u c l e o s i l l O C N (Table 7.2, Figs.7.4 and 7.5)

L i c h r o s o r b Si60 5 p m

Nucleosi 1 10C18

Q

C.M, Na, No, P .T

52

n-Hexane-CH2Cl2-EtOH-DEA(30O:30:

rum (Table 7.7.Fig.7.12)

A n a l y s i s M i n opium

C .M, No, P, T,

51

q0:0.5)

Papaver b r a c t e a t u m and P . s o m n i f e -

C.M,T,norM

C,M,No,P,T

300x4

46

Determination i n raw m a t e r i a l s

Bondapax CX/Corasil

A n a l y s i s o f poppy straw concent r a t e (Fig.7.9)

UBondapak Phenyl

Zorbax NH2 A n a l y s i s P a p a v e r somniferum p l a n t m a t e r i a l and l a t e x ( F i g . 7 . 7 )

MeOH-2M NH OH-1M NH NO (30:2:1) ACN-0. 005M4aq. ( NHI)%COi( 4:6)

.-

68

I

ACN-O.02M NH OH(3:2) ACN-O.02M te?rabutylamnoni urn hydrox i d e (3:7),(2:3) 70 1 5 0 ~ 4 . 6 1,2-Dichloroethane-MeOH-AcOH-DEAH20(80:20:1:0.5:1) 71

1%aq. NH OAc(pH 6.3)-ACN-dioxane (79: 16:5)4 1%aq. NH40Ac(pH 5.8)-ACN-dioxane f8:l:l) i%aqI'NH OAc(pH 5.8)-ACN(4:1), (3:2), ( 7 :$) 1%aq. NH OAc(pH 5.8)-ACN(7:3), 300x4 ( 3 :2 ) ,(65435 ) 78 1 0 0 0 ~ 2 . 1 ACN-phosphate b u f f e r ( p H 4.5)(3:7) 103 A. ACN-H 0(5:95) 250x5 B. ACN-H{0(20:80) b o t h c o n t a i n i n g l m l / l AcOH and 0.04 m l / 1 N, N-d i m e t h y l o c t y 1ami ne 117 L i n e a r g r a d i e n t : A t o B(20 min) 2 5 0 ~ 4 . 6 ACN-0.025M KH2P04(3:1) 119 300x4'

*For a b b r e v i a t i o n s see f o o t n o t e Table 7.18 w

E!

TABLE 7.16

w

N

HPLC ANALYSIS OPIUM ALKALOIDS I N PHARMACEUTICAL PREPARATIONS

AIM

ALKALOIDS*

OTHER COMPOUNDS

OxyC,caf,hmtropine

Acsa1,phen. hexobarb

C.diHCone,Eph,S, methylhomatropine

Various a n t i - A n a l y s i s o f cough-cold mixt u s s i v e s ,anti - t u r e s h i s t a m i n i c s and analgesics

C o r a s i l C18 o r C o r a s i l Phenyl

Promethazine. phenobarbi t a l

A n a l y s i s pharmaceuticals

Spherosil 5

C,M,No,cephaeline, emeti ne, Eph

Separation as d a n s y l d e r i v a t i v e s (Fig.7.15)

S i l i c a gel Silo0

M

Determination M i n p a r e g o r i c VBondapak C18 USP

STATIONARY PHASE

COLUMN DIM. LxID(mn)

MOBILE PHASE

REF.

1 0 0 0 ~ 2 . 1 1.5M Na SO 0.005M $NO:

Q u a n t i t a t i v e a n a l y s i s m u l t i - Zipax WAX component drugs

pm

9

1 2 2 0 ~ 2 . 3 ACN-0.1% a;.(NH ) CO (pH 8.5)(1:1) ACN-0. 1%aq. ( NH4)2C03(pH 8.6) (3: 2 ) ACN-0. 1%aq. ( NH4)2C03(pH 8.9) (9: 1) ACN-1% aq.(NH )dA2 (3H 7.04)(1:4) ACN-1% aq.(NH4)OAc (pH 7.4)(3:2) ACN-1% aq.(NH:)OAc (pH 7.58)(4:1) 14 1 0 0 ~ 4 . 8 EtOAc-MeOH (9: 1) 1 5 0 ~ 4 . 8 EtOAc-MeOH-50% aq. ethylamine (97: 2.94: 0.06)

25

2 5 0 ~ 2 . 8 ( I s o p r ) 0-isoprOH-conc.NH OHQ8:2:0.3) ( I s o p r ) 2 0 s a t . w i t h c0nc.~NH~0HisoprOHf99: 1 ) 29 300x4

0.1M KH2P04 b u f f e r i n H20-MeOH (93:7)

33

No,Dmethphan.caf

A n t i t u s s i v e s . A n a l y s i s a n t i c o l d drugs expectorants, antihistaminics

Porous s t y r e n e - d i v i n y l no detailsMeOH-NH40H benzene polymer o r polymethacrylate g e l

C,P,hthphan,atropine, homat r o p i ne,S, Q, Qd ,Had, scopo .eph, xanthines

Various drugs

A n a l y s i s pharmaceuticals

P a r t i s i l 10

Separation (Table 7.4)

VBondapak C18

300x4

MeOH-H 0(1:1),(2:3) containing 0.005M2heptanesulfonic a c i d idem i n ACN-H20(1:3)

40

A n a l y s i s expectorants

VBondapak C18

300x4

0.05M KH2P04 i n H70-MeOH(87:13)

43

2 5 0 ~ 4 . 6 CH2C12-MeOH(1:3)

36,37 w i t h 1%conc.NH40H 39

C,M,T,H,oripavine, 16 s y n t h e t i c d e r i vatives C

pm

Various drugs

No,Dmethphan

A n t i t u s s i v e s . A n a l j s i s pharmaceuticals expectorants, antihistaminics

Polymethacrylate

No

Analgesics

P a r t i s i l 5 pm

A n a l y s i s w i t h electrochemical detector

no d e t a i l s

~.

MeOH-28% NH40H(99:1) 45

1 5 0 ~ 4 . 8 EtOAc-MeOH-H 0-ethylamine (782: 200: 6: 23

48

M

Methylparaben A n a l y s i s M i n i n j e c t a b l e s propylparaben

uBondapak C18

300x4

A. MeOH B. 0.1% aq.NaH PO s o l u t i o n cont a i n i n g 5% MeOfi(pfi 4.0), A+B(3:2)

A n t i t u s s i v e s , Separation on porous polymer expectorants, r e s i n s antihistaminics various o t h e r s I d e n t i f i c a t i o n i n pharmaC,Na .No .P, EtM.Q .S, c a f .A,Eph ,aconi t i ne, c e u t i c a l s (Fig.7.14) scopo,homatropine, emeti ne,cephael ine

Styrene-divinylbenzene 500x5 methyl m e t h a c r y l a t e co- o r polymer, s u b s t i t u t e d 500x3 w i t h hydroxyrnethyl groups P a r t i s i l PXS 5/25

2 5 0 ~ 4 . 6 E t 0 s a t . w i t h 50-100% H20 0.8% DEA

C.M.No.P,T.EtM,oxyC, diHC,diHCone,diHM, d i HMone ,c a f

A n a l y s i s pharmaceuticals

P a r t i s i l 7 um S i l i c a RP18 10 um

2 5 0 ~ 4 . 4 MeOH-2M NH OH-1M NH NO (30:2:1) 250x4 ACN-0. 005M4aq. (NH4j2Ca3 (4: 6)

A n a l y s i s analgesics

uBondapak C18

300x4

A n a l y s i s syrups

uBondapak C18 o r RPlOA

No.caf.hethphan

59

+

0.05-

60 68

Acsal.Salam, phen,par

C,caf

50

MeOH-NH OH(99:l) MeOH-H28-NH40H(95:5: 1)

C,M.EtM

0.01M KH PO b u f f e r i n H20-MeOH (81:19) $H 9.3 o r 4.85

83 300x4 0.01M NH NO i n ACN-H 0(375:625) 2 5 0 ~ 4 . 6 c o n t a i n i flg d.005M d i o ? t y l s u l fosucc i n a t e , pH 3.3 w i t h AcOH 84 ACN-O.015M Na2HP04(pH 3 .O) (1:3 ) 300x4 85 2 5 0 ~ 2 . 1 CH C 1 -MeOH-2-hexane-NH40H(232:44: 723: If 86

M,H,COAcM.coc, benzoylecgonine

Benzoic a c i d

S t a b i l i t y H and COC i n phar- uBondapak C18 maceutical dosage form

C,M,apoM.nalo,nalox

Par,4-aminophenol

A n a l y s i s pharmaceuticals

L i c h r o s o r b Si60 10 um

Analysis i n i n j e c t i o n s

P a r t i s i l SCX

2 5 0 ~ 4 . 6 0.7% KH PO i n MeOH-H 0(1'3)(pH 2.0) A. 0.15% PO i n Me$H-H'0(1:19) B. 1.5% KH $0 4 i n MeOH-H 6(2:3) l i n e a r g r a 8 i e f l t A t o B (PO% B/min) 88

Determination i n t a b l e t s

UBondapak Phenyl

300x4

M,pseudoM.M

N-ox,

COC

Ap0M.N-2-propyl norapoM Chlorocresol, methyl 4-hydroxybenzoate C ,M ,No, P ,T ,met hy 1homatropine

C,M.No M.H.60AcM.M pseudoM

N-ox.

d

THF-0.05M KH PO 0.001M methanes u l f o n a t e ( 15?85j' MeOH- ACN-O.05M KH2P04( 5: 15:80)

Determination M i n i n j e c t i o n s L i c h r o s o r b RP18 10

1 0 0 ~ 4 . 6 ACN-0.75% aq. NH40Ac( pH 7.0) (3: 7), (1:4)

Determination i n t a b l e t s and i n j e c t i o n s Determination i n pharmaceuticals

1 2 0 ~ 4 . 6 ACN-O.01M aq. phosphate b u f f e r (pH 5.0)(2:3) (2x) 5 0 0 ~ 2 . 1 ACN-O.lM KH2P04(pH 4.8)

89

90

Determination degradation products M and H

N u c l e o s i l 5C8 Eondapak CX/Corasi 1

101 H y p e r s i l ODS 5 y m U l t r a s p h e r e ODS 5 urn

*For a b b r e v i a t i o n s see f o o t n o t e Table 7.18

92

100x5

0.01M aq. pentanesul fonate-ACN-H3P04 (69.5: 30:O.S) (pH 2.0) 2 5 0 ~ 4 . 6 idem (69.95:30:0.05)(pH 2.6) 116

rp

g

0

P Lo

C,Eph,hnethphan

Antitussives, antihistaminics

Determination i n cough-cold preparations

uSondapaK C18

300x4

5.89 Na d i o c t y l s u l f o s u c c i n a t e i n MeOH-H 0-THF-85% H3P04(680:290: 40:1)(1H 3.8)

C,diHCone,diHMone

Par

Determination i n t a b l e t s

,,Bondapak C18

300x4

MeOH-O.01N KH2P04, O.05M KN03 b u f f e r pH 4.5(1:3) 123

C

Par,acsal

Determination i n t a b l e t s and s u p p o s i t o r i e s

N u c l e o s i l 10C18

300x4

MeOH-H 0(9:16) c o n t a i n i n g Na o c t y l sulfonite 124

122

TABLE 7.17 HPLC ANALYSIS OPIUM ALKALOIDS I N BIOLOGICAL MATERIAL ALKALOIDS

OTHER COMPOUNDS

AIMS

STATIONARY PHASE

COLUMN D I M . LxID(mn)

MOBILE PHASE

REF.

~~

C.M,coc.caf,Tp

Detection i n u r i n e

M,di HM

D e t e c t i o n i n u r i n e u a b l e 7 . 1 2 ) P a r t i s i l 7 urn

Opiates

Opioid peptides

BOP(no f u r t h e r d e t a i l s )

I o n - p a i r chromatography w i t h VBondapak C18 TFA

Heptane-prOH(9: 1) 16 20,28 2 5 0 ~ 4 . 6 MeOH-2M NH40H-1M NH NO (3:2:1) 4. 3 5mM TFA(pH 2.5) i n l i n e a r g r a d i e n t 300x4 o f 30-50% ACN(10 min, 2ml/min) 1 M HCOOH-MeOH(1:l) 5mM TFA-MeOH(1:l) 49 MeOH-ACN-buffer(0.02M KH PO -0.03M c i t r i c acid, pH 3.25, c o i t a l j n i n g 0. O O l M dodecyl sul f a t e ) (36: 9 :55 1 53,58,62

ApoM, N-n-propyl norapoM ,ap& isoa poC, boldine

Determination i n serum

uBondapak Phenyl

300x4

Various drugs C, H. M, oxyM, 60AcM, d i HM,di HMone ,Medi HM, Nalox, keto, lev,norlev, dextrorphan.cephae1 ine, p s i 1o c i n,caf ,LSD

Electrochemical d e t e c t i o n , analysis M i n blood

S i l i c a S y l o i d 74 7 um

2 0 0 ~ 4 . 6 MeOH-NH4N03 b u f f e r pH 10.2(9:1)

ApoM

Determination i n plasma o r tissues

L i c h r o s o r b S i l o 0 10 um

200x3

Barbiturates

XAD2 e x t r a c t i o n method

XAD2

Various sol vents

65

Chlorpheni r a mine

Determination i n plasma

P a r t i s i l OOS 10 urn

MeOH-1% aq. AcOH and 0.005M hept a n e s u l f o n i c a c i d (55:45)

67

Par,benzodiazepi nes

Determination M i n serum, electrochemical d e t e c t i o n

L i c h r o s o r b RP18

300x4

MeOH-O.01M KH2P04(85:15)

C,M,diHCone

Determination i n u r i n e

Spherisorb ODS 5 um

250x4

0.1M NaH2P04 i n ACN-H20(1:3)

P ,ethavari ne

Determination i n plasma

P a r t i s i l ODS 10

2 5 0 ~ 4 . 6 MeOH-0.1% aq.KH2P04(65:35)

C ,M .na l o , Meth

61 CH2C12-MeOH-1M HC104(956:40:4) 63

69 72 75

r

2 B P

P 0 4-

0

P,Losine,caf,Tb,Tp

C h l o r t h i a z i d e s Determination i n u r i n e and t e t r a c y c l i n e s plasma Amperometric d e t e c t i o n , det e r m i n a t i o n i n plasma

C8 reversed-phase 10

2 5 0 ~ 4 . 6 MeOH-0.015M Na-borate b u f f e r pH 8.5 (58:42) 76

PBondapak C18

300x4

C.M ,nalo,oxyM,nalox, pentazocine,naltrexone C,norC,C N-ox,M.norM, M N-ox ,6OAcC,di HC ,di HM, M-3-gl ucuroni de,C-6-gl uc u r o n i de ,nal o

Combined HPLC-inmunoassay H y p e r s i l OOS 5 method f o r a n a l y s i s M, C and metabolites i n biological f l u i d s (Table 7.1)

C,M,Mep,caf

Amphetamines, barbiturates, diazepines

Identification i n urine

uBondapak C18

300x4

P,H,ethaverine,coc, S.caf ,yohimbine

Various b a s i c compounds

Determination P i n blood

Micropak CN-10

300x4

C.M.P.norC,tj-isoprop y l C ,caf C.M,H,EtM,norM.M-3and M-6-glucuronide

Par,phen,ibuprofen

Determination C i n plasma

pBondapaK C18

A n a l y s i s M i n p l a s m and urine

U l t r a s p h e r e OOS 5

M

Monoarni ne transmi t t e r s

Determination i n mouse brains

U l t r a s p h e r e ODS 5 p m

MeOH-H 0(1:4) c o n t a i n i n g 0.05M Bu4N, $H 6.3, a d j u s t e d w i t h H3P04 77

1 0 0 ~ 4 . 6 MeOH-O.01M phosphate b u f f e r , 0.1M KBr(pH 3 ) ( 1 : 7 )

pm

80 MeOH-(aq. 125ml 0.1M KH PO +20.3ml 0.1M NaOH, d i l . t o 750 21 $ i t h H20. pH 6.2 w i t h NaOH or H3P04)(6:4) 82

n-Hexane-CH C1 -ACN-propylamine T50:25:25:0?1)2 3 0 0 ~ 3 . 9 MeOH-H20-H PO (21:79:1.5) 3 4

pm

97

108 1 5 0 ~ 4 . 6 ACN-O.01M NaH PO b y f f e r pH 2.1 (26:74) contaPni8g 0.001M dodecylsulfate 110 2 5 0 ~ 4 . 6 0.05M C i t r a t e b u f f e r pH 4.25 111 c o n t a i n i n g 1%THF KH2P04. O.Snt4

M,nalo

Determination i n c e r e b r o s p i - VBondapak C18 n a l f l u i d and plasma

300x4

ACN-MeOH-aq.0.07M EDTA(5:8:87)

C,M,H,60AcM,AcCpxyC. Various drugs oxyM,diHCone,diHMone. rnep,normep ,meth,prop, nalo,nalox,lev,Q,caf,

Determination H and metabol i t e s i n blood

L i c h r o s o r b Si60 5

300x4

ACN-MeOH-(MeOH-NH OH(2:1))-(AcOHMeOH( 1: 1 ) ) (75:25:d.D40:D. 216)

C,M, H,COAcM,norC,norM, AcC, EtM,diHM,M-3-g1 ucuronide,nal o

Determination M i n b i o l o g i cal f l u i d s , using fluorescence d e t e c t i o n

S i l i c a g e l 60 5 prn P a r t i s i l 10 ODS o r Zorbax ODS 8 p m

200x4

MeOH-2M NH OH-1M NH NO ( 3 : Z : l ) MeOH-O.1M f!Br(12.5:fl7.3) pH 3 w i t h H3P04

M, na lox, n a l trexone

Determination i n r a t b r a i n s

R S i l C18HL 10 pm

2 5 0 ~ 4 . 6 MeOH-H 0(1:3) c o n t a i n i n g 0.05M tetram&hylamnonium, pH 6.1 w i t h H3P04

112

113

COC

100x4

114

115

0 m N

TABLE 7.18

0 m N

HPLC ANALYSIS DRUGS OF ABUSE I N DRUG SEIZURES AND AS PURE COMPOUNDS

ALKALOIDS*

OTHER COMPOUNDS

H. 60AcM,M

Procaine

C,M,P,T,H.coc,Q, c i nchoni ne,caf C.M.No,P,T,H,crypt. 6OAcM,meth ,c a f

M, H,60AcM,meth

Acsal,phen, p-aminophenol, Par

AIMS

STATIONARY PHASE

REF

P o r a s i l T 37-50 urn

Separation by dynamic coat i n g HPLC

Heptane-EtOH(10:l) w i t h d i f f e r e n t C o r a s i l I and I1 coated % o f s a t u r a t i o n w i t h Poly 6-300 w i t h Poly 6-300(2%) 1000x1 2,3 1 0 0 0 ~ 2 . 1 0.24 NaOH pH 9.5 w i t h b o r i c acid, Zipax SCX 37-44 pm 0.2M KNO , 4% ACN, 1%prOH 0.15M NadH pH 9.8 w i t h b o r i c a c i d 0.04M NaOH pH 9.3 w i t h b o r i c acid, 12% ACN, 2% prOH 4 Zipax SAX 37-44 ptn 1 0 0 0 ~ 2 . 1 0.15M NaOH pH 8.8 w i t h b o r i c a c i d

Separation by ion-exchange HPLC

Q u a n t i t a t i v e a n a l y s i s and identification

Zipax SCX 37-44

pm

Zipax SAX

C,H,meth,prop,coc, LSD, S,Q.mesc

Amphetamines, I d e n t i f i c a t i o n s t r e e t d r u g s benzodi azepi nes , barbiturates, THC,procaine, various others

C o r a s i l I1 37-50 urn

A n a l y s i s s t r e e t drugs Barb,par,ligA n a l y s i s i l l i c i t H samples nocaine,proca- ( F i g .7.2) ine

*For a b b r e v i a t i o n s see f o o t n o t e a t t h e end o f t h i s Table

1 0 0 0 ~ 4 . 5 CHC13-MeOH(4:1) 1

Methapyrilene, A n a l y s i s drugs o f abuse anileridine, p r o c a i ne

C,M,H,60AcM,diHC, caf.S.Q.eph ,cot

MOBILE PHASE

Q u a n t i t a t i o n and i d e n t i f i cation

C,M.No,P.T,H.crypt. Losine,EtM,oxyM.diHCone. d i HMone ,apoM, meth, isometh ,mep,Q, Qd,coc

M. H. OAcM,S ,caf ,Q ,LSD

COLUMN DIM. LxID(mn)

1000x2

1ooox2.1

0.08M NaOH pH 9.5 0.15M NaOH pH 9.8 2% ACN 0.04M NaOH pH 9.3 12% ACN, 2% prOH 0.15M NaOH pH 8.8

with boric acid w i t h b o r i c acid, w i t h b o r i c acid, with boric acid

5

A. 0.01M b o r i c a c i d pH 9.5 w i t h NaOH B. 0.01M KH PO pH 6.0 w i t h 1M NaOH g r a d i e n t A+a(88:15) t o B ( l i n e a r 5 o r lO%/min) 6

5 0 0 ~ 2 . 3 Cycl ohexane-MeOH-cycl ohexyl ami ne (983: 15: 2), (945:45: 1) A1 0 Woelm B18 18-30um 5 0 0 ~ 2 . 3 Cycl ohexane-cycl ohexyl arni ne( 988:Z) 5 0 0 ~ 2 . 3 A. S k e l l y B-95% EtOH-dioxaneCoFaSil I1 37-50 um cyclohexylami ne(99.1: 50:25: 13) B. idem (686:100:200:14) 11 l i n e a r gradient A t o B no d e t a i l s

no d e t a i l s Zipax SCX

1200x2.1

15

A. 0.2M b o r i c a c i d pH 9.3 w i t h NaOH B. 0.2M b o r i c a c i d pH 9.8 w i t h NaOH, 12% ACN, 2% prOH l i n e a r g r a d i e n t 0-100% B (2ml/min, 6 min) 18

C,M.Na,No,P.T,H.cot, Losine,prot,EtM,mep, oxyC .di HC ,di HM,oxyM, diHCone,diHMone,Acd i HCone ,COAcM,nalo. A, coc, c a f ,n i c, Q, Qd, S,Tp,LSD,isoLSO. Lysac,Lysam

Amphetamines, Separation v a r i o u s drugs o f diazepines. abuse (Table 7.8, 7.9 and l o c a l anaesth- Fig.7.16) etics,analges i c s .various others

Partisil 6

un

2 5 0 ~ 4 . 6 MeOH-2M NH OH-1M NH N03(27:2:1) MeOH-0.2M WH4N03(3:?)

22

M,H.meth,coc

Screening drugs o f abuse

Bondapak ClE/Corasil

610x2

E v a l u a t i o n ion-exchange and reversed-phase columns f o r a n a l y s i s drugs

P a r t i s i l SCX 10 um

2 5 0 ~ 4 . 6 (NH ) H PO b u f f e r s pH 3, 5 o r 7, ionqc % t r & g t h 0.5, 0.1, 0.05 o r 0.01M. w i t h 0, 20, 40 o r 60% MeOH 300x4 0.025M NaH PO o r Na HPO b u f f e r s pH 3, 5, 720r49, w i t 6 0,420, 40, 60 o r 80% MeOH 21,30

ACN-H 0(9:1),(65:35),(1:1) w i t h 6.1% by weight (NH,),CO,

. -

M,mep .coc, n i c, eph ,Q, caf,tubocurarine

Amphetamines, analgesics, sulfas,barbiturates

VBondapak C18

Identification

C,M.No.P,EtM,coc

all 24 I

0.2M NaOH+5% prOH, 1%KN03 and 2% ACN (pH 9 )

Zipax SCX UBondapak C18

300x4

0.005M Heptanesulfonic a c i d i n MeOH-AcOH-H20(40:1:59)(pH 3.5)

M,Na, P,H,AcC ,60acM, ca f

Amphetamines. I o n - p a i r HPLC o f drugs o f b a r b i t u r a t e s . abuse (Table 7.3) l o c a l anaesthetics Determination i n s t r e e t drugs

pBondapak C18

300x4

ACN-H20-1% aq. (NH4)2C03(140:156:4)

M .H ,OAcM, AcC.Q

Procaine

I d e n t i f i c a t i o n s t r e e t drugs

P a r t i s i l 10 OOS

250x4

ACN-H 0(7:3).(6:4) (NH4)303

t o t a l l y 101 drugs o f f o rensic interest

I d e n t i f i c a t i o n by means o f dual wavelength d e t e c t i o n (Table 2.2, 2.3)

uBondapak C18

3 0 0 ~ 3 . 9 0.025M NaH2P04 i n MeOH-H20(2:3) DH 7.0 3 0 0 ~ 3 . 9 MeOH-2M NH OH-1M NH NO (27:2:1) CH2C12-con?. NH40H( fOOd:2)

C ,M, No, P ,T. H ,OAcM,

AcC.Q,S ,Tp .caf ,COC, ergot alkaloids

C,M,No,P,H,diHC,EtM, diHCone.diHMone.nic, oxyC.oxyM,meth,caf, Tb,Tp,eph,mesc.coc. Q,S.lobeline

34

38.73 44

PPorasi 1

c o n t a i n i n g 0.1%

56

M, H ,60AcM, 30AcM

Separation H from i t s hydro- uBondapak C18 l y s i s products

3 0 0 ~ 3 . 9 ACN-aq. 0.015M KH2P04(3:7)(pH 3.5)

C,M, P,H. AcC,OAcM,oxyC. oxyM,diHCone,diHMone, noroxyM, n a l o

Separation o f illi c i t samples (Table 7.5)

300x4

C ,M ,H ,Et M ,oxyC .oxyM , diHC,diHCone,diHMone, lev,dmethphan,pentazo-

E s t i m a t i o n r e t e n t i o n i n d i c e s UBondapak C18 from Hansch s u b s t i t u e n t constants

cine,levallorDhan.Dhenazoci ne

47

pBondapak C18

57 MeOH-aq. 0.01M Bu N pH 7.5(47:53) MeOH-aq. 0.01M hefltanesulfonic a c i d pH 3 (35:65) changing to(55:45) a f t e r 20 ml 64

3 0 0 ~ 3 . 9 MeOH-H20(2:3) c o n t a i n i n g 1.65 g K HPO and 2.19 KH2P04/1 MgOH-ft 0(7:3) c o n t a i n i n g 0.82 g K2HP042and 1.059 KH2PO4/ 1

w

74

r:

C,M,No.P,T,H,AcC OAcM,caf,S

Analysis i l l i c i t heroin sampl es

dondapak C18

C.M,Na.No,P,T.narcoto1ine.1 audani dine.oxydi morphine

Separation

Bondapak CX C o r a s i l

-

300x4

ACN-0.75% aq. NH40Ac(65:35) 79 ACN-O.1N aq. Ca-phosphate b u f f e r pH 4.8 (3:7) ACN-O.1N aq, Ca-phosphate b u f f e r pH 7.5 (3:7) 0.1N Na-phosphate b u f f e r pH 7.5

Bondapak C18 C o r a s i l P-cell ulose

87

A n a l y s i s h e r o i n seizures (Table 7.11)

uPorasi 1

300x4

Cyclohexane- (CHCl 3-MeOH-NH40H (800:200:1))(3:1)

C,M,No,P,T,H.AcC,EtM, 30AcM,60AcM, c a f ,Q ,S

Analysis i l l i c i t h e r o i n samples ( F i g. 7.6)

S5 NH2, aminopropyl

250x4

ACN-0.005M t e t r a b u t y l amnonium phosphate(85: 15)

C,M .No, P

S t a t i s t i c a l optimization method f o r HPLC system development

dondapak C18

300x4

0.001M Phosphate b u f f e r and 0.015M camphorsulfonic a c i d i n MeOH-H20 94 (41:59)(pH 2.0)

C .M ,No, P.T,norM

Ion-exchange and s t r a i g h t -phase p a r t i t i o n HPLC on s i l i c a g e l (Fig. 7.17)

L i c h r o s o r b Si60 5

M.nalox,lev.ethorphine

C.M.No,P.H,60AcM. AcC.mep.meth.caf. COC,Q,Qd 3s

Hypnotics,ana1gesics.local anaesthetics

91

m

93

1 5 0 ~ 4 . 6 ACN-H O-A~OH-DEA(l0:90:0.5:0.5) THF-M$OH-AcOH-0EA-H2O( 80:ZO:O. 5: 0.5:l)

95

D e t e c t i o n by means o f f l u o r - Supelco LC18 escence

ACN-aq. phosphate b u f f e r pH 5.05 (65: 35)

96

I o n - p a i r HPLC o f drugs o f forensic interest

0.005M A l k y l s u l f o n a t e ( C .C ,C ) i n &OH-H O-AcOH(40:59:1) .(30:69:1), (20: 79?1) (pH 3.5) 98s 99.100

C,M,No,P.T,H.AcC. AcM,caf,Tp,coc,eph, S,Q,Qd,mesc,LSO, isoLSD

Barbiturates, amphetamines, l o c a l anaesthetics

M,No,P,H,60AcM,AcC

Procaine.Acsa1 D e t e c t i o n Ac-procaine i n Ac-procaine, heroin(Fig.7.8) ascorbic a c i d

dondapak C18

M, P,T,H,60AcM,AcC, caf

Procaine

Multichannel detection i n a n a l y s i s h e r o i n samples

APS-hypersil 5

H

A n a l y s i s s t r e e t samples

no d e t a i l s a v a i l a b l e

T, H.AcC, c a f

Analysis i l l i c i t h e r o i n sam- C 8 - s i l i c a g e l 10 p l e s w i t h GCMS and HPLC

C,M,No,P,T,H.AcC, Analgesics,an- A n a l y s i s h e r o i n seizures AcM,mep,meth,caf,Tp, t i h i s t a m i n i c s . (Table 7.6) A.Q,S,eph.coc. t r o p a - hypnotics ,loCOC c a l anaesthetics , various o t h e r s

Sondapak C18, dondapak Phenyl o r dondapak CN

m

Sondapak C18 o r P a r t i s i 1 10-00s-3

V-I

300x4

ACN-H 0(91.5:8.5) c o n t a i n i n g 0.008% tris(gydroxymethy1 )aminomethane 105

100x5

ACN-O.005M t e t r a b u t y l a m n o n i um phosphate (85:15)

106 107

2 5 0 ~ 4 . 6 ACN-0.75% NH40Ac(55:45)

120

ACN-H 0-H PO (12:87:1) c o n t a i n i n g 0.02M2metfian&ulfonic a c i d pH 2.2 121

329

*Abbreviations used i n Tables 7.13-7.18

A AcC Acdi HCone Acsal ApoC ApoM B Barb C Ca f COC

cot Crypt DiHC DiHCone D i HM D i HMone Dmethphan

E;!

H Keto Lev Losi ne M MeP

Referenca p. 310

Atropine Acetyl codeine

Mesc Meth Acetyldihydrocodeinone(thebacone) Na Acetylsalicylic acid Nal o Apocodeine Nalox N ic Apomorphine Brucine No Barbital 30AcM Codeine 60AcM Caffeine oxyc Cocaine OxyM P Cotarnine Cryptopi ne Par D i hydrocodei ne Phb D i hydrocodei none Phen Dihydromorphine Prop D i hydromorphi none Prot Dextromethorphan PseudoM Q Ephedrine Ethylmorphi ne Qd S Heroin Ketobemi done Salam Levorphanol Scop Laudanosi ne T Morphine Tb Meperidi ne TP

Mescal ine Methadone Nar c e ine Nalorphi ne Naloxone Nicotine Noscapi ne 3-0-Acetylmorphi ne 6-n-Acetylmorphi ne Oxycodone Oxymorphone Papaveri ne Paracetamol Phenobarbi t a l Phenacetine Propoxyphene Protopine Pseudomorphi ne Quinine Quin i d i n e S t r y c h n i ne Salicylamide Scopol ami ne Thebaine Theobromine Theophyl 1i n e

This Page Intentionally Left Blank

331

Chapter 8 TERPENOID INDOLE ALKALOIDS AND SIMPLE INDOLE ALKALOIDS

...............................................................

8.1. Ion-exchange HPLC.. 8.2. Reversed-phase HPLC ............................................................... 8.3. Ion-pair HPLC ..................................................................... 8.4. Straight-phase HPLC 8.5. Detection ......................................................................... References..

............................................................... ...........................................................................

331 331 335 338 344 344

The indole a l k a l o i d s form the g r e a t e s t group of a l k a l o i d s . Several hundreds of a l k a l o i d s containing an indole skeleton a r e known so f a r . Rich in indole a l k a l o i d s a r e t h e p l a n t fami l i e s Apocynaceae. Loganiaceae and Rubiaceae. Many of the indole a l k a l o i d s have c h a r a c t e r i s t i c pharmacological a c t i v i t i e s and a r e , therefore, used i n therapy. HPLC has been extensively applied i n the a n a l y s i s of indole a l k a l o i d s for quite differ e n t purposes. A s e r i e s of papers on the analysis of drugs of abuse includes t h e a n a l y s i s of one o r more indole a l k a l o i d s , p a r t i c u l a r l y strychnine, a common a d u l t e r a n t of heroin (Table 8.16). Moreover strychnine and brucine have often been used a s t e s t compounds f o r a number of

-

separation systems 112’9’16921y32*40s43 a s well as i n t e r n a l standards10y13. Comparison of HPLC and TLC in the analysis of indole a l k a l o i d s has been p e r f ~ r r n e d ~ ’ ~ ~ ’ ~ ~ ’ 34141344, as well a s t h a t f o r HPLC and GLC4. Semipreparative aspects o f HPLC in the i s o l a t i o n of indole alkaloids have been Because of the poor v o l a t i l i t y and i n s t a b i l i t y of many high molecular indole a l k a l o i d s . such as reserpine and psilocybin, HPLC lends i t s e l f t o the a n a l y s i s of such a l k a l o i d s , as compared w i t h GLC 6,11,20,23,27,30,37,46 A review on the i d e n t i f i c a t i o n of drugs of abuse, including Psilocybe a l k a l o i d s , has been 68 given by Gough and Baker .

8.1. ION-EXCHANGE HPLC

6 Rodgers used a p e l l i c u l a r strong cation-exchange packing f o r the separation o f some Rauwolfia alkaloids. An amnonium phosphate buffer (pH 7 . 0 ) containing methanol was used a s e l u e n t . An improved separation was obtained with gradient e l u t i o n . described ligand-exchange chromatography o f a l k a l o i d s on ion-exWal ton and change materials loaded w i t h metal ions (see Chapter 7 ) . Perkal e t a l . 4 6 separated psilocybin and p s i l o c i n on a cation-exchanger - P a r t i s i l SCX - using methanol - water (1:4) containing 0.2% ammonium phosphate and 0.1% potassium c h l o r i d e (pH 4.5) a s mobile phase (Fig.8.1). 8.2. REVERSED-PHASE HPLC

Wu and S i g g i a l S 2 described a dynamic coating technique f o r the a p p l i c a t i o n of a l i q u i d s t a t i o n a r y phase - Poly 6-300 - on a s i l i c a gel support. With heptane - ethanol ( 1 O : l ) sat u r a t e d w i t h the s t a t i o n a r y phase. strychnine and brucine could be separated. Polyethylene glycol coated s i l i c a gel as support has. however, been superseded by chemically bonded s t a tionary phases.

Reference# p. 344

332 Chemically bonded octadecyl groups on a p e l l i c u l a r support was used as s t a t i o n a r y phase 4 water ( 4 : l ) f o r the separation o f a s e r i e s o f Mitragyna oxindole a l k a l o i d s w i t h methanol

-

as mobile phase. P h i l l i p s o n e t a l .77 compared the r e t e n t i o n behaviour o f nine heteroyohimbine and e i g h t oxindole a l k a l o i d s on an octadecyl bonded phase w i t h p r e v i o u s l y obtained r e s u l t s on s t r a i g h t -phase TLC (Table 8.1).

I f the r e t e n t i o n on s i l i c a gel could be explained i n terms o f the

a v a i l a b i l i t y o f the N-4 lone-pair electrons, such a c o r r e l a t i o n could n o t be concluded f o r the r e t e n t i o n i n the reversed-phase system. Reserpine has been analyzed i n pharmaceutical preparations w i t h d i u r e t i c and antihypertens i v e drugs on p e l l i c u l a r octadecyl o r phenyl

packing^^^'^^.

A s e r i e s o f mobile phases was used

t h a t could meet the d i f f e r e n t a n a l y t i c a l demands. I n s o l v e n t systems w i t h a pH o f over 8. several peaks were observed i n a d d i t i o n t o reserpine. They were assumed t o be due t o the decomp o s i t i o n o f reserpine during the analysis. For the r a p i d q u a l i t a t i v e and q u a n t i t a t i v e analysis o f t h e r a p e u t i c a l l y used Catharanthus alkaloids, GSrog e t a1.26 applied reversed-phase HPLC on an o c t y l type column. With acetonitrile

- 0.01

M ammonium carbonate (47:53) a f a i r l y good separation was achieved, both i n terms

o f analysis time and r e s o l u t i o n (Table 8.2, Fig.8.2).

Catharanthus a l k a l o i d s i s o l a t e d from

t i s s u e c u l t u r e s have been separated on an octadecyl column w i t h methanol 50 amine mixtures

.

-

water

-

triethyl-

Verzele e t a1.60 p r e f e r r e d a g r a d i e n t e l u t i o n (from 50 t o 85% methanol i n water containing 0.1% ethanolamine) t o separate the wide range o f d i f f e r e n t a l k a l o i d s present i n vinca rosea. I t was found t h a t column e f f i c i e n c y was improved by increasing the temperature t o 50’ C (doub-

l e d p l a t e number i f compared w i t h 5’ C). Szepesi and G a ~ d a gstudied ~~ the separation o f 22 eburnane a l k a l o i d s on an o c t y l and octadecyl type o f column. Optimal r e s u l t s were obtained on both columns w i t h the mobile phase acetonitrile

-

0.01 M aqueous ammonium carbonate (3:2). The octadecyl s t a t i o n a r y phase gave

b e t t e r r e s o l u t i o n than the o c t y l packing (Table 8.3, F i g . 8.3). The reversed-phase system was capable o f separating the three d i f f e r e n t groups o f eburnane a l k a l o i d s as w e l l as separating the e s t e r homologues and some stereoisomers. S t r u c t u r a l and stereoisomers were, however, separated b e t t e r w i t h straight-phase HPLC systems (see below)55. The a p p l i c a t i o n s o f the developed methods t o various separation problems i n v o l v i n g eburnane a l k a l o i d s have been discussed75. Oubruc e t a l . 5 3 reported the analysis o f vincamine i n plasma. Analysis was performed on an octadecyl bonded s t a t i o n a r y phase. Large volumes (0.5 m l ) o f the sample i n a non-eluting s o l vent (0.02 M aqueous potassium phosphate) were used f o r automatic operation o f the analysis.

As mobile phase: a c e t o n i t r i l e - a c i d i c phosphate b u f f e r was used. D i l u t e samples o f u r i n e and plasma have been i n j e c t e d d i r e c t l y on an octadecyl column f o r the determination o f the vincamine aqueous ammonium carbonate was employed. For mobile phase methanol Hussey and Newlon” analyzed vindesine by means o f a m i c r o p a r t i c u l a t e octadecyl column and

-

a mobile phase o f methanol

-

water

-

diethylamine (1000:600:3).

i n the presence o f high l e v e l s o f 6.7-dihydrovindesine.

For the analysis o f vindesine

the r a t i o (835:600:3) was preferred.

For pharmaceutical preparations containing vindesine, a 0.5 M potassium phosphate b u f f e r i n combination w i t h a higher amount o f methanol could be used i n s t e a d o f diethylamine t o g i v e prolonged column l i f e .

To avoid column degradation due t o high pH, Crouch and Short” octadecyl column w i t h an a c i d i c mobile phase: methanol

-

analyzed strychnine on an

0.005 M potassium dihydrogen phos-

333 TABLE 8.1 RETENTION TIMES OF HETEROYOHIMBINE- AND OXINDOLE-TYPE ALKALOIDS77

-

Column, Spherisorb ODS 5 pm (250x4 mn ID), mobile phase. S1 methanol water (80:20), 52 methanol water conc. anmonia (8O:ZO:l). 53 a c e t o n i t r i l e 1%amnonium carbonate (60:40), 1%amnonium carbonate (8O:ZO). f l o w r a t e 2 mllmin, detection UV 254 nm. 54 methanol

-

-

-

-

tr (min) i n solvent system

A1k a l o i d s

51

52

s3

54

5.1 10.9 5.8 1.0 4.3 2.0 1.6

4.8 8.6 4.6 1.0 3.4 2.0 1.6

6.2 10.0 6.5 5.2

2.9

7.7 12.7

3.9 4.9

-

3.6 10.0 4.2

3.8 3.8 3.7 3.7 3.4 3.7

3.8 3.2 3.0 3.0 3.2 3.0

3.7 2.8 2.8 2.7 3.0 2.7

2.9 2.3 2.2 2.2 2.4 2.0

3.0 6.2

3.0 4.0

3.3 5.1

3.3 4.2

~~~

P e n t a c y c l i c heteroyohimbines

T e t r ahydroa 1st onine Rauniticine Akuamni g i ne 3-Iso-rauni t i c i n e Ajmalicine 3-Iso-ajmal i c i n e 19-Epi -3-isoajmal i c i n e T e t r a c y c l i c heteroyohimbines

D i hydrocorynantheine Hirsutine

3.7

Pen t a c g c l i c oxindol es

Isopteropodi ne Pteropodine Speciophyllline Uncarine F Mitraphyll ine Isomitraphyl l i n e Tetracyclic oxindoles

Isorhynchophyll i n e Rhynchophyl 1ine TABLE 8.2

RETENTION DATA FOR SOME CATHARANTHUS ALKALOIDS AND SEMISYNTHETIC DERIVATIVES (Fig.8.2)26 Column, Lichrosorb RP8 (250x4 mn ID), mobile phase, a c e t o n i t r i l e (47:53), flow r a t e 1.5 ml/min. detection UV 298 nm. A1 k a l o i ds

Substituted a t

Monomeric a l k a l o i d s

Lochnerine Ajmalicine Tetrahvdroal stoni ne Catharkthine Vindoline d e r i v a t i v e s

Vindol i n e

166 OH

OH OH OH OCOCH3 OH

D i m e r i c alkaloids V i n b l a s t i n e and d e r i v a t i v e s N

16a COOCH COOCH3 COOCH3 COOCH3 COOCH3 COOCH3 CH20H3

17 OCOCH3

166

17 OCOCH,

Vinblastine

CR-

OH

H CH CHd CHO

OH

DesacetoXyvinblastine Vincristine Leorosi ne Formyl l e u r o s i ne

References p. 344

Retention time (min) 3.29 7.90 14.65 9.75

OH

Vindolinol Vindorosine

- 0.01

HCH3 CHO

OH OH OH

OH OCOCH C1

OCOCH2N(CH ) OCOCHzNH(Ca3f OCOCH: OH

OCOCH3 H OCOCH3

OH

6.16 4.71 9.39 5.84 4.30 23.22 4.44 7.90

12.37

10.04 17.15 7.22 4.87 15.65 11.89 9.75

M amnonium carbonate

TABLE 8.3

0 J, 0

CAPACITY RATIOS, k' , FOR EBURNANE ALKALOIDS ON OCTADECYL AND OCTYL SILICA PPCKINGS WITH DIFFERENT ELUENTS" Column pBondapak C18 ( 3 0 0 ~ 3 . 9 mm ID) or 1@m Lichrosorb RP8 ( 2 5 0 ~ 4 . 6 n ID), flow rate 1 ml/min, detection UV 280 nm. A1 kaloid

Ratio o f acetonitrile and 0.01 I1 (NH4)2C03 4:6

5:5

6 :4

7:3

8:2

pBondapak C18

4:6

5:5

6:4

7:3

10 um LiChrosorb RP-8

~~

(+)-cis-Apovincaminic acid (+)-ci s-Vincaminic acid (+)-cis-Dehydroepivincamine (+)-cis-Dehydrovincamine

(+)-trans-Vincaminic acid ethyl ester (+)-cis-Epivincamine (-)-cis-Epivincamine (+)-cis-Epivincaminic acid ethyl ester (+)-trans-Epivincaminic acid ethyl ester (+)-cis-Vincamine (-)-ci s-Vincami ne (+)-cis-Vincamone (+)-cis-Vincanole (+)-cis-Isovincanol e (+)-cis-Vincaminic acid ethyl ester (+)-cis-Apovincamine (+)-trans-Apovincaminic acid ethyl ester (+)-cis-Apovincaminic acid ethyl ester (+)-cis-Vincamenine (+)-cis-Apovincaminic acid phenyl ester (+)-cis-10-Bromovincamine

(+)-cis-11-Bromovincamine

0.05

0.10 4.65 6.03 8.93 9.23 9.23 12.4 14.4 12 . A 12.4 13.7 15.6 18.8 16.9 23.9 30.3 32.5 46.5 56.1 13.4 13.4

0.05 0.10 2.57 3.21 4.29 4.71 4.71 6.36 6.36 6.36 6.36 7.21 8.07 9.43 8.47 14.1 18.5 20.2 31.2 36.8 9.11 9.11

0.0 0.07 1.57 1.86 2.29 2.71 2.71 3.43 3.43 3.43 3.43 4.04 4.29 5.43 4.38 6.78 7.76 9 .oo 14 .O 14.0 4.86 4.86

0.0 0.05 1.04 1.31 1.32 1.79 1.79 1.97 1.79 2.14 2.14 2.57 3.14 3.71 2.57 4.11 3.97 6.20 8.71 7.07 3.54 3.54

0 .o 0 .o 0.57 0.57 0.57 0.75 0.75 0.95 0.75 0.90 0.90 1.43 1.71 1.71 i .43 2.64 2.03 3.27 5.57 4.00 1.70 1.70

0.0

0.0

0.0

0.0

4.61 5.65 10.81 0.02 6.02 8.45 19.8 10.9 10.9

2.14 2.60 4.43 2.60 2.60 3.60 7.04 3.53 3.53 5.51 3.60 4.30 5.03 9.42 21.8 13.6 19.2 26.4 7.32 7.32

12.4

0.0

0 .o 1.30 1.55 2.19 1.55 1.55 1.92 3.26 1.87 1.87 3.19 2.26 2.83 2.57 4.79 8.64 6.36 10.4 9.80 3.57 3.57

0.0 0.0 0.66 0.98 1.30 1.04 1.04 1.30 1.83 1.30 1.30 1.87 1.55 1.87 1.55 2.66 4.23 3.47 5.26 4.81 2.11 2.11

335 TABLE 8.4 SEPARATION OF SOME ALKALOIDS ON A POROUS POLYMER43 Column, H i t a c h i Gel 3010 (macroporous s t y r e n e - d i v i n y l b e n z e n e copolymer), 10 pm ( 2 2 0 ~ 4 . 6 n I D ) , m o b i l e phase, S 1 a c e t o n i t r i l e - w a t e r (3:7) c o n t a i n i n g 0.02 M t e t r a b u t y l a n o n i u m h y d r o xide, S2 a c e t o n i t r i l e - w a t e r (6:4) c o n t a i n i n g 0.02 M tetrabutylammonium h y d r o x i d e , S3 acet o n i t r i l e - w a t e r ( 6 : 4 ) c o n t a i n i n g 0.02 M ammonia, f l o w r a t e 1 ml/min. d e t e c t i o n UV 254 nm. k ’ i n S 1 (55’

Compound Tyrami ne Norephedri ne Ephedrine Atropine Amphetamine Methamphetamine Strychnine C i nchoni ne Quinine

C)

k ’ i n S2 (25’ C)

Compound Y o h i mb ine Reserpine

0.4 0.8 1.8 3.9 4.0 7.3 8.4 10.4 12.6

2.6 11.7 k ’ i n S3 (65’

2.4,6-Trimethylp y r i d i ne 2-Methylquinol i n e 5,6-Dibenzoquinoline Acridine

C)

3.2 4.5 12.5 13.0

S t r y c h n i n e and b r u c i n e were n o t s e p a r a t e d i n t h i s system.

p h a t e b u f f e r (PH 3 . 0 ) ( 2 : 3 ) .

Sasse e t a l . 4 5 r e p o r t e d t h e a n a l y s i s o f harmane a l k a l o i d s i n c e l l suspension c u l t u r e s b y

means of HPLC. An o c t y l column and a m o b i l e phase o f methanol

-

w a t e r - f o r m i c a c i d (166:34:1)

b u f f e r e d a t pH 8.5 w i t h t r i e t h y l a m i n e was used f o r t h e s e p a r a t i o n o f t h e a l k a l o i d s ( F i g . 8 . 4 ) . F o r t h e a n a l y s i s o f e l l i p t i c i n e i n b i o l o g i c a l samples an o c t a d e c y l s t a t i o n a r y phase i n combination with acetonitrile

-

0.01 M sodium dihydrogenphosphate b u f f e r has been used7’.

To en-

s u r e r e s o l u t i o n between e l l i p t i c i n e and 9 - h y d r o x y e l l i p t i c i n e a r a t i o o f 1:3 had t o be used (Fig.8.5).

I n t h e case where o n l y e l l i p t i c i n e i s p r e s e n t , a r a t i o o f 36:64 was used.

T y m e ~r e~p o~r t e d a c o l l a b o r a t i v e s t u d y o f t h e a n a l y s i s o f p h y s o s t i g m i n e i n p h a r m a c e u t i c a l p r e p a r a t i o n s u s i n g o c t a d e c y l s t a t i o n a r y phases w i t h t h e m o b i l e phase: a c e t o n i t r i l e

-

0.05 M

ammonium a c e t a t e ( 1 : 1 ) . Aramaki e t a l . 4 3 made a p r e l i m i n a r y s t u d y o f t h e s e p a r a t i o n o f some a l k a l o i d s on a macroporous s t y r e n e - d i v i n y l b e n z e n e polymer ( T a b l e 8.4, F i g . 8 . 6 ) .

An advantage o f t h e s e columns i s

t h e i r s t a b i l i t y , a l s o under t h e s t r o n g b a s i c c o n d i t i o n s a p p l i e d f o r t h e a n a l y s i s o f a l k a l o i d s . It was assumed t h a t a l k a l o i d s would be b e s t r e t a i n e d under b a s i c c o n d i t i o n s , t h e a l k a l o i d s b e i n g i n t h e uncharged, non-protonated form. I O N - P A I R HPLC

8.3.

Reversed-phase i o n - p a i r chromatography has been a p p l i e d f o r t h e a n a l y s i s o f drugs o f abuse

-

i n c l u d i n g ~ t r y c h n i n e ~ ~ ’ ~ ~ F’o r~ a~ more ’ ~ ~ d’e~t a~i l e. d d i s c u s s i o n see Chapter 7 ( T a b l e

7.6). r e p o r t e d t h e d e t e r m i n a t i o n o f r e s e r p i n e i n plasma by i o n - p a i r chromatography. To o b t a i n h i g h s p e c i f i t y and t o i n c r e a s e t h e s e n s i t i v i t y r e s e r p i n e - a f t e r e x t r a c t i o n f r o m p l a s ma w i t h benzene - was o x i d i z e d t o 3 - d e h y d r o r e s e r p i n e w i t h vanadium p e n t o x i d e i n c o n c e n t r a t e d p h o s p h o r i c a c i d , t o which 9 m l methanol

were added. The f l u o r e s c e n t compound was s u b s e q u e n t l y

analyzed on an o c t a d e c y l column w i t h methanol - 0 . 0 0 1 M h e p t a n e s u l f o n a t e i n w a t e r (65:35) as m o b i l e phase. Reserpine and rescinnamine c o u l d n o t be d i s t i n g u i s h e d by t h i s method. E l l i p t i c i n e and i t s d e r i v a t i v e s as w e l l as r e l a t e d q u a t e r n a r y ammonium compounds have been s e p a r a t e d on an o c t a d e c y l column w i t h methanol - w a t e r

References p. 344

m i x t u r e s , t o which 1 - h e p t a n e s u l f o n a t e

336 TABLE 8.5.

Structures and retention times o f six ellipticines

36

Column pBondapak C18 (300x4 mm ID), mobile phases: S1 methanol water (7:3) 52 methanol water (7:3) containing 0.005 M 1-heptanesulfonate 0.032 M acetic acid 53 methanol - water (7:3) containing 0.005 M 1-pentanesulfonate - 0.032 M acetic acid 54 methanol water (3:l) containing 0.005 M 1-heptanesulfonate 0.032 M acetic acid S5 methacol water (3:l) containing 0.02 M ammonium acetate Flow rate 1.2 ml/min, detection UV 254 nm and 313 nm. fluorescence (excitation 305 nm. emission 470 nm).

-

-

-'

R

ComDound

t.. (min)

~~~

~

s1

~~

s3

s4

55

.>30 3.8 >30 4.5 >30 7.1 OCH3 ~ 3 0 7.4 Br >30 13.5

3.5 3.8 5.2 5.7 8.2

3.5 4.0 5.4 5.7 8.2

4.9 5.5 9.3 9.5

-**

3.9

4.1

4.2

NH2

? CH3

~

52

>30

4.6

-

CH3

:.,9-Hydroxy-2-methyl Ellipticine el 1 iptici ni

um

TABLE 8.6. Structures and retention times of some ellipticine quaternary ammonium derivatives36 Column VBondapak C18 (300x4 nun ID), mobile phase methanol - water (75:25) containing 0.005 M sodium 1-heptanesulfonate and 0.032 M acetic acid, flow rate 1.2 mlhin, detection UV 254 nm and 313 nm. fluorescence (excitation 305 nm, emission 470 nm).

R

Compound CH3

H

% OCOSH, NO Br2 CH :$5H n-C3H7 n-C4H9 CH 6C@CH2

CHECH~OH R. R!

tr(min) 5.7 3.7 6.0 5.1 5.6 8.4 4.1 4.5 5.1 5.9 7.6 4.7 3.4 4.3 4.6 4.8 5.4 8.7 16.0 11.8

n

0

R1=

CH,CH,N

R2=

CH2CH,N3

R3=

c H* C

u

~3

H ~

331 TABLE 8.7 HPLC ANALYSIS P I LOCARPINE ,PIjSOST I G M I NE ,I T S DEGRADATION PRODUCTS AN0 PRESERVATIVES I N PHARMACEUTICAL PREPARATIONS Column, VBondapak C18 ( 3 0 0 ~ 3 . 9 n I D ) , m o b i l e phase, methanol - w a t e r (2:3) c o n t a i n i n g 0.005 M h e p t a n e s u l f o n i c a c i d (pH 3.6), f l o w r a t e 1 ml/min. d e t e c t i o n UV 235 nm and 292 nm ( f o r rubreserine). ~

~~~

Compound Rubreseri ne P i 1o c a r p i ne Sa 1 ic y 1 a t e Phenethyl a l c o h o l Methyl y h y d r o x y b e n z o a t e Physostigmi ne

k'

Sample conc.(pg)

1.7 2.3 2.9 3.5 4.2 5.1

0.02 80 8 10 0.8 8

Detection l i m i t (ug) 0.001 0.02

0.003

was added as c o u n t e r i o n ( T a b l e 8.5, 8.6)36. Ammonium a c e t a t e was used as i o n i c component i n t h e m o b i l e phase. K n e c ~ k eanalyzed ~~ pharmaceutical p r e p a r a t i o n s c o n t a i n i n g p i l o c a r p i n e and p h y s o s t i g m i n e as w e l l as t h e i r d e g r a d a t i o n p r o d u c t s and p r e s e r v a t i v e s on an o c t a d e c y l column u s i n g i o n - p a i r chromatography ( T a b l e 8.7, Fig.8.7). T h o m ~ o nr e~p~o r t e d t h e reversed-phase i o n - p a i r s e p a r a t i o n o f p s i l o c y b i n and p s i l o c i n . Because b o t h a l k a l o i d s e x i s t as z w i t t e r - i o n s .

c a t i o n i c and a n i o n i c p a i r i n g i o n s can be used.

A l k y l s u l f o n a t e s (C5-C8) and t e t r a a l k y l ammonium (C3-C6) i o n s were f o u n d u n s a t i s f a c t o r y f o r p s i l o c y b i n . Good r e s u l t s were o b t a i n e d w i t h a l o n g c h a i n q u a t e r n a r y ammonium i o n , cetrimonium. Optimal c o n d i t i o n s f o r q u a n t i t a t i v e a n a l y s i s on an o c t a d e c y l s t a t i o n a r y phase were 0.15% p a i r i n g i o n i n methanol

-

0.4% aqueous phosphate b u f f e r (pH 7.2).

Some o t h e r q u a t e r n a r y i n d o l e

a l k a l o i d s have a l s o been separated b y means o f i o n - p a i r HPLC. P a r k i n 6 1 analyzed t h e b i s q u a t e r n a r y a l k a l o i d a l c u r o n i u m i n b i o l o g i c a l f l u i d s . A f t e r an i o n - p a i r e x t r a c t i o n , t h e a l k a l o i d was analyzed on an o c t a d e c y l column w i t h t h e m o b i l e phase: methanol

-

w a t e r ( 4 : l ) c o n t a i n i n g 0.25%

a c e t i c a c i d and 0.005 M d o d e c y l s u l f a t e . N - p r o p y l a j m a l i n e has been analyzed by means o f s t r a i g h t - p h a s e i o n - p a i r HPLC. A s i l i c a g e l column loaded w i t h 0.2 M p e r c h l o r i c a c i d and 0.9 M sodium p e r c h l o r a t e i n c o m b i n a t i o n w i t h t h e m o b i l e phase: _?-butan01

-

1 , Z - d i c h l o r o e t h a n e - hexane (15:40:45) was s u i t a b l e f o r s e p a r a t i n g a3

t h e two C-21 epimers, as e x t r a c t e d f r o m plasma

.

Szepesi e t a l . 7 3 r e p o r t e d an i o n - p a i r s e p a r a t i o n o f eburnane a l k a l o i d s on a c h e m i c a l l y bonded cyanopropyl s t a t i o n a r y phase. As c o u n t e r - i o n , di-(2-ethylhexyl)phosphoric a c i d o r (+)-10-camphorsulfonic

a c i d were used i n a m o b i l e phase c o n s i s t i n g o f hexane - c h l o r o f o r m

a c e t o n i t r i l e m i x t u r e s ( T a b l e 8.8, 8.9).

-

Because o f t h e p o o r s o l u b i l i t y o f t h e l a t t e r p a i r i n g

i o n , d i e t h y l a m i n e ( T a b l e 8 . 9 ) was added t o t h e m o b i l e phase. A d d i t i o n o f d i e t h y l a m i n e cons i d e r a b l y reduced t h e k ' o f t h e a l k a l o i d s , due t o s u p p r e s s i o n o f t h e i o n i z a t i o n o f t h e a l k a l o i d s . However, due t o t h e s t r o n g a c i d i c c h a r a c t e r o f t h e p a i r i n g i o n . i o n - p a i r s were s t i l l formed under these c o n d i t i o n s . The c a m p h o r s u l f o n i c a c i d c o n t a i n i n g m o b i l e phases were f o u n d t o be v e r y u s e f u l f o r t h e s e p a r a t i o n o f o p t i c a l isomers ( T a b l e 8.10,

8.11,

F i ~ . 8 . 8 ) ~ I~t.

was a l s o found t h a t t h e s e l e c t i v i t y o f t h e system c o u l d be a l t e r e d by choosing d i f f e r e n t med i u m - p o l a r i t y s o l v e n t s (moderator s o l v e n t s ) as dioxane, c h l o r o f o r m o r t e t r a h y d r o f u r a n . The p o l a r component o f t h e s o l v e n t system a f f e c t e d peak shape. Based on t h e s e o b s e r v a t i o n s , a method was developed t o analyze t h e o p t i c a l p u r i t y o f vincamine and v i n p o c e t i n e . F o r t h e ana-

References p. 344

338 TABLE 8.8 DEPENDENCE OF THE CAPACITY RATIOS ( k ’ ) ME9$$IREO FOR EBURNANE ALKALOIDS ON THE 01-(2-ETHYLHEXYL)PHOSPHORIC A C I D (OHP) CONCENTRATION Column. uBondapak CN (300~3.9mn 10). e l u e n t f l o w r a t e 1 ml/min, d e t e c t i o n UV 280 nm. E l u e n t compos it i o n ( %)

Compound

Hexane 65 C h l o r o f o r m 20 A c e t o n i t r i l e 15 OHP ( m o l / l )

-

Hydrocortisone Predni s o l one (+)-cis-Epivincamine (+) - c i s-Vincami ne (+)-cis-Apovincami n i c a c i d ethyl ester (t) - c i s- V i ncameni ne (t)-cis-Apovincamime (+)-cis-Vincamome ( t ) - c i s-Vi ncanol (-)-cis-Vincanol (t)-cis-Vincamini c a c i d ethyl ester I t ) - c i s-I s o v i n c a n o l

1.89 2.52 2.93 1.93 0.45 0.93 0.55 0.28 3.34 3.34 1.52 5.72

65 20 15 0.0005

65 20 15 0.001

65 20 15 0.005

65 20 15 0.01

65 20 15 0.025

60 23 17 0.005

70 17 13 0.005

1.89 2.50 2.36 1.75

1.97 2.52 4.07 5.34

1.92 2.53 2.36 6.57

1.93 2.55 2.31 7.46

1.94 2.59 3.44 9.04

1.48 1.89 1.96 5.78

3.00 3.90 3.00 10.1

0.43 0.54 0.32 2.75 2.75

2.24 3.00 2.72 1.62 5.83 5.83

3.36 3.64 4.14 3.50 4.21 4.21

4.25 3.92 4.42 4.12 3.58 3.58

4.78 4.78 5.30 4.63 6.33 6.33

2.78 3.30 3.56 3.19 3.67 3.67

4.26 4.56 5.44 4.70 5.07 5.07

1.54 4.18

4.14 8.69

6.50 10.0

5.43 9.08

6.93 10.4

4.78 7.52

7.19 12.7

0.86

l y s i s o f v i n c a m i n i c a c i d i n pharmaceutical p r e p a r a t i o n s , a c e t o n i t r i l e - aqueous 0.001 M ammonium carbonate (7:3) has been used, c o n t a i n i n g 0.001 M t r i o c t y l m e t h y l a m o n i u m as p a i r i n g ion

75 ,

8.4. STRAIGHT-PHASE HPLC For s t r o n g b a s i c a l k a l o i d s such as s t r y c h n i n e , b r u c i n e , s e r p e n t i n e and a l s t o n i n e , t h e add i t i o n o f a base t o t h e m o b i l e phase reduces t a i l i n g due t o c h e m i s o r p t i o n on t h e s i l i c a

A m o b i l e phase c o n s i s t i n g o f d i e t h y l e t h e r

-

methanol

-

d i e t h y l a m i n e proved t o

ble be u s e f u l i n t h e s e p a r a t i o n o f S t r y c h n o s a l k a l o i d s on a s i l i c a g e l c o l ~ m n ~ ’ ~ ~ ( T a8.12,

Fig.8.9). A s i m i l a r system was a p p l i e d by Gimet and F i l l o u x 3 ’ f o r t h e a n a l y s i s o f v a r i o u s a l k a l o i d s i n pharmaceutical p r e p a r a t i o n s (Fig.7.14). I t was f o u n d t h a t an i n c r e a s e d s a t u r a t i o n o f t h e d i e t h y l e t h e r w i t h w a t e r l e d t o reduced r e t e n t i o n t i m e s ; a s i m i l a r e f f e c t was found f o r t h e amount o f d i e t h y l a m i n e added t o t h e s o l v e n t . F o r t h e s e p a r a t i o n o f Strychnos a l k a l o i d s , good r e s u l t s were a l s o o b t a i n e d w i t h t h e m o b i l e phase: e t h y l a c e t a t e ammonia i n combination w i t h s i l i c a g e l

column^^^'^^'^^.

-

methanol

-

K i n g s t o n and Li17 r e p o r t e d f o r Tabernaemontana a l k a l o i d s t h a t s i l i c a g e l i n c o m b i n a t i o n w i t h ammonia c o n t a i n i n g s o l v e n t systems o r aluminium o x i d e i n c o m b i n a t i o n w i t h n e u t r a l s o l vents c o u l d be used f o r p r e p a r a t i v e HPLC. The HPLC system d e s c r i b e d by Bushway e t a1.14 s i l i c a g e l column and c h l o r o f o r m - methanol

-

(9:l) as m o b i l e phase

-

had o n l y l i m i t e d a p p l i -

c a b i l i t y f o r the analysis o f strychnine, since i t d i d not separate strychnine from brucine. A j m a l i c i n e ( r a u b a s i n e ) analyses have been performed on s i l i c a g e l w i t h a n e u t r a l s o l v e n t system - n-hexane

-

d i i s o p r o p y l e t h e r - methanol ( g t 1 : 1 0 : 3 ) ~ ~ ’The ~ ~ .HPLC method was found

t o be more c o n v e n i e n t than a TLC d e n s i t o m e t r i c and a UV s p e c t r o p h o t o m e t r i c method.

TABLE 8.9 DEPENDENCE OF THE CAPACITY RATIOS ( k ' ) IIEASURED FOR EBURNANE ALKALOIDS ON THE (+)-CAMPHORSULFONIC ACID (CSA) CONCENTRATION73 Conditions as in Table 8.8. Compound

Eluent mixtures (hexane-isopropanol, 8:2) DEP ( M ) 0.001 0.001 0.001 0.001 0.001 0.001 0.000250.00050.001 0.002 CSA (I?) - 0.0005 0.001 0.0015 0.003 0.005 0.002 0.002 0.002 0.002 0.002 Hydrocortisone 1.97 1.95 1.95 2.05 2.01 2.00 Prednisolone 2.29 2.18 2.19 2.23 2.26 2.30 2.20 2.27 2.13 2.23 2.16 (+)-cis-Epivincamine 0.82 1.21 6.08 7.32 6.75 6.23 7.10 6.60 6.33 6.03 6.00 (-)-cis-Epivincamine 0.82 1.21 6.75 8.14 7.64 7.00 7.87 7.30 7.10 6.70 6.67 (+)-cis-Vincamine 0.65 1.00 8.23 11.1 10.1 8.97 10.9 10.0 9.50 8.93 8.73 (-)-cis-Vincamine 0.65 1.00 8.94 11.5 10.5 9.40 11.4 10.4 9.93 9.33 9.10 (+)-cis-Apovincaminic 0.41 0.59 6.50 7.46 6.89 6.20 7.20 6.53 6.80 6.23 6.00 acid ethyl ester 6.80 6.23 6.00 (-1-cis-Apovincaminic 0.41 0.59 6.50 7.46 6.89 6.20 7.20 6.53 acid ethyl ester 13.2 17.0 16.5 14.5 (+)-trans-Apovincaminic acid ethyl ester 13.6 17.6 17.2 15.1 (-)-trans-Apovincaminic acid ethyl ester 0.15 0.31 5.52 6.57 5.71 5.20 (+)-cis-Vincamenine (+)-cis-Apovincamine 0.41 0.66 7.80 8.93 7.75 7.10 (+)-cis-Vincamone 0.32 0.52 5.71 7.14 6.71 5.97 (-)-cis-Vincamone 0.32 0.52 5.71 7.14 6.71 5.97 (+)-cis-Vincanol 0.53 0.62 5.28 7.25 6.75 6.00 (-)-cis-Vincanol 0.53 0.62 5.71 7.50 7.04 6.17 (+)-cis-Vincaminic acid 0.47 0.72 8.35 8.71 8.50 7.30 ethyl ester (-)-cis-Vincaainic acid 0.47 0.72 8.72 9.21 9.04 7.70 ethyl ester

0.0021 0.00215 0.0022 0.003 0.004 0.002 0.002 0.002 0.002 0.002 2.23 5.81 6.48 8.48 0.87 5.65

2.11 5.97 6.68 8.77 9.10 5.45

2.20 2.26 2.26 1.65 1.65 0.55

2.23 0.94 0.94 0.74 0.74 0.42

2.23 0.87 0.87 0.65 0.65 0.42

5.65

5.45

0.55

0.42 0.42

W IP 0

TABLE 8.10 SEPARATION PPT1CP.L I S O I I E R I C EBURNANE ALKALOIDS; DEPENDENCE OF CAPACITY FACTORS ( k ' ) AND SEPARATION FACTORS (rji) CONCENTRATION OF CHLOROFORI" AN0 ALCOHOLS I N THE

ON THE

Column, N u c l e o s i l 10 CN (250xn.6 mn 1.0.); f l o w - r a t e : 1 ml/min; d e t e c t i o n a t 280 nm. A 1 1 volume o f e l u e n t c o n t a i n s 2x10-3 mole o f (+)-10-camphorsulfonic a c i d and 10-3 m l e o f DEA. Compound

Eluent mixture

Hexane-chloroform-methanol 80:18:2 70:27:3 60:36:4

Hexane-chloroform-ethanol

k'

k'

-80:18:2 70:27:3

(+)-cis-Vincaminic a c i d ethyl ester (-)-cis-Vincaminic acid ethyl ester (+)-cis-Epi v i ncamine (-)-cis-Epivincamine (+)-cis-Vincamine ( - ) - c i s-Vincami ne ( + ) - t r a n s - E p i v i ncami ne (-)-trans-Epivincamine (+)-trans-Vincamine ( - )-trans-Vincamine ( + ) - c i s-Vincamone (-)-cis-Vincamone (+)-trans-Vincamone (-)-trans-Vincamone (+)-cis-C.povincaminic acid ethyl ester (-)-cis-Apovincaminic acid ethyl ester (+)-trans-Apovincaminic acid ethyl ester (-)-trans-Apovincaminic acid ethyl ester Hydrocortisone Prednisolone

rji

5.24

k'

rji

1.55 1.05

5.50

k'

r.. J1

0.65 1.06

1.65

rji

6.55 1.00

;:;

1.10

98:;

1.08

;:; 1.10 ;::: 1.08

>20

::::

1.06

3.86

1 .oo

3.86

i:;: 1.04 1.20 2.60

1.03

1 .no

0.45

1.20

11.0

0.45

1.00

'20

1 .oo

;:it

1.00

17.9 19.2 1.07 5.38 1.04 5.57 18.7

0.91 1.00

J1

0.75

2.10

>20

1.00

J1

2.40

2.40

1.60

1 .oo

1.70

1.03

k'

.14

'ji

1.10

3.48 .09 2:01 1 90 1.06 3.81 4.24 -13 2.70 3.00 1.10 4.81 6.33 > 20 >20 >20 6.90 '.09 >20 > 20 10.2 >20 11.1 1-08 >20 3.67 .oo 11:80 80 1.00 '.O0 3.67 >20 > 20 '20 >20 12.5 3.90 .04 1.05 13.2 4.10

1.04 1.14 1.09

;:;

1.00

2.81 3.00 0.55

1.07 1 .oo

0.55

4.85 1.04

3.48

J1

0.90 l*oo 1.11

33:57 38 1.06

1.60

60:36:4

10.9

1.05

1.07

70:27:3 k' r..

0.83

;::;1.04

;:::

J1

10.6

1.06

1.00

80:18:2 k' r..

1.10

1.11

> 20

;:

r..

1.90 1.10

7.20

0.65

k'

Hexane-chloroform-isopropanol

60:36:4 k' r..

1.95

3.95

1.87

11.3 12.6 1'12 14.8 16.0 '.14 > 20

;:;: >20

> 20

19.7 1.06

1.00

1.03

11.4

2.60

0.91

19.5

5 .OO

1.95

>20

>20

20.9

10.3 14.2

3.40 4.60

1.36 2.09

12.5 17.2

3.70 5.10

1.76 2.35

20.0 >20

6.14 9.00

2.52 3.85

341 TABLE 8.11 OPTIMAL SEPARATION SYSTEMS FOR OPTICAL I S O M E R I C EBURNANE ALKALOIDS7' Column, I Nucleosil CN 5 p m ( 1 5 0 ~ 4 . 6nun I D ) , I 1 Nucleosil CN 10 p m ( 2 5 0 ~ 4 . 6mn ID), mobile phase, A hexane - dioxane - n-butanol (70:25:5). B hexane - chloroform - ethanol (70:27:3), C hexane - dioxane - ethoxyeThano1 (57.5:37.5:5), a l l three solvent systems contain 0.002 M (+)-10-camphorsulfonic a c i d and 0.001 M diethylamine, f l o w r a t e 1.5 ml/min. d e t e c t i o n UV 280 nm.

H(mn)

RS

Asf*

-

A 6 A 8 6 6

7.95 1.78 8.90 2.21 7.04 9.04

8.70 1.95 10.05 2.37 7.42 9.51

1.07 1.10 1.13 1.07 1.05 1.05

0.060 0.023 0.064 0.026 0.041 0.054

1.35 1.26 1.60 1.16 1.14 1.20

0.98 1.15 1.27 1.14 1.25 2.08

-

A

7.78

8.83

1.14

0.061

1.77

0.92

1 - 8 I1 - c I1 - c

4.83 2.83 10.2

4.97 4.10 10.8

1.03 1.45 1.06

0.097 0.084 0.112

1.22 3.11 1.15

1.44 1.20 2.26

Compound

HPLC system

(+)-ci s-Epivincami ne

I1 1 I1 1 1 1

-

I1

( 5 ) - c i s-Vincamine (5)-trans-Epivincamine (+)-trans-Vincamine (2)-cis-Apovincaminic a c i d ethyl ester (2)-trans-Apovi ncami n i c a c i d ethyl ester (+)-ci s-Vi ncamone (+I-trans-Vi ncamone

*

-

-

k'(t)

r..

k'(-)

.I1

Asf = back p a r t o f the peak / f r o n t p a r t o f the peak

TABLE 8.12 SEPARATION

OF SOME STRYcHNoS A L K A L O I D S ~ ~

Column, Merckosorb Si60 5 p m (300x2 mm ID), mobile phase S 1 d i e t h y l e t h e r - d i e t h y l amine (99:1), f l o w r a t e 2.00 ml/min, 52 d i e t h y l e t h e r - methanol ( l : l ) , f l o w r a t e 1.15 ml/min, detection UV 254 nm. A1 kal o i d I c a j i ne Vomi c i ne Pseudostrychnine Strychnine 4-Hydroxystrychnine a-Col ubrine

Retention time (min) s1 s2 4.2 4.6 6.8 7.2 7.6 8.8

Alkaloid

2.6 1.6

Spermostrychnine 6-Col u b r i ne O i abol ine Brucine Serpentine A1 s t o n i ne

12.4 14.3

Retention time (min) s1 52

9.8 10.3 16.0 18.4 >20 >20

10.2 10.9 17.6

TABLE 8.13 RETENTION DATA OF RESERPINE ,HYDROCHLOROTHIAZIOE, AND RELATED Column, Lichrosorb Si60 5 pm ( 2 5 0 ~ 2 . 1nun ID), mobile phase n-hexane - isopropanol form - diethylamine (77:18:5:0.01), f l o w r a t e 1.5 ml/min. d z t e c t i o n UV 254 nm. Comoound

Retention k' time [sec) 68 1.0 50 0.5

Reserpine 3-Isoreserpi ne 3,4-Dehydroreserpi ne

3.4,5,6-tetradehydroreserpine(lumi-) Deserpidine Rescinnami ne 3,4,5-Trimethoxybenzoic t

Retained peak

References p. 344

acid

-

-

61 82

0.8 1.4

-

-

-

Comoound

Retention time (sec) 3,4,5-Trimethoxycinnamic a c i d

l-Amino-3-chloro-4.6-benzenedisulfonamide Hydrochlorothiazide Chlorothiazide Methychlothiazide Bendroflumethiazide Po 1y t h ia z i de

chlorok'

-

210 320 200 114 59 108

5.2 8.5 4.9 2.4 0.7 2.2

TABLE 8.14 CAPACITY RATIOS, k ' , FOR EBURNANE ALKALOIDS USING FOUR-COf!PONENT ELUENT Column Lichrosorb Si60, 5 pm (250~4.6nnn ID), flow rate 1 ml/min, detection UV 280 nm. ffobile phases hexane - chloroform acetonitrile methanol in the ratios 1, (55:20:25:3); 2, (55:25:20:3); 3, (55:22.5:22.5:3); 4, (60:20:20:3); 2, (60:24:15:3); 6, (60:22.5:17.5:3); 7, (65:17.5:17.5:3); 8, (65:20:15:3); 2, (65:15:20:5); lo, (55:25:20:1);-fi, (55:25:20:5).

-

A1 kaloid ~~

+)-trans-Apovincaminic acid ethyl ester +)-trans-Epivincaminic acid ethyl ester +)-trans-Vincaminic acid ethyl ester +)-cis-Vincamone +)-ci s-Apovi ncami nic acid phenyl ester +)-cis-Apovincaminic acid ethyl ester +) -ci s-Apovi ncami ne +)-cis-Vincamenine +)-cis-Dehydrovincamine +)-cis-Oehydroepivincamine +)-cis-10-Bromovincamine

+)-cis-11-Bromovincamine +)-cis-Vincaminic acid ethyl ester +)-cis-Vincamine -)-cis-Vincamine +)-cis-Epivincaminic acid ethyl ester +)-cis-10-Methoxyvincamine +)-cis-Epivincamine + ) - c i s-Vincanol e +)-cis-Isovincanol e

Eluent 1 0.32 0.53 0.68 0.88 0.92 1.00 1.13 1.29 1.40 1.55 1.62 1.77 2.17 2.35 2.35 3.09 3.09 3.43 3.43

3.96

2 0.18 0.36 0.49 0.62 0.68 0.76 0.85 0.97 1.05 1.15 1.26 1.39 1.65 1.79 1.79 2.56 2.35 2.79 2.79 3.13

3 0.22 0.40 0.54 0.68 0.72 0.78 0.85 0.92 1.00 1.10 1.25 1.37 1.61 1.75 1.75 2.36 2.32 2.61 2.61 2.97

4 0.20 0.47 0.56 0.71 0.78 0.82 0.98 0.98 1.25 1.35 1.48 1.62 1.90 2.15 2.15 2.86 2.86 3.24 3.09 3.58

5 0.15 0.33 0.58 0.63 0.68 0.76 0.82 0.91 1.15 1.27 1.37 1.51 1.74 1.98 1.98 2.97 2.62 3.26 3.26 3.54

6 0.20 0.45 0.58

0.68 0.75 0.82 0.88 0.96 1.25 1.35 1.46 1.62 1.93 2.18 2.18 3.06 2.84 3.35 3.35 3.85

7 0.25 0.57 0.74 0.78 0.82 0.85 0.98 0.98 1.50 1.62 1.74 1.89 2.18 2.49 2.49 3.39 3.39 3.92 3.54 4.04

8 0.20 0.45 0.68 0.64 0.62 0.64 0.71 0.71 1.23 1.33 1.43 1.57 1.71 1.99 1.99 2.94 2.66 3.24 2.94 3.24

9 0.35 0.65 0.74 0.78 0.78 0.78 0.88 0.77 1.25 1.35 1.50 1.63 1.76 2.04 2.04 2.63 2.63 3.08 2.63 3.08

10 0.10 0.39 0.61 0.82 0.86 0.96 1.14 1.27 1.52 1.64 1.76 1.93 2.35 2.60 2.60 4.64 3.60 5.34 4.28 4.88

11 0.05 0.27 0.39 0.40 0.40 0.40 0.50 0.50 0.60 0.65 0.80 0.88 1.04 1.14 1.14 1.59 1.47 1.73 1.78 1.88

343 TABLE 8.15

, FOR

CAPACITY RATIOS, k '

EBURNANE ALKALOIDS ON SONDAPAK CN WITH DIFFERENT ELUENTS55

Column, Sondapak CN ( 3 0 0 ~ 3 . 9 mm I.D.), f l o w - r a t e 1 ml/min. d e t e c t i o n UV, 280 nm. m o b i l e phases: A = hexane-chloroform-acetonitrile (65:20:15); B = 70:ZO:lO; C = 75:20:5. A1 k a l o i d

Eluent

(+)-cis-Vincamone (+)-cis-Apovincaminic acid e t h y l e s t e r (t) - c i s-Apovincami ne (+)-cis-Vincamenine (+)-cis-Vincaminic a c i d e t h y l ester (t)- c i s - V i ncami ne (-)-cis-Vincamine (+)-cis-Epivincaminic a c i d e t h y l e s t e r (t)-cis-10-Methoxyvi ncami ne (+)-cis-Epivincamine ( - ) - c i s - Epi v incami ne (+)-cis-Vincanole (t ) - c i s - I s o v i ncanole

0.11 0.23

A

0.25 0.28 0.72 1.00 1.00 1.15

1.28 1.34 1.34 1.57 2.62

F o r t h e a n a l y s i s o f r e s e r p i n e and h y d r o c h l o r o t h i a z i d e ,

B

C

0.27 0.32 0.40 0.43 0.86 1.07 1.07 1.20 1.61 2.06 2.06 2.ld 3.00

0.38 0.57 0.64 0.64 1.36 1.57 1.57 1.80 1.95 3.79 3.79 3.86 4.57

B u t t e r f i e l d e t a1 .30 p r e f e r r e d a

s t r a i g h t - p h a s e s e p a r a t i o n . Because o f t h e low c o n c e n t r a t i o n o f t h e a l k a l o i d compared t o t h e t h i a z i d e drug, i t was d e s i r a b l e t o maximize t h e response o f t h e f o r m e r by e l u t i n g i t f r o m t h e column f i r s t . I t was achieved by t h e s t r a i g h t - p h a s e s e p a r a t i o n t e c h n i q u e . On a m i c r o p a r t i c u l a t e s i l i c a g e l column, t h e drugs mentioned and a number o f d e c o m p o s i t i o n p r o d u c t s c o u l d be analyzed ( T a b l e 8.13), To a n a l y z e t h e ha1 1ucinogeni c a1 k a l o i d s i n Psilocybe mushrooms Whi t e 3 7 used a p o l a r m o b i l e phase

-

methanol - w a t e r

-

aqueous 1 M ammonium n i t r a t e (24:5:1)

t o w h i c h some ammonia was

added (pH 9.7) - and a s i l i c a g e l column ( F i g . 8 . 1 0 ) . By i n c r e a s i n g amounts o f ammonia, t h e s e p a r a t i o n of p s i l i c y b i n and b a e o c y s t i n ( d e m e t h y l p s i l o c y b i n ) c o u l d be improved. S i m i l a r s o l v e n t systems have been a p p l i e d t o t h e a n a l y s i s o f drugs o f abuse

-

including

(Table 7.8). C h r i s t i a n s e n e t a1.59'66'78

m o d i f i e d t h e method r e p o r t e d by White37. By u s i n g a d i f f e r e n t

s o l v e n t r a t i o , t h e s e p a r a t i o n o f t h e a l k a l o i d s was improved, t h e k ' o f p s i l o c i n p a r t i c u l a r l y was changed ( F i g . 8 . 1 1 ) .

The two-step e x t r a c t i o n method employed guaranteed a t l e a s t a 98%

e x t r a c t i o n o f p s i l o c y b i n . As e x t r a c t i n g s o l v e n t , 10% 1

M ammonium n i t r a t e i n methanol was

used. Hara e t a l . 4 2 demonstrated a s y s t e m a t i c approach t o s o l v e n t system o p t i m i z a t i o n w i t h t h e Gardneria a l k a l o i d s .

s e p a r a t i o n o f a s e r i e s o f uncaria and

Because t h e s e p a r a t i o n o f some eburnane a l k a l o i d s c o u l d n o t be a c h i e v e d i n a reversed-phase s e p a r a t i o n o f t h e s e a l k a l o i d s 55

HPLC system54, normal-phase HPLC was a l s o i n v e s t i g a t e d f o r t h e

Using s i l i c a g e l w i t h a two-component m o b i l e phase ( c h l o r o f o r m - m e t h a n o l ) r e l a t i v e l y poor s e p a r a t i o n s were o b t a i n e d . A d d i t i o n o f hexane t o t h e m o b i l e phase gave o n l y a s l i g h t improvement. Four-component systems gave t h e b e s t r e s u l t s . A hexane - c h l o r o f o r m methanol (55:25:20:3)

-

acetonitrile

-

e l u e n t was found t o be o p t i m a l . By choosing an a p p r o p r i a t e m i x t u r e ,

d i f f i c u l t s e p a r a t i o n s o f s t e r e o and s t r u c t u r a l isomers c o u l d be o b t a i n e d ( T a b l e 8.14). Also, group s e p a r a t i o n was o b t a i n e d on s i l i c a g e l . A cyano-group bonded s t a t i o n a r y phase gave s i m i -

References p. 344

344

l a r r e s u l t s t o s i l i c a gel (Table 8.15). The a p p l i c a t i o n s o f some straight-phase systems t o separation problems w i t h Vinca a l k a l o i d s have been shown (Fig.8.12)

.

75

WhelptonB4 reported the analysis o f physostigmine i n plasma using a s i l i c a gel column w i t h the mobile phase methanol

-

1 M amonium n i t r a t e (pH 8.6)(9:1)

i n combination w i t h an e l e c t r o -

chemical detection method.

8.5. DETECTION Verpoorte and Baerheim Svendsen 7 determined the optimum wavelength o f d e t e c t i o n f o r some indole a l k a l o i d s f o r a f i x e d wavelength detector equipped f o r 254 and 280 nm detection. The r a t i o between the absorbance a t 254 and 280 nm, which i s c h a r a c t e r i s t i c f o r each compound, was used f o r the i d e n t i f i c a t i o n of drugs o f abuse 82 2 . 3 1 ~ ~the . r a t i o 220/254 nm has a l s o been used

.

-

i n c l u d i n g strychnine (Table 2.2,

Samz7 oxidized reserpine p r i o r t o i t s HPLC-analysis. The 3-dehydro d e r i v a t i v e formed was detected f l u o r i m e t r i c a l l y ( e x c i t a t i o n 390 nm, emission 470 nm). This allowed a more s p e c i f i c and s e n s i t i v e detection. E l l i p t i c i n e and d e r i v a t i v e s can be detected a t t h e i r UV-maximum o f about 300 nm w i t h a det e c t i o n l i m i t o f about 1 ng. For some a l k a l o i d s , fluorescence d e t e c t i o n i s even more sensitive

-

a l l o w i n g d e t e c t i o n i n pg amounts ( e x c i t a t i o n 305 nm, emission 470 nm). A poorly f l u o -

rescent compound such as 9-hydroxy-2-methylellipticinium acetate could be transformed t o i t s fluorescent acetoxy d e r i ~ a t i v e ~Bykadi ~. e t a1 .70 a l s o detected e l l i p t i c i n e f l u o r i m e t r i c a l l y ( e x c i t a t i o n 360 nrn, emission 455 nm). Sasse e t a1.45 used the d i f f e r e n c e i n fluorescence maxima o f harmane a l k a l o i d s i n t h e quant i t a t i o n o f the a l k a l o i d s a f t e r t h e i r separation (Fig.8.4).

F l u o r i m e t r i c d e t e c t i o n w i t h a de-

t e c t i o n l i m i t o f about 10 pg was about 100 times more s e n s i t i v e than UV detection. Fluorescence detection has a l o been applied i n t h e analysis o f Psilocybe a l k a l o i d s (Fig.8.11).

46.59

The minimum detectable amount was f o r f l u o r i m e t r i c d e t e c t i o n 250 pg ( e x c i t a t i o n

267 nm. emission 335 nm) and f o r UV d e t e c t i o n 30 ng (267 nm) f o r p s i l o c y b i n , f o r p s i l o c i n the detection l i m i t s were 7 ng ( e x c i t a t i o n 260 nm, emission 312) and 150 ng (267 nm). P s i l o c i n - a phenolic compound

-

can a l s o be detected w i t h an electrochemical detector3’.

Lang e t a1.80 reported the use o f a post-column air-segmented r e a c t o r t o enable f l u o r i m e t r i c detection o f reserpine. As o x i d i z i n g reagent: s u l f u r i c a c i d which converted reserpine i n t o 3,4-dehydroreserpine.

-

sodium n i t r i t e was used,

The r e a c t i o n product was detected by

measuring emission above 470 nm a f t e r e x c i t a t i o n a t a wavelength o f 395 nm. For the detection o f N-propylajmaline, f l u o r i m e t r i c d e t e c t i o n was employed ( e x c i t a t i o n 242 nm, emission 320 nm)8 3 . WhelptonE4 detected physostigmine electrochemically, using an o x i d a t i o n p o t e n t i a l o f 0.8

V vs SSCE. The capacitance c o n d u c t i v i t y detector as described by Hashimoto e t a1.32 was tested on a series o f a l k a l o i d s

-

i n c l u d i n g some i n d o l e a l k a l o i d s .

REFERENCES

1 C.Y. Wu and s. Siggia, A n a l . Chem., 44 (1972) 1499. 2 C . Y . NU, Diss. Abstr. I n t . 8 , 33 (1973) 4166. 3 J.O. Wittwer and J.H. Kluckhohn, J. Chromatogr. S c i . , 11 (1973) 1.

546

4 5 6 7 8

9 10 11 1

G.H. J o l l i f f e and E.J. S h e l l a r d , J . C h r o m a t o g r . , 81 (1973) 150. M.L. Chan, C. W h e t s e l l and J.D. McChesney, J . C h r o m a t o g r . s c i . , 12 (1974) 512. D.H. Rodgers, J . C h r o m a t o g r . S c i . , 12 (1974) 742. R . V e r p o o r t e and A. Baerheim Svendsen, J . C h r o m a t o g r . , 100 (1974) 227. R. Verpoorte and A. Baerheim Svendsen, J . C h r o m a t o g r . , 100 (1974) 231. H.F. Walton, J . C h r o m a t o g r . , 102 (1974 57. I . L . Honigberg, J.T. S t e w a r t and A.P. k t h , J. P h a r m . S c i . , 63 (1974) 766. I . L . Honigberq, J.T. S t e w a r t , A.P. Smith, R.O. P l u n k e t t and D.W. Hester. J . P h a r m . S c i . ,

63 (1974)-1762. 12 P.J. T w i t c h e t t , Chem. B r . , 11 (1975) 443. H.W. Z i e g l e r , T.H. Beasley and D.W. Smith, J. dssoc. off. A n a l . C h e m . , 58 (1975) 888 13 14 R.J. Bushway, C.W. Cramer, A.R. Hanks and B.M. C o l v i n , J . dssoc. off. A n a l . Chem., 58 (1975) 957. 15 P.J. T w i t c h e t t , J . C h r o m a t o g r . , 104 (1975) 205. 16 E. Murgia and H.F. Walton, J . C h r o m a t o g r . , 104 (1975) 417. 17 0.1. K i n g s t o n and B.T. L i , J . C h r o m a t o g r . , 104 (1975) 431. 18 R. V e r p o o r t e and A. Baerheim Svendsen, J . C h r o m a t o g r . , 109 (1975) 441. 19 I . Jane, J . C h r o m a t o g r . , 111 (1975) 227. 20 I . L . Honigsberg, J.T. Stewart, A.P. Smith and D.W. Hester. J . Pharm. S c i . , 64 (1975) 1201. 21 E.O. Murgia, D i s s . d b s t r . I n t . B , 36 (1976) 3911. 22 8.B. Wheals, J . C h r o m a t o g r . , 122 (1976) 85. 23 J.W. Robinson, EDRO SARdP R e s . T e c h . R e p . , 2 (1977) 1035. CA 88 (1978) 141759h. 24 I . L u r i e , J . Assoc. Off. A n a l . Chem., 60 (1977) 1035. 25 R.G. A c h a r i and E.E. Theimer, J. C h r o m a t o g r . S c i . , 15 (1977) 320. 26 5. Gdrog. 8. Herenyi and K. Jovanovics, J . C h r o m a t o g r . , 139 (1977) 203. 27 R. Sams. A n a l . L e t t . , 611 (1978) 697. 28 E. Leverd, D. B e z i a t and P. H a t i n g u a i s . ~ 0 1 1 . C h i m . F a r m . , 117 (1978) 27. 29 M.D. Crouch and C.R. S h o r t , J. Assoc. Off. A n a l . C h e m . , 6 1 (1978) 612. 30 A.G. B u t t e r f i e l d , E.G. L o v e r i n g and R.W. Sears, J . Pharm. S c i . , 67 (1978) 650. 31 R.L. Hussey and W.M. Newlon, J . P h a r m . S c i . , 67 1978) 1319. 32 Y. Hashimoto, M. Moriyasu, E. Kato, M. Endo. M. i i y a m o t o and H. Uchida, M i k r o c h i m . d c t a 2 (1978) 159. 33 R. Verpoorte, E.W. Kodde and A. Baerheim Svendsen, P l a n t a M e d i c a , 34 (1978) 62. 34 J . Gleye, E. Lavergne de Cerval, E. S t a n i s l a s , E. Leverd, 0. B e z i a t and P. H a t i n g u a i s , A n n . Pharm. F r a n c . , 37 (1979) 217. 35 J.K. Baker, R.E. S k e l t o n and Ch.Y. Ma, J . C h r o m a t o g r . , 168 (1979) 417. 36 G. Muzard and J.B. Le Pecq, J . C h r o m a t o g r . , 169 (1979) 446. 37 P.C. White, J . C h r o m a t o g r . , 169 (1979) 453. 38 R. Gimet and A. F i l l o u x , J . C h r o m a t o g r . , 177 (1979) 333. 39 M.M. White, J . C h r o m a t o g r . , 178 (1979) 229. 40 E. Soczewinski and T. Dzido, J . L i g . C h r o m a t o g r . , 2 (1979) 563. 41 R. Verpoorte and A. Baerheim Svendsen, Z b l . P h a r m . , 118 (1979) 563. 42 S. Hara, N. Yamauchi, C. Nakae and S. Sakai. A n a l . C h e m . , 52 (1980) 33. 43 K. Aramaki, T. Hanai and H.F. Walton, A n a l . C h e m . , 52 (1980) 1963. 44 R. Verpoorte, i n I n d o l e a l k a l o i d s a n d b i o g e n e t i c a l l y r e l a t e d a l k a l o i d s , e d i t e d b y J.D. P h i l l i p s o n a n d M . H . Z e n k , d c a d e m i c P r e s s , L o n d o n , 1980, p. 91. 45 F. Sasse, J . H a n e r and J. B e r l i n , J . C h r o m a t o g r . , 194 (1980) 234. 46 M. P e r k a l , G.L. Blackman, A.L. O t t r e y and L.K. Turner, J . C h r o m a t o g r . , 196 (1980) 180. 47 M. Kneczke, J . C h r o m a t o g r . , 198 (1980) 529. 48 J.L. Love and L.K. P a n n e l l , J . F o r e n s i c S c i . , 25 (1980) 320. 49 B.M. Thomson, J . F o r e n s i c S c i . , 25 (1980) 529. 50 W . Kohl, H. Vogelmann and G. H o f l e , P l a n t a M e d . , (1980) 283, A b s t r a c t s I n t e r n a t i o n a l con51 52 53 54 55 56 57

58 59 60

gress on N a t u r a l P r o d u c t s as M e d i c i n a l A g e n t s , S t r a s s b o u r g , 1 9 8 0 . J.D. W i t t w e r , Forensic S c i . I n t . , 18 (1981) 215. P.8. Baker and T.A. Gough, J . C h r o m a t o g r . S c i . , 19 (1981) 483. C. Dubruc, H. Caqueret and G . B i a n c h e t t i , J . C h r o m a t o g r . , 204 (1981) 335. G . Szepesi and M. Gazdag, J . C h r o m a t o g r . , 204 (1981) 335. G . Szepesi and M. Gazdag, J . C h r o m a t o g r . , 205 (1981) 57. M.W. Beug and J . Bigwood, J . C h r o m a t o g r . , 207 (1981) 109. B. Zsadon. M. S z i l a s i . F. Tudos and J. S z e j t l i , J . C h r o m a t o g r . . 208 (1981) 109. P . P i e t t a , s A . Rava and-E. Catenacci, J . C h r o m a t o g r . , 210 (1981)-149. A.L. C h r i s t i a n s e n , K.E. Rasmussen and F. Tonnesen, J . C h r o m a t o g r . , 210 (1981) 163. M. Verzele, L. de Taeye, J. van Oyck. G. de Decker and C. de Pauw, J. C h r o m a t o g r . ,

.

1981 95

,

214

61 J.E. b a r k j n , J . C h r o m a t o g r . , 225 (1981 240. 62 G. Hoogewijs, Y . M i c h o t t e , J. Lambrech and D.L. Massart, J . C h r o m a t o g r . , 226 (1981) 423. 63 I.S. L u r i e and S.M. Oemchuk, J. L i q . C h r o m a t o g r . , 4 (1981) 337.

2

346

64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84

I . S . L u r i e and S.M. Demchuk, J. L i q . C h r o m a t o g r . , 4 (1981) 357. I.S. L U r i e , J. L i g . C h r o m a t o g r . , 4 (1981) 399. A.L. C h r i s t i a n s e n , K.E. Rasmussen and K. HBiland, Plants M e d . , 42 (1981) 229. N.W. Tymes, J. ASSOC. Off. A n a l . Chem., 6 5 (1982) 132. T.A. Gough and P.8. Baker, J. C h r o m a t o g r . sci., 20 1982) 289. R.R. B r o d i e and L.F. Chasseaud, J . C h r o m a t o q r . , 228 11982) 413. G. Bykadi, K.P. F l o r a , J.C. Cradock and G.K. P o o c h i k i a n , J . C h r o m a t o g r . , 231 (1982) 137. L. A l l i o t . G. B r y a n t and P.S. Guth, J . C h r o m a t o g r . , 232 (1982) 440. J.G. Umans. T.S.K. Chiu, R.A. Lipman, N.F. S c h u l t z , S.U. S h i n and C.E. I n t u r r i s i , J. Chromatogr., 233 (1982) 213. G. Szepesi. M. Gazdag and R. I v a n c s i c s , J . C h r o m a t o q r . , 241 (1982) 153. M. Kozma, P. P u d l e i n e r and L. Vereczkey, J. C h r o m a t o g r . , 2 4 1 (1982) 177. M. Gazdag, G. Szepesi and K. Csomor, J. Chromatogr., 243 (1982) 315. G. Szepesi. M. Gazdag and R. I v a n c s i c s , J . C h r o m a t o q r . , 244 (1982) 33. J.D. P h i l l i p s o n , N. S u p a v i t a and L.A. Anderson, J . C h r o m a t o q r . , 244 (1982) 91. A.L. C h r i s t i a n s e n and K.E. Rasmussen, J . C h r o m a t o q r . , 244 (1982) 357. R.J. Flanagan, G.C.A. S t o r e y , R.K. Bhamra and I. Jane, J . C h r o m a t o q r . , 247 (1982) 15. J.R. Lang, I.L. Honigberg and J.T. Stewart. J . C h r o m a t o q r . , 252 1982) 288. T. E g l o f f , A. N i e d e t w i e s e r , K. P f i s t e r , A. Otten, B. Steinmann, S t e i n e r and R . G i t z e l mann, J. C l i n . Chem. Clin. B i o c h e m . , 20 (1982) 203. I.S. L u r i e , S.M. S o t t o l a n o and S . B l a s o f , J. F o r e n s i c Sci., 27 (1982) 519. I. Grundevik and B.A. Persson, J. L i g . C h r o m a t o g r . , 5 (1982) 141. R. Whelpton, J. C h r o m a t o g r . , 272 (1983) 216.

4.

TABLE 8.16 INDOLE ALKALOIDS I N THE CONTEXT OF HPLC ANALYSIS OF DRUGS OF ABUSE (CHAPTER 7 and 9 ) A1 k a l o i ds S* S

S B S S

Psi l o c i n S S

S S S S

Ref. 3 5 12 13 15 19 22 24 35 39 48 51 52 63,64,65 72 82

Ref. i n Chapter 7

1 11 15 17 18( F i g .7.2) 22(Table 7.8,Fig. 7.16) 32

38 56 61 79 91(Table 7.111 9 3 ( F i g . 7 -6) 98,99,100 113 121(Table 7.6)

-

* A b b r e v i a t i o n s used i n Tables 8.16 Ajmal Alst 8 a-C

8-C

D

I 40HS Phys PSS

Ref. i n Chapter 9

Ajmal ine A1 s t o n i ne Brucine a-Col u b r i ne 8-Colubrine Diabol i n e I c a j i ne 4-hydroxystrychnine Physostigmi ne Pseudostrychnine

8.20

RSC RSP S Sarp THalst

V VCR VLB Yoh

Resci nnami ne Reserpine S t r y c h n i ne Sarpagine T e t r a h y d r o a l s t o n i ne Vomi c i ne V i n c r i s t i ne V i n b l a s t i ne Yohi mbi ne

I

TABLE 8.17

3

HPLC ANALYSIS VARIOUS COMPOUNDS INCLUDING INDOLE ALKALOIDS

f rn

P 0

P

ALKALOIDS *

OTHER COMPOUNDS

STATIONARY PHASE

AIMS

COLUMN DIM. LxIO fmm)

P

S ,B,xanthi nes

Dynamic c o a t i n g HPLC

S,B ,RSP ,yoh,THal s t , ajmal ic i ne ,serpent i ne,al s t , 16 o t h e r s

Analysis a l k a l o i d s

S,opium a l k a l o i d s , q u i nine,ci nchoni ne, n i c o t i n e , a t r o p i ne, cocaine S,B,RSP,yoh,Cinchona a1 k a l o i ds ,scopol amine, emeti ne , c a f f e i ne B,codeine,narceine, c i nchoni dine,col c h i c i ne,aconi t i n e ,caffeine

REF.

C o r a s i l I o r 11, dyn a m i c a l l y coated w i t h 1.1%Poly 6-300 Merckosorb Si60 5 urn

500x1

Heptane-EtOH(10:l) s t a t i o n a r y phase

300x2

C H C l -MeOH(9:1),(8:2),(7:3) Et2D3MeOH(8:2), ( 7 : 3), (6:4)

Separation on ion-exchange r e s i n s (ligand-exchange LC)

Hydrolyzed Porggel PT loaded w i t h Cu Bio-Rad rC20 loaded w i t h Cu'

4 7 0 ~ 6 . 3 0.06M 0.2 M 4 7 0 ~ 6 . 3 0.05M 0.03M

D e t e c t i o n w i t h conductance detector

S i l i c a g e l 10 urn

E f f e c t s o l v e n t composition on r e t e n t i o n

L i c h r o s o r b RP2 10

S e o a r a t i o n on s t v r e n e - d i v i nyibenzene copo1;mer (Table 8.4, Fig.8.6)

H i t a c h i g e l 3010 10 m 2 2 0 ~ 4 . 6 ACN-O.OZM NH OH(6:4) ACN-0.02M t e f r a b u t y l amnoni umhydroxide(3:7),(6:4)

S e p a r a t i o n b a s i c drugs w i t h non-aqueous i o n i c s o l v e n t s

S p h e r i s o r b S5W silica

sat. w i t h 1,2

7 NH OH NH4DH NH40H NHO :H

in in in in

33% E t O H 33% E t O H 33% EtOH 33% EtOH

9,16,21

CHCl 3-MeOH-hexane(7:3: 10) 32

Santonine

pin

1 2 0 ~ 3 . 5 MeOH-H20(1:4),(2:3),(3:2),(4:1) MeOH 40

S, RSP,yoh ,opi um a1 kaloids,cinchonine,quin i ne,atropi ne,ephedrine,acridine

Ajmal i n e , v a r i o u s others

MOBILE PHASE

43 Various drugs

2 5 0 ~ 4 . 9 MeOH-hexane(85:15) 0.02% HC104

containing 79

TABLE 8.18 HPLC ANALYSIS INDOLE ALKALOIDS I N PLANT MATERIAL AND I N NATURALLY OCCURRING MIXTURES ALKALOIDS*

AIMS

STATIONARY PHASE

Speciopbylline,uncarine F,isopteropodine.mi t r a p h y l 1 ine,iso-

Separation Y i t r a g y n a a l k a loids

Corasil

mitraphylline,rhynchophylline, *For a b b r e v i a t i o n s used see f o o t n o t e Table 8.16

Cis

COLUMN DIM. LxID (mm)

MOBILE PHASE

5 3 2 ~ 1 . 7 !1eOH-H20(4: 1)

REF

isorhynchophylline.rotundifo1ine isorotundifoline,rhynchociline, ci 1 iaphyl line Separation (Fi9.8.9) S.B.serpenti ne ,a1 s t Semipreparative separation Tabernaemontana a1 ka 1 oi ds

S,B,a-C,B-C.D,40HS,PsS,I.V,

spermoS,alst,serpentine alkaloids 26 Catharanthus alkaloids

Rauwolfia

qimal i ci ne Bisnordihydrotoxiferine,bisnor-C-alkaloid H.caracurine V Ellipticine and derivatives

Psi locybin,psi 1 oci n.baeocysti n RSP,yoh,mcaris and Gardneria alkaloids

Harmol.hatmine.harmalo1 ,hatmaline Psilocybin,psilocin Psi 1 ocybi n ,psi loci n a 1 ka 1 oi ds 22 Eburnane alkaloids

Ca tharan t h u s

Separation Strychnos alkaloids (Table 8.12) Analysis in plant material Quality control (Table 8.2. Fig.8.2) Analysis in plant material Isolation from s t r y c h n o s species Separation (Table 8.5,8.6)

Merckosorb Si60 5 Porasil C Porasil B C18

pm

A1 707 Merckosorb Si60 5 No details available Lichrosorb RP8 pPorasi1 ,Porasil ,,Bondapak C18

300x2 Et20-MeOH-DEA(90:10:1),(70:30:1) 6 1 0 ~ 9 . 5 CHCl3-MeOH-NH40H(99:O.8:0.Z) 6 1 0 ~ 9 . 5 MeOH MeOH-H 0-NH OH(90:5:5) 610~9.5 CHCl 3-gexan8(9: 1 ) 300x2 Et OIDEA(99:l) Et;O-MeOH(l:l) 250x4

4 8

17 18 23

ACN-O.01M aq. (NH4)2C03(47:53)

300x4 _?-he~ane-(isopr)~O-MeOH(90:10:3) 300~3.9 EtOAc-MeOH-25% NH40H(90:10:1), (80:25 :2) 300x4 MeOH-0.005M heptanesulfonic acid, 0.032M AcOH( 7:3), (3: 1) MeOH-0.OOSM pentanesulfonic acid, 0.032M AcOH(7:3) MeOH-0.02M NH40Ac(3:1) 250~4.6 MeOH-H20-1M NH4N03(24:5:l)(pH 9.7)

26 28.34 33 41.44 9

36

Analysis Psilocybe mushrooms Partisil 5, 6 (Fig.8.10) 37 n-hexane-THF, CH C1 -DEA, CHC13-MeOH. Solvent system optimization Spherosil XOA-600. 5 or Wakogel LCH-10, 10pm 100~0.5 THF-MeOH in vari&~s~ratios procedure Stepgradient CH C1 -MeOH-DEA(100:O.l: 0.01) to (lOO:l?0.6l) 42 250x3 MeOH-H O-HCOOH(l66:34:1) buffered at Analysis in tissue culture of Lichrosorb RP8 7 P e q a n u m harmala (Fig.8.4) pH f1.5~with TrEA 45 Analysis Psilocybe mushrooms Partisil SCX-10 250~4.6 MeOH-H 0(1:4) containing 0.2% NH446 (Fig.8.1) phosphite and 0.1% KCI, pH 4.5 Analysis Psilocybe mushrooms ,Bondapak C18 300x4 MeOH-0.4% phosphate buffer(pH 7.2) -cetrirnoniurn bromide(40:60:0.15) 49 Isolation from tissue culture Lichrosorb RP18 MeOH-H20-TrEA( 75:25:0.1) ,(95:5:O. 5) 50 300~3.9 ACN-O.01M (NH ) C03 (2:3),(1:1), Separation eburnane alkaloids ,Bondapak C18 or Li chrosorb RP18 250~4.6 (3:2),(7:3),(4:?) 54 (Table 8.3. Fig.8.3)

!3?

21 Eburnane a l k a l o i d s

Separation eburnane a l k a l o i d s Micropak Si 10 o r L i c h r o s o r b Si60 5 (Table 8.14. 8.15) pBondapak CN

Psilocybin.psi1ocin.baeocystin

Analysis Psilocybe mushrooms

uBondapak C18

3 0 0 ~ 3 . 9 0.05M h e p t a n e s u l f o n i c a c i d i n H 0-MeOH(3:l) o r H20-ACN(3:l).pH 3.5 w?th AcOH 0.05M tetrabutylamnonium i n H20-MeOH 56 ( 3 : l ) pH 7.5 w i t h H3P04

Tabersonine, ( - ) and (+)-vincad i f f o r m i ne,vi ncami ne,apo- and

Separation w i t h i n c l u s i o n LC

6-Cyclodextrin polymer

280x16

Psilocybin.psilocin

A n a l y s i s Psilocybe mushrooms (Fig.8.11)

P a r t i s i l 5. 6 um

2 5 0 ~ 4 . 6 MeOH-H 0-1M aq. NH4N03(pH 9.6) (22: 7 :?)

VCR.VLB.vindo1 ine.vindo1 inine. c a t h a r a n t h i ne . l e u r o s i ne ,cOrOn a r i d i ne

Separation vinca rosea a l k a 1o i ds

RSil-C18-HL-0 10 urn

17 Eburnane a l k a l o i d s

Separation eburnane a1 k a l o i d s vBondapak CN (Table 8.8, 8.9)

I

rn

P ca P

250x2 Hexane-CHC1 -ACN-MeOH(55:25:20:3) 2 5 0 ~ 4 . 6 and v a r i o u s 3 0 t h e r r a t i o s 3 0 0 ~ 3 . 9 Hexane-CHC13-ACN( 65: 20: 15), (7:2: 1 ) (75: 20: 5 )

55

pH 5 c i t r a t e b u f f e r

ethylapo-vincamine,quebrachamine,N-methylquebrachamine.

57

( - ) - v i ncadi ne

59,66

2 5 0 ~ 4 . 6 g r a d i e n t MeOH-H O ( 1 : l ) t o (85:15) c o n t a i n i n g 0. 112ethanolamine

60 3 0 0 ~ 3 . 9 Hexane-CHC1 -ACN-di-(2-ethylhexyl)phosphoric a c i d i n various r a t i o s

Hexane-isoprOH-DEA-camphorsulfonic a c i d i n various r a t i o s

73

Analysis i n vinca e x t r a c t s ( F i g . 8.12)

L i c h r o s o r b Si60 5 urn Micropak S i 10 uBondapak C18

2 5 0 ~ 4 . 6 Hexane-CHC1 -MeOH(8:1:1) 250x2 CHCl -EtOH(d5:5) 3 0 0 ~ 3 . 9 ACN-d. 01M ( NH4)2C03( 3: 2)

18 Eburnane a1 k a l o i ds

Separation stereoisomers (Table 8.10, 8.11, Fig.8.8)

N u c l e o s i l 5CN o r Nucleosil lOCN

1 4 0 ~ 4 . 6 Hexane-CHC1 -MeOH, hexane-CHC1 -EtOH 250~4.6 o r hexane-Ck1 -isoprOH (80:18?2), (70:27:3) o r (80:36:4) c o n t a i n i n g 0.002M camphorsulfonic acid, 0.001M DEA 76

9 Heteroyohimbine and 8 o x i n dole alkaloids

Separation on reversed phase Spherisorb ODS 5 urn HPLC (Table 8.1)

250x4

Psi 1ocybin.psi 1ocin.baeocystin

A n a l y s i s Psilocybe mushrooms

2 5 0 ~ 4 . 6 *OH-H 0-1M NH NO (pH 9.6)(22:7:1) M ~ O H - H ~ ~ O -NH:OA~ ~ M (PH 9.6) (24: 5: 1 )

Vincamine.vincine,vincanole, isovincanole,vincamone,vincamenine

P a r t i s i l 5, 6 urn

MeOH-H O(4:l) MeOH-H20-conc. NH OH( 80: 20: 1) ACN-IX~(NH co (4:2) MeOH-1%(N H $ ~ C O i ( 4: 1)

75

77 78

ca

TABLE 8.19

EIl 0

HPLC ANALYSIS INDOLE ALKALOIDS I N PHARMACEUTICAL PREPARATIONS

ALKALOIDS

*

OTHER COMPOUNDS

RSP,RSC.sarp,yoh, ajmal

AIMS

STATIONARY PHASE

COLUMN DIM. LxID(m)

MOBILE PHASE

REF.

A n a l y s i s Rauwolfia a l k a l o i d s

ION-X-SC

1 0 0 0 ~ 2 . 6 0.02M (NH4)2HP04 i n MeOH-H20(3:7) pH 7.0 0.05M (NH ) HPO i n MeOH-H20(3:7) pH 8.0 g r a d i e n t from 0.01M. pH 7.0 t o 0.05M pH 8.0

Analysis i n pharmaceutical preparations

C o r a s i l C18

1 2 2 0 ~ 2 . 3 MeOH-0.5% NH4C1(55:45)(pH 5.6) 1 2 2 0 ~ 2 . 3 ACN-0.1% NH OA~(4:1),(3:2).(2:3),(1:1) MeOH-1% NH dAc(1:l) ACN-1% NH h(4:1),(1:1) MeOH-0.5X4NH C l ( 1 : l ) ACN-0.2% (NH4) CO (1:l) NOH-0. 2%(NH4)2C03( 1:1) MeOH-1% (NH4f2zO3fl: 1) 20

6

RSP

Chlorothiazide. phenantrene

RSP

Chlorothiazide, Analysis diuretic-antihypertriamterene.hyt e n s i v e drugs d r o c h l o r o t h i a z i de, hydra1a z i ne ,guanethidine.methy1dopa

C o r a s i l C18 o r Phenyl

S,12 o t h e r s

Various drugs

Analysis pharmaceuticals (Table 2.5)

P a r t i s i l 10 m

2 5 0 ~ 4 . 6 CH2C12-MeOH(1:3) w i t h 1%NH40H

Ajmalicine

Acetani 1ide

Analysis i n pharmaceuticals and p l a n t m a t e r i a l

UPorasil

300x4

RSP,RSC,degradat i o n products

Diuretics

Analysis i n t a b l e t s (Table 8.13)

L i c h r o s o r b Si60 5 urn

2 5 0 ~ 2 . 1 n-Hexane-isopr0H-CHCl3-DEA (77:18: 3:O.Ol) 30

Vindesine.dehydroPhenantrene vindesine,vindesine N-oxide

A n a l y s i s b u l k drug

VBondapak C18

3 0 0 ~ 3 . 9 MeOH-H20-OEA(1000:600:3), 600:3)

S,various

I d e n t i f i c a t i o n i n pharmaceut i c a l s ( F i g . 7.14)

P a r t i s i l PXS 5/25

2 5 0 ~ 4 . 6 E t 0 s a t . w i t h 50-100% H20 0.65-0.8% DEA

VBondapak C18

3 0 0 ~ 3 . 9 MeOH-0.005M h e p t a n e s u l f o n i c a c i d i n H20 (pH 3.6)(2:3)

others

Various drugs

11

25

~-Hexane-(isopr)20-MeDH(90:10:3) 28

(835: 31

Phys ,r u b r e s e r i ne, p i 1ocarpi ne

Methyl -,ethyl -, Determination i n aqueous sol u t i o n s (Table 8.7, Fig.8.7) p r o p y l -p-hydroxybenzozte

Phys

F1urazepam,benzylalcohol

C o l l a b o r a t i v e study o f d e t e r - VBondapak C18 m i n a t i o n phys i n pharmaceutical s

*For a b b r e v i a t i o n s used see f o o t n o t e Table 8.16

+ 38

47 3 0 0 ~ 3 . 9 ACN-0.05M NH4DAc (1:l) 67

2 3

2 P I w

P

e

Vinpocetine,vi ncam i ne.epi v i ncami ne, vincamini c acid, vincamone

Diazepam

A n a l y s i s i n pharmaceuticals (content u n i f o r m i t y )

Nucleosi 1 10C18

2 5 0 ~ 4 . 6 ACN-O.01M (NH4) CO (9:1),(7:3)which c o n t a i n 5 0 .OO l M 2 t r j o c t y l m e t h y l ammonium c h l o r i d e 250x2 CHC13-EtOH(95:5) 75

Micropak S i 10

RSP.methy1 r e s e r p a t e

Post-col umn r e a c t o r f o r flu o r i m e t r i c d e t e c t i o n

Phenyl bonded phase

not given

ACN-0.005M NaH2P04 b u f f e r ( p H 6) (7:3)

80

TABLE 8.20 HPLC ANALYSIS INOOLE ALKALOIDS I N BIOLOGICAL MATERIAL AN0 ALKALOIDS;

OTHER COMPOUNDS

IN

TOXICOLOGICAL ANALYSIS

AIMS

STATIONARY PHASE

COLUMN DIM. Lx I D (inn)

MOBILE PHASE

-

REF.

S

Determination i n g r a i n b a i t s

pPorasil

300x4

CHCl,-MeOH(9:1)

14

RSP

D e t e r m i n a t i o n i n plasma

pBondapak C18

300x4

27

S

Determination i n g r a i n b a i t s and stomach c o n t e n t s

pBondapak C18

300x4

MeOH-O.01M aq. heptanesulfonate (65: 35) MeOH-0.005M KH2P04 b u f f e r ( p H 3 ) (3:2)

E l l i p t i c i n e and de r iv a t ives

A n a l y s i s i n b i o l o g i c a l f l u i d s pBondapak C18 (Table 8.5, 8.6)

300x4

Vincamine,desoxyv i ncami nami de

A n a l y s i s i n plasma

S p h e r i s o r b ODS 5

Vincamine,l4-epi-, and apo-vincamine, papaverine

Determination i n b i o l o g i c a l fluids

UBondapak C18

3 0 0 ~ 3 . 9 MeOH-O.01M ( NH4)2C03( 3: 1)

A1 c u r o n i um, tubocurarine

Determination i n b i o l o g i c a l fluids

UBondapak C18

3 0 0 ~ 6 . 4 MeOH-H O ( 4 : l ) c o n t a i n i n g 0.25% AcOH and 0.605M Na-dodecylsulfate 61

D e t e r m i n a t i o n papaverine i n blood

Micropak CN 10

300x4

n-Hexane-CH C1 -ACN-propylamine 750: 25: 25: 0?1)‘

11-bromovincamine, vincamine

D e t e r m i n a t i o n i n plasma

u Bondapak C18

300x4

ACN-0.1% NaH2P04(35:65)(pH 3.5)

E l 1i p t i c i n e , g - h y d r o xyell ipticine,ll-dem e t h y l e l 1 ip t i c i ne

Analysis i n b i o l o g i c a l samples

S,yoh ,cocaine,papaverine,heroin

pm

29

MeOH-H 0(7:3), idem c o n t a i n i n g O.OO5M’heptane- o r pentanesulfonate. and 0.032M AcOH MeOH-H O(3:l) c o n t a i n i n g 0.005M heptangsulfonate and 0.032M AcOH MeOH-H20(3:1) c o n t a i n i n g 0.02M NH40Ac 36

1 5 0 ~ 4 . 6 ACN-O.OZM phosphate b u f f e r ( p H 2.3) (1:l) 53

58

Various drugs

62 69

*For a b b r e v i a t i o n s used see f o o t n o t e Table 8.16

p

Bondapak C18

300x4

ACN-O.01M NaH2P04(36:64), (25:75) pH 3.5

70

UI I-

w N VI

S.qui n i n e

Determination S i n b i o l o g i c a l S i l i c a g e l 10 pm fluids

2 5 0 ~ 2 . 6 MeOH-conc. NH40H(99.25:0.75)

Vinpocetine,vincami n i c a c i d.apovi ncami n i c a c i d

Determination i n plasma

L i c h r o s o r b RP8 10 um

2 0 0 ~ 4 . 6 ACN-0.0075M phosphate b u f f e r (pH 3.5)(28:72)

74

V i n p o c e t i ne.apovi ncami ne

Determination i n plasma

L i chrosorb RP8

2 5 0 ~ 4 . 6 ACN-O.01M (NH4)*C03

75

S,B

Determination i n u r i n e and tissue extracts

L i c h r o s o r b Si60 7 um

2 5 0 ~ 4 . 6 MeOH-H20-conc. NH40H(85:14.2:0.8)

N-propylajmaline

Determination i n plasma

L i c h r o s o r b S i l o 0 5 um 1 5 0 ~ 4 . 5 n-BuOH-1.2dichloroethane-hexane 715 :40: 45) coated w i t h 0.2M HC104 and 0.8M NaCIOO

Phys, r u b r e s e r i ne, e s e r o l ine

Determination i n plasma

Spherisorb 5 um

71

81

83

2 5 0 ~ 4 . 5 MeOH-1M NH4N03(pH 8.6)(9:1) 84

353

1

4

0

5

10

15

0

5

10

15

0

5

15

10

5

0

10

15

min

Fig. 8.1. Separation o f Psilocybe alkaloids46 Column Partisil SCX-10 (250~4.6mn ID), protected with a precolumn ( 3 0 ~ 2 . 8mn ID) packed with 30 pm pellicular beads, mobile phase methanol - water (1:4) containing 062% amnonium phosphate and 0.1% potassium chloride (pH 4.5). flow rate 1 ml/min. temperature 50 C. Peaks: 1, psilocybin; 2. psilocin; 3, dimethyltryptamine (internal standard). Chromatogram a: standard mixture, detection UV 267 nm; chromatogram b: mushroom extract, detection UV 267 nm; chromatogram c: standard mixture, fluorescence detection ( excitation 267 nm, emission 335 nm); chromatogram d: mushroom extract, f 1 uorescence detection. 5

I

I

min

2'0

1'5

Ib

6

r -

o

I

0

.

3

.

7.8.9.10

. 9. 12. 15. I8, 21. 24. 27, 30. . man

6

26 Fig. 8.2. Separation of some Catharanthus alkaloids Column Lichrosorb RP8 (250x4 mn ID), mobile phase acetonitrile 0.01 M amnonium carbonate (47:53), flow rate 1.5 ml/min, detection UV 298 nm. Peaks: 1. lochnerine; 2, vindoline; 3, vincri s tine; 4, ajmal i ci ne; 5. catharanthi ne; 6, vinbl as tine; 7, tetrahydroalstoni ne; 8. leurosi ne; 9, desacetoxyvinblas tine. Fig. 8.3. Separation of eburnane alkaloids on an octadecyl column54 Column VBondapak C18 (300~3.9mn 10). mobile phase acetonitrile - 0.01 M amnonium carbonate (6:4). flow rate 1 ml/min. detection UV 280 nm. Peaks: 1, (+)-cis-apovincamine; 2, (+)-cis-dehydroepivincamine; 3, (t)-ci s-dehydrovincami ne; 4, (+)-trans-vi ncami ni c acid ethyl ester; 5, (+)-ci s-epivi ncami ne; 6, (-)-ci s-epi vi ncami ne; 7. (+)-cis-vincamine; 8, (-)-ci s-vi ncami ne ; 9, (+)-cis-epivincaminic acid ethyl ester; 10, (+)-trans-epivincaminic acid ethyl ester; 11, (+)-cis-vincamone; 12. (+)-cis-vincanole; 13, (+)-cis-vincaminic acid ethyl ester; 14, (+)-ci s-i sovi ncanole; 15, (+)-cis-apovincamine; 16, (+)-trans-apovincami ni c acid ethyl ester; 17, (+)-cis-apovincaminic acid ethyl ester; 18, (+)-cis-apovincaminic acid phenyl ester; 19. (+)-ci s-vincameni ne.

-

Referencca p. 944

364

075

355

A330

211

Lh

c rnin

Fig. 8.4. Separation of harmane alkaloids45 Column Lichrosorb RP8 7 (250x3 nun ID), mobile phase methanol - water - formic acid (166: 34:l) buffered with triethylamine at pH 8.5, flow rate 1 ml/min, detection UV 330 nm (a), fluorescence (excitation 304 nm, emission 355 nm)(b) and fluorescence (excitation 396 nm, emission 475 nm)(c). Peaks: 1, harmol; 2. harmalol; 3, harmine; 4, harmaline. Fig. 8.5. Separation of ellipticine and related alkaloids, isolated from mouse blood7' Column pBondapak C18 ( 3 0 0 ~ 4 . 0nun ID), protected with a precolumn (50~4.6 mn ID) packed with a 40 Lim pellicular material, mobile phase acetonitrile 0.01 M sodium dihydrogen phosphate (1:3)(pH 3.5 with 2 N phosphoric acid). flow rate 1.4 ml/min, detection UV 300 nm. Peaks: 1, el 1 ipti ci ne; 2, 9-hydroxyel l ipti ci ne; 3, 11-demethylel 1 ipti cine (internal standard).

-

f

L

6

1 mllrnin l

0

0

15 mm

'

I

L

.

I

'

8

2rnllmin I

.

12

1

1

'

18

1

.

22

I

-

26

I

30min

Fig. 8.6. Separation of some alkaloids on a porous polymer43 Column Hitachi Gel 3010 (macroporous styrene-divinylbenzene copolymer), 10 Vim (220~4.6mn ID), mobile phase acetonitrile - water (6:4) containing 0.02 M ammonia, flow rate 2 ml/min. detection UV 254 nm. (see also Table 8.4). Peaks: 1, morphine; 2, codeine; 3, papaverine; 4, yohimbine; 5, noscapine; 6, reserpine.(reproduced with permission from ref. 43. by courtesy of the American Chemical Society) Fig. 8.7. HPLC analysis pilocar ine, physostigmine. its degradation products and preservatives in pharmaceutical Column VBondapak C18 ( 3 0 0 ~ 3 . 9nun ID), mobile phase methanol - water (2:3) containing 0.005 M heptanesulfonic acid (pH 3.6). flow rate 1 ml/min, detection UV 235 nm and 292 nm. Peaks: 1, pilocarpine; 2, salicylate; 3, methyl-E-hydroxybenzoate; 4. physostigmine; 5, propyl-p-hydroxybenzoate. (see also Table 8.7)

preparation^^^

366 3

min 25 20 1'5 io 0 Fig. 8.8. Separation o f e i g h t o p t i c a l isomers o f vincamine 76 Column Nucleosil 5CN ( 1 5 0 ~ 4 . 6mn I D ) , mobile phase hexane - chloroform ethanol (70:27:3) containing 0.002 M (+)-10-camphorsulfonic a c i d and 0.001 M diethylamine, f l o w r a t e 1 ml/min, d e t e c t i o n UV 280 nm. Peaks: 1. (+)-cis-epivincamine; 2, (-)-cis-epivincamine; 3, ( + ) - c i s - v i ncamine; 4, ( - ) - c i s-vincami ne; 5, (+)-trans-epivi ncami ne; 6, (-)-trans-epi vincami ne; 7, (+)-trans-vincamine; 8, (-)-trans-vincamine.

5

-

1

/I

A 6 5 L 3 2 1 0

mtr:

Refemlrs. P. 944

37 Fig. 8.10. Separation o f Psilocybe a l k a l o i d s Column P a r t i s i l 5. 6 (250~4.6 mn ID), mobile phase methanol 1 M ammonium n i t r a t e (24:5:1) b u f f e r e d a t pH 9.7 w i t h water amnonia, f l o w r a t e 2 ml/min. d e t e c t i o n UV 254 nm. Peaks: 1, p s i l o c i n ; 2. p s i l o c y b i n ; 3, baeocystin ( i n mushroom e x t r a c t ) .

-

-

3 56 I01

L

Ib'

i

Fig. 8.11. Separation o f Psilocybe a l k a l o i d s 59 water - 1 M ammonium n i t r a t e Column P a r t i s i l 5. 6 urn ( 2 5 0 ~ 4 . 6n I D ) , mobile phase methanol (22:7:1) buffered t o pH 9.6 w i t h amnonia, f l o w r a t e 1 ml/min, detection UV 254 nm ( a ) , f l u o rescence ( e x c i t a t i o n 267 nm. emission 335 nm) ( b ) . Peaks: 1, 2 and 3, unknown; 4 , p s i l o c y b i n ; 5, p s i l o c i n .

-

Fig. 8.12. Separation o f vincanole and isovincanole i n production mother l i q o ~ r ~ ~ Column Micropak Si-10 (250x2 n I D ) , mobile phase chloroform - ethanol ( 9 5 : 5 ) , f l o w r a t e 20 ml/h, detection UV 280 nm. Peaks: 1, toluene; 2, unknown; 3, vincamone; 4, vincamenine; 5. unknown; 6, unknown; 7, isovincanole.

357

Chapter 9 ERGOT ALKALOIDS

9.1. Reversed-phase HPLC ...............................................................

9.2. I o n - p a i r HPLC.....................................................................

............................................................. ......................................................................... .............................................................................

9.3. Straight-phase HPLC.. 9.4. Detection References

357 362 362 366 368

The e r g o t a l k a l o i d s can be d i v i d e d i n t o two main groups: the l y s e r g i c a c i d a l k a l o i d s and the c l a v i n e a l k a l o i d s . The a n a l y t i c a l problems concerning the l y s e r g i c a c i d a l k a l o i d s can be summarized as f o l l o w s :

1. Analysis o f LSD and r e l a t e d compounds.

2. Separation o f ergometrine and the ergotamine and the ergotoxine groups o f a l k a l o i d s . 3. Separation o f the i n d i v i d u a l a l k a l o i d s w i t h i n each o f the groups mentioned.

4. Separation o f t h e C-8 stereoisomers ( - i n e / i n i n e ,

normal/iso-).

5. Separation o f t h e dihydroderivatives of the ergotoxine group o f a l k a l o i d s and ergotamine. For each o f the separations mentioned, HPLC has been used. The analysis o f LSO i s sometimes described i n connection w i t h t h e analysis o f drugs o f Reviews on HPLC analysis o f LSD have been given16’49959. L u r i e and Weber32’49 described a semipreparative HPLC separation o f LSD t o enable f u r t h e r i d e n t i f i c a t i o n o f i t by means of d i f f e r e n t spectral data. T w i t c h e t t e t a1.28 reported the analysis o f LSD i n body f l u i d s by means o f a combination o f HPLC, f l u o r i m e t r y and radioimnuno-assay. The separation o f the n a t u r a l l y occurring e r g o t a l k a l o i d s derived from l y s e r g i c a c i d i n groups has been i n v e s t i g a t e d i n a long series o f studies

19,25,34,35,36,41.43.50,52,57,58,60,61

and so has the separation o f the various components w i t h i n each group, p a r t i c u l a r l y w i t h i n the ergotoxine group 25.34s36,41,43’51’60161.The C-8 stereoisomers can u s u a l l y be separated w i t h o u t d i f f i c u l t y 10.14,17 9 19~25~26,34,36,41~43947,51~52960~61~ t h e separation of t h e d i hydrod e r i v a t i v e s of the ergotoxine group of a l k a l o i d s has a l s o been achieved6g11*19g25~33r34g41s60~ 61, S t a b i l i t y studies o f ergotamine17, ergometrine31 and d i h y d r ~ e r g o t a m i n ehave ~ ~ been p e r f o r med using HPLC f o r the separation o f the a l k a l o i d and i t s degradation products. Schauwecker e t a1.22 developed a method f o r the t r a c e enrichment o f ppb q u a n t i t i e s o f e r g o t a l k a l o i d s i n urine. al.

HPLC analysis o f c l a v i n e a l k a l o i d s has been c a r r i e d o u t by Wurst e t a1.29 and Eckers e t 57,58

9.1.

REVERSED-PHASE HPLC Reversed-phase HPLC has been widely used f o r the analysis o f both LSD and e r g o t a l k a l o i d s .

The f i r s t reversed-phase separation o f e r g o t a l k a l o i d s was reported by Jane and Wheals3 i n connection w i t h the analysis o f LSD. P e l l i c u l a r beads w i t h chemically bonded octadecyl groups were used i n combination w i t h methanol

-

0.1% aqueous amnonium carbonate (3:2) as mobile phase.

However, the mobile phase proposed by V i v i l e c c h i a e t a1.6 f o r the separation o f the dihydroergotoxine a1 kaloids on octadecyl columns ( a c e t o n i t r i l e

Relerrncu p. 3138

-

aqueous amnonium carbonate) has been

368 TABLE 9.1 CAPACITY FACTORS ( k ' ) AN0 SEPARATION FACTORS FOR ERGOT ALKALOIDS ON REVERSED-PHASE PACKINGS" '34 Co1umn:Lichrosorb RP2 (5 p m ) , RP8 (10 pin) o r RP18 (10 pm)(250x2 o r 4 mn 10). mobile phase a c e t o n i t r i l e - 0.01M aqueous amonium carbonate (2:3), d e t e c t i o n UV 320 o r 280 nm. A1 k a l o i ds

Lichrosorb RP2 (I

Lysergic a c i d Ergometri ne ma1eate Ergometrini ne Ergotamine t a r t r a t e

0.16

Ergocrypti ne Ergocornine

67.8 *24

Ergocri s t i n e Ergotami n i ne Ergocorninine Ergocrypti nine Dihydroergotamine t a r t r a t e Oihydroergocornine methanesulfonate Oihydroergocryptine methanesulfonate O i hydroergocristine methanesul fonate

8.45

1.08

9*87 12.91 14.50

1.53 1.12

1*54 2'27 5.34

7.15 9.11 9.82

Lichrosorb RP8 Lichrosorb RP18 k' (I k (I 0.20

:::: :::: :::::

1.47 2.35

1.27 1.08

''11

16:66

0.10 2.11 3.21 1.46 1.37 1.10 1.75 1.47

9.43

1.44

::;;

OS8'

20.50

::::!

2.27 3.95 1.63 1.57 1.10

29.6

1.86

6":;

~~:~~ 1.10 o:;i

7.94 9.7

1.58

1.50 1.20

TABLE 9.2 SEPARATION OF ERGOTAMINE AN0 ITS DECOMPOSITION R e l a t i v e capacity f a c t o r s (k;=k;/kh) o f decomposition product [ ( r e l a t i v e t o ergotamine E) Column pBondapak C18 (300x4 m ID), mobile phase a c e t o n i t r i l e - 0.01M aqueous ammonium carbonate, l i n e a r gradient i n 15 min from 3:37 t o 1:1, f l o w r a t e 8.0 ml/min, d e t e c t i o n UV 320 nm. Alkaloid

kb

Rel. standard d e v i a t i o n [%I 0.3 0.4 0.3 1.0

Ergotaminine 1.18 Aci -ergotami ne 0.77 0.98 Aci-ergotaminine Lysergic a c i d amide 0.45 I s o l y s e r g i c a c i d amide 0.61 Lysergic a c i d 0.08 Isolysergic acid 0.18

0.8 1.2 1.5

TABLE 9.3 SEPERATION OF SOME ERGOT Column Lichrosorb RP18. 10 pm (250x4 mn ID), mobile phase 0.1% ammonium acetate i n aceton i t r i l e - water (35:65). f l o w r a t e 3.5 ml/min, d e t e c t i o n UV 254 nm. A1 kal oids

Retention time(min)

Ergometrine Ergometri n i ne Papaverine(interna1 standard) Ergosi ne Ergotami ne Ergocorni ne Ergocrypti ne Ergocri s t i n e Ergotami n i ne Ergocorninine Ergocryptinine Ergocristinine

1.6 2.4 4.0 6.2 6.6 10.0 11.4 16.2 27.0 42.0 48.0 56.0

369

TABLE 9.4

SEPARATION

OF ERGOTOXINES

___________

AND OIHYDROERGOTOXINES~~ _______~

~~~

~

A1 k a l o i d

~

k‘

Ergocornine O i h y d r o e r g o c o r n i ne a-Ergocryptine D i h y d r o - a - e r g o c r y p t i ne 8 - E r g o c r y p t i ne Dihydro-B-ergocryptine

4.22 4.38 5.00 5.44 5.78 5.91

Column L i c h r o s o r b RP18 10 pm ( 2 5 0 ~ 4 . 6 mm I D ) , m o b i l e phase t e t r a h y d r o f u r a n ammonium a c e t a t e ( 2 : 3 ) , f l o w r a t e 1.5 ml/min, d e t e c t i o n UV 322 nm (Fig.9.5).

-

0 . 0 1 M aqueous

found t o have w i d e r a p p l i c a t i o n s f o r t h e s e p a r a t i o n o f e r g o t a l k a l o i d s . The s o l v e n t was t e s t e d by D o l i n a r ”

and Szepesy e t a1 .25934 i n c o m b i n a t i o n w i t h d i f f e r e n t reversed-phase packings.

O o l i n a r found t h a t t h e p a c k i n g w i t h c h e m i c a l l y bonded o c t y l groups was t h e most s u i t a b l e s t a t i o n a r y phase f o r t h e s e p a r a t i o n o f e r g o t a l k a l o i d s w i t h a wide range o f p o l a r i t i e s (Fig.9.1). The s e p a r a t i o n s o b t a i n e d on an o c t a d e c y l column were s i m i l a r ( F i g . 9 . 2 ) . column (RP2) and a s p h e r i c a l o c t a d e c y l column ( S p h e r i s o r b ODS),

On a d i m e t h y l s i l y l

t h e less p o l a r a l k a l o i d s were

n o t as w e l l s e p a r a t e d as on t h e above-mentioned columns; however, t h e more p o l a r a l k a l o i d s were b e t t e r separated ( F i 9 . 9 . 3 ) . Szepesy e t a 1 . 2 5 * 3 4 i n v e s t i g a t e d t h e e f f e c t o f t h e c h a i n l e n g t h o f t h e c h e m i c a l l y bonded a l k y l groups on t h e s e p a r a t i o n o f t h e a l k a l o i d s . B e s t r e s u l t s f o r t h e e r g o t o x i n e and d i h y d r o e r g o t o x i n e groups of a l k a l o i d s were o b t a i n e d w i t h t h e o c t a d e c y l s t a t i o n a r y phase. An i n c r e a s e o f c h a i n l e n g t h o f t h e alkanes caused an i n c r e a s e o f t h e c a p a c i t y f a c t o r s o f t h e a l k a l o i d s and, t o some e x t e n t , a l s o an i n c r e a s e i n t h e s e l e c t i v i t y ( T a b l e 9 . 1 ) . The f o u r a l k a l o i d s o f t h e e r g o t o x i n e group, as w e l l as t h r e e d i h y d r o e r g o t o x i n e a l k a l o i d s , c o u l d be separated. S i m i l a r sys tems have a1 s o been used i n o t h e r inves t i g a t i o n s l 7 * 18’21’22937’39950. F o r t h e q u a n t i t a t i v e a n a l y s i s o f ergotamine and i t s decomposition p r o d u c t s ( T a b l e 9.2). Bethke e t a1 , I 7 f o u n d t h a t a l i n e a r g r a d i e n t was necessary when a c e t o n i t r i l e

-

aqueous amno-

nium carbonate was used as m o b i l e phase. A s t e p w i s e g r a d i e n t as used b y E r n i and F r e i 1 8 c o u l d be used, b u t some problems arose w i t h t h e q u a n t i t a t i o n o f t h e chromatogram. However. t h e s t e p wise gradient procedure o f f e r e d p o s s i b i l i t i e s f o r the analysis o f very low concentrations o f d i h y d r o e r g o t o x i n e a l k a l o i d s (ppb r a n g e ) . I n j e c t i o n volumes o f up t o s e v e r a l hundred m i l l i l i t r e s c o u l d be a p p l i e d : t h e a l k a l o i d s were adsorbed on t h e t o p o f t h e column and t h e y were subseq u e n t l y e l u t e d by means o f t h e s t e p w i s e W e h r l i e t a l .24 i n v e s t i g a t e d t h e i n f l u e n c e o f o r g a n i c bases ( p r i m a r y , secondary. t e r t i a r y and q u a t e r n a r y amines) on t h e s t a b i l i t y o f c h e m i c a l l y bonded reversed-phase m a t e r i a l s . I t was found t h a t w i t h sodium h y d r o x i d e and q u a t e r n a r y amines, t h e s i l i c a t e s t r u c t u r e o f t h e p a c k i n g was r a p i d l y a t t a c k e d , making columns u s e l e s s w i t h i n 1-3 days. Decomposition o f such m a t e r i a l s i s m a i n l y due t o d i s s o l u t i o n o f t h e s i l i c a g e l . However, when w o r k i n g w i t h h i g h pH values, p r i m a r y , secondary and t e r t i a r y a l k y l a m i n e s can be used w i t h o u t a n o t i c e a b l e decrease i n t h e q u a l i t y o f t h e column. T r i e t h y l a r n i n e was found t o be a good choice. I n c r e a s e i n t h e w a t e r content o f the eluents tested ( a c e t o n i t r i l e

-

water

-

base) gave an i n c r e a s e i n t h e d i s s o l u -

t i o n o f t h e s i l i c a g e l . F o r t h e a n a l y s i s of e r g o t a l k a l o i d s . a c e t o n i t r i l e was p r e f e r r e d above methanol because o f a b e t t e r s e l e c t i v i t y , a h i g h e r p l a t e number, and a l o w e r p r e s s u r e drop. The v a r i o u s amines t e s t e d were compared f o r t h e i r i n f l u e n c e on t h e r e t e n t i o n b e h a v i o u r o f t h e

Referencea p. 368

360 TABLE 9.5 SEPARATION OF ERGOT ALKALOIDS AND LSDZ8 S1: column Spherisorb 5-ODS ( 1 0 0 ~ 4 . 6mn ID), mobile phase methanol - 0.025M disodium hydrogen phosphate (65:35)(pH 8). f l o w r a t e 1.0 ml/min. S2: column Spherisorb S5W (150~4.6 mn ID), mobile phase methanol - aqueous ammonium n i t r a t e (3:2), f l o w r a t e 1 ml/min. Detection UV 280 nm o r fluorescence ( e x c i t a t i o n 320 nm, emission 400 nm).

s1

A1 k a l o i d

s1

s2

l.OO(4.9ml) 2.04 0.38 0.23 0.61 0.53 1.15 1.68 2-0xo-LSD 0.65 D-Lysergic a c i d monoethylamide 0.52 D i hydroergocorni ne 2.60 D i hydroergocri s t i n e 3.68 D i hydroergocryptine 2.63

D-LSD Iso-LSD 0-Lysergami de D-Lysergi c a c i d Lysergol Lumi-LSD

1.00 (3.4ml) 1.28 2.76 0.65 0.76 0.85 0.59

0.81 0.74 0.76 0.71

D i hydroergotami ne Ergocorni ne Ergocri s t i n e Ergocrypti ne Ergocrypti nine Ergometri ne Ergometri nine Ergosi ne Ergosinine Ergotamine Ergothioneine Methysergi de Methylergometrine

s2

0.81

2.48 1.39 2.31 1.86 2.08 0.26 0.47 1.22 1.22 1.57 >6 0.65 0.39

0.79

0.81 0.78 0.72 0.79 0.79 0.78 0.75 0.81 >2.35 0.85 0.76

~~

ergot alkaloids. Hartmann e t a1 .33 found t h a t some reversed-phase columns showed i n s u f f i c i e n t s t a b i l i t y against a mobile phase such as a c e t o n i t r i l e

-

aqueous ammonium carbonate. However, by r e -

p l a c i n g amnonium carbonate by a 0.33 M phosphate b u f f e r (pH 7.5) s i m i l a r r e s u l t s could be obtained for the separation o f the dihydroergotoxine a l k a l o i d s , and the problem concerning the i n s t a b i l i t y o f the s t a t i o n a r y phase was avoided. S i m i l a r r e s u l t s were a l s o obtained w i t h triethylamine. A mobile phase c o n s i s t i n g o f water

-

acetonitrile

-

t r i e t h y l a m i n e (32:8:1)

and an octadecyl column gave a complete r e s o l u t i o n o f a l l f o u r dihydroergotoxine a l k a l o i d s (Fig.9.4).

A s i m i l a r method was applied by A l i and Strittmatter3’.

-

(35:65) containing 0.1% ammonium acetate and tetrahydrofuran

Acetonitrile

-

water

0.01 M amnonium acetate (3:2)

have been used i n combination w i t h octadecyl columns t o separate, respectively. various e r g o t a l k a l o i d s (Table 9.3)36 and some ergotoxine-type a l k a l o i d s (Fig.9.5. Table 9.4) 60 . To avoid decomposition o f reversed-phase co\umn m a t e r i a l by b a s i c solvents. Sondack31 preferred acetonitrile

-

acetic acid

- water

(30:1:79)

i n combination w i t h an octadecyl s t a t i o -

nary phase t o separate ergometrine and i t s decomposition products. T w i t c h e t t and co-workers (12,27,28.46)

found t h a t a mobile phase c o n s i s t i n g o f methanol

- 0.025

M aqueous disodium

hydrogen phosphate (65:35)(pH 8) i n combination w i t h an octadecyl column was b e t t e r s u i t e d f o r the analysis o f LSD than a normal-phase (Table 9.5). I n connection w i t h the development o f a post-column reactor, G f e l l e r e t a1.42 used a s t a t i o n a r y phase w i t h chemically bonded d i o l groups, which allowed the use o f e x c l u s i v e l y aqueous mobile phases (0.01 M, pH 3, phosphate b u f f e r ) . A s e r i e s o f a l k a l o i d s was separated, v i z . d i hydroergotamine and bromocryptine. Wurst e t a1 .29’43 reported the separation o f clavines and l y s e r g i c a c i d d e r i v a t i v e s on a s t a t i o n a r y phase containing alkylamine groups i n combination w i t h n e u t r a l organic solvents (Tables 9.6 and 9.7). For the clavines, the best separation was obtained w i t h d i e t h y l e t h e r

- ethanol

(84:16);

-

under i s o c r a t i c conditions

-

a g r a d i e n t e l u t i o n was, however, found

t o g i v e b e t t e r r e s u l t s . For the a l k a l o i d s derived from l y s e r g i c acid, d i e t h y l e t h e r

-

ethanol

361 TABLE 9.6 RELATIVE RETENTIONS

OF CLAVINE ALKALOIDS AND SIMPLE DERIVATIVES OF LYSERGIC ACID2’

Column Micropak NH , 10 pm (250x2 mn ID). f l o w r a t e 1 ml/min. d e t e c t i o n UV 225 and 240 nm. Mobile phase S1: d?ethyl e t h e r ethanol (84:16) S2: d i e t h y l e t h e r - ethanol (80:20) 53: d i e t h y l e t h e r isopropanol (70:30) 54: d i e t h y l e t h e r - isopropanol (60:40) S5: chloroform - isopropanol (9O:lO) S6: chloroform isopropanol (80:20)

-

-

A1 kal o i d Paspacl a v i ne I s o s e t o c l a v ine Lysergene Setoclavi ne I s o l y s e r g i c a c i d amide Lysergi ne Agroclavi ne Pyroclavine Festucl a v i ne Penniclavine Lysergic a c i d amide Pal ic l a v i n e Elymoclavine Lysergol Chanocl a v i ne r e t e n t i o n volume agroc l a v i n e (ml)

s1

s2

s3

54

55

S6

0.11 0.25 0.38 0.55 0.83 0.88 1.00 1.45 2.08 2.80 2.90 3.12 3.22 3.50

0.11 0.26 0.39 0.61

8.00

1.49 1.98 2.65 2.51 2.25 2.90 2.99 6.03

0.11 0.27 0.46 0.67 0.69 1.20 1.00 1.80 2.37 2.97 2.29 1.66 3.17 3.48 8.56

0.14 0.26 0.45 0.67 0.64 1.14 1.00 1.63 2.01 2.72 1.85 1.26 2.72 2.83 6.33

0.12 0.70 0.58 0.98 0.58 1.24 1.00 1.65 1.46 5.05 4.00 3.85 4.48 5.80 9.83

0.12 0.70 0.63 1.05 0.55 1.30 1.00 1.71 1.47 3.85 2.95 2.99 3.05 4.30 6.03

4.67

3.00

4.90

3.50

2.67

1.90

0.80

0.87 1.00

TABLE 9.7 RELATIVE RETENTIONS OF ERGOT ALKALOIDS43 Column Micropak NH , 10 pm (250x2 mn ID)(A) and Lichrosorb NH2. 10 r a t e 1 ml/min, d e t g c t i o n UV 310 nm. Mobile phase S1: d i e t h y l ether - ethanol (93:7) column B S2: d i e t h y l e t h e r ethanol (88:12) column A S3: d i e t h y l ether - ethanol (84:16) column A 54: d i e t h y l ether isopropanol (60:40) column A S5: chloroform isopropanol (9O:lO) column A

-

A1 k a l o i d Ergocryptine Ergocryptini ne Ergocorni ne Ergocorni nine Ergocristine Ergocri s t i n i n e Ergostine Ergostinine Ergosine Ergos in i ne Ergotami ne Ergotaminine Ergometrine Ergometri n i ne Lysergic a c i d amide I s o l y s e r g i c a c i d amide 8-Hydroxy-ergotamine Agroclavine r e t e n t i o n volume agroc l a v i n e (ml)

Rsferenca p. 368

-

s1

s2

53

54

55

2.82 1.59 3.06 2.00 3.76 2.35 7.38 3.15 8.47 3.09 12.44 4.56 12.82 3.82

0.50

0.47 0.21 0.56 0.30 0.78 0.38 1.30 0.57 1.21 0.46 1.94 0.88 4.16 1.00

0.31 0.14 0.32 0.18 0.45 0.26 0.82 0.37 0.71 0.28 1.08 0.52 2.55 0.57 1.85 0.64

0.13 0.03 0.12 0.05 0.15 0.04 0.33 0.05 0.47 0.07 0.53 0.07 4.93 0.98 4.00 0.58

-

0.25 0.64 0.34 0.89 0.45 1.85 0.75 1.83 0.63 2.25 0.94 5.12 1.05

2.90

8.88 1.00

-

0.83

1.00

1.00

1.00

1.00

1.10

6.00

4.67

3.50

2.67

pm

(250x2 mn IO)(B). f l o w

362

(88:12) o r (93:7) gave very good separation. However, i n this case a gradient e l u t i o n was a l s o preferred. The components of the ergotoxine group were best separated w i t h diethyl e t h e r ethanol (97.5:2.5). Harzer62 used a column switching technique t o confirm the i d e n t i t y of LSO. An octyl type of stationary phase and a straight-phase s i l i c a gel column were used i n combination with the solvent methanol - 0.3% potassium dihydrogen phosphate i n water (pH 3 ) ( 1 : 1 ) .

9.2. ION-PAIR HPLC Fluorimetric ion-pair chromatography has been applied f o r ergotamine, ergotaminine and dihydroergotamine i n connection w i t h the analysis of pharmaceutical preparations containing tropane a l k a l ~ i d s ~ ~ (Chapter ’ ~ ~ ’ ~4 )~. ’ ~ ~ Lurie described ion-pair chromatography of LSD. As pairing-ion heptanesulfonic acid (0.005 M ) ” or methanesulfonic acid (0.005 M)32’49 was used in a mobile phase of water - methanol a c e t i c acid (59:40:l)(pH 3.5) and a microparticulate octadecyl material as s t a t i o nary phase. For semipreparative work, the concentration of the pairing-ion was increased t o 0.04 M i n order t o reduce t a i l i n g and t o eliminate peak s p l i t t i n g ( see a l s o Chapter 7 ) 54-56 . Post column derivatization i n connection w i t h ion-pair chromatography was used by Lawrence e t al.48 f o r , e.g., ergotamine (Chapter 4 ) . Ali and Strittmatter3’ studied the separation of some dihydroergot alkaloids on reversed-phase columns (octadecyl and octyl) w i t h a c e t o n i t r i l e - water mixtures containing s a l t s ( c i t r a t e , acetate or bromide) as mobile phase. Best r e s u l t s were obtained with a mobile phase of pH 7 i n combination w i t h an octyl column. Under such conditions the dihydroergotoxine alkaloids and dihydroergotamine could be separated with three d i f f e r e n t salt-containing solvents (Table 9. 8 ) . In acidic media the separations were inadequate. The a- and B-isomers of dihydroergocrypt i n e could only be separated a t a h i g h pH (12.3) w i t h a c e t o n i t r i l e - methanol - diethylamine (375:65:21) as mobile phase, i . e . a solvent system s i m i l a r t o t h a t mentioned by Hartmann e t

.

a l . 33 A normal-phase ion-pair chromatographic method f o r the separation of ergot alkaloids has been reported by Szepesi e t ale6’. The behaviour of some alkaloids upon changes i n the d i - ( 2 -ethylhexyl)phosphoric acid concentration i s shown i n Table 9.9. The increase of the concent r a t i o n of the counter-ion showed a nearly l i n e a r relationship with the increase of the capac i t y factor of the ergot alkaloids. Due t o the strong interaction of 10-camphorsulfonic acid and the ergot alkaloids, systems w i t h t h i s counter-ion were unsuitable f o r ergot alkaloids (Table 8 . 9 ) . Under basic conditions t h e alkaloids could a l s o be separated using t h i s s o r t of mobile phase (Table 9.10). 3.3. STRAIGHT-PHASE HPLC Wittwer and Kluckhohn’ analyzed LSD and a s e r i e s of ergot alkaloids on s i l i c a gel with a c e t o n i t r i l e - diisopropyl e t h e r a s mobile phase. A s i m i l a r system was used by Perchalski et a1.7 f o r the determination of ergotamine i n plasma. Heacock e t a1.‘ performed some preliminary investigations on the analysis of ergot alkaloids on p e l l i c u l a r s i l i c a gel packings. By means o f chloroform - methanol - ethyl a c e t a t e a c e t i c acid (60:20:50:3), reasonable r e s u l t s were achieved. Water-deactivated columns in combination with chloroform - methanol were a l s o used w i t h good r e s u l t s .

-

363

TABLE 9.8 CAPACITY FACTORS AN0 SEPARATION FACTORS PHASE COLUtIN39

(a)

FOR SOME ERGOT ALKALOIDS ON A REVERSED

Column, Lichrosorb RP8. 7 rn (250x4.6), mobile phases, S 1 a c e t o n i t r i l e - water - t r i ethanolamine - c i t r i c a c i d (45 m l t 60 m l t 0.4 m l + 0.166 g), pH = 7.1; S2 a c e t o n i t r i l e water - triethanolamine - sodium acetate (45 m l t 60 m l t 1 m l t 0.3 g), pH = 7.1, adjusted by a d d i t i o n o f two drops a c e t i c acid; S3 a c e t o n i t r i l e - water tetradecyl t r i m e t h y l amnonium bromide (45 m l t 60 m l t 0.1 g ) , pH = 7.1, adjusted by a d d i t i o n o f two drops triethanolamine; S4 a c e t o n i t r i l e water - amnonium carbonate (45 m l t 60 ml t 0.04 9). pH = 8.3; 5 5 a c e t o n i t r i l e water diethylamine (375:625:21), pH = 12.3.

-

-

-

ALKALOIDS

MOBILE PHASE

s1 d i hydroergotamine d i hydroergocornine dihydro-a-ergocryptine d i hydroergocristine d i hydro-8-ergocrypti ne

k' 2.68 3.45 4.92

52

1.29 1.43 1.12

5.50

s3

k'

a

3.29 4.00 5.71 6.24

54

k'

a

1.21 1.43 1.09

2.08 2.65 3.84 4.22

k'

a

1.27 1.45 1.10

55

k'

a

1.30 1.75 2.15 3.05

1.35 1.23 1.42

a

4.66 5.72 8.21 9.26

1.23 1.44 1.13

TABLE 9.9 DEPENDENCE OF CAPACITY RATIOS MEASURED FOR SOME ERGOT ALKALOIDS ON DI-(2-ETHYLHEXYL) PHOSPHORIC ACDI ( OHP) CONCENTRATION^^ Conditions: $ondapak CN column ( 3 0 0 ~ 3 . 9mm I.D.); d e t e c t i o n a t 280 nm. Compound

1 cm3/min;

Eluent composition (%) Hexane 65 65 Chloroform 20 20 A c e t o n i t r i l e 15 15 DHP ( t f ) .

8-Ergocryptinine a-Ergocrypti n i ne Ergocorni n i ne Ergocri s t i n i n e 6-Ergocryptine a-Ergocrypti ne Ergocornine Ergocri s t i ne Ergotamine Ergotaminine Ergometrine O i hydro-6-ergocrypti ne D i hydro-a-ergocrypti ne Dihydroergocornine D i hydroergocristine D i hydroergotami ne

e l u e n t flow-rate,

I

-

65 20 15 0.0005 0.001 0.005

0.90 0.89 0.90 0.89 0.90 0.89 1.07 1.07 1.07 1.18 1.07 1.18 1.28 1.36 1.41 1.50 2.72 2.36 1.48 1.39 12.0 12.0 2.79 1.75 2.79 1.75 3.28 2.00 3.55 2.21 4.17 3.71

65 20 15

65 20 15

65 20 15 0.01 0.025

60 70 23 17 17 13 0.005 . . 0.005

1.17 4.71 1.17 4.71 1.41 6.46 1.41 7.21 1.69 1.96 1.69 1.96 1.93 2.21 2.10 2.21 3.62 2.93 5.55 15.9 15.2 1.59 1.41 1.59 1.41 1.85 1.71 1.97 1.71 3.28 2.64

10.4 10.4 12.9 14.3 2.38 2.38 2.77 2.77 3.54 25.9

3.70 3.70 4.63 5.22 2.41 2.41 2.78 2.78 4.11 16.8

4.15 4.15 4.48 5.52 1.48 1.48 1.67 1.67 2.37 12.0

9.67 9.67 12.7 14.6 3.37 3.37 3.89 4.15 5.35 28.3

1.69 1.69 2.00

2.00 2.00 2.26 2.26 3.37

1.15 1.15 1.30

2.70 2.70 3.10 3.10 4.63

2.00

3.00

1.30

1.96

-

TABLE 9.10 INFLUENCE OF DHP CONCENTRATION I N THE PRESENCE OF DEA I N THE ELUENT ON THE CAPACITY RATIOS ( k ' ) AN0 SELECTIVITY FACTORS ( r i j ) OBTAINED FOR NATIVE AND HYDROGENATED ERGOT PEPTIDE ALKALOIDS~~ Conditions as i n Table 9.9 Compound DEA (H) DHP (H)

Eluent, hexane-isopropanol

zXy3 10k'

Hydrocortisone Predni sol one 6-Ergocryptinine a-Ergocryptinine Ergocornin i ne Ergocri s t i nine 6-Ergocryptine

1.87 2.10 2.29

:;1.22

a-Ergocryptine Ergocornine Ergocristine

1'26

Ergotarninine Ergotarnine

4.81 4*35

1.11

Dihydroergocornine Dihydroergocristine Dihydroergotarnine

2.48 3.06 4.06

k'

'ij

1.85 2.10

22.94 *29 3.58 2.06 '.06 2.42 3.06

Dihydro-6-ergocryptine 2.10 D i hydro-a-ergocrypti ne 2.10

7.5~10-4 10-3

10-3 rij

l.oo

1.17

o.lo

:::!

1.00

$:1;

1.25 1.32

5::;

4.48

:;I: ;:!;

o.lo

;:;

43.47 3;:

1.18

r.

'ij

1.93 2.10 1.07 1.43

l.o

1.05

3.53

:46.07 3

7.5~10-4 4x10-3

k'

'i j

k'

1.10

1.90 2.13 10.4 11.5

'050

4.27 4*87 4.87 7.20

1'48

1.98

&gg

2.77

i:

:::; A:

1.87 2.12 1.10 1.35

z:

6.15 8.60 4.40

::;:

4.53

2:;

i:::

1.47

7.80

1.62

;i!:

1.72

:i!2

l.oo

2.94

o.lo

;2:

1.15

3.26 3.58

1.11 '03*

5.88

k'

1j

7.5~10-4 2x10-3

1.20

1'39

X;; 4.52

k'

7.5~10-4 1.5~10-3

1.87 2.01

2*40 3.95 2.74

(80:20)

;:;

4.31 4.81

1.10 1.33

$i;

7.51

1.43

i::: 6.03

1.47

'ij

;;::

3.07 3.70

1.48

7.13

365

TABLE 9.11 CAPACITY FACTORS ( k ' ) OF ERGOT ALKALOIDS ON SILICA GEL PACKINGS WITH DIFFERENT Column Lichrosorb Si60, 10 um (250x2 n ID), mobile phase S 1 n-hexane - chloroform - ethanol (40:40:10), 52 chloroform - methanol (95:5), S3 chloroform-etlianol (95:5), d e t e c t i o n UV 280 o r 320 nm ~~

~~~

s1

s2

S3

17.41 10.00 6.27 1.90 1.90 1.90 1.40

-

-

A1 k a l o i d Ergometrine maleate Ergometrinine Ergotamine t a r t r a t e Ergocornine Ergocrypti ne Ergocristine Ergotami nine

1.80 0.50 0.50 0.50 0.28

0.60 0.60 0.60 0.37

~

~~

Alkaloid

s1

s2

53

Ergocorninine Ergocryptinine Ergocri s t i n i ne Dihydroergocorninea Dihydroergocryptinea Dihydroergocristinea

0.93 0.93 0.93

0.25 0.25 0.25 1.75 1.65 1.50

0.13 0.13 0.13

-

-

a As methanesulfonate s a l t TABLE 9.12 CAPACITY FACTORS ( k ' ) AND SEPARATION FACTORS (a) FOR ERGOT ALKALOIDS ON SILICA GEL PACKINGS WITH DIFFERENT ELUENTS51 Column Lichrosorb Si60, 5 um (250x4.6), mobile phase S 1 hexane - chloroform - a c e t o n i t r i l e (60:25:15), 52 idem i n r a t i o (56:22:22), 53 idem i n r a t i o (55:20:25), S4 hexane - chloroform - a c e t o n i t r i l e - methanol (55:20:25:3), f l o w r a t e 100 ml/h, d e t e c t i o n UV 320 nm.

s1

Alkaloid 6-Ergocrypti n i ne a-Ergocryptinine Eraocristinine Ergocorninine Ergotami n i ne 6-Ergocrypti ne a-Erqocryptine Ergoirisitine Ergocornine Ergometri n i ne

k' 2.04 2.54

1.00 1.08 1.32 1.40 2.78 3.29 3.67 4.78 4.40

1'24 1.28

3.24 2.31 7.48 9.28

s3

52

k'

a

1'24 1.16

10.80

a

k'

i.:!

0.95 1.00

I."'

"05 1.22

1.22 2.25

'"' 4.67

Ergotami ne Ergometrine

s4

20.54

2.75 3.09

"" 1.15

3.54

k'

a

0.77 0.77 0.77 0.77 1.01 1.21 1.21 1.21 1.21 2.11

:g;

a

i::

::;; 2.32

TABLE 9.13 CAPACITY FACTORS OF SOME ERGOT ALKALOIDS ON A POROUS POLYSTYRENE STATIONARY PHASE41 Column H i t a c h i Gel no 3011-0, 5-7 m ( 5 0 0 ~ 4 . 6 nun ID). mobile phase S 1 n-hexane t r i e t h y l a m i n e (70:30:0.5), 52 cyclohexane - ethanol - t r i e t h y l a m i n e (7n:30:0.5), - chloroform - t r i e t h y l a m i n e (5:95:0.5), d e t e c t i o n UV 280 nm. Alkaloids

k' i n

E rgornet r ine a-Ergocrypti ne Ergocornine Ergocristine a-Ergocryptinine Ergocorni n i ne Ergocristinine Ergotami n i ne Oihydro-aergocryptine Oihydroergocornine Dihydroergocristine

Referenew p. 366

S1

s2

8.68 1.47 1.85 3.07 1.78 2.45 4.31 5.57 1.13 1.48 2.38

5.15 0.73 0.88 1.31 0.89 1.15 1.82 2.41 0.57 0.71 1.00

'

s3 26 0.76 0.84

1.11 2.14 2.86 3.23 3.41 1.53 1.96 2.37

-

ethanol S3 _?-hexane

366

Jane 9 separated a wide range of drugs of abuse on microparticulate s i l i c a gel w i t h polar mobile phases containing ammonium n i t r a t e solutions. LSD could be analyzed w i t h methanol 0.2 M ammonium n i t r a t e solution (3:2)(Fig.9.6). The system has been successfully applied t o the analysis of LSD13’15’16’28’45’46, but f o r the analysis of ergot alkaloids the above men-

tioned reversed-phase system gave b e t t e r resul ts28’46 (Table 9.5). Szepesy e t a l . 25’34’35 found microparticulate s i l i c a gel t o be very useful f o r the separation of the alkaloids present in the ergometrine, ergotamine. ergotoxine and dihydroergotoxine groups. The stereoisomers could be separated on s i l i c a gel (Table 9.11). To obtain a good separation of the various alkaloids within each group, reversed-phase chromatography was found to be best (Table 9.1). Later51, a straight-phase system was developed f o r the separation o f the ergotoxine alkaloids (Table 9.12. Figs. 9.7 and 9.8). By means of a solvent system consisting o f hexane - chloroform - a c e t o n i t r i l e - methanol (50:20:25:3), a rapid group separation could be achieved (Fig.9.9). Aigner e t al.14 investigated the separation of some drugs, e.g. ergotamine and ergotaminine, on s i l v e r iodide impregnated s i l i c a g e l . Multi-component drugs could be separated with chloroform - diethylamine as mobile phase on a 1.09% s i l v e r iodide impregnated s i l i c a gel column. Quercia e t a1 .I1 used a microparticulate aluminium oxide column to separate the dihydroderivatives of some ergot alkaloids and pentane - methanol (98:2) o r (97:3) as mobile phase. Yoshida e t al.41 isolated ergotoxine and ergotoxinine from ergot by means of a s i l i c a gel column and cyclohexane - acetone ( 1 : l ) as mobile phase. The ergotoxinine group o f alkaloids was separated on a porous polystyrene, modified by hydroxymethyl groups by using _I-hexane ethanol - triethylamine (70:30:0.5) as mobile phase. The ergotoxine group alkaloids and their dihydro derivatives could be separated in t h i s system (Table 9.13). For the LC-MS analysis of clavine alkaloids in ergot fermentation broth, Eckers e t a1.57*58 separated the alkaloids on s i l i c a gel with the mobile phase dichloromethane - methanol - concentrated ammonia (95: 5:O. 1) (Fig .9.10). 9.4. DETECTION The intense fluorescence of LSD provides the basis f o r a very s e n s i t i v e and s e l e c t i v e detection o f t h i s compound - 10 pg. The excitation wavelength used i s about 325 nm13*15g16’ 21’28’53 and the emission i s measured a t 389 nm53, 400 nm4128 o r 420 nm13’15916’21. Prolonged i r r a d i a t i o n - 5 min - of LSD trapped in a scanning fluorimetric detector, r e s u l t s i n the conversion i n t o a non-fluorescent lumi-derivative. The disappearance of the fluorescence upon UV-irradiation has been used t o distinguish LSD from other fluorescent compounds t h a t do not exhibit t h i s b e h a v i o ~ r ~ ~ ’The ~ ~same ’ ~ ~principle . has been used f o r the i d e n t i f i c a t i o n o f ergot alkaloids by Scholten and Frei44. A photochemical reaction detector was designed i n which the eluted ergot alkaloids were i r r a d i a t e d f o r 20 sec with 327 nm UV-light - and the emission was measured a t 410 nm. Dihydroderivatives were i r r a d i a t e d a t 280 nm and the fluorescence measured a t 340 nm. Under such conditions a 90-99% decrease o f the fluorescence was found f o r the 17 alkaloids investigated. The influence of the solvent on the quenching of the fluorescence was studied by Heacock e t al.‘. From Table 9.14 i t i s c l e a r t h a t only chloroform causes considerable quenching. The fluorescence maxima of some ergot alkaloids a r e l i s t e d in Table 9.15‘.

367

TABLE 9.14 INFLUENCE OF SOLVENT ON FLUORESCENCE INTENSITY OF E R G O T A M I N E ( ~ . ~ X ~ O - ~ M ) ~ hex= 350 nm

Solvent

Re1a t i ve i n t e n s i t y ( % )

100

100% Ethanol 50% D i i s o p r o p y l e t h e r a 50% Cyclohef;anea 50% Acetong 50% Hexane 50% Benzenea 50% Chloroforma

130 113 96 91 109 13

a 50% m i x t u r e s w i t h a b s o l u t e e t h a n o l TABLE 9.15 EMISSION MAXIMA AND RELATIVE FLUORESCENCE INTENSITIES FOR SOME ERGOT ALKALOIOS I N ACETONE SOLUT ION^.

xPy=

350 nm, c o n c e n t r a t i o n = 100 ppm.

A1 k a l o i d

,Amau

emission

Relative intensity(%)

~~

Ergotami ne Ergotami n i n e E r g o c r i s t i ne E r g o c r y p t i ne E r g o c r y p t i n i ne Ergocornine Ergocorninine Ergosi ne b L y s e r g i c a c i d amide I s o l y s e r g i c a c i d amide 0-Lysergic a c i d Ergotrate Isosetoclavifle Elymoclavine Agrocl a v i n e 0-Lysergic acid diethylamide

400 397 400 403 39 7 402 398 398 393 393 392 403 393 394 392 397

40.2

100.0 21.0 22.1 62.2 18.3 83.8 56.1 1.1 61.5 32.8 28.9 82.2 0.3 0.5 62.2

b C o n c e n t r a t i o n = 20 ppm, due t o low s o l u b i l i t y o f compound i n acetone. Fluorescence d e t e c t i o n has found w i d e a p p l i c a t i o n i n t h e a n a l y s i s o f e r g o t a l k a l ~ i d s ~ ’ ’ ~ ’

36*50. S c o t t and Lawrence”

found t h a t f o r t h e f l u o r i m e t r i c d e t e c t o r used i n t h e i r i n v e s t i -

g a t i o n s , t h e optimum wavelength f o r e x c i t a t i o n was 235 nm. Baker e t a1.4 compared a f l u o r i m e t r i c d e t e c t o r and d e t e c t o r s w i t h v a r i a b l e wavelength (334 nm) and s i n g l e wavelength (254 nm) f o r t h e a n a l y s i s o f i l l i c i t LSO samples. The f l u o r i m e t r i c and t h e v a r i a b l e wavelength d e t e c t i o n a l l o w e d a n a l y s i s w i t h o u t sample p r e t r e a t m e n t , whereas s e v e r a l peaks i n t e r f e r e d w i t h LSO by d e t e c t i o n a t 254 nm. For a s p e c i f i c d e t e c t i o n o f t h e e r g o t o x i n e a l k a l o i d s , Szepesy e t a1.25 used a wavelength o f 320 nm, f o r t h e i r d i h y d r o d e r i v a t i v e s 280 nm. Bethke e t a1.17 a p p l i e d s i m u l t a n e o u s d e t e c t i o n a t 280 and 320 nm f o r ergotamine and i t s decomposition p r o d u c t s . D e t e c t i o n a t 241 nm 52

has a l s o been employed

.

S i n c e t h e l u m i a l k a l o i d s have o n l y v e r y s m a l l a b s o r p t i o n a t 320 nm, t h e y do n o t i n t e r f e r e w i t h t h e d e t e c t i o n o f t h e e r g o t a l k a l o i d s w i t h a 9,lO-double

References p. 368

bond. The d i h y d r o and t h e l u m i

368 a l k a l o i d s can be detected a t 280 nm. the lumi a l k a l o i d s w i t h an increase o f s e n s i t i v i t y by a f a c t o r o f 1000 when compared w i t h the 320 nm d e t e c t i o n l i m i t . Szepesy e t al.25 used t h e wavelength 320 nm and 280 nm f o r t h e d e t e c t i o n o f the ergotoxine and the dihydroergotoxine alkaloids, r e s p e c t i v e l y . For q u a n t i t a t i v e determinations o f e r g o t a l k a l o i d s , Wurst e l a l , 2g’43 made use o f several wavelengths

-

310. 282, 254. 240 and 225 nm. I n t h a t way, p a r t i a l l y resolved a l k a l o i d s could

be q u a n t i f i e d . White45 developed an electrochemical d e t e c t i o n method f o r t h e HPLC, a l s o s u i t a b l e f o r the analysis o f LSD and some phenothiazine d e r i v a t i v e s . Santi e t a1.10s26 as w e l l as G f e l l e r e t a1 .30’38 studied t h e s e l e c t i v e d e t e c t i o n o f ergotamine as p i c r a t e i o n - p a i r i n mu1 ti-component preparations. This method i s discussed i n Chapter 4. Frei4’

has reviewed r e a c t i o n l i q u i d chro-

matography, i n c l u d i n g the method mentioned above.

A post column f l u o r e s c e n t i o n - p a i r i n g technique has been developed f o r e r g ~ t a m i n e ~The ~. major advantage o f t h i s technique i s an improvement o f the s e l e c t i v i t y , as no i n t e r f e r i n g peaks due t o non-ion-pairing compounds are observed i n u r i n e analyses (see Chapter 4). Lawrence e t a1.48 described a s i m i l a r technique, b u t they used an organic e l u e n t i n com42

b i n a t i o n w i t h a s i l i c a gel column, instead o f an aqueous e l u e n t as used by G f e l l e r e t a l . (see Chapter 4).

Eckers e t a1.57*58 reported the analysis o f c l a v i n e a l k a l o i d s i n e r g o t fermentation b r o t h by means o f LC-MS (Fig.9.10). than CI-MS;

I n general. EI-MS was found t o g i v e more i n f o r m a t i v e r e s u l t s

however, the a l k a l o i d s p a l l i c l a v i n e decomposed during EI-MS, n e c e s s i t a t i n g the

l a t t e r method f o r the analysis o f t h a t a l k a l o i d . Various conditions f o r the mass spectrometry were studied. REFERENCES

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L. Szepesy, I . Feher, G. Szepesi and M. Gazdag. J . C h r o m a t o g r . , 149 (1978) 271. J.M. Huen, R.W. F r e i , W. Santi and J.P. Thevenin, J . C h r o m a t o g r . , 149 (1978) 359. P.J. Twitchett, P.L. Williams and A.C. Moffat, J . C h r o m a t o g r . , 149 (1978) 683. P.J. Twitchett. S.M. Fletcher, A.T. S u l l i v a n and A.C. Moffat. J . C h r o m a t o g r . , 150 (1978) 73. M. Wurst, M. F l i e g e r and Z. Rehacek. J . C h r o m a t o g r . . 150 (1978) 477. J.C. G f e l l e r , J. Huen and J.P. Thevenin, J . C h r o m a t o g r . . 166 (1978) 133. D.L. Sondack, J . C h r o m a t o g r . , 166 (1978) 615. I.S. L u r i e and J.M. Weber, J . L i q . C h r o m a t o q r . , 1 (1978) 587. W. Hartmann, M. Rodiger, W. Ableidinger and H. Bethke, J . Pharm. S c i . , 67 (1978) 98. L. Szepesy, I . Feher, G. Szepesi and M. Gazdag, Magy. Kem. ~ o l y ,84 (1978) 375. P. Horvath, G. Szepesi and A. Kassai, P l a n t a Med.. 33 (1978) 407. P. Hatinguais, 0. Beziat, P. Negol and R. Tarroux, Trav. SOC. Pharm. M o n t p e l l i e r , 38 (1978)

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

G. Megges, A r c h . K r i m i n o l . , 164 (1979) 25. J.C. G f e l l e r , J.M. Huen and J.P. Thevenin, C h r o m a t o g r a p h i a , 12 (1979) 368. S.L. A l i and T. S t r i t t m a t t e r , I n t . J . P h a r m . , 4 (1979) 111. R.W. F r e i , J . C h r o m a t o g r . , 165 (1979) 75. A. Yoshida, S. Yarnazaki and T. Sakai, J . C h r o m a t o g r . , 170 (1979) 399. J.C. G f e l l e r . G. Frey, J.M. Huen and J.P. Thevenin, J . C h r o m a t o g r . , 172 (1979) 141. M. Wurst, M. F l i e g e r and 2 . Rehacek, J . C h r o m a t o q r . , 174 (1979) 401. A.H.M.T. Scholten and R.W. F r e i , J . C h r o m a t o q r . , 176 (1979) 349. M.W. White, J . C h r o m a t o q r . . 178 (1979) 229. R.E. Ardrey and A.C. Moffat. J. F o r e n s i c Sci. Soc., 19 (1979) 253. F. Luccioni, A. B a r l a t i e r and 0. Lena, P h a r m . d c t a Helv., 54 (1979) 69. J.F. Lawrence, U.A.T. Brinkman and R.W. F r e i , J . C h r o m a t o g r . , 185 (1979) 473. I.S. Lurie, I n t e r n a t i o n a l Laboratory. (1980) 61. P.M. Scott and G.A. Lawrence, J . d g r i c . Food Chem., 28 (1980) 1258. G. Szepesi, M. Gazdag and L. Terdy, J. C h r o m a t o g r . , 191 (1980) 101. R. Fankel and I . Slad, z. d n a l . Chem., 303 (1980) 208. P.O. Edlund, J. C h r o m a t o g r . , 226 (1981) 107. I.S. L u r i e and S.M. Demchuk. J . L i o . C h r o m a t o -u r . . . 4 (1981) 337. I.S. L u r i e and S.M. Demchuk; J . L i g . C h r o m a t o g r . , 4 (1981j 357. I.S. Lurie, J . L i q . C h r o m a t o g r . , 4 (1981) 399. C. Eckers, D.E. Games, D.N.B. Mallen and B.P. Swann, A n a l . P r o c . , 8 (1982) 133. C. Eckers, O.E. Games, D.N.B. Mallen and 8.P. Swann, B i o m e d . M a s s S p e c t r o m . , 9 (1982)

329.

162. 59 T.A. Gough and P.8. Baker, J . C h r o m a t o q r . S c i . , 20 (1982) 289. 60 8. Herlnyi and S. Gorog. J . C h r o m a t o g r . , 238 (1982) 250. 61 G. Szepesi, M. Gazdag and R. Ivancsics, J . C h r o m a t o g r . , 241 (1982) 153. 62 K. Harzer, J . C h r o m a t o q r . . 249 (1982) 205.

.u

370

11

101

1 I

I

I

1

1

I

1

0

L

8

12

16

20

min

Fig. 9.1. Separation o f some ergot a l k a l o i d s 1 9 Column Lichrosorb RP8 5 pm ( 1 2 5 ~ 4 . 2 mn 10). mobile phase a c e t o n i t r i l e - 0.02% aqueous ammonium carbonate ( 2 : 3 ) , f l o w r a t e 100 ml/h, d e t e c t i o n UV 254 nm. Peaks: 1, l y s e r g i c acid; 2, i s o l y s e r g i c acid; 3, l y s e r g i c a c i d amide and ergometrine; 4 , i s o l y s e r g i c a c i d amide; 5, e r gometrinine; 6, ergosine; 7, ergotamine; 8, ergocornine; 9, ergocryptine ( a and 8 ) ; 10, ergoc r i s t i n e ; 11, ergosinine; 12, ergotaminine; 13, ergocorninine; 14, ergocryptinine; 15, ergoc r i s t i n i n e ; x, unknown. (reproduced w i t h permission from r e f . 19, by the courtesy o f F r i e d r . Vieweg & Sohn, Wiesbaden) 19 F i g . 9.2. Separation o f some e r g o t a l k a l o i d s Column Lichrosorb RP18 10um ( 1 2 5 ~ 4 . 2 mm I D ) , mobile phase a c e t o n i t r i l e - 0.02% aqueous ammonium carbonate (42:58), f l o w r a t e 100 ml/h, d e t e c t i o n UV 254 nm. Peak numbering as i n Fig. 9.1. (reproduced w i t h permission from r e f . 19, by the courtesy o f F r i e d r . Vieweg E Sohn, W i esbaden)

11 5

13

1

0

1

I

L

8

I

12

16

I

min

F i g . 9.3. Seperation o f some ergot a l k a l o i d s 1 9 ( 1 2 5 ~ 4 . 2 mm I D ) , mobile phase Column Spherisorb ODS 5 a c e t o n i t r i l e - 0.02% aqueous ammonium carbonate (42:58), f l o w r a t e 100 ml/h. d e t e c t i o n U V 254 nm. Peak numbering 1-8 as i n Fig. 9.1.. peak 9, ergosinine; 10, ergocryptine; 11, e r g o c r i s t i n e , ergocorninine and ergotaminine; 12, e r gocryptinine; 13, e r g o c r i s t i n i n e ; x, unknown. (reproduced w i t h permission from r e f . 19, by the courtesy o f F r i e d r . Vieweg & Sohn. Wiesbaden)

371

I I

0

5

10

15

min

50

40

30

20

10

0

min

Fig. 9.4. Separation of dihydroergotoxine alkaloids33 Column Lichrosorb RP18 5 pin (150x3 mm ID), mobile phase water - a c e t o n i t r i l e - triethylamine (32:8:1). flow r a t e 1.0 ml/min, detection UV 280 nm. Peaks: 1, dihydroergocornine; 2 , dihydro- a-ergocryptine; 3, dihydroergocristine; 4, dihydro-B-ergocryptine. (reproduced with permission from ref. 33, by the courtesy o f Journal Pharmaceutical Sciences) Fig. 9.5. Analysis of ergocornine and ergocryptine i n fermentation liqour 60 Column Lichrosorb RP18 10 pm ( 2 5 0 ~ 4 . 6mn ID), mobile phase tetrahydrofuran - 0.01 M aqueous ammonium acetate (2:3), flow r a t e 1.5 ml/min, detection UV 322 nm. Peaks: 1, ergocornine; 2. a-ergocryptine;3,6-ergocrypti ne; 4, ergocorni nine; 5 , a-ergocrypti nine; 6 , 6-ergocrypti ni ne; 7 , ergometrine; 8 , ergometrinine.

i

li

'"1

I

_A_ L

O

Fig. 9.6. HPLC analysis of LSD and some related compounds9 Column P a r t i s i l 6 p m ( 2 5 0 ~ 4 . 6mm ID), mobile phase methanol - 0.2 M aqueous ammonium n i t r a t e ( 3 : 2 ) , flow r a t e 1 ml/min, detection UV 320 nm. Peaks: 1, lysergic a c i d ; 2 , lysergamide; 3 , LSD; 4, isoLSD. Fig. 9.7. Separation of ergotoxine alkaloids 51 Column Lichrosorb Si60 5 pm ( 2 5 0 ~ 4 . 6mm I D ) , mobile phase hexane - chloroform - a c e t o n i t r i l e (56:22:22), flow r a t e 100 ml/min, detection UV 320 nm. Peaks: 1, ~ e r g o c r y p t i n i n e ; 2 , cr-ergocryptinine; 3, ergocorninine; 4, B-ergocryptine; 5, a-ergocryptine; 6 , ergocornine; x, unknown.

References p. 368

312

3

I

2

51 F i g . 9.8. S e p a r a t i o n o f f o u r isomers o f e r g o c r i s t i n e Column L i c h r o s o r b S i 6 0 5 pm ( 2 5 0 ~ 4 . 6mn I D ) , m o b i l e phase hexane - c h l o r o f o r m - a c e t o n i t r i l e (56:22:22), f l o w r a t e 100 ml/h. d e t e c t i o n UV 320 nm. Peaks: 1, e r g o c r i s t i n i n e ; 2, a c i - e r g o c r i s t i n i n e ; 3, e r g o c r i s t i n e ; 4, a c i - e r g o c r i s t i n e ; x, unknown. F i g . 9.9. S e p a r a t i o n o f e r g o t a l k a l o i d s 5 1

Column L i c h r o s o r b S i 6 0 5 pm ( 2 5 0 ~ 4 . 6 mn ID), m o b i l e phase hexane - c h l o r o f o m - a c e t o n i t r i l e methanol (55:20:25:3), f l o w r a t e 100 ml/h, d e t e c t i o n UV 320 nm. Peaks: 1, e r g o c r i s t i n i n e , e r g o c o r n i n i n e , a- and 8 - e r g o c r y p t i n i n e ; 2, e r g o t a m i n i n e ; 3, e r g o c r i s t i n e , ergocornine,a- and 8 - e r g o c r y p t i n e ; 4, e r g o m e t r i n i n e ; 5, ergotamine; 6, e r g o m e t r i n e ; x, unknown.

.O

10

20

30

LO

5’0

60 min

F i g . 9.10. Reconstructed t o t a l i o n c u r r e n t t r a c e o b t a i n e d by E I LC/MS of e r g o t Column S p h e r i s o r b 5W s i l i c a g e l (250x5 mn I D ) , m o b i l e phase d i c h l o r o m e t h a n e - methanol c o n c e n t r a t e d ammonia (95:5:0.1). f l o w r a t e 1 ml/min. Peaks: 1, s o l v e n t f r o n t ; 2, a g r o c l a v i n e ; 3, s e t o c l a v i n e ; 4 , f e s t u c l a v i n e ; 5, p a l l i c l a v i n e o r isomer; 6, p a l l i c l a v i n e o r isomer; 7 , N-noragrocl a v i n e ; 8, elymoclavine; 9, penni c l a v i n e ; 10, isochanoclavine; 11, n o r c h a n o c l a v i n e s I and 11; 12. c h a n o c l a v i n e I ; 13, c h a n o c l a v i n e 11. (reproduced w i t h p e r m i s s i o n o f John W i l e y & Sons, L t d . )

TABLE 9.16 HPLC ANALYSIS ERGOT ALKALOIDS I N AN0 FROM FUNGI ALKALOIDS *

AIMS

Lysac, Ergta,Ergtaine,Ergct,Ergctine diHErgct,Ergcp,Ergcpine, diHErgcp,Ergco,Ergcoine, di HErgco ,Ergm,Ergmine Aocl ,Chcl ,Elcl ,Fcl ,Pcl ,Pycl, Scl,isoScl,paspaclavine, pal iclavine,Lysam, isolysam, Lysgol ,Lysg,lysergen Ergta ,Ergtaine,Ergct,Ergcti ne

Analysis plant extracts,fermented Lichrosorb Si60,lO wn products and pharmaceutical preparations (Table 9.1 and 9.11) Lichrosorb RP2,RP8 and RP18 Analysis clavines and simple Micropak NH2,lO fl 1 ysergi c acid derivatives (Table 9.6)

COLUMN D I M . MOBILE PHASE LxID mm

STATIONARY PHASE

Analysis in crude Ergot Ergta ,Ergtaine,Ergs ,Ergsine, Analysis in scl erotia ,cul ture Ergct,Ergctine,Ergcp,Ergcplne, media and pharmaceutical proErgco,Ergcoine,Ergm,Ergmine ducts (Table 9.3) papaverine Analysis in crude Ergot and pharErgtaine,Ergct,Ergctine, di HErgct ,a-Ergcp, a-Ergcpi ne, maceuti cal preparations diHErgcp,Ergco,Ergcoine, (Table 9.13) diHErgco,Ergm Agcl ,Lysam,isolysam Analysis in fermentation media Ergta,Ergtaine,B-OH-Ergta, and pharmaceutical preparations Ergs,Ergsine,Ergst,Ergstine, (Table 9.7) Ergct ,Ergctine,Ergcp, Ergcpi ne, Ergco,Ergcoine,Ergm,Ergmine Ergta ,Ergtaine ,Ergs ,Ergsine, Analysis ergot a1 kaloids in flour Ergct ,Ergcti ne,Ergcp ,Ergcpine, Ergco .Ergcoine,Ergm, Ergmine Ergta,Ergtaine,Ergct,ErgctAnalysis ergotoxine alkaloids (Table 9.12 and Fig.9.7.9.8 ine,u-, 8-Ergcp,a-,B-Ergcpine, Ergco,Ergcoine,Ergm,Ergmine and 9.9)

Micropak Si-10 Li chrosorb RP18,lO un

Ergta,Ergtaine,Ergct,Ergcti-

Silica gel C8, 5 1 ~ n

Analysis in sclerotium material

ne ,a-Ergcp ,8-Ergcp ,Ercni ne, Ergco ,Ergcoi ne *For abbreviations see footnote Table 9.19

Hitachi Gel no 3301-0 5-7 un Lichrosorb NH2,10 fl or Micropak NH2, 10 pm

250x2

REF.

n-hexane-CHC13-EtOH(4:4:1) -CHC13-MeOH(95:5) CHCl2-EtOH(95 :5)

250x2 250x2

250x2 250x4

CHCl3-isoprOH(9: 1) ,(8: 2) Et20-isoprOH(7:3). (6:4) EtzO-EtOH(84:16) .(8:2) also gradient elution CHCl3-EtOH( 95 :5) 0.1% (NH4)OAc in ACN-H20 (35:65)

29 35

36 500~4.6 n-hexane-EtOH-TrEA( 70:30 :0.5) n-hexane-CHC13-TrEA(5:95:0.5) cycl ohexane-EtOH-TrEA( 70: 30:O. 5) 41 CHC13;isoprOH(9:l) 250x2 EtpO-1 soprOH( 6:4) EtzO-EtOH(84: 16) (88:12), (93: 7) also gradient elution 43 250~4.6 ACN-O.02M aq. (NH )2CO3(43:57), (35 :65), (48 :72) 50 250~4.6 Hexane-CHC1 -ACN(56:22:22), (60:25:15),?55:20:25) Hexane-CHC13-ACN-MeOH 51 (55:20:25 :3) 250x4 ACN-H20( 55:45) containing 0.04% (NH4)CO? 52 3

Lichrosorb RP8, 5

rrn

Lichrosorb Si60,5 ~n

w 4 w

Agcl,norAgcl,Chcl I and 11, Analysis i n e r g o t f e r m e n t a t i o n b r o t h w i t h LC-MS (Fig.9.10) isoChcl 1,norChvl I and 11, Elcl,Fcl,Pcl,Pycl,Scl ,isoScl, norisoScl .palliclavine

Spherisorb 561

250x5

00s-type 5 um

250x5

NH2-tyne,

5 ,jn

not given

CH2C12-MeOH-conc. NH40H (95:5:0.1) MeOH-HpO-conc.NH OH (60:40:0.!) isooctane-CH2C12-MeOH( 5:4: 1 ) 57.58

Ergm,Ergmi ne ,Ergco,Ergcoine, a-Ergco, 6-Ergcoine, E-Ergcp, a-Ergcnine,diHErgco ,di H- aErgcn,diH- &Ergco

Separation ( F i g .9.5 ,Table 9.4)

L i c h r o s o r b RP18, 10 un

250~4.6 THF-O.01M aq NH40Ac(2:3)

15 E r c o t a l k a l o i d s

Separation w i t h s t r a i n h t phase i o n - p a i r HPLC (Tables 9.9, 9.10)

rSondapak CN

3 0 0 ~ 3 . 9 Hexane-CHC1 ACN-di-(2-ethylhexyl)phosp&ric a c i d i n various r a t i o s

60

61

TABLE 9.17 HPLC ANALYSIS ERGOT ALKALOIDS I N PHARMACEUTICAL PREPARATIONS ALKALOIDS*

OTHER COnPOUNDS

AIMS

STATIONARY PHASE

Agcl ,Elcl Pcl .isoScl ,LSD, Lysg,isoLysg,Ergta, Ergtaine,Ergs, Ergs ine, E r g c t , E r g c t i n e ,Ergcp, Ergcpine,Ergco, Ergcoi ne ,Ergm

Preliminary investigation o f s e p a r a t i o n w i t h HPLC

diHErgct,diHErgcp, diHErgco

Separation on small p a r t i c l e UBondapak C18 s i z e column packings Separation as i o n - p a i r s S p h e r o s i l XOB,5-10 rn loaded w i t h 0.03M p i c r i c a c i d and b u f f e r DH 5 S i l i c a g e l 100, 5 fl loaded w i t h 0.06M p i c r i c a c i d and b u f f e r PH 5 A n a l y s i s i n pharmaceutical Micropsk A1-5 preparations

Ergta .atroDine, scopolamine

diHergta,diHErgct, diHergcp,diHErgco

Corasil

COLUMN DIM. LxID(mn)

MOBILE PHASE

1 0 0 ~ 2 . 4 CHCl3-MeOH-EtOAc-AcOH(60: CHCl3-MeOH( 100:4)

REF. 20: 50: 3 )

2

*For a b b r e v i a t i o n s see f o o t n o t e Table 9.19

300x4

ACN-O.01M aq.(NH4)2C03(2:3) 6

CHC13 s a t . w i t h 0.05M p i c r i c 1 0 0 ~ 2 . 8 a c i d i n pH 5 b u f f e r CHC13.sat.with 0.06M p i c r i c 1 0 0 ~ 2 . 8 a c i d i n pH 5 b u f f e r 250x2 Pentane-MeOH(98:2) ,( 97:3)

10

11

P

; I

Ergta,Ergtaine atropine,scopo1 ami ne ,caffeine

Butalbital , phenobarbital

Separation on silver impregnated silica gel

B

P 0 m m

Ergta ,Ergtaine, aciErgta ,aciErgtaine,Lysam,isoLysam,Lysac, isoLysac.lumiErgta Ergta .Ergtaine aci Ergta ,aci Ergtaine Lysam,isoLysam, Lysac,isoLysac Ergta,Ergtaine,Ergs, Ergsine,Ergct,Ergctine,diHErgct, a-, B-Ergcp,Ergcpine.diHErgcp, Ergco,Ergcoi ne, Ergm,Ergmine, Lysam,isoLysam, Lysac,isoLysac Ergct,a-. kErgcp, Ergco

Qua1 ity control ergotamine preparations (Table 9.2)

14

17 ACN-O.OlM(NH )2CO3 stepwise graiient : 8% ,15%,30%, 40%,50% and 60%

Separation witn stepwise gradient system

Nucleosil C18 5 m

150x3

Separation with reversed phase HPLC (Fig.9.1,9.2, 9.3)

Spherisorb ODS,5 un

125~4.2

Lichrosorb RP2,5 un Lichrosorb RP8.5 im Lichrosorb RP18,5 im

125~4.2 ACN-O.O2%aq (NH4)2C03( 15:85) 125~4.2 ACN-O.O2%aq(NH4)2C03(2:3) 125~4.2 ACN-0.02%aq( NH4)2C03( 2: 3), (38:62)

18

Influence of organic bases Lichrosorb RP18,5 run on the stability reversed phase stationary phases Lichrosorb Si60,lOim Analysis pharmaceutical preparations (Table 9.1 and 9.10)

Ergta,Ergtaine, Ergct ,Ergctine, diHErgct, Ergcp ,Ergcpine, di HErgcp Ergco,Ergcoine, diHErgco, Ergm,Ergmine,Lysac Ergta,Ergtaine, butalbita1,pheno- Separation with ion-pair diHErgta,tropane barbita1,barbitchromatography alkaloids,caffeine a1,pizotifene Ergm,Ergmi ne, Lysac

CHCl3-DEA( 99.99 :O .01) Lichrosorb Si100,5 un imp.with 1.09% AgI not given gradient A CHC13-hexane(l:l) B CHC13-MeOH-DEA(90:10:0.5) linear gradient from 16 to 92% B i n A (1.5-2.5 min) ACN-O.OlM(NH4)2C03(2:1),(1:1) uBondapak C18 or 300x4 300~4.6 Nucleosil C18,lO un

Analysis ergometrine preparations

19

250~4.6 0.05M TrEA in ACN-H20(1:1)

24 250x2

n-hexane-CHC1 EtOH (4:4 : 1 ) iTHCl3-MeOH(95?;) CHC13-EtDH(95:5)

Lichrosorb RP2,RP8, RP18

250x2

ACN-O.01M aq. (NH4)2C03( 2: 3 )

Lichrosorb Si100,5 pin loaded with 0.06M picric acid and buffer pH 6 UBondapak C18

150x3

CHC13 sat.with O.O€M picric acid and buffer pH 6

25,34 26.30, 38 300x4

ACN-AcOH-H20(20: 1:79) 31 0 4

cn

Separation dihydroergot o x i n e i n pharmaceutical p r e p a r a t i o n s (Fig.9.4) A n a l y s i s i n pharmaceutical p r e p a r a t i o n s (Table 9.8)

diHErgct,diHa-Ergcp, d i Hs-Ergcp .diHErgco diHErgta,diHErgct, d i Ha-Ergcp, d i Hg-Ergcp,diHErgco

L i c h r o s o r b RP18,5 p

150x3

HzO-ACN-TrEA(32:8:1) H20-MeOH-TrEA( 25 :3.6: 1)

L i c h r o s o r b RP18,lO pm L i chrosorb RP8,7 rm

250x4 ACN-HzO-DEA(375:625:21) 2 5 0 ~ 4 . 6 ACN-H20( 45:60 )+0.049( NH4)eC03 ACN-H 0 TrEA(45:60:0.4)+citric acid i 6 6 9 ) .pH 7.1

33

(6.

ACN-H20-TrEA(45:60:1)+0.3g sodium acetate,pH 7.1 ACN-H20(45:60)+0. l g t e t r a d e c y l t r i r n e t h y l a m o n i um bromide pH 7.1 Ergtaine,Ergct, E r g c t i ne, d i Ergct, a-Ergcp ,a-Ergc p i ne,di Ha- Ergcp Ergco ,Ergcoine, diHErgco,Ergm diHErgta,bromocryptfne.atropine, emetine,ephedrine

Analysis i n pharmaceutical p r e p a r a t i o n s (Table 9.13)

H i t a c h i Gel no 3011-0 5-7 ldn

cyclohexane-EtOH-TrEA(70:30:0.5) 41 P i n d o l o l ,guanfacin,ketotifen, p i z o t i f e n ,clemastine

Post column d e r i v a t i z a t i o n L i c h r o s o r b DIOL.10 m using the f l u o r i m e t r i c L i c h r o s o r b RP8.10 um i o n - p a i r technique

wn

Ergta ,Ergtaine, a c i E r g t a ,aciErgtaine,Ergct,Ergcp, Ergco

D e t e c t i o n w i t h a photochemical r e a c t i o n d e t e c t o r

L i c h r o s o r b RP18,5

diHErgta

S t a b i l i t y studies

uBondapak C18

Detection i o n w i t h postcolumn d e r i v a t i z a t i o n

L i c h r o s o r b Si60.5 vm

Ergta,atropine

39

5 0 0 ~ 4 . 6~-hexane-EtOH-TrEA(70:30:0.5) n-hexane-CHC1 3-TrEA( 5:95: 0.5)

250x4 0.1M phosphate b u f f e r (pH 3 ) 1 0 0 ~ 4 . 6 MeOH-0.02M aq. phosphate b u f f e r (PH 3)(3:2)

42

1 2 0 ~ 4 . 6ACN-O.01M NaHC03(42:58), (38:62),pH 2.2 o r 8.5 44

Hydroxyatrazine

300x4 60x3

ACN-H20(2:3)

47

0.1M b u t y r i c a c i d i n CHC13MeOH( 9: 1 )

48

?

$

TABLE 9.18

E

HPLC ANALYSIS LSD AND RELATED COMPOUNDS I N DRUG SEIZURES AND AS PURE COMPOUNDS

0

ALKALOIDS

P P

*

OTHER COMPOUNDS

AIMS

STATIONARY PHASE

COLUMN D I M LxlD(mn)

MOBILE PHASE

Sil-X

C o r a s i l I1

500~2.3 610~2.3

ACN-( isopr)20 ( 4 :6) ACN-(isopr)pO (25:75)

m

LSD,i soLSD,Lysac, Lysam, isolysam

STP,strychnine, phencycl i d i ne

A n a l y s i s i11i c i t preparations

REF.

Ergta,Ergtaine,diHErgta, Ergct,Ergctine,diHErgct Ergcp ,Ergm ,MeErgm, MeMeErgm

, 1

LSO ,isoLSD,Lysac, Lysam,Lysgol .Ergta, Ergs ,Ergsi ne,Ergct, Ergctine,diHErgct. Ergcp.Ergcpine. diHErgcp, Ergco ,di HErgco , Ergm, Ergmine. MeErgm,MeMeErgm

I d e n t i f i c a t i o n LSO i n i l l i c i t p r e p a r a t i o n s by HPLC and GLC

C o r a s i l C18

1200x2.2

MeOH-0.1% aq. (NH4)2C03 (3:2)

L SD

Comparison o f photometric d e t e c t o r s f o r HPLC Identification street drugs

Zorbax S i l

250~2.1

CH2C1 2-MeOH-AcOH( 70:30:0.1)

C o r a s i l II,37-5Om

500~2.3

Separation drugs o f abuse (Fig.9.6)

Partisil 6

un

250~4.6

MeOH-0.2 M NHqN03(3:2)

A n a l y s i s i l l i c i t samples

P a r t i s i l 5 um

250~4.9

MeOH-O.3% aq. ( NH4)2C03 (3:2)

300x4

0.005M h e p t a n e s u l f o n i c a c i d i n MeOH-AcOH-H20 (40:1:59), pH 3.5

LSO,codeine,heroin, methadon,cocaine, strychnine,mescaline,quinine

3.8

Barbiturates,amphetami nes , various other drugs o f abuse

LSO ,isoLSD,Lysac, Lysam LSD LSD,isoLSD,Lysac, Lysam,Ergta,Ergm

Phencyclidine, N-methylpropylamide,various o t h e r drugs o f abuse

9

I o n - p a i r chromatography f o r pbondapak C18 t h e s e p a r a t i o n o f t h e drugs o f abuse

*For a b b r e v i a t i o n s see f o o t n o t e Table 9.19

4 Cyclohexane-cyclohexylamine (98.8:O .2 ) g r a d i e n t e l u t i o n (1inear) A to B A s k e l l y B-95%EtOH-dioxanecyc 1oh exy 1ami ne (99. I :50:25 :13) B idem (686:100:200:14) 5

16

20

LSD

Fluorescence d e t e c t i o n f o r HPLC-analysis LSD

Micropak MCH-10

250~2.2

ACN-O.1M (1:l)

LSD

Photochemical d e t e c t i o n f o r i d e n t i f i c a t i o n LSD

Spherisorb DDS

100~4.6

MeOH-aq. 0.025M Na2HP04 (65:35) pH 8

LSD,isoLSD

Semipreparative HPLC f o r uBondapak C18 the i d e n t i f i c a t i o n o f drugs o f f o r e n s i c i n t e r e s t P a r t i s i l - 1 0 ODS

300~4.4 250~9.4

aq.(NH4)2C03

21 27,E

0.005M methanesulfonic a c i d i n MeOH-AcOH-H20(40:1:59), pH 3.5 0.04M methanesulfonic a c i d i n MeOH-AcOH-H20(40:1:59), 32, pH 3.5 49

LSD,lysergic a c i d methylpropylamide

Benzocaine

A n a l y s i s i l l i c i t preparations

Bondapak C18

300x4

ACN-H 0-1% aq.(NH4)2C03 (4OO:E72: 28)

LSD

Phenothiazine type drugs

Electrochemical d e t e c t o r

S i l i c a C S y l o i d 74,

2DOx4.6

MeOH- pH 10.2 NH4N03 b u f f e r (9:l) 45

Analysis i l l i c i t prepar a t i o n s w i t h TLC, HPLC and MS (Table 9.5)

Sherisorb 5 ODS

100~4.6

Spherisorb S5W

15Dx4.6

MeOH-0.025M Na2HP04(65: 35) PH 8 MeOH-O.2M NH4N03

37

LSD,isoLSD,Lysac, Lysam,Lysgol , Ergta,diHErgta, Ergs ,Ergsine, Ergct,diHErgct, Ergcpine,diHErgcp, Ergco,diHErgco, Ergm,MeErgm,

7m

AK

HPLC ANALYSIS ERGOT ALKALOIDS I N BIOLOGICAL MATERIAL ALKALOIDS*

OTHER COMPOUNDS

AIMS

STATIONARY PHASE

E r g t a i ne

Reserpine

Determination i n plasma

S i l i c a g e l 10

LSD,isoLSD

Detection i n b i o l o g i c a l f l u i d s , i n combination w i t h TLC

Partisi

diHErgct ,diHErgcp, diHErgco

Trace enrichment technique

Nucleos I C18, 5 un

Determination i n i n t e s t i n a l homogenate

Partisi

Ergta

Quinine

UII

6un

COLUMN D I M LxID(mn)

MOBILE PHASE

REF.

250~2.2

( i s o p r ) 0-ACN-MeOH (69.5: 36:O. 5 )

250~4.6

MeOH-0 .2M aq (11:9)

100x3

ACN-O.1M (2:3)

250~4.6

ACN-1% a c e t a t e b u f f e r pH 6.5 (55:45)

7

. NH4N03 15,13

10/25 ODS

aq.(NH4)2C03

22 23

P

f

I

P 0

m m

0-LSD,isoLSD,Lysac, Lysam,Lysgol , lumiLS0,Z-oxoLSD, Lysac monoethylamide,Ergta, diHErgta ,Ergs, E r g s i ne,Ergct, diHErgct,Ergcp, Ergcpine,diHErgcp, Ergco ,diHErgco, Ergm,Ergmi ne, MeErgm,MeMeErgm. Ergothioneine diHErgta,bromoc r y p t ine ,m e t ine , hyoscyamine, ephedr ine

A n a l y s i s LSO i n body f l u i d s Spherisorb 5 ODS (Table 9.5) Spherisorb S5W

100~4.6

D e t e c t i o n w i t h p o s t - c o l umn L i c h r o s o r b RP8 f l u o r i m e t r i c i o n - p a i r technique Lichrosorb Oiol

100x4 .6

28 Various drugs

5 um

E r g t a ,Ergtaine, MeErgm ,MeMeErgm, Ergct

A n a l y s i s i n plasma

H y p e r s i l OOS,

LSO

O e t e c t i o n i n serum and urine

L i c h r o s o r b RP8,7w w i t h column s w i t c h to L i c h r o s o r b S i 60, 5 Irn

*Abbreviations used i n Tables 9.16 Agcl Ccl Chcl E lc l Fcl Fucl Mcl Pcl Pycl Scl Lysam

150~4.6

MeOH-0.025M NazHP04(65:35) PH 8 MeOH-0.2M NHqN03(3:2)

250~4.6

MeOH-0.02M phosphate b u f f e r (PH 3)(3:2) 0.1M phosphate b u f f e r (PH 3 ) 42

250~4.6

ACN-O.OlM (1:l)

(NH4)2C03( 3:7),

250x4

A MeOH-0.3% KH2PO4 (pH=3)

53

-

(1:l)

B MeOH-1% (NH4)2C03(3:2) 125x4

s o l v e n t A f o r column s w i t c h system 62

9.19:

Agrocl a v i ne Costaclavine Chanocl a v i n e Elymoclavi ne F e s t u c l a v i ne Fumiclavine Molliclavine Penni c l a v i n e Pyrroclavine Setoclavine L y s e r g i c a c i d amide ( e r g i n e )

Lysac LSD LYsg Lysgol Ergm Ergmi ne Ergta E r g t a i ne MeErgm MeMeErgm Ergst

Lysergic a c i d 6-Lysergide Lysergine Lysergol Ergometrine (ergobasine o r ergonovine) Ergometri n i ne Ergotamine Ergotami n i n e Methylergometrine 1-Methylmethylergometrine (methysergide) E r g o s t i ne

Ergs t i n e Ergs Ergs ine Ergtox Ergct Ergctine Ergcp Ergcpi ne Ergco Ergcoine d i H-

E r g o s t i n i ne Ergosi ne Ergosi n i n e E r g o t o x i ne( ErgcttErgcptErgco) E r g o c r i s t i ne Ergocri s t i n i n e E r g o c r y p t i ne Ergocrypti nine Ergocornine Ergocorni n i ne dihydro-

w -a W

This Page Intentionally Left Blank

381

Chapter 10 STEROIDAL ALKALOIDS

..................................................................... ........................................................................

10.1. HPLC systems 10.2. Detection References

.............................................................................

The s t e r o i d a l k a l o i d s comprise a l a r g e number o f h e t e r o c y c l i c p l a n t bases

-

381 382 382

some o c c u r r i n g

as the aglycone p o r t i o n of a g l y c o a l k a l o i d bonded t o one o r more hexoses (Solanurn a l k a l o i d s ) , others as esters (Veratrurn a l k a l o i d s ) . So f a r , HPLC has had o n l y l i m i t e d a p p l i c a t i o n i n the f i e l d o f s t e r o i d a l k a l o i d analysis - mainly because o f the l a c k o f a strong chromophore i n most o f such a l k a l o i d s , thus n e c e s s i t a t i n g R I d e t e c t i o n o r UV d e t e c t i o n a t 200-210 nm. A s h o r t wavelength, UV d e t e c t i o n l i m i t s the number e l u t i o n solvents t o those having low UV absorption: water, a c e t o n i t r i l e . ethers, alcohols and hydrocarbons. Fluorescent i o n - p a i r i n g techniques o r d e r i v a t i z a t i o n techniques have so f a r n o t been used t o improve the d e t e c t i o n p r o p e r t i e s o f steroid alkaloids. 10.1. HPLC SYSTEMS Hunter e t a1

.'

developed a straight-phase preparative HPLC method f o r some s t e r o i d a l k a l o i d s

i s o l a t e d from a Solanurn species. I t was l a t e r improved by using m i c r o p a r t i c u l a t e s i l i c a gel as stationary

The improved method was applied t o the separation o f a s e r i e s o f non-gly-

c o s i d i c Solanurn and n o n - e s t e r i f i e d Veratrurn a l k a l o i d s (Figs.lO.1 and 10.2). Ppior t o the HPLC 6 analysis the Solanurn a l k a l o i d s were f r a c t i o n a t e d by means o f a chrornatofuge . Reversed-phase HPLC o f potato s t e r o i d g l y c o a l k a l o i d s was reported by Bushway e t a1 .2'5. An octadecyl s t a t i o n a r y phase was used t o separate the g l y c o a l k a l o i d s according t o the number o f g l y c o s i d i c bonded hexoses i n the molecule, and a s t a t i o n a r y phase c o n t a i n i n g alkylamino groups was used t o separate the glycoalkaloids a-chaconine,

8-chaconine and a-solanine (Fig.

10.3). However, f o r the analysis o f the g l y c o a l k a l o i d s i n potato tubers, peels, and sprouts, a "carbohydrate a n a l y s i s column" was preferred, since no i n t e r f e r i n g peaks were observed (Fig. 3 10.4). The same k i n d o f column, as w e l l as an octadecyl column, were used by Crabbe and F r y e r f o r the separation o f solasodine, solasodine glycoal k a l o i d s and solasodiene. The i n f l u e n c e o f varying r a t i o s o f the solvents on the k ' was investigated. Buffered solvent systems (pH 7-7.5 w i t h 0.01 M T r i s b u f f e r ) were found t o g i v e b e t t e r r e p r o d u c i b i l i t y . Glycoalkaloids o f the solasodine and solasodiene types were separated on the octadecyl column. Increasing the water content i n the mobile phase from 10% t o 25% l e d t o separation o f the g l y c o a l k a l o i d s i n three groups: mono-, d i - and t r i - g l y c o s i d i c a l k a l o i d s (Fig.10.5).

On r e p l a c i n g methanol by aceto-

n i t r i l e i n the mobile phase, an increase o f the s e l e c t i v i t y and o f t h e number o f t h e o r e t i c a l p l a t e s was achieved. The composition o f the solvent had, i n such a case, a g r e a t e r e f f e c t on the k ' than i n the case o f methanol

-

water systems. The "carbohydrate a n a l y s i s column" behaved

as a normal-phase column and gave increased r e t e n t i o n w i t h increased p o l a r i t y o f t h e a l k a l o i d s . This column could be used t o separate a l k a l o i d s w i t h the same number o f g l y c o s i d i c bonded hexoses (Fig.10.6).

Referencw p. 382

382 The amino-type s t a t i o n a r y phase was a l s o used i n more recent studies by B u ~ h w a y ~ ’ ~For ’~~. the separation o f a-chaconine and a-solanine i n potato extracts, the mobile phase tetrahydrofuran

-

acetonitrile

-

water

-

methanol (50:25:15.5:9.5)

was used, r e s u l t i n g i n a considerably

reduced analysis time when compared t o the p r e v i o u s l y reported methods. For a n a l y s i s o f the a l k a l o i d s and t h e i r metabolites. t h e mobile phase r a t i o was changed t o (55:30:10:5)(Fig.l0.7). For separation o f the lower glycosides from a-chaconine and a-solanine,

a carbohydrate ana-

l y s i s column was used i n combination w i t h the mobile phase tetrahydrofuran - water - acetonitri l e (55:8:37) (Fig. 10.8)”. Morris and Lee7 analyzed potato a1 kaloids on o c t y l and octadecyl-type s t a t i o n a r y phases. Using a mobile phase c o n s i s t i n g o f a c e t o n i t r i l e

-

water t h a t contained small amounts o f etha-

nolamine ( l e s s than 0.1%). d e t e c t i o n a t 200 nm was possible. The separation o f a-chaconine and a-solanine could be achieved on an octadecyl column w i t h a c e t o n i t r i l e amine (45:55:0.1)(Fig.10.9e) 45:0.1)(Fig.10.9f).

-

water

-

ethanol-

o r on an o c t y l column w i t h the same s o l v e n t i n the r a t i o (55:

The a l k a l o i d s could a l s o be separated on s i l i c a gel w i t h t h i s mobile phase

i n the r a t i o (77.5:22.5:0.05)(Fig.l0.9g).

I n the case when solanidine was present i n the ex-

t r a c t s , the s i l i c a gel column was preferred. Hydrolysates o f the a-chaconine and a-solanine could a l s o be analyzed w i t h the octadecyl column (Fig.lO.9a-d). used f o r the analysis o f potato e x t r a c t s (Fig,10.9e-g).

The systems could a l s o be

For a t o t a l g l y c o a l k a l o i d analysis,

the normal-phase system gave the f a s t e s t r e s u l t s (Fig.10.9h). 10.2. DETECTION Most o f the s t e r o i d a l a l k a l o i d s have UV absorption o n l y a t about 200 nm. Using UV detectors w i t h v a r i a b l e wavelength, i t i s possible t o analyze microgram amounts o f such a l k a l o i d s . For f u r t h e r ’ i d e n t i f i c a t i o n o f the separated s t e r o i d a l k a l o i d s . Bushway e t a1 .2’5 used UV absorption r a t i o s a t 215, 225, 235 and 245 nm. For the detection, a wavelength o f 215 nm was p r e f e r r e d t o 208 nm because o f a more s t a b l e baseline, and w i t h s u f f i c i e n t s e n s i t i v i t y . The s e n s i t i v i t y could be increased by using a c e t o n i t r i l e

-

water as mobile phase. Such a s o l v e n t

system allowed d e t e c t i o n a t 200 nm. Morris and Lee7 detected the potato glycoalkaloids a t 200 nm, which they found t o be 960% more s e n s i t i v e than detection a t 215 nm and 55% more s e n s i t i v e than d e t e c t i o n a t 205 nm; the detection l i m i t was 0.1 ug, which a l s o compared favorably w i t h R I d e t e c t i o n ( d e t e c t i o n l i m i t

5

pg).

A t 200 nm there was no i n t e r f e r e n c e o f sugars as was observed a t 195 nm. The ethanol-

amine i n the mobile phase had t o be kept below 0.1%, otherwise the background absorption o f the mobile phase became too high. REFERENCES

1 I . R . Hunter, M.K. Walden, J.R. Wagner and E. Heftmann, J . C h r o m a t o g r . , 119 (1976) 223. 2 R.J. Bushway, E.S. Barden, A.W. Bushway and A.A. Bushway, J . C h r o m a t o g r . , 178 (1979) 533. 3 P.G. Crabbe and C. Fryers, J . C h r o m a t o g r . , 187 (1980) 87. 4 I . R . Hunter, M.K. Walden and E. Heftmann, J . C h r o m a t o g r . , 198 (1980) 363. 5 R.J. Bushway, E . S . Barden, A.M. Wilson and A.A. Bushway, J . F O O ~sci., 45 (1980) 1088. 6 W.D. Nes, E. Heftmann, I . R . Hunter and M.K. Walden, J . Liq. C h r o m a t o g r . . 3 (1980) 1687. 7 S. Morris and T.H. Lee, J . C h r o m a t o g r . , 219 (1981) 403. 8 R.J. Bushway, J . C h r o m a t o g r . , 247 (1982) 180. 9 R.J. Bushway and R.H. Storch, J . Liq. C h r o m a t o g r . , 5 (1982) 731. 10 R.J. Bushway, J. Lig. Chromatogr., 5 (1982) 1313.

383

6

mllmin-

2L

16mIlm0n,

I

L0

72

,

,

,

96 120 I L L

min

Fig. 10.1. Separation o f some s t e r o i d a l alkaloids 4 Column Zorbax-Sil 6 urn ( 5 0 0 ~ 4 . 6mn ID), mobile phase n-hexane - methanol - acetone (18:l:l). flow r a t e 0.3 ml/min (60 min), followed by 1.5 ml/min, detection UV 213 nm. Peaks: 1, tomat i l l i d i n e ; 2, solanidine; 3, tomatidine; 4, 5-tomatidenol; 5, solasodine; 6 , veramine; 7, rubi j e r v i n e . 4 Fig. 10.2. Separation of some s t e r o i d a l a l k a l o i d s Column Zorbax-Sil 6 pm ( 5 0 0 ~ 4 . 6nnn ID), mobile phase n-hexane - ethanol - acetone (18:l:l). flow r a t e 1 . 0 ml/min (36 min), followed by 1.6 ml/minT detection UV 213 nm. Peaks: 1, isorubijervine; 2, r u b i j e r v i n e ; 3, muldamine; 4, veratramine; 5 , verarine; 6. cyclopamine; 7. j e r v i ne. 1

0

1

2

3

1

5

5

7

0

8

1

2

3

L

5

6

7

min

m n

2

Fig. 10.3. Separation of some glycoal kaloids Column UBondapak NH (300x4 n ID), mobile phase tetrahydrofuran - 0.025 M potassium dihydrogen phosphate - a c e ? o n i t r i l e (2:l:l). flow r a t e 1 ml/min, detection refractometer and UV 208 nm. Peaks: 1, 8-chaconine; 2, a-chaconine; 3, tomatine; 4 , a-solanine. Fig. 10.4. Separation o f glycoalkaloids present in d r i e d potato peels 2 Column UBondapak Carbohydrate (300x4 mn ID), mobile phase tetrahydrofuran - water - acetonit r i l e (56:14:30), flow r a t e 2 ml/min. detection UV 215 nm. Peaks: 1. a-chaconine; 2. a-solanine.

References p. 382

384 2 1

1

I

0

5

10 min

7

I

20

15

5

0

I

10

15

-

0

6 mln

L

min

3 Fia. 10.5. Seoaration solasodine alvcosides Coiumn UBondapak C18 (300~3.9 mm f6). mobile phase methanol 0.01 M T r i s b u f f e r (3:1), f l o w r a t e 2 ml/min, d e t e c t i o n UV 205 nm. Peaks: 1, a-glycosides; 2. 8-glycosides; 3. y-glycosides; 4, solasodine.

-

Fig. 10.6. Separation o f solasodine glycosides3 Column VBondapak Carbohydrate (30063.9 n I D ) . mobile phase isopropanol - methanol (7:3). f l o w r a t e 2 ml/min, temperature 40 C. d e t e c t i o n UV 205 nm. Peaks: 1, solasodine and solasodienei 2. 8- and y-solamargine; 3. y-solasonine; 4. a-solamargine; 5, 6-SOlaSOnine; 6, a-solasonine. 8 Fig. 10.7. Separation o f g l y c o a l k a l o i d s from a p o t a t o meal e x t r a c t Column Radial-Pak PBondapak NH (100x8 n I D ) , mobile phase t e t r a h y d r o f u r a n - a c e t o n i t r i l e water - methanol (55:30:10:5).2flow r a t e 3.0 ml/min, d e t e c t i o n UV 215 nm. Peaks: 1, y-chaconine; 2, B2-chaconine; 3, Brchaconine; 4, a-chaconine.

-

6

I

r

0

.

.

.

,

4

.

.

r

.

8

min

I

. .

I

12

.

r

.

t

16

10 Fig. 10.8. Separation o f metabolites of a-chaconine and a-solanine acetoniColumn pBondapak Carbohydrate (300x4 mn ID), mobile phase t e t r a h y d r o f u r a n - water t r i l e (55:8:37), f l o w r a t e 1.1 ml/min, d e t e c t i o n UV 215 nm. Peaks: 1, y-chaconine; 2, y-solanine; 3, B2-chaconine; 4, B1-chaconine; 5, unknown; 6 , a-chaconine; 7, E,-SOlanine. ireproduced w i t h permission from r e f . 10. by t h e courtesy o f Marcel Dekker Inc.)

-

b

a

-

0

2

L

C

I

.

0

2

,

min

6

f

-

0

2

min

1

0

-

,

L

0

2

min

1

6

0

2

man

L

min

9

2

L min

0

. -

2

L ' 6 min

0

2

L

min

Fig. 10.9. Separation o f glyco-alkaloids o f the a-chaconine and a-solanine s e r i e s 7 Chromatogram a, b and e:column Radial-Pak C18 (100x8 n I D ) . mobile phase a c e t o n i t r i l e - water ethanolamine (45:55:0.1). f l o w r a t e 3 ml/min, d e t e c t i o n UV 200 nm. Chromatogram c. d, g and h: column Radial-Pak S i l i c a (100x8 mn I D ) , mobile phase a c e t o n i t r i l e water - ethanolamine (775:225:0.5), flow r a t e 3 ml/min, d e t e c t i o n UV 200 nm. ethaChromatogram f: column Radial-Pak C8 (100x8 mn I D ) , mobile phase a c e t o n i t r i l e - water nolamine (55:45:0.1). f l o w r a t e 3 ml/min, d e t e c t i o n UV 200 nm. Peaks: 1, a-solanine; 2, P s o l a n i n e ; 3, y-solanine; 4, a-chaconine; 5, bl-chaconine; 6, 62 -chaconine; 7, y-chaconine; 8, solanidine; 9, solanadiene.

-

-

Refarenem p. 382

TABLE 10.1 HPLC ANALYSIS STEROIDAL ALKALOIDS AIMS

ALKALOIDS

STATIONARY PHASE

COLUMN DIM. MOBILE PHASE

REF.

T o m t i dine.so1 anidine,solasodine, r u b i j e r v i ne.veratramine, jervine

P r e p a r a t i v e HPLC

P o r a s i l A 37-75 m,

a- and s-chaconine,a-solanine,

Analysis p o t a t o a l k a l o i d s (Fig.10.3 and 10.4)

J3ondapak C18 300x4 300x4 ,Bondapak NH ,Bondapak Cagbohydrate 300x4

THF-H O-ACN(5:3:2) THF-O?025M KH PO -ACN(2:1:1),(5:3:2) THF-H O-ACN(58:14:30) ,(6:1:3) ACN-H:0(85: 15)

Analysis p l a n t m a t e r i a l and hydrolyzed g l y c o s i d e s ( F i g . 10.5 and 10.6)

,,Bondapak C18

MeOH-O.01M T r i s buffer(88:12).(3:1) ACN-O.01M T r i s buffer(7:3).(2:3) MeOH-isoprOH(3:7)

tomati ne

Solasodine,solasodine glycosides,solasodiene

2440x12 OD g r a d i e n t e l u t i o n A. Me CO-n-hexane(2:l) B . 97% aqT MeC ,O

-

,,Bondapak

300x4

Carbohydrate 300x4

IsoprOH-cyclohexane(4:1) 2 5 0 ~ 4 . 6 1-Hexane-MeOH-Me CO( 18: 1:l) -n-Hexane-EtOH-Me$O( 18: 1: 1)

14 so~anumand Veratrum a l k a loids

Separation s t e r o i d a l a l k a l o i d s ( F i g . l O . 1 and 10.2)

Zorbax S i l 6

a-Chac0nine.a-solanine

Quantitative analysis potato alkaloids

UBondapak Carbohydrate 300x4

Solasodine,various

Analysis i n Solanum species

Zorbax S i l 6

3 0 0 ~ 4 . 6 p-Hexane-MeOH-Me2CO(18:l:l)

Analysis p o t a t o a l k a l o i d s (Fig.10.9)

Radial-Pak C8

100x8

Radi a1 -Pak C18 Radi a1 -Pak S i 1ica

100x8 100x8

steroids

a-,61-,@2- and y-chac0nine.a-, 6- and y-sol anine,solani dine,

sol anadi ene

3 4 5

6

ACN-H 0-ethanolamine( 50:50:0.2), (55:43:0.1) Idem (36:65:0.2) ,(45:55:0.1) Idem (77.5:22.5:0.5).(45:55:0.01)

7

THF-ACN-H O-MeOH(55:30:10:5), (50: 25: 15?5:9.5)

8

and y-chac0nine.a-, 8- and v-solanine

Analysis p o t a t o a l k a l o i d s ( F i g . 10.7)

Radi a1 -Pak NH2

100x8

a-Chaconi ne,u-solanine,commersonine,demissine

Semipreparative HPLC p o t a t o glyco-alkaloids

Zorbax NH2

2 5 0 ~ 9 . 4 THF-H20-ACN((55:20:25)

Determination m e t a b o l i t e s o f p o t a t o glyco-a1 k a l o i d s ( F i g . 10.8)

UBondapak Carbohydrate 300x4

, B ~ - and v-chaconine,a-, and v-solanine

2

THF-H20-ACN(53: 17: 30)

a-.B1-,

a-

1

9 THF-H20-ACN(55:8:37) 10

387

Chapter 11 XANTHINE ALKALOIDS

................................................

11.1. Theophylline i n b i o l o g i c a l f l u i d s 11.2. Sample preparation f o r xanthine assays i n b i o l o g i c a l f l u i d s 11.3. Ion-exchange HPLC.. 11.4. Reversed-phase HPLC.. 11.5. I o n - p a i r HPLC 11.6. Straight-phase HPLC.. 11.7. Oetection References

387 388 389 390 393 393 394 395

...................... .............................................................. ............................................................ .................................................................... ............................................................ ........................................................................ .............................................................................

The a n a l y t i c a l problems concerning xanthine d e r i v a t i v e s can be grouped as f o l l o w s :

1. Analysis o f theophylline i n b i o l o g i c a l f l u i d s (Table 11.8).

2. Analysis o f pharmaceutical preparations containing c a f f e i n e (Table 11.7). 3. Analysis o f drugs o f abuse containing c a f f e i n e as a d u l t e r a n t (Table 11.5). 4. Analysis o f caffeine, theobromine and t h e o p h y l l i n e i n food and beverages (Table 11.9).

11.1 THEOPHYLLINE I N BIOLOGICAL FLUIDS I n the numerous studies on the analysis o f t h e o p h y l l i n e i n b i o l o g i c a l f l u i d s , o n l y a few HPLC systems have been used. However, a v a r i e t y o f sampling techniques have been described, ranging from d i r e c t i n j e c t i o n of plasma t o solvent e x t r a c t i o n techniques. These techniques w i l l be discussed b r i e f l y l a t e r (see below). Whereas series o f drugs has been reported n o t r t o interfere w i t h the analysis of theophyl 1 ine18,27,33935s48,68,75,96,i04,1059 1069 111I 1 122’1263127’lZ8’ 156*160y183’191’193’ lg6, some drugs i n t e r f e r e , e.g. ampici 11i n and methic i 11in5491409187, c e p h a l o ~ p o r i n s ~ ~ ~acetazolamide82’101, ~ ~ ’ ~ ~ ~ ’ ~ ~ ~ , s u l fadiazine”’,

procain-

a ~ n i d e ’ ~ ~and , sulfamethoxazole 128’1399140. The i n t e r f e r e n c e o f c a f f e i n e metabolites (theop h y l l ine, theobromi ne. l17-dimethylxanthi ne (paraxanthine) , 3-methyl xanthine, 7-methylxanthi ne, I-methyl uric a c i d and 1.3-dimethyl u r i c acid)51 363*67’144’165’ 1773 185y186s

lg6, theophyll i n e

metabolites and o t h e r xanthine metabolites has been d e a l t w i t h i n a number o f studies56’63y76y

80’95’96’981106’112’116*128112g, The xanthine d e r i v a t i v e d y p h y l l i n e has a l s o been reported t o i n t e r f e r e w i t h the analysis o f t h e o p h y l l i n e

183,187

For the q u a n t i t a t i o n o f theophylline, an i n t e r n a l standard has i n most cases been used, e. g. 8-chloro-theophyll ine. 8-hydroxyethyl -theophyll i n e and 8-hydroxypropyl-theophyl l ine. Several authors have noted peak anomalies f o r t h e o p h y l l i n e i n t h e case when samples d i s solved i n a c e t o n i t r i l e (used f o r deproteination) were analyzed on an octadecyl t y p e o f c o l umn112,159.196 For d i r e c t a n a l y s i s of t h e o p h y l l i n e i n plasma and serum, the use o f pre-columns has been recornended inorder to prolong column 1 ife18s63.91,99,109, 111,122.138.146,177,192,196. ~~t~~ and B e r n ~ t e i nreported ~~ a method i n which no pre-column was necessary. UV spectrometry 26.27,33,35,59,100, Comparison o f HPLC w i t h GLC27’53’98’100’104a113~128~12g, 128,154

EMIT69,72,97,

100,110,127.135,154,157,196

FIA179

and RIA154,

155,165 has been made,

regarding t h e i r a p p l i c a b i l i t y t o the analysis o f t h e o p h y l l i n e i n b i o l o g i c a l f l u i d s . Because of interference i n UV spectrometry, HPLC was p r e f e r r e d by a l l authors. The time-consuming

Reference. p. 395

388

preparation of samples necessary f o r GLC, makes HPLC preferable. The r e s u l t s of EMIT and HPLC had generally good correlation69~72y100,127,136~15791g6.However, Sheen e t a1 .97 and Tin e t al.l1° reported t h a t significantly higher values were found w i t h EMIT. HPLC was found t o be more precise, b u t EMIT had the advantage of s h o r t e r analysis time, and the analysis was e a s i e r t o perform. Reviews of theophylline monitoring have been given50’78*831132’188. 11.2. SAMPLE PREPARATION FOR XANTHINE ASSAYS IN BIOLOGICAL FLUIDS For the assay of theophylline i n biological f l u i d s the following types of sample preparation have been described: 1. Direct injection of plasma, serum o r s a l i v a . 2. Oeproteination of plasma, serum o r s a l i v a p r i o r to injection. 3. Solvent extraction of the d r u g from biological f l u i d s p r i o r t o injection. 4. Preconcentration of the drug on adsorbent columns. 1. The f i r s t method has the disadvantage of very short column l i f e due t o contamination w i t h plasma proteins72. By using precolumns, which were replaced every 30-40 samples, the problem could be s ~ l v e d l ~ Saliva ’ ~ ~ . samples could be injected d i r e c t l y without problems 26 . Manno e t a1.l” injected serum, plasma and s a l i v a d i r e c t l y on the column. By regularly cleaning of the column by subsequently pumping water, methanol - water (1:l) and methanol through the column, i t could be used f o r 400-500 samples. Yoshida e t a l . l E 1 treated an octadecyl type column with human plasma. This column could then be used f o r drug monitoring by d i r e c t injection of plasma samples, giving good reproducibility and complete recovery of the drugs. 2. Several methods have been described t o remove the proteins from the sample. Addition of trichloroacetic acid26968’72’120’147 or perchloric acid65 followed by centrifuging has been reported, as well as addition of organic solvents, e.g. acetonitrile55~56976.80.86,105,122, 138’1401176’177’191’1g6 and m e t h a n 0 1 ~ ~i n’ ~which ~ ~ the internal standard has been dissolved, a l s o followed by centrifuging, whereafter the c l e a r supernatant was used f o r i n j e c t i o n . Oeproteination can a l s o be achieved by molecular Oesiraju e t a l . 63 preferred such f i l t r a t i o n above addition of t r i c h l o r o a c e t i c acid, acetone, a c e t o n i t r i l e o r ammonium s u l f a t e . The former two additives caused i n t e r f e r i n g peaks, the l a t t e r two were not effective. Van den Bemd e t a1.95 found f i l t r a t i o n and the addition of methanol were compatible. Bateman e t al.74 used u l t r a f i l t r a t i o n of s a l i v a prior t o d i r e c t i n j e c t i o n . 3. The most widely used procedure is extraction of the drug from the biological material by means of an organic solvent a t various pH. Chloroform - isopropanol (95:5) has been successf u l l y used t o e x t r a c t theophylline from plasma, serum and s a l i v a samples. The samples were e i t h e r extracted directly26’35’54’98’112~12g~156’160~187o r a f t e r the addition o f an 72395*99, amnonium s u l f a t e 19,51,67,110,156.165,168 or buffers: pH 6.5339127 or pH 7.444. Ex-

traction a t pH 8.6, with chloroform - isopropanol ( 9 : l ) . avoids interference of acetazolamide i n the HPLC analysis”’. Theophylline was extracted from brain homogenates w i t h chloroform a t pH 7113. Dyphylline was extracted from serum o r plasma w i t h chloroform - isopropanol (9:l) a f t e r the addition of sodium hydroxidelZ5. Other organic solvents used f o r the extraction of theophylline and other xanthine derivadiethyl e t h e r (4:7)71’96; dichloromethane - isopropanol (4:1)lo2; tives are: dichloromethane dichloromethane - methanol ( 9 : l ) o r dichloromethane alone, a f t e r the addition of d i l u t e hydroc h l o r i c acid126g136; d i ~ h l o r o m e t h a n e l ~=-butanol ~; - !-hexane (2:3) a f t e r the addition of

-

389

-

-

ammonium sulphate4’; t-pentanol chloroform 0.1% hydrochloric acid (80:19.9:0.1)75; ethyl acetate499183; and J-pentanol - ethylene chloride a t pH 15.9~’. Ion-pair extraction with t e t r a butylamonium as counter-ion a t pH 6 enabled the extraction i n one s t e p of both theophylline and u r i c acid derivatives ( f o r the analysis of theophylline and i t s metabolites i n urine146). A similar method (pH 11.0) has been used f o r caffeine and i t s Jusko and Pol iszczuk26 compared an extraction and a deproteination procedure and found t h a t extraction was more s e n s i t i v e than deproteination with t r i c h l o r o a c e t i c acidz6 o r a ~ e t o n i t r i l e ~ ’ .Soldin and Hill54 preferred extraction t o d i r e c t serum injection because of the possible interference of ampicillin and methicillin in the l a t t e r case. Interference of cephalosporins can be resolved by using an extraction step p r i o r t o HPLC77. Extraction y i e l d s cleaner samples than deproteination. but i s more time consuming7’. 4 . Concentration o f the drug on adsorbent columns has been done f o r the analysis of theophylline and i t s metabolites in urine“ and f o r some other xanthine derivatives i n urine and plasma6’. Thompson e t a1.16 used an anion-exchange resin f o r pre-fractionation i n a xanthine and u r i c acid fraction. Bye and Brown62 collected the xanthine derivatives on a metal-chelate resin column.

metabolite^^^'.

The solvent used f o r the injection of theophylline plasma samples was shown t o a f f e c t c r i t i c a l l y the chromatographic r e s ~ l t s ~ ~ ‘When ’ ~ ~the ~ .mobile phase was used as solvent ( a c e t o n i t r i l e - sodium acetate buffer (8:92)) the best r e s u l t s were obtained. Pure acetonit r i l e resulted in poor peak shape. Jowett15’ found t h a t a c e t o n i t r i l e used during deproteination had to be removed p r i o r to HPLC analysis, otherwise extra peaks were observed in the chromatogram of theophylline. Muir e t a1 .lg6 found t h a t the complete removal of a c e t o n i t r i l e prior to the HPLC analysis was e s s e n t i a l . Mannlo3 found t h a t the pH of the injected sample influenced the retention times of theophylline and 8-chlorotheophylline. By adding a c e t i c acid to the sample p r i o r t o i n j e c t i o n , variations i n the retention times of the alkaloids in the serum samples could be avoided. The extraction methods can generally be regarded as most s e n s i t i v e and s p e c i f i c . The deproteination methods require regular cleaning of the column and the use of replaceable precolumns; but they a r e more simple and rapid than the extraction methods. 11.3. ION-EXCHANGE HPLC For the analysis o f theophylline and i t s metabolites i n biological f l u i d s , an Aminex A-5 cation-exchange resin has been used in combination with a mobile phase consisting of 0.45 M ammonium dihydrogen phosphate buffer (pH 3.65) (Table ll.l)16’18’g7. Jusko and PoliszczukZ6 reported the analysis of theophylline in biological f l u i d s by using a chemically bonded strong cation-exchanger on p e l l i c u l a r beads (Table 11.2). 0.66% Aqueous a c e t i c acid was used as mobile phase. The same mobile phase was applied in combination w i t h 49 a microparticulate chemically bonded strong cation-exchanger by Peng e t a l . Walton e t a1.l” separated xanthines on a 4% crosslinked cation-exchange resin. The e f f e c t of the counter-ion of the cation-exchanger, the pH. and the percentage of organic solvent in the aqueous mobile phase, was studied and the analysis of xanthines i n serum and beverages was described. Analgesics, including some xanthines, have been analyzed by ion-exchange chromatography ( r e f . 1.2.3,6,8,10,12,13,15,29,38,89). Murgia e t a1.l0, Walton15, Murgia3’ and Hanai e t al.”

.

-

References p. 396

390 TABLE 11.1 SEPARATION

OF SOME

U R I C A C I D AN0 XANTHINE DERIVATIVES16

Column Aminex A-5 cation-exchange r e s i n , 13 vm ( 6 6 5 ~ 3 . 2n ID), mobile phflse 0.45M ammonium dihydrogen phosphate b u f f e r (pH 3.65), f l o w r a t e 10 ml/hr, temperature 55 C, d e t e c t i o n UV 280 nm. Compound

Retention time(mi'n)

Uric acid 3-Methyluric a c i d Xanthine 1-Methyl u r i c a c i d 3-Methyl xanthi ne 7-Methyl xanthine 1,3-Dimethyluric a c i d

10.2 min 11.3 13.3 13.7 14.8 15.2 15.6

Compound

Retention time(min)

17 .O

Theobromine 1-Methylxanthine Hypoxanthi ne Theophyll ine Paraxanthine Caffeine

17.3 19.2 20.0 22.8 26.0

TABLE 11.2 RELATIVE

RETENTION

OF VARIOUS

XANTHINE DERIVATIVES

RELATED TO THEOPHYLLINE~~

Column Zipax SCX ( 1 0 0 0 ~ 2 . 1mn ID), mobile phase 0.66% aqueous a c e t i c acid, d e t e c t i o n UV 254 and 280 nm. column pressure 1200 p s i . Compound

Relative retention

Compound

Re1a t i ve r e t e n t i on ~~

Theophyll ine Caffeine Theobromi ne 1-Methylxanthine 3-Methylxanthine Xanthine Hypoxanthi ne Paraxanthi ne 8-Chl oroxanthi ne

1.00 1.66 0.97 0.97 0.79 0.79 1.59 1.76 0.79

Uric acid 1-Methyluric a c i d 1.3-Dimethyluric a c i d 8-Nitrotheophylline 8-Chlorotheophyll i n e 8-Bromotheophyll ine D i hydroxypropyl theophyll ine B-Hydroxypropyl theophyll ine Phenobarbital

0.76 0.76 0.80 0.90 0.90 0.90 0.86 1.41 0.68

t e s t e d d i f f e r e n t ion-exchange r e s i n s f o r the separation o f analgesics. The i n f l u e n c e o f the counter-ion, the pH. the degree o f c r o s s l i n k i n g , and solvent composition on the separation, were investigated. I t was concluded t h a t no ion-exchange was involved, b u t t h a t t h e dominant mechanism o f r e t e n t i o n was adsorption on the r e s i n m a t r i x . The b e s t r e s u l t s were obtained w i t h amnonium o r c h l o r i d e counter-ions f o r , respectively, c a t i o n - and anion-exchange r e s i n s . Non-ionogenic resins were also tested. Ligand-exchange chromatography was used by Wolford e t a1 .3 t o separate oxypurines. Wal ton15 has given a review on ligand-exchange and m a t r i x - a f f i n i t y chromatography. 11.4. REVERSED-PHASE HPLC For the analysis o f theophylline i n b i o l o g i c a l f l u i d s , a reversed-phase separation on a m i c r o p a r t i c u l a t e s t a t i o n a r y phase w i t h chemically bonded octadecyl groups, and a mobile phase c o n s i s t i n g o f a 0.01 M sodium acetate b u f f e r (pH 4.0) c o n t a i n i n g 4-10% a c e t o n i t r i l e , i s w i d e l y 27

used (Table 11.3 and F i g . l l . 1 )

.

The reversed-phase system allows the d i r e c t i n j e c t i o n o f deproteinated serum o r plasma samples, whereby the analysis time i s considerably reduced. However, under these conditions, '~~~, a number o f o t h e r drugs, e . g . a m p i c i l l i n , m e t h i ~ i l l i n ~c ~e ,p h a l o ~ p o r i n s ~ ~acetazolamide ( r e f . 82,101) and t r i s u l f a p y r i m i d i n e s ' "

i n t e r f e r e d w i t h the analysis o f theophyl l i n e i n t h e

391 TABLE 11.3

’

SEPARATION OF SOME XANTH I NE DERI VAT IVES54’

381 58

S1: Column VBondapak C18 (300x4 mn I D ) , mobile phase a c e t o n i t r i l e -5g.02M sodium acetate b u f f e r (pH 4.0)(1:9), f l o w r a t e 1.8 ml/min, d e t e c t i o n UV 254 nm 52: Column Hypersil ODS 5 pm (100x5 mn ID), mobile phase acetonitrilplZ 0.02M sodium acetate b u f f e r (pH 4.0)(8:92), flow r a t e 1.5 ml/min, d e t e c t i o n UV 273 nm 1M sodium 53: Column Spherisorb ODS 10 pm (250~4.6 mm ID), mobile phase a c e t o n i t r i l e acetate b u f f e r (pH 4.0)(15:85), f l p y 8 r a t e 1.0 ml/min, d e t e c t i o n UV 273 nm 3Q . 54: As system S3, s o l v e n t ratio(18:82) . 55: Column Ultrasphere ODS 5 ~m ( 1 5 0 ~ 4 . 6nun I D ) , mobile phase acetonitrile1580.01M sodium acetate b u f f e r (pH 6.5)(9:91), f l o w r a t e 1 ml/min, detection UV 273 nm .

.

.

Compound Uric acid Xanthi ne Hypoxanthi ne 3-Methyluric a c i d 7-Methyluric a c i d 1-Methyl u r i c a c i d 3-Me thy 1xa nt h ine 7-Me thy1 xan t h i ne 1-Methylxanthi ne 1.3-Dimethyluric a c i d Theobromi ne Theophyll ine Paraxanthi ne Caffeine B-Hydroxypropyl t h e o p h y l l i n e

s1

s2

1.78 2.00 2.00 2.00

1.00 0.95 0.95

2.37 2.65

1.15 1.35

2.93 3.23 3.65 4.88

1.40 1.65 1.65 2.50 2.50 4.65

8.47 8.90

s3

s4

s5

2.88 3.04 3.08

2.79 3.05 3.10

1.03 1.35 1.32

3.45 3.51 3.66 3.97 4.26 3.83 4.78 5.76 5.96 10.08 7.98

3.07 3.13 3.59 3.77 3.82 3.75 4.47 5.14 5.31 8.33 6.60

1.06 1.12 1.84 1.75 2.00 1.47 2.60 3.85 3.85 7.53 9.02

p

3.20 2.15 29.0

6-Hydroxyethyltheophylline Paracetamol Phenobarbi t a l

method described. E x t r a c t i o n o f t h e o p h y l l i n e from the b i o l o g i c a l m a t e r i a l t o be analyzed eliminated the i n t e r f e r e n c e o f the compounds mentioned. Also, a c a f f e i n e metabolite, 1,7-dimethylxanthine, can i n t e r f e r e w i t h t h e theophyl l i n e analysisg5. Some studies reported no s i g n i f i c a n t amounts o f t h i s metabolite are t o be found i n the plasma o r serum98’112’129, whereas others reported considerable amounts present, and causing e r r o r s o f up t o 30% ( i n the case where no separation was obtained between t h e o p h y l l i n e and the metabol ite185a186a196 1. By the a d d i t i o n o f tetrahydrofuran t o the above mentioned mobile phase and by i n c r e a s i n g the pH o f the b u f f e r t o 5 the i n t e r f e r e n c e o f 1.7-dimethylxanthine

could be

The same was the case w i t h the o t h e r drugs mentioned above ( a m p i c i l l i n , e t c . ) . Also, by decreasing the percentage o f a c e t o n i t r i l e i n the mobile phase, the i n t e r f e r e n c e o f a m p i c i l l i n could be eliminated1”,

With ageing o f the columns, Marion e t

found t h a t procainamide

i n t e r f e r e d w i t h theophylline. The pH o f the b u f f e r i s important f o r the separation. The r e t e n t i o n o f 8-chlorotheophylline i n p a r t i c u l a r i s g r e a t l y dependent on the pH, and i t can be varied a t w i l l by changing the pH35,112,129 Also. f o r the analysis o f c a f f e i n e , the sodium acetate b u f f e r c o n t a i n i n g a c e t o n i t r i l e (as mentioned above) has been applied136s138. b i o l o g i c a l fluids57’102y123’147.

It has a l s o been used f o r d y p h y l l i n e assays i n

Due t o the i n t e r f e r e n c e o f benzoic a c i d (among others present

i n c a f f e i n e i n j e c t i o n s ) Blanchard e t a1

modified an e a r l i e r reported method138 (Table

11.3). Changing the pH o f the acetate b u f f e r t o 6.5 and decreasing the amount o f a c e t o n i t r i l e i n the mobile phase lead t o a separation o f benzoic a c i d and c a f f e i n e . The separation o f the c a f f e i n e metabolites i n t h i s system i s , however, n o t as good as under the conditions o r i g i n a l -

Refennew p. 396

392 l y reported.

-

A gradient o f a c e t o n i t r i l e

aqueous a c e t i c a c i d has been used i n connection w i t h an octa-

decyl column f o r assays o f c a f f e i n e and i t s metabolites i n b i o l o g i c a l f l u i d s 1079133. The system has a l s o been used t o e l i m i n a t e t h e i n t e r f e r e n c e o f sulfamethoxazole i n the a n a l y s i s o f 139 theophylline i n serum

.

George and Patel'''

i n v e s t i g a t e d some reversed-phase column packing m a t e r i a l s f o r the pre-

sence o f a c t i v e s i l a n o l groups, by using the columns i n the adsorption mode w i t h 1-heptane as solvent. Four materials were t e s t e d f o r the a n a l y s i s o f t h e o p h y l l i n e and c a f f e i n e . These two compounds were w e l l separated on a l l m a t e r i a l s with the mobile phase a c e t o n i t r i l e

- acetic acid

(95:5:0.2).

- water

However. the sequence o f c a f f e i n e and the i n t e r n a l standard 6-hydro-

xypropyl t h e o p h y l l i n e varied f o r the columns. Broussard'"

reported the analysis o f t h e o p h y l l i n e i n serum using a m o d i f i e d method o f

Adam e t a1.35. As mobile phase, water

- methanol -

acetonitrile

-

10 m l / l a c e t i c a c i d (788:

180:16:16) was used i n combination w i t h an octadecyl type o f column. A s e r i e s o f n o n - i n t e r f e r i n g drugs was reported. According t o Jonkman e t a1 .185*186lr7-dimethylxanthine co-elutes w i t h theophylline i n t h i s system and causes e r r o r s o f up t o 30% (see a l s o r e f 196, Fig.11.8). Phosphate b u f f e r s has been used i n place o f acetate b u f f e r s i n some c a s e s 5 5 s 8 0 ~ 1 0 3 ~ 1 5 2 ~ 1 7 8 ~

181'183'187D197 (Fig.11.2).

V o l a t i l e b u f f e r s have been used t o avoid corrosion157. Non-buffered

mixtures o f water and a c e t o n i t r i l e have given good r e s u l t s i n t h e analysis o f t h e o p h y l l i n e ( r e f . 76,113.161)

and i n the analysis o f dyphylline130 i n b i o l o g i c a l f l u i d s .

To avoid the t o x i c a c e t o n i t r i l e . methanol has been preferred. Peat e t al.59 analyzed theop h y l l i n e on an octadecyl column w i t h methanol

- 0.01

M sodium acetate b u f f e r (1:3).

In this

case too the pH o f the b u f f e r had great i n f l u e n c e on t h e r e t e n t i o n o f 8-chlorotheophylline. The optimum pH f o r the analysis was found t o be 4. S i m i l a r systems were found t o be useful f o r e l i m i n a t i n g the i n t e r f e r e n c e o f such drugs as a m p i c i l l i n , m e t h i c i l l i n , e t c . (mentioned above)104,140,191 Phosphate b u f f e r (pH 4.7)

-

methanol (88:12) was used i n the analysis o f t h e o p h y l l i n e and

i t s metabolites on an octadecyl column (Fig.11.3)63.

Anderson and Murphy46 used a b u f f e r o f

pH 6.0 i n 25% methanol t o o b t a i n a separation o f some xanthines. M i l l e r and Tuckerlog p r e f e r r e d a phosphate b u f f e r o f pH 2.3 i n 40% methanol f o r the serum analysis o f a s e r i e s o f drugs

-

i n c l u d i n g some xanthine d e r i v a t i v e s . Several o t h e r r e l a t e d systems have been a p p l i e d f o r the

~ ~ the ~ ~ automated ~ ~ ~ ~ ~ HPLC ~ ~ a. n a l y s i s o f t h e o p h y l l i n e i n serum. analysis of t h e ~ p h y l l i n e For

-

0.0025 M sodium dihydrogen phosphate (14:86) containing 0.065% t r i e t h y l a m i n e (pH 6.6) has been used i n combination w i t h an o c t y l type o f s t a t i o n a r y phaselg3. Two hundred drugs

methanol

were tested f o r i n t e r f e r e n c e i n t h i s system. To separate theophylline from 1.7-dimethylxanthine, formamide

-

Rodriguez e t a1 .177 used methanol

potassium dihydrogen phosphate (0.05 M)(22:11.5:66.6)(pH

-

5.8) i n combination w i t h

an octadecyl type o f column. The already mentioned i n f l u e n c e o f the pH o f the b u f f e r on t h e r e t e n t i o n o f d-chlorotheop h y l l i n e was i n v e s t i g a t e d by H i d 4 , who found t h a t an optimum separation o f xanthine derivat i v e s was obtained a t a pH o f 5.0 f o r a mobile phase c o n s i s t i n g o f 1%p r o p i o n i c a c i d

-

methanol

(8:2) on an octadecyl column. For a simultaneous a n a l y s i s o f theophylline. p r o x y p h y l l i n e and o t h e r xanthine d e r i v a t i v e s i n serum, Nielsen Kudsk and K i r s t e i n Pedersen6'

-

-

found t h a t aceto-

0.02 M potassium chlor i d e (pH 2 ) (3:7) as mobile phase i n connection w i t h an octadecyl column (Fig.11.4).

nitrile

acetate b u f f e r s were unsatisfactory. and p r e f e r r e d methanol

393 Weidner e t a1.lZ8, on the o t h e r hand, performed analyses o f t h e o p h y l l i n e on an octadecyl

-

column w i t h isopropanol

0.1 M phosphate b u f f e r (pH 3.8)(4:96)(Fig.11.5).

They e x t e n s i v e l y

discussed the i n t e r f e r e n c e o f other drugs i n the a n a l y s i s . Rosenbaum13 analyzed analgesics by means o f m i c r o p a r t i c u l a t e octadecyl columns, because he found t h a t the columns had a longer l i f e under t h e conditions used .for the a n a l y s i s than d i d ion-exchange columns

.

Jandera e t a1.118 studied the i n f l u e n c e o f various g r a d i e n t systems o f methanol

-

water,

i n combination w i t h octadecyl columns, on the r e t e n t i o n behaviour o f xanthines, w h i l e i s o c r a t i c e l u t i o n w i t h such solvents was applied f o r the analysis o f crude drugs c o n t a i n i n g 131 xanthine d e r i v a t i v e s

.

The peak broadening caused by the sample solvent i n the analysis o f analgesics was invest i g a t e d by Williams e t a1 .145. For reversed-phase systems, the b e s t r e s u l t s were obtained w i t h solvents t h a t had about the same p o l a r i t y as t h e mobile phase used (Fig.11.6). For the analysis o f xanthine d e r i v a t i v e s i n food and beverages, octadecyl type o f columns have been used i n combination w i t h the mobile phase methanol 142,205,207 ( r e f . 86,141,148). o r s i m i l a r r a t i o s

-

water

-

a c e t i c a c i d (20:79:1)

Jonkman e t a1.156 reported the use o f a non-polar mobile phase (chloroform absolute ethanol

- water

-

a c e t i c a c i d (600:400:32:1.5:0.8))

- heptane -

i n combination w i t h an octadecyl

type o f s t a t i o n a r y phase. The advantage o f t h i s method i s t h a t chloroform

-

isopropanol ex-

t r a c t s o f serum can be i n j e c t e d d i r e c t l y on the column. To optimize a chromatographic assay, Weyland e t a1.‘02

reported the a p p l i c a t i o n o f an

operational research technique c a l l e d non-linear p r o g r a m i n g . 11.5.

ION-PAIR HPLC

Muir e t a1.146 determined theophylline and i t s metabolites i n u r i n e by i o n - p a i r HPLC. The method was based on a combination o f l i q u i d - l i q u i d and i o n - p a i r l i q u i d - l i q u i d e x t r a c t i o n , followed by reversed-phase i o n - p a i r separation. I t allowed simultaneous analysis o f xanthine and u r i c a c i d d e r i v a t i v e s . The method was f u r t h e r improved f o r the analysis o f c a f f e i n e and i t s metabolites (Fig.11.7)lg2.

I t was a l s o modified f o r the a n a l y s i s o f t h e o p h y l l i n e i n plasma

and s a l i v a l g 6 . This method had the advantage t h a t t h e i n t e r f e r e n c e o f the c a f f e i n e metabolite. 1.7-dimethylxanthine

(paraxanthine), was avoided (Fig.11.8).

The authors reported t h a t a ty-

p i c a l number o f cups o f coffee may r e s u l t i n apparent plasma t h e o p h y l l i n e concentrations o f ca 3 mg/l,

i f the 1.7-dimethylxanthine

i s n o t separated from theophylline. were found

Methods p r e v i o u s l y described f o r the analysis o f t h e o p h y l l i n e i n

t o be less s u i t a b l e because of i n t e r f e r i n g peaks, even i n blank u r i n e . I n a study on i o n - p a i r chromatography o f multicomponent drugs, Huen e t a1

included c a f f e i n e (see Chapter 4).

Because o f t h e i r weak basic properties, the xanthine d e r i v a t i v e s do n o t form i o n - p a i r s w i t h alkylsulfonates. and are thus l i t t l e a f f e c t e d by changes i n t h e concentration o r i n the a l k y l - c h a i n length o f the pai r i n g - i o n ~ ~ 171. ~ ~ ’ ~ ~ ~ ’ 11.6. STRAIGHT-PHASE HPLC S i t a r e t a1.l’

used straight-phase HPLC t o analyse t h e o p h y l l i n e i n b i o l o g i c a l f l u i d s . A

m i c r o p a r t i c u l a t e s i l i c a gel column and a mobile phase c o n s i s t i n g o f chloroform

References p. 385

-

isopropanol

-

394

TABLE 11.4 STRAIGHT-PHASE HPLC SEPARATION OF SOME XANTHINE DERIVATIVES51’53’67 (250x3 nnn ID), m o b i l e phase dichloromethane - methanol c o n t a i n Column L i c h r o s o r b Si60, 5 i n g 0.029 ammonium formate and 0.017ml 97% f o r m i c a c i d p e r l O O m l (978:22), f l o w r a t e Zml/min, d e t e c t i o n UV 280 nm.

.

Retent i on t i m e (mi n)

Compound

Theobromi ne Theophyl 1ine Paraxanthi ne Prednisolone(interna1 standard) a c e t i c a c i d (84:15:1) chloroform

-

methanol

2.4 2.9

Compound

R e t e n t i on t i m e ( m i n)

3 - M e t h y l x a n t h i ne 7-Methylxanthine 1-Methylxanthine

3.6

7.4 8.4

9.8

4.4

was used. S i m i l a r systems have been d e s c r i b e d where o t h e r r a t i o s o f

- a c e t i c a c i d have been

Midha e t a1.51 added up t o 40%

hexane t o such a s o l v e n t system i n o r d e r t o compensate f o r v a r i a t i o n s i n column r e t e n t i v i t y , keeping t h e k ’ o f t h e o p h y l l i n e a t about 7-8. As a second s o l v e n t system f o r t h e a n a l y s i s o f c a f f e i n e m e t a b o l i t e s , dichloromethane

- ammonium f o r m a t e b u f f e r

i n methanol was

( T a b l e 11.4). Evenson and Warren33 developed an assay f o r t h e o p h y l l i n e u s i n g m i c r o p a r t i c u l a t e s i l i c a g e l columns and a m o b i l e phase o f w a t e r - s a t u r a t e d c h l o r o f o r m - heptane - a c e t i c a c i d (300:200:0.4) t o which 6 % e t h a n o l was added. Heptane was necessary t o achieve a s e p a r a t i o n o f theobromine and t h e o p h y l l ine, Boeckx e t a1 .127 found t h a t t h e advantage o f t h e assay mentioned, o v e r methods based on reversed-phase s e p a r a t i o n s , was t h a t s i l i c a g e l columns used i n c o m b i n a t i o n w i t h a s i m p l e e x t r a c t i o n had a l o n g e r l i f e than reversed-phase columns used i n c o m b i n a t i o n w i t h a d i r e c t i n j e c t i o n o f d e p r o t e i n i z e d samples. To a v o i d t h e i n t e r f e r e n c e o f 1 , 7 - d i m e t h y l x a n t h i n e ,

a caf-

f e i n e m e t a b o l i t e , i n t h e a n a l y s i s o f t h e o p h y l l i n e i n b i o l o g i c a l f l u i d s , Van Aerde e t a1.165 developed a s e p a r a t i o n on s i l i c a gel. As t h e m o b i l e phase, c h l o r o f o r m - d i o x a n e (99.5: 4.5:O.Ol)

-

formic acid

was used.

I n a d d i t i o n t o a c i d i c s o l v e n t s , n e u t r a l s o l v e n t s have been used i n t h e a n a l y s i s o f t h e o ~ h y l l i n e ~ and ~ ’ c a~ f f ~e i n’ e 1~ 4 4~ i~n b i o l o g i c a l f l u i d s

-

as w e l l as b a s i c

Analgesics have been analyzed on s i l i c a g e l columns w i t h a c i d i c s o l v e n t ~ y s t e m s ~ ~w h’ i~l e~ , x a n t h i n e d e r i v a t i v e s p r e s e n t i n food and beverages have been analyzed on s i l i c a g e l columns with neutral solvent

system^^^'^^

(Fig.11.9).

A i g n e r e t a l . 4 3 impregnated t h e s i l i c a g e l w i t h

s i l v e r c h l o r i d e t o improve t h e s e p a r a t i o n o f x a n t h i n e s . 11.7. DETECTION The m e t h y l x a n t h i n e d e r i v a t i v e s a r e u s u a l l y d e t e c t e d a t t h e i r a b s o r p t i o n maximum

-

273 nm -

b u t d e t e c t i o n a t 254 nm has a l s o been done. a l t h o u g h t h e a b s o r p t i o n i s much l e s s a t t h i s wavelength

-

f o r t h e o p h y l l i n e i t i s o n l y 35% o f t h a t a t 273 nm. Peat e t a l ? ’

used a d e t e c t o r

w i t h a f i x e d wavelength o f 280 nm and found i t more s e n s i t i v e t h a n d e t e c t i o n a t 273 nm. However, o t h e r a u t h o r s r e p o r t e d t h e opposite116. A disadvantage o f d e t e c t i o n a t 254 nm i s t h e presence o f more s p u r i o u s peaks t h a n a t 280 nm146. W i t h a simultaneous d e t e c t i o n a t 254 nm and 280 nm van den Bemd e t a1.g5 c o u l d c o r r e c t t h e t h e o p h y l l i n e plasma l e v e l s f o r i n t e r f e rence o f 1,7-dimethylxanthine.

F o r i d e n t i f i c a t i o n purpose

-

so f a r m a i n l y a p p l i e d f o r drugs

396 o f f o r e n s i c i n t e r e s t - the absorbance r a t i o a t 254 nm and 280 nm has been used, i n combination w i t h the r e l a t i v e r e t e n t i o n times o f the compounds i n question115 (see Chapter 2, Table 2.2). Lewis and Johnson8' detected methylxanthines u s i n g amperometric methods. Electrochemical d e t e c t i o n enabled determination o f t h e o p h y l l i n e i n the presence o f 1,7-dimethylxanthine,

which

had the same r e t e n t i o n time i n the HPLC system employed. As t o the s e n s i t i v i t y f o r theophyll i n e , a pulse amperometric d e t e c t i o n compared favorably w i t h UV d e t e c t i o n a t 254 nm. A combined photometric and amperometric d e t e c t i o n o f f e r e d the advantage o f an increased s e l e c t i v i t y and the p o s s i b i l i t y of observing unexpected i n t e r f e r e n c e of o t h e r compounds i n the anal y s i s . Greenberg and Mayer116 also studied electrochemical d e t e c t i o n methods f o r t h e a n a l y s i s o f theophylline. An increased s e n s i t i v i t y , as w e l l as the p o s s i b i l i t y o f s e l e c t i v e a t t e n u a t i o n o f i n t e r f e r e n c e o f o t h e r compounds, were reported as advantages o f electrochemical d e t e c t i o n when compared w i t h UV detection. Hashimoto e t a l .94 developed a capacitance c o n d u c t i v i t y detector, which was found u s e f u l f o r a l k a l o i d analysis. Caffeine was included i n the studies. Kit0

e t a1.1g7 reported the s e l e c t i v e d e t e c t i o n o f hypoxanthine and xanthine w i t h an i m -

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F i g . 11.1. S e p a r a t i o n o f some x a n t h i n e d e r i v a t i v e s 56 Column VBondapak C18 (300x4 m ID), m o b i l e phase a c e t o n i t r i l e - 0.01 M sodium a c e t a t e b u f f e r (pH 4.0) (7:93). f l o w r a t e 2.0 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, 1 - m e t h y l u r i c a c i d ; 2, 3 - m e t h y l x a n t h i n e ; 3, 1,3-dimethylu r i c a c i d ; 4, theobromine; 5, t h e o p h y l l i n e ; 6, E-hydroxye t h y l t h e o p h y l l i n e ; 7, p h e n o b a r b i t a l ; 8, c a f f e i n e ; 9, 8- c h l o r o t h e o p h y l l i n e . (reproduced w i t h p e r m i s s i o n f r o m r e f . 56, by t h e c o u r t e s y o f C l i n i c a l C h e m i s t r y )

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80 F i g . 11.2. S e p a r a t i o n of some x a n t h i n e d e r i v a t i v e s and u r i n a r y m e t a b o l i t e s Column VBondapak C18 (300x4 mm I D ) , m o b i l e phase a c e t o n i t r i l e - 0.1 M d i s o d i u m hydrogen phosphate and 0.1 M sodium d i h y d r o g e n phosphate i n w a t e r (2:38), f l o w r a t e 1.5 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, u r i c a c i d ; 2, c r e a t i n i n e ; 3, 1 - m e t h y l u r i c a c i d , 3 - m e t h y l u r i c a c i d and 7 - m e t h y l u r i c a c i d ; 4. x a n t h i n e ; 5, 7-methylxanthine; 6, 1 , 3 - d i m e t h y l u r i c a c i d ; 7, 3-methylx a n t h i ne; 8, 1-methyl x a n t h i ne; 9, theobromi ne ; 10, 8 - c h l o r o t h e o p h y l 1 ine; 11, t h e o p h y l 1 ine and 1 , 7 - d i m e t h y l x a n t h i n e ( p a r a x a n t h i n e ) ; 12, d y p h y l l i n e ; 13, c a f f e i n e . (reproduced w i t h p e r m i s s i o n from r e f . 80, by c o u r t e s y o f C l i n i c a l C h e m i s t r y )

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I

0 mtn

Fig. 11.3. Separation of a standard mixture of xanthine derivatives in serum63 Precolumn Bondapak C18-Porasil 8 (40~2.3n I D ) , column pBondapak C18 (300~3.9nun I D ) , mobile phase methanol - 0.05 M potassium dihydrogen phosphate buffer (pH 4.7) (12:88), flow rate 1.1 ml/min, detection UV 254 nm. Peaks: 1, uric acid; 2, hypoxanthine; 3, xanthine; 4 , l-methyluric acid; 5. 3-methylxanthine; 6, 1-methylxanthine; 7, 1,3-dimethyluric acid; 8, theobromine; 9, theophylline; 10, dyphylline. (reproduced with permission from ref. 63, by the courtesy of Journal Chromatographic Science) Fig. 11.4. Analysis of theophylline and proxyphylline in serum68 Column pBondapak C18 (300~3.9nim I D ) , mobile phase methanol - 0.02 M aqueous potassium chloride (pH 2 adjusted with hydrochloric acid)(3:7), flow rate 1.6 ml/min, detection UV 280 nm. Peaks: 1, theobromine; 2. theophylline; 3, proxyphylline; 4, caffeine; 5 , 8-chlorotheophylline; xl and x2 are unidentified compounds constantly present in serum. (reproduced with permission from ref. 68, by courtesy of Acta Pharmacologica et Toxicologica)

Fig. 11.5. Analysis serum of patient receiving theophylline caffeine and theobromine are added to the sample126 Column Micropak MCH-10, mobile phase isopropanol - 0.1 M potassium dihydrogen phosphate buffer (pH 3 . 8 , adjusted with phosphoric acid), flow rate 1 ml/min, detection UV 277 nm. Peaks: 1, solvent front; 2, theobromine; 3 , theophylline; 4, 8-chlorotheophylline; 5, caffeine. (reproduced with permission from ref. 128, by the courtesy of the American Journal of Clinical Pathology)

401 3

5

14 11

L

-

0

min

I

20

0

I 10

I

20

I

1

30 min

145 F i g . 11.6. S e p a r a t i o n o f some a n a l g e s i c s Column P a r t i s i l PXS 10/25 ODs2 ( 2 5 0 ~ 4 . 6 n I D ) , m o b i l e phase a c e t o n i t r i l e - a c e t i c a c i d w a t e r (25:5:70), f l o w r a t e 1 ml/min, d e t e c t i o n UV 275 nm. Peaks: 1, paracetamol (acetaminophen); 2, c a f f e i n e ; 3, a c e t y l s a l i c y l i c a c i d ; 4, s a l i c y l i c a c i d ; 5, p h e n a c e t i n . F i g . 11.7. S e p a r a t i o n o f some x a n t h i n e d e r i v a t i v e s 1 9 2 Precolumn L i c h r o s o r b RP2 10 p m ( 4 0 ~ 2 . 1mm I D ) , column U l t r a s p h e r e ODS 5 !Jm ( 2 5 0 ~ 4 . 6 n ID), mobile phase q r a d i e n t w i t h s o l v e n t A: 0.01 M sodium a c e t a t e and 0.005 M tetrabutylammonium hydrogen s u l f a t e i n w a t e r (pH 4.9),-solvent B: same s a l t c o n c e n t r a t i o n s i n 50% methanol (pH 4.8). G r a d i e n t 0 - 7 . 5 m i n 0% B, 7.5-15 m i n 0-T5% B, 15-25 m i n 15-30% B, 25-33 min 30-32% B, 33-38 min 32-45% B and 38-41 min 45-0% B. D e t e c t i o n UV 280 nm. Peaks: 1, x a n t h i n e ; 2, u r i c a c i d ; 3, 3 - m e t h y l u r i c a c i d ; 4, 7-methylxanthine; 5, 3 - m e t h y l x a n t h i n e ; 6, 1 - m e t h y l x a n t h i n e ; 7, theobromine; 8, 3 , 7 - d i m e t h y l u r i c a c i d ; 9, 7 - m e t h y l u r i c a c i d ; 10, 1 - m e t h y l u r i c a c i d ; 11, 1 , 3 - d i m e t h y l u r i c a c i d ; 12, 1 , 7 - d i m e t h y l x a n t h i n e ; 13, t h e o p h y l l i n e ; 14, B - h y d r o x y e t h y l t h e o p h y l l i n e ( i n t e r n a l s t a n d a r d ) ; 15, 1 , 7 - d i m e t h y l u r i c a c i d ; 16,, 1 , 3 , 7 - t r i m e t h y l u r i c a c i d ; 17, c a f f e i n e . (reproduced w i t h p e r m i s s i o n from r e f . 192, by t h e c o u r t e s y o f J o u r n a l Chromatograp h i c Science) 1

1

7

196 F i g . 11.8. A n a l y s i s o f t h e o p h y l l i n e i n t h e presence o f c a f f e i n e and i t s m e t a b o l i t e s Precolumn L i c h r o s o r b RP2 ( 4 5 ~ 2 . 0 mm I D ) , column U l t r a s p h e r e ODS 5 Vm ( 1 5 0 ~ 4 . 6 mm I D ) , m o b i l e phase 0.01 M sodium a c e t a t e and 0.005 M tetrabutylammonium hydrogen s u l f a t e i n w a t e r (pH 4.75 w i t h 0 . 1 M a c e t i c a c i d o r sodium h y d r o x i d e ) t o w h i c h 12.5% methanol i s added, f l o w r a t e 1.5 ml/min, d e t e c t i o n UV 274 nm. Peaks: 1, u r i c a c i d ; 2, 3 - m e t h y l x a n t h i n e ; 3, 1 - m e t h y l x a n t h i n e ; 4 , 1 - m e t h y l u r i c a c i d ; 5, 1 , 3 - d i m e t h y l u r i c a c i d ; 6, 1,7-dimethylxanthine; 7, t h e o p h y l l i n e ; 8, 8-hydroxyethyltheophylline ( i n t e r n a l s t a n d a r d ) ; 9, c a f f e i n e ; x, unknown.Chromatogram A: s t a n d a r d m i x t u r e ; chromatogram 8: b l a n k plasma; chromatogram C: plasma from p a t i e n t t a k i n g t h e o p h y l l i n e and d r i n k i n g c o f f e e .

References p. 395

402

1

24,37 F i g . 11.9. Straight-phase separation o f xanthine d e r i v a t i v e s Column Lichrosorb S i 6 0 5 um (300x3 mn I D ) , mobile phase d i c h l o r o methane - ethanol - water (936:47:17), f l o w r a t e 70 ml/h. d e t e c t i o n UV 275 nm. Peaks: 1 , c a f f e i n e ; 2. t h e o p h y l l i n e ; 3 , theobromine.

2

5

0 min

403

TABLE 11.5 XANTHINE ALKALOIDS I N THE CONTEXT OF HPLC ANALYSIS OF DRUGS OF ABUSE (CHAPTER 7 ) A1 k a l o i ds*

Ref

Caf Caf Caf Caf Caf Caf Caf Caf,Tp Ca f Caf Caf ,Tb .Tp Caf

5 9 17 21 22 23 45 60 73 114 115 143

Ref i n Chapter 7 2 4 15 18( F i g . 7.2) 21 ZZ(Fiq.7.16, 30' 38 44 55 56 73

A b b r e v i a t i o n s used i n Tables 11.5

-

Caf Ca f Caf Caf Caf Table 7.8) Caf Caf Caf Caf ,Tp Caf Caf ,Tp

11.9

Caffeine Theobromine Theophyl 1 i n e 1-Methylxanthine 3-Methylxanthine 7-Methylxanthine Hypoxanthine Paraxanthine(l,7-dimethylxanthine) Xanthine Dyphyl 1 i n e

Caf Tb TP lMeX 3MeX 7MeX hypox paraX X dY P prox OHEtTp 8C1 Tp UA lMeUA 3MeUA 7MeUA 1,3diMeUA 1,7diMeUA 3,7di MeUA 1,3,7tri MeUA

Uric acid 1-Methyluric a c i d 3-Methyluric a c i d 7-Methyluric a c i d 1,3-Dimethyl u r i c a c i d lI7-Dimethy1 u r i c a c i d 3,7- O i me t h y 1 u r i c ac id 1,3.7-Trimethyl u r i c a c i d

A Acsal hPY Antp Benzac EPh Par Ph Phbarb Sal Salam Scop

Atropine Acetyl s a l i c y l i c a c i d Aminopyrine Antipyrine Benzoic a c i d Ephedrine Paracetamol(acetaminophen) Phenacetin Phenobarbital Salicylic acid Sa 1icy 1 ami de Scopolamine

References p. 395

Alkaloids

Proxyphylline(6-hydroxypropyltheophylline) 8-Hydroxyethyltheophylline 8-Chlorotheophyl l ine

Ref

Ref i n Chapter 7

149

79 82 9 l ( T a b l e 7.11) 93 97 106 108 113 118 120 l Z l ( T a b 1 e 7.6)

i5i

...

163 164 167 182 184 199 201 203 204

TABLE 11.6

a P 0

HPLC ANALYSIS OF VARIOUS COMPOUNDS INCLUDING XANTHINE DERIVATIVES ALKALOIDS *

OTHER COMPOUNDS

AIMS

Caf .Tb.Tp,strychnine, brucine

Separation by means o f dynamic c o a t i n g HPLC

Caf,various

D e t e c t i o n w i t h conductance d e t e c t o r

others

Caf,Tb,Tp,hypoX,X Caf, codeine,brucine, c o l c h i c i ne.aconi t i ne, n a r c e i ne ,cinchoni d i ne

U r a c i l s , b a r b i t u r a t e s R e t e n t i o n i n RP g r a d i e n t e l u t i o n LC Santoni ne E f f e c t s o l v e n t composition on r e t e n t i o n

Caf,Tp,various loids

alka-

COLUMN DIM. LxIDlnnn)

C o r a s i l I o r 11. coated w i t h 1.1% Poly 6-300 S i l i c a g e l 10 pm

1000x1

MOBILE PHASE

Heptane-EtOH(20:1),(10:1)

REF. sat.

w i t h s t a t i o n a r y phase C H C l -MeOH-hexane(7:3:lO) 3

L i c h r o s o r b Si100, 3 0 0 ~ 4 . 2MeOH-H20 i n v a r i o u s r a t i o s 10 m, C18 coated L i c h r o s o r b RP2 10 urn 1 2 0 ~ 3 . 5MeOH-H20(1:4).(2:3),(3:2),(4:1) MeOH

4.7 94

118

121

Caf,tropane a l k a l o i d s , codeine,papaverine,quinidine.ephedrine

Caf .Tp

STATIONARY PHASE

0.OOSM Heptanesulfonic a c i d i n H20-MeOH-AcOH(50:49:1)

R e t e n t i o n behaviour b a s i c drugs i n i o n - p a i r HPLC

pBondapak C18, VBondapak Phenyl, pBondapak CN, ~ B o n d a g e,l Chromegabond C8 o r Chromegabond C6H ll

300x4

Par.Acsa1 ,various hypnotics

T o x i c o l o g i c a l drug screen

PE/HS-5 C8

Various drugs

S e p a r a t i o n b a s i c drugs w i t h S p h e r i s o r b S5W s i l non-aqueous i o n i c s o l v e n t s i c a

1 2 5 ~ 4 . 6 G r a d i e n t o f 20-60% ACN i n 0.05 M phosphate b u f f e r ( p H 4.4) 194 2 5 0 ~ 4 . 9MeOH-hexane(85:15) c o n t a i n i n g 0.02% HC104 201

150

TABLE 11.7 HPLC ANALYSIS XANTHINE DERIVATIVES I N PHARMACEUTICAL PREPARATIONS ALKALOIDS*

OTHER COMPOUNDS

Caf

Acsal,Salam,Ph,Par

Analysis a n a l g e t i c t a b l e t s

Zipax anion-exchanger

1 0 0 0 ~ 2 . 10.005M NH4N03 i n pH 9.2 b u f f e r

Ca f

Acsal,Salam,Ph,Par

Analysis analgetic t a b l e t s

LFS p e l l i c u l a r anion-exchange r e s i n

3000x1

*For a b b r e v i a t i o n s see f o o t n o t e Table 11.5

AIMS

STATIONARY PHASE

COLUMN DIM.

MOBILE PHASE

REF

1 1.OM T r i s b u f f e r (pH 9.0)

2

r

I B c

P

Caf ,Tb,Tp,lMeX,3MeX, 7MeX,X,hypoX,UA

Acsal ,Ph

Caf .Tb,Tp .hypoX

Caf

Acsal ,Salam,Ph,Par, Separation anal gesi cs Sa1,aminophenols.phenol,acetanilides, ethylbenzoates,aminobenzoic acid methylester Acsal,Salam,nicoti- Separation analgesics nic acid,trigonel lin Analysis analgeti c tab1ets Ph .propyphenazone Acsal,Ph.hexobarbi- Analysis analgesics tal Acsal,Ph,Par,Benzac, Analysis analgesics butal bi ta1,p-chloroacetani lide Acsal ,Salam,Ph,Par Analysis analgesics

Ca f

Ph ,Ampy

0

Caf .Tb ,Tp ,hypoX,X ,UA Caf Caf ,homatropi ne,oxycodone Caf

Separation by ligand-exchange LC

Analysis analgesics

Chelex 100, 200500x10 400 mes?+loaded with Cu Q-150s-NH cation-exchange$ 1500x2 AG 1-X8-C1 1000x2 PVP resin

1M NH40H, 3M NH40H 3 25% EtOH

25% EtOH IsoprOH 6

Aminex 50W-X4

440x6

25% EtOH

10,15,38 11 Merckosorb Si60 20,m 200x2 CHCI3 1000~2.1 1.5M Na2S04,0.005M HN03 in H20 Zipax WAX 30 un 12 1220~2.30.01M Na-borate,O.OlM NH4N03 in Zipax SAX H O uBondapak C18 300x4 A8N-O.OlX aq. (NH4)2C03 13 Pore glass CPG-10240A,200-400 mesh 600~2.1 CHCl -AcOH(92:8) 600~2.1CHC13-AcOH(92:8) Corasil I 1 25 CHCl ;-CH2C1 2-AcOH(42: 50: 8) 1000~2.1 A. 0.15M Na2S04,0.05M NaOH in Zipax SCX H O B. ~%/min linear 29 gradient 100% A to A+B(91:9) 150~4.8(Isopr) 0-MeOH-50% aq. ethylSpherosil 5 bm ami ne (93:6.86:O. 14) EtOAc-MeOH-50% aq. ethylamine (97:2.94:0.06) 31 36 ODS Sil-X-I1 500~2.6 MeOH-H20( 85: 15), ( 1:4) Lichrosorb Si60 5 wn not 0.45% AgCl imp. given CHCl -n-hexane-MeOH( 6:4:0.5) Li chrosorb Si 100 lOpm CHCl3-DEA(99.99:0.01) 1.09% AgCl imp. A. C k l -n-hexane(1:l) B. CHCl 3-FleOH-DEA(90:10:0.5) 43 gradien? 16-92% B in A no de- MeOH-NH40H Porous styrenedi vi nyl benzene tails co-polymer ava i 1 able 52,58 Partisil 10 250~4.6 CH C1 -MeOH(1:3)with 1% 29% NH;OH' 61 $

MPOH

Caf .Tp,papaverine

Ampy,nicotinamide, Phbarb

Analysis pharmaceutical preparations

Antp,Ph,acetanilide Caf Caf ,Tb,Tp,X, A, scop.er- Butalbi tal gotamine,ergotaminine

Analysis analgesics Separation on silver impregnated si 1 ica

Caf,noscapine.dextromorphane

Various compounds

Caf.Tb,Tp,various alkaloids

Various drugs

Separation antitussives, expectorants and antihistamins Separation various drugs

Caf

Caf Caf,tropane alkaloids

and e r g o t

Acsal,Sal,Salam,Ph, Par

Analysis analgesics

Amber1 i t e XAO-7

3 0 0 ~ 2 . 8 E t 0-hexane i n v a r i o u s r a t i o s CH8l -hexane i n v a r i o u s r a t i o s EtOH3hexane i n v a r i o u s r a t i o s 70

Salam,Ph.fluoren

Analysis analgesics

S i l i c a g e l Si60

240x10

Hexane-dioxane-HCOOH(45:40:2)

Phbarb,butalbital, barbital ,pizotifene

S e p a r a t i o n by i o n - p a i r chromatography

Lichrosorb Silo0 loaded w i t h 0.06M p i c r i c acid(pH 6 )

150x3

C H C l s a t . w i t h 0.06M p i c r i c acid3(pH 6)

Caf,X

Acsal ,Sal ,Salam.Par, , Separation a n a l g e s i c s Ph,p-ami nobenzoi c-, w i t h cation-exchange n i c z t i n i c - , c i nnamic-. r e s i n s acid, t r i g o n e l l i n

Caf ,Tb,Tp,8ClTp,aminop h y l l ine,A,Eph

Phenazone

Caf

S e p a r a t i o n muscle-relaxant- pBondapak CN Acsal,Par,Ph,meprobarnt e ,methoca rbamol ,-analgesic m i x t u r e s carisoprodol ,chlorzoxazone

Caf ,noscapine

Acsal,Sal.Par,ethoxybenzamide,various neuroleptics

Analysis analgesics

85

aa

Aminex 50W-X4-NHd o r 2 0 0 ~ 6 . 3 25% E t O H i n 0.1M b u f f e r o f 250x4 v a r i o u s pH TSK-LS110 90 H y p e r s i l ODS 5 pm

100x5

MeOH-aq. KH2P04 b u f f e r ( p H 6.0) (1:4) 92

p P o r a s i l 10

300x4

eluedt;-l?'2-ahd

S e p a r a t i o n on porous p o l y mer r e s i n s

DVB-MCL-0 and H i t a c h i g e l 3011. 3011-0 and 3030

500x5

MeOH-NH OH(99:l) ACN-NH4eH(99: 1)

3

Caf, tropane and o p i um alkaloids,quinine,emetine.cephaeline,Eph, strychnine

I d e n t i f i c a t i o n o f pharmac e u t i c a l s ( F i g . 7.14)

P a r t i s i l PXS 5/25

2 5 0 ~ 4 . 6 E t 0 s a t . w i t h 50-100% H20 t 0.65%-0.8% DEA

Caf .opi um a1 k a l o i d s

A n a l y s i s opium a l k a l o i d s

P a r t i s i l 7 wn

2 5 0 ~ 4 . 5MeOH-2M NH40H-1M NH4N03 (30 :2: 1)

93

117

119 124

Caf

Ampy,Phbarb ,ni c o t i n - S e p a r a t i o n ami de

S p h e r o s i l XOA 600

Caf

Acsal .Sal .Ph,Par

Analysis analgesics

P a r t i s i l PXS 10/25 OOS2

250~4.6

Caf ,codei ne

Acsal,Salam,Ph,Par

Analysis analgesics

uBondapak C18

300x4

0.01M KH PO i n H 0 w i t h 19% MeOH pH #.8f o r a g j u s t e d 2.3 153

Caf, Tp

S-carboxymethyl-L-cysteine

A n a l y s i s i n t a b l e t s and supposirories

Micropak M-CH 10

300x4

MeOH-H 0(3:7) c o n t a i n i n g 0.005 M t e t r % b u t y l a n o n i u m ( p H 7.5) 162

50x4

I s o o c t a n e - ( i s o p r ) 0-MeOH-H20 (35: 50: 15: 0.2: 0.78)

130

ACN-H20-ACN(25: 70: 5) 145

6 0)

i? T

TP ,EPh

Phbarb

Analysis i n tablets

P a r t i s i l ODS I 1 I T P

250x4

MeOH-0.007M KHZP04(pH 2.3) (37:63)

172

8 P

TP ,EPh

Phbarb

Analysis i n tablets

uBondapak C18

300x4

ACN-O.01M phosphate b u f f e r (pH 7.8)(24:76)

173

Caf

Na-benzoate

Analysis i n pharmaceuticals N u c l e o s i l C18

TP s EPh

Hydroxyzine

Analysis i n t a b l e t s and syrups Analysis i n tablets

Caf

Saccharin.benzoic acid

O p t i m i z a t i o n s e p a r a t i o n by non-linear programing

1 0

I

Tp.dioxyfyl1 i n e

n o t a v a i - MeOH-phosphate b u f f e r

L i c h r o s o r b RP18 l O u m

lable

ACN-H20(3:7)

uBondapak C18

3 0 0 ~ 4 . 1 ACN-0.1% aq. (NH4)C03(pH 7.0) (1:l)

L i c h r o s o r b RP8 1 0 m

2 5 0 ~ 4 . 6 ACN-MeOH-H20 i n various r a t i o s

189 190

200 202

TABLE 11.8 HPLC ANALYSIS OF XANTHINE DERIVATIVES I N BIOLOGICAL FLUIDS ALKALOIDS*

OTHER COMPOUNDS

Caf,Tb,Tp,lMeX,7MeX

Oxazepam

AIMS

STATIONARY PHASE

COLUMN DIM. LxID(mn)

MOBILE PHASE

Assay o f Tp i n b i o l o g i c a l fluids

Durapak OPN

Caf .Tb,Tp,lMeX,3MeX, 7MeX, hypoX.paraX ,XI UA, lMeUA,3MeUA,1,3diMeUA

Determination Tp and i t s m e t a b o l i t e s i n u r i n e and serum(Tab1e 11.1)

Aminex A-5 c a t i o n -exchanger

6 6 5 ~ 1 . 8 0.45M NH4H2P04(pH 3.65)

Caf ,Tb.Tp ,3MeX ,hypoX,X, OHEtTp.Dyp.BClTp.Tp-7- a c e t i c acid,UA. 1MeUA. 1.3diMeUA

Assay o f Tp i n serum

Aminex A-5 c a t i o n -exchanger

8 5 0 ~ 1 . 6 0.45M NH4H2P04(pH 3.65)

Caf .Tb, Tp, 3MeX. 1,3di MeUA

Assay o f Tp i n plasma

Micropak S i l o 1Oum

500x3

Determination i n u r i n e

BOP(no f u r t h e r d e t a i l s )

Caf .Tp

2 0 0 0 ~ 4 . 2 Hexane-isoprOH g r a d i e n t (86:14) t o (78:22)

REF.

14 16

18 Morphine.codeine, cocaine

19

Heptane-prOH(9:l)

20

Caf ,Tb,Tp, lMeX.3MeX.X, Phbarb hypoX, parax. dyp ,prox. 8BrTp,8ClTp,8NO Tp ,8ClX, UA, lMeUA, 1.3didUA

Assay o f fluids

p i n biological

Zipax SCX

Caf .Tp, lMeX ,3MeX, 7MeX, hypoX. X. UA. lMeUA

Assay o f

p i n plasma

PBondapak C18

*For a b b r e v i a t i o n s see f o o t n o t e Table 11.5

CHC13-isoprOH-AcOH(84:15:1)

1 0 0 0 ~ 2 . 10.66% aq. AcOH

26 300x4

ACN-O.01M NaOAc(pH 4.0)(1:9) 27

P 0 4

W 0

Tp,OHEtTp Caf ,Tp

Phbarb

Caf ,Tb,Tp,BClTp,dyp, prox

Assay o f Tp i n serum Assay o f Tp i n plasma

pBondapak C18 P a r t i s i l 10

300x4 450x2

ACN-O.01M NaOAc(pH 4.0)(7:93) CHZCl2-MeOH-28% NH40H(92:7:1)

Assay o f Tp i n plasma

Zorbax S i l 6-8

UII

2 5 0 ~ 2 . 1 H 0 s a t . C H C l -heptane-EtOHA2OH (600:400 8:64)

33

13

urn

2 5 0 ~ 2 . 6 ACN-H20-1% aq. AcOH(1:48:1) (PH 4.5)

35

:a.

Caf,Tb,Tp,3MeX,hypoX,X, OHEtTp,EClTp, 1,3diMeUA

Assay Tp i n serum

ODS S i l - X - I

Caf,Tb,Tp,OHEtTp,8ClTp

Assay Tp i n b l o o d and saliva

~8ondapak C18

300x4

ACN-O.01M NaOAc(pH 4)(7:93), (4:96)

Caf ,Tb ,Tp ,prox, dyp

Phbarb

Assay Tp and dyp i n serum

P a r t i s i l 10

2 5 0 ~ 4 . 6 C H C l -n-heptane-MeOH(39:56: f o l l a w z d by (37:54:5)

Caf ,Tb.Tp ,3MeX,X

Purine nucleotides

Analysis i n b i o l o g i c a l extracts

uBondapak C18

300x4

MeOH-0.05M NH4H2P04(pH 6.0) (1:3)

Caf ,Tb,Tp,3MeX,lMeUA, 1.3diMeUA

Assay Tp i n plasma

uPorasi 1

300x4

n-Hexane-sec-BuOH-MeOH-H20

Caf,Tb,Tp

Assay Tp i n plasma and paliva

P a r t i s i l SCX

2 5 0 ~ 4 . 6 0.66% aq. AcOH

Caf ,Tb,Tp .paraX

I d e n t i f i c a t i o n caf metab o l i t e s i n plasma(Tab1e 11.4)

L i c h r o s o r b Si60 5 urn 250x3

TP

Predni s o l one

28 30

39,109 5)

T69: 25: 5 : r

44 46 48 49

CHCl -isoprOH-AcOH(92:7:1) + up t o 48% n-hexane CH C 1 -70.29 NH formate and 15 ~1'80% HCOOH in4100 m l MeOH) (98:2) 51

L i c h r o s o r b Si60 5 um 2 5 0 ~ 3 . 2 CHCl -isoprOH-AcOH(94:5:1) Assay o f Tp i n plasrna,comCH C? -(0.2% NH formate and p a r i s o n w i t h GLC(Tab1e 11.4) 0.62%2HCOOH i n ieOH)(98:2)

53

Caf.Tb,Tp,1MeX,3MeXaX, hypoX,prox.UA.lMeUA. 3MeUA,1,3diMeUA

Assay Tp i n serum (Table 11.3)

PBondapak C18

, TP TP,~ Y P 8C1 Caf,Tb,Tp,3MeX,OHEtTp, 8ClTp,lMeUA,1,3diMeUA

Assay Tp i n plasma

P a r t i s i l 10

Assay Tp i n plasma, serum and s a l i v a ( F i g . l l . 1 )

$ondapak

C18

300x4

ACN-O.01M NaOAc(pH 4.0)(7:93) o r (5:95)

56

Tb.Tp.dyp.prox

Assay dyp i n serum

PBondapak C18

300x4

ACN-O.01M NaOAc(pH 4.0)(7:93)

57

Caf .Tb, Tp,8C1 Tp,dyp

Assay Tp i n whole b l o o d and Spherisorb OOS 10 ,m 2 5 0 ~ 2 . 6 MeOH-O.01M NaOAc(pH 4)(1:3) serum Determination i n plasma and Micropak S i 10 um 500x2 A. Hexane-isoprOH(10:l) B. Hexane-isoprOH-conc.NH40H urine (5:50:4:1), 67% B i n A

HypoX.X.al1 u p u r i no1 , o x i p u r i no1

Phbarb

300x4

ACN-O.02M NaOAc(pH 4.0)(1:9) 54

ODS

2 5 0 ~ 4 . 6 ACN-O.01M KH2P04(9:1)

55

59

62

?

3 0 0 ~ 3 . 9MeOH-0.05M KH2P04(pH 4.7) 112:88)

Tb,Tp ,1MeX,3MeX .hypoX, X, dyp,UA,lMeUA,1,3diMeUA

Analysis Tp and m e t a b o l i t e s u8ondapak C18 i n serum. s a l i v a and u r i n e ( F i g . 1l.j)

Caf ,Tb.Tp .8C1 Tp

Assay Tp i n serum

UBondapak C18

300x4

MeOH-1% p r o p i o n i c a c i d i n H 0 (pH 5.0 w i t h NaOH)(1:4)

64

Caf ,Tb ,Tp

Assay Tp i n serum

UBondapak C18

300x4

MeOH-O.01M NaH,PO,(1:4)

65

Assay c a f m e t a b o l i t e s i n plasma (Table 11.4)

L i c h r o s o r b Si60 5

250x3

CH C1 -(0.029 NH formate and 0 . 6 1 7 2 ~ 1 97% HCOdH i n 100 m l MeOH) (978: 22)

67

Caf,Tb,Tp,SMeX,prox, dyp ,8C1Tp

Assay xanthines i n serum ( F i g .11.4)

UBondapak C18

3 0 0 ~ 3 . 9 MeOH-0.02M KCl(pH 2 w i t h HC1) (3:7)

Tp,prox

Comparison method r e f . 26 w i t h EMIT

Zipax SCX

Caf ,Tp

Assay Tp i n b i o l o g i c a l fluids

Spherisorb OOS 5

Caf .Tb .Tp ,OHEtTp

Comparison Tp assays (HPLC and EMIT)

H y p e r s i l ODs 5 iim

100x5

Tp ,8ClTp

Assay Tp i n s a l i v a

ODs- I

2 5 0 ~ 2 . 6 MeOH-0.2% H3P04(pH 4 w i t h NaOH) (1:4) 74

Assay o f Tp i n serum

Partisil 5

Assay o f Tp i n plasma

OD5 HC S i 1-X-1 pBondapak C18

63

W 0

Prednisolone

Caf, Tb. Tp, IMeX. 3MeX, 7MeX.paraX

Caf,Tb,Tp,lMeX,3MeX,X,

UA, lMeUA, 3MeUA, 1,3di MeUA

-

Furosemide.phbarb, s a l ,s u l f a t h i azol e , c h l o r o t h i azide

Caf ,Tb,Tp ,3MeX, dyp , 8ClTp.lMeUA.l,3diMeUA

68

1 0 0 0 ~ 2 . 10.66% aq. AcOH 69 pm

1 5 0 ~ 4 . 6 MeOH-H20(2:3) 71 ACN-O.01M NaOAc(pH 4.0)(7:93) 72

vim

2 5 0 ~ 2 . 1Hexane-isoprOH-H20(80:19:1) 75 n o t g i v e n ACN-H 0(1:9) n o t g i v e n ACN-H:0(6:94)

76

I n t e r f e r e n c e o f Tp a n a l y s i s P a r t i s i l 10 OOS

2 5 0 ~ 4 . 6 ACN-O.lM

Caf .Tb,Tp ,OHEtTp

Assay o f Tp i n serum

,,Bondapak C18

300x4

ACN-0.026M NaOAc(pH 4.0)(1:9)

Caf.Tb.Tp.lMeX.3MeX. Creatinine 7MeX .paraX,X .dyp ,8C1 Tp , UA, lMeUA.3MeUA.7MeUA. 1,3diMeUA

Amperometric d e t e c t i o n o f

JIondapak C18

300x4

ACN-(O.1M Na2HP04, 0.1M NaH2P04) (2:38) ACN-O.1M NaH2P04(21:400)

I n t e r f e r e n c e o f Tp a n a l y s i s pBondapak C18

300x4

ACN-O.01M NaOAc(pH 4.0) (7:93)

TP

Cephalosporin a n t i biotics

Tp ,OHEtTp

Cephal o s p o r i ns

Tp, OHEtTp

Acetazol amide

NaOAc(pH 4.0)(1:9) 77

methylxanthines(Fig.ll.2)

I n t e r f e r e n c e o f Tp a n a l y s i s vBondapak C18

300x4

ACN-O.OlM

TP

Assay o f Tp i n serum

,,Bondapak

300x4

ACN-O.01M NaOAc(pH 4.0)(8:92)

Tb 9 TP ,hYpoX.dyp s8C1TP

Assay o f Tp i n serum

P a r t i s i l 10 ODS

C18

NaOAc(pH 4.0)(7:93)

2 5 0 ~ 4 . 6 MeOH-0.025M KH2P04(pH 2.5) (35:65)

79

80 81 82 84 91

4

Assay o f Tp in serum Assay o f Tp in serum and saliva Comparison Tp assays (HPLC and EMIT) Comparison Tp assays (HPLC and GLC)

uBondapak C18 300x4 ACN-O.01M NaOAc(pH 4.0)(1:9) 95 Lichrosorb Si60 7 vm 1 0 0 ~ 2 . 8 CH C1 -(0.02% NH formate. 0.017 % k O 6 H in MeOH)?99:1) 96 Aminex A5 cation8 5 0 ~ 1 . 6 0.45M NH4H2P04(pH 3.65) -exchange resin 97 100x5 ACN-O.OZM NaOAc(pH 4.0)(8:92) Hypersil ODs 5 wn

Assay of Tp in plasma

uBondapak C18

Comparison Tp assays (HPLC and EMIT) Interference o f Tp analysis Interference of dyp analysis Assay of Tp in plasma

Micropak MCH-10

Caf ,Tb,Tp, lMeX.3MeX. 7MeX,OHEtTp,lMeUA, 1.3diMeUA

Assay of Tp in serum

uBondapak C18

Caf ,Tb ,Tp,3MeX,dyp3 IMeUA.1.3diMeUA Caf ,Tb, Tp .parax, OHEtTp

Assay of Tp in plasma

PSX 10/25 ODS

Interference Tp assay by caf metabolite (parax) Caf metabolism in newborns

PBondapak C18

Tp,paraX.OHEtTp Caf, Tb, Tp ,paraX

Chloramphenicol

TP Caf .Tb,Tp ,lMeX,3MeX, 7MeX, hypoX,paraX. X, OHEtTp, 1MeUA93MeUA, 1.3MeUA Caf ,Tp ,8C1 Tp

Par.heptabarbi tal phbarb

98

TD Tp ,OHE tTp Tp.dyp ,OHEtTp Caf.Tb,Tp,dyp.BClTp

Caf ,Tb,Tp, 1MeX. 3MeX, 7MeX,paraX,lMeUA,3MeUA. 7MeUA, 1,3di MeUA, 1.7di MeUA,3,7di MeUA, 1,3.7tri MeUA

Acetazolamide Sulfadi azi ne

uBondapak C18 uBondapak C18 uBondapak C18

uBondapak C18 (RP-10 in ref

300x4

MeOH-0.05M NH4H2P04(pH 5.2) (74: 26) not given CHC13-isoprOH-AcOH(4:5:1)

99

100 ACN-0.003M NaOAc(pH 4.5)(8:92)101 ACN-O.OlM NaOAc(pH 4.0)(7:93) 102 ACN-1M KH2P04(pH 4.0)-H20(50: 5:895) 103 300x4 MeOH-O.02M NaDAc(pH 3.5) (15:851 104 not given 95% EtOH-HzD(1:4) 105 300x4 MeOH-THF-D.01M NaOAc(pH 5.0) (95:4: 1) 106 300x4 ACN-0.5% AcOH, concave gradient from (15:985) to (75:925) 133) 300x4 300x4 300x4

107,133

Tp, 1MeX93MeX,8C1Tp, lMeUA, 1,JdiMeUA Caf ,Tb ,Tp .dyp, prox

Tp metabolism

Caf .Tb,Tp

Comparison Tp assays in serum,saliva and spinal fluids with HPLC or EMIT

Caf .Tb ,Tp ,OHE tTp ,8C1Tp

Assay o f Tp in serum,plasma and saliva

Assay o f Tp in serum

C-18 stationary 150x4 ACN-O.01M NaOAc(pH 4.0) phase(no details) 5”m (12:88) Partisil 10 ODs 2 5 0 ~ 4 . 6 MeOH-0.049M phosphate buffer (pH 2.3)(2:3) Lichrosorb Si60 5 urn 250~3.2 CHCl3-isopr0H-AcOH(96:2:2)

108 109

110 UBondapak C18

300x4

ACN-O.01M NaOAc(pH 4.0)(7:93) or (4:96)

111

Caf,Tb,Tp,3MeX,paraX, OHEtTp ,prox,BClTp, lMeUA,1.3diMeUA Caf,l-propylTp Caf ,Tb,Tp,BClTp Caf ,Tp,hypoX Caf,Tb,Tp,lMeX,paraX, OHEtTp,dyp,lMeUA, 1,3diMeUA Oyp ,OHEtTp Caf ,Tb.Tp,dyp,OHEtTp

Caf ,Tb,Tp,lMeX,3MeX, dyp ,8C1 Tp,prox,UA Caf ,Tb,Tp ,lMeX,3MeX,X, hypox,EClTp,UA,lMeUA, 3MeUA Caf,Tb.Tp,OHEtTp Caf ,Tb,Tp ,8C1Tp

Ampicillin

Assay of Tp in serum, com- Hypersil OOS 5 ym 100x5 ACN-O.OZM NaOAc(pH 4.0) parison with GLC (Table (8:92) 11.3) Caf determination in plasma 00s-Sil-X-1 10 pn 250~2.6ACN-H20(3:I) or tissues Electrochemical detection Lichrosorb C8 10 w 250~3.2EtOH-NaOAc buffer(pH 4.0) TP (8:92) Analysis on cation-exchange Aminex 50W-X4 150~4.6 0.05M Na2HP04(pH 7.5) resins Partisil PXS 5/25OOS 250x5 MeOH-THF-O.01M NaOAc(pH 5.0) Assay of Tp (92:7: 1) MeOH-THF-O.OlM NaOAc(pH 5.0) RCM with Radial Pak A (C18) (928:60:12) Urinary excretion of dyp UBondapak C18 300~3.9ACN-O.01M NaOAc(65:935) Assay of dyp in plasma, UBondapak C18 300x4 ACN-H20(9:91) urine and saliva 100~4.6MeOH-H20(1:4). MPLC RP18 Assay of Tp in plasma, urine and saliva Comparison Tp assays(HPLC Micropak SI 5 5 rm 250x2 CHCl -heptane-AcOH-EtOH and EMIT) (300?200:0.4: 32) not given IsoprOH-O.1M KH2P04(pH 3.8) Assay of Tp in serum and Micropak MCH-10 (4:96) possible interferences (Fig.11.5) 100x5 ACN-NaOAc buffer(pH 4.0) Assay of Tp in body fluids Hypersil OOS 5 wn (8:92) Simultaneous assay caf, Tb S5 OOS 150~4.6ACN-O.01M NaOAc(pH 4)(12:88) and Tp in plasma, comparison with EMIT Assay of caf in plasma Spherisorb ODs 10 un 250~4.6ACN-O.01M NaOAc(pH 4.0)(15:85), (Table 11.3) (18:82)

Caf,Tb,Tp,lMeX,3MeX, 7MeX. hypoX .paraX,X,dyp, prox,UA,lMeUA,7MeUA, 1,3diMeUA Sulfamethoxazole Interference Tp assay TP Sulfamethoxazole,am- Interference Tp assay Caf .Tb ,Tp,8C1Tp pici 1 lin,par.Acsal , sal ,various antibiotics Caf,Tb,Tp,lMeX,paraX, Carbamazepine Assay of caf in serum lMeUA,l,3diMeUA

Lichrosorb RP8 Li chrosorb RP8

250~4.6ACN-0.005M AcOH(1:3) 250~4.6MeOH-0.02M NaOAc(pH 5.5)(1:4)

Partisil 5 5 un

100~4.6THF-CH2C12(1:4)

112 113 116 120

122 123 125 126 127 128 129 136

138 139 140,191 144

E

Ip

I-

N

Caf,Tp,lMeX,3MeX,paraX, OHEtTp,lMeUA,1,3diMeUA

Assay of Tp and m e t a b o l i t e s U l t r a s p h e r e OOS 5 m i n urine

Caf.Tb,Tp,dyp,OHEtTp

Assay Tp and dyp i n plasma Separation and a n a l y s i s i n plasma

Caf ,Tb,Tp,bami f y l 1 ine, dyp ,prox. 1omi f y l 1ine, p e n t i f y l l ine,pentoxifylline

p8ondapak C18 Spherisorb Spherisorb Spherisorb Zorbax S i l

300x4 ACN-O.01M NaOAc(6:94) ACN-O.01M phosphate b u f f e r C6 5 p, CN 5 pm o r (pH 2.7) (1:4), (28: 72) OOS 5 pm 2 0 0 ~ 4 . 6 7 fl 2 0 0 ~ 4 . 6 Hexane-CHC13-isoprOH-AcOH (50:43:5:2)

Tp.30Hpropyl Tp

Comparison Tp-assays (HPLC and R I A )

RP-8

Caf,Tb,Tp,3MeX,paraX, 1MeUA,3MeUA

Assay Tp i n serum and saliva

Spherisorb ODS 10

Caf, Tb,Tp, lMeX .3MeX. X, Par,ethosuximi de hypoX,OHEtTp,8ClTp,UA, lMeUA.3MeUA

Simultaneous a n a l y s i s i n plasma and comparison w i t h EMIT

pBondapak C18

Caf,Tb,Tp,lMeX,3MeX, Benzoic a c i d 7MeX,hypoX ,X,paraX.prox,

I n t e r f e r e n c e benzoic a c i d w i t h method ref.138 (Table 11.3)

Ultrasphere

UA,lMeUA,7MeUA,1,3diMeUA

147

152

125~4.6 ACN-0.04M NaOAc(pH 4.0)(5:95) 155 pm

250x3

Heptane-CHC1 -EtOH-H20-AcOH (400:600: 32:?. 5:O. 8 )

156

3 0 0 ~ 3 . 9 ACN-H 0-TrEA o r N-ethylmorpho1ine -?AcOH( 1:11:O .008: 0.006) 157

OOS 5 m 1 5 0 ~ 4 . 6ACN-O.01M a c e t a t e b u f f e r (pH 6.5) (9:91) 158

A r t e f a c t s w i t h acetoni t r i l e Radial-Pak C18 formed by d e p r o t e i n a t i o n o f serum

Tp,OHEtTp

2 5 0 ~ 4 . 6 A. 0.01M NaOAc, 0.005M t e t r a butylamnonium s u l f a t e i n H20 B. idem i n 50% MeOH g r a d i e n t from 4.5-23% MeOH 146

100x8

ACN-O.01M NaOAc(l:9) 159

Caf,Tb,Tp,3MeX,8ClTp

Par,salicylate,mephensine,acetophenetidine

Assay o f Tp i n serum

Spherisorb ODS 10pm 250x3

ACN-MeOH-H O - l O m l / l ( 16 :180 :788 :16 )

Caf, Tb,Tp

Ampici 11i n

Assay of Tp i n serum and s a l iva

pBondapak C18

ACN-H20(8:92)

aq.AcOH 160

300x4

161

Caf,Tb,Tp,paraX,3-isob u t y l -lMeX

Assay of Tp i n plasma, corn- RSi1, s i l i c a g e l 5 pm parison w i t h R I A

1 0 0 ~ 2 . 8CHCl3-dioxane-HCOOH(995:45:0.l)

Caf ,Tb ,Tp .paraX ,OHE tTp

Determination c a f and i t s m e t a b o l i t e s i n dogs plasma

VBondapak C18

3 0 0 ~ 3 . 9 ACN-MeOH-THF-0.005M NaOAc b u f f e r ( p H 5)(28:30:17:925) 167

Caf .Tb ,Tp, lMeX, 3MeX, OHEtTp,UA,1,3diMeUA

Assay of Tp i n plasma

UPorasil

3 0 0 ~ 3 . 9 Hexane-EtOH(76:24)

TP TP

Assay o f Tp i n plasma

No d e t a i l s a v a i l a b l e

I n t e r f e r e n c e i n Tp assay

uBondapak C18

165

168 Procainamide

174 3 0 0 ~ 3 . 9 ACN-O.OIM NaOAc b u f f e r ( p H 4.0) (7:93) 176

Tp.paraX

Interference paraX in Tp assay

Lichrosorb RP18 7,m

300x4

MeOH-FMA-0.05M KHZPO4(22:11.5: 66.6)(pH 5.8) 177 Caf ,Tp .8C1 Tp Analysis Tp in biological ODs- type no details ACN-D.1M phosphate buffer(pH fluids available 5.3)(1:9) 178 Direct injection of plasma TSK LS-410. 20-32 60x4 ACN-phosphate buffer(pH 7.4) TP samples on column m, treated with human plasma 181 Caf,Tb,Tp,3MeX,paraX. Par.acetazo1amide. Fast analysis Tp i n serum C18 type 5 um 125~4.6ACN-O.OZM phosphate buffer OHEtTp ,dyp .8C1 Tp, procainamide,various (pH 3.6)(95:905) others 183 Caf,Tb,Tp,3MeX,OHEtTp, Par.pr0cainamide.NSimultaneous assay of some VBondapak C18 300~3.9 ACN-O.1M phosphate buffer -Ac-pr0cainamide.N- drugs i n serum 8ClTp.dyp (pH 4.0)(97.5:902.5) -propionylprocainamide,Sal ,Acsal 187 Caf ,Tb,Tp. lMeX ,3MeX, Assay for simultaneous Ultrasphere ODS 5um 250~4.6 A. 0.01M NaOAc, 0.005M tetra7MeX,paraX,X,OHEtTp. quantitation xanthines and butylamnonium in H O(pH 4.9) lMeUA.3MeUA.7MeUA.1.3uric acids in urine B. as A in 50% M e O H d H 4.8) diMeUA.3.7diMeUA.1.7dinon-1 inear gradient'elution MeUA,1,3,7triMeUA 192 Automated HPLC-analysis in Fast LC-8 5 urn Caf,Tp Par 150~4.6 MeOH-0.0025M NaH PO (14:86) serum with 0.065% TrEAfpH46.6) 193 Caf .Tb,Tp,3MeX ,OHEtTp Analysis caf i n biological ODS 5 pm 250~4.6 ACN-H3P04-H20(260:1:1739) samples and coffee 195 Analysis Tp in plasma and Ultrasphere ODS 5um 150~4.6 MeOH-O.01M NaDAc, 0.005M teCaf, Tb,Tp, lMeX ,3MeX, paraX.OHEtTp,UA, lMeUA, saliva in the presence of trabutylamnonium(pH 4.75) 1.3diMeUA caffeine and its metabolites (1:7) 196 HypoX.X,UA Specific detection withxan- Nucleosil 5 C18 200x4 ACN-O.01M phosphate buffer thine oxidase reactor (pH 5.5)(1:99) 197 9MeX,hypoX,X,UA,adenine, Hypersil ODS 3 pm Analysis in biological 150~4.6 0.02M KH2P04(pH 3.65) guani ne,al lopurinol fluids 198 Caf ,Tp,OHEtTp Comparison HPLC columns @ondapak C18 3 0 0 ~ 4 . 0ACN-H20-AcOH(5:95:0.2) Hi-Chrom Reversible 250~4.6 00s-C18 5 pnl Partisil PXS 5/25 DDS 250~4.6 Zorbax ODS 5 pm 250~4.6 208

P

0 c

TABLE 11.9 HPLC ANALYSIS XANTHINE DERIVATIVES I N FOOD AND BEVERAGES

*

AIMS

STATIONARY PHASE

Caf.Tb .Tp. lMeX,3MeX,7MeX, X, hypoX ,UA

Determination c a f i n beverages

Chelex 100, 200-400 500x10 mesh, loaded w i t h Cu++

Caf.Tb,Tp,X

Determination food a d d i t i v e s

Zipax SAX

Caf .Tb.Tp.hypoX.X,UA. tri gone1 1in,ni c o t i n i c a c i d

Analysis coffee

Aminex 50W-X4,

Caf ,Tb ,Tp

A n a l y s i s o f food and beverages (Fig.11.9)

Caf

Caf,Tb,Tp.hypoX,trigonel-

ALKALOIDS

REF.

COLUMN D I M . MOBILE PHASE LxID(mn) 1M NH OH 3M NH$i

3

1 0 0 0 ~ 2 . 1 0.01M Na-borate buffet-(pH 9.2)

8

440x6

NH4-formate b u f f e r ( p H 3.65)

L i c h r o s o r b Si60 5 urn o r N u c l e o s i l 50 5 pin

300x3 300x3

CH Cl2-EtOH-H2O(936:47:17) phgse

A n a l y s i s i n food

ODS S i l - X - 1

2 5 0 ~ 2 . 6 ACN-H20(2:98)

Analysis i n coffee

Zipax SCX

Caf

Determination c a f , s a c c h a r i n and sodi urn benzoate i n beverages

VBondapak C18

300x4

5% aq. AcOH

Caf

A n a l y s i s c o f f e e and t e a

Zipax SCX

300x4

0.01M HN03(pH 2)

Caf, Tb ,Tp

Analysis tea constituents

pBondapak C18

300x4

MeOH-O.1M c i t r a t e - p h o s p h a t e b u f f e r (pH 7.0)(1:4)

Caf,Tb

A n a l y s i s cocoa and chocolate

pBondapak C18

300x4

MeOH-H20-AcOH(20:79: 1 )

Caf ,Tb ,Tp ,XI adeni ne

Analysis tea

Dowex AG-50W-XE (H') c a t i on-exchanger

600x9

25% EtOH

Caf.Tb

A n a l y s i s i n cocoa beans

pBondapak C18

300x4

ACN-H20(15:85) c o n t a i n i n g l%(NH4)2HP04 90

Caf .Tb.Tp.EClTp

A n a l y s i s i n pharmaceutical raw products (tea, c o l a nuts, e t c . ) Ana 1y s is i n cocoa

pBondapak C18

3 0 0 ~ 4 . 6 MeOH-H20( 2:3)

NH4+

i n 25% EtOH 10.15.38 organic 32

1 0 0 0 ~ 2 . 1 aq. HN03(pH 1.56) 40

1i n . n i c o t i n i c a c i d

Caf.Tp

24.37

41 42 47 86,141.148 87

131 134

no d e t a i l s a v a i l a b l e 2 5 0 ~ 4 . 6 MeOH-4% aq. AcOH(1:4)

142

Tb

A n a l y s i s i n animal f e e d i n g s t u f f s P a r t i s i l PXS 10/25

Caf ,Tb ,Tp ,isobuty 1X

I n f l u e n c e brewing method on c a f content coffee

P a r t i s i l PXS 10/25 ODS 2 5 0 ~ 4 . 6 MeOH-H20(1:4)

Caf

Analysis decaffeinated coffee

pBondapak C18

400x6

MeOH-H20(7:3)

180

Caf .Tb ,Tp

Analysis animal d i e t s

Radial-Pak C18

100x8

MeOH-H20-AcOH(25:74: 1)

205

Caf .Tb .Tp

Analysis coffee.tea and c o l a n u t s L i c h r o p r e p Si60

240x10

CHCl3-EtOH-25%NH40H(9O:l0:O.25)

206

300x4

MeOH-H20-AcOH(19:80: 1 )

Analysis cocoa c o n t e n t i n m i l k Caf,Tb. Tp ,3MeX, X, dyp , powder products OHEtTp * F o r a b b r e v i a t i o n s see f o o t n o t e Table 11.5

175

pBondapak C18

207

415

Chapter 12 DITERPENE ALKALOIDS

...................................................................... ............................................................................

12.1. HPLC-systems

415

References..

415

12.1. HPLC SYSTEMS Only a few d a t a have been p u b l i s h e d on t h e HPLC o f a c o n i t i n e t y p e o f a l k a l o i d s ( T a b l e 12.1). Sheu e t a 1 . l a n a l y z e d a c o n i t i n e i n crude drugs on an o c t a d e c y l s t a t i o n a r y phase u s i n g methanol

- aqueous phosphate b u f f e r (pH 7.52) (85:15) as m o b i l e phase. The i n f l u e n c e o f t h e w a t e r

-

methanol r a t i o i n t h e m o b i l e phase on t h e r e t e n t i o n o f a l k a l o i d s , i n c l u d i n g a c o n i t i n e , has 3 been i n v e s t i g a t e d on a reversed-phase p a c k i n g

.

H i k i n o e t a1.4 developed two HPLC methods f o r t h e a n a l y s i s o f a c o n i t i n e and r e l a t e d a l k a l o i d s i n crude drugs. I n t h e f i r s t one t h e a l k a l o i d s were s e p a r a t e d on an o c t a d e c y l column u s i n g t e t r a h y d r o f u r a n - 0.05

M phosphate b u f f e r (11:89) as m o b i l e phase. M o b i l e phases con-

t a i n i n g methanol and a c e t o n i t r i l e gave b r o a d e r peaks and l e s s r e s o l u t i o n o f some o f t h e a l k a l o i d s . I n t h e pH range 2-5, l i t t l e v a r i a t i o n i n k ' was found f o r t h e a l k a l o i d s , b u t above pH 5 some a l k a l o i d s showed i n c r e a s e d k ' values. B e s t r e s u l t s were o b t a i n e d a t pH 2.7 ( F i g . 1 2 . 1 ) . A second method was developed t o m i n i m i z e t h e r i s k o f i n t e r f e r e n c e o f c o - e l u t i n g compounds f r o m t h e p l a n t m a t e r i a l . The same t y p e o f column as above was used i n t h e r e v e r s e d -phase i o n - p a i r mode, and, as p a i r i n g - i o n , 0.01 M h e x a n e s u l f o n a t e was added t o t h e m o b i l e phase ( t e t r a h y d r o f u r a n

-

0.05 M phosphate b u f f e r (pH 2 . 7 ) ( 1 5 : 8 5 ) ) .

A s t r a i g h t - p h a s e HPLC method was developed by Gimet and F i l l o u x ' .

The a l k a l o i d s ,

including

a c o n i t i n e , were s e p a r a t e d on s i l i c a g e l u s i n g a m o b i l e phase o f w a t e r s a t u r a t e d d i e t h y l e t h e r , t o which some d i e t h y l a m i n e was added (see Chapter 7, F i g . 7 . 1 4 ) . The d e t e c t i o n o f a c o n i t i n e - t y p e a l k a l o i d s has been performed a t 254 nm, a l t h o u g h t h e maximum UV a b s o r p t i o n i s a t 235 nm. REFERENCES

1 S.J. Sheu, C. Chen, Y.P. Chen and H.Y. Hsu, Chung-Kuo Nung Yeh Hua Hsueh Hui C h i c h . 17 (1979) 71. CA 9 1 (1979) 1 2 9 0 7 7 ~ . 2 R. Gimet and A. F i l l o u x , J. C h r o m a t o g r . , 177 (1979) 333. 3 E. Soczewinski and T. Dzido, J . L i g . Chromatogr., 2 (1979) 511. 4 H. H i k i n o , C. Konno, H. Watanabe and 0. Ishikawa, J. Chromatogr., 211 (1981) 123.

TABLE 12.1 HPLC ANALYSIS ACONITINE AND RELATED ALKALOIDS ALKALOIDS

OTHER COMPOUNDS

AIMS

STATIONARY PHASE

Aconi ti ne

Analysis crude drugs

Zorbax OOS

Aconi tine,opium-, Sulfanilamide tropane alkaloids, phenytoine, strychnine,quini- phenobarbital ne.caffeine.emetine.cephaeline Aconitine,colchi- Santonine cine,caffeine,narceine,codeine.brucine,cinchoni dine Aconitine.mesaconi tine,desoxyaconi tine.hypaconi tine,jesaconi tines and the correspondi nq benzovl aconi nes

Identification in pharmaceuticals(Fig.7.14)

Partisil PXS 5/25

Effect solvent composition on retention

Li chrosorb RP2 10 ,ni

Analysis crude drugs(Fig. 12.1)

TSK Gel LS 410 ODS

1i

REF.

250~4.6 MeOH-aq. phosphate buffer(pH 7.42) (85:15) 250~4.6 Et 0 sat. with 50-100% H20 + 0.05-0.8% OE8

T20

LO

SO

man

1

2 120~3.5 MeOH-H20( 1 :4),(2:3), (3:2), (4:1) MeOH 3

Sil 5

300x4

THF-0.05M phosphate buffer(pH 2.7) (11:89) 0.01M hexanesulfonate in THF-O.05M phosphate buffer(pH 2.7)(15:85)

4

10

10

30

COLUMN DIM. MOBILE PHASE LxIDlmnl

4 Fig. 12.1. Separation of some aconite alkaloids Column TSK Gel LS410 ODS Sil 5 um (300x4 mn ID), mobile phase tetrahydrofuran - 0.05 M aqueous phosphate buffer (pH 2.7) (11:89), flow rate 0.9 ml/min, detection UV 254 nm. Peaks: 1, benzoylmesaconine; 2, benzoylaconine; 3, benzoylhypaconine; 4, benzoyldesoxyaconine; 5 , benzoyljesaconine; 6, mesaconitine; 7, hypaconitine; 8, aconitine; 9, desoxyaconitine; 10, jesaconitine.

417

Chapter 13 COLCHICINE AND RELATED ALKALOIDS

..................................................................... ...........................................................................

13.1. HPLC systems

417

References..

417

13.1. HPLC SYSTEMS Forni and Massaranil described the analysis o f c o l c h i c i n e and colchicoside i n p l a n t mat e r i a l , They used a s i l a n i z e d s i l i c a gel as s t a t i o n a r y phase and a g r a d i e n t o f a c e t o n i t r i l e and water as eluent. Assay o f t h e a l k a l o i d s i n p l a n t m a t e r i a l has been performed on a microp a r t i c u l a t e o c t y l column w i t h methanol

-

water as mobile phase2. The best separation o f a

series o f c o l c h i c i n e d e r i v a t i v e s was obtained on an octadecyl column w i t h a c e t o n i t r i l e thanol

- phosphate b u f f e r as mobile

-

me-

phase ( F i ~ . 1 3 . 1 ) ~ ’ ~ ’ ~ .

Optimum pH f o r the separation was 6, and the a d d i t i o n o f methanol improved the peak shape The method had t o be modified f o r the analysis o f colchiceinamide and i t s demethylated metab o l i t e ~ ~By. using smaller p a r t i c l e s f o r the s t a t i o n a r y phase, the r e s o l u t i o n could be i m proved. Peak shape was improved by adding t r i e t h y l a m i n e hydrochloride t o the mobile phase. A c i d i f y i n g the mobile phase t o pH 2.2 r e s u l t e d i n r e s o l u t i o n o f c o l c h i c i n e and c o l c h i c e i n amide. J a r v i e e t a1.3 used a m i c r o p a r t i c u l a t e s i l i c a g e l column f o r the determination o f c o l c h i c i n e i n plasma. I o r i o e t a1.6 i s o l a t e d and i d e n t i f i e d some i m p u r i t i e s o f c o l c h i c i n e by means o f HPLC, TLC and MS. A l k a l o i d separation was performed on s i l i c a gel w i t h a g r a d i e n t system o f chloroform

-

methanol.

Detection o f c o l c h i c i n e and d e r i v a t i v e s i s best performed a t i t s UV absorption maxima 3 a t 350 nm2*5*7 o r 240 nm

.

REFERENCES G. Forni and G. Massarani, J . Chromatogr., 131 (1977) 444. P. P e t i t j e a n , L. van Kerckhoven, M. Pesez and P. B e l l e t , Ann. Pharm. F r . , 36 (1978) 555. D. Jarvie, J. Park and M.J. Stewart, C l i n . T o x i c o l . , 14 (1979) 375. E. Soczewinski and T. Dzido. J. L i q . Chromatogr., 2 (1979) 511. A.E. K l e i n and P.J. Davis, A n a l . Chem.. 52 (1980) 2432. M.A. I o r i o . A. Mazzao-Farina. G. Cavina. L. B o n i f o r t i and A. Brossi. H e t e r o c u c l e s , 14 (1980) 625: P.J. Davis and A.E. Klein, J . Chromatogr., 188 (1980) 280. A.E. K l e i n and P.J. Davis, J . Chromatogr., 207 (1981) 247. J.T. Hughes and P.J. Davis, J . Chromatogr., 219 (1981) 321.

lb

TABLE 13.1

w

m

HPLC ANALYSIS COLCHICINE AND DERIVATIVES COLUMN DIM. L x I D (mn)

MOBILE PHASE

ALKALOIDS

AIMS

STATIONARY PHASE

Colchicine,colchicoside

Analysis c o l c h i cum seeds

Silanized Lichrosorb Si60 30 pm

500x3

A. ACN 8. ACN-H 0(1:9) gradient2B t o 30% A i n B

Colchi cine.co1 chicoside. 3-demethylcolchicine

Analysis o f p l a n t m a t e r i a l

L i c h r o s o r b RP8 10 vm

250

MeOH-H20(1:2)

Colchi c i n e

Determination i n plasma

H y p e r s i l 5 pm

100x5

CH,C1 ,-isoprOH-NH40H(ratio

Colchicine,caffeine,narceine, codeine,brucine,cinchonidine,

E f f e c t s o l v e n t composition on r e t e n t i o n

L i c h r o s o r b RP2 10

Determination i n m i c r o b i a1 cultures

L i chrosorb RP18

REF.

1

2 pm

unspecified)3

1 2 0 ~ 3 . 5 Me6H-H20( 1:4) ,( 2:3) ,( 3:Z) ,(4: 1) MeOH

4

aconi t i n e Colchi c i ne.colchiceine.demeco1-

cine.N-methyl-colchiceinamide.

1 0 0 ~ 4 . 1 ACN-MeOH-phosphate b u f f e r ( p H 6 ) (16: 5: 79)

col c h i sine.ethy1 i s o c o l c h i c i n a t e . ethylcolchicinate

5

Col c h i cine, 17-hydroxycolchi c i - I d e n t i f i c a t i o n i m p u r i t i e s i n colchi cine ne,col c h i c i 1i n e , 6 - l u m i c o l c h i c i ne.8-formyl -desacetyl c o l c h i c i ne Col c h i c i ne ,demcol c i ne.N-des-

acetylcolchicine.3-demethylcolchicine.2-demethylcolchi-

A n a l y s i s i n b i o l o g i c a l material(Fig.13.1)

L i c h r o s o r b S i l o 0 10 um 2 5 0 ~ 4 . 5 A. CHCl s a t . w i t h H 0 B. CHC13-MeOH(95:5) Eat. w i t h H20 gradien2 10% B t o 100% B i n A 300x4

VBondapak C18 pBondapak Phenyl L i c h r o s o r b RP8 10 vm L i c h r o s o r b RP18 10 p m

ACN-MeOH-0.022M phosphate b u f f e r 300x4 (pH 6)(16:5:79) 150x4 250 100~4.1

U l t r a s p h e r e ODS 5 um

2 5 0 ~ 4 . 6 ACN-MeOH-O.1M TrEA.HC1 i n 0.02M phosphate b u f f e r ( p H 2.2)(200:85:715)

7

cine, 1-demethylcolchicine Colchi c i ne.demeco1cine.N-desSeparation c o l chine d e r i v a a c e t y l c o l c h i cine.3-demethyl c o l - t i v e s c h i cine.2-demethyl c o l c h i cine,

1-demethylcolchicine.ethylco1chicinate,ethylisocolchicinate,

8

N-methylcolchi ceinamide C o l c h i c i ne,col cheinamide.N-met h y l - and N,N-dimethylcolchic e i nami de

6

ACN-MeOH-phosphate b u f f e r ( p H 6.0) (16: 5: 79)

pBondapak C18

Determination i n m i c r o b i a l cultures

9

419 2

10

20

man

7

Fig. 13.1. Separation o f c o l c h i c i n e and some d e r i v a t i v e s Column UBondapak C18 (300x4 nun I D ) , mobile phase a c e t o n i t r i l e methanol - phosphate b u f f e r (0.038 M potassium dihydrogen phosphate, 0.005 M dipotassium hydrogen phosphate. pH 6.0) (16:5:79). f l o w r a t e 2 ml/min, d e t e c t i o n UV 350 nm. Peaks: 1. 3-demethylcolchicine; 2, 2-dem e t h y l c o l c h i c i n e ; 3, N-desacetylcol c h i c i ne; 4, 1-demethyl c o l c h i c i ne; 5, N-methyl -N-desacetylc o l c h i c i ne; 6, c o l c h i c i ne.

-

Rafermnca p. 417

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421

Chapter 14 IMIDAZOLE ALKALOIDS

14.1. HPLC systems ..................................................... References.

............................................................

..... .....

421 422

14.1. HPLC SYSTEMS Pilocarpine i s widely used in ophtalmology in eye-drops.However, in solution i t may undergo epimerization t o isopilocarpine, and even hydrolysis t o pilocarpic acid. In b o t h cases loss of pharmacological activity i s the result. Therefore. the analysis of ophtalmic solutions for pilocarpine and i t s decomposition products have been the subject of several investigations. The f i r s t methods described for the analysis of pilocarpine used a cation-exchange resin in combination with a basic DeGraw e t a 1 . l and Urbanyi e t al.3 used a TRIS buffer, 2 b u t i t was found t o degradecolumn performance . A sodium phosphate buffer - t r i e d instead of the TRIS buffer - caused problems as to the reproducibility of the separation 2 . Khalil 4 used an octadecyl and a cyanopropylsilane column in s e r i e s in order t o analyze pilocarpine in the presence of preservatives in ophtalmic preparations. Tetrahydrofuran - borate buffer (pH 9.2)(3:7) was used as mobile phase. Noordam e t a1 .5’9 reported the separation of pilocarpine. and the decomposition products mentioned above, on an octadecyl column with a mobile phase consisting of a mixture of water and methanol (97:3) containing 5%potassium hydrogen phosphate (pH 2.5)(Fig.14.1). I t was found t h a t increasing the s a l t concentration and lowering the pH lead t o improved s e l e c t i v i t y and peak shape. Kennedy and McNamara” found that replacing the octadecyl type of column in the method of Noordam e t a1.5’9 with a phenyl type of stationary phase reduced the analysis time, whilst improving peak shapes and resolution of pilocarpine and i t s degradation products. As mobile phase, 5%potassium dihydrogen phosphate (pH 2.5) in water was found t o be the most suitable. Kneckze6 applied ion-pair HPLC t o analyze ophtalmic solutions containing pilocarpine, physostigmine and rubreserine (see Chapter 8, Table 8.7, Fig.8.7). To improve the sensitivity of the pilocarpine analysis - enabling determinations in biological fluids - Mitra e t a1.’ developed a derivatization technique by means of which pilocarpine was quaternarized with the aid of e-nitrobenzylbromide (0.25 mg/ml - 24 hrs a t 4OoC). The quaternary derivatives were analyzed on an octadecyl column using 0.001 M sodium octanesulfonate in methanol - water ( 4 : l ) as mobile phase. the derivatization technique described was also applicable t o other amines. Straight-phase HPLC was used by Dunn e t a l . l O , whereby the alkaloids were separated on s i l i c a gel with hexane - 2% amnonia in propanol (7:3). To avoid interference of UV absorbing n i t r a t e , samples were passed through an ion-exchange column prior t o HPLC. Bundgaard and tiansen” separated pilocarpine and i t s degradation products on a s i l i c a gel column using methanol - 2 M phosphoric acid - water (3:5:92) containing 3% sodium s u l f a t e as mobile phase (Fig.14.2). The poor separation of pilocarpine and isopilocarpine a t a column temperature of 20-25’ C was improved by increasing the temperature t o 40’ C.

References p. 4 2 2

422

Detection o f p i l o c a r p i n e i s most s e n s i t i v e a t i t s UV maximum o f 215 nm. However. d e t e c t i o n a t 220 nm improves the s t a b i l i t y o f the baseline, whereas only a minor decrease - 5% o f the

-

peak h e i g h t o f p i l o c a r p i n e i s observed as compared w i t h 215.11m~~.The d e t e c t i o n l i m i t a t 215 nm i s about 0.04 pg, the d e t e c t i o n l i m i t obtained f o r the r e f r a c t i v e index method i s about 9 6 pg . REFERENCES

1 J . I . DeGraw, J.S. Engstrom and E. W i l l i s , J. P h a r m . Sci., 64 (1975) 1700. 2 J.D. Weber, J. ASSOC. Off. Anal. C h e m . , 59 (1976) 1409. 3 T. Urbanyi, A. Piedmont, E. W i l l i s and G. Manning, J . P h a r m . s c i . , 65 (1976) 257. 4 S. K.W. K h a l i l , J. P h a r m . Sci., 66 (1977) 1625. 5 A. Noordam, K. Waliszewski, C. Olieman, L. Maat and L. Beyerman, J . C h r o m a t o g r . , 153 (1978) 271. 6 M. Kneczke, J . C h r o m a t o g r . . 198 (1980) 529. 7 A.K. M i t r a , B.L. Baustian and T.J. Mikkelson. J. P h a r m . Sci., 69 (1980) 257. 8 J.J. O'Donnell. R. Sandman and M.V. Drake, J . P h a r m . Sci., 69 (1980) 1096. 9 A. Noordam, L. Maat and H.C. Beyerman, J . P h a r m . S c i . , 70 (1981) 96. 10 D.L. Dunn, B.S. S c o t t and E.D. Dorsey, J . P h a r m . Sci.. 70 (1981) 446. 11 J.M. Kennedy and P.E. McNamara, J . C h r o m a t o g r . , 212 (1981) 331. 12 H. Bundgaard and S.H. Hansen. rnt. J . P h a r m . , 10 (1982) 281. 13 M.V. Drake, J.J. O'Donnell and R.P. Sandman, J . P h a r m . S c i . , 71 (1982) 358.

3

2

min

20

10

0

o

1

8

1'2

min

Fig. 14.1. Separation o f p i l o c a r p i n e and degradation products5 Column Lichrosorb RP18 10 um (300x4 m I D ) , mobile phase 5% potassium dihydrogen phosphate i n water - methanol (97:3)(pH 2.5), f l o w r a t e 1.5 ml/min, d e t e c t i o n w i t h d i f f e r e n t i a l r e fractometer. Peaks: 1, pilocarpine; 2, i s o p i l o c a r p i n e ; 3, p i l o c a r p i c acid; 4, i s o p i l o c a r p i c acid. Fig. 14.2. Separation o f p i l o c a r p i n e and degradation products" Column Lichrosorb Si60 5 um (250~4.6 mn ID), mobile phase methanol - 2 M phosphoric a c i d water (3:5:92) containing 3% o f anhydrous sodium s u l f a t e , f l o w r a t e 1.2 ml/min. d e t e c t i o n UV 214 nm. Peaks: 1, pilocarpine; 2, isopilocarpine; 3, p i l o c a r p i c acid; 4. i s o p i l o c a r p i c acid; 5. n i t r a t e ; 6, unknown decomposition product.

-

P2 t 3

TABLE 14.1 HPLC ANALYSIS PILOCARPINE AN0 RELATED ALKALOIDS

P

P

M

ALKALOIDS*

OTHER COMPOUNDS

AIMS

STATIONARY PHASE

COLUMN D I M . LxIO(mm)

0.2M T r i s b u f f e r ( p H 9.2) i n 5% isoprOH

1 2

100x6

0.2M T r i s buffer-isoprOH(95:5)(pH 9 )

3

300x4 300x4

THF-borate b u f f e r ( p H 9 . 2 ) ( 3 : 7 )

Analysis synthesized p i 1

P i 1, i s o p i l ,pi l a c

A n a l y s i s ophtalmic s o l u t i o n s Aminex-7 7-11 um

Pi1,isopil

A n a l y s i s i n pharmaceuticals

Aminex-7 7-11 urn

A n a l y s i s i n pharmaceuticals

uBondapak C18 and WBondapak CN i n series

A n a l y s i s i n pharmaceuticals ( F i g . 14.1)

LichrosorbRP18 l O ~ m 3 0 0 x 4 o r N u c l e o s i l C18 150x4

A n a l y s i s i n pharmaceuticals (Table 7.7, Fig.7.7)

UBondapak C18

P i 1 .benzal k o n i um

Hydroxypropylmethylcellulose

P i 1, isopi 1 ,pi l a c .

is o p i 1ac P i 1 .physostigmi ne. r u b r e s e r i ne

-

Pi1,isopi Pi1,isopi isopilac

,pilac,

Salicylate,phenethyl alcohol, methyl paraben

100x6 65x5.5

4 5% KH2P04 i n H20-MeOH(97:3)(pH 2.5) 59 9

3 0 0 ~ 3 . 9 MeOH-0.005M aq. h e p t a n e s u l f o n i c a c i d (pH 3.6)(2:3) 6

A n a l y s i s i n b i o l o g i c a l f l u i d s UBondapak C18 3 0 0 ~ 3 . 9 MeOH-H O ( 4 : l ) c o n t a i n i n g 0.001M Na-ocby d e r i v a t i z a t i o n p i 1 tanesubonate A n a l y s i s i n pharmaceuticals L i c h r o s o r b RP18 lOpm250x4.6 5% KH2P04 i n H20-MeOH(97:3)(pH 2.5)

wn

P i 1. i s o p i

A n a l y s i s i n pharmaceuticals

Si60 Hibar 5

Pi1,isopi ,pilac, isopilac

A n a l y s i s i n pharmaceuticals

UBondapak Phenyl

P i 1 , i s o p i l ,pi l a c , isopilac

A n a l y s i s p i 1 degradation p r o d u c t s i n b a s i c aqueous solutions(Fig.14.2)

L i c h r o s o r b Si60 5um 2 5 0 ~ 4 . 6 MeOH-2M H PO -H 0(3:5:92) 3% Na2S043

*

REF.

0.1M Na-phosphate b u f f e r ( p H 9.0) i n 5% isoprOH

Pi1,isopil

Aminex A-7

MOBILE PHASE

2 5 0 ~ 4 . 6 Hexane-2% NH40H i n isoprOH(7:3)

7 8,13 10

3 0 0 ~ 3 . 9 5% KH2P04 i n H20(pH2.5) 11 containing

12

p i l = p i l o c a r p i n e , i s o p i l = i s o p i l o c a r p i n e , p i l a c = p i l o c a r p i c acid, i s o p i l a c = i s o p i l o c a r p i c a c i d

P

N w

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426

Chapter 15

QUATERNARY AMMONIUM COMPOUNDS

.....................................................................

15.1. HPLC systems References .............................................................................

425 427

15.1. HPLC systems The analysis of quaternary nitrogen compounds by HPLC requires chromatographic systems, such as ion-pai r and reversed-phase chromatography, which a r e able t o separate highly polar Both straight-phase (adsorption compounds. Reviews on ion-pair HPLC have been and/or p a r t i t i o n ) and reversed-phase ion-pair chromatography have been used. Eksborg and Schill’ separated a s e r i e s of alkylamines on a c e l l u l o s e column loaded with a 0.06 M aqueous p i c r a t e solution, of pH 11.2, using as mobile phase: chloroform - 1-pentanol ( 1 9 : l ) . Addition of the alcohol t o the mobile phase was necessary t o reduce t a i l i n g . Celite and porous s i l i c a gel were found t o be l e s s s u i t a b l e as s o l i d support f o r aqueous p i c r a t e solutions. A t the low pH necessary t o ionize t e r t i a r y , secondary and primary amines, p i c r a t e bled from the column (pHc6). Better r e s u l t s could be obtained f o r the amines mentioned w i t h 6-naphtalenesulfonate as counter-ion 2 A cellulose o r c e l i t e support could be impregnated with 0.1 M aqueous solution of t h i s counter-ion (pH 2.4), and by using chloroform - 1-pentanol, saturated w i t h the stationary phase (as mobile phase) reproducible r e s u l t s were obtained. Eksborg 3 reported the separation o f alkylammonium compounds as ion-pairs, using chloride as counter-ion. The support, diatomaceous earth, was coated with 0.05 M hydrochloric acid and chloroform - 1-pentanol (19:l) was used as mobile phase. To permit s e n s i t i v e UV Qetection of the alkylamines, a second indicating column was coupled with the separating column. On the indicating column the chloride ions were exchanged by strong UV absorbing B-naphtalenesulfonate counter-ions. Diatomaceous earth was used in the indicating column as s o l i d support and a solution of 0.1 M 8-naphtalenesulfonate in 0.1 M hydrochloric acid was used as stationary phase. microparticulate Optimization of the system has been described by C r ~ n n n e n ~ ’H~e ~used . low surface s i l i c a gel as s o l i d support f o r a solution of 6-naphtalenesulfonate i n a phosphate buffer of pH 2.3. The s o l i d support was impregnated with the stationary phase by pumping the stationary phase through the s o l i d support, when packed i n the column. After the column i s completely f i l l e d with the stationary phase, the mobile phase saturated w i t h the s t a tionary phase - i s pumped through the column until a c l e a r eluent comes from the column. Injection of the stationary phase until droplets of i t were eluted from the column was a l s o used in order t o impregnate the s o l i d support w i t h the stationary phase. Amino acids, dipept i d e s and alkylamines could be separated and s e n s i t i v e l y detected by t h i s method. For quaternary alkylamines. a. 0.1 M naphtalesulfonate solution i n choline c i t r a t e buffer (pH 3 . 8 ) has been used as stationary phase on low surface s i l i c a g e l , and w i t h chloroform 1-pentanol ( 9 : l ) saturated with the stationary phase as mobile phase”. Because a change of the stationary phase i s time consuming, the retention of the compounds t o be analyzed i s usually regulated by changing the r a t i o of the solvent used i n the mobile phase (Fig.15.1).

.

-

Refenncea p. 4 21

426

Hackzell and S c h i l l ”

l a t e r found t h a t an alkanediol modified s i l i c a gel was more s u i t a b l e

as support f o r the aqueous s t a t i o n a r y phase. Best r e s u l t s were obtained w i t h r a t h e r high loads o f s t a t i o n a r y phase Fig.15.2). analyzed some basic drugs and quaternary ammonium compounds by means Greving e t o f i o n - p a i r chromatography i n the straight-phase adsorption mode. Bromide o r perchlorate were

used as counter-ions i n connection w i t h m i c r o p a r t i c u l a t e s i l i c a gel columns. Chloride and i o d i d e were less s u i t a b l e as counter-ions, because they caused, r e s p e c t i v e l y , corrosion o f the equipment o r a too strong UV absorption background o f the mobile phase. Methanol was used as mobile phase, containing 0.01-0.1 M o f the counter-ion. Homologous series o f quaternary alkylamines have been separated on porous microspherical

polystyrene-divinylbenzene gel by means o f methanol

-

containing p e r c h l o r i c , s u l f u r i c o r hy-

d r o c h l o r i c a c i d as mobile phase (Figs.15.3 and 15.4)4. The r e t e n t i o n behaviour o f the a l k y l amines could be explained i n terms o f p a r t i t i o n chromatography. Reversed-phase l i q u i d - l i q u i d i o n - p a i r chromatography has been used t o separate some t e r t i a r y and quaternary ammonium drugs15. M i c r o p a r t i c u l a t e porous s i l i c a gel was used as s o l i d support. I t was impregnated w i t h l i p o p h i l i c alcohols (1-pentanol,

butanol). The aqueous mobile phase

contained buffered s a l t s o l u t i o n s and 1%o f the l i p o p h i l i c alcohol. Retention o f i o n i c samples could be increased by ions o f the opposite charge ( i o n - p a i r e f f e c t ) and decreased by ions o f the same charge (competition e f f e c t ) . Various c a t i o n i c and a n i o n i c components were discussed. Perchlorate. n i t r a t e and 10-camphorsulfonate were used as p a i r i n g - i o n (see a l s o Chapter 4, Fig. 4.7). Johansson e t a1.* separated some organic ammonium compounds by means o f i o n - p a i r chromatography using m i c r o p a r t i c u l a t e chemically bonded octadecyl columns, t h a t were impregnated w i t h 1-pentanol or dichloromethane as s t a t i o n a r y phase. The aqueous mobile phase phate b u f f e r saturated w i t h the s t a t i o n a r y phase

-

-

a pH 2.2 phos-

contained the counter-ion. T a i l i n g could

be reduced by adding a long chain t e r t i a r y o r quaternary ammonium compound t o t h e mobile phase.

D i hydrogen phosphate, bromide, cyclohexyl sul famate, d i cycl ohexyl s u l famate and o c t y l sul f a t e were used as p a i r i n g - i o n s . Antidepressiva, n e u r o l e p t i c amines and r e l a t e d quaternary ammonium compounds were analyzed

9

.

E l l i p t i c i n e and r e l a t e d quaternary a l k a l o i d s have been analyzed on an octadecyl column using heptane- o r pentanesulfonate as p a i r i n g - i o n (see Chapter 8, Tables 8.5 and 8.6). Some quaternary a c e t y l c h o l i n e esterase i n h i b i t o r s were analyzed by the Ruyter e t a l . 1 7 on an o c t y l column using heptanesulfonic a c i d containing mobile phase (Fig.15.5).

Best column

performance was observed a t lower pH. A d d i t i o n o f tetramethylammonium t o the mobile phase reduced t a i l i n g on some o f the t e s t e d reversed-phase column m a t e r i a l s . Tetramethylamnonium could a l s o be used t o r e g u l a t e the r e t e n t i o n o f the compounds analyzed. The quaternary compounds were i s o l a t e d from b i o l o g i c a l m a t e r i a l by means o f i o n - p a i r e x t r a c t i o n . Van der Maeden e t a1.6 determined tubocurarine i n curare samples. The i n f l u e n c e o f cations and pH on the separation o f the a l k a l o i d s on an octadecyl column were studied. Optimum pH was found t o be 4. B e t t e r peak performance and increased r e s o l u t i o n was obtained by u s i n g t e t r a methylamnonium as c a t i o n i n the mobile phase, as compared w i t h mobile phases c o n t a i n i n g potassium o r ammonium ions. Optimum separation was obtained w i t h gradient e l u t i o n (see Chapter 6, Fig.6.3). Berberine has been analyzed on an octadecyl column as i o n - p a i r w i t h dodecylsulfate 20,22,26 Reversed-phase separation on an octadecyl column has been used f o r the analysis o f berberine, using a mobile phase o f a c e t o n i t r i l e

-

phosphate b u f f e r (pH 5.2)(3:2)7.

427

REFERENCES

1 S. Eksborg and G. S c h i l l . A n a l . C h e m . , 45 (1973) 2092. 2 S. Eksborg, P.O. Lagerstrom, R. Modin and G. S c h i l l , J. C h r o m a t o g r . , 83 (1973) 99; 3 S. Eksborg, A c t a P h a r m . S u e c . . 12 (1975) 43. 4 A. Nakae, K. K u n i h i r o and G. Muto, J . C h r o m a t o g r . , 134 (1977) 459. 5 J. Crommen, 8. Fransson and G. S c h i l l , J . C h r o m a t o g r . , 142 (1977) 283. 6 F.P.8. van d e r Maeden, P.T. van Rens, F.A. Buytenhuys and E. Buurman, J . C h r o m a t o g r . , 142 (1977) 715. 7 T. H a t t o r i , N. Kamiya, M. Inoue and M. Hayakawa, Yakugaku Z a s s h i , 97 (1977) 1305. CA 88 (1978) 79161b. 8 I.M. Johansson, K.G. Wahlund and G. S c h i l l , J. C h r o m a t o g r . , 149 (1978) 281. 9 K.G. Wahlund and A . S o k o l w s k i , J . C h r o m a t o g r . , 151 (1978) 299. 10 E. Tomlinson, T.M. J e f f e r i e s and C.M. R i l e y , J . C h r o m a t o g r . . 159 (1978) 315. 11 F. Debros and A.J. Gissen, A n e s t h e s i o l o g y , 51 (1979) 5265. 12 J. Crommen, A c t a Pharm. S u e c . , 16 (1979) 111. 13 G. Muzard and J.8. Le Pecq, J. C h r o m a t o g r . , 169 (1979) 446. 14 J.E. Greving, H. Bournan, J.H.G. Jonkman, H.G.M. Westenberg and R.A. de Zeeuw, J . C h r o m a t o g r . . 186 (1979) 683. 15 J . Crommen, J . C h r o m a t o g r . , 186 (1979) 705. 16 8.A. B i d l i n g m e y e r , J . C h r o m a t o g r . S c i . , 18 (1980) 525. 17 M.G.M. de Ruyter, R. C r o n n e l l y and N. C a s t a g n o l i , J . C h r o m a t o g r . , 183 (1980) 193. 18 J . CrOImen, J . C h r o m a t o g r . , 193 (1980) 225. 19 J.E. Greving. J.H.G. Jonkman, H.G.M. Westenberg and R.A. de Zeeuw, Pharm. Wkbld., S c i . E d . 2 (1980) 81. 20 Y. Akada, S. Kawano and Y . Tanase, Yakugaku Z a s s h i , 100 (1980) 766. CA 93 (1980) 2 4 5 5 8 8 ~ . 21 L. H a c k z e l l and G. S c h i l l , A c t a P h a r m . S u e c . , 18 (1981) 257. 22 Y. Hashimoto, K. Kawanishi, H. Tomita. Y . Uhara and M. Moriyasu, Anal. L e t t . , 14 (1981) 1525. 23 P. M a j l a t , P. Helboe and A . K . K r i s t e n s e n , r n t . J . P h a r m . , 9 (1981) 245. 24 J.E. P a r k i n , J. Chromatogr., 225 (1981) 240. 25 A. Meulemans, J . Mohler. 0. Henzel and Ph. D u v a l d e s t i n . J . C h r o m a t o g r . , 226 (1981) 255. 26 T. M i s a k i , K. Sagara. M. Ojima, S . Kakizawa, T. Oshima and H. Yoshizawa. Chem. P h a r m . ~ u l i . , 30 (1982) 354. 27 G. Bykadi, K.P. F l o r a , J.C. Cradock and G.K. Poochikian, J . C h r o m a t o g r . , 231 (1982) 137.

428

1 1

0021

2

[OOOsA

i

3

0

2

,

I

,

,

L

6

8

10 min

3 1

r

I

8

10

6

L

2

rnin

0

Fig. 15.1. Separation of some quaternary amnonium ions 18 Column Lichrosorb Si500 10 urn, coated with 0.1 M naphtalene-2-sulfonate i n 0.1 M choline c i t r a t e buffer (pH 3 . 8 ) ( 2 5 0 ~ 4m I D ) , mobile phase chloroform - 1-pentanol ( 9 : l ) saturated with the stationary phase, flow r a t e 1.0 ml/min, detection UV 254 nm. Peaks: 1, tripropylbutyl amnoni um bromide; 2 , tetrapropylammoni um bromide; 3, tripropylmethyl ammoni um iodide. Fig. 15.2. Separation of some quaternary amnonium compounds21 Column Lichrosorb Diol 10 ( 1 5 0 ~ 3 . 2mn ID) loaded w i t h 0.1 M naphtalene-2-sulfonate i n 0.1 M aqueous phosphate buffer (pH 2 . 1 ) by subsequently pumping 30 ml of phosphate buffer (pH 2.1) and 50 ml of the stationary phase through the column, followed by the mobile phase until no more droplets could be observed in the e l u a t e (ca. 20 ml). Finally the column was recycled with 500 ml of mobile phase. Mobile phase chloroform - n-propanol ( 9 : l ) and chloroform - n-propanol ( 9 : l ) saturated w i t h the stationary phase mixea in a r a t i o (1:9), flow r a t e 0.5 ml/min, detection UV 254 nm. Peaks 1, tetrabutylammonium; 2. tributylmethylammonium; 3 , tetrapropylamnonium; 4. tripropylrnethylamnonium. (reproduced w i t h permission from r e f . 21, by the courtesy of Acta Pharmaceutica Suecica). 1

I

1

1’0

b

1

io 20 min Fig. 15.3. Separation of alkylbenzyldimethylamonium chlorides 4 Column Hitachi Gel 3011 (500x4 mm I D ) , mobile phase 0.5 M perchloric acid in methanol, flow r a t e 1.1 ml/min. column temperature 30’ C, detection UV 220 nm. Peaks: 1, decyl- ; 2 , dodecyl- ; 3, tetradecyl- ; 4, hexadecyl- ; 5, octadecyl-benzyldimethylammonium chloride. Fig. 15.4. Separation of alkylpyridinium chlorides 4 Column Hitachi Gel 3011 (500x4 mm 10). mobile phase 0.5 M perchloric acid in methanol, flow r a t e 1.1 ml/min, column temperature 30’ C , detection UV 260 nm. Peaks: 1, decyl- ; 2 , dodecyl- ; 3, tetradecyl- ; 4 , hexadecyl- ; 5, octadecylpyridinium chloride. O

2’0

iomin

429

0

2

L

6

8

10 12min

Fig. 15.5. Analysis o f neostigmine in serum sample 17 Column 5 pm Ultrasphere Octyl ( 1 5 0 ~ 4 . 6 mn ID), mobile phase 0.01 M heptanesulfonate, 0.01 M sodium dihydrogen phosphate and 0.0025 M tetramethylammonium chloride in acetonitrile - water (1:4)(pH 3.0), flow rate 2 ml/min, detection UV 214 nm. Peaks: 1, interference; 2. edrophonium (internal standard); 3, neostigmine.

Reference# p. 427

TABLE 15.1

IP

0 w

HPLC ANALYSIS QUATERNARY AMMONIUM COMPOUNDS COMPOUNDS Quat. a1 k y l amines

A1 k y l ami nes

STATIONARY PHASE

AIMS

COLUMN DIM. MOBILE PHASE LxID(mn)

Separation by i o n - p a i r p a r t i t i o n C e l l u l o s e 30-65 prn 300~2.7 loaded w i t h 25% 0.06 chromatography M picrate solution (pH 11.2) I o n - p a i r chromatography o f orga- C e l l u l o s e 30-65 urn o r C e l i t e 37-74 o r 15-37 n i c compounds pm, loaded w i t h 0.06M p i c r a t e s o l u t i o n ( p H 11.2) o r 0.1M naphtalenesulfonate s o l u t i o n ( p H 2.4) 3 0 0 ~ 2 . 7

300

Alkylamines

I o n - p a i r chromatography separat i o n f o l l o w e d by t r a n s f o r m a t i o n i n t o UV-absorbing i o n - p a i r

Dia-Chrom 37-44 prn loaded w i t h 25% 0.05M HC1

Alkylbenzyldimethylamno-

Separation (Fig.15.3,

H i t a c h i g e l 3011 10-15 500x4

15.4)

n i um- ,a1 k y l py r id i n i um ha1ides

REF.

CHCl -n-hOH(19:1) statqoiiary phase

sat. with the

CHCl -n-hOH(19:1) s t a t q o n a r y phase

sat. w i t h the

CHCl -n-AmOH(19:1) statqonary phase

sat. w i t h t h e

1

2

3 0.5M HC104 i n MeOH

um

4

A1 kylamines.amino acids, dipeptides

I o n - p a i r chromatography w i t h h i g h l y UV-absorbing counter i o n s

Lichrospher S i l o 0 10pm 1 5 0 ~ 4 . 5 CHCl -n-hOH(95:5),(9:1) loaded w i t h 0.01M naphstatqonary phase t a l e n e s u l f o n a t e i n pH 2.2 phosphate b u f f e r

Tubocurarine,chondrocura-

Analysis curare

pBondapak C18

300x4

0.025M tetramethylamnonium phosphate i n A. MeOH-H 0(1:3)(pH 4) B. MeOH-H20(9:ll)(pH 4) i n 30 min l i n e a r g r g d i e n t A+B(9:1) t o (3:17) 6

B e r b e r i ne

Analysis i n C o p t i s species

pBondapak C18

300x4

ACN-phosphate b u f f e r ( p H 5.2)(3:2)

Phenylethy1amines.neuro-

Separation by reversed-phase ion-pai r HPLC

1 5 0 ~ 3 . 2 0.028M Dimethyloctylamine i n 0.1M pH 2.2 L i c h r o s o r b RP8 10 um dynamically coated w i t h phosphate b u f f e r , s a t . w i t h n-hOH n-AmOH 0.043M NaBr, 0.028M d i m e t h y l z c t y l a m i n e i n 0.1M pH 2.0 phosphate b u f f e r , s a t . w i t h n-AmOH 8.9 pBondapak C18 300x4 ACN-H20(1B:82) c o n t a i n i n g 0.2M HC104 11 (PH 5.4)

r i ne,curari ne,isochondrodendrine,curine

l e p t i c amines ,quaternary amnoni um compounds

Tubocurarine,isochondoden- Analysis i n plasma drocurine

sat. w i t h the 5,12

7

Ellipticine and quaternary derivatives

Separation(Tab1e 8.5, 8.6)

UBondapak C18

Quaternary amnonium derivatives and basic drugs Atropine,scopolamine,methylatropine,various drugs

Separation by ion-pair adsorption chromatography Separation by reversed-phase ion-pair HPLC(Fig.4.7) Analysis in biological fluids

Lichrosorb Si60, SilOO 300 or 150x4 or Lichrospher 5 un Lichrospher SilOO lOum 200x4

300x4

MeOH-O.OO5M heptanesulfonic acid, 0.032M AcOH(7:3),(3:1) MeOH-0.005M pentanesulfonic acid, 0.032M AcOH(7:3) MeOH-0.02M NH40Ac(3:1) Various conc. NaBr or NaC104 in MeOH

13

14.19 0.1M Na-phosphate buffer(pH 2.2)+ 1.9% AmOH 15 Pyridostigmine,neostigmiUltrasphere Octyl 5 um 150~4.6 0.01M Heptanesulfonate, 0.01M NaH PO ne,edrophonium and their and 0.0025M tetramethylammonium c&lo?i3-hydroxy metabolites de in ACN-H20(1:4),(17:83)(pH 3) 17 Quaternary a1 kyl ami nes Separation(Fig .15.1) Li chrospher Si 500 10Um 250x4 CHCl -n-AmOH(g:l) sat. with the statioloaded with 0.1M naphnary3p5ase talenesulfonate in pH 3.8 choline citrate buffer 18 Berberine,acrinol Analysis pharmaceuticals Zorbax ODS 250 0.005M Oodecylsulfate in ACN-H20(95:5) 20 Quaternary alkylamines Determination by using UV-absor- Lichrosorb DIOL 10 m 150~3.2 CHCl -prOH(9:1) sat. with the statiobing counter-ions(Fig.15.2) loaded with 0.1M naphnary3phase to various degrees talenesulfonate in aq. phosphate buffer(pH 2.1) LI Berberine,palmatine Histochemical chromatography Lichrosorb RP8 not given ACN-THF-O.1N tartaric acid-Na dodecylsulfate(20:20:59.5:0.5) 22 Methylhomatropine.codeine, Analysis pharmaceuticals Nucleosil 5C8 120~4.6 ACN-O.01M phosphate buffer(pH 5.0) morphine,noscapine,papave(2:3) rine,thebaine 23 Alcuronium,tubocurarine Analysis in biological fluids uBondapak C18 300~6.4 MeOH-H O(4:l) containing 0.25% AcOH and 0.605M Na-dodecylsulfate 24 Tubocurarine Analysis in plasma Radial-Pak C18 100x5 MeOH-(TrEA(lOg/l)-pentanesulfonic acid 25 (lml )-H3p04(2ml )-H20 ad 11 ) (2:3) Berberine,palmatine,copAnalysis in plant material TSK gel LS-410 5 urn 150x4 ACN-MeOH-O.1N tartaric acid-Na dodecyltisine 26 sulfate(40:10:49.5:0.5) El 1 i pti ci ne,g-hydroxy-el- Analysis in biological material uBondapak C18 300x4 ACN-O.01M NaH2P04(36:64).(25:75) liptine,ll-demethylellip(PH 3.5) ti ci ne 27

^.

P N 0

Appendix D e s c r i p t i o n o f some s t a t i o n a r y phases, which have been r e p o r t e d i n t h i s book. The l i s t i s n o t a complete l i s t o f a v a i l a b l e s t a t i o n a r y phases, f o r such a sumnary i s r e f e r r e d t o r e f s 1 and 2. Ion-exchange packings Name

Panufacturer/Suppl ie r

Pminex A7

Bio-Rad

7-11

polystyrened i v i c y l benzene r e s i n

N u c l e o s i l SA

I'acherey Nagel E Co

5,lO

s i l i c a gel

particle size urn

materials

(SO3 (SO3 P a r t i s i l SCX

Whatman

10

ion-exchange c a p a c i t y nieq/g

5

1 1

1 1

s i l i c a gel

(SO3

1

Vydac TP401 SCX

I'acherey Nagel & Co

10

s i l i c a gel ( so3- 1

Zipax SAX

DuPont

25-37

1a u r y l methacryl a t e polymer coated pelliculars ( N R ~ + )

Zipax SCX

DuPont

25-37

SO3-, p e l l i c u l a r

Zipax \!AX

DuPont

25-37

laurylmethacrylate Dolymer coated D e l l i -

1

Reversed-phase packings Name

Ilanufacturer/Suppl i e r

particle size

urn

bonded f u n c t i o n a l group

p a r t i c l e shape % C 1oadi ng

*

VBondapak C18

Waters Associates

10

octadecylsilyl

irr

C o r a s i l C18

Waters Associates

37-50

octadecyl s i l y l

pel1

H y p e r s i l ODS

Shandon

6

octadecylsi l y l

spher

L i c h r o s o r b RP18 Micropak #CH

E. t!erck

5,lO

Varian

5.10

octadecyl s i l y l octadecyl s i l y l

irr irr

10% 9X.endcapped

22% 12%

P

cn w

Nucleosil C18 ODs Sil-X-I ODS Si1-X-I1 Partisil 00s-1 Partisil ODS-2 Partisil ODs-3 Spherisorb ODS U1 trasphere ODS Zipax OOS Zorbax ODS Chromegabond C8 Lichrosorb RP8 Nucleosil C8 U1 trasphere Octyl Spherisorb C6 Chromegabond C6Hll Hypersil SAS Lichrosorb RP2 Corasi 1 Phenyl ~rBondapa k Phenyl

tlacherey Nagel & Co Perkin Elmer Perkin Elmer Whatman Whatman Whatman Phase Separation Ltd A1 tex Oupont Dupont ES Industries E .t!erck Yacherey Nagel & Co A1 tex Phase Separation Ltd ES Industries Shandon E.I!erck Ma ters Associates Waters Associates

5.7.5.10 13 35~15 5,lO 10 10 5,lO 395 25-37 6-8 5,lO 5,lO 5,7.5,10 5 5

10 3.5.10 5.10 37-50 10

octadecylsilyl octadecyl silyl octadecylsilyl octadecylsilyl octadecylsilyl octadecylsilyl octadecylsilyl octadecylsilyl oc tadecy 1 si 1 yl octadecylsilyl. octylsilyl octyl s i 1 yl octyl si lyl octyl si lyl hexyl si lyl cyclohexylsilyl short alkylsilyl dimethyl silyl diphenylsilyl diphenylsilyl

spher i rr pel 1 i rr i rr i rr spher spher pel 1 spher i rr irr spher spher spher i rr spher i rr Pel 1 i rr

15-16%

5%

15% lO%.endcapped 7%,endcapped 12%,endcapped 15% 15% 13-14% 10-11% 6.5l.endcapped endcapped 10% 3%

10%

Polar chemically bonded packings Name

Manufacturer/Suppl ier

VBondapak CII Lichrosorb CN Nucleosil CN Srherisorb CN VBondapak NH2

Waters Associates E .Yerc k Pacherey Nagel & Co Phase Separation Ltd Waters Associates

particle size 10 5.10 5.10 5 10

bonded functional cyano cyano cyano cyano alkylamino

particle shape % C i rr i rr spher spher i rr

9%

9% e w

Nicropak NH2

Varian

Nucl eosi 1 NH2

Kacherey Nagel & Co

Lichrosorb Diol

E.t!erck

uBondapak Carbohydrate

Waters Associates

Ourapak OPN

Waters Associates

Zipax ETH Hami 1t o n P R P l

H i t a c h i g e l 3011

P 0 P

10

a1 k y l ami no

i rr

5,lO

alkylamino

spher

hydroxy

irr

10 10 37-75

oxydipropionitrile

irr

Oupont Hami 1t o n

25-37

e t h e r groups

pel 1

10-15

polystyrenedivinylbenzene resin(CH2OH f u n c t i o n a l groups)

Hitachi

10-15

polystyrenedivinylbenzene resin(CH2OH f u n c t i o n a l groups

irr

Normal-phase packings Name

t!anufacturer/Suppl ie r

Hypers i 1

Shandon

L i c h r o s o r b S i 60

E.Perck

L i c h r o s o r b S i 100

E.t?erck

Lichrospher S i 100

E.tlerck

Lichrospher S i 500

E.Herck

Kicropak S i ( i d e n t i c a 1 w i t h L i c h r o s o r b Si 60)

Varian

N u c l e o s i l 50

tlacherey Nagel & Co

Nucl eos i1 100

t!acherey Nagel & Co

Partisil

Whatman

Porasil A

Waters Associates

T

Waters Associates

Porasil UPorasi 1

Waters Associates

RSi1

A1 1t e c h

Sil-X-1

P e r k i n Elmer

particle size vm

pore s i z e nm

6 5,7,10,30 5,7,10,30 5,10,20 10 5,lO

10 6 10 12 50 6

5,7.5,10 5,7.5,10 5,10,20 37-75 15-25,25-37 10 5,lO 13

5 10 4-5 10 15 10 6 10

p a r t i c l e shape s u r f a c e area m2/g spher

200

irr irr

475 278

spher

2 56

spher irr spher spher irr spher spher

irr

i rr i rr

45 475

500 300 400 350-500 300 400 550 400

pore volume m l /g

0.76 1.02 1.20 0.88 0.76 0.80 1 .oo 0.70

1.05

1.00

Spherisorb SW Spherosil XOA 800 Spherosil XOA 600 Spherosil XOA 400 (identical with Porasil A ) Spherosil XOA 200 Vydac TP Adsorbent Zorbax Sil

*

Phase Separation Ltd Rh6ne-Progil Rh8ne-Progil Rh6ne-Progil

3,5,10 5,lO 5,10,20 10

Rh6ne-Propil Kacherey Nagel & Co DuPont

10 10 6

8

3 6 10 15 33 7.5

spher spher spher spher

220 830 600 350-500

0.6 0.4-0.6 0.7-1 1.05

spher i rr spher

125-250 100 300

0.9 0.6

irr=irregular,pell=pellicular,spher=spherical

References: 1. K.K.Unger, Porous Silica,J.Chromatogr.Library

2. R.E.Majors,J.Chromatogr.

Vo1.16, Elsevier, Amsterdam,l979.

,Sci. .18(1980)488.

P cn w

This Page Intentionally Left Blank

437

INDEXES How to find your way

The various subjects discussed i n the chapters are presented i n the Subject Indexes, as w e l l as the b o t a n i c a l names o f p l a n t s mentioned i n t h e t e x t . Solvent systems, d e t e c t i o n methods and various chromatographic techniques (ion-exchange,

reversed-phase,

ion-pair,

straight-phase) are n o t included i n the Subject Index. These subjects are d e a l t w i t h separately f o r each group o f a l k a l o i d s and may be found i n the l i s t o f contents o f each chapter.

A l l compounds w i t h a p e r t i n e n t reference t o r e t e n t i o n times o r d e t e c t i o n methods e i t h e r i n the t e x t , f i g u r e s o r tables are l i s t e d i n the compound index. F o r the various groups o f a l k a l o i d s , tables summarizing a l l the a v a i l a b l e l i t e r a t u r e are presented a t t h e end o f each chapter. Compounds l i s t e d i n these tables are n o t included i n the Compound Index. Hence t o f i n d a l l data r e l a t i n g t o a p a r t i c u l a r compound, i t may be necessary t o c o n s u l t both the Compound Index and the appropriate tables.

SUBJECT INDEX GAS-L I QUID CHROMATOGRAPHY dcronychia a1 k a l o i d s

93-95

151, 152 d e r i v a t i z a t i o n 23, 151, 152 GC-MS 151

Amaryllidaceae a l k a l o i d s

Aporphine a l k a l o i d s

Caffeine analysis i n biological material

191- 194 a n a l y s i s i n food and beverages

114, 115, 128,

129, 147-150

190, 191 a n a l y s i s i n pharmaceutical

analysis i n biological material

128, 129, 148-150 d e r i v a t i z a t i on 23, 148-150 GC-MS

148, 150

GC-MS

53-56 dspidosperma a l k a l o i d s 166, 167

GC-MS

166

15-19, 40, 45, 46, 53, 81,

61-72 Cactus a l k a l o i d s 97-101 analysis i n b i o l o g i c a l m a t e r i a l 101 analysis i n p l a n t m a t e r i a l

97-101 Cactus a l k a l o i d s derivatization

101

89, 90, 128, 136-138, 165, 166, 187, 189, 208 B-Carboline d e r i v a t i v e s 156, 157,

61-72

Belladonna a l k a l o i d s

GC-MS

187, 192, 208

c a p i l l a r y gas chromatography

A r t e f a c t formation, see vol. 23A

Atropd a l k a l o i d s

194-198 187, 189, 208

preparations c a p i l l a r y GC

170 Cephalotaxus a l k a l o i d s

213-215

analysis i n plant material GC-MS 213

213, 214

Cinchona a l k a l o i d s 87-91 a n a l y s i s i n b i o l o g i c a l m a t e r i a l 87-89 analysis i n food and beverages 89 a n a l y s i s i n pharmaceutical prepara-

22, 101

t i o n s 87-90 c a p i l l a r y GC

89, 90

430

derivatization 22, 87-89, 91 GC-MS 89 Clavine alkaloids 176, 177 Cocaine metabolites 75-80 Coca leaves 73, 80, 8 1 Conium a1 kaloids 54 Datura a1 kaloids 61-72 Deactivation solid support 9-13 Derivatization of alkaloids f o r gas chromatography 21-24 Dihydroergotoxine alkaloids 177, 178, 180, 182

Drugs of abuse 113, 116, 127-136, 138- 14 1 analysis drug seizures 129-134,

142-144

173-176

Lupine alkaloids 55-59 Nicotine metabolites 42, 43, 45, 46, 47

Opium alkaloids 111-146 analysis i n biological material

analysis i n pharmaceutical preparations 125, 126 analysis in plant material 113, capillary GC 128, 136-138 derivatization 22, 23, 114-124, 126, 128, 129, 136, 137.

106-108

analysis in pharmaceutical preparations 105, 106 analysis in plant material 105 derivatization 22, 104 Ergot alkaloids 173-184 analysis in biological material 179

analysis in pharmaceutical perparations 176, 182, 183 derivatization 23, 24, 173-177 GC-MS 179. 180 Erythrina alkaloids 153, 154 derivatization 153, 154

142-144

GC-MS 116. 121-123, 127, 128 Papaveraceae a1 kaloids (see O p i u m alkaloids) 111-146 Physostigma a1 kaloids 166 Piperidine alkaloids 53, 54 analysis in plant material 53, 54

capillary GC 53 Piper nigrum alkaloids 53 Pseudotropine alkaloids 73-85 analysis in biological material 75-80

analysis in drug seizures 73,

153

Fused s i l i c a columns 15, 19, 81, 89, 90, 137. 138. 188

Glycoalkaloids 185, 186 Heteroyohimbine alkaloids

analysis i n opium 116-120, 125,

117

116, 134, 135

alkaloids 103-109 analysis in biological material

Ephedra

158, 159,

81

analysis in pharmaceutical preparations 74, 80 analysis in plant material 73, 80, 8 1

168-170 Hyoscyamus

154

LSO and derivatives

126

116, 127, 135, 136

capi 1 lary GC 136-138 derivatization 128-133, 135-137,

GC-MS

239-245)

Isoquinoline alkaloids 97-101, 111-

115, 116, 120-128, 136, 137

137

analysis in biological material

GC-MS

Imidazole alkaloids 217, 218 derivati zation 24 Indole alkaloids derivatization 23 Isolation from biological materials 388, 389 (see also vol. 23A 51-58,

alkaloids 61-72

capillary GC 8 1

439

derivatization GC-MS

21, 73-80, 83, 84

78-80

Pyridine alkaloids

33-52

42-47 35, 38-40, 41 34, 40-42

analysis i n plant material

21, 37, 43

35, 38-40,

45, 47 29-32

analysis i n plant material

c a p i l l a r y GC 31

21, 31, 32

Quinolizidine alkaloids

55-59

57, 58 159-162

160, 161

61-85 61-72

a n a l y s i s i n p h a n a c e u t i c a l preparations

analysis i n plant material

161

160

64-69

analysis i n plant material derivatization

Sample p r e p a r a t i o n

231, 388, 389

(see a l s o v o l . 23A 51-58, 239-245) Solanaceae a1 k a l o i d s ( s e e t r o p i n e 61-72

GC-MS

155-159, 175

uncaria a1 k a l o i d s

165-166

d e r i v a t i z a t i o n 24, 185, 186 61-72

Strychnos a1 k a l o i d s

162-165

168-170 165-166

analysis i n biological material

185, 186

Stramonium a l k a l o i d s

61-69

21, 65-69

67

Tryptamines

Vinca a l k a l o i d s

185, 186

Steroidal alkaloids

c a p i l l a r y GC

165, 166

derivatization GC-MS

analysis i n biological material 165

165

165

X a n t h i ne d e r i v a t i v e s

187-21 1

a n a l y s i s i n b i o l o g i c a l m a t e r i a1

a n a l y s i s i n pharmaceutical preparations

Tropane a l k a l o i d s Tropine a l k a l o i d s 67

analysis i n biological material

Solanurn a l k a l o i d s

21, 37, 43

45, 47

analysis i n biological material

Rauwolfia a1 k a l o i d s

alkaloids)

40, 45. 46

derivatization GC-MS

55-59

analysis i n plant material

GC-MS

34,

41

a n a l y s i s i n smoke 34, 40-42

P y r r o l i z i d i n e a1 k a l o i d s

GC-MS

42-47

40, 45, 46

derivatization

187. 208

Tobacco a l k a l o i d s analysis i n biological material

a n a l y s i s i n smoke derivatization

187, 189, 190,

202-208 GC-MS

34,

analysis i n plant material

c a p i l l a r y GC

189, 208

derivatization

analysis i n biological material

GC-MS

r a t i o n s 200 c a p i l l a r y GC

164

analysis i n plant material

162,

163 Symphytum a l k a l o i d s

190-194, 200-208 a n a l y s i s i n f o o d and beverages 190, 191 analysis i n phanaceutical pre-

31, 32

Theophyl 1i n e analysis i n biological material 190, 200-208 a n a l y s i s i n pharmaceutical prepa-

parations c a p i l l a r y GC derivatization

194-198, 200 187, 189, 208 24, 187, 189.

190, 199, 202-208 GC-MS

187, 192, 208

440

SUBJECT INDEX HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Abbreviations f o r solvents

222

Aconite a l k a l o i d s 415, 416 A1 k y l ami nes 228 Aluminium oxide 229 Amaryllidaceae a l k a l o i d s 230, 287. 290,

291 A r t e f a c t formation (see v o l . 23A 53-56) Atropine and decomposition products

357-360,

362-368. 371 D i s s o l u t i o n s t a t i o n a r y phase 225.

228, 359 Diterpene a l k a l o i d s 415, 416 Drugs o f abuse 260-268, 297-330 analysis i n biological material

305, 308, 309, 324, 325 a n a l y s i s drug seizures 297, 300,

249-251. 254 Belladonna a l k a l o i d s 249-259 8 i sbenzyl isoquinol i n e a1 k a l o i d s 287,

288, 289, 292, 293 Cactus a l k a l o i d s 289, 290 Caffeine analysis i n b i o l o g i c a l f l u i d s

387-394, 399-401, 407-413 analysis i n food and beverages

389, 393, 394, 402, 414 metabolites 387-394, 399-401.

407-413 Catharanthus a l k a l o i d s

D i hydroergotoxine a1 k a l o i d s

332, 333,

347-349, 353 Chemically bonded s t a t i o n a r y phases c h a r a c t e r i z a t i o n 432-434 Chemisorption 225, 228 Cinchona a l k a l o i d s 269-285 analysis i n b i o l o g i c a l m a t e r i a l

269-273, 275-279, 283-285 analysis i n food and beverages

271. 272, 278, 285 analysis i n pharmaceutical preparat i o n s 272, 282, 283 C i r c u l a r dichroism detector 230, 290 Clavine a l k a l o i d s 360, 361. 367, 372 Cocaine metabolites 260, 265, 268 Colchicine and r e l a t e d a l k a l o i d s

417-419 Corrosion o f s t a i n l e s s s t e l l by mobile phase 226 Curare a l k a l o i d s 287, 288, 292 Detection methods 230, 231

301, 303, 304-310. 313-315, 326-328 Dynamic ion-exchange HPLC 227 Eburnane a l k a l o i d s 332, 334, 337-343, 347-353. 355. 356 Electrochemical d e t e c t i o n 230 E l l i p t i c i n e d e r i v a t i v e s 335, 336, 344 E l u o t r o p i c s e r i e s o f solvents f o r macroporous polymer r e s i n s 227 E l u o t r o p i c s e r i e s o f solvents f o r straight-phase chromatography (see v o l . 23A 5-7) E l u o t r o p i c s e r i e s o f solvents i n reversed-phase HPLC 224. 225 Ergometrine and decomposition products 357, 360 Ergot a l k a l o i d s 357-379 analysis i n biological material

357, 359, 362, 378-379 357, 358, 360, 361, 366. 368, 371-374

a n a l y s i s i n fungi

a n a l y s i s i n pharmaceutical preparations 357-360, 362, 366, 374-376 Ergotamine and decomposition products

357-359, 367 Ergotoxine a l k a l o i d s 357-361, 363-

368, 370-372 Fluorescence d e t e c t i o n 230 Free s i l a n o l groups i n s t a t i o n a r y phases 224 Glycoalkaloids 381-386

441

225, 229. 231

Guard columns

material

335, 354 Heroin metabolites 308 Harmane a l k a l o i d s HPLC-MS

231, 273. 291, 366, 368, 372

I d e n t i f i c a t i o n by using UV-absorption ratio

230. 237. 238. 302, 304

421-423 Indole a l k a l o i d s 331-379

Papaveraceae a l k a l o i d s

(see opium

297-330 287. 289. 290

P i l o c a r p i n e and decomposition products 421-423

Ion-exchange s t a t i o n a r y phases

432 I o n - p a i r HPLC 227, 228

P o l a r chemically bonded s t a t i o n a r y

characterization

phases

288. 290 I s o l a t i o n from b i o l o g i c a l material Ipecac a l k a l o i d s

388, 389 (see a l s o vol 51-58, 239-245)

. 23A

characterization

433. 434

Post-column d e r i v a t i z a t i o n 230,

249, 250-253,

290. 310, 360, 368 230, 251,

Pre-column d e r i v a t i z a t i o n

223. 287-330,

analysis i n biological material

288, 290, 296 a n a l y s i s i n pharmaceutical prepar a t i o n s 295, 296 a n a l y s i s i n p l a n t material

288.

290, 294, 295

252, 290 Pseudotropine a1 k a l o i d s

260-268

analysis i n b i o l o g i c a l m a t e r i a l

260, 265, 268 a n a l y s i s i n pharmaceutical prepar a t i o n s 264, 265

225, 226, 300

a n a l y s i s i n s t r e e t drugs Psilocybe a l k a l o i d s

analysis i n b i o l o g i c a l m a t e r i a l

357, 378, 379 a n a l y s i s i n drug seizures

357,

360, 366, 367, 371, 377, 378 Lumi-ergot a l k a l o i d s 360, 366 227, 235, 300, 316. 335, 354, 365, 426, 428

Macroporous polymer r e s i n s

Masking o f s i l a n o l groups

225, 226

331, 337, 343,

344, 347-349, 353, 355, 356 P y r i d i n e a l k a l o i d s 241-248 analysis i n b i o l o g i c a l m a t e r i a l 242, 246 analysis i n drugs of abuse analysis i n plant material

241-248

analysis i n plant material Quaternary alkylamines

Normal-phase s t a t i o n a r y phases

Quaternary amnonium compounds

434, 435

On-column preconcentration 231, 232,

359 223, 297-330

analysis i n biological material

303, 305, 308-310, 324, 325 analysis i n opium and p l a n t

245 243.

246 Pyrrolizidine alkaloids

297-330 Morphine metabolites 299. 305

Morphinan a l k a l o i d s

Opium a l k a l o i d s

260,

261. 266. 267

LSD and d e r i v a t i v e s

characterization

264

analysis i n plant material

Long chain a l k y l amines as m o d i f i e r i n solvents

305, 306, 310, 317,

Phenylethylamines

HPLC 223

Isoquinoline alkaloids

rations 322-324 alkaloids)

Imidazole a l k a l o i d s Ion-exchange

299, 300, 305, 306,

316, 317, 320, 321 a n a l y s i s i n pharmaceutical prepa-

241,

245 425, 428 250,

251, 254, 255, 287, 288, 290, 292, 336, 337. 425-432 Quinidine metabolites 269-273, 275-277, 279 Quinoline alkaloids

269-285

analysis i n biological material

269-273, 275-279, 283-285

44 2

analysis i n food and beverages 271, 272, 278, 285 analysis i n pharmaceutical preparations 272, 282, 283 Rauwolfia alkaloids 331. 335, 338, 341, 344 Reversed-phase HPLC 224-227 Reversed-phase ion-pai r HPLC 227, 228 Reversed-phase stationary phases characterization 432-433 Sample preparation 231, 388, 389, (see a l s o vol. 23A 51-58, 239-245) Senecio-a1 kaloids 241, 243, 244 S i l i c a gel 228, 229 S i l i c a gel pH 226, 229 S i l i c a gel stationary phases characterization 434, 435 Soap chromatography 227 Solanurn a1 kaloids 381-386 Solvent s e l e c t i v i t y (see vol. 23A 5-8) Stationary phases characterization 432-435 Steroidal a1 kaloids 381-386 Straight-phase HPLC 228 Straight-phase ion-pair HPLC 228 Strychnos a1 kaloids 331, 338, 341, 347-349, 355 Symphyturn-alkaloids 242, 244. 245 Tabernaemontana a1 kaloids 338 Terpenoid indole a1 kaloids 331-356 analysis i n biological material 332, 335, 337, 344, 351, 352, 354 analysis i n Pharmaceutical preparations 332, 337, 338, 343. 344, 350, 351, 354

analysis i n plant material 332, 335. 338, 343, 344, 347-349, 353, 355, 356 Theophyl l i n e analysis i n biological materials 387-394, 399-401, 407-413 metabolites 387-394, 399-401, 407413 Tobacco alkaloids 241-248 analysis i n biological material 242, 246 analysis i n drugs of abuse 245 analysis i n plant material 243, 246 Trace enrichment 231, 232, 359 Tropane alkaloids 249-268 Tropine alkaloids 249-259 analysis i n biological material analysis i n pharmaceutical preparations 249-252, 254. 255, 257259 analysis i n plant material Uric acid derivatives 387, 390, 391, 399-401 veratrurn alkaloids 383 Vinca alkaloids 332, 334, 337-343, 347-353, 355, 356 Xanthine alkaloids 387-414 analysis i n biological materials 387-394, 399-401, 407-413 analysis i n food and beverages 389, 393, 394, 402, 414 analysis i n pharmaceutical preparations 389, 393, 394, 401, 404407

443

COMPOUND INDEX

Acetaminophen (paracetamol)

195,

196, 237, 304, 307, 309, 391, 401 Acetazolamide

387, 388, 390

14-Acetoxycodeinone

Acetyl thebaol

137

Aci-ergocristine

372

Aci-ergocristinine

113

Aci-ergotamine

14-Acetoxydi hydrocodeinone

113

14-Acetoxydi hydrodesoxycodei ne

Aci-ergotaminine 112

Aconitine Acridine

335

Acronine

93, 94

Acetylcephalotaxine

Acronycidine

213, 214

94

Acronycine

94

134. 137, 261, 301-304, 308,

Adiphenine

255

314, 315

Agroclavine

17, 113, 129, 130-

A c e t y l d i hydrocodeinone 307 0-acetyldihydromorphine

302

4-0-acetyl-3,6-dimethoxy-5-( 2-Nmethylacetamido) - e t h y l phenantrene 137 0-acetylethylmorphine Acetyl intennedine

-

Acetyllycopsamine N-acetylmescaline

159, 168, 333, 338,

353 Ajmaline

16, 23, 161, 162 159, 168, 333

Akuamnigine N-oxide

31, 32

Alcuronium A1 l a n t o i n

307

31, 32

Acetyl-lycopsamine N-oxide

Ajmalicine

Akuamnigine

131, 132

6-0-acetyl isopropylmorphine

23, 24, 176, 177,

361. 367, 372

131, 132

0-Acetyldi hydrothebainone

358

240, 317, 415, 416

7-Acetoxy-l-methoxymethyl-l,2dehydro-&r-pyrrolizidine 30 Acetylcodeine

372

358

245

168

288, 337 245

Allococaine

261

A1 lopseudococaine

261

A1 lopseudocodeine

112

1-A1 l y l theobromine

97

188

5, 113, 129,

Alphenal

5, 17, 19,

Alstonine

239, 338, 341, 355

22, 113, 114, 129-137, 261,

Ambelline

151, 152

299. 302, 304, 307-310, 313-

Amethocaine

315, 318

4 - h i noacetophenone

3-0-acetylmorphine 131, 132, 135 6-0-acetylmorphine

Alpinigenine

6-0-acetylmorphi ne glucuronide 136

306

307

Acetylprocaine

89

300. 301, 304,

308, 315

Aminophenazone

131, 132

a-Aminopyridine 35

132 Acetylsalicylic acid 237, 304, 401

Aminopyrine

35

304, 308

5-Aminoquinoline

3-0-acetyl -6-0-propionylmorphi ne

Amitriptyline Amobarbital

194, 198,

341 197

8-Aminopyridine

3-0-acetyl -6-0-propionyl d i hydro-

107

1-Amino-3-chl oro-4.6-benzenedisulfonamide

N-acetylprocainamide

morphine

200, 205

191

133, 134 126. 195, 196,

200. 205 Amphetamine

237-239,

261, 335

444

A m p i c i l l i n 387, 390, 391, 392 h y d r i c a i n e 261 Amylocaine 261, 304 Anabasine

16, 17, 34-39, 41, 46,

48, 243 Anagyrine

56, 58

Anatabine 34-36, 38, 39, 48, 243 7 - A n g e l y l h e l i o t r i d i n e 30 7-Angelyl retronecine 30 Angustidine 170 A n g u s t i f o l i n e 57, 58 Angustine 170 Angustoline 170 Anhalamine 97, 99, 100 Anhalidine 97, 99, 100 Anhalinine 97, 99, 100 Anhalonidine 97-100 Anhalonine 97, 99, 100 Anhydroplatynecine 30 Antazoline 239, 307 Antipyrine 198, 261, 272, 304,

308 Apoatropine 5, 61, 64, 68, 249-

251, 254, 255 Apocodeine 128, 129. 150, 301 Apanorphine 22, 23, 114, 115,

128, 129, 133, 143, 150, 301, 303, 310 Aposcopolamine 61. 65, 68, 69, 254 Apovincamine (and stereoisomers)

166, 334, 338, 339, 342, 343, 353 Apovincaminic a c i d 165, 166, 334, 353 Apovincaminic a c i d phenyl e s t e r (and stereoisomers) 166, 334, 338-

343, 353 Aprobarbi t a l 200. 205 Arbutin 161, 162 A r i c i n e 159 A r i zoni ne 289 Ascorbic a c i d 304, 315 Aspidocarpine 167 Aspidospermidine 167 Aspidospermine 16, 167

Atropic a c i d 65, 71, 250, 251,

254 Atropine (hyoscyamine) 5, 16, 17,

21, 36, 61-69, 71, 230, 239, 240, 249-252, 254, 255, 304, 307, 317, 335 Baeocystin 343 B a p t i f o l i n e 56 B a r b i t a l 304, 307, 313 B a r b i t u r i c a c i d 237 a-Belladonnine 251 W e l l a d o n n i n e 251 Benactyzine 255 Bendroflumethiazide 341 Benzocaine 73, 80, 261, 304,

307, 308 Benzoic a c i d 197, 391 Benzoylaconine 416 Benzoyldesoxyaconine 416 Benzoylecgonine 21, 22. 66,

74-77, 79, 80, 83, 84, 260, 261, 268 Benzoylecgonine n-ethyl e s t e r Benzoylecgonine n-propylester

79 78

Benzoyl hypaconine 416 Benzoyl jesaconine 416 Benzoylmesaconine 416 Benzoyltropeine 304 Benzphetamine 238 Benztropi ne 251, 307 Berberine 16, 148, 287, 288,

426 Boldi ne 147-149, 301 9-Bromoapovi ncami n i c a c i d 166 Bromocryptine 360 I-Bromodi hydrocodeinone 302 1-Bromodi hydrothebainone 302 Bromodi phenhydrami ne 307 10-Bromovi ncami ne 166, 334, 342 11-Bromovincamine 166, 334, 342 8-Bromoxanthine 390 Brompheniramine 239 Bromural 237 Brucine 16, 162-164, 176, 239,

446

305, 331, 335, 338, 341, 355 Bufotenine 16, 156-159 Bulbocapnine 150 Butacaine 261, 307

278, 280, 317, 335 Cineverine 57 cis-Cinnamoylcocaine

Butani 1i c a i n e 261

21, 73,

74, 260-262 trans-Cinnamoylcocaine

Caffeine 16, 17, 24, 57, 58, 80, 131, 133, 134, 137, 237, 239, 240, 261, 304, 307, 308, 313315, 317, 318, 387, 389-395, 399-402 Candicine 98 Carnegine

239, 270-272, 275, 277,

97, 100, 289

Catalauverine 57 Catharanthine 333, 353 Cephaeline 239, 240, 287, 288, 290. 317 Cephalosporin 387, 389, 390 Cephalotaxine 213, 214

21,

73, 74, 260-262 Cocaine 19, 21, 22, 62, 7381, 83, 130, 238, 239, 251, 260-262, 268, 304, 307, 308, 313 Cochlearine 66 Codeine 5, 19, 22, 23, 62, 64, 77, 112-120, 123-127, 129-137, 142-144. 195, 200, 201, 237-240, 261, 299, 301-304, 306-310, 313-318, 354 Codeine methyl e t h e r Codeine N-oxide 299

112

Cephalotaxinone 213, 214 a-Chaconine 185, 186, 381-385

Codeinone

B-Chaconine 385

Col c h i ceinamide 417 Colchicine 16, 417, 419

185, 186, 381, 383-

Chanoclavine I

177, 361, 372

Chanoclavine I1 177, 361, 372 Chlordiazepoxide 237, 307

113

Colchicos i d e 41 7 a-Colubrine 163, 341 B-Colubrine 163, 341 Conessine 16

2-Chloroaporphine 147 Chloroprocaine 261 Chloroquine 89

Conhydrine 54 y-Coniceine 54

8-Chlorotheophyll i n e 188, 387,

Coniine

389, 391, 392, 399, 400

54

Coptisine 288 Coreximine 148, 149

Chlorothiazide 341 8-Chloroxanthine 390

Corydine

Chlorpheniramine 65, 197, 239,

Corynantheidine

307 Chlorphentennine 42 Chlorproethazine 78

Corynoceine 170 Corynoxi ne 170

Chlorprornazine 307 Chlorthioxantone 197 Chondrocurine 292 C i l i a p h y l l i n e 170 Cinchocaine 261

Costaclavine 177 Cotarnine 114, 307 Cotinine 34-36, 42, 43, 45-47,

Corytuberine

Cinchonidine 16, 87, 88, 90, 91, 239, 270-272. 275-280 Cinchonine

16, 87-91,

148, 149

129,

170

147

242, 243 Coti n i ne-1-oxide Creatinine 399

46

Criglaucidine 151, 152 Crinamine 151, 152

446

Crispatine 31 C r i w e l l i n e 151, 152 Cryptopine 113, 299, 306, 313, 314 Cupreidine 275 Curarine 292 Curine 292 Cyclizine 195, 196, 261 Cycloclavine 177 Cycl omethycai ne 261 Cyclopamine 383 Cyheptamine 200-202 C y t i s i n e 55, 56, 58 Deacetylbowdensine 151, 152 Deacetyl p y r i f o l i d i n e 167 Oecy 1benzy 1dimet hy 1amnoni um 428 Decylpyridinium 428 1,2-Dehydroaspi dospermidine 167 12-Dehydrodeacetylpyrifol i d i n e 167 Dehydroglaucine 288 5 A -Dehydrolupanine 58 All-Dehydrolupanine 58 3-Dehydroreserpine 335, 341, 344 Dehydrothal icarpine 293

Desoxycodeine 112 Desoxymorphine 114 Desoxyretronecine 30 Dextromethorphan 238, 239 Dextropoxyphene 307 Oiaboline 162, 163, 341 Oiacetyldi hydromorphine 131, 132 Oiacetylnalorphine 138 Diacetylnorcodeine 137 Diazepam 237, 307, 308 5.6-Di benzoquinol i n e 335 N.N-dibenzylbenzamide 261 1,7-Di bromodi hydrocodei none 1,7-Di bromodihydrothebainone 302 Dibucaine 125 Dichloralphenazone 237 Diethazine 307 N.N-diethyl tryptamine 155 Oihydroacetylcodeine 132 Dihydrocinchonidine 88, 91 Dihydrocinchonine 88, 91 Dihydrocodeine 112-114, 131, 132, 237, 238, 299, 302. 307 Dihydrocodeinone 112, 114, 126, 132, 237, 238, 302, 307

Oehydrovincamine (and stereoisomers) 334, 342, 353 Oemethoxyaspidospermi ne 167 Demethylaspidospermine 167

D i hydrocorynantheine 168, 333 D i hydrodesoxycodeine 112 D i hydrodesoxythebainone 112 Dihydroergocornine 178, 182, 183, 358-360, 363-365 Oihydroergocorninine 178

1-Demethyl col c h i c i ne 419 2-Oemethyl c o l c h i c i ne 419 3-Oemethyl c o l chi c i ne 419 11-Demethyl e l 1ip t i c i ne 354 Demissine 24, 185, 186 Oeoxyharringtonine 213, 214 Desacetoxyvi nbl a s t i ne 333, 353 N-desacetylcolchicine 419 Deserpidine 161, 341 Oesmethyl cephal o t a x i none 213, 214 Desoxyaconitine 416

Dihydroergocristine 177, 178, 182, 183, 231, 358, 360, 363-365, 371 Dihydroergocryptine 177, 178, 182, 183, 358-360, 362-365, 371 D i hydroergocryptinine 178 Dihydroergotamine 179, 357-360. 362-364 D i hydroergotaminine 179 Dihydroergotoxine 177, 178, 182, 183, 357-360, 362-365, 371 D i hydroerysodi ne 153, 154

441

D i hydroethylmorphine

132

O i hydrogambi r t a n n i ne

170

D i hydrohydroxymorphone

Oihydroisocodeine

cis- 1.2-Dimethyl -4- hydroxy-6.7-

dimethoxytetrahydroisoquinoline

309

112

Dihydrolysergic a c i d amide a-Dihydrolysergol

182

100

177

N,N-dimethyl-4-hydroxy-3-methoxy-

Dihydrometanicotine 35, 46 Dihydromorphine

phenyl e t h y l ami ne

114, 131, 132.

tetrahydro-6-carboline

113, 114, 131,

2,6, -dime thy1 p i p e r i d i ne

132, 237, 238, 302, 307, 309 Dihydroquinidine

N.N-dimethyl tryptamine

88, 89, 91, 239,

1,3-Dimethyluric a c i d

88, 91, 272, 278,

Oihydrosinomenine

1,7-Oimethyluric

113

Dihydro-6a-thebainol methyl ether

387, 390, 391

acid

401

3,7-Dimethyluric a c i d

401

1,7-Dimethyl xanthi ne (paraxanthi ne)

112

387, 390-395, 399, 401

Dihydro-6pthebainol methyl e t h e r 112 Dihydrothebainone

Diphenhydramine 113. 302

Diphenylamine

Dihydrothebainone methyl e t h e r 6,7-Dihydrovindesine

112 113

2.1 1 - D i hydroxy- 10-methoxy-N-methyl aporphine

-

150

3.6-di hydroxytropane and esters

62,

63

238. 304, 307 197

Diphenyl hydantoin Dipipanone

332

8,14-Di hydroxydihydrocodeinone

237

261

D i propionyl d i hydromorphi ne Dipropionylmorphine a,a'-Dipyridyl

35

a ,B' - D i p y r i d y l

35

131, 132

114, 131, 132

Dodecyl benzyl dimethyl amnoni um

Dimenhydrinate Dimethocaine

237

Dodecylpyridinium

261

Dopamine

2 ,lo-Dimethoxy-11-hydroxy-N-methylaporphine

150

98, 100

237, 238 213, 214

Dyphylline (dihydroxypropyltheophylline)

3,5-0imethoxy-4-hydroxyphenyl e t h y l -

428

428

100, 287, 292

Doxylamine Orupacine

3,4-Dimethoxy-5-hydroxyphenylethyl-

amine

155-159, 175,

399-401

280

amine

156, 157 54

237, 238, 353

269-273, 275, 280 Dihydroquinine

100

1.2-Dimethyl-6-methoxy-1,2,3,4-

299, 302, 307, 309, 310 Dihydromorphinone

100

trans-1,2-Dimethyl-4-hydroxy-6,7dimethoxytetrahydroisoquinoline

187, 189, 387, 388,

390-392, 399-400

98, 100

3,4-Dimethoxyphenylethylamine

98-

6,7-Dimethoxy-1 ,2 ,3,4-tetrahydroisoquinoline

0,N-dimethylanhalonidine

198 100

N,N-dimethylanil i n e 7 N .N-dimethyl-3,4-dimethoxyphenyl100

167

21, 22, 66, 74-77, 84,

262 Echimidine

289

Dimethyl ami noanti p y r i n

ethylamine

Eburnamonine Ecgonine

100

31

Echimidine N-oxide Echinatine 30 Echiumine

31

Ectylurea

237

Edrophonium

429

244, 245

448

Ellipticine

335, 336, 344, 354,

426 Elymoclavine 177. 361, 367. 372 Emetine 239. 240. 287, 288, 290,

317 5, 16, 22, 103-108, 126, 200, 237-240, 261, 304, 317 19-Epia jmal i c i n e 168 Epinephrine 239 E p i q u i n i d i n e 88, 91. 280 Epiquinine 88, 91, 280 Epirauvanine 159 Ergocornine 178-181, 358-361, 363-365. 367, 370-372 Ergocorninine 178, 358. 361, 363365, 367, 370-372 E r g o c r i s t i n e 178-181, 358. 360. 361, 363-365, 367, 370, 372 E r g o c r i s t i n i n e 178. 358, 361, 363-365, 370, 372 Ergocryptine 178-181, 358-361. 363365, 367. 370-372 E r g o c r y p t i n i n e 178. 358. 360, 361, 363-365, 367, 370-372 Ergometrine 24, 176, 357, 358, 360. 361, 363, 365. 366. 370-372 Ergometrinine 176, 358. 360. 361, 365. 370-372 Ergosine 179-181, 358-361. 367, 370 Ergosinine 360, 361, 370 Ergostine 179-181, 361 E r g o s t i n i n e 361 Ergotamine 179-181, 250. 255, 357-368. 370, 372 Ergotaminine 179, 255, 358. 361367, 370, 372 Ergothioneine 360 Ergotoxine 178-181, 357-367. 370-372 E r g o t r a t e 367 Erysodine 153, 154 E r y s o f l o r i n o n e 153. 154 E r y s o l i n e 153, 154 Ephedrine

153, 154 153, 154 Erysosalvine 153. 154 Erysosalvinone 153, 154 E r y s o t i n e 153, 154 Erysotinone 153. 154 E r y s o t r i n e 153, 154 Erysovine 153, 154 E r y t h r a l i n e 153, 154 E r y t h r a t i d i n e 153, 154 E r y t h r a t i n e 153, 154 E r y t h r a t i n o n e 153, 154 E r y t h r a v i n e 153, 154 a - E r y t h r o i d i n e 153, 154 8 - E r y t h r o i d i n e 153. 154 Ethaverine 239 Ethinamate 238 Ethoheptazine 238 Ethopropazine 307 Ethylmorphine 112, 114. 116, 123, 128, 131, 132, 135. 137, 237, 238, 240, 301, 303. 307, 317 N-ethyl normorphi ne 123 N-ethyl n o r n i c o t i ne 47 Europine 30 Erysonine Erysopine

197 177. 361, 372 Fluoranthene 200, 202, 203 F l u o r e s c e i n 237 Flurazepam 237 N-formyldihydrothebainone 302 Formyl l e u r o s i ne 333 Fenalamide

Festuclavine

N-formyl-1-methyldi hydrothebainone

302

N-formyl -norni c o t i ne Fulvine

Fumigaclavine A Fumigaclavine B Gambi r ine

176, 177 177

168

Gambi r t a n n i ne G i g a n t i n e 289 Glaucine

243

31

288

170

449

Gluthethimide 192, 237 Gnoscopine 306 Gramine 16 Grantianine 31 H a n a l i n e 170, 354 Hatmalol 354 Hannan 170 Harmine 170. 354 Harmol 354 Harringtonine 213, 214 Heleurine 30 Heliosupine 31 H e l i o t r i d a n e 30 H e l i o t r i d i n e 30 H e l i o t r i n e 30 H e l i o t r o p i n e 24 Heptabarbital 200-204, 206 Hernandaline 293 Hernandal i n o l 293 Hernandine 147 Heroin 5, 17, 19, 111. 113, 114. 129-135, 137, 237239, 261, 297-309, 313315, 318 Hexadecylbenzyl dimethyl amnoni um 428 Hexadecylp y r i d i n i urn 428 Hexobarbital 191, 192, 200 1-n-Hexyl theobromine 188 H i rsuteine 168 H i r s u t i n e 168, 333 H o l s t i i n e 163 Hornatropine 16. 17, 36, 61. 62, 64, 65, 69, 71, 239, 240, 251, 252, 317 Homatropine methylbromide 66 Honwharringtonine 213, 214 Homoveratrylamine 97 Hordenine 98-100, 159 Hydrochlorothiazide 341, 343 Hydrocotarnine 289 Hydroxyamphetami ne 237, 238 11-Hydroxycephalotaxi ne 213, 214

14-Hydroxycodeine

113

14-Hydroxycodeinone 113 3-Hydroxycoti n i ne 46 14-Hydroxydesoxycodeine 112 5-Hydroxy-N,N-diethyl tryptamine 156 14-Hydroxydi hydrocodeine 113, 114 14-Hydroxydi hydrocodei none 113, 114, 132, 237. 238, 302, 307 14-Hydroxydihydrodesoxycodeine 112 14-Hydroxydi hydroi socodeine 113 14-Hydroxydi hydromorphinone 113, 114, 132, 237, 238, 302, 307. 309 14-Hydroxydi hydronorcodeinone 113

Hydroxydihydrothebainone 113. 302 4-Hydroxy-N ,N-dimethyl tryptami ne 156 5-Hydroxy-N ,N-dimethyl tryptami ne (bufotenine) 156-159 6-Hydroxy-N ,N- d i met h y l t r y p tami ne 156 7- Hydroxy- N ,N-d imet hy 1t r y ptami ne 156 6-Hydroxydopami ne 239 9-Hydroxyell i p t i c i n e 335, 336, 354 8-Hydroxyergotami ne 361 B-Hydroxyethyl theophyll i n e 187189, 387, 390-392, 399, 401 Hydroxyheliotridane 30 13-Hydroxylupanine 55-58 7- Hydroxy- 1-methoxyme thy1 -1,2dehydropyrrolizidine 29. 30 3-Hydroxy-4-methoxyphenyl e t h y l amine 100, 289

-

4-Hydroxy-3-methoxyphenylethylamine 100, 289

7-Hydroxy-l-methyl-l,2-dehydro-8u-pyrrolizidine

30

9-Hydroxy- Z-me t hy 1e 11ipt ic in iurn

336, 344

460

7-Hydroxy- 1-methyl ene-&a-pyrrol iz i dine

30

58

I s o l y s e r g i c a c i d 358, 370

7-Hydroxy- 1-methyl ene-85-pyrrol iz i dine

a-Isolupanine

30

I s o l y s e r g i c a c i d amide

1-Hydroxymethyl-1 .2-epoxy-&a-pyrrolizidine

30

Isolysergi c a c i d diethylamide (IsoLSD)

2-Hydroxymethyltropine

76, 77, 83

5-Hydroxy-N-methyl tryptamine 14-Hydroxymorphine

157

113

14-Hydroxymorphinone

173, 175, 360, 371

I s o l y s e r g i c a c i d dimethylamide 173, 175 I s o l y s e r g i c a c i d dipropylamide

113

173,

175

10-Hydroxy-N-n-propyl noraporphi ne 150

I s o l y s e r g i c a c i d ethylpropylamide 173, 175

11-Hydroxy- N-n-propyl noraporphi ne 150

I s o l y s e r g i c a c i d methylpropylami de 173, 175

8-Hydroxypropyl theophyll i n e (proxyphylline)

Isolysergol

187-189, 387,

391, 392. 400 3-Hydroxyquinidine

270-272, 275-

277, 279

177

Isomitraphylline

170, 333

Isomi t r a p h y l l i n e

N-oxide

I s o n ic o t i nami de

304

Isopenni c l avine

177

13-Hydroxysparteine

55, 56

Isopilocarpic acid

4-Hydroxystrychni ne

341

Isopilocarpine

14-Hydroxytetrahydrodesoxycodei ne 112

421, 422

Isopropyl a n t i p y r i n

195

170, 333

Hypaconitine 416

Isopteropodine

N-oxide

Hypoxanthine

I s o q u i n o l i n e 38

390, 395, 400

3-Isorauni t i c i n e Ibogaine 175 163, 341

Intermedine

31

333

I s o r e s e r p i 1 ine

159

3-Isoreserpine

341

I s o r e s e r p i n i n e 159 Isoretronal

31, 32

5-Isatropic acid

30

Isorhynchophylline

251

170, 333

anti-Isorhynchophyl l i n e N-oxide

I s o a j m a l i c i n e 159, 168, 333

170

Isoajmaline 162 Isoapocodeine

170

I s o r a u n i t i d i n e 159

I n d i c i n e 30 Integerrimine

170

16, 217, 421, 422

Isopteropodine

Icajine

358, 361,

367, 370

128, 129, 150, 301

3- Isobutyl-1-methylxanthine

200,

I s o r o t u n d i f o li n e I s o r u b ij e r v i n e

170 383

I s o s a l s o l i n e 289

205-208 Isochanoclavine I 177. 372

Isosetoclavine

177, 361, 367

Isochondrodendrine 292

Isosophoramine

58

Isocodeine

a-Isosparteine

55

5-Isosparteine

58

112

Isocorynantheidine

170

Isothebaine

Isocprynoxeine 170 Isocorypalmine

148, 149

3-Iso-19-epi-ajmal i c i n e Isoharringtonine

306

168, 333

213, 214

Jacobine

31, 244

Jacoline

31. 244

461

Jaconine

31, 244

Jacozine

31, 244

Javaphylline Jervine

Lysergic a c i d methylcarbinolamide 176

170

Lysergic a c i d methylpropylamide 173,

416

Lysergic a c i d monoethylamide

383

175

Jesaconitine

Lysergine 8-Ketosparteine Lamprolobine

58

Lysergol

58

177, 360, 361

Macromerine 98, 100

Lasiocarpine 31

Mandelic a c i d

Latifoline

Matrine

31

117, 306, 307

Laudanosine Leucinocaine

71

55, 58

Meclophenoxate

261

Mecloqualone

307 261

Leurosine 333, 353

Meconin

Levallorphan

Medazepam 200, 204

133, 237. 238

Levorphanol Lidocaine

237, 238

137

M e l i c o p i c i n e 94

45, 197, 198, 261,

Melicopidine

304, 308

94

Melicopine

94 238, 304, 307, 308

Lignocaine 42, 261, 307

Meperidine

Lobeline 237

Mephenesin 237

Lochnerine

Mephenoxalone

333, 353

Lophocerine Lophophorine

100

237

Mephenteramine 238

97, 99, 100

Mephentermine

LSD ( l y s e r g i c a c i d diethylamide)

237

Mepivacaine 193, 261

23, 173-177, 303, 357, 360,

Mesaconitine 416

362, 366-368, 371

Mescaline

Lumiergometrine 176

22, 97-101,

175, 237,

238, 290

Lumi-LSO 360, 366

Metanicotine 34-36, 41, 46

Lumi reserpine( 3,4,5,6-tetradehydro-

Meteloidine

reserpine) Lupanine

341

55-58

307. 308

Lycorine Lysergene

Methapyrilene

31, 32

Lycopsamine N-oxide

Methaqualone

245

Methazolamide

177, 361

Methicillin 176, 358,

Methocarbamol

360. 361, 367, 370, 371

Methohexital

218 387, 390, 392 237 237

156-159 N-( 2-methoxyethyl ) - n o r n i c o t i n e

362, 366-368, 371 Lysergic a c i d dimethylamide 173, 175 Lysergic a c i d dipropylamide 173, 175 Lysergic a c i d ethylpropylami de

237, 261, 307, 308

5-Methoxy-N ,N-dimethyl tryptami ne

Lysergic a c i d diethylamide (LSD) 23, 173-177, 303, 357, 360,

175

126, 238, 301, 304,

307, 308

151, 152

Lysergic a c i d amide

21, 66

Methadone 77. 130, 238, 304,

Lupinine 55,56,58 Lycopsamine

360

177, 361

173,

47

8-Methoxy-14-hydroxydi hydrocodei none 113

l-Methoxynethyl-l,2-dehydropyrrol izidine

29, 30

462

1-Methoxymethyl-1.2-epoxypyrrol i z i d i n e 30 5-Methoxy-N-methyl tryptamine 156, 157, 159 4-Methoxyphenylethylamine 99 5-Methoxytryptamine 155-157, 159 3-Methoxytyramine 98 10-Methoxyvincamine 342, 343 Methychlothiazide 341 Methylamphetamine 237, 238, 261, 335 Methylanabasine 35, 46, 47 0-methylanhalidine 99, 100 N-methyl anha i n i n e 97 0-methylanhalonidine 97, 99, 100 Methyl aposcopol ami ne 254 Methylatropine 251, 255 4-Methylbenzoic a c i d 251 N-Methylconiceine 54 N-Methylconi i n e 54 0-methylcorypalline 289 Methylcytisine 55, 56 Methylcytisine N-oxide 55 N-methyl -N-desacetyl col chi c i ne 419 1-Methyldihydrocodeine 302 1-Methyldi hydrocodei none 302 Methyldihydromrphine 114, 307, 309 1-Methyldi hydrothebainone 302

N-methyl-3,4-dimethoxyphenylethylamine 98, 100 N-methyl 7,8- d i methoxy- 1,2,3.4-

-

tetrahydroisoquinoli ne 289 Methylecgonidine 73, 262 Methylecgonine 79, 80 1-Methylenepyrrol i z i d i n e 30 N-Methylephedrine 240, 317 Methylergometrine 360 Methyl p-hydroxybenzoate 337, 354 cis-1-Methyl -4-hydroxy-6.7dimethoxytetrahydroisoqui no1ine 100 trans-1-Methyl -4-hydroxy-6,7-

dimethoxytetrahydroi soq u i n q l i n e 100 7- (N-methyl -N- hydroxyethy1)3-ami no-2-hydroxypropy1)t h e o p h y l l i n e 188 N-methyl-4-hydroxy-3-methoxyphenyl-

ethylamine 100 N-me t hy 1-4- hydroxy pheny 1e thy 1ami ne 100 N-methylmescal i n e 97, 99, 100 N-methyl-6-methoxy-7,8-methyl ene-

dioxy-l,2,3,4-tetrahydroisoq u i n o l i n e 289 2-Methyl -6-methoxy-1,2 ,3,4-tetrahydro-8-carboline 156, 157 N-methylmorphine 115, 143 Methylmyosmine 35 Methyl nicotineamide 35, 36 8-0-methyloxedrine 100 0-methylpellotine 97 Methylphenidate 237, 238 3-Methyl-3-phenylpiperidine 42 Methyl 3-pyridylacetate 47 Methyl N-( 3-pyridylacetyl ) g l y c i nate Methyl 4- (3-pyri dyl ) b u t y r a t e 46 Methyl 4-(3-pyridyl)-l-hydroxybut y r a t e 46 Methyl 4- (3-pyri dyl ) -4-oxobutyrate 46 Methylpyrrole 67 2-Methylquinoline 39, 335 N-methyl sal sol ine 289 Methylscopolamine 250, 251, 254 2-Methyl tetrahydro-8-carboline 156, 157 2-Methyl tetrahydroharmine 157 3' -0-methyl tetrahydropapaverol ine 292 4' -0-methyl tetrahydropapaverol ine 292 6-0-methyl tetrahydropapaverol ine 292 7-0-methyl tetrahydropapaverol ine 292 2-0-methyl tetrahydroxyberbine 292

453

3-0-methyltetrahydroxyberbine

292

10-0-methyl tetrahydroxyberbine

Neomycine 250

292

Neophytadiene

11-0-methyltetrahydroxyberbine 292 3-Methyl-5-triazolophtalazine 277

Neopine 112

5-Methyl tryptamine

159

Nicotine

N-methyltryptamine

156, 157, 159

Neostigmine

40

429

16, 17, 21, 33-48,

N-methyl tyramine

98-100

Nicotine-1 , l ' - d i o x i d e

0-methyltyramine

100

N i c o t i ne-1'-oxi de

1-Methyluric a c i d

387, 390, 391.

Nicotyrine

399-401 7-Methyluric a c i d 1-Methylxanthine

237

390, 391, 399, 401

N i trazepam 307

391. 399, 401

8-Nitroxanthine

199, 390, 391, 394,

399-400 387, 390, 391, 394,

399-401

390

N-noragroclavine Norapocodei ne

3-Methylxanthine

Norcarnegine

372

128 100

Norchanoclavine I

7-Methylxanthine

387, 390, 391, 394,

399, 401

372

Norchanoclavine I 1 Norcodeine

372

22, 23, 112, 114, 115,

Methysergide 360 M i t r a c i 1i a t i n e 170

Norcotinine

Mitragynine

170

N-nor-dihydrothebainone

Mitrajavine

159. 168

Norecgoni ne

Mitraphylline

126, 135, 143, 299

170, 333

M i t r a p h y l l i n e N-oxide

46

Norephedrine 170

104, 105, 107 94

Modaline 44, 45

Normelicopidine

94

Monocrotaline

Nomlicopine

Morphine

Nonorphine

29, 31 5, 12, 19, 22, 23, 111-125,

127, 129-137, 142-144, 237-239,

261,

298-306, 308-310, 313-318, 354 120, 121. 136,

299 22, 23, 115, 143,

299

136. 143, 299, 309, 310, 318 Normorphine glucuronide Nornicotine

54

Mu1dami ne

383

17, 34-39, 41, 46, 48, 243

Myosmine Nalorphine

114, 118. 119, 121-124,

136, 142, 299, 302, 307. 309, 310

104, 105, 107

Noscapine

5, 113, 114, 118, 126,

238-240, 299-301, 304, 306, 307, 308, 310, 313-318, 354 162, 163

N u t t a l i n e 58 N y l i d r i n e 237, 238

128, 237, 238

Nal trexone

128

Naphazoline

237, 238, 239

113, 240, 306. 307, 314,

315. 317 Neblinine

302

Norpseudoephedrine

Novacine

Narceine

136

17, 34-41, 46, 48, 243

Nortropine 66

Morphol i n e

Naloxone

94 22, 23, 114, 115, 135,

Noroxymorphone

Morphine N-oxide

302

262

Normelicopicine

Morphine glucuronide

46

46

17, 34-37. 39. 46

N i kethamide

3-Methyluric a c i d

237,

241-243, 307

Octacaine

261

Octadecylbenzyl dimethylamnonium 428 Octadecylpyridinium

167

Octahydronicoti ne

428 37

454

Oc topami ne

100

Oripavine

Phendimetrazine 42, 107, 238

302

Orthocaine

Phenethyl alcohol

261

Otosenine

31

69, 105, 200,

Phentermine

170

17-Oxolupanine

Phenylbutazone

58

113, 114, 132, 237,

Oxyethyl theophyll i n e

187, 189

237, 238, 239

Oxymorphone 113, 114, 132, 237,

Phenylethylamine

238, 302, 307, 309 Oxypropyl theophyl l i n e 17-Oxysparteine

Phenytoine

240, 317

Pholcodine

114

Physostigmine

187. 189

23, 166, 335, 337,

16, 24, 217, 218, 337,

Pilocarpine

354, 421, 422

P i 1ocerei ne

56, 58

Piminodine Piperidine

361, 368, 372

54, 67 16, 17, 19, 53

Piperine

288

100 238

16, 113, 114, 116, 118,

Piperocaine

261

125-128, 133, 164, 237-240, 288,

Platynecine

30

299-302, 304, 306-308, 313-318,

P l a t y p h y l l i n e 31 P o l y t h i a z i d e 341

354, 358 Paracetamol (acetaminophen)

195,

Pramoxine

200, 201

196, 237, 304, 307, 309, 391,

Prilocaine

261. 304

401

Primaquine

272, 279

Paspaclavine Pellotine

65, 237-239

P i l o c a r p i c a c i d 421, 422

Oxypyrronium 255

Papaverine

100

344, 354

Oxyphenoni um 255

Palmatine

237-239

Phenylpropanolamine

238, 302, 307 Oxymethazoline

178. 304

Phenylephrine

276

Palliclavine

304

Phenylanaline-proline lactam 179

Z-OXO-LSO 360

Oxycodone

Phenobarbital

317, 390. 391. 399

100

Oxogambirtannine

Oxprenolol

105, 237, 238

206. 207, 240, 304, 308,

Oxazepam 237 Oxedrine

337

Phenmetrazine

361

97, 99, 100, 289

Penniclavine

177, 361, 372

Pentazocine

237, 238, 307

Probenecid p r o p y l e s t e r Procainamide Procaine

200, 204

89, 387, 391

16, 73, 80, 129, 191,

237, 238, 261, 301, 304. 307,

Persedon 195

100

Peyophorine

Probenecid 204, 205

308, 315

Phenacaine 261

Promethazine

Phenacemide

N-propal g y l - 14-hydroxydi hydronorcodei

Phenacetin

237 193-195, 197, 198. 237,

304, 307, 308, 401 Phenaglycodol

237

Phenazocine 237. 238, 307 Phenbutrazate

307

Phencycl idene 307 Phencyclidine 237, 238, 304

none

238

113

l-Propin-(Z' ) - y l theobromine 7-Propin- ( 2 ' ) - y l theophyl 1i n e

188 188

3-0-propionyl-6-0-acetyldi hydromorphine

132

3-0-propionyl-6-0-acetylmorphine 132, 133

-

455

Propionylcodeine 131. 132 Propionyl di hydrocodeine 132 0-propionyldi hydromorphine 132 Propionylethylmorphine 131, 132 0-propi onylmorphi ne 132, 133 Propoxycaine 261 Propoxyphene 197 N-propyl ajmal i ne 337, 344 Propyl hexedri ne 238 Propyl -p-hydroxybenzoate 354 N-n-propylnorapocodeine 150 N-n-propylnorapomorphine 129, 150, 301 Protopine 306, 307 Proxymetacaine 261 Pseudococaine 261 Pseudocodeine 112 Pseudocodeinone 112 Pseudoecgonine 21, 66 Pseudoephedrine 22, 103-108 Pseudoheroin 131 Pseudomorphine 22, 23, 115, 309, 310 Pseudostrychnine 163, 341 Pseudotropine 63,66, 67 Psilocin 331, 337, 343, 344, 353, 355, 356 Psilocybin 175, 331, 337, 343, 353, 355, 356 Pteropodine 170, 333 Pteropodine N-oxide 170 Pyridine 34, 67 3-Pyridylacetic acid 47 N- ( 3-pyr i dy 1 acety 1 ) g 1 yc i ne 4 7 4-(3-Pyridyl)butyric acid 46 3-Pyridyl ethyl ketone 34, 36 4- ( 3-Pyridyl ) -4-hydroxybutyri c acid 46 3-Pyridyl methyl ketone 34, 35 4-(3-Pyridyl)-4-oxobutyramide 46 4-(3-Pyridyl)-4-oxobutyric acid 46 3-Pyridyl n-propyl ketone 17. 34-37 5-(3-Pyridyl )tetrahydrofuran-2one 46

Pyrifolidine 167 Pyrilamine 239, 304 Pyroclavine 177, 361 Pyrrocaine 261 Pyrrole 67 Pyrrolidine 67 Quinidine 17-19, 22, 87-91, 239, 269-273, 275-280, 301, 304, 307, 308 Quinidine-l0,ll-dihydrodiol 277 Quinidine N-oxide 276, 277, 279 Quinidinone (quininone) 88, 280 2'-Quinidinone 272, 275-277, 279 Quinine 17-19, 87-91, 129, 130, 134, 165, 191, 238-240, 269-273, 275, 277, 278, 280, 301, 304, 307, 308, 313, 315, 317, 335 Quinoline 44, 45 Ouinotoxine 88 Racemethorphan 132 Racemorphane 132 Raubasine 239 Raumitorine 159 Rauniticine 159, 168, 333 Raunitidine 159 Rauvanine 159 Rescinnamine 23, 160, 161, 335, 341 Reserpi 1 i ne 159 Reserpine 23, 160, 161, 239, 331, 332, 335, 341, 343, 344, 354 Reserpinine 159 Retamine 56 Retronecanol 30 Retronecine 30 Retroneci ne trachel anthate 30 Retronecine vi ri di f lora te 30 Retrorsine 31, 243, 244 Retuline 162, 163. Retusamine 31 Retusine 31 Rhombifoline 58 Rhynchociline 170 Rhynchofol i ne 170

466

Rhynchophylline 170, 333 Rhynchophylline N-oxide 170 R i ddel 1iine 31 Rinderine 30 Rosmarinine 31 Rotundifoline 170 Roxburghine C 170 Roxburghine D 170 Roxburghine E 170 Rubi j e r v i n e 383 Rubreserine 337 Salicylamide 195, 196, 237, 304, 307 S a l i c y l i c a c i d 304, 337, 354, 401 S a l s o l i d i n e 97, 289 Salsoline 289 Sal sol ino1 292 S a l u t a r i d i n e 306 Sandwichine 162 Sarracine 30 Sceleratine 31 Scopolamine 16, 17, 21, 36, 61-69, 71, 239, 240, 249255, 317 Scopolamine N-oxi de 251 Scopoline 21, 65-67, 69, 71, 249, 252 Scoulerine 148, 149 Sedormid 195 Senecionine 29, 31, 243, 244 Seneci phyl 1ine 31, 243, 244 Senkirkine 31 Serotonin

155, 156, 157 Serpentine 230, 239, 338, 341, 355 Setoclavine 177, 361, 372 Sinomenine 113 Sinomenine methyl e t h e r 113 Solamargine 384 Solanidine 185, 186, 382, 383, 385 Solanine 24. 185, 186, 381-385 Solasodiene 381, 384, 385 Solasodine 381, 383, 384

Solasonine 384 Sparteine 55-58 S p e c i o c i l i a t i n e 170 Speciofoline 170 Speciogyni ne 168 Speciophylline 170. 333 Speci ophyll ine N-oxide 170 Spectabiline 31 Spermostrychnine 163, 341 Strychnine 16, 126. 128, 130, 137, 162-165, 238-240, 279, 304, 307, 308, 313, 314. 317, 318, 331, 332, 335. 338, 341, 343, 344, 355 Strychnospermine 163 Stylopine 148, 149 Sulfacetamide 239 Sulfadiazine 387 Sulfamethoxazole 387, 392 Sulfanilamide 237, 240, 317 Supinidine 30 Supinine 30 Symphytine 31, 32 Symphytine N-oxide 244, 245 T a r t a r i c a c i d 304 Tetrabutylamnonium 428 Tetracaine 73, 260, 261, 304, 308

Tetradecylbenzyldimethylammonium 428 Tetradecyl pyridinium 428 Tetrahydroalstonine 159, 168, 239, 333, 353 Tetrahydroberberine 148 Tetrahydrodesoxycodeine 112 Tetrahydroharmine 157 Tetrahydropapaveroline 292 Tetrahydropiperine 53 1,2,9,1O-tetrahydroxyaporphi ne 148, 149 1.2,1O,ll-tetrahydroxyaporphi ne 148, 149 2,3,9,10-Tetrahydroxyberbine 148, 149, 292

467

2,3,10,11 -Tetra hydroxyberb ine 148, 149, 292 Tetrahydrozoline 237, 238 T e t r a p h y l l i n e 159, 162 T e t r a p r o p y l a m n i u m 428 Thalicarpine 290, 293 Thebaine 113, 114, 118, 126, 127, 133, 239, 299-301, 304-307, 314-316, 318 Theobromine 24, 187-189, 199, 200, 203, 204-206, 207, 237, 240, 387, 390, 391, 395-402 Theophylline 19. 24, 105, 187190, 199, 200, 202-208, 237, 239, 275, 304, 307, 387-395, 399-402 Thermopsine 56, 58 Thiamylal 237 Thiobarbi t a l 200, 202 Thiopental 237 Thioridazine 238 5-Tomatidenol 383 Tomatidine 383 Tomatine 186, 383 Tomati 11i d i ne 383 T r i b u t y l m e t h y l a m n i u m 428 Trichocereine 98-100 T r i g l a u c i n e 151, 152 Z,lO,ll-Tri hydroxy-#-methyl aporphine 150 2,4,6-Trimethylpyridine 335 1,3,7-Trimethyluric a c i d 401 T r i pel ennami ne 304 Tripropylbutylamnonium 428 T r i s u l f a p y r i m i d i n e 390 Tropacocaine 304 Tropanol 67 Tropic a c i d 21, 65, 68, 69, 249-251, 254 Tropi cami de 239 Tropine 16, 17, 21, 62, 63, 66, 67, 249, 252 Tropinone 16, 17

Tryptamine 155-159 Tubocurarine 287, 288, 290, 292, 426 Tyramine 98-100, 155, 335 Uncarine A 170 Uncarine B 170 Uncarine F 170, 333 Uncarine F N-oxide 170 U r i c a c i d 390, 391, 399-401 Veramine 303 Verarine 383 Veratramine 383 V i n b l a s t i n e 333, 353 Vincamenine 166, 334, 338, 339, 341, 342, 343, 353, 356 Vincamine (and stereoisomers) 23, 165, 166, 332, 334, 337-343, 353, 355 Vincaminic a c i d (and stereoisomers) 166, 334 Vincaminic a c i d e t h y l e s t e r (and stereoisomers) 166, 334, 338340, 342, 343, 353 Vincamone (and stereoisomers) 338-343, 353. 356

334,

Vincanole (and stereoisomers) 166, 334, 338, 339, 342, 343, 353, 356 V i n c r i s t i n e 333, 353 Vindesine 332 Vindoline 333, 353 V i n d o l i n o l 333 Vindorosine 333 Vinpocetine 165, 337 Vomicine 162. 163, 341 Xanthine 199, 390, 391, 395, 39940 1 Xylometazoline 238, 239 Yohimbine

239, 335, 354

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