Analytical Profiles of Drug Substances and Excipients Volume 6

Analytical Profiles n

Of

Drug Substances Volume 6 Edited b y

Klaus Florey The Squibb Institute for Medical Research New Brunswick, New Jersey Contributing Editors

Norman W. Atwater Salvatore A. Fusari Glenn A. Brewer, Jr. Bruce C. Rudy Bernard Z.Senkowski Jack P. Comer

Compiled under the auspices of the Pharmaceutical Analysis and Control Section Academy of Pharmaceutical Sciences

Academic Press New York San Francisco London A Subsidiary of Harcourt Brace Jovanovich. Publishers

1977

EDITORIAL BOARD Norman W. Atwater Jerome I. Bodin Glenn A. Brewex, Jr. Lester Chafetz Edward M. Cohen Jack P. Comer Klaus Florey Salvatore A. F h u i

Erik H. Jemen k e n T. Kho Arthur F. Michaelis Gerald J. Papariello Bruce C. Rudy Bernard 2. Senkowski Frederick Tiehler

Academic Press Rapid Manuscript Reproduction

COPYRIGHT 0 1977, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWI

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 70-1 8 7 2 5 9

ISBN

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PRINTED IN THE UNITED STATES O F AMERICA

AFFILIATIONS OF EDITORS AND CONTRIBUTORS

H. Y. Abooul-Enein, Riyadh University, Riyadh, Saudi Arabia I. M. Asher, Food and Drug Administration, Washington, D.C. N. W.Atwuter, E. R. Squibb and Sons, Princeton, New Jersey S. A. Benezra, Burroughs Wellcome Co., Greenville, North Carolina

J. I. Bodin, Carter-Wallace Inc., Cranbury, New Jersey G. A. Brewer, The Squibb Institute for Medical Research, New Brunswick, New Jersey

L. Chufetz, Warner-Lambert Research Institute, Morris Plains, New Jersey G. P. Chrekian, Lederle Laboratories, Pearl River, New York

P. J. Cloes, University of Leuven, Leuven, Belgium E. M. Cohen, University of Southern California, Los Angeles, California J. L. Cohen, University of Southern California, Los Angeles, California

J. P. Comer, Eli Lilly and Company, Indianapolis, Indiana M. Dubost, R h h e Poulenc, Vitry-sur-Seine, France M. G. Ferrunre, Schering-Plough Corp., Bloomfield, New Jersey

K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey S. A. Fusuri, Parke, Davis and Company, Detroit, Michigan

vii

AFFILIATIONS OF EDITORS A N D CONTRIBUTORS

E. H.Jensen, The Upjohn Company, Kalamazoo, Michigan B. T. Kho, Ayerst Laboratories, Rouses Point, New York B. Krei1g;;rd. Royal Danish School of Pharmacy, Kobenhagen, Denmark

A. F. Michuelis, Sandoz Pharmaceuticals, East Hanover, New Jersey G. W. Michel, The Squibb Institute for Medical Research, New Brunswick, New Jersey

G. J. Pupriello, Wyeth Laboratories, Philadelphia, Pennsylvania R. Rucki, Hoffman-LaRoche, Inc., Nutley, New Jersey B. C. Rudy,Burroughs Wellcome Co., Greenville, North Carolina

W.C. Suss, Parke, Davis and Company, Detroit, Michigan R. E. Schwmer, Eli Lilly and Company, Indianapolis, Indiana G. Schwrtzmn, Food and Drug Administration, Washington, D.C.

B. Z. Senkowski, Hoffmann-LaRoche, lnc., Nutley, New Jersey

F. Tishler,CibaGeigy, Summit, New Jersey USASRG, Food and Drug Administration, Washington, D.C.

H. Vanderhaeghe, University of Leuven, Leuven, Belgium

C. K. Ward, Eli Lilly and Company, Indianapolis, Indiana D. B. Whigun, The Squibb Institute for Medical Research, New Brunswick, New Jersey

W. C. Window, Hoffmann-LaRoche, Inc., Nutley, New Jersey R. D. G. Woolfenden, The Squibb Institute for Medical Research, Moreton, Wirral, England

V. Zbinovsky, Lederle Laboratories, Pearl River, New York

viii

PREFACE Although the official compendia list tests and limits for drug substances related to identity, purity, and strength, they normally do not provide other physical or chemical data, nor do they list methods of synthesis or pathways of physical or biological degradation and metabolism. For drug substances important enough to be accorded monographs in the official compendia such supplemental information should also be made readily available. To this end the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences, has undertaken a cooperative venture to compile and publish Analytical Profiles of Drug Substances in a series of volumes of which this is the fifth. The concept of analytical profiles is taking hold not only for cornpendial drugs but, increasingly, in the industrial research laboratories. Analytical profiles are being prepared and periodically updated to provide physicochemical and analytical information of new drug substances during the consecutive stages of research and development. Hopefully, then, in the not too distant future, the publication of an analytical profile will require a minimum of effort whenever a new drug substance is selected for cornpendial status. The cooperative spirit of our contributors has made this venture possible. All those who have found the profiles useful are earnestly requested to contribute a monograph of their own. The editors stand ready to receive such contributions.

Klaus Florey

ix

AMPHOTERICIN B

Irvin M. Asher George Schwartzman and the USASRG *

*The U.S. Antibiotics Standards Research Group (USASRG) is an ad hoc collaboration of antibiotics researchers, a t the U.S. Food and Drug Administration and other Public Health Service Laboratories. Contributors t o this monograph include T. Alexander (BD) I. Asher ( 0 s ) B. Baer (NIH) B. B a r o n (BD) W. Benson (BD) W. Brannon (BD) J. Blakelp (BD) R. Bradky (NM)

M. Bunow (NIH)

G. Schwartzman (BD)

S. Delgado(BD) V. Folen (BD) C. Graichen (BF) R. Gryder ( 0 s ) I. Levin (NIH) M. Maienthal (BD) G. Mazzola (BF)

E. Sheinin (BD) B. Smith (EDRO) J. Staffa(0S) J. Taylor (BD) L. Wayland (BD) A. Wong (NIH) C. Zervos(0S)

The USASRG was formed at the request of P. Weiss, the National Center for Antibiotic Analysis, FDA, and is presently coordinated by the Office of Science, FDA. Individual contributions are referenced where possible.

2

IRVlN M. ASHER e t a / .

TABLE OF CONTENTS 1, Description 1.1 Drug Properties 1.2 Chemical Properties 1.3 The U.S. Standard 1.4 Chemical Composition 1.5 Structure 1.6 Physical Description 2. Physical Properties 2.1 Thermal Properties (DTA, TGA) 2.2 X-Ray Powder Diffraction 2.3 Solubility 2.4 Acid-Base Properties 2.5 Aggregation 3. Spectral Properties (Optical) 3.1 Ultraviolet Absorption 3.2 Infrared Absorption 3.3 Raman Scattering 3.4 ORD, CD, Specific Rotation 3.5 Fluorescence 4 . Spectral Properties (Other) 4.1 Proton NMR 4.2 13C-NMR 4.3 Mass Spectrometry 5. Chromatography 5.1 Paper 5.2 Thin Layer 5.3 High Pressure Liquid 5.4 Gas 5.5 Electrophoresis 6. Isolation 7. Stability 8. Antimicrobial Properties and Assays 9. Amphotericin A 1. DESCRIPTION 1.1 Drug Properties Amphotericin B is a macrocyclic, polyene antibiotic produced by streptomycetes nodosus (M4-575). It was originally isolated from a soil culture from the Orinoco River region, Venezuela (1). Used topically as a cream, or parenterally as a Na-desoxycholate suspension (Fungizone), it is effective against a broad variety of fungi and yeasts, and some protozoans (1-3; see Section 8 ) . The possibility that Amphotericin B combines with

AMPHOTERICIN B

3

c h o l e s t e r o l t o form i o n - t r a n s p o r t i n g c h a n n e l s a c r o s s c e l l membranes i s b e i n g w i d e l y i n v e s t i g a t e d (4-6). The a b s e n c e o f membrane s t e r o l s would t h u s e x p l a i n t h e i n a b i l i t y o f Amphot e r i c i n B t o a f f e c t b a c t e r i a l growth. I n canine experiments (7) , o r a l l y a d m i n i s t e r e d Amphotericin B induced a 20-45% r e d u c t i o n i n serum c h o l e s t e r o l , s u g g e s t i n g a p o s s i b l e f u t u r e r o l e as a h y p o c h o l e s t e r o l e m i c a g e n t . Amphotericin B h a s a l s o b e e n used (8) t o t r e a t c a n i n e 30% r e d u c t i o n i n gland s i z e ) . p r o s t a t i c hyperplasia However, t h e t o x i c i t y of t h e b i l e s a l t complex ( 9 , l O ) may d i s c o u r a g e s u c h a p p l i c a t i o n s i n humans. Work on less t o x i c d e r i v a t i v e s i s underway ( 3 ) . I n mice, i n t r a p e r i t o n e a l LD5ois 280 mglkg f o r Amphotericin B ( 3 , 1 1 ) , 8 8 mg/kg f o r Fungizone and 1320 mglkg f o r t h e m e t h y l e s t e r . The c o r r e s p o n d i n g i n t r a v e n o u s dosages a r e o v e r an o r d e r of magnitude lower ( 3 ) . (d

1.2

Chemical P r o p e r t i e s Amphotericin B i s an a m p h o t e r i c , m a c r o c y c l i c h e p t a e n e w i t h a mycosamine s u g a r head group. I t y i e l d s a v o l a t i l e b a s e i n c o n c e n t r a t e d NaOH and c a n b l e a c h KMnO4 o r Br2-CC14 (1). Its o r i g i n a l s e p a r a t i o n w a s b a s e d on i t s s o l u b i l i t y p r o p e r t i e s (1; see S e c t i o n 6 ) . Amphotericin B i s a p a r t i c u l a r l y d i f f i c u l t a n t i b i o t i c t o c h a r a c t e r i z e a n a l y t i c a l l y . I t i s i n s o l u b l e i n many solvents (Section 2 . 3 ) . Vibrator grinding dramatically a f f e c t s X-ray powder d i f f r a c t i o n p a t t e r n s ( S e c t i o n 2 . 2 ) and infrared absorption s p e c t r a (Section 3.2). pH d r a m a t i c a l l y a f f e c t s ORD and s p e c i f i c r o t a t i o n ( S e c t i o n 3 . 4 ) . H20 o r C02 ( o r b o t h ) may b e a s s o c i a t e d w i t h t h e l a t t i c e ( S e c t i o n 1 . 4 ) . Such c o n t i n g e n c i e s have l e d t o i r r e p r o d u c i b l e r e s u l t s and c o n f l i c t s i n t h e l i t e r a t u r e . T h i s r e p o r t t r i e s t o a n a l y z e some of t h e p i t f a l l s , b u t c o n s i d e r a b l e c a u t i o n (and o f t e n i n g e n u i t y ) i s s t i l l r e q u i r e d € o r a meaningful a n a l y s i s .

1.3

The U . S. S t a n d a r d The c u r r e n t U . S . a n t i b i o t i c s t a n d a r d (Ampho. B - 2 ; 111271 74) w a s o b t a i n e d from Squibb which m a r k e t s t h e d r u g under t h e name Fungizone. The f i n a l s t a g e s of m a n u f a c t u r e i n c l u d e p r e c i p i t a t i o n from aqueous m e t h a n o l (pH c o n t r o l l e d by H C 1 t h e n NaOH), washing w i t h a c e t o n e , d r y i n g , and f o r c i n g through a s i z i n g s c r e e n . The s t a n d a r d is s t o r e d i n l o t s of 250 mg a t -20°C, p r o t e c t e d from l i g h t and m o i s t u r e . Samples were d r i e d f o r 3 h o u r s a t 6OoC ( 45 mm p r e s s u r e ) b e f o r e measuring p o t e n c y , u l t r a v i o l e t a b s o r p t i o n , o r s p e c i f i c r o t a t i o n . T h e r e i s a l s o an Amphotericin B-1 (Amphotericin B-2 f u r t h e r r e c r y s t a l l i z e d w i t h v a r i o u s s o l v e n t s and s a l t s ) f o r which no U. S . s t a n d a r d e x i s t s ; i t i s n o t f u r t h e r

4

IRVlN M. ASHER etel.

c o n s i d e r e d h e r e . There is a l s o an i n t e r n a t i o n a l s t a n d a r d (WHO) f o r Amphotericin B (12). 1.4

Chemical Composition E m p i r i c a l Formula and Molecular Weight 1.41 (1% = 12.000) (a)

c47 H73 N017 MW = 923.62

i n agreement w i t h r e c e n t x-ray (13) and mass s p e c t r o m e t r i c (14) measurements; a c c e p t e d by USP-XIX ( 1 5 ) , s u p e r s e d e s : (b)

c46 H73 NO20 MW = 959.62

r e p o r t e d i n Reference (11,16). 1.42

Elemental Composition (a)

C 61.12%

H 7.96%

C47 H73 NO17 r e q u i r e s : N 1.52%

0 29.45%

N 1.62%

--

Reference 1 found: C 60.40%

H 8.38%

w i t h n e g a t i v e r e s u l t s f o r h a l o g e n s , s u l f u r , and a c e t y l and methoxyl groups, f o r samples p r e p a r e d by t h e methods of Reference 1. (b) C 57.58%

H 7.67%

C46 H73 NO20 r e q u i r e s : N 1.46%

0 33.34%

N 1.20%

0 29.98%

and Reference 1 7 found: C 57.17%

H 7.80%

f o r u n t r e a t e d U.S. s t a n d a r d Amphotericin B , c o n s i s t e n t w i t h t h e CHN r e s u l t s of R e f e r e n c e s 18,19. ( I n t h e l a t t e r Amphot e r i c i n B w a s d r i e d 3 h o u r s a t 80°C p r i o r t o a n a l y s i s . ) Other measurements (20) on d r i e d samples of t h e U.S. s t a n d a r d ( 3 h o u r s , 60°C) gave r e s u l t s (C 59.61%, H 8.32%, N 1.43%) c l o s e r t o t h o s e of R e f e r e n c e 1. N o t i c e t h a t t h e oxygen c o n t e n t of Reference 17 is c o n s i s t e n t w i t h 1 . 4 1 ( a ) r a t h e r t h a n 1 . 4 1 ( b ) .

AMPHOTERICIN 6

5

The f u l l CHNO a n a l y s i s of Reference 1 7 is c o n s i s t e n t w i t h t h e hydrochloride s a l t of 1 . 4 1 ( a ) p l u s 1 . 5 waters of h y d r a t i o n . ( V a r i a t i o n i n w a t e r c o n t e n t a l o n e can only p a r t i a l l y r e s o l v e t h e d i s c r e p a n c i e s noted above.) However, tests (20) f o r C1 i n t h e U.S. s t a n d a r d were n e g a t i v e (60.11%). The Karl F i s h e r t e s t gave 6 . 3 6 % (c) water c o n t e n t f o r t h e u n t r e a t e d U.S. s t a n d a r d (21). The s t a n d a r d e x h i b i t s a 4-5% loss on d r y i n g a t 6OoC under a vacuum. A t atmospheric p r e s s u r e , thermal g r a v i m e t r i c a n a l y s i s (Section 2.12) indicates an 3.5% weight l o s s between 60100°C. Although some of t h i s w a t e r may b e adsorbed, some appears t o b e i n c o r p o r a t e d i n t o t h e l a t t i c e ; t h e Amphotericin B d e r i v a t i v e i n v e s t i g a t e d i n Reference 1 3 i n c o r p o r a t e d t h r e e t e t r a h y d r o f u r a n molecules and one water molecule p e r u n i t cell. 1.5

Structure The following s t r u c t u r e is based on x-ray c r y s t a l l o g r a p h i c s t u d i e s o f N-iodoacetyl Amphotericin B , trit e t r a h y d r o f u r a n monohydrate c r y s t a l (13). It corresponds t o formula 1 . 4 1 ( a ) .

COOH

AMPHOTERICIN B The r i g i d heptaene c h a i n e l o n g a t e s t h e macrocycle, such t h a t one s i d e (polyene) is hydrophobic, w h i l e t h e o t h e r s i d e ( a l i p h a t i c ) is h y d r o p h i l l i c due t o t h e p r e s e n c e of s e v e n hydroxyl groups and an ester carbonyl group. This may account f o r i t s a b i l i t y t o a c t as an ion-channel i n membranes (4-6). A mycosamine r e s i d u e is a t t a c h e d t o one end, providi n g a f r e e amino group. There is an i n t e r n a l hemi-ketal r i n g . I t has been suggested (14) t h a t t h e ketal-form may b e i n e q u i l i b r i u m w i t h an open keto-form i n s o l u t i o n . However, r e c e n t 13C-NMR r e s u l t s (22) confirm t h e presence o f t h e ketal-form i n DMSO s o l u t i o n ( S e c t i o n 4.2), and provide no evidence f o r a keto-form i n t h a t environment. This s t r u c t u r e s u p e r s e d e s an earlier, p a r t i a l s t r u c t u r e by Cope, e t a l . , (23) which is i n c o r r e c t i n several details.

6

IRVIN M. ASHEA e t a / .

1.6

Physical Description B r i g h t yellow powder. Microscopic examination r e v e a l s prisms o r n e e d l e s f o r samples f r e s h l y r e c r y s t a l l i z e d from dimethylformamide ( 1 1 ) ; b u t t h i n , i r r e g u l a r fragments (roughly 5-15 l o n g , less t h a n 0 . 3 p t h i c k ) i n t h e U.S. s t a n d a r d (25). The fragments tend t o clump i n t o l a r g e ( + 8 0 d~ i a m e t e r ) c l u s t e r s . The g r i n d i n g p r o c e s s used i n d r u g manufacture may a l s o c o n v e r t some c r y s t a l s t o an amorphous form (24; S e c t i o n 2 . 2 ) . A t y p i c a l photomicrograph of t h e s t a n d a r d i s shown i n F i g u r e 1. PHYSICAL PROPERTIES 2.1 Thermal P r o p e r t i e s 2.11 D i f f e r e n t i a l Thermal A n a l y s i s (DTA) DTA s c a n s (25) show a g r a d u a l , approximately l i n e a r d e c r e a s e from 35 t o 135OC w i t h peaks n e a r 157 and 209°C ( F i g u r e 2 ) . The sample b e g i n s t o decompose above 2 O O 0 C , w i t h o u t m e l t i n g . The 157°C t r a n s i t i o n i s accompanied by a change i n c o l o r from b r i g h t yellow t o brown-orange which. b e g i n s around 130°C, and i n c r e a s e s p r o g r e s s i v e l y . T h i s presumably r e f l e c t s an endothermic chemical change i n v o l v i n g t h e chromophore. 2.

2.12

Thermal G r a v i m e t r i c A n a l y s i s (TGA) TGA s c a n s (25) show an N 3.5% weight l o s s s t a r t i n g below 65°C which r e a c h e s completion n e a r 90°C ( F i g u r e 2 ) . A f u r t h e r r e d u c t i o n i n weight b e g i n s n e a r 18OoC and l e v e l s o f f n e a r 220"C, w i t h maximum s l o p e n e a r 205OC. These changes may r e f l e c t l o s s of r e s i d u a l s o l v e n t and decomposition r e s p e c t i v e l y . 2.13

Melting Point We f i n d no e v i d e n c e of t h e m e l t i n g i n Amphotericin B up t o 250°C, a t which t e m p e r a t u r e t h e a n t i b i o t i c h a s a l r e a d y decomposed. This i s c o n s i s t e n t w i t h Reference (l), b u t perhaps n o t Reference (16,18). V a p o r i z a t i o n is d e t e c t e d (26) above 25OoC i n a mass s p e c t r o meter (vacuum 4 torr) Trimethylsilyl-ether derivatives of Amphotericin B may v a p o r i z e as low as 180°C (26).

.

2.2

X-Ray Powder D i f f r a c t i o n The X-ray powder d i f f r a c t i o n p a t t e r n of " u n t r e a t e d " (unground, unheated) U.S. s t a n d a r d Amphotericin B demons t r a t e s d e f i n i t e c r y s t a l l i n e s t r u c t u r e . The observed ds p a c i n g s are g i v e n i n Table 1 and F i g u r e 3 ( s o l i d c u r v e ) . Unground samples h e a t e d 1 5 minutes a t 158OC produce a p a t t e r n w i t h less i n t e n s e p e a k s , s l i g h t l y s h i f t e d d-spacings and i n c r e a s e d background ( F i g u r e 3 , d o t t e d c u r v e ) . These

U

Figure 1.

Photomicrograph (x100) of U.S. standard Amphotericin B. The final stages of the manufacturing process break the thin needles characteristic of the freshly recrystallized antibiotic.

U

J \

e E

/

DTA 70 I

100%

I

157

210 m

90 %

AMPHOTERICIN B

80% I

40

I

I

80

I

I

120

I

1

160

200

240

TEMPERATURE ( " C )

Figure 2.

Differential thermal analysis (DTA) and thermal gravimetric analysis (TGA) scans of Amphotericin B.

280

AMPHOTERICIN B

TABLE 1 X-Ray Powder D i f f r a c t i o n Data f o r Amphotericin B ( U n t r e a t e d Sample)

*

T

d (A)

111,

d (A)

18.0 9.30 7.73 7.42 6.30 5.82 5.14 4.82 4.65

23 6 12 10 91 21 33 17 7 5 46 90 100

3.87 3.79 3.49 3.33 3.22 2.925 2.775 2.460 2.370 2.315 2.240 2.040

=

I/Io

B B B B B

17 16 12 16 13 11 9 4 4 4 11 7

triplet

B = broad

*

= t h r e e most i n t e n s e l i n e s

TABLE 2 S o l u b i l i t y of Amphotericin B (MG/ML) dimethyl s u l f o x i d e (1) formamide ethylene glycol dimethyl formamide (1) a c e t i c a c i d (1) propylene g l y c o l (1) pyridine methanol * isoamyl a l c o h o l water benzyl alcohol 1.4-dioxane ethanol ethyl ester acetone ethyl acetate e thylene-C 1 isoamyl a c e t a t e

cs2

methyl e t h y l k e t o n e isopr. alcohol

CHC1-j

benzene c-hexane pet. ether CCl4 t o l uene iso-octane X0.2

-

30. 6.40 2.60 2. 1. 1. 1.75 1.60 1.05 0.75 0.75 0.55 0.50 0.50 0.35 0.30 0.30 0.30 0.24 0.16 0.11 0.08 0.06 0.02 0.01 0.002 0.0 0.0

-

40. 4. 2. 2.

0 . 4 mg/ml f o r anhydrous methanol i n R e f e r e n c e 1.

9

AMPHOTERICIN B

632

I

1

24

I

20

i

1

I

16

12

28(DEG R E ES 1

Figure 3 .

X-ray powder diffraction patterns of "untreated" (unheated, unground) Amphotericin B ) and an aliquot heated to 158' C for 15 minutes (----). Both patterns taken at ( a z e n t temperature using a Philips wide-angle diffractometer equipped with a theta compensating slit and a focusing monochromator. The decreased peak intensities and elevated background of the heated material indicate some loss of crystallinity ( % 3 0 % ) . Ordinate for the magnified (x2.5) insert i s 4 x lo2 cps.

AMPHOTERICIN B

11

changes indicate the introduction of additional strain in the crystal lattice and an increase in the amorphous (noncrystalline) fraction of the sample ( 2 4 ) . Otherwise, the two patterns are highly similar. In contrast, the diffraction pattern of vibratorground Amphotericin B (ground at room temperature in 2 mg. aliquots, 3 minutes each) displays only a few broad, weak peaks with a high background (Figure 4). Such a pattern is characteristic of amorphous powders, and demonstrates that the original crystalline powder has mostly undergone a transition to an amorphous form. This polymorphism explains the variations previously observed in infrared spectra (Section 3.2).

A complete structural determination of the N-iodoacetyl derivative (tri-tetrahydrofuran monohydrate crystal) is given in Reference 13 (see Section 1.5). Solubility As seen from its structure (Section 1.51, Amphotericin B is amphoteric with both polar (acidic and amino head groups) and nonpolar portions. It thus dissolves poorly in most pure solvents; exceptions are dimethylsulfoxide and dimethylformamide. The solubility data of Table 2 , unless otherwise noted, are part of a previous FDA study ( 2 7 ) . Ionization of the acidic and amino groups often aids solvation (1,ll): 2.3

neutral acidic basic

insoluble 0.1 mg/ml 0.1 mg/ml

CH30H

dimethylformamide

0 . 2 - 0 . 4 mg/ml 3-5 mg/ml 2-3 mg/ml

2-4 mg/ml 60-80 mg/ml

Water solubility can be greatly increased by adding Na-lauryl sulfate (19) or Na-desoxycholate (as in commerical injectable Fungizone). Amphotericin B also dissolves in lecithin-cholesterol vesicles and sterolcontaining natural membranes ( 4 - 6 ) . Acid-Base Properties Titration (28) of 66% aqueous dimethylformamide solutions of Amphotericin B with methanolic HC1 and KOH yields pK's near 5 . 7 and 10.0. Comparison with N-acetylAmphotericin B (pK=6.5) and Amphotericin B-methyl ester (pK=8.8) assigns the two pK's to carboxyl and amino groups respectively. Amphotericin B is found to be almost completely zwitterionic in this solution (tautomeric equilibrium 2.4

AMPHOTERICIN B (VIBRATOR GROUND) is

. I

24

Figure 4.

-

-

- -

__

-~

I

20 16 12 SCATTERING ANGLE, 28(DEGREES)

-~

I

8

X-ray powder diffraction of Amphotericin B ground in a vibrator (3 min., 2 mg. at a time). The dramatic decrease in peak heights and increase in background demonstrate a phase transition to an amorphous form; little crystalline Amphotericin B remains.

AMPHOTERICIN B

13

c o n s t a n t Kt = 1000 with r e s p e c t t o t h e n e u t r a l m o l e c u l e ) . 2.5

Aggregation Measurements (29) of t h e u l t r a v i o l e t a b s o r p t i o n of aqueous s o l u t i o n s of Amphotericin B as a f u n c t i o n of concent r a t i o n do n o t obey t h e Beer-Lambert law. Subsequent Rayleigh l i g h t s c a t t e r i n g measurements (29) i n d i c a t e t h a t Amphotericin B forms very l a r g e , l a b i l e a g g r e g a t e s of N 2 x 106 M.W. i n 10-4 - 10-5 M aqueous s o l u t i o n s (pH 7 . 9 , i n t h e presence of Na+-desoxycholate and phosphate). The a g g r e g a t e mass is approximately u n a f f e c t e d by t h e a d d i t i o n of up t o 35% C2H50H, b u t drops p r e c i p i t o u s l y t h e r e a f t e r . S i m i l a r e f f e c t s a r e observed i n t h e i n t e n s i t y of t h e 349, 367, 386, 409 nm u l t r a v i o l e t a b s o r p t i o n bands; however, t h e 328 nni band i s a f f e c t e d by even 10% C2H50H. The d a t a a r e e x p l a i n e d i n terms of e x c i t o n i c i n t e r a c t i o n s between t h e heptaene chromophores of t h e a g g r e g a t e . The a g g r e g a t e mass was c a l c u l a t e d u s i n g a (measured) v a l u e of 290. ml/mg f o r dn/dc, t h e change i n t h e index of r e f r a c t i o n with c o n c e n t r a t i o n of Amphotericin B. 3.

SPECTRAL PROPERTIES (OPTICAL) 3.1 Ultraviolet Amphotericin B h a s a h i g h l y c h a r a c t e r i s t i c u l t r a v i o l e t a b s o r p t i o n spectrum i n DMSO, CH30H s o l u t i o n s ( F i g u r e 5). The s h a r p , i n t e n s e bands a r i s e from t r a n s i t i o n s of t h e heptaene chromophore. The same spectrum o c c u r s i n h e a t e d samples (15 minutes, 158"C), b u t with 25% less a b s o r b t i v i t y . The i n t e n s e 406, 382, 363, 345 nm. q u a r t u p l e t of Amphotericin B s h i f t s t o 318, 304, 291, 289 nm. i n Amphotericin A (1,18). Thus, an u l t r a v i o l e t s p e c i f i c a t i o n is p a r t of t h e F e d e r a l R e g i s t e r (30) c r i t e r i a of a c c e p t a b i l i t y f o r Amphotericin B. S p e c t r a of Amphotericin B i n aqueous s o l u t i o n ( s o l u b i l i z e d by DMSO o r Na+-desoxycholate) a r e c o n s i d e r a b l y d i f f e r e n t (Figure 6 ) , and change f u r t h e r upon t h e a d d i t i o n o f l e c i t h i n and/or c h o l e s t e r o l (31,32). These changes appare n t l y r e f l e c t t h e presence of l a r g e , l a b i l e a g g r e g a t e s i n such aqueous s o l u t i o n s ( s e e S e c t i o n 2.4). A more d e t a i l e d account of Amphotericin B u l t r a v i o l e t a b s o r p t i o n s p e c t r a i n v a r i o u s H20: C2H50H systems may be found i n Reference (29). U l t r a v i o l e t r e f l e c t i o n s p e c t r a of Amphotericin B monolayers on w a t e r y i e l d t h r e e c o n c e n t r a t i o n - s e n s i t i v e bands (33). The t r a n s i t i o n moment ( o r i e n t e d along t h e heptaene c h a i n ) l i e s w i t h i n 6" of t h e w a t e r i n t e r f a c e ; t h e a d d i t i o n of c h o l e s t e r o l t i l t s t h i s upward t o approximately 35".

n*

3.2

Infrared L i t e r a t u r e s p e c t r a of Amphotericin B are contrad i c t o r y (1,18,34). Two b a s i c types of s p e c t r a a r e s e e n (Figures 7 a , b ) . We f i n d t h a t both t y p e s can b e o b t a i n e d a t

14

IRVlN

M. ASHER e r a / .

I

1

'

1

'

1

'

I

'

6

AMPHOTERICIN B (DMSO/CH30H)

363

303

n

AMPHOTERICIN

''

WAVELENGTH (nm) Figure 5.

Ultraviolet absorption spectra of Amphotericins B and A in DMSO/CH30H solution (concentrations respectively 5.45, 8.32 pg/ml).

15

AMPHOTERICIN B

1.4

a.

H ~ O

b.

H;O H20

C.

3

+ CHOLESTEROL + CH30H

I

I

I I

I

I I I

I I

a.

I

I

I 1

I I I I I I I I I I I I I I

I

I

I

1.2

IC.

I

I I I I

I

I

I I

I

I

I

I

I

I

I

I I

I

I

I

I

1

I I

I I

I

I

I I I I

0.8

I I

d I I I I I I

1

I I

I I I I I

I I I

I I

I I I

I

I I I

I I

I I

I I

I

I

I

0.4

I

I I

300

350

400

450

WAVELENGTH (nm)

Figure 6. Ultraviolet absorption spectra of Amphotericin B (1 IJM) solutions: (a) water, (b) water and cholesterol (10 v M ) , (c) water and methanol (1:1 v/v) , (From Reference 32).

16

IRVIN M. ASHER e r a / .

I

I

I

I

AMPHOTERICIN B

I R A B S O R P T I O N F R E Q U E N C Y (crn-1:

Figure 7. Infrared absorption spectra of Amphotericin B: (a) hand-ground powder, (b, c) vibrator ground powder pressed into KBr disks, (d) DMSO solution (saturated). Note the changes in the C=C and C=O stretch regions resulting from differences in sample preparation.

AMPHOTERICIN B TABLE 3 Infrared Spectra Type I

Type I1

(625) 664 69 7 (732) 762 79 5 812

T e n a t i v e Assignment

OH O u t - o f - p l a n e

(sh) (792) (804)sh

Bend ( ? )

Pyranose Ring B r e a t h i n g ( C )

818 (837)sh 851 (878) 889 (898)sh 9 16 (9 31) s h (953) s h (972)sh (981)sh 1009 1041 1 0 70 1109 1132 1164sh 1186 1210sh 1233sh 1272sh (sh) 1324 (1338) s h (1371)sh 1381 1401 1448 1556* (1628)B 1692" (1710)sh+

CH Bend (GI

888

'

}

?:6"

1130 (1173)B (1188)B

}

1712B* 2859* 2925*

2940d (2960)sh 2978 3009 (3370) 3390B

}

1

(29 79 ) s h 3015 3 39 OB

CO Asym. S t r e t c h (COC, COH)

COC Asym. S t r e t c h (COC=O)

(1230) s h 1269 (1291) 1322

(sh) 2918d

Pyranose Ring V i b r a t i o n ( C )

CH O u t - o f - p l a n e Bend (trans p o l y e n e )

1010 1040

(1385) B (1400)B 1449 1566* 1628sh

CH Bend, CH3 Rock

1

CH2 Wag, Bend ( s k e l e t a l )

CH3 Sym. Bend, OH d e f o r m a t i o n CH I n - p l a n e Bend ( p o l y e n e ) CH2,CH3 Asym. Bend P o l y e n e C=C S t r e t c h NH2 I n - p l a n e Bend C-0 S t r e t c h CH2,CH3 Symm. S t r e t c h CH2 Asym. S t r e t c h

CH3 Asym. S t r e t c h CH S t r e t c h ( p o l y e n e ) OH S t r e t c h ( S t r o n g l y H-bonded)

NOTES:

B

broad, s h = shoulder, sl = s l a n t , S = s o l v e n t peaks, f r e q u e n c y u n c e r t a i n , sym = s y m m e t r i c , asym = asymmetric, = f r e q u e n c y c h a r a c t e r i s t i c of Type I o r Type 11, and + = may a r i s e from s l i g h t a d m i x t u r e o f Type 11. =

( ) = weak,

*

17

18

IRVlN M. ASHER e t a / .

room temperature, i n t h e same medium ( i . e . , KBr p e l l e t o r Nujol mull) depending on t h e method of sample p r e p a r a t i o n (24). Handground powders t y p i c a l l y y i e l d t y p e I s p e c t r a (Figure 5a; Reference 1 , 1 8 ) , w h i l e v i b r a t o r ("wigglebug") ground powders y i e l d t y p e I1 s p e c t r a ( F i g u r e 5b; Reference 34) o r a more even mixture of t h e two t y p e s ( F i g u r e 7c). Type I s p e c t r a are c h a r a c t e r i z e d by a s h a r p C=O s t r e t c h band a t 1692 crn-l, a 1556 cm-l C=C s t r e t c h band and c o n s i d e r a b l e s u b s t r u c t u r e ( e . g . , 800-950 c m - l r e g i o n ) . Type I1 s p e c t r a a r e c h a r a c t e r i z e d by a broad C=O s t r e t c h band n e a r 1 7 1 2 cm-1, a 1566 c m - l C=C s t r e t c h band and l e s s - r e s o l v e d s u b s t r u c t u r e . I n "mixed" spectra ( F i g u r e 5 c ) , superp o s i t i o n g i v e s a C=O 1692, 1710 c m - 1 d o u b l e t . S p e c t r a of DMSO s o l u t i o n s c o n t a i n a C=O s i n g l e t near 1715 cm-l. X-ray powder d i f f r a c t i o n s t u d i e s ( S e c t i o n 2.2) show t h a t t y p e I1 s p e c t r a r e p r e s e n t an amorphous phase induced by v i b r a t o r g r i n d i n g (24); similar polymorphism h a s been observed i n t h e Cinchona a l k a l o i d s (35). The broad s h o u l d e r observed n e a r 1710 cm-I i n F i g u r e 7a, may i n d i c a t e an amorphous f r a c t i o n i n t h e s t a n d a r d ( c f . 1 . 3 ) . Handg r i n d i n g of a l l samples would seem p r e f e r a b l e i n t h e f u t u r e , e s p e c i a l l y when preceded by f r e s h r e c r y s t a l l i z a t i o n . Heating t h e sample t o 120°C h a s l i t t l e e f f e c t on t h e spectrum. I n c o n t r a s t , t h e s p e c t r a of samples h e a t e d above t h e chemical t r a n s i t i o n n e a r 157°C ( S e c t i o n 2.1) resemble Type T I , even when handground. This i s c o n s i s t e n t w i t h t h e -30% i n c r e a s e i n t h e amorphous f r a c t i o n observed u s i n g x-ray powder d i f f r a c t i o n ( 2 4 ) . The i n f r a r e d a b s o r p t i o n f r e q u e n c i e s of Amphotericin B and t h e i r t e n t a t i v e i d e n t i f i c a t i o n are g i v e n i n Table 3 . F o u r i e r t r a n s f o r m i n f r a r e d s p e c t r a confirm t h e e x i s t e n c e o f many of t h e weaker peaks. The 1692 cm-1 peak i s a c t u a l l y a very c l o s e d o u b l e t . The a d d i t i o n of Amphotericin B t o aqueous suspensions of l e c i t h i n : c h o l e s t e r o l ( 3 : l ) v e s i c l e s s h i f t s t h e midpoint of t h e "melting" t r a n s i t i o n of t h e l e c i t h i n s i d e c h a i n s from 41°C t o r ~ 4 5 " C( a s monitored by frequency s h i f t s i n t h e CH s t r e t c h r e g i o n ; Reference 3 6 ) . Because of t h e h i g h i n f r a r e d a b s o r p t i v i t y of w a t e r , such measurements r e q u i r e t h e u s e of narrow, IRTRAN sample c e l l s . 3.3

RAMAN Laser Raman s p e c t r a o f Amphotericin B (37) a r e p r e s e n t e d i n Figure 8 and Table 4. The p r e s e n c e of a s t r o n g v i s i b l e a b s o r p t i o n r e s o n a n t l y enhances modes coupled t o the chromaphore. The i n t e n s e peak n e a r 1562 cm-l corresponds t o

AMPHOTERICIN B

19

CH 3 OH Solution

1

1800

ls00

1

1

1

1400

lz00

loo0

WAVE NUMBER DISPLACEMENT (cm' t

Figure 8. Resonance Raman spectra of Amphotericin B powder. Spectra taken with the 48808 line of an Argon ion laser (incident power -50 mw). Only those vibrations coupled t o the polyene chromophore are enhanced sufficiently to be seen. There is a -1 4-fold increase in the intensity of the 1564 cm line upon changing from 514.5 nm to 457.9 nm.

20

IRVlN M. ASHER e t a / .

TABLE 4 Resonance Raman S p e c t r a (cm-l)

Powder

Ref. (36)

CH30H

KBr P e l l e t

(9 80 1

922

Assignment C=CC, HCC inp l a n e Bend

995

1007

1011

1007 (1014)sh 1142

1136sh

1140sh

1131sh

1136sh

1159

1156

1161

1152

1156

1202 1298 1562 1608

119 8 (1298) 1559 1602

1201

(1198)

1562 1607

1635

1639

1640

(1195) 1287 1554 (1597) 1624 1636

}

CC S t r e t c h ,

In-plane HCC Bend (mixed with' C=C S t r e t c h ) C=C S t r e t c h

(intense) C=O S t r e t c h

(mixed w i t h C=C S t r e t c h )

(1645) sh (1666)

(1661)

AMPHOTERICIN B

21

almost p u r e C-C s t r e t c h , whereas t h e weak 1635-1645 c m - 1 modes a l s o c o n t a i n c o n s i d e r a b l e C=O s t r e t c h c o n t r i b u t i o n s . However, t h e numerous nonresonant modes could n o t b e o b s e r v e d , even u s i n g a dye l a s e r . N o t i c e t h a t s e v e r a l of t h e Raman modes are n o t i n f r a r e d a c t i v e (compare S e c t i o n 3 . 2 ) . S o l i d - s t a t e s p e c t r a d i f f e r o n l y s l i g h t l y from t h o s e i n CH30H o r DMSO s o l u t i o n (37). However, o u r r e s u l t s d i f f e r markedly from p r e v i o u s o b s e r v a t i o n s of w e t Amphoteri c i n B powder smeared on f i l t e r paper ( 3 8 ) ; i n p a r t i c u l a r , The supposed a b s e n c e of a w e observe a peak n e a r 1010 cm-1. 1010 cm-1 Amphotericin peak i n p r e v i o u s s p e c t r a w a s used t o i n t e r p r e t carotenoid s p e c t r a (38). S p e c t r a of h e a t e d Amphotericin B powder (15 minutes a t 158°C) d i s s o l v e d i n CH30H (pH 5.) appear normal, d e s p i t e t h e change i n sample c o l o r ( S e c t i o n 2 . 1 ) . However, lowering t h e pH t o ( 1 c a u s e s immediate decomposition i n t o a p r o d u c t i n which t h e i n t e n s i t y of t h e prominent 1156, 1559 cm-1 peaks is markedly reduced. 3.4

ORD, CD, S p e c i f i c R o t a t i o n The s p e c i f i c r o t a t i o n , [& ] ~ 2 4 cof Amphotericin B has been given as -33.6' and +333' i n 0.1N m e t h a n o l i c H C 1 and " a c i d i c " DMF r e s p e c t i v e l y ( 1 , l l ) . However, c l o s e r i n v e s t i g a t i o n (39) shows t h a t t h e s p e c i f i c r o t a t i o n i s h i g h l y pH dependent. I t i s approximately +285 and pH 1 . 0 , and +413 a t pH 2 . 1 , i n DMF (2.5 mg/ml) (The "pH" was measured w i t h a Beckman pH-meter w i t h one g l a s s and one K C 1 e l e c t r o d e ) . C i r c u l a r d i c h r o i s m (CD) s p e c t r a of Amphotericin B i n H 2 0 , CH30H/H20, and H 2 0 / c h o l e s t e r o l (32) are g i v e n i n F i g u r e 9 . The c o r r e s p o n d i n g o p t i c a l r o t a t o r y d i s p e r s i o n (ORD) s p e c t r a i n CH30H (0.1N HC1) and DMJ? (pH 2.2) s o l u t i o n s (40) are g i v e n i n F i g u r e 10. A l l CD peaks i n CH30H/H20 c l o s e l y match Amphoteri c i n B u l t r a v i o l e t a b s o r p t i o n f r e q u e n c i e s ; t h e peak r o t a t i o n s are p o s i t i v e f o r t h e s t r o n g 340-420 nm. q u a d r u p l e t , and n e g a t i v e f o r t h e weak 260-290 nm. t r i p l e t ( F i g u r e 9 c ) . The CD s p e c t r a of DMSO-solubilized Amphotericin B i n H20 and H 2 0 / c h o l e s t e r o l are less complex, o p p o s i t e i n s i g n and an o r d e r of magnitude more i n t e n s e . P r e p a r a t i o n s of Squibb Fungizone (Amphotericin B s o l u b i l i z e d i n H 2 0 by Na+-desoxychol a t e ) are s i m i l a r b u t even more o p t i c a l l y a c t i v e ( F i g u r e 9 a , b). The o p t i c a l r o t a t i o n i n a c i d i c CH30H (40) d i s p l a y s a p p r e c i a b l e changes only i n t h e 260-300 nm r e g i o n , whereas i n a c i d i c DMF both r e g i o n s show c o n s i d e r a b l e changes. In acidic DMF, t h e r o t a t i o n n e a r 2 7 1 , 392, 413 nm. is p o s i t i v e and t h e maximum n e a r 290 nm. becomes a minimum ( F i g u r e l o ) . ORD measurements (41) i n n e u t r a l CH30H somewhat resemble t h o s e i n a c i d i c DMF; however, t h e 286 nm. band is a s s i g n e d t o an

.

+ 2000 0.

a. n20

t

1500

+

1000

b. n 2 0 + CHOLESTEROL C.

5

B

H2Ot CH30H

u D OI

4

+ 500

250

300

350

400

450

WAVELENGTH (nm)

Figure 9. Circular dichroism (CD) spectra of Amphotericin B (1 PM) solutions: (a) water, (b) water and cholesterol (10 uM), and (c) water and methanol (1:l v/v) . (32b) Preparations of Squibb Fungizone (Amphotericin B solubilized in H20 by Na+desoxycholate) are similar but even more optically active (Figure 9 a , b ) .

AMPHOTER IClN B

23

Figure 10. Optical rotatory dispersion (ORD) of Amphotericin B in acidic methanol (a,b) and acidic DMF (c) with base lines (-.-.- ) . Vertical units are (a) O.0lo, (b) 0.04', (c) O.lOo. There may be some spectral change in the 20 minute interval required to obtain the spectrum (a,b). Amphotericin B undergoes a chemical change in 0.1N HC1-methanol (40). The optical rotation appears to be +87.7O soon after dissolution (0.2 mg/ml) , but decreases approximately linearly from +80.5 to - 3 0 . 2 O in 12 minutes in another experiment (2.0 mg/ml). Thus, the values given in References 1,11 should be viewed with caution.

24

IRVlN M. ASHER e t a / .

i m p u r i t y . Reduction w i t h Na-borohydride h a s l i t t l e e f f e c t on t h e ORD s p e c t r a , s u g g e s t i n g t h e absence of t h e k e t o n e (and p r e s e n c e of t h e hemi-ketal) form i n n e u t r a l methanol.

3.5

Fluorescence The f l u o r e s c e n c e spectrum of Amphotericin B (8.35 fl i n s a l i n e T r i s b u f f e r ) i s g r e a t l y enhanced by i n c o r p o r a t i o n i n t o l e c i t h i n v e s i c l e s (31). T h i s e f f e c t is s u b s t a n t i a l l y reduced i n t h e p r e s e n c e of e p i c h o l e s t e r o l b u t n o t c h o l e s t e r o l o r e r g o s t e r o l . The f l u o r e s c e n c e e m i s s i o n f o r 340 nm e x c i t a t i o n is c o n s i d e r a b l e between 410-500 run, w i t h broad maxima n e a r 427, 451, 472 nm. The most e f f e c t i v e e x c i t a t i o n wavelengths f o r 480 nm emission l i e between 300345 nm, w i t h broad maxima n e a r 310, 333 n m ( 3 1 ) . I n f r e e aqueous s o l u t i o n (10 J.M, 5OoC) t h e a d d i t i o n of c h o l e s t e r o l s l i g h t l y lowers t h e p a r t i a l quantum e f f i c i e n c y (355 nm e x c i t a t i o n , 475 nm d e t e c t i o n ; Reference 42).

4.

SPECTRAL PROPERTIES (OTHER) P r o t o n NMR A t y p i c a l 60 MHz p r o t o n NMR spectrum of Amphot e r i c i n B i n DMSO-db s o l u t i o n (43) i s p r e s e n t e d i n F i g u r e l l a . The broad s i g n a l s can o n l y b e l o o s e l y i d e n t i f i e d w i t h s p e c i f i c chemical groups. S u b s t r u c t u r e is p r e s e n t ( c . f . t h e 1.19 pprn broad m u l t i p l e t ) b u t d i f f i c u l t t o r e s o l v e i n t h e 60 MHz spectrum. Amphotericin B h a s 13 exchangeable p r o t o n s (10 hydroxyl, 2 amino, 1 a c i d ) . Rapid exchange between H20 and Amphotericin p r o t o n s g i v e s rise t o a combined OH s i n g l e t . Its p o s i t i o n i s h i g h l y v a r i a b l e and depends upon t h e e x t e n t of Amphotericin-H20 hydrogen bonding, and t h u s H20 concent r a t i o n . P o s i t i o n s between 3.8 and 4.7 ppm a r e t y p i c a l (19,

4.1

43).

The 220 MHz spectrum ( F i g u r e l l b ) r e s o l v e d c o n s i d e r a b l e d e t a i l (e. g . , more t h a n 10 r e s o n a n t s i g n a l s between 0.7 - 1.7 ppm), a l t h o u g h t h e complexity of t h e molecule makes d e t a i l e d assignments d i f f i c u l t (44).

4.2

I3C-NMR 13C-NMR s p e c t r a of Amphotericin B and i t s N-acetyl and methyl ester d e r i v a t i v e s c l e a r l y d e m o n s t r a t e t h e pres e n c e of a hemi-ketal r i n g i n DMSO-d6 s o l u t i o n (22) c o n s i s t e n t w i t h t h e s o l i d - s t a t e conformation of Reference 1 3 . In There is no evidence o f an e q u i l i b r i u m w i t h a keto-form. u n - d e r i v a t i z e d Amphotericin B, t h e hemi-ketal and hemia c e t a l (mycosamine C-1) carbons appear a t 9 7 . 1 and 95.9 ppm r e s p e c t i v e l y ; they are r e s p e c t i v e l y a s i n g l e t and a d o u b l e t i n of f-resonance measurements.

The l a c t o n e and COO- c a r b o n y l

i\

AMPHOTERICIN B

60 MHz 0

N

u1

1

CH

OH D M S

7.50

Figure 11.

6.75

I

I

I

1

5.00

3.75

2.50

1.25

60 MHz and 200 MHz proton nuclear magnetic resonance spectra of Amphotericin B in

d6-DMSO.

The complex substructure can be resolved in the latter.

26

IRVlN M. ASHER e t a / .

carbons appear a t 170.6, 177.6 ppm r e s p e c t i v e l y . A t y p i c a l spectrum of t h e U.S. s t a n d a r d (45) a p p e a r s i n F i g u r e 1 2 .

Mass Spectrometry E a r l y mass s p e c t r o m e t r i c a t t e m p t s a t s t r u c t u r a l e l u c i d a t i o n were n o t completely s u c c e s s f u l (23). More r e c e n t s t u d i e s (14; photo p l a t e d e t e c t o r ) of t h e per-TMS and perdg-TMS d e r i v a t i v e s are c o n s i s t e n t w i t h s t r u c t u r e 1.5 (TMS = t r i m e t h y l - s a l i n e ) . The f r a g m e n t a t i o n p a t t e r n of Amphotericin B i s f a r more complex t h a n t h a t of n y s t a t i n , d e s p i t e t h e i r c l o s e chemical resemblance. A d d i t i o n a l mass s p e c t r a (46; e l e c t r i c a l d e t e c t o r c a l i b r a t e d t o m / e 1800) of t h e TMS-ether d e r i v a t i v e are p r e s e n t e d i n Table 5. D e s p i t e g e n e r a l agreement s e v e r a l c h a r a c t e r i s t i c i o n s d i f f e r by 1-2 amu, o r are n o t observed (Table 6 ) . The M-150 fragment (m/e 1637) r e p r e s e n t s t h e l o s s of C02 CH3, and TMS:OH from t h e m o l e c u l a r i o n ; fragments f , g , h , i r e p r e s e n t t h e l o s s of a d d i t i o n a l TMS:OH. Fragment 1 (m/e 1346) r e p r e s e n t s M-150 minus a doubly s u b s t i t u t e d mycosamine fragment (m/e 201). F u r t h e r l o s s e s of TMS:OH from fragment 1 y i e l d fragments m, n , 0 , q , r. The g l y c o s i d e l i n k a g e i s p a r t i c u l a r l y v u l n e r a b l e t o f r a g m e n t a t i o n (46). The t r i p l y TMS-substituted mycosamine-ester fragment g i v e s rise t o an intense m / e 362 (80.5%) peak; c h a r g e r e t e n t i o n on t h e o p p o s i t e s i d e o f t h e l i n k a g e w a s less common (m/e 378, 4.05%). No s u g a r fragments were found w i t h a l l f o u r l a b i l e hydrogens r e p l a c e d (m/e 434, 450). 4.3

5.

CHROMATOGRAPHY 5.1 Paper The o r i g i n a l method ( 1 ) u t i l i z e d Whatman No. 1 p a p e r p r e t r e a t e d w i t h 0.3M K3PO4 b u f f e r (pH 3 . 0 ) . Spot developed 6-7 h o u r s w i t h 80% p r o p a n o l . The m o b i l i t y w a s Rf(B) = 0 . 5 f o r Amphotericin B and Rf(A) = 0.7 f o r Amphot e r i c i n A. However, t h e low pH damaged t h e a n t i b i o t i c s , p r e v e n t i n g l o n g e r development. High-pressure l i q u i d t e c h n i q u e s ( S e c t i o n 5 . 3 ) are p r e f e r a b l e f o r a u t o m a t i o n , quan t i t a t i o n , and co 1l e c t i o n . Alternate methods (51) u t i l i z e Whatman No. 1 paper p r e t r e a t e d with McIlvaine's b u f f e r , equibrated over s o l v e n t f o r 1 h o u r , and developed f o r 5 hours. The r e s u l t s are : Solvents Sec-butanol: H20: C a C 1 2 (20 m l : 80 m l : 200 mg)

Rf(A)

Rf(B)

pH

0.82

0.64

3.2

T(OC) 37

"C-NMR Amphotencin B (DMSO)

ppm

Figure 1 2 .

200

100

13C-NMR spectrum of Amphotericin B in DMSO-d6 solution (saturated).

0

28

IRVlN M. ASHER e t a / .

TABLE 5 High Mass Portion of the Spectrum of Amphotericin B-TMSI I/BASE 0.74% 0.14% 0.51% 1.44% 1.40% 1.75% 0.96% 0.76% 3.72% 0.89% 3.16% 0.09% 1.04% 1.56% 2.70% 3.14% 0.63% 3.11% 1.15% 0.61% 1.08% 0.31% 0.50% 1.88% 0.16% 1.04% 2.88% 0.09% 1.61% 0.80% 0.36% 2.28% 1.27% 1.28% 0.98% 0.95% 2.29% 1.61% 1.90% 1.26% 0.74% 1.05% 0.61%

MAS s 706.5 707.3 708.4 711.3 715.6 716.4 720.5 722.3 723.5 724.2 726.1 729.8 731.5 734.5 735.4 737.6 738.5 741.4 745.3 746.3 747.7 749 * 3 751.8 754.5 756.7 760.3 761.3 762.7 763.8 765.2 766.4 768.7 769.3 770.8 771.3 773.0 777.5 778.4 781.6 782.5 785.5 788.8 790.2

I/BASE 0.11% 0.71% 1.30% 1.33% 0.24% 0.87% 0.41% 3.63% 2.83% 2.64% 0.29% 1.05% 0.58% 0.87% 3.23% 2.71% 0.21% 1.05% 2.08% 3.24% 2.52% 2.05% 1.96% 1.23% 0.18% 0.53% 0.40% 0.71% 1.36% 1.76% 0.71% 0.92% 0.56% 2.53% 0.56% 1.50% 0.28% 0.76% 0.16% 0.51% 1.96% 2.79% 1.52%

MASS 791.5 793.5 794.3 796.3 798.7 804.3 805.5 806.6 807.3 810.5 811.3 813.7 815.5 817.1 818.2 819.6 820.3 823.3 826.5 835.2 836.1 837.2 838.4 839.3 840.4 841.1 844.0 846.8 848.5 851.0 852.0 853.2 857.0 861.5 865.4 866.3 867.5 868.3 868.9 869.6 877.0 881.3 882.4

AMPHOTERICIN B

0.44% 1.84% 1.23% 0.66% 0.31% 0.31% 1.29% 1.31% 1.06% 2.95% 0.09% 0.57% 0.74% 0.74% 0.61% 0.74% 1.12% 1.30% 1.40% 3.25% 0.40% 1.03% 2.34% 0.50% 0.62% 1.65% 1.98% 0.18% 1.25% 0.20% 0.33% 0.96% 0.17% 1.57% 0.08% 0.64% 2.12% 0.16% 0.09% 0.83% 1.63% 1.61% 1.55% 0.48% 0.69% 1.04% 2.21% 1.67% 1.32%

884.5 888.8 890.8 891.3 892.2 893.1 894.5 897.9 899.3 907.4 908.4 910.4 912.7 916.3 918.6 921.1 922.6 924.3 933.1 936.4 943.0 943.8 952.5 954.0 957.8 960.8 965.8 967.5 969.4 974.1 976.1 978.1 980.2 982.9 985.6 987.7 993.4 1000.8 1003.2 1004.8 1006.3 1016.3 1019.1 1024.0 1041.2 1044.8 1046.6 1050.3 1056.9

1.32% 1.56% 0.13% 0.31% 0.50% 0.77% 1.24% 1.21% 0.50% 0.06% 0.42% 0.79% 0.99% 0.95% 0.40% 0.12% 0.26% 0.78% 2.04% 1.07% 0.06% 0.56% 1.26% 2.08% 2.11% 0.47% 0.53% 0.37% 0.38% 0.44% 0.51% 1.51% 1.33% 0.81% 0.93% 0.45% 0.67% 0.45% 0.75% 0.21% 0.28% 0.26% 0.90% 0.52% 0.37% 0.64% 0.67% 1.84% 1.77%

1056.9 1058.1 1059.9 1061.2 1064.6 1072.0 1076.5 1094.3 lliO.l 1122.1 1123.1 1134.0 1148.5 1151.3 1153.1 1155.6 1171.3 1178.2 1204.3 1207.7 1209.3 1216.7 1223.0 1226.5 1228.6 1229.7 1232.8 1241.8 1247.6 1249.8 1250.6 1255.8 1257.3 1260.4 1268.0 1278.0 1280.5 1293.2 1300.5 1312.9 1319.1 1323.8 1332.0 1334.8 1340.8 1345.6 1351.5 1363.5 1366.4

29

30

I R V l N M. ASHER e r a / .

0.13% 0.94% 0.35% 1.00% 0.37% 1.01% 0.72% 0.84% 1.41% 0.73% 0.05% 0.13% 0.48% 0.98% 1.25% 0.36% 0.13% 0.35% 0.29% 0.41% 2.06% 1.57% 1.82% 0.93% 0.21% 0.43% 0.072

1360.5 1374.0 1393.9 1406.5 1412.8 1417.5 1423.6 1431.1 1433.1 1441.8 1445.0 1449.5 1451.3 1455.5 1491.1 1499.1 1500.8 1516.8 1532.4 1539.3 1549.3 1572.8 1594.5 1607.9 1641.2 1650.5 1652.5

TABLE 6 Comparison of C h a r a c t e r i s t i c Ions o f Ampho t e r i c i n B-INSi Reference 14

R e f e r e n c e 46

mfe Mt M-TMSi M- 150 ( e ) (f)

(8)

fh ) (1)

(1)

(m) (n) (0)

(j)

(4) (P) (r) (k)

*

m/e

X R.

Intensity

1787 1714

1b37 1624 1567 1534 1457 1444 1367 1346 1277 125h 1166 1076 988 986

89 h

aot

1549.3* 1532.4* 1455.5* 1 4 4 5 .O* 1366.4 1345.6 1278.0* 1255.8 1076.5 987.7 985.6 897.9* 806.6

71b

Measurements d i f f e r by

1 mu.

Not Observed N o t Observed 2.0 0.3 0.9 0.05 1.8 0.6

0.5 1.5 Not Observed 1.2 0.6 0. I 1.3 3.6 Not Observed

AMPHOTERICIN 6

Same ( p a p e r n o t equilibrated) A c e t o n e : H 2 0 (8:Z)

0.86

0.41

3.2

37

0.77

0.59

4.6

25

31

The l o c a t i o n o f t h e a n t i b i o t i c s was d e t e r m i n e d by b i o a u t o graphy u s i n g Candida t r o p i c a l i s (SC 1 6 4 7 ) , u s i n g t h e method f o r n y s t a t i n (52). 5.2

T h i n Layer (TLC) Most u s a b l e s o l v e n t s y s t e m s f o r t h i n - l a y e r chromatography (TLC) of A m p h o t e r i c i n B c o n t a i n a l c o h o l (Table 6 ) . Solvent system G should s e p a r a t e Amphotericin B (Rf 0.32) from Amphotericin A. S o l v e n t s y s t e m s G,J s h o u l d s e p a r a t e Amphotericin B (Rf = 0 . 3 2 , 0.18 r e s p e c t i v e l y ) from n y s t a t i n (Rf 0 . 6 5 , 0.54 r e s p e c t i v e l y ) . O t h e r r e f e r e n c e s are found i n R e f e r e n c e 3. 5.3

High-pressure L i q u i d (HPLC) U s i n g a Waters A s s o c i a t e s ( M i l f o r d , M a s s , ) p , c18 column, h i g h - p r e s s u r e l i q u i d chromatography (HPLC) c o u l d s e p a r a t e s o l u t i o n s of A m p h o t e r i c i n B from small amounts of a n accompanying d e g r a d a t i o n p r o d u c t i n a v a r i e t y o f a c i d i c methanol s y s t e m s . The c o n t a m i n a n t r a n g e d from 0.7% i n f r e s h s o l u t i o n s t o ~ 3 i n%o l d s o l u t i o n s u s i n g t h e s o l v e n t s y s t e m s of R e f e r e n c e 53. The u s e f u l s e p a r a t i o n of A m p h o t e r i c i n A and B i s more d i f f i c u l t , b u t c a n b e a c h i e v e d u s i n g t h e f o l l o w i n g p r o c e d u r e (53): 20% CH30H/80% DMF t o 100% CH30H o v e r 5 m i n u t e s , s t r a i g h t o r concave g r a d i e n t , 1 . 5 ml/min f l o w , a b s o r p t i o n m o n i t o r e d a t 280 nm. S e p a r a t i o n r e q u i r e s l e s s t h a n 20 m i n u t e s . Maximum r e s o l u t i o n ( n a r r o w e s t p e a k s ) w a s o b t a i n e d f o r a concave g r a d i e n t ( F i g u r e 1 2 ) . S e p a r a t i o n w a s n o t a c h i e v e d i n CH30H, d e s p i t e e a r l i e r r e p o r t s o f s u c c e s s w i t h less e f f i c i e n t columns ( 5 4 ) . The B/A u l t r a v i o l e t a b s o r b a n c e r a t i o is 0.6 n e a r 280 nm. The r e t e n t i o n times found b y o t h e r w o r k e r s (55) u s i n g VYDAC-RP (30-44 pm) columns w i t h H20:CH30H:tetrahydrof u r a n (420:90:45) f o r A m p h o t e r i c i n B (3.4 m i n u t e s ) a n d n y s t a t i n ( 3 . 0 , 3.4 m i n u t e s ) are t o o s i m i l a r t o d i f f e r e n t i a t e between them. The method o f R e f e r e n c e 5 3 i s a l s o u n a b l e t o s e p a r a t e Amphotericin B and n y s t a t i n . 5.4

Gas

C o n t r o l l e d p y r o l y s i s f o l l o w e d by g a s chromatography o f t h e r e s u l t i n g f r a g m e n t s ( > 30) g a v e d i s t i n c t " f i n g e r p r i n t s " f o r n y s t a t i n and A m p h o t e r i c i n B ( 5 6 ) .

5.5

Electrophoresis E l e c t r o p h o r e t i c m o b i l i t i e s of A m p h o t e r i c i n B ,

HPLC (,+tC18)

280 nm AMPHOTERICIN A

AMPHOTERICIN

B7 r

11

AMPHOTERICIN B

A

10.60

N W

TIME (MIN) Figure 13. High-pressure liquid chromatograms of: (a) Amphotericin B dissolved in acidic methanol (1% v/v acetic acid), (b) Amphotericin A dissolved in neutral methanol, and (c) mixture of solutions (a) and (b). The standard samples contained (a,c) 20 pg of Amphotericin B and (b,c) 11 pg of Amphotericin A at a concentration o f 1. mg/ml. A Waters p c18 column was used with a methanol/dimethylforamide solvent system as described in the text. The absorption o f effluent was monitored at 280 nm.

AMPHOTERICIN B

TABLE 7 Solvent Systems for Thin Layer Chromatography System

Solvent

€!!

Reference

A

CHC1-j:CH30H:Borate Buffer (7:5:1) 0.60 pH 8.3

B

N-b utano 1:C2H50H :CH3COOH:H20 (50:1 5 : 15:20)

0.6

50

C

N-butanol:CH$OOH:H20

0.5

50

D

CH30H:Acetone:CH3COOH (8:l: 1)

0.45

48

E

CHC13 :CH30H:20% NaOH (2 :2 :1)

0.4

50

F

Pyridine: ethylacetate: H20 (25:16: 7)

0.4

50

G

Butan-l-ol:pyridine:H20 (3:2:1)

0.32

49

H

N-butanol (H20 saturated)

0.2

50

I

C2H5 OH: ammonia:dioxan-H20 (8:l:l:l)

0.19

49

J

CH30H:propan-2-ol:CH3COOH (90: 10:1)

0.18

48

K

Butan-l-ol:ammonia:methanol:H~0

0.07

47

(20: 1:2:4)

(3:l: 1)

47

33

34

I R V l N M. ASHER e t a / .

TABLE 8 Minimal I n h i b i t o r y C o n c e n t r a t i o n (MIC) of Amphotericin B

Candida a l b i c a n s Cand i da t r o p i c a l i s Candida pseudo t r o p i c a l is Candida p a r a k r u s e i Cryptococcus neoformans Epidermophyton floccosum F u s a r ium b u l b igenum Microsporum canis Microsporum a u d o u i n i Rhodotorula g l u t i n i s Rhodotorula mucilagenosa Saccharomyces c e r e v i s i a e Sporotrichum s c h e n c k i i ( y e a s t phase) T r i c h o p h y t o n megnini T r i c h o p h y t o n mentagrophytes Trichophyton g a l l i n a e T r i c h o p h y t o n rubrum Trichophyton t o n s u r a n s Monosporium apiospermum MIC was

A s p e r g i l l u s fumigatus Candida p a r a p s i l o s i s Cephalosporium r e c i f e i Cladosporium c a r r i o n i i Cladosporium w e r n e c k i Fonsecaea p e d r o s o i Fonsecaea compactum Geotrichum s p . Note:

> 40 P g f m l

1.9 25.0 7.3 1.1 0.2 0.2

14.7 7.3

0.9 1.9 1.8 0.07 0.9 2.4 7.3 7.3 4.9 30.0

for:

Microsporum gypseum Nocardia a s t e r o i d e s N o c a r d i a a s t e r o i d e s mexicana Nocardia b r a s i l i e n s i s N o c a r d i a madurae Philaophora verrucosa Sporotrichum s c h e n c k i i (mycelial phase)

From R e f e r e n c e 1; M I C ()lg/ml) measured on s e c o n d day a f t e r i n n o c u l a t i o n of a g a r medium.

AMPHOTERICIN B

35

Amphotericin A , and several o t h e r a n t i b i o t i c s i n v a r i o u s e l e c t r o l y t e systems have been r e p o r t e d (57).

6.

ISOLATION I n t h e o r i g i n a l method of Vandeputte, e t a l . , ( l ) , Streptomyces nodosus (M 4575) whole b r o t h i s mixed w i t h i s o p r o p a n o l ( 1 : l ) and a d j u s t e d t o pH 10.5. The f i l t r a t e i s n e u t r a l i z e d , t h e a l c o h o l e v a p o r a t e d , and t h e r e s u l t i n g powder (40-70% pure) washed w i t h water and a c e t o n e , and vacuum d r i e d . S l u r r y i n g w i t h a 2 % C a C 1 2 methanol s o l u t i o n s e p a r a t e s Amphotericin A ( f i l t r a t e ) and Amphotericin B ( p r e c i p i t a t e ) . The B f r a c t i o n i s t h e s l u r r i e d w i t h a c i d i c DMF, followed by d i l u t i o n of t h e f i l t r a t e i n methanol and p r e c i p i t a t i o n w i t h w a t e r w h i l e m a i n t a i n i n g pH 5. The p r e c i p i t a t e (75-80% p u r e ) i s a g a i n d i s s o l v e d i n a c i d i c DMF, d i l u t e d w i t h p u r e methanol, and p r e c i p i t a t e d w i t h water. Amphotericin A (65-70%) r e s u l t s from adding water t o t h e A f i l t r a t e , and d r y i n g t h e p r e c i p i t a t e . (Methanolic C a C 1 2 s o l u b i l i z a t i o n and water p r e c i p i t a t i o n can be r e p e a t e d t o remove t h e remaining Amphotericin B . )

7.

STABILITY Dry Amphotericin B powder a p p e a r s s t a b l e f o r l o n g p e r i o d s of t i m e a t room temperature (1,ll). Isopropanol:H20(1:1) s o l u t i o n s a r e s t a b l e f o r days a t pH 6-8, less s t a b l e a t pH 4 , 10 and decompose r a p i d l y a t pH 1 2 (1). The s t a b i l i t y a t 70°C (pH 7) i s h a l f t h a t a t 3OoC ( 1 ) . S o l u t i o n s i n phosphatec i t r a t e b u f f e r ( 5 < p H < 7 ) are a p p a r e n t l y s t a b l e ( 5 8 ) . I n d e x t r o s e i n f u s i o n s a t room t e m p e r a t u r e , Amphotericin B a g g r e g a t e s i n t h e presence of N a C l (25% r e d u c t i o n of a c t i v i t y within 4 hours). The a c t i v i t y of aqueous, c l i n i c a l l y prepared d e x t r o s e s o l u t i o n s ( p H > 4 ) d i d n o t d e c r e a s e a p p r e c i a b l y d u r i n g an 8hour exposure t o 100-foot c a n d l e s of ambient f l u o r e s c e n t l i g h t (59). A f t e r 3 days exposure t o l i g h t i n o t h e r e x p e r i ments, b i o l o g i c a l ( b u t n o t c o l o r i m e t r i c ) a s s a y s showed a 26% l o s s i n a c t i v i t y ( 60) . Heating d r y samples f o r 16 hours a t 105°C r e s u l t s i n only ~ 1 7 l%o s s of potency. I n c o n t r a s t , 1 5 minutes a t 158OC (above t h e chemical t r a n s i t i o n of S e c t i o n 2 . 1 1 ) i s s u f f i c i e n t t 3 cause an ~ 2 1 l%o s s of potency ( 2 1 ) . Vibrator g r i n d i n g o f t h e sample a t room temperature causes an 2, 30% l o s s of potency (average a c t i v i t y 688 mcg/min, r a t h e r t h a n 986 mcg/min; Reference 61) as measured by t h e Saccharomyces Cervisiae a s s a y o f Reference 30.

8.

ANTIMICROBIAL PROPERTIES AND ASSAYS Minimal i n h i b i t o r y c o n c e n t r a t i o n s (MIC) of Amphotericin B are given i n Table 8 f o r s e v e r a l organisms (1). Stock

36

IRVlN M. ASHER etal.

s o l u t i o n s were made i n DMSO (4 mg/ml) and d i l u t e d i n d i s t i l l e d water; t h e f u n g i were p l a t e d on a g a r ( b r o t h d i l u t i o n a s s a y s g i v e somewhat d i f f e r e n t r e s u l t s ) . The d a t a of Table 8 are f o r t h e second day of o b s e r v a t i o n . Assay procedures u t i l i z i n g Saccharomyces c e r e v i s i a e , Candida a l b i c a n s , o r Candida t r o p i c a l i s are d e s c r i b e d i n References ( 1 , 3 , 1 6 ) . The Code of F e d e r a l R e g u l a t i o n s (30) prescribes a microbiological agar d i f f u s i o n assay s u i t a b l e f o r p h a r m a c e u t i c a l f o r m u l a t i o n s u s i n g Saccharomyces c e r e v i s i a e (ATCC 9763). A d d i t i o n a l b i o l o g i c a l a s s a y s can b e found i n Reference 3 and are summarized i n T a b l e 9 . The b i n d i n g of Amphotericin B t o 5. c e r e v i s i a e h a s been i n v e s t i g a t e d u s i n g f l u o r e s c e n c e ( 6 2 ) . Weak, r e v e r s i b l e b i n d i n g o c c u r s even a t O°C and i n t h e p r e s e n c e of m e t a b o l i c i n h i b i t o r s ; i t a p p e a r s t o a f f e c t o n l y t h e o u t s i d e of t h e membrane. I n c o n t r a s t , antimicrobial a c t i o n involves the l o s s of e s s e n t i a l c e l l u l a r c o n s t i t u e n t s as a r e s u l t of s t r o n g , i r r e v e r s i b l e b i n d i n g t o t h e membrane. This s t r o n g b i n d i n g , which can b e blocked by c o o l i n g t o O°C o r by m e t a b o l i c i n h i b i t o r s , a p p a r e n t l y d i s r u p t s t h e deeper hydrophobic p o r t i o n s of t h e membrane. Enhanced f l u o r e s c e n c e a s s a y s are r e p o r t e d t o b e l i n e a r i n t h e r a n g e 0 . 1 - l 0 . p (62). Serum and u r i n e can b e assayed by a g a r d i f f u s i o n f o r Amphotericin B a c t i v i t y w i t h a s e n s i t i v i t y of about 0 . 0 1 mcg/ml (63). An e q u a l l y s e n s i t i v e t u r b i d i m e t r i c microbiol o g i c a l a s s a y (64) h a s been developed f o r u s e w i t h small samples ( e . g . , 2 5 ~ of ~ 1serum o r s p i n a l f l u i d ) . These methods are summarized i n Table 9. Feces l e v e l s can be determined by s p e c t r o p h o t o m e t r y of s i m p l e DMSO e x t r a c t s , making u s e o f a c o r r e c t i o n f o r t h e h i g h b a s e l i n e a b s o r p t i o n (64).

9.

AMPHOTERICIN A Amphotericin A (C46C73N019, Reference 13) i s i s o l a t e d from Streptomyces Nodosus, along w i t h Amphotericin B which i t c l o s e l y resembles ( 1 ) . I t i s , however, a t e t r a e n e ( l i k e n y s t a t i n ) and is t h u s r e a d i l y d i s t i n g u i s h e d from Amphotericin B by i t s u l t r a v i o l e t a b s o r p t i o n spectrum: 2 2 8 , 280, 291, 304, 318 nm ( 1 , 1 8 ) . I t s s p e c i f i c r o t a t i o n [ 0~ (-9.9" i n 0.1N methanolic HC1; +32" i n " a c i d i c " DMF) is a l s o d i s t i n c t i v e ( 1 , 3 ; b u t see S e c t i o n 3.4). In contrast, i n f r a r e d s p e c t r a ( 1 , 1 8 , 3 4 ) are h i g h l y s i m i l a r , b u t n o t i d e n t i c a l t o Amphotericin B. Amphotericin A i s f a r more s o l u b l e i n CH30H, DMF, waters a t u r a t e d propanol o r b u t a n o l , and CH3COOH t h a n Amphotericin B ( 1 ) . Unlike Amphotericin B , i t forms a water s o l u b l e sodium s a l t i n methanolic -NaOH and a methanol s o l u b l e C a C 1 2 complex; t h e l a t t e r p r o p e r t y w a s used i n i t s o r i g i n a l

37

AMPHOTERICIN B

TABLE 9 M i c r o b i o l o g i c a l Assay Methods f o r Amphotericin B

Type of Sample Formulated and unformulated products

Body F l u i d s

Animal Feeds

Method

T e s t Culture

Reference

Diffusion

S a cch a romy ces cerevisiae N.C.Y.C. 87

65

Diffusion

Saccharomyces cerevisiae ATCC 9763

66

Turbidimetric

Candida tropicalis ATCC 13803

64

D if f us ion

Paecilomyces v a r i o t i MSSC 5605 N I A I D

63

Turbidimetric (Micro s c a l e )

Candida tropicalis ATCC 13803

64

Diffusion

Sac c h a r omy c es cerevisiae ATCC 9763

67

38

IRVlN M. ASHER e t a / .

i s o l a t i o n (1). Amphotericin A can (presumably) b e s e p a r a t e d from Amphotericin B and n y s t a t i n by t h e t h i n - l a y e r chromatog r a p h i c methods of References 49 and 68 r e s p e c t i v e l y . I t can be r e l i a b l y s e p a r a t e d from Amphotericin B by h i g h - p r e s s u r e l i q u i d chromatography ( S e c t i o n 5 . 3 ) . Amphotericin A i s s e v e r a l times l e s s a c t i v e than Amphot e r i c i n B (59) and i s u s u a l l y encountered a s a contaminant of t h e l a t t e r . Amphotericin A i s c o n s i d e r a b l y more s e n s i t i v e t o c a t a l y t i c h y d r o l y s i s , and is t h u s less s t a b l e i n aqueous i s o p r o p a n o l (1).

39

AMPHOTERICIN B

REFERENCES

1. J . V a n d e p u t t e , J. L. W a c h t e l , and E. T. S t i l l e r , A n t i b i o t i c s Annual , 1955-1956, 579 (1956).

2.

P h y s i c i a n s Desk R e f e r e n c e , Med. Econ. I n c . , ( O r a d e l l , N J , 1970).

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A. H. Thomas, The A n a l y s t ,

101,321,

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4. S . C . Kinsky i n A n t i b i o t i c s , Vol. I , D . G o t t l e i b and P. D . Shaw e d . , ( S p r i n g e r V e r l a g ; B e r l i n , 1 9 6 7 ) , pp. 122-141. 5. A. Cass, A. F i n k e l s t e i n and V . K r e s p i , J. Gen. P h y s i o l . , 56:lOO (1970); R. Holz and A. F i n k e l s t e i n , J . Gen. P h y s i o l . , 56: 125 (1970).

6. B. D e K r u i j f f , W . J . G e r r i t s e n , A . Oerlemans, R. A. D e m l , and L. L. Mivan Deenen, Biochem. Biophys. Acta, =:30 (1974); i b i d , 44; 44; B. D e K r u i j f f and R. A. D e m l , Biochem. Biophys. Acta, 339:57 (1974). 7. C . P . S c h a f f n e r and H. W. Gordon, P r o c . N a t . Acad. S c i . (USA) , 61, 36, 1968.

8. H. W. Gordon and C . P. S c h a f f n e r , P r o c . Nat. Acad. S c i . (USA), 60, 1201, 1968. 9.

J . M. T . H a m i l t o n - M i l l e r ,

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10. F. R. K e i m , J . W. P o u t s i a k a , J . K i r p a n and C . H. K e y s s e r , S c i e n c e , 179, 584, 1973. 11. The Merck I n d e x , Merck & Co.,

(Rahway, N J , 1 9 6 8 ) .

12. J . W. Lightbown, P. d e R o s s i and P. I s a a c s o n , B u l l . World H e a l t h Org., 47,343, 1972. 13. W. M e c h l i n s k i , C. P. S h a f f n e r , P. G a n i s , and G. A v i t a b i l e , T e t r a h e d r o n L e t t . , H : 3 8 7 3 (1970); P. G a n i s , G. A v i t a b i l e , W. M e c h l i n s k i , and C . P . S c h a f f n e r , J . Am. Chem. SOC., 2: 4560 (1971).

1 4 . K. D. Haegele and D . M. D e s i d e r i o , Biomed. Mass S p e c . ,

1:20 (1974). 1 5 . The U. S. Pharmacopeia, 1 9 t h Ed. ( R o c k v i l l e , MD, 1 9 7 5 ) .

, USP

Convention, I n c .

,

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16. Encyclopedia of I n d u s t r i a l Chemical A n a l y s i s , Volume 5 , F. D. S n e l l and C. L . H i l t o n , Ed., I n t e r s c i e n c e Pub. (New York, 1966).

1 7 . C. G r a i c h e n , BF, FDA, u n p u b l i s h e d d a t a (1976). 18. I n d e x of A n t i b i o t i c s from Actinomycetes, H. Umezawa, e d . , Un. Park Press ( S t a t e C o l l e g e , PA, 1967). 1 9 . E. R . Squibb & Sons, I n c . , u n p u b l i s h e d d a t a (1972).

20. A. Wong and B. Baer, N I H , u n p u b l i s h e d d a t a . 2 1 . S. Delgado and L. Wayland, BD, FDA, u n p u b l i s h e d d a t a .

2 2 . R. C. Pandey and K. L. R i n e h a r t , J r . , Un. I l l i n o i s , manuscript submitted. 23. A. C . Cope, J . Am. Chem. SOC., 3 : 4 2 2 8 (1966) 2 4 . G. Schwartzman, I . M. Asher, V. F o l e n , W. J . T a y l o r , FDA, m a n u s c r i p t s u b m i t t e d .

Brannon, and

25. M. M a i e n t h a l , BD, FDA, u n p u b l i s h e d d a t a (1976). 26. W. Barron, BD, FDA, u n p u b l i s h e d d a t a (1976). 27. M. L . Andrew and P . J . Weiss, A n t i b i o t i c s and Chemot h e r a p y , 9:277 (1959).

2 8 . E. D . E t i n g o v , G. V . Kholodova, V. 0. Kul'bakh, and A. I. K a r n a t u s h k i n a , A n t i b i o t i k i , 17,301 (1972). 29. J . Lematre, H. R i n n e r t , and G. Dupont, i n p r e s s .

30. "Code of F e d e r a l R e g u l a t i o n s , ' ' T i t l e 2 1 , Food and Drugs, Parts: 436.10, 436.105, 449.4, 449.104, 449.204, 449.504, U.S. Government P r i n t i n g O f f i c e , Washington, D . C. (1976). 31. R. B i t t m a n , W. C. Chen, and 0 . R. Anderson, B i o c h e m i s t r y , 13: 1364 (1974).

3 2 . J . Lematre and H. Moulki, C. R. Acad. S c i . P a r i s , Ser. C , 280:481 (1975); J . tematre, p r i v a t e communication. 33. N . Ockman, Biochim. Biophys. Acta, 373:48 (1974). 34. L. Wayland and P. J . Weiss, i n I R and UV S p e c t r a of Some

AMPHOTERICIN B

Compounds of P h a r m a c e u t i c a l I n t e r e s t , A.O.A.C. D. C . , 1972).

41

(Washington,

35. A. L. Hayden and 0. R . Sammul, J. Am. Pharm. A s s o c . , 49: 497, 1960. 36. I . M. Asher, FDA, I. L e v i n , N I H , m a n u s c r i p t i n preparation. 37. M. Bunow, I . A s h e r , and I. L e v i n , u n p u b l i s h e d d a t a ( 1 9 7 6 ) . 38. L . R i m a i , M. E . Heyde and D . G i l l , J . Am. Chem. S O C . , 95:4493 (1973).

39. S . Delgado, BD, FDA, m a n u s c r i p t i n p r e p a r a t i o n . 40. K. W. Henry, EDRO, FDA, u n p u b l i s h e d d a t a ( 1 9 7 6 ) .

41. C . N . Chong and R. W. R i c h a r d s , T e t r a h e d . L e t t . , 5053, 19 72. 42. F. S c h r o e d e r , J . F. Holland and L . L. B i e b e r , Biochemi s t r y , 11,3105 (1972). 43. E. S h e i n i n , BD, FDA, u n p u b l i s h e d d a t a ( 1 9 7 6 ) . 44. R. B r a d l e y , N I H , u n p u b l i s h e d d a t a (1976). 45. G . Mazzola, BD, FDA, u n p u b l i s h e d d a t a ( 1 9 7 6 ) . 46. R. B a r r o n , BD, FDA, u n p u b l i s h e d d a t a (1976). 47. M. K a l a s z , V. S z e l l , J . Gyimesi, K. Magyar, I. H o r v a t h , and I. Szabo, Acta M i c r o b i o l . Acad. S c i . Hung., 19, 111, 1972. 48. L . Dryon, J. Pharm. B e l g . ,

2,433,

49. S . Ochab, D i s s n e s Pharm. Pharmac.,

1966.

22,

351, 1970.

50. J . B l a k e l y , BD, FDA, u n p u b l i s h e d d a t a (1976). 51. J . Semar, The Squibb I n s t i t u t e f o r M e d i c a l R e s e a r c h , u n p u b l i s h e d d a t a (1964). 52. E. Meyers and D . S m i t h , 3. Chromatog.,

14,129

53. B. Smith, BD, FDA, m a n u s c r i p t i n p r e p a r a t i o n .

(1964).

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54. Waters Associates, private communication. 55. W. Mechlinski and C. P. Schaffner, J . Chromat., 9 9 , 619 (1974). 56. H. J. Burrows and D. H. Calm, J. Chromat., 53, 566 (1970). 57. S. Ochab, Diss. Pharm. Pharmacol. , 24, 205 (1972): C. A. 77:44438t. 58. J . M. T. Hamilton-Miller, J . Pharm. Pharmac., 1973.

25, 401,

59. S. Shadomy, D. L. Brumer and A. V. Ingroff, Am. Rev. of Respir. Dis. , 107, 303 (1973). 60. J . F. Gallelli, Drug Intell.,

1,1 0 2 ,

1967.

61. S.L. Caldwell and E. Tarcza, BD, FDA, unpublished data. 62. J. Kotler-Brajtburg, G. Medoff, D. Schlessinger, and G. S. Kobayashi, Antimicrobial Agents and Chemotherapy, 6: 770 (1974). 63. S . Shadomy, J . A. McCoy, and Microbiol., 17,497, 1969.

S.

I. Schwartz, Applied

64. T. B. Platt, J. D. Levin, J. Gentile, and M. A. Leitz in Kavanagh, F. editor, "Analytical Microbiology," Vol. 11, Academic Press, New York and London, 1972. 65. "British Pharmacopoeia 1973," HM Stationary Office, London, 1973 p A102. 66. "Code of Federal Regulations," Title 21, Food and Drugs, Part 141.101, U . S . Government Printing Office, Washington, D. C. (1976). 67. T. B. Platt and A. G. Itkin, J. Assoc. Off. Analyt. Chem.,

5 7 , 536, 1974. 68. T . Ikekawa, F . Iwami, E. Akita, and H. Umezama, JAntibiot., ,&I 5 6 , 1963. 69. W. Gold, H. A. Stout, J. F. Pagano, and R. Donovick, Antibiotics Annual, 1955-1956, 579 (1956).

BETAMETHASONE DIPROPIONATE

Michael G. Ferrante and Bruce C.Rudy

44

MICHAEL G. FERRANTE AND BRUCE C. RUDY

INDEX

Analytical P r o f i l e

-

Betamethasone D i p r o p r i o n a t e

1.

Description 1.1 Name, Formula, M o l e c u l a r Weight 1 . 2 Appearance

2.

Physical P r o p e r t i e s 2.1 I n f r a r e d Spectrum 2 . 2 N u c l e a r Magnetic Resonance Spectrum 2 . 3 Mass Spectrum 2.4 U l t r a v i o l e t Spectrum 2.5 O p t i c a l R o t a t i o n 2.6 M e l t i n g Range 2.7 D i f f e r e n t i a l Scanning C a l o r i m e t r y 2.8 T h e r m o g r a v i m e t r i c A n a l y s i s 2.9 S o l u b i l i t y 2.10 Xray D i f f r a c t i o n

3.

Synthesis

4.

Stability

5.

Method of A n a l y s i s 5.1 Elemental A n a l y s i s 5.2 T h i n Layer Chromatographic A n a l y s i s 5 . 3 L i q u i d Chromatographic A n a l y s i s 5.4 Direct S p e c t r o p h o t o m e t r i c A n a l y s i s 5.5 C o l o r i m e t r i c A n a l y s i s

6.

References

BETAMETHASONE DIPROPIONATE

1.

45

Description

Name, F o r m u l a , M o l e c u l a r Weight The c h e m i c a l name f o r b e t a m e t h a s o n e d i p r o p i o n a t e i s 9a-fluoro-11B-hydroxy-l6~-methyl-l7~2l-dipropionyloxy-pregna1,4-diene-3,20-dione.

1.1

2 8H37Fo 7

1.2

2.

Molecular Weight

504.6

Appearance B e t a m e t h a s o n e d i p r o p i o n a t e i s a w h i t e t o cream c o l o r e d powder.

Physical Properties I n f r a r e d Spectrum (IR) The i n f r a r e d s p e c t r u m o f b e t a m e t h a s o n e d i p r o p i o n a t e i s p r e s e n t e d i n F i g u r e 1 . The s p e c t r u m was o b t a i n e d a s a m i n e r a l o i l m u l l on a P e r k i n - E l m e r Model 1 8 0 g r a t i n g i n f r a r e d s p e c t r o p h o t o m e t e r . The a s s i g n m e n t s f o r t h e c h a r a c t e r i s t i c bands i n t h e i n f r a r e d spectrum are l i s t e d i n Table I. 1 2.1

Figure 1

INFRARED SPECTRUM OF BETAMETHASONE DIPROPIONATE 2.5

1001

WAVELENGTH, MICRONS 6 7 8

3

4

5

I

I

I

I

I 2000

1

I

I

9

10

I

I

12 14 I

1

18 22

I I 1 1 1 1

35 50

I I

P

m

0

4Ooo

3500

3000

2500

1

I

I

I

I

1700

1400

1100

800

500

FREOUENCY (CM-’1

200

BETAMETHASONE DIPROPIONATE

47

Table I I R Assignments f o r Betamethasone D i p r o p i o n a t e

Frequency (cm-l)

3300 3025, 3000 1755, 1728 1660 1620, 1608 1189 1068

*

Intensity

C h a r a c t e r i s t i c of 0-H s t r e t c h C-H s t r e t c h , A 1 y 4 C=O s t r e t c h , 17,21-diprop i o n a t e , 20-ketone C=o s t r e t c h , 3-ketone C=C s t r e t c h , A1,4-diene C-0 s t r e t c h , p r o p i o n a t e ester C-0 s t r e t c h , 11-hydroxyl

m W

s,d s

s,d S

m

* s = s t r o n g , m=medium, w=weak, d = d o u b l e t 2.2

N u c l e a r Magnetic Resonance Spectrum (NMR) The 100 MHz F o u r i e r t r a n s f o r m p r o t o n NMR s p e c t r a of betamethasone d i p r o p i o n a t e , F i g u r e 2 , was o b t a i n e d on a V a r i a n XL-100-15 s p e c t r o m e t e r a t ambient t e m p e r a t u r e i n CDC13 s o l v e n t w i t h a c o n c e n t r a t i o n of 20 mg/ml. Chemical s h i f t s a r e r e p o r t e d i n ppm ( 6 ) d o w n f i e l d from i n t e r n a l t e t r a m e t h y l s i l a n e (TMS) i n T a b l e I I . 2 T a b l e I1 NMR Assignments f o r Betamethasone D i p r o p i o n a t e

8

21CH20CCH2CH3

I

0 //

Figwe 2 NMR SPECTRUM OF BETAMETHASONE DIPROPIONATE ,..... . ,

P

cn

.,

,

, ....

...

'.'.

:".

"

~

'

'

'

'

~

'

'

: ' ' .'. ~ , ,

I.

. ,

.;.

.

BETAMETHASONE DIPROPIONATE

Proton

*

C 13-CH 3 C16-CH3 C10-CH3 1 la-H 21-H 21'-H 118-0-H C4-H C2-H

Chemical S h i f t (6) 0.92 1.27 1.52 4.30 4.45 4.80 5.52 6.04 6.26

C1-H 7.30 C17 and C21 P r o p i o n a t e 1.05 and 1 . 0 9 methyls C17 and C21 P r o p i o n a t e 2.42 methylenes

49

Mu1t i p l i c i t y Singlet Doublet Sing l e t Mu1t i p l e t Doublet Doublet Doublet Broad s i n g l e t D o u b l e t of doublets J1, =10 Hz; J 2 , -1.5 Hz Doublet Triplet Quartet

*Chemical s h i f t and c o u p l i n g c o n s t a n t v a r y w i t h concent r a t i o n and t e m p e r a t u r e , b u t d i s a p p e a r s when D20 i s added.

2.3

Mass Spectrum The mass s p e c t r u m of b e t a m e t h a s o n e d i p r o p i o n a t e w a s o b t a i n e d a t 7 0 e i on a V a r i a n MAT CH5 medium r e s o l u t i o n s i n g l e focusing (magnetic s e c t o r ) i n s t r u m e n t , i n t e r f a c e d w i t h a V a r i a n SS-1OOC d a t a s y s t e m , a t a p r o b e t e m p e r a t u r e of 170°C and a s o u r c e t e m p e r a t u r e o f 25OoC. The d a t a system u t i l i z e d t h e o u t p u t of t h e s p e c t r o m e t e r t o d e t e r mine t h e masses, t h e n compared t h e i r i n t e n s i t i e s t o t h e b a s e peak (100% i n t e n s i t y ) and produced t h e b a r g r a p h i n Figure 3 . 3 A l i s t i n g of t h e p r o m i n e n t f r a g m e n t s and t h e i r r e s u l t i n g masses a r e g i v e n i n T a b l e 111.

-

50

~

BETAMETHASONE DIPROPIONATE

51

Table 111 Mass Spectrum Assignments for Betamethasone Dipropionate Mass -

Ion

505

M+ 1

484

M-20

Fragments Lost

HF

P

M-87

CH~O~CH~CH~

410

M-94

HF+CH3CH2COOH

343

M-161

CH20CCH CH +CH3CH2COOH 2 3

336

M-168

HF+2CH3CH2COOH

333

M-171

C O C H ~ OC~H:~ C H ~ +2 C

315

M-189

0 COCH~O~CH~CH~+CH~CH~COOH

295

M-209

277

M-227

267

M-237

417

223

147

R

9

~

4

fl

~

COCH20CCH2CH3+CH CH COOH+HF 3 2 C O C H ~ O ~ C H ~ C H ~CH + C CHO O H + H ~ O 3 2

9

91

COCH20CCH2CH3+CH3CH2COOH+C0

~

52

MICHAEL G. FERRANTE AND BRUCE C. RUDY

T a b l e 111

(Continued)

Mass Spectrum Assignments f o r Betamethasone D i p r o p i o n a t e Ion -

Mass -

Loss -

2.4

U l t r a v i o l e t Spectrum (UV) When t h e u l t r a v i o l e t s p e c t r u m o f b e t a m e t h a s o n e d i p r o p i o n a t e was scanned from 350 t o 210 nm, a s i n g l e maxima was o b s e r v e d a t 238 nm @ = 1 . 5 7 ~ 1 0 4 ) . The s p e c t r u m i n F i g u r e 4 was o b t a i n e d from a s o l u t i o n of 3.056 mg of b e t a methasone d i p r o p i o n a t e i n 100.0 m l of m e t h a n o l . 2.5

Optical Rotation Betamethasone d i p r o p i o n a t e e x h i b i t e d t h e f o l l o w i n g specific rotations:4

26'

2'7

BETAMETHASONE DIPROPIONATE

Figum 4 ULTRAVIOLET SPECTRUM OF BETAMETHASONE DIPROPIONATE

NAN0 METERS

53

54

MICHAEL G. FERRANTE AND BRUCE C. RUDY

2.6

M e l t i n g Range Betamethasone d i p r o p i o n a t e m e l t s i n a 3' r a n g e between 1700 and 179OC w i t h d e c o m p o s i t i o n , when t h e USP XvIT.1 c l a s s I a p r o c e d u r e i s u s e d . 5

2.7

D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y (DSC) The DSC c u r v e f o r b e t a m e t h a s o n e d i p r o p i o n a t e obt a i n e d a t a s c a n r a t e of 10°C/min. i s shown i n F i g u r e 5 . The c u r v e was r e c o r d e d w i t h a DuPont 900 D i f f e r e n t i a l Thermal Analyzer u n d e r a n a t m o s p h e r e of n i t r o g e n f l o w i n g a t 200 c c / m i n . A s i n g l e endotherm w a s o b s e r v e d , t h e e x t r a p o l a t e d o n s e t o f m e l t i n g o c c u r r e d a t 175OC.6

2.8

T h e r m o g r a v i m e t r i c A n a l y s i s (TGA) The TGA c u r v e f o r s t a n d a r d b e t a m e t h a s o n e d i p r o p i o n a t e e x h i b i t e d no w e i g h t l o s s on a s c a n from 27O t o 175OC a t 10°C/min.7 2.9

Solubility The s o l u b i l i t y d a a f o r b e t a m e t h a s o n e d i p r o p i o n a t e is l i s t e d i n Table IV.

s

Table I V Betamethasone D i p r o p i o n a t e S o l u b i l i t y Measurements Solubility mglml, 25OC

Solvent Ac e t o n e Benzene Chlorof orm Dimethylformamide D i m e t h y l s u l f ox i d e E t h a n o l (USP) E t h a n o l ( U S P ) 85% Water 15% ( v / v ) Ether E t h y l Acetate Methanol Mineral O i l Petroleum Ether P o l y e t h y l e n e G l y c o l 400 Propylene Glycol Water

-

>loo 30

>loo >loo >loo 45 30 5 70 55
26 7 <0.04

BETAMETHASONE D IPROPIONATE

Figure 5 DSC OF BETAMETHASONE OIPROPIONATE

55

MICHAEL G. FERRANTE AND BRUCE C. RUDY

56

2.10

Xray D i f f r a c t i o n The x r a y d i f f r a c t i o n s p e c t r u m of b e t a m e t h a s o n e d i p r o p i o n a t e is p r e s e n t e d i n T a b l e V.9 The d a t a were c o l l e c t e d on a P h i l i p s APD-3500 u t i l i z i n g Cu Ka r a d i a t i o n (1.54188). Table V Xray Data f o r Betamethasone D i p r o p i o n a t e

I/I'

I/I'

41.72 39.112 38.024 36.392 34.864 33.944 32.880 30.234 28.685 25.022 23.909 9.700 9.291 9.664 8.046 7.047 6.082 5.703 3.

50 52 52 51 52 50 49 46 43 31 26 22 54 40 45 15

5.290 4.862 4.622 4.602 4.572 4.520 4.507 4.465 4.421 4.405 3.948 3.893 3.845 3.596 3.370 3.359

100

3.030

71

3.020

77 55 12 13 14 12 12 18 24 24 28 40 26 20 37 37 24 24

Synthesis Betamethasone d i p r o p i o n a t e i s p r e p a r e d by t h e f o l l o w i n g s y n t h e s i s . Betamethasone is r e a c t e d w i t h e t h y l o r t h o p r o p i o n a t e and t o l u e n e - p - s u l p h o n i c a c i d t o y i e l d betamethas o n e 17,21-ethylorthopropionate.~O T h i s compound i s t h e n r e a c t e d w i t h a c e t i c a c i d t o y i e l d b e t a m e t h a s o n e 17propionate.llThis intermediate product is then t r e a t e d with p r o p i o n y l c h l o r i d e a t OOC, d i l u t e d w i t h water and a c i d i f i e d with d i l u t e hydrochloric acid. This y i e l d s t h e crude d i e s t e r which when r e c r y s t a l l i z e d y i e l d s t h e f i n a l p u r e form of b e t a methasone d i p r o p i o n a t e . 12

BETAMETHASONE D IPROPIONATE

57

4.

Stability Betamethasone d i p r o p i o n a t e h a s a h i g h s t a b i l i t y i n aqueo u s s u s p e n s i o n s a s compared t o o t h e r c o r t i c o s t e r o i d s . T h i s may b e a t t r i b u t e d t o i t s d i e s t e r s t r u c t u r e and c o r r e s p o n d i n g l y low s o l u b i l i t y i n water. The compound is most s t a b l e a t pH 4 , w i t h any h y d r o l i z a t i o n r e s u l t i n g i n t h e f o r m a t i o n o f b e t a m e t h a s o n e a l c o h o l . 13 A t t h e e x t r e m e s of pH, l a r g e amounts of more p o l a r p r o d u c t s were o b s e r v e d which a l t h o u g h n o t i d e n t i f i e d , c a n b e assumed t o b e f u r t h e r breakdown prod u c t s of t h e d i h y d r o x y a c e t o n e s i d e c h a i n . 1 4 Betamethasone d i p r o p i o n a t e i s s t a b l e towards a i r oxidat i o n i n t h e s o l i d s t a t e . H e a t i n g of t h e compound a t 75 C f o r 6 months i n t h e p r e s e n c e of a i r shows no change i n c o l o r o r i n t h e t h i n l a y e r chromatogram. 15 Over l o n g p e r i o d s of e x p o s u r e t o f l o u r e s c e n t l i g h t , t h e r e i s m i n o r d e g r a d a t i o n of t h e d r u g . 1 6 It s h o u l d a l s o b e exp e c t e d t h a t s o l u t i o n s of b e t a m e t h a s o n e d i p r o p i o n a t e a r e s u b j e c t t o p h o t o l y t i c d e g r a d a t i o n s i n c e p h o t o l y t i c degradat i o n of t h e A-ring of s t e r o i d a l 1,4-diene-3-ones h a s been r e p o r t e d i n l i t e r a t u r e . 17

5.

Method of A n a l y s i s 5.1

Elemental A n a l y s i s The r e s u l t s o f e l e m e n t a l a n a l y s i s on a sample of s t a n d a r d b e t a m e t h a s o n e d i p r o p i o n a t e a r e p r e s e n t e d below. 18 Element C H F

5.2

Theory

Found

66.65

66.54

7.40

7.18

3.77

3.65

T h i n L a y e r Chromatographic A n a l y s i s (TLC) A TLC s y s t e m which i s used i n t h e a n a l y s i s of b e t a methasone d i p r o p i o n a t e i s as f o l l o w s . The sample i s a p p l i e d t o a s i l i c a g e l GF p l a t e and s u b j e c t e d t o a s c e n d i n g chromatog r a p h y u s i n g ch1oroform:acetone (7: 1) a s t h e d e v e l o p i n g s o l vent.

MICHAEL G. FERRANTE AND BRUCE C. RUDY

58

A f t e r t h e s o l v e n t i s allowed t o ascend 1 5 cm, t h e p l a t e i s a i r d r i e d . T h i s p l a t e i s t h e n viewed u n d e r a s h o r t w a v e u l t r a v i o l e t l i g h t t o i d e n t i f y and l o c a t e t h e b e t a m e t h a s o n e d i p r o p i o n a t e band. The a p p r o x i m a t e Rf v a l u e is 0.5.'9 5.3

Liquid Chromatographic A n a l y s i s A h i g h p r e s s u r e l i q u i d chromatography s y s t e m f o r t h e s e p a r a t i o n and d e t e c t i o n of b e t a m e t h a s o n e d i p r o p i o n a t e was d e v e l o p e d u s i n g t h e p a r a m e t e r s l i s t e d below i n T a b l e V I . 20 Table V I Permaphase ODs* (DuPont) packed i n a I m x 2mm ( i . d . ) s t a i n l e s s s t e e l column. D e t e c t o r : U l t r a v i o l e t d e t e c t o r a t 254 nm. Mobile P h a s e : A c e t o n i t r i 1 e : w a t e r ( 1 : 3 ) ( d e g a s s e d f o r 5 m i n u t e s u s i n g vacuum) P r e s s u r e : 600 p s i , a d j u s t a b l e Flow Rate: 0.5 ml/min Q u a n t i t y I n j e c t e d : 0.14 mg R e t e n t i o n T i m e s ( m i n u t e s ) : Betamethasone m o n o p r o p i o n a t e s 5 Betamethasone d i p r o p i o n a t e 7 Column:

*ODs

-

Octadecylsilane

Direct S p e c t r o p h o t o m e t r i c A n a l y s i s Direct UV a b s o r b a n c e s may b e c a r r i e d o u t on b e t a methasone d i p r o p i o n a t e . A s o l u t i o n of b e t a m e t h a s o n e d i p r o p i o n a t e i s p r e p a r e d c o n t a i n i n g a p p r o x i m a t e l y 0 . 0 2 mg/ml i n methanol. The a b s o r p t i o n s p e c t r u m of t h i s s o l u t i o n i s t h e n r e c o r d e d between 350 and 220 nm and compared t o a s i m i l a r s o l u t i o n of t h e s t a n d a r d . 2 1 5.4

5.5

Colorimetric Analysis The c o l o r i m e t r i c a n a l y s i s f o r b e t a m e t h a s o n e d i p r o p i o n a t e i n v o l v e s u t i l i z a t i o n of t h e Mader-Buck r e a c t i o n . 22 A s o l u t i o n of b e t a m e t h a s o n e d i p r o p i o n a t e i s p r e p a r e d c o n t a i n i n g a p p r o x i m a t e l y 0.016 mg/ml i n e t h a n o l (USP). To 2 0 . 0 m l of t h i s s o l u t i o n i s added 2 . 0 m l of b l u e t e t r a z o l i u m s o l u t i o n (125 mg of b l u e t e t r a z o l i u m r e a g e n t i n 25 m l of USP e t h a n o l ) and 2.0 m l of tetramethylammonium h y d r o x i d e s o l u t i o n ( 1 0 m l of tetramethylammonium h y d r o x i d e , l o % , d i l u t e d t o 100 m l w i t h USP e t h a n o l ) . T h i s s o l u t i o n i s t h e n h e a t e d a t 45OC i n a water b a t h f o r 4 5 m i n u t e s . A f t e r h e a t i n g , 1 . 0 m l of g l a c i a l a c e t i c a c i d i s added and t h e s o l u t i o n i s a l l o w e d t o c o o l . The a b s o r p t i o n s p e c t r u m of t h i s v i o l e t c o l o r e d s o l u t i o n i s r e a d between 600 and 4 5 0 nm (hmax = 5 2 5 m).23

BETAMETHASONE DIPROPIONATE

6.

59

References

1.

E c k h a r t , C . and M c G l o t t e n , J . , S c h e r i n g - P l o u g h P e r s o n a l Communication.

2.

B r a m b i l l a , R. a n d M c G l o t t e n , J . , S c h e r i n g - P l o u g h C o r p . , P e r s o n a l Communication.

3.

B a r t n e r , P . and M c G l o t t e n , J . , S c h e r i n g - P l o u g h P e r s o n a l Communication.

Corp.,

4.

E c k h a r t , C. and McGlotten, J . , Schering-Plough P e r s o n a l Communication.

Corp.,

5.

Rosenkrantz, B., Commun i c a t i o n .

6.

G l i s s o n , R. and R o s e n k r a n t z , B . , C o r p . , P e r s o n a l Communication.

7.

Ibid.

8.

Rosenkrantz, B., Communication.

Schering-Plough

Corp., Personal

9.

S a n c i l i o , F. D . , Communication.

Schering-Plough

Corp.,

10.

Schering-Plough

Corp.,

Corp.,

Personal

Schering-Plough

E l k s , J . , May, P. J . , a n d Weir, N . G . ,

Personal

US P a t e n t

3,312,591 (1967). 11.

E l k s , J . , May, P. J . , a n d Weir, N . G . ,

US P a t e n t

3,312,590 (1967). 12.

Ibid.

13.

Rosenkrantz, B., Communication.

14.

Gut tman, D . ,

15.

Rosenkrantz, B., Communication.

16.

Ibid.

Schering-Plough

J . An.

Pharm. ASSOC.,

Schering-Plough

Corp.,

Personal

47,773 Corp.,

(1958).

Personal

60

MICHAEL G. FERRANTE AND BRUCE C. RUDY

17.

Hamlin, W. E., Chulski, T., Johnson, R. H., and Wagner, J. G., J. Am. Pharm. ASSOC., Sci. Ed.

49, 253 (1963)

18. Evans, B. and McGlotten, J., Schering-Plough Corp., Personal Communication. 19.

Rosenkrantz, B., Schering-Plough Corp., Personal Communication.

20.

Bole, V. and Upton, L., Schering-Plough Corp., Personal Communication.

21.

Rosenkrantz, B., Schering-Plough Corp., Personal Communication.

22.

Mader, W. J. and Buck, R. R., Anal. Chem. 24, 666-667,

23.

(1952).

Rosenkrantz, B., Schering-Plough Corp., Personal Communication.

CLONAzEPAM

Walter C. Window

62

WALTER C . WINSLOW

Contents Analytical Profile - Clonazepam 1.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

2.

Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Scanning Calorimetry 2.8 Thermogravimetric Analysis 2.9 Solubility 2.10 Crystal Properties 2.11 Dissociation Constant

3.

Synthesis

4.

Stability Degradation

5.

Drug Metabolism and Pharmacokinetics

6.

Toxicology

7.

Methods of Analysis 7.1 Elemental Analysis 7.2 Phase Solubility Analysis 7.3 Chromatographic Analysis 7.4 Electron Capture Gas Liquid Chromatography 7.5 Spectrophotometric Analysis 7.6 Polarographic Analysis 7.7 Titrimetric Analysis

8.

Acknowledgements

9.

References

CLONAZEPAM

1. Description 1.1 Name, Formula, Molecular Weight Clonazepam is (5-[2-chlorophenyl]-l,3-dihydro-7nitro-2H-1,4-benzodiazepin-2-one)

Y

CLONAZEPAM CiSHioCl N 3 0 3

M.W.

315.7

1.2 Appearance, Color, Odor

Light yellow, crystalline powder which is practically odorless. 2.

Physical Properties 2.1

Infrared Spectrum The infrared spectrum of a mineral oil suspension of reference standard clonazepam is presented in Figure l.[l] The spectral assignments are listed in Table 1. Table 1

1.

NH stretching: 3250-3100 CM-I

2.

Aromatic CH stretching: 3076, 3056 CM-’

3.

Carbonyl stretching: 1696 CM-’

4.

Aromatic Ring:

5.

Asymmetric NO2 stretching:

6.

Symmetric NO2 stretching:

7.

Aromatic CH out-of-plane bendin : 4 adjacent free H’s: 750 CM2 adjacent free H ’ s : 844 CM-I

1615, 1582 CM-’ 1540 CM-l

1339 CM-’

?

63

I \

I (0

0 0

0

*

I 0

33NVllIWSNWl% 64

I

8

CLONAZEPAM

2.2

65

Nuclear Magnetic Resonsance Spectrum (NMR) The NMR spectrum o f clonazepam is shown in Figure 2. The spectrum was determined on a JEOL C-60 HL spectrometer at ambient temperature (ca 25°C). The sample was dissolved in DMF-d, containing ?T.IS as an internal reference. The spectral assignments are listed in Table 2. [ * ]

Table 2 Proton a b

Chemical Shift 6 (ppm)

4.48 7.55-7.85

C

7.93

d

8.50

e

11.30

Multiplicity

Coupling Const.J (Hz)

Singlet

---

Mu1t iplet

---

Doublet

2.5

Doublet (2 Sets) 2.5 (meta coupling) Broad Singlet

8.6 (ortho coupling)

2.3 Ultraviolet Spectrum The W spectrum of clonazepam (1 mg of clonazepam in 100 ml of 7.5% methanol in isopropanol) in the region of 230 to 400 nm exhibits maxima at 248 nm ( E = 1.45 x l o 4 ) and 310 nm ( E = 1.16 x l o 4 ) . Minima are observed at 239 and 279 nm. The spectrum is shown in Figure 3. [ 3 ]

a ;d N

a PI 0

c

(D

u

a

a,

m

L

66

4

4

f

67

CLONAZEPAM

FIGURE 3 UV Scan of Clonazepam

0.1

0.i

0.E

0.5

w

0

2

a

E 0.4

E:m a

0.3

0.2

0.I

0 1

1

1

250

300

350

NANOMETERS

1 400

68

WALTER C . WINSLOW

2.4

Mass Spectrum The low resolution mass spectrum of clonazepam is shown in Figure 4. [4] The spectrum was run on a Varian CH 5 spectrometer interfaced with a Varian data-handling system. The computer calculates ion masses and compares their peak intensities to the base peak, This information is then automatically plotted as a series of lines whose heights are proportional to the peak intensities. The largest mass was observed at m/e = 315. The other characteristic peaks observed were: Mass (m/e)

Species M+ M+ - H M+ OH M+ - CHO M+ - C1 314 - NO2 280 - CO, 280 268-CO 280-NO

315 314 298 286 2 80 268 252 2 40 2 34 205

-

-

CHzN

240-C1

A high resolution scan confirmed the results of the low resolution spectrum. [ 4] The elemental

composition for the characteristic masses determined in the high resolution scan are shown in Table 3 . Table 3

Mass Observed 151.0541 177.0596 205.0763 213.0354 2 34.0 797 240.0437 252.0531 252.0760 268.0433 2a0.0701 286.0370 287.0308 298.0362 314.0294 315.0364

Mass Calculated _ C _ H 151.0548 1 2 7 177.0578 1 3 7 205.0767 1 4 9 213.0327 1 4 3 234.0794 15 10 240.0455 1 4 9 252.0536 1 4 8 252.0773 14 10 268.0404 1 5 9 280.0723 15 10 286.0383 1 4 9 287.0349 15 1 0 298.0381 1 5 9 314.0333 1 5 9 315.0411 15 10

_N 0 0 1 2 3 2 2 2 3 2 3 3 1 3 3 3

0 0 0 0 1 0 3 2 1 3 2 3 2 3 3

0 0 0 0 0 1 0 0 1 0

1 1 1 1 1

69

5

a at N

2

0

m

m

2

WALTER C. WINSLOW

70

2.5

Optical Rotation Clonazepam exhibits no optical activity.

2.6

Melting Range Clonazepam melts between 237°C and 240°C when tested according to the USP XIX Class I procedure. [ J

2.7 Differential Scanning Calorimetry The DSC thermogram of clonazepam at a of 10°C/minute is shown in Figure 5. endothermic transition, corresponding ing of the compound, i s observed from 240.2"C. [ 6 ]

heating rate A single to the melt238.6"C to

2.8 Thermogravimetric Analysis

The TGA of clonazepam exhibited a single S shaped weight loss as a function of temperature. The loss started at ca. 195"C., reached 15% at 285°C. and then leveled off at 355°C. at which point 34% of the sample weight had been lost. Gradual weight loss continued until 500°C. (upper limit of instrument). 2.9

Solubility Approximate solubilities in various solvents, as determined gravimetrically from solutions equilibrated for 3 hours at 25"C, are given in Table 4 . Table 4 Solvent Water 95% Ethanol Absolute Ethano1 Methanol Isopropanol Chloroform Ethyl Ether Benzene Ace tone Ethyl Acetate Propylene Glycol

Solubility mg/ml


15 0.7 0.5 31 10 5.2

71

U

vl

n

72

WALTER C. WINSLOW

2.10 C r y s t a l P r o p e r t i e s The X-Ray powder d i f f r a c t i o n p a t t e r n of clonazepam is p r e s e n t e d below. [ 7 l Instrument C o n d i t i o n s Instrument

GE Model XRD-6 G e n e r a t o r

Camera

Guider-DeWolff Sample Screen

X-Ray T a r g e t

Chromium (CrK, = 2.2909A)

Focus

Line

Voltage

50 KV

Current

12.5 mA

Atmosphere

Helium

Exposure T i m e

2 Hrs.

Film

I l f o r d X-Ray Film I n d u s t r i a l G

11, w i t h Pt-Rh 0

0

28 17.89 22.30 22.58 23.33 26.30 25.02 27.49 27.93 30.21 30.84 33.59 34.39 34.72 36.16 36.78 38.91 39.47 41.50 42.04

d (A)

*

7.37 5.92 5.85 5.67 5.03 5.29 4.82 4.75 4.40 4.31 3.96 3.88 3.84 3.69 3.63 3.44 3.39 3.23 3.19

I/Io**

0.54 1.00 0.57 0.32 0.11 0.10 0.29 0.29 0.47 0.49 0.19 0.54 0.60 0.38 0.76 0.15 0.56 0.27 0.16

*d ( i n t e r p l a n a r d i s t a n c e ) = nX/(2 S i n e ) = r e l a t i v e i n t e n s i t y based on a maximum of 1.00

**I/Io

CLONAZEPAM

73

2.11 Dissociation Constant The pKa values for clonazepam have been determined spectrophotometrically to be 1.5, corresponding to deprotonation of the nitrogen in the 4 position and 10.5 for the nitrogen in the 1 position.[’] 3.

Synthesis Clonazepam may be prepared by the reaction scheme shown in Figure 6 . [ 9 ] o-Chlorobenzoyl chloride is reacted with p-nitroaniline in a modified Friedel-Crafts reaction to The aminoyield 2-amino-5-nitro-2’-chlorobenzophenone. ketone is then condensed with bromoacetyl bromide to form 2-bromoacetamido-5-nitro-2’-chlorobenzophenone. This compound is isolated and converted to the corresponding acetamido compound by reacting it in solution with ammonia. The ammonium bromide by-product is separated and the solvent removed. The residue is taken up in 5N anhydrous hydrogen chloride in methanol to form the hydrochloride salt which is then taken up in boiling ethanol. Pyridine is added which catalyzes ring closure to clonazepam. [lo]

4.

Stability Degradation Degradation of clonazepam occurs principally via hydrolysis. Decomposition by this route is illustrated in Figure 7. The major breakdown products are 2-amino-2’chloro-5-nitrobenzophenone (I) and 3-amino-4-(2-chlorophenyl)-6-nitrocarbostyril (111). [ 11,121 The latter is presumably formed via the aminoacetamido intermediate(I1). Formation of the benzophenone results in a reduction in the absorptivity at 310 nm when measured in isopropanol, while formation of the carbostyril leads to an increase in the absorptivity. [ 12]

5. Drug Metabolism and Pharmacokinetics Clonazepam is an antiepileptic drug useful in the treatment of minor motor seizures which probably acts by potentiating inhibitory mechanisms in the subcortical brain structure responsible for the propagation of seizure activity , Clonazepam, even in pg doses, protected mice from pentetrazole induced convulsions, and elevated the threshold for electroshock seizures in mice and cats. At very low doses clonazepam suppressed amygdalohippocampal evoked potentials in the cat and elevated the threshold for the

+

74

8 z

O

E f

w

8 z

TI

FIGURE 7

Decomposition of Clonazepam v i a H y d r o l y s i s

7

%N

_Ic

\ CLONAZEPAM U

v1

02Na&+i"2 COOH

CI \

I

NH2

76

WALTER C . WINSLOW

generation of thalmic, but not cortical, after-discharges. On the spinal level, clonazepam depressed various motor reflex pathways and potentiated presyna tic inhibition as measured by the dorsal root potential. [p3] The principle pathways of biotransformation were shown by Eschenhoff[14] (Figure 8 ) to be reduction of the nitro group to an amine, subsequent acetylation of the amine and oxidative hydroxylation at C S which results in the elimination of these products as their glucuronides and/or sulfate conjugates. The half-life of the parent compound varies from 18 to 50 hours in humans and the major route of excretion is in the urine. [15,16] The two most prevalent metabolites of clonazepam have been found to be amino clonazepam and acetylamino clonazepam. Analytical procedures for detecting these compounds in body fluids, including differential pulse polarography[ 16] and electron capture glc [16,17,18],have been reported. 6.

Toxicology The chronic tolerance of clonazepam in laboratory animals is excellent. The LDSo for rats and mice: >4000 mg/kg by oral o r i.p. administration and no fetotoxic effects were observed. [ 3]

7.

Methods of Analysis 7.1

Elemental Analysis The elemental analysis of a sample of reference standard clonazepam is presented in Table 5. [ 19] Table 5 Element

% Theory

C

57.37

57.07

H

3.17

3.19

c1

11.28

11.23

N

13.43

13.31

0

14.75

1 5 . 2 0 (by

% Found

difference)

0

I ’

\ /

-

F

0

(u

-

y p

I ’

\ /

w

a

77

I

2-X

78

WALTER C. WINSLOW

7.2 Phase Solubility Analysis Phase Solubility Analysis is carried out using methanol as a solvent. A typical example, listing the experimental conditions, is shown in Figure 9 . 7.3

Chromatographic Analysis Thin Layer Chromatography The following TLC systems are useful for identification and evaluation of clonazepam. System I[2o, 2 1 ] is a mixture of acetone:heptane 6 0 : 4 0 v/v. System 11[ 2 1 ] is ethyl acetate:carbon tetrachloride 50:50. In both systems, 20 p 1 of sample solution, containing 0.5 mg of clonazepam in acetone, is applied to a silica gel GF plate and subjected to ascending chromatography. After development for about 15 cm the plates are removed and air dried. Detection is by examination of the plates under shortwave ultraviolet light. The plates are subsequently sprayed with 10% sulfuric acid and heated at 105'C for 15 minutes followed by diazotization and reaction with Bratton-Marshall reagent. The limit of detection for all species listed is at the 0.5 pg level ( 0 . 1 % ) . Approximate R values for f clonazepam and related compounds are given below. System I

Rf

Rf

Clonazepam

.46

.43

Bromacetamido Impurity

.56

Aminoacetamido Impurity

.64

Species

7.4

System I1

Carbostyril

.60

Benzophenone

.90

Electron Capture Gas Liquid Chromatography Methods for the determination of clonazepam in blood and urine have been reported which measure clonazepam directly, [ 17] as its benzophenone[ 16] and as its N-1-methyl derivative. ['*I Each of these methods is reported to have a sensitivity of approximately 1 ng/ml. [ 2 2 ]

FIGURE 9

Phase S o l u b i l i t y A n a l y s i s of Clonazepam

e

n

"

SOLVENT METHANOL SLOPE -0.02°/0 EQUILIBRATION 20 HOURS AT 25°C EXTRAPOLATED SOLUBILITY 10.72 mg/g SOLVENT

0

I

10

I

20

1

30

1

40

I

50

1

60

1

70

I

80

SYSTEM COMPOSITION: rng SOLUTE/g SOLVENT

I

90

I

80

WALTER C.WINSLOW

7.5

Spectrophotometric Analysis S p e c t r o p h o t o m e t r i c a n a l y s i s of clonazepam may be c a r r i e d o u t d i r e c t l y u t i l i z i n g t h e W maximum a t 310 nm i n i s o p r o p a n o l , [ 1 2 ] however, as h y d r o l y s i s p r o d u c t s o f clonazepam may a f f e c t t h e a b s o r p t i v i t y a t t h i s wavelength ( s e e s t a b i l i t y s e c t i o n ) , t h e absence of t h e s e s p e c i e s a t a p p r e c i a b l e l e v e l s should b e confirmed by TLC.

7.6

P o l a r o g r a p h i c Assay Clonazepam e x h i b i t s a d u a l r e d u c t i o n wave which may b e a t t r i b u t e d t o t h e r e d u c t i o n of t h e 4,5-azomethi n e and n i t r o groups. S e n k ~ w s k i [ ~e ~ t ]a l . showed t h a t t h e p o l a r o g r a p h i c r e d u c t i o n of t h e s e groups f o r v a r i o u s 1 , 4 - b e n z o d i a z e p i n e s i n 0.1N H C 1 i n 20% methanol are s u f f i c i e n t l y s e p a r a t e d f o r q u a n t i t a t i v e work based on t h e r e d u c t i o n of t h e azomethine group a t a b o u t - 0.6V v s . SCE. L i n e a r i t y w a s obt a i n e d between sample c o n c e n t r a t i o n and t h e d i f f u s i o n c u r r e n t . The p o l a r o g r a p h i c a s s a y of clonazepam h a s been performed i n aqueous systems by D e S i l v a e t a 1 . , [ 1 6 ] w i t h a s e n s i t i v i t y of 0.5 - 0 . 7 5 pg/ml.

7.7

T i t r i m e t r i c Analysis Clonazepam i s a s s a y e d by d i s s o l v i n g t h e sample i n a c e t i c a n h y d r i d e and t i t r a t i n g w i t h 0.1N p e r c h l o r i c a c i d (HC104) i n g l a c i a l a c e t i c a c i d . The e n d p o i n t may b e determined p o t e n t i o m e t r i c a l l y u s i n g a g l a s s calomel e l e c t r o d e system o r , a l t e r n a t i v e l y , by a d d i n g 5 d r o p s of N i l e Blue hydroc h l o r i d e i n d i c a t o r (1% i n glacial acetic acid) to t h e sample and t i t r a t i n g t o a yellow-green e n d p o i n t . Each m l of 0.1N p e r c h l o r i c a c i d is e q u i v a l e n t t o 31.57 mg of clonazepam.

8 . Acknowledgements

The a u t h o r w i s h e s t o acknowledge t h e a s s i s t a n c e of D r . K. Blessel, D r . R . I . F r y e r and t h e photog r a p h i c and g r a p h i c s e r v i c e s d e p a r t m e n t s of Hoffmann-LaRoche i n t h e p r e p a r a t i o n of t h i s p r o f i l e .

CLONAZEPAM

81

9. References 1. Waysek, E. and Go, M.V., Hoffmann-La Roche Inc., Personal Communication 2. Johnson, J., Hoffmann-La Roche Inc., Personal Communication 3. Data on File, Hoffmann-La Roche Inc. 4. Benz, W., Hoffmann-La Roche Inc., Personal Communication 5. Data on File, Hoffmann-La Roche Inc. 6. Ramsland, A., Hoffmann-La Roche Inc., Personal Communication 7. Chiu, A.M., Hoffmann-La Roche Inc., Personal Communication 8. Kaplan, S.A., Alexander, K., Jack, M.L., Puglisi, C.V., DeSilva, J.A.F., Lee, T.L., Wenfeld, R.E. Journal of Pharmaceutical Sciences, 63, 527 (1974) 9. Propper, R. and Niemczyk, H., Hoffmann-La Roche Inc., Internal Report 10. Chase, G., Hoffmann-La Roche Inc., Internal Report 11. Mayer, W., Erbe, S., Wolf, G., and Voigt, R., Phmmazie, 29, 700-707, (1974); CA, 82:1601636 (1975) 12. Johnson, J.B. , Hoffmann-Lzoche Inc. , Internal Report 13. Blum, J.E. , Haefely, W. , Jalfre, M. , Polc, P . , and Schaerer, K. , Arzneim. -Forsch., 23, 377-389 (1973) , CA, 79: 190 t (1973) 14. Eschzoff , E. , Arzneim. -Forsch., 23, 390 (1973) 15. Data on File, Hoffmann-La Roche Inc. 16. DeSilva, J.A.F., Puglisi, C.V., Munno, N., Journal of Pharmaceutical Sciences, 63, 520 (1974); CA, %:99138h (1974) 17. Naestoft, J. , Larsen, N.E. , Journal o f Chromatography, 93, 113-122 (1974) 18. DeSilva, J.A.F. , Bekersky, I., Journal of Chromatography, 3,447-460 (1974) 19. Scheidl, F., Hoffmann-La Roche Inc., Personal Communication 20. Guastella, J. and Laureano, C., Hoffmann-La Roche Inc., Internal Report 21. Gomez, R., Hoffmann-La Roche Inc., Internal Report 22. Brooks, M.A. , DeSilva, J.A.F. , Talanta, 22, 849-860 (1975) 23. Senkowski, B . Z . , Levin, M.S., Urbigkit, J.R., Wollish, E.G. , Analytical Chemistry, 36, 1991 (1964)

CYCLIZINE

Steven A . Benezra

STEVEN A. BENEZRA

04

INDEX Analytical Profile

1.

- Cyclizine

DESCRIPTION 1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color, Odor

2.

PHYSICAL PROPERTIES 2.1 2.2 2.3 2.4 2.5 2.6 2.7

Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Melting Range Differential Scanning Calorimetry Solubility

3.

SYNTHESIS

4.

STABILITY

5.

DRUG METABOLISM AND PHARMACOKINETICS

6.

METHODS OF ANALYSIS 6.1 6.2 6.3 6.4 6.5 6.6 6.7

Elemental Analysis Nonaqueous Titration Thin Layer Chromatography Gas Chromatography High Pressure Liquid Chromatography Fluorimetry Colorimetry

CYCLlZlNE

1.

DESCRIPTION

1.1 Name, Formula, Molecular Weight Cyclizine is 1-(diphenylmethyl)-4-methylpiperazine .

H

Mol. Wt. 266.40

C18H22N2 1.2 Appearance, Color, Odor

Cyclizine is a white, odorless, crystalline powder. 2.

PHYSICAL PROPERTIES 2.1

Infrared spectrum

The infrared spectrum of cyclizine in KBr is shown in Figure 1. The following assignments are given to the bands in Figure 1. 3058 cm;; 1448 cm-l 1372 cm 745,698 cm-I

aromatic C-H stretch C-C skeletal vibration C-N stretch (tertiary amine) mono-substituted benzene

Numerous other bands are in agreement wif h the published spectrum of N,N-dimethylpiperazine. 2.2

Nuclear Magnetic Resonance Spectrum

The 100 MHz NMR spectrum is shown in Figure 2. The spectrum was taken as a 3 mgl0.5 ml solution of cyclizine in CDC13 containing tetramethylsilane. The following assignments can be made for the observed signals.

85

86 1.

Figure 1.

Infrared Spectrum of Cyclizine

I

1 10 0

9

I

8

1

I

I

7

6

5

-rI

4

I

3

PPm Figure 2.

100 MHz NMR Spectrum of Cyclizine

I

I

I

2

I

0

88

STEVEN A. BENEZRA

Proton Position

No. of Protons

a b

3 8 1 10

C

d

Chemical Shift (ppm)

Multiplicity

2.27 2.43 4.21 7.19-7.44

1 H

singlet singlet singlet mu1tiplet

(C

(b)

n (a) @l No N-CH3 (!I

\

/

(d) 2.3

Ultraviolet Spectrum

The UV spectrum in 0.1 N HC1 is shown in Figure 3 . The maxima and minima are listed in Table 1 along with the molar extinction coefficients at the A The values o tained are in good agreement withm%ose reported by Siek.9

.

TABLE 1 UV Absorption Data for Cyclizine in 0.1 N HC1

Wavelength of Maximum (nm) 269 263 258 253 (sh) 225

Molar Absorptivity 540 742 694 548 1.13 lo4

Wavelength of Minimum (nm) 267 260 244

2.4 Mass Spectrum The low resolution mass spectrum obtained at 70 ev electron energy is represented by the bar graph in

WAVELENGTH (nm)

Figure 3.

Ultraviolet Absorption Spectrum of Cyclizine

90

STEVEN A. BENEZRA

Figure 4 . The molecular Ion of m/e 266 is present but is not the base peak. The base peak in the mass spectrum occurs at m/e 99, the N-methyl piperazine fragment. The species at m/e 167 is the molecular ion minus the N-methyl piperazine radical. Ions at m/e 194, 195, 207, and 208 are from the rearrangement and fragmentation of the N-methyl piperazine moiety. 2.5

Melting Range

The melting range reported in the N.F. XIV for cyclizine is 106°C to 109°C using the class I procedure.3 2.6

Differential Scanning Calorimetry

The DSC scan for cyclizine is shown in Figure 5. An endotherm caused by melting was observed at 103°C

(uncorrected) when the temperature program was lO"/minute. The AHf was 7.1 kcal/mole. 2.7

Solubility

The solubility of cyclizine at 25°C is as follows:4

3.

Solvent

Solubility gm/ml

Water Ethanol Chloroform Ether

<0.1 mg/ml 0.17 1.1 0.17

SYNTHESIS

Cyclizine may be synthesized by the reaction scheme shown in Figure 6. Diphenylcarbinol is reacted to give the benzhydryl chloride which in tu n is reacted with N-methyl piperazine to give cyclizine.3 4.

STABILITY

Cyclizige is stable up to 5 years at room temperature. At 1 0 5 ° C cyclizine suspensions at pH 11.5 decompose to N-methylpiperazine, benzhydrol and benzophenone.7

91

= 7.1

I

I

I

I

I

caI/mole

I

TEMPERATURE OC Figure 5.

DSC Thermogram of Cyclizine

I

I

OH I

CI I

w

(0

Figure 6 .

Synthesis of Cyclizine

STEVEN A. BENEZRA

94

5.

DRUG METABOLISM AND PHARMACOKINETICS

8 Kuntzman and coworkers have determined that cyclizine is metabolized to its demethylated derivative, norcyclizine, which has little activity compared to cyclizine. Both the parent drug and its metabolite, norcyclizine, are distributed in plasma and tissues. The highest concentrations of drug and its metabolite were found in lung, spleen, liver, and kidney. The average half-life of norcyclizine in man was indicated to be less than 1 day wh n cyclizine was administered 5 50 mg t.i.d. for 6 days. 6.

METHODS OF ANALYSIS

6.1 Elemental Analysis Theoretical (%)

C

H N

Found ( X ) 10

81.33 8.32 10.52

80.93 8.33 10.50

6.2 Nonaqueous Titration Dissolve 0.3 g in 75 ml glacial acetic acid. Titrate with 0.1 N perchloric acid using crystal violet indicator. Each ml of 0.1 N perchloric acid is equivalent 3 to 0.01332 g of cyclizine. 6.3

Thin Layer Chromatography

A variety of thin layer chromatographic systems have been used for cyclizine. They are given in Table 11. All visualization was done with short wave W .

CYCLlZlNE

95

TABLE I1

Thin Layer Chromatograph Systems for Cyclizine Ref -

Ad so rbent

Mobile Phase

silica gel

cyc1ohexane:diethylamine: benzene (95:15:5)

0.55

11

silica gel

benzene:ethanol:NH OH 4 (95:15 :5)

0.61

11

silica gel

methano1:chloroform (1:2)

0.60

11

silica gel

ethy1acetate:methanol:NH OH

0.67

11

silica gel

ch1oroform:isopropyl alcohol: 0.45 5% aq. NH40H (74:25:0.6) cyc1ohexane:benzene: 0.55 diethylamine (75:15:10)

11

0.1 M NaOH coated Si02 plates

(17:2 :1)

R

-f-

4

12

0.1 M NaOH coated Si02 plates

methanol

0.46

12

0.1 M NaOH coated Si02 plates

acetone

0.27

12

0.41

12

0.16

12

0.1 M KHSO coated methanol 4 SiO plates 2 0.1 M KHS04 coated 95% ethanol SiO plates 2

6.4 Gas Chromatography Cyclizine will elute off a 2 meter 0.07% SE-30 column, a 0.08% phenyldiethanolamine succinate polymer column, a 1.07% XF1150 column, and a 1.08% Carbowax 20M column in 3.2 min, 4.9 min, 6.2 min, and 4.8 min 13 respectively. The columns were maintained at 175OC.

96

STEVEN A. BENEZRA

6.5

High Pressure Liquid Chromatography

Cyclizine as the hydrochloride salt has a retention time of approximately 6 minutes on a DuPont strong anion exchange column (37-44 p ) 1 meter x 2.1 mm i.d. A mobile phase of 0.1% sodium borate at 1 ml/min, ampient ternperature is used, Detection is U.V. at 254 nm.

6.6

Fluorimetry

Cyclizine when treated with 3%H202 solution at the 0.1 mg/ml level has fluorescence maxima at 417 andl5 449 nm when excited at 305 nm and 335 run respectively. 6.7

Colorimetry

Tissue levels of cyclizine were determingd by complexation of cyclizine with methyl orange.

CYCLl Zl NE

97

REFERENCES 1. 2.

3.

4.

5. 6. 7.

8. 9.

10.

11. 12.

13.

14. 15.

P.J. Hendra and D.B. Powell, J . Chem. SOC., 5705 (1960). T.J. Siek, J . Forensic Sci. 19, 193 (1974). National Formulary XIV, 157 (1975). U.S.P. XIX, 1st Supplement p. 68 (1975). U . S . Patent #2,630,435 J. Murphy, Burroughs Wellcome, personal communication T . J . Coombers, Burroughs Wellcome, personal communication R. Kuntzman, A. Klutch, I. Tsai, and J.J. Burns, J . Pharmacol. and Exp. Ther. 140, 29 (1965). R. Kuntzman, I. Tsai, and J.Jxurns, J. Pharmacol. and Exp. Ther. 158,332 (1967). S. Hurlbert, Burroughs Wellcome, personal communication C-H. Yang, unpublished data W.W. Fike, Analyt. Chem. 38, 1697 (1966). A . MacDonald and R.T. Pflaum, J. Pharm. Sci. 53, 887 (1964). M. Franklin, Burroughs Wellcome, personal communication R.E. Jensen and R.T. Pflaum, J . Pharm. Sci. 2, 835 (1964)

.

DIPERODON

Jordan L . Cohen

100

JORDAN L. COHEN

Table of Contents

1.

2.

3. 4. 5. 6.

7. 8.

Description 1.1 Name: Diperodon 1.2 Formula and Molecular Weight 1.3 Hydrates 1.4 Salts 1.5 Appearance, Color, Odor and Taste Physical Properties 2.1 Spectra 2.11 Infrared Spectrum 2.12 Nuclear Magnetic Resonance Spectrum 2.13 Mass Spectrum 2.14 Ultraviolet Absorption Spectrum 2.2 Optical Rotation 2.3 Melting Range 2.4 Solubility 2.5 Dissociation Constant 2.6 Dipole Moment 2.7 X-Ray Diffraction Synthesis Isolation and Purification Stability and Compatibility Methods of Analysis 6.1 Identification Tests 6.2 Quantitative Analytical Methods 6.21 Elemental Analysis 6.22 Ultraviolet Spectrophotometry 6.23 Titrimetry 6.24 Chromatography Analysis in Biological Fluids and Pharmacokinetics References

DIPERODON

1.

101

Description

1.1

Name: Diperodon Diperodon ~ s L i~s Jd e s i g n a t e d by Chemical A b s t r a c t s as 3-piperidino-1,2-propanediol d i c a r b a n i l a t e monohydrate. I t i s a l s o known as 1 , 2 - p r o p a n e d i o l , 3 - ( l - p i p e r d i n y l ) - , b i s (phenycarbamate) monohydrate. 1.2

Formula and M o l e c u l a r Weight

415.49

1.3

Hydrates Diperodon h a s b e e n r e p o r t e d t o e x i s t i n b o t h t h e monohydrate and anhydrous orms w i t h t h e f o r m e r b e i n g t h e p h y s i c a l l y s t a b l e compound

i.

1.4

Salts The h y d r o c h l o r i d e s a l t i s t h e o n l y r e p o r t e d s a l t of p h a r m a c e u t i c a l i n t e r e s t 5 . 1.5

Appearance, C o l o r , Odor and Taste Diperodon o c c u r s as a f i n e , w h i t e c r y s t a l l i n e , o d o r l e s s power w i t h a c h a r a c t e r i s t i c a l l y b i t t e r teste f o l l owed by a s e n s e o f numbness. 2.

Physical Properties 2.1

Spectra 2.11

I n f r a r e d Spectrum The I R s p e c t r u m od d i p e r o d o n h y d r o c h l o r i d e r e c o r d e d i n a K B r p e l l e t i s shown i n F i g u r e 1.6 S t r u c t u r a l a s s i g n m e n t s from t h i s s p e c t r u m are p r e s e n t e d i n T a b l e I .

Figure 1.

Infrared Spectrum of Diperodon Hydrochloride

DIPERODON

103

Table I Infrared Spectrum of Diperodon HC1

-1 IR Absorption Band (cm )

Assignment N-H(H-bonded)stretch H-C1, stretch C=O, stretch C=C, Aromatic, stretch N-H, bending C-0 vibration monosubstituted aromatic

3400,3200 2630,2530 1730 1590,1490 1540 1200 690

This spectrum is consistent with the drug structure and is in good agreement with the literature infrared spectrum for diperodon. 2.12 Nuclear Magnetic Resonance Spectrum The 60 MHZ magnetic spectrum of dip rodon run in deuterodimethylsulfoxide is shown in Figure 2,' The structural assignments are illustrated in Table 11. Table I1

NMR Spectral Assignments for Diperodon Chemical Shift

(T)

No. -

Proton Assignment

-CH2-(aliphatic ring)

6

8.2

Impurity

-

7.5

-H- CH

4

6.7

-1-0-CH 2

2

5.7

-C-0-CH-C

1

4.5

-CH- ( aromatic)

5

2.7

0

2

9

0

11

-0C-NH-C H + 65

- C-N- C H

2

0.1,0.2

1

-1.0

Figure 2 .

Nuclear Magnetic Resonance Spectrum of Diperodon

DIPERODON

105

2.13

Nass Spectrum The low r e s o l u t i o n mass s p e c t r u m of d i p e r o d o n from a s o l i d p r o b e i n s e r t i o n i s d e p i c t e d i n F i g u r e 3. The e x t r e m e l y weak i n t e n s i t y of t h e p a r e n t i o n p e a k a t 397 m / e i s t y p i c a l of c a r b a m a t e s which undergo t h e r m a l a n d / o r e l e c t r o n impact induced i s o c y a n a t e e l i m i n a t i o n . Other s t r u c t u r a l a s s i g n m e n t s t o t h i s f r a g m e n t a t i o n are shown i n T a b l e 111. T a b l e I11

Mass S p e c t r a l F r a g m e n t a t i o n of Diperodon MassICharge

(m/e)

Assignment

A

98

119

Lb-CH2-

0

0

-NH-t-

124

-CH2CHCN

C C

N -CH CH-CHOH

141

N-CH2-C=CHOH

158

+

l o s s of 119 and H

260

No c o m p a r a t i v e l i t e r a t u r e s p e c t r u m is a v a i l a b l e .

2.14

U l t r a v i o l e t A b s o r p t i o n Spectrum The u l t r a v i o l e t a b s o r p t i o n s p e c t r u m of a ~ x ~ O - s~ oMl u t i o n of d i p e r o d o n i n H C 1 i s shown i n F i g u r e 4. Maximal a b s o r t i o n o c c r e d a t 233 nm w i t h a m o l a r a b s o r 8 p t i v i t y of 2 . 6 ~ 1 01 mole -'cm-l. The a b s o r p t i o n s p e c t r u m w a s a l s o r e c o r d e d i n h e p t a t e with-? A-Tax of 234 nm and a m o l a r a b s o r p t i v i t y of 3 . 9 ~ 1 0 1 mole c m Although t h e r e i s no c o m p a r a t i v e l i t e r a t u r e d a t a t h e X max i s i n agreement w i t h t h a t r e p o r t e d f o r diperodon i n t h e o f f i c i a l a s s a y procedure of t h e N a t i o n a l Formulary!

.

2.2

Optical Rotation The o p t i c a l r o t a t i o n o f d i p e r o d o n h a s a n assymetric c e n t e r and t h e m e t h y l e t h y l k e t o n e s o l v a t e s of t h e

K1

Ln n!

0

d ! .

0

106

0

30h

38'2 395

3hS

32s

30s 382

092 Oh2

022 002 08 I 09 1 OhI

0.2 00 08

09 Oh

02 0

Q

0

2

s

0

0 0

33NVWOS9V

107

0

0

-.

C

0

0

a

0

w

108

JORDAN L. COHEN

d- and 1- forms were r e p o r t e d 8 t o be [aID i- 14.5 and [aID-14.3" r e s p e c t i v e l y . Water w a s t h e s o l v e n t . 2.3

Melting Range The o r i g i n a l s y n t h e t i c l i t e r a t u r e ' r e p o r t e d a m e l t i n g p o i n t of 106.5"C f o r diperodon and a range of 197198°C f o r i t s h y d r o c h l o r i d e s a l t . C u r r e n t compendia3,101ist t h e m e l t i n g range f o r t h e h y d r o c h l o r i d e between 195 and 200°C w i t h decomposition.

2.4

Solubility Diperodon i s p r a c t i c a l l y i n s o l u b l e i n water b u t i s moderately s o l u b l e i n a l c o h o l and v e r y s o l u b l e i n most non-polar s o l v e n t s . The h y d r o c h l o r i d e s a l t i s s o l u b l e i n a l c o h o l , s l i g h t l y s o l u b l e i n e t h y l a c e t a t e , a c e t o n e and water ( l e s s t h a n 1%) and i n s o l u b l e i n most o r g a n i c s o l v e n t s such as benzene and e t h e r . 2 Its s o l u b i l i t y i n water is r e p o r t e d l y i n c r e a s e d by t h e a d d i t i o n of sodium ~ h l o r i d e .Like ~ many o t h e r t e r t i a r y amino a n e s t h e t i c s , dlperodon i s r e p o r t e d t o form 1:l s o l u b l e complexes w i t h 1 , 3 , 5 - t r i n i t r o b e n z a n e .I2 These i n t e r a c t i o n s are p o s t u l a t e d t o i n v o l v e t h e t e r t i a r y amino group and a r e probably c h a r g e - t r a n s f e r and hydrophobic i n n a t u r e . A s i g n i f i c a n t s p e c t r a l change i s observed a t 475 nm. 2.5

D i s s o c i a t i o n Constant Diperodon i s a t e r t i a r y amine and i s weakly b a s i c . Aqueous s o l u t i o n s of 1%diperodon h y d r o c h l o r i d e have a pH of 5 . 1 . l ' Although t h e d i s s o c i a t i o n c o n s t a n t i s n o t s p e c i f i c a l l y r e p o r t e d i n t h e l i t e r a t u r e a pKa of 8 . 4 4 can be e s t i m a t e d from t h i s i n f o r m a t i o n . 2.6

Dipole Moment The d i p o l e morlrent of diperodon i s n o t a v a i l a b l e from t h e l i t e r a t u r e .

2.7

X-Ray D i f f r a c t i o n The x-ray d i f f r a c t i o n p a t t e r n f o r diperodon h y d r o c h l o r i d e h a s been determined and i s summarized i n Table IV . I 3

DIPERODON

109

Table I V

X-Ray D i f f r a c t i o n P a t t e r n of Diperodon H C 1 28 2.26 2.95 3.13 3.22 3.49 3.64 3.94

111,

28

1/10

-13 -16 - 34 -18 -27 -28 -25

-50 -23

4.29 4.58 5.10 5.89 7.06 9.39 11.42

-50 -60 -23 -22 -100

3.

Synthesis Diperodon i s one of s e v e r a l p h e n y l u r e t h a n e d e r i v a t i v e s of d i a l k y l amino a l c o h o l s which h a v e d e m o n o s t r a t e d s i g n i f i c a n t l o c a l a n e s t h e t i c a c t i v i t y . 1 4 The o r i g i n a l s y n t h e ~ i s , ~ ’ ~ ~ w h i c h h a s been P a t e n t e d , 16involves t h e c o n s e n d a t i o n of p i p e r i d i n e w i t h g l y c e r o l c h l o r o h y d r i n ( I ) i n t h e p r e s e n c e of a l k a l a i and t h e n c o n d e n s a t i o n of t h e r e s u l t i n g 1-piperidinopropane-2,3d i o l (11) w i t h p h e n y l i s o c y a n a t e ( 1 1 1 ) . The s y n t h e s i s i s o u t l i n e d below. NHR2 i s p i p e r i d i n e .

NHR2 HOCH2CHOHCH 2C1-,>

(1)

R2NCH2 CHOHCH 20H

(11)

(diperodon) 4.

I s o l a t i o n and P u r i f i c a t i o n Diperodon i s g e n e r a l l y a v a i l a b l e as t h e h y d r o c h l o r i d e s a l t which can b e r e c r y s t a l l i z e d from a m i x t u r e of a c e t o n e and e t h y l a c e t a t e . The f r e e b a s e can t h e n b e o b t a i n e d by addi n g a n e x c e s s of a l k a l a i t o an aqueous s o l u t i o n o f t h e hydroc h l o r i d e s a l t and e x t r a c t i n g w i t h e t h e r . The e t h e r must b e d r i e d o v e r anhydrous sodium s u l f a t e , f i l t e r e d and e v a p o r a t e d .

JORDAN L. COHEN

110

The r e s u l t i n g diperodon i s r e c r y s t a l l i z e d from h i g h b o i l i n g petroleum e t h e r .

5.

S t a b i l i t y and C o m p a t i b i l i t y Diperodon h y d r o c h l o r i d e i s r e a d i l y n e u t r a l i z e d by t r a c e q u a n t i t i e s of a l k a l a i and s o l u t i o n s s h o u l d be s t o r e d i n nona l k a l i n e g l a s s c o n t a i n e r s . Even t r a c e s of a l k a l a i w i l l l e a d t o p r e c i p i t a t i o n of t h e i n s o l u b l e f r e e b a s e and g e n e r a l l y a trace of a c i d is added t o s o l u t i o n s o r d i l u t i o n s t o i n s u r e s o l u b i l i t y . 1 2 The removal of a c i d by f i l t e r p a p e r can a l s o l e a d t o p r e c i p i t a t i o n of t h e f r e e b a s e and less of potency of diperodon h y d r o c h l o r i d e s o l u t i o n s . S o l u t i o n s of t h e hydroc h l o r i d e a l s o a p p e a r t o decompose o v e r t i m e t o produce t r a c e amounts of a n i l i n e . T h i s i s a c c e l e r a t e d by h e a t i n d u r i n g s t e r i l i z a t i o n and a l s o by t h e a d d i t i o n o f a l k a l a i . g 8 A maximal pH of 4 . 8 i s recommended f o r diperodon h y d r o c h l o r i d e s o l u t i o n s and s o l u t i o n s w i t h t r a c e s of c l o u d i n e s s o r c o l o r should n o t be used. Diperodon monohydrate, which i s n o t i n compatible w i t h t r a c e s of a l k a l a i h a s been u t i l i z e d more rec e n t l y i n n o n - s o l u t i o n dosage forms i n c l u d i n g l o t i o n s and ointments.5 Methods o f A n a l y s i s 6.1 I d e n t i f i c a t i o n Tests Diperodon h a s been q u a l i t a t i v e l y i d e n t i f i e d by i n f r a r e d s p e c t r o p h o t o m e t r y *' C o n d i t i o n s f o r p a p e r e l e c t r o p h o r e s i s 1 9 a n d paper and t h i n - l a y e r chromatography20have a l s o been established. The x-ray d i f f r a c t i o n p a t t e r n h a s a l s o been reSeveral s p e c i f i c chemical t e s t s ported13(see s e c t i o n 2.7). have been r e p o r t e d t o d i s t i n g u i s h diperodon h y d r o c h l o r i d e from o t h e r a n e s t h e t i ~ s .A~ w h i t e p r e c i p i t a t e is formed upon t h e a d d i t i o n of s i l v e r n i t r a t e which i s s o l u b i z e d by e x c e s s ammo n i a . A d d i t i o n of H C 1 , sodium n i t r a t e and b e t a n a p h t h o l produces a w h i t e p r e c i p i t a t e which d a r k e n s t o y e l l o w and t h e n orange upon s t a n d i n g . Diperodon h y d r o c h l o r i d e r e a c t s w i t h c h l o r i d e t o g i v e a n organge-yellow p e r c i p i t a t e . 6.

6.2

Q u a n t i t a t i v e A n a l y t i c a l Methods

5 Elemental A n a l y s i s C h l o r i d e i s determined by g r a v i m e t r i c a n a l y s i s f o l l o w i n g t h e a d d i t i o n of s i l v e r n i t r a t e t o a n ammonia s o l u t i o n . N i t r o g e n is a n a l y z e d u s i n g a modified K j e l d a h l determination. Selenium o x y c h l o r i d e i s used i n p l a c e of copp e r s u l f a t e a s a c a t a l y s t and a f o u r h o u r , r a t h e r t h a n two h o u r , d i g e s t i o n i s used.

6.21

DIPERODON

111

U l t r a v i o l e t Spectrophotometry The o f f i c i a l a s s a y p r o c e d u r e f o r d i p e r o d o n o i n t m e n t i n v o l v e s a c h r o m a t o-g r a- p h i c s e p a r a t i o n o f t h e v e h i c l e from t h e d r u g u s i n g a n a l u m i n a column and a 1:l m i x t u r e of hexane and i s o p r o p y l a l c o h o l a s t h e e l u a n t . Q u a n t i t a t i o n i s performed by m e a s u r i n g t h e u l t r a v i o l e t a b s o r p t i o n a t 235 and 300 nm. The s u b s t a n t i a l m o l a r a b s o r p t i v i t y of d i p e r o d o n a l l o w s a t h e o r e t i c a l s e n s i t i v i t y i n t h e low microgram / m l range t o be achieved. 6.22

6.23

Titrat ion The compendia1 a s s a y f o r d i p e r o d o n i n v o l v e s t i t r a t i o n i n a c e t i c a c i d u s i n g p e r c h l o r i c a c i d and c r y s t a l v i o l e t as t h e i n d i c a t o r . Each m l of 0 . 1 N H C l O i s e q u i v a l e n t 4 Diperodon h y d r o c h l o r i d e c a n b e t o 39.75 mg of d i p e r o d o n . t i t r a t e d w i t h H C l O i n a c e t i c a c i d f o l l o w i n t h e a d d i t i o n of 4 mercuric a c e t a t e t o produce t h e f r e e base.28 M e t h y l v i o l e t i n monochlorobenzene i s used as t h e i n d i c a t o r . 6.24

Chromatography A quantitative thin-layer

chromatographic method u s i n g p h o t o d e n s i t o m e t r y h a s b e e n r e p o r t e d . 20

7.

Analysis i n Biological Fluids a g . P h a r m a c o k i n e t i c s Diperodon h a s n o t been a d m i n i s t e r e d i n t e r n a l l y and no d a t a concerning 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 , metabolism o r p h a r m a c o k i n e t i c s i s a v a i l a b l e from t h e l i t e r a t u r e .

Acknowledgement The a u t h o r would l i k e t o e x p r e s s h i s a p p r e c i a t i o n t o D r . W i l l i a m L. D a v i e s of t h e Norwick Pharmacology Company f o r p r o v i d i n g v a l u a b l e d a t a on diperodon.

112

1. 2. 3. 4. 5. 6.

7.

8. 9. 10. 11. 12.

13.

14. 15. 16. 17.

18. 19. 20. 21.

JORDAN L. COHEN

References The N a t i o n a l Formulary, XlV, p.232 ( 1 9 7 5 ) . Merck, I n d e x , 8 t h Ed., p.385 ( 1 9 6 8 ) . Remington's P r a c t i c e of Pharmacy, 1 4 t h Ed., p.1076 (1970). J . S . S c a n l o n , General P r a c t i c e , 27, 1 3 ( 1 9 6 4 ) . Diperodon H y d r o c h l o r i d e B r o c h u r e , S.B. P e n i c k and Co., New York, N . Y . ( 1 9 6 2 ) . These s p e c t r a were k i n d l y p r o v i d e d b y D r s . K. F l o r e y , B. T o e p f i t z and A. Cohen, Squibb M e d i c a l R e s e a r c h , New Brunswick, N . J . K.P. O ' b r i e n , and R.C. S u l l i v a n , B u l l . N a r c o t i c s , 22, 35 (1970). M.S. Raasch a n d W.R. Brode, J. Am. Chem. SOC., 64,1 1 2 (1942) T.H. R i d e r , J . Am. Chem. S O C . , 52, 2115 (1930) U n i t e d S t a t e s D i s p e n s a t o r y , 2 7 t h Ed., p.439 ( 1 9 7 3 ) . T.H. R i d e r , J . o f Lab. C l i n . Med., 2,771 (1934). T.H. R i d e r , J . P h a r m a c o l . Exper. T h e r a p . , 255 (1933) R.C. S u l l i v a n and K.B. O ' b r i e n , B u l l . N a r c o t i c s , 2 0 ( 3 ) , 3 1 (1968). T.H. R i d e r , J . Am. Chem. SOC., 52, 2583 (1930) T.H. R i d e r and A . J . H i l l , J . Am. Chem. SOC., 5 2 , 1528 (1930) U.S. P a t e n t 2 004,132 (1935) E.S. Cook, K . Bambach and T.H. R i d e r , J . Am. Pharm. Assoc. 24, 269 (1935 E.S. Cook and T.H. R i d e r . J. Am. Pharm. ASSOC., 26, 222 (1937). 0. S c h e t t i n o , Farmac Ed. P r a t . 20, 4 0 ( 1 9 6 5 ) ; C.A. 62: 89353 ( 1 9 6 5 ) . V . J o k l and A. Muchora, Acta. F a c . Pharm. Behemoslov., 1 1 : 2 3 ( 1 9 6 5 ) ; C.A. 64: 14024g ( 1 9 6 6 ) . J . W . B e c h e r , The Assay a n d S o l u b i l i t y of Diperodon, Ph.D. T h e s i s , U n i v e r s i t y of Maryland, S c h o o l of Pharmacy, 1 9 6 2 . The l i t e r a t u r e s u r v e y f o r t h i s monograph was from 1 9 3 0 t o J u l y , 1976 i n c l u s i v e .

.

5,

.

,

ERGOTAMINE TARTRATE

Bo Kreilgard

114

BO K R E I L G ~ R D

CONTENTS 1.

Description

1.1 N a m e 1 . 2 Formula a n d M o l e c u l a r Weight 1 . 3 Appearance, C o l o r , Odor a n d T a s t e 2.

Physical Properties 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

Infrared Spectra N u c l e a r M a g n e t i c Resonance S p e c t r u m U 1t r a v i o l e t Spectrum F 1uo re sc e n ce and P ho spho re s ce nce Mass Spectrum Optical Rotation M e l t i n g Range S o l u b i l i t y , P a r t i t i o n C o e f f i c i e n t s and Mo l e c u l a r Complexes Dissociation Constants

3.

P r o d u c t i o n and S y n t h e s i s

4.

Degradation o f Ergotamine T a r t r a t e 4.1 4.2

Chemistry o f Ergotamine Degradation S t a b i l i t y i n P h a r m a c e u t i c a l Dosage Forms

5.

Drug Metabolism

6.

Methods o f A n a l y s i s 6.1 6.2 6.3

Identification Tests Element A n a l y s i s Spectrophotometric Analysis 6.3.1 6.3.2 6.3.3

6.4 6.5

Ultraviolet Colorimetric Fluorescence

Non-Aqueous T i t r a t i o n Chromatographic A n a l y s i s 6.5.1 6.5.2 6.5.3 6.5.4

P a p e r Chromatography T h i n L a y e r Chromatography Column Chromatography High P r e s s u r e L i q u i d Chromatography

7.

Determination i n B i o l o g i c a l Systems

8.

Determination i n Pharmaceutical Preparations

9.

References

ERGOTAMINE TARTRATE

1.

115

Description 1.1

Name

Ergotamine Tartrate (1-3) is the (+)-tartrate salt of (6aRI9R)-N-((2R,5S,1OaS,l0bS)-5-phenylmethyl-lOb-hydroxy-2-methyl-3,6-di0~0-2,3,5,6~ 9,10,10a,10b-octahydro-8H-oxazolo[3,2-alpyrrolo [2,1-c]pyrazin-2-yl) 7-methy1-4,6,6at7,8,9-hexahydro-indolo[4,3 -fglquinoline-9-carboxamide. 1.2

Formula and Molecular Weight

r

COOH I

CHOH I CHOH I

COOH

2 (C33H3s05N5)2"24H606 1.3

Molecular Weight: 1313,43

Appearance, Color, Odor and Taste

Ergotamine tartrate occurs as colorless crystals or white yellowish white, crystalline, odorless powder with a slightly bitter taste. 2.

Physical Properties 2.1 Infrared Spectra The infrared absorption spectrum of ergo-

116

BO K R E I L G ~ R D

t a m i n e t a r t r a t e i s p r e s e n t e d i n F i g u r e 1. The spectrum w a s t a k e n i n a KBr p e l l e t w i t h a P e r kin-Elmer G r a t i n g S p e c t r o p h o t o m e t e r , Model 457. The I R s p e c t r u m o f e r g o t a m i n e b a s e u s i n g t h e K B r as w e l l a s t h e n u j o l t e c h n i q u e h a s b e e n rep o r t e d by Cromp a n d Turney ( 4 ) a n d Hofmann ( 5 ) . 2.2

N u c l e a r M a g n e t i c Resonance S p e c t r u m

The H 1-NMR s p e c t r u m shown i n F i g u r e 2 w a s o b t a i n e d by d i s s o l v i n g e r g o t a m i n e t a r t r a t e ( p r e v i o u s l y d r i e d a t 6OoC below 1 mm Hg f o r 2 hours) i n deuterated dimethylsulfoxide contain i n g t e t r a m e t h y l s i l a n e as a n i n t e r n a l r e f e r e n ce. The s p e c t r u m w a s r e c o r d e d o n a J e o l JNMC-60HL i n s t r u m e n t . The s p e c t r a l a s s i g n m e n t s o f some of t h e p r o t o n s a r e p r e s e n t e d i n Table 1. A d e t a i l e d spectral a n a l y s i s of setoclavine, which h a s a s t r u c t u r e s i m i l a r t o t h a t o f l y s e r g i c a c i d h a s b e e n r e p o r t e d ( 6 ) . The 13C-NMR spectrum o f e r g o t a m i n e a n d e r g o t a m i n i n e h a v e b e e n p u b l i s h e d by Bach e t a l . ( 7 ) . 2.3

U l t r a v i o l e t Spectrum

The u l t r a v i o l e t s p e c t r u m o f e r g o t a m i n e t a r t r a t e i n t a r t a r i c a c i d s o l u t i o n (1% w/v) i s shown i n F i g u r e 3 ( 8 ) . The s p e c t r u m of e r g o t a mine s a l t s and e r g o t a m i n e i t s e l f e x h i b i t s a c h a r a c t e r i s t i c f l a t maximum a t a b o u t 317 nm a n d a minimum a t a b o u t 270 nm. Maximum w a v e l e n g t h s a n d molar a b s o r p t i v i t i e s a r e p r e s e n t e d i n Tabl e 2. 2.4

F l u o r e s c e n c e and P h o s p h o r e s c e n c e

E r g o t a l k a l o i d s of t h e l y s e r g i c a c i d a n d i s o l y s e r g i c a c i d t y p e a r e known t o e x h i b i t f l u o r e s c e n c e when i r r a d i a t e d w i t h u l t r a v i o l e t l i g h t . Loss o f t h e 1 0 , l O a d o u b l e b o n d c o n j u g a t e d w i t h t h e i n d o l e g r o u p c a u s e s loss o f t h e fluorescence ( 1 1 , 1 4 1 . Fluorescence s p e c t r a of e r g o t a m i n e i n aque0u.s s o l u t i o n (pH 2 . 1 and 10.8) There i s a n d i n e t h a n o l a r e shown i n F i g u r e 4 . a h y p s o c h r o m i c s h i f t i n moving from t h e a l k a l o i d s a l t t o t h e b a s e a n d from a q u e o u s t o e t h a n o l i c s o l u t i o n ( 1 7 , 1 8 ) . Heacock e t a1 ( 1 9 ) des c r i b e d t h e i n f l u e n c e o f some o r g a n i c s o l v e n t s on t h e f luor escence i n t e n s i t y of ergotamine. The f l u o r e s c e n c e i n t e n s i t y of e r g o t a m i n e i n

1 117

118

ERGOTAMINE TARTRATE

119

Table 1 NMR S p e c t r a l Assignments o f Ergotamine T a r t r a t e

Proton

Number of p r ot o n s

*

Mu1t i p 1i c i t y

Chemical S h i f t (ppm)

(indole

1

10.9

Broad s i n g l e t

NH ( a m i d e )

1

9.5

Broad s i n g l e t

6.9-7.4

Mu1t i p l e t

NH

*

Aromatic p r o t o n s

+ . ,

C-lO'b,-OH

1

NE-CH

3 Tartrate,-OH H2° C-10 ' a , -H C-5' ,-H C - 2 ' ,-CH

*

3

10

5

J

*

6.3-6.7

1

6.3

Triplet

1

4.5

Triplet

3

1.5

Broad s i n g l e t

~~

Exchangeable w i t h D 0 2

~

~~

~

~~

16

12

4

240

260

280

300

320

340

Wavelength, nm Figure 3 .

U l t r a v i o l e t Spectrum of Ergotarnine T a r t r a t e in 1%T a r t a r i c A c i d Solution ( 8 ) .

Table 2 Ultraviolet Spectral Characteristics

A

p!

A

max, nm

Reference

Compound

Solvent

Ergotamine t a r t r a t e

1%t a r t a r i c a c i d

2 4 0 , 318

7 . 7 2 ( a t 318 nm)

8

Ergotamine t a r t r a t e

1%t a r t a r i c a c i d

317

7.34

9

Ergot a m i ne t a r t r a t e

0.01 N H C 1

317

Ergo tamine t a r t r a t e

0.01 M t a r t a r i c acid

317.5

8.00

10

Ergotamine

Ethanol

318

7.24

11

Ergo t a m i ne

Methylene c h l o r i d e

308-310

8.59

12

Ergo tamine

Ethanol

311

8.60

13

-7.50

2

BO K R E I L G ~ D

122

7.

- 5'

c @

2 W

t

2

W

0 2 W

0

v) W

a2 0 3

Y

0 WAVELENGTH

(nm) F i g u r e 4.

E x c i t a t i o n s p e c t r a ( l e f t ) of e r g o t a m i n e in: (1) water a t pH 2 . 1 ( A e m 435 m ) : ( 2 ) water a t pH 1 0 . 8 ( A e m 4 2 2 nm; ( 3 ) e t h a n o l (Aern 4 0 2 nm). E m i s s i o n s p e c t r a ( r i g h t ) o f e r g o t a m i n e i n (1) water a t pH 2 . 1 (Aex 325 nm) : ( 2 ) w a t e r a t 10.8 ( A e 318 nm); ( 3 ) e t h a n o l (Aex 318 my (17).

ERGOTAMINE TARTRATE

123

aqueous s o l u t i o n i s h i g h l y dependent on t h e p H o f t h e s o l u t i o n showing a l m o s t e q u a l i n t e n s i t y i n t h e pH-range 1 - 9 a n d maximum i n t e n s i t y a t pH -11 ( 1 7 , 1 8 1 . Data o n f l u o r e s c e n c e o f e r g o t a m i n e a r e summarized i n Table 3 . The p h o s p h o r e s c e n c e s p e c t r u m o f e r g o t a m i n e i n e t h a n o l a t 7 7 O K showed Xmax a t 5 1 6 , 5 5 8 a n d 6 1 3 nm ( 1 3 ) . 2.5

Mass SDectrum

S e v e r a l a u t h o r s h a v e r e p o r t e d o n t h e mass spectrum of ergotamine ( 2 0 - 2 3 ) . The low r e s o l u t i o n mass s p e c t r u m o f e r g o t a m i n e i s shown i n F i g u r e 5 ( 2 2 ) . The m o l e c u l a r i o n ( p a r e n t p e a k ) i s a b s e n t i n t h e spectrum o b t a i n e d b y 7 0 e V e l e c t r o n impact i o n i z a t i o n ( 2 1 - 2 3 ) , w h i l e a "reasonable-sized" parent i o n i s observed using h i g h r e s o l u t i o n mass s p e c t r o s c o p y a t 1 6 e V ( 2 0 ) . The i o n s b and c o r i g i n a t e from t h e m o l e c u l a r i o n by s p i i t t i n g o f t h e bond b e t w e e n t h e C-9 carboxamide n i t r o g e n and t h e q u a r t e r n a r y c a r b o n ( C - 2 ' ) , followed by t h e hydrogen a t o m t r a n s f e r from t h e p e p t i d i c p a r t t o t h e l y s e r g a m i d i c part. Ion b (m/e = 267) i s i d e n t i c a l with t h e m o l e c u l a r i o n o f l y s e r g i c a c i d amide whose f r a g m e n t a t i o n i s known ( 2 4 ) . The s u b s t a n t i a l p a r t o f t h e i o n c u r r e n t ( 8 0 - 9 0 p e r c e n t ) comes from i o n s from t h e p e p t i d i c p a r t o f t h e molec u l e , w h i l e i o n b and i t s f r a g m e n t s form 1 0 - 2 0 per cent of t h e t o t a l ion current ( 2 3 ) . Other i m p o r t a n t f r a g m e n t s a r e shown i n Scheme I ( 2 1 , 23). C h a r a c t e r i z a t i o n of ergotamine r e l a t i v e t o o t h e r e r g o t a l k a l o i d s of t h e p e p t i d e t y p e i s based on t h e i o n s 1,& a n d t h e t r o p y l i u m i o n s i n c e t h e s e fragments i n c l u d e t h e methyl group a t C-2' and t h e benzyl group a t C-5'. High r e s o l u t i o n mass s p e c t r o s c o p y o f e r g o tamine has a l s o been r e p o r t e d ( 2 0 , 2 1 1 .

c,

2.6

Optical rotation

Carbons 6a, 9 , 2 ' , 5 ' , 1 0 ' a and 1 0 ' b o f e r g o t a m i n e a r e a s y m m e t r i c , r e s u l t i n g i n 6 4 poss i b l e c o n f i g u r a t i o n a l isomers. However, o n l y i s o m e r s w i t h c h a n g e d c h i r a l i t y a t C-9 a n d C - 2 ' o c c u r 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 . The rot a t i o n o f e r g o t a m i n e a n d some o f i t s i s o m e r s

Table 3 D a t a on Fluorescence of Ergotamine

Solvent

Temper a t u r e

E x c i t a t i o n wavelength

(nm)

-3

Maximal Emission wavelength (nm)

Reference

Ethanol

25OC

3 20

404

13

Water

2 5OC

3 20

432

13

Ethanol

77OK

3 20

383

13

Water (pH 2.1)

ambient

325

43 5

17

Water (pH 1 0 . 5 )

ambient

318

422

17

Ethanol

ambient

318

402

17

Water (pH 2-6)

ambient

335

43 5

18

Water ( p H 8 - 1 4 )

ambient

325

425

la

Acetone

ambient

350

400

19

loor '0(i)

80

[L

125 (h)

[L

3 V

-6

E

I

2

a I-

0

-4

bp

120(k)

50 F i g u r e 5.

w 100

150

200

Mass Spectrum of Ergotarnine ( 2 2 ) .

250

+ U

0

244 (d)

300

M'E

0

I

q2

0

0

I

qj+-

-

0

a

-

.-

126

+*

I

1

+

f

4J

k

m0 w

0

+I

ro

.lJ

c,

H

H

w X

U

m

ERGOTAMINE TARTRATE

127

are l i s t e d i n T a b l e 4 . The o p t i c a l r o t a t o r y d i s p e r s i o n spectrum of ergotarnine i n methanol h a s been r e p o r t e d (25) and i s reproduced i n Figure 6. 2.7

Melting range

T h e f o l l o w i n g m e l t i n g p o i n t s h a v e b e e n report ed:

(1)

18OoC (decomp. -190Oc

(2)

203OC (decomp. ) 2.8

(29)

S o l u b i l i t y , P a r t i t i o n C o e f f i c i e n t s and Mol e c u l a r Complexes

The s o l u b i l i t y o f e r g o t a r n i n e t a r t r a t e i s a s follows: Solvent

Water I1

Approximate solubility rng/rnl

Temperature

2.5

30

-2

Ambient

Reference

(33) (112)

0 , l N HC1

3.5

30

(33)

0 , l M phosphate b u f f e r ( p H 6.65)

0.01

30

(33)

Ethanol

2- 3

Chloroform

-1

Ambient

(2,341

Ambient

(2)

The b a s e , e r g o t a r n i n e , h a s b e e n r e p o r t e d t o be s o l u b l e 1 : 3 0 0 i n e t h a n o l , 1:70 i n m e t h a n o l , 1:150 i n a c e t o n e , f r e e l y s o l u b l e i n c h l o r o f o r m a n d a l m o s t i n s o l u b l e i n w a t e r a t room t e m p e r a ture (29). D i s t r i b u t i o n of e r g o t a m i n e b e t w e e n a q u e o u s a n d o r g a n i c s o l v e n t s h a s b e e n s t u d i e d b y several a u t h o r s (17,35-37). Virtually quantitative e x t r a c t i o n o f ergotarnine from aqueous a l k a l i n e s o l u t i o n s (pH -8-11) i n t o b e n z e n e , e t h e r a n d c h l o r o f o r m h a s b e e n observed ( 1 7 , 3 6 1 . Beran a n d Sermonsky ( 3 8 ) r e p o r t e d o n c o u n t e r c u r r e n t d i s t r i b u t i o n of e r g o t a m i n e i n t h e s y s t e m of

128

BO KREILGXRD

Table 4 O p t i c a l R o t a t i o n f o r Ergotamine a n d some of i t s i s o m e r s

Compound Ergotamine

[al

A , r-lm 589

-166'

Conditions CHC13(c=l) ,25 0C CHC13 ( c = l ),20 0C

Reference 26

Ergo tamine

-181O

546.1

Ergotamine

-150°

589

C H C 1 3 ( c = 1 ) ,20°C

28

Ergo tamine

-160°

589

CHC13 ( c = l ),2O0C

29

Ergo tamine

-192O

546.1

CHC13 ( c = l ),2O0C

29

589

p y r i d i n e (c=l. 0) 200C

29

546.1

p y r i d i n e ( c = l . O ) 2OoC

29

Ergo tamine

-12.7O

Ergotamine

-8.6O

27

Ergo tami ne

-155O

589

CHC13, 20°C

30

Ergo t a m i ne

+40°

5 89

e t h a n o l ,2OoC

30

Ergotamine

-466O

365

CHC13 (c=O. 6 ) ,20°C

31

Ergo t a m i ne

-375O

405

CHC13 ( c = 0 . 6 ) ,2O0C

31

Ergotamine

-309O

436

C H C l ( c = O . 6) ,2O0C

31

Ergo t a m ine

-174O

546

CHC13 ( c = 0 . 6 ) ,20°C

31

Ergotamine

-152O

578

31

Ergotamine

-145O

589

CHC13 (c=O. 6 ) ,20°C CHClj(c=0.6) ,20 0C

546.1

CHC13, 20°C

27

589

C H C l (c=O.5 ) ,2O0C

29

546.1

C H C l ( c = 0 . 5 ) ,2O0C

29

Ergotaminine +4 50° 0

Ergotaminine +369

0

E r g o t a m i n i n e +462

3

3

Ergotaminine +49 7O

589 546.1

3 0 pyridine (c=O .5) , 2 O C p y r i d i n e ( c = 0 . 5 ) , 2 0 0C

E r g o t a m i n i n e +381°

589

CHC13, 2O0C

0

Ergotaminine +397

Ac i - e r g o tamine

-32O

Aci-ergo tamine

+258O

589

31

29 29 30

p y r i d i n e ( c = 1 . 2 ) ,20 0C

32

p y r i d i n e ( c = 1 . 2 ) ,20 0C

32

129

a-

0 0

0 Lo

m

0 c*l

m

0

00

(v

0

=r (v

0 0 (v

E

C I ICY

r

W

W

w e

3

BO K R E I L G ~ R D

130

aqueous t a r t a r i c a c i d o r t a r t r a t e s o l u t i o n s chloroform. Using t h e p h a s e s o l u b i l i t y t e c h n i q u e i t h a s been shown t h a t e r g o t a m i n e t a r t r a t e forms m o l e c u l a r complexes w i t h x a n t h i n e d e r i v a t i v e s ( 3 3 , 3 7 ) . The o b s e r v a t i o n s made do n o t p e r m i t c a l c u l a t i o n s of s t a b i l i t y c o n s t a n t s . 2.9

Dissociation Constants

Due t o t h e low s o l u b i l i t y o f e r g o t a m i n e i n w a t e r t h e a c i d d i s s o c i a t i o n c o n s t a n t , pKa, c o u l d n o t b e d e t e r m i n e d by c o n v e n t i o n a l t i t r a t i o n m e t h o d s . An a p p a r e n t pKa v a l u e o f 6 . 4 a t 24O w a s obtained p o t e n t i o m e t r i c a l l y u t i l i z i n g a s o l u t i o n o f e r g o t a m i n e i n 2% c a f f e i n e ( 3 9 ) i n a c c o r d a n c e w i t h a v a l u e of 6 . 3 u t i l i z i n g t h e A pKa v a l u e o f 5 . 6 i n 8 0 s o l u b i l i t y method. p e r c e n t a q u e o u s m e t h y l c e l l o s o l v e h a s b e e n reported ( 5 ) . 3.

P r o d u c t i o n and S y n t h e s i s

E r g o t a m i n e w a s o r i g i n a l l y p r o d u c e d by i s o l a t i o n o f t h e a l k a l o i d from t h e f u n g u s C l a v i c e p s Purp u r e a ( 3 0 , 4 0 ) . Methods o f i s o l a t i o n o f e r g o t a m i n e and p r e p a r a t i o n o f t h e t a r t r a t e s a l t h a v e b e e n deThe c o m p l e t e s y n t h e s i s o f scribed (i.e. : 5,29,42) e r g o t a m i n e was n o t r e p o r t e d u n t i l 1 9 6 1 ( 4 3 ) (Scheme 11) M e t h y l b e n z y l o x y m a l o n i c a c i d - h e m i - e s t e r (I) i s r e a c t e d i n p y r i d i n e w i t h L-phenylalanyl-L-prolinel a c t a m (11) The r e s u l t i n g a c y l a t e d d i k e t o p i p e r a z i n e (111) i s v e r y l a b i l e a n d i s t h u s i m m e d i a t e l y t r e a t e d w i t h Pd/H2 t o c l e a v e t h e b e n z y l g r o u p (IV) (IV) c y c l i z e s s p o n t a n e o u s l y t o t h e c y c l o l s t r u c t u r e (V) Using f r a c t i o n a l c r y s t a l l i z a t i o n t h e stereoisomer w i t h t h e d e s i r e d c h i r a l i t y a t C - 2 ' i s i s o l a t e d . The c a r b e t h o x y g r o u p a t C - 2 ' i s t r a n s f o r m e d i n t o a n amino g r o u p ( V 1 ) t h r o u g h a C u r t i u s r e a c t i o n . The h y d r o c h l o r i c s a l t of t h e p e p t i d e p a r t i s react e d w i t h t h e h y d r o c h l o r i c s a l t of l y s e r g i c a c i d c h l o r i d e (VII) i n c h l o r o f o r m a n d t r i b u t y l a m i n e t o form e r g o t a m i n e . The f i r s t s y n t h e s i s of l y s e r g i c a c i d was r e p o r t e d by Kornblum e t a 1 ( 4 4 ) . An i m p r o v e d s y n t h e s i s o f e r g o t a m i n e u s i n g ( S ) - ( - 1 -methyl-benzyloxy-malonic-hemi-acid c h l o r i d e h a s b e e n reported (45).

.

.

.

.

.

ERGOTAMINE TARTRATE

1.

-

COOH

2 . - Cocl

HCI

*

V

3.

'

H2N

- CON3 CH2C6H5

sc - C I

$i

N-CH3

H

8

HCI

VI

H

ERGOTAMI N E

SCHEME I1

Synthesis of Ergotarnine ( 4 3 ) .

131

BO K R E I L G ~ D

132

D e g r a d a t i o n of e r g o t a m i n e t a r t r a t e

4.

4.1

1

C h e m i s t r y of e r g o t a m i n e d e g r a d a t i o n

The p o s s i b l e pathways o f d e g r a d a t i o n of e r g o t a m i n e t a r t r a t e a r e summarized i n Scheme I11 ( 8 ) .

$

Ergot;:e Aci-ergotamine

z

Ergot;y;ine

1

Lumi compounds

Aci-ergotaminine I

i c

Lysergic acid amide

I lsolysergic acid amide I 1

t c

ie

Lysergic acid lsolysergic acid

Oxidation products

Scheme 111. D e g r a d a t i o n scheme f o r e r g o t a m i n e . b: r e v e r a : r e v e r s i b l e e p i m e r i z a t i o n a t C-9. s i b l e e p i m e r i z a t i o n a t C-2 ' ( t h e a c i - i n v e r c: h y d r o l y s i s . d: f o r m a t i o n o f l u m i sion) e: o x i d a t i o n . compounds.

.

E p i m e r i z a t i o n a t C-9 w i t h f o r m a t i o n o f t h e i s o l y s e r g i c a c i d d e r i v a t i v e , ergotaminine, i s t h e m o s t i m p o r t a n t r o u t e of d e g r a d a t i o n ( 4 6 48). I n acidic solutions ergot alkaloids epim e r i z e a t c-2', t h e so-called aci-inversion ( 3 2 , 4 7 , 4 9 ) . H y d r o l y s i s of t h e f o u r e r g o t a m i n e isomers w i l l r e s u l t i n f o r m a t i o n o f e i t h e r l y s e r g i c a c i d o r l y s e r g i c a c i d amide o r t h e corr e s p o n d i n g is0 compounds ( 2 7 , 4 7 , 4 8 , 5 0 - 5 3 ) . Upon e x p o s u r e t o l i g h t , p a r t i c u l a r l y U V - l i g h t , e r g o t a l k a l o i d s add a molecule w a t e r t o t h e 1 0 , l 0 a - d o u b l e b o n d ( 1 5 , 5 5 1 a s shown:

ERGOTAMINE TARTRATE

133

CO-R

r?

N-CH,

CO-R

J 7

N- CH,

N-CH, HO H

A l l compounds m e n t i o n e d a b o v e are a b l e t o un-

d e r g o o x i d a t i o n ( 5 6 ) . One of t h e e x p e c t e d deg r a d a t i o n p r o d u c t s i s t h e 2-0x0-3-hydroxy-2,3dihydro d e r i v a t i v e s ( R = peptide p a r t ) :

Ergotamine t a r t r a t e i n s o l i d s t a t e d e g r a d e s when e x p o s e d t o l i g h t , humid c o n d i t i o n s and h i g h t e m p e r a t u r e (34). 4.2

S t a b i l i t y i n P h a r m a c e u t i c a l Dosage Forms

S e v e r a l s t u d i e s o n t h e s t a b i l i t y of e r g o t a m i n e t a r t r a t e i n a q u e o u s s o l u t i o n h a v e been done ( 4 7 , 4 8 , 5 5 , 5 7 , 5 8 , 5 9 , 6 0 , 6 6 ) . However, i n some of t h e s t u d i e s n o n - s p e c i f i c methods o f a n a l y s i s were u s e d . Due t o t h e f a c t t h a t e p i m e r i z a t i o n a t C-9 p r o c e e d s r a t h e r f a s t a t t h e pH of o p t i m a l s t a b i l i t y ( 4 7 ,4 8 ) i n j e c t i o n s c o n t a i n i n g t h e d r u g

124

BO K R E I L G ~ R D

a r e f o r m u l a t e d t o c o n t a i n a n e q u i l i b r i u m mixt u r e of t a r t r a t e s o f ergotamine a n d e r g o t a m i nine (1,2,63). I n accordance with t h i s , inves t i g a t i o n of some l i q u i d f o r m u l a t i o n s of e r g o t a m i n e showed a c o n t e n t of o n l y 50-60 p e r c e n t o f e r g o t a m i n e ( 6 1 , 6 2 ) . A t p H = 3 . 6 s u c h a mixt u r e a p p e a r s t o b e s t a b l e when s t o r e d p r o t e c t e d a g a i n s t l i g h t i n a r e f r i g e r a t o r ( 4 8 ) . The r a t e of a c i - i n v e r s i o n i n c r e a s e s w i t h d e c r e a s i n g pH w h i l e h y d r o l y s i s i n t o t h e a c i d o r amide i s a t a m i n i m u m a t pH - 3 ( 4 8 ) . The i n f l u e n c e of b u f f e r s u b s t a n c e s on t h e r a t e of l i g h t - c a t a l y z e d f o r m a t i o n o f l u m i compounds h a s a l s o b e e n i n v e s t i gated (55). I f s o l u t i o n s containing ergotamine a r e p r o t e c t e d a g a i n s t l i g h t and s t o r e d under i n e r t g a s f o r m a t i o n of l u m i compounds and o x i d a t i o n are very s l o w processes (47,481. Ergotamine t a r t r a t e i s n o t s t a b l e f o r prolonged p e r i o d s i n t a b l e t s . In t a b l e t s containing ergotamine t a r t r a t e , p h e n o b a r b i t a l and a m i x t u r e o f t r o p a n e a l k a l o i d s t h e c o n t e n t o f erg o t a m i n e was o b s e r v e d t o d e c r e a s e g r a d u a l l y dur i n g t i m e of s t o r a g e ( 6 4 ) . I n a c c o r d a n c e w i t h t h i s t h e c o n t e n t of e r g o t a m i n e i n commercial t a b l e t s i n g e n e r a l i s less t h a n t h e d e c l a r e d amount ( 6 1 , 6 2 1 El-Shami e t a 1 ( 6 5 ) s u g g e s t t h a t e r g o t a mine t a r t r a t e i n s u p p o s i t o r i e s i s s t a b l e f o r a b o u t 2 y e a r s when 4 mg t a r t a r i c a c i d b l e n d e d w i t h 4 0 mg l a c t o s e were u s e d a s s t a b i l i z i n g a g e n t . The a u t h o r s , however, u s e d a method which o n l y d e t e c t e d l o s s of a c t i v e d r u g t h r o u g h oxidation.

.

5.

Drug M e t a b o l i s m

Very l i t t l e i n f o r m a t i o n i s a v a i l a b l e on t h e abs o r p t i o n , metabolism and e x c r e t i o n of e r g o t a m i n e (66). E r g o t a l k a l o i d s of t h e p e p t i d e t y p e a r e i n gen e r a l p o o r l y and i r r e g u l a r l y a b s o r b e d from t h e gas t r o i n t e s t i n a l t r a c t a n d a l a t e n t p e r i o d o f -30 m i nutes w a s observed (32,66). Caffeine i n c r e a s e s t h e r a t e and e x t e n t of a b s o r p t i o n of e r g o t a m i n e t a r t r a t e and r e d u c e s t h e l a t e n t p e r i o d (32). The a l k a l o i d d i s a p p e a r s v e r y r a p i d l y from t h e b l o o d a f t e r i n t r a venous i n j e c t i o n ( 6 7 - 6 9 ) . Only a m i n o r amount o f t h e d r u g i s e x c r e t e d i n t h e u r i n e , i n d i c a t i n g det o x i f i c a t i o n by t h e l i v e r ( 3 2 , 6 6 , 6 8 , 6 9 ) . N o i n f o r -

ERGOTAM IN E TARTRATE

135

m a t i o n on m e t a b o l i s m o f e r g o t a r n i n e seems t o b e available i n the literature. 6.

Methods of a n a l y s i s 6.1

I d e n t i f i c a t i o n tests

Ergotamine t a r t r a t e c a n be i d e n t i f i e d by v i r t u e o f i t s U V , I R , NMR a n d f l u o r e s c e n c e s p e c t r a , a s w e l l a s i t s o p t i c a l r o t a t i o n (see section 2) V a r i o u s c h r o m a t o g r a p h i c methods s u c h a s TLC ( s e c t i o n 6 . 6 . 2 1 , PC ( s e c t i o n 6 . 6 . 1 ) and HPLC ( s e c t i o n 6 . 6 . 4 ) p r o v i d e a l t e r n a t e m e thods f o r purposes of i d e n t i f i c a t i o n . A b l u e c o l o r i s p r o d u c e d when 0 . 3 mg a l k a l o i d i s dissolved i n 1 . 0 m l g l a c i a l acetic a c i d ( c o n t a i n i n g 0 . 5 p e r c e n t F e ( I I 1 ) a s FeCl 3 a n d 0 . 1 % g l y o x y l i c a c i d ) and 1 . 0 m l 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 i s added ( 5 ) . T h i s s o - c a l l e d K e l l e r r e a c t i o n which, modified s l i g h t l y , i s u s e d i n USP X I X (1) i s b a s e d o n a r e a c t i o n between t h e a l k a l o i d and g l y o x y l i c a c i d which i s almost always p r e s e n t as a n i m p u r i t y i n g l a c i a l acetic acid. The van Urk r e a c t i o n i s b a s e d o n c o n d e n s a t i o n between two m o l e c u l e s of an e r g o t a l k a l o i d and o n e m o l e c u l e p - d i m e t h y l a m i n o b e n z a h dehyde f o l l o w e d by a F e ( I I 1 ) - c a t a l y z e d o x i d a t i o n of t h e c o n d e n s a t e ( 5 , 7 1 , 7 2 ) . Other c o l o r t e s t s h a v e been r e p o r t e d by C l a r k e ( 7 3 ) . E r g o t a m i n e t a r t r a t e h a s b e e n i d e n t i f i e d by means o f TLC of i t s u l t r a v i o l e t d e g r a d a t i o n p r o d u c t s ( 7 4 ) a n d by o s c i l l o p o l a r o g r a p h y ( 7 5 ) .

.

6.2

Elemental a n a l y s i s

The e l e m e n t a l c o m p o s i t i o n of e r g o t a m i n e t a r t r a t e p r e v i o u s l y d r i e d f o r t w o h o u r s a t 60° and a t a p r e s s u r e below 1 mm Hg t o remove water is: Carbon

64.01%

Hydrogen

5.83%

Nitrogen

10.66%

Oxygen

19.49%

136

80

KREILGARD

6.3 6.3.1

Spectrophotometric Analysis Ultraviolet

The u l t r a v i o l e t a b s o r p t i o n o f e r g o t a m i ne t a r t r a t e c a n b e used f o r q u a n t i t a t i o n ( 8 , 9 , 1 2 , 7 6 , 7 7 ) , b u t t h e p o s s i b i l i t y of i n t e r f e r e n c e from r e l a t e d a l k a l o i d s , d e g r a d a t i o n p r o d u c t s and e x c i p i e n t s r e q u i r e s t h a t t h e a l k a l o i d b e i s o l a t e d from t h e s e o t h e r s u b s t a n c e s p r i o r t o measurement. The a b s o r b a n c e of t h e f i n a l sampl e i s g e n e r a l l y measured i n t h e r e g i o n 310-320 nm d e p e n d i n g on t h e s o l v e n t ( s e e s e c t i o n 2 . 3 ) . E r g o t a m i n e t a r t r a t e i n a t a r t a r i c a c i d sol u t i o n o b e y s B e e r ' s l a w a t 271 a n d 318 run i n (8,761. t h e c o n c e n t r a t i o n r a n g e (1-10) x The c o n t e n t o f n a t i v e e r g o t a l k a l o i d s a s impur i t i e s i n h y d r o g e n a t e d a l k a l o i d s c a n be d e t e r mined by measurement of t h e u l t r a v i o l e t absorb a n c e ( 1 0 ) . By r e a d i n g t h e a b s o r b a n c e a t 2 7 1 , 283 and 318 nm o f a d e g r a d e d e r g o t a m i n e t a r t r a t e s o l u t i o n t h e e x t e n t o f f o r m a t i o n of l u m i compounds a n d o f o x i d a t i o n p r o d u c t s c o u l d b e estimated ( 8 ) . 6.3.2

Colorimetric

The m o s t w i d e l y u s e d c o l o r i m e t r i c method f o r a n a l y s i s of e r g o t a m i n e i s t h e r e a c t i o n w i t h p-dimethylaminobenzaldehyde ( 8 t 7 1 r 7 2 t 7 9 - 9 7 ) . I n t h e l i t e r a t u r e t h e r e a g e n t u s e d i s named a s e i t h e r t h e v a n Urk r e a g e n t ( 7 1 ) I t h e M a u r i c e Smith r e a g e n t ( 8 9 ) o r t h e A l l p o r t r e a g e n t ( 8 0 ) depending on v a r i o u s minor m o d i f i c a t i o n s . Sev e r a l a g e n t s have been s u g g e s t e d t o b r i n g a b o u t t h e o x i d a t i o n of t h e c o n d e n s a t e ( s e e sect i o n 6 . 1 ) s u c h a s l i g h t ( 8 9 , 9 3 , 9 5 ) , FeC13 ( 8 0 , 9 3 , 9 5 ) , hydrogen p e r o x i d e ( 8 0 , 9 6 , 9 7 ) and s o d i um n i t r i t e ( 9 5 ) . The a b s o r b a n c e o f t h e f i n a l The sample i s g e n e r a l l y m e a s u r e d a t 550 nm. method i s n o t s p e c i f i c f o r e r g o t a m i n e . A l l compounds w i t h i n t a c t l y s e r g i c o r i s o l y s e r g i c a c i d s t r u c t u r e as w e l l a s l u m i d e r i v a t i v e s w i l l i n t e r f e r e . M e a s u r i n g a b s o r b a n c e a t 546 and 586 nm f o l l o w i n g t h e v a n U r k r e a c t i o n a n d c a l c u l a t i o n s assuming a two-component s y s t e m w i l l l e a d t o a n e s t i m a t e o f t h e amount o f l u m i d e r i v a t i v e s i n a given ergotamine t a r t r a t e sample ( 8 ) . I t h a s b e e n s u g g e s t e d t o u s e m e t a l d e h y d e r e a g e n t r a t h e r t h a n a p-dimethylami-

ERGOTAMINE TARTRATE

137

nobenzaldehyde r e a g e n t due t o improved s e n s i t i v i t y and s p e c i f i c i t y ( 9 8 , 9 9 ) . E r g o t a m i n e t a r t r a t e h a s a l s o been a n a l y s e d c o l o r i m e t r i c a l l y by r e a c t i o n w i t h m i d o p y r i m i d i n e ( 1 0 0 ) . Ionp a i r f o r m a t i o n between e r g o t a m i n e a n d t r o p a e o l i n 0 0 0 ( t h e sodium s a l t of 4 - [ ( 2 - h y d r o x y napthy1)azolbenzol-sulphonic a c i d ) h a s b e e n u s e d i n t h e q u a n t i t a t i v e a n a l y s i s of e r g o t a m i n e (101). 6.3.3

Fluorescence

A f l u o r i m e t r i c a n a l y s i s f o r ergotamine t a r t r a t e i n t a b l e t s h a s b e e n d e s c r i b e d by Hoop e r e t a 1 ( 1 7 ) . The t a b l e t s were e x t r a c t e d w i t h a n a c i d i c a q u e o u s s o l u t i o n , which a f t e r b e i n g made a l k a l i n e w a s e x t r a c t e d w i t h benzen e . A f t e r e v a p o r a t i o n of b e n z e n e t h e r e s i d u e was d i s s o l v e d i n e t h a n o l a n d t h e f l u o r e s c e n c e i n t e n s i t y w a s r e a d w i t h a n e x c i t a t i o n wavel e n g t h o f 318 nm and a n e m i s s i o n w a v e l e n g t h o f 4 0 2 nm. The minimum d e t e c t a b l e c o n c e n t r a t i o n was r e p o r t e d t o b e 0 . 0 0 2 ug p e r m l a n d t h e s t a n d a r d c u r v e w a s l i n e a r up t o 5 pg p e r m l ( 1 7 ) . It is reported t h a t the standard curve f o r ergotamine t a r t r a t e i n t a r t a r i c a c i d solut i o n i s n o n - l i n e a r i n t h e r a n g e 1 0 - 6 0 ug p e r m l (102). In order t o increase sensitivity f l u o r i m e t r i c d e t e c t o r s h a v e b e e n u s e d i n anal y s i s o f e r g o t a m i n e by h i g h p e r f o r m a n c e l i q u i d chromatography ( 1 9 , 1 0 3 ) . F o r d e t e r m i n a t i o n of ergotamine t a r t r a t e i n pharmaceutical dosage forms by q u a n t i t a t i v e t h i n l a y e r c h r o m a t o g r a phy, e l u t i o n f o l l o w e d by f l u o r i m e t r i c a n a l y s i s o f t h e e l u a t e h a s been u s e d (61). The f l u o r e s c e n c e i n t e n s i t y o f e r g o t a m i n i n e i s 2.5 f o l d g r e a t e r than t h a t of ergotamine ( 1 9 ) . F l u o r i m e t r y h a s been u s e d t o d e t e r m i n e t h e amount of n a t u r a l e r g o t a l k a l o i d s i n hydrogenated alkal o i d s e.g. dihydroergotamine ( 7 8 , 1 0 5 ) .

6.4

Non-Aqueous T i t r a t i o n

Ergotamine t a r t r a t e d i s s o l v e d e i t h e r i n a m i x t u r e of a c e t i c a n h y d r i d e and g l a c i a l a c e t i c a c i d (1) o r a m i x t u r e o f d i o x a n e a n d g l a c i a l a c e t i c a c i d ( 2 ) can be t i t r a t e d with perchlor i c a c i d i n g l a c i a l a c e t i c a c i d . The e n d p o i n t c a n b e o b s e r v e d p o t e n t i o m e t r i c a l l y o r by u s i n g

138

BO K R E I L G ~ R D

c r y s t a l v i o l e t as i n d i c a t o r .

Each m l o f 0 . 0 5

N p e r c h l o r i c a c i d i s e q u i v a l e n t t o 32.84 mg o f ergotamine t a r t r a t e . I s o l a t i o n o f t h e a l k a l o i d b a s e by c h l o r o form e x t r a c t i o n o f a n a l k a l i n e a q u e o u s s o l u t i o n and s u b s e q u e n t t i t r a t i o n w i t h p e r c h l o r i c a c i d o r p-sulphonic a c i d h a s been d e s c r i b e d (84). 6.5 6.5.1

Chromatography Paper chromatography

Several paper chromatographic systems f o r e r g o t a m i n e are summarized i n T a b l e 5 , and methods f o r v i s u a l i z i n g t h e s p o t s a r e o u t l i n e d O t h e r r e p o r t s o n PC of e r g o t a m i n e i n Table 6. a r e found i n r e f e r e n c e s ( 9 2 , 1 0 6 , 1 0 7 , 1 5 9 ) . Q u a n t i t a t i o n o f e r g o t a m i n e f o l l o w i n g pap e r chromatography i s d e s c r i b e d i n r e f e r e n c e s (47,108-110). 6.5.2

T h i n L a y e r Chromatography ( T L C )

A v a r i e t y o f TLC s y s t e m s h a v e b e e n dev e l o p e d f o r e r g o t a m i n e and most of t h e s e are summarized i n T a b l e 7 . Methods u s e d f o r d e t e c t i o n a n d i d e n t i f i c a t i o n of e r g o t a m i n e o n t h e p l a t e a r e summarized i n T a b l e 8. Q u a n t i t a t i o n o f e r g o t a m i n e f o l l o w i n g TLC i s done u s i n g e i t h e r e l u t i o n t e c h n i q u e ( 8 , 111-114,120) o r i n s i t u s c a n n i n g ( 6 2 , 1 1 5 - 1 1 9 ) . The most s u i t a b l e s o l v e n t t o u s e as e l u t i n g a g e n t i s a w a t e r - m e t h a n o l m i x t u r e t o which a n i n o r g a n i c o r o r g a n i c a c i d i s added ( 1 1 2 , 1 2 0 ) . Measurement o f t h e e l u a t e i s done u s i n g e i t h e r W - s p e c t r o p h o t o m e t r y o r c o l o r i m e t r y . The in s i t u measurement i s d o n e u s i n g U V - r e f l e c t a n c e (1161, fluorimetry (62,115,117) o r transmiss i o n of p l a t e s s p r a y e d w i t h t h e van Urk t y p e r e a g e n t ( 1 1 9 ) . TLC s y s t e m s h a v e a l s o b e e n mentioned i n r e f e r e n c e s ( 5 8 , 7 8 , 8 1 , 8 5 , l O l , l l l , 115,121-127)

.

6.5.3

Column Chromatography

I n t a c t e r g o t a m i n e and t h e most import a n t degradation product ergotaminine can be d e t e r m i n e d i n p h a r m a c e u t i c a l s by u s i n g t w o se-

Table 5 P a p e r Chromatography S y s t e m s f o r E r g o t a m i n e No. Paper -

Impregnation

S o lv en t system

Rf -

Appl i ca t i o n

Reference

1.

S c h l e i c h e r and S c h h l l no. 2043b

Ace t o n e - f o r m a m i de (6:4)

Benzene

0.17

S e p a r a t i o n of ergot alkaloids

12,15

2.

Whatman n o . 1

Dimethylpthalate

Formami d e - w a t e r ( 4 : 6 ) (pH=5.2 w i t h Formic a c i d )

0.37

S e p a r a t i o n of ergot alkaloids

16

3.

S c h l e i c h e r and S c h i i l l no. 2043b

C i t r i c acid-phosphate buffer (pH=5.6)

Benzene-ethanol ( 9 5 % )(9:l)

0.35

41

4.

S c h l e i c h e r and S c h i i l l no.2043b

Ethanol-formamide (1:l)

Benzene

0.05

54

5.

Whatman n o . 1

E t h a no 1- f o r m a m i de

Chloroform

0.86

70

2

W w

(1:l) 6.

S c h l e i c h e r and S c h i i l l no.2043bM

D i m et h y l pt h a l a t e

Formamide-O.066M Na2HP04 s o l u t i o n (4:6)

0.05

S e p a r a t i o n from d e g r a d a t i o n products

47

7.

S c h l e i c h e r and S c h G l l no. 2043bM

Dimethylpthala t e

Formamide-citrate b u f f e r (pH 4 . 4 ) (2:8)

0.38

S e p a r a t i o n from d e g r a d a t i o n products

47

Paper

Impregnation

S o lv en t system

Rf -

Application

8.

S c h l e i c h e r and S c h i i l l No.2043bM

D i m e t h y l p t h a l at e

Formami de0 . 1 N KOH ( 2 : 8 )

0.00

Separation from d e g r a d a tion products

47

9.

S c h l e i c h e r and S c h i i l l no.2043bM

Formamide- b e n z o i c acid (25:l)

Ether

0.24

Separation f r o m de g r a d a tion products

47

10. Whatnian no. 1

5% sodium d i h y d r o gen c i t r a t e

2.4 g o f c i t r i c a c i d i n water-butanol (65:435)

0.65

I d e n t if i c a t i o n

78

11.

Whatman n o . 1

None

Methylethylketoneacetone-formic acid w a t e r (40:2:1:6)

0.80

Identification

104

12.

Whatman no. 1

None

Methylethylketonediethylamine-water (921:2:77)

0.91

104

13.

Whatman no. 1

None

1 0 p a r t s of m e t h y l isobutylketone satur a t e d w i t h 1 p a r t of 4% formic a c i d

0.16

104

14.

Whatman n o . 1

None

10 p a r t s of c h l o r o form s a t u r a t e d w i t h a m i x t u r e of 1 p a r t of m e t h a n o l a n d 1 p a r t of 4% formic a c i d

0.47

104

No. -

4

Reference

P

0

ERGOTAMINE TARTRATE

Table 6 V i s u a l i z a t i o n o f Ergotamine o n P a p e r Chromatograms No. -

Treatment

Result

Reference

1.

Ultraviolet light ( A = 254 or 366 nm)

Blue

15,78,104

2.

p-dimethylaminobenzaldehyde r e a g e n t ( v a r i o u s modifications)

Blue-violet

16,54,78, 104

3.

mnO4 (1% aqueous s o l u t i o n )

78,104

4.

2,6-dibromoquinone-4-chloroimide (0.5% s o l u t i o n i n d i oxane-acetone ( 4 : l ) )

104

141

Table 7 T h i n Layer Chromatography Systems f o r Ergotamine Sorbent

Rf -

A p p l i c a t i o n and comment

1. Heptane-tetrahydrofurantoluene-chloroform (5:4:1:5)

S i l i c a g e l (Merck G ) 0.2% NaOH impregnated

0.04

Separation o f e r g o t alkaloids

128

2. Heptane-tetrahydrofurantoluene- ethy1 a c e t a t e (10:8:3:9)

S i l i c a g e l (Merck G ) , 0.2% NaOH impregnated

0.14

S e p a r a t i o n of e r g o t alkaloids

128

3. H e p t a n e - t e t r a h y d r o f u a n t o l u e n e (2:4:5)

S i l i c a g e l (Merck GI, 0.2% NaOH impregnated

0.05

Separation of ergot alkaloids

128

4. Heptane-tetrahydrofurantoluene (2:4:1)

S i l i c a g e l (Merck G ) 0.2% NaOH impregnated

0.08

Separation of e r g o t a 1ka l o i d s

128

5. Heptane-tetrahydrofurantoluene (1:4:1)

S i l i c a g e l (Merck G ) 0.2% NaOH impregnated

0.16

S e p a r a t i o n of e r g o t alkaloids

128

6. T e t r a h y d r o f u r a n - t o l u e n e (4:l)

S i l i c a g e l (Merck G ) 0.2% NaOH impregnated

0.27

S e p a r a t i o n of e r g o t alkaloids

128

7. Benzene-cyclohexaned i e t h y lamine ( 5 :2 :0.0 1 )

S i l i c a g e l (Merck G ) 0.2% NaOH i m p r e g n a t e d

0.05

S e p a r a t i o n of e r g o t alkaloids

128

S o l v e n t system

'p N

Reference

Sorbent

Rf

A p p l i c a t i o n and comment

S i l i c a g e l , formamide impregnated

0.17

S e p a r a t i o n of e r g o t a 1k a l o i d s

129

S i l i c a g e l , formamide imp r e g na t e d

0.29

S e p a r a t i o n of e r g o t alkaloids

129

Silica gel

0.39

S e p a r a t i o n from o t h e r l y s e r g i c a c i d type compounds

130

S i l i c a gel

0.25

S e p a r a t i o n from o t h e r l y s e r g i c a c i d type compounds

130

1 2 . Benzene-chloroform ethanol (2:4:1)

Silica gel G

0.62

Quantitative analysis

131

1 3 . Methanol-chloroform ( 2 : 8)

Silica gel G

0.65

I d e n t i f i c a t i o n of ergot alkaloids

132

1 4 . Diethylamine-chloroform (1:9)

Silica gel G

0.09

I d e n t i f i c a t i o n of ergot alkaloids

132

S o l v e n t system

8. Di-isopropylether-tetrahydrof uran-die thylamine (80: 20: 0 . 2 )

9. Dibuthylether-dichlorome t h a n e - d i e t h y l a m i n e (60:40:0.2) s a t u r a t e d w i t h f ormamide 1 0 . Chloroform-methanol

P

(9:l)

11. Chloroform-methanolc o n c e n t r a t e d ammonia

(18:l:O.Ol)

Reference

Sorbent

Rf

Application and comment

Reference

15. Methanol-chloroformconcentrated ammonia (20:80:0.2 )

Silica gel G

0.75

Identification of ergot alkaloids

132

16. Chloroform-ethanol (96:4)

Aluminiumoxide G

0.58

Identification of ergot alkaloids

132

17. Ethylacetate-N,N-dimethyl formamide-ethanol (13:1.9:0.1)

Silica gel G

0.31

Quantitative'analysis

114

18. Benzene-N ,N-dimethylformamide (13:2 )

Silica gel G

0.31

Quantitative analysis

114

19. Chloroform-diethylether-

Aluminium oxide G

0.01 Quantitative analysis

114

20. Benzene-n-propanol-NH (1 M) 3 (100:10:2)

Silica gel

0.29

Quantitative analysis

158

21. Chloroform-ethanol-acetone (6:4:4)

Silica gel G

0.51

Quantitative analysis

120

22. ChLoroform-ethanol (9:1)

Silica gel G

0.27

Quantitative analysis

120

23. Dichloromethane-methanol (92.7:7.3)

(Merck) 0.56 Silica g e l GF 254 0.1 N Na CO

Usefulness of azeotropic mixtures in TLC

133

Solvent system

P

water (7:1:2)

2

irnpregnated

3

S o l v e n t system 24. Chloroform-ethanol

(92: 8)

Sorbent

Rf

A p p l i c a t i o n and comment

S i l i c a g e l GF254(Merck) 0.1 N Na2C03

0.44

Usefulness of azeotropic m i x t u r e s i n TLC

133

0.16

U s e f u l n e s s of a z e o t r o p i c m i x t u r e s i n TLC

133

0.31

Usefulness of azeotropic m i x t u r e s i n TLC

133

Reference

impregnated 25. Chloroform-2-butanon (17:83)

S i l i c a g e l GF254(Merck) 0 . 1 N Na2COj impregnated

26. Acetone-cyclohexane (67.5:32.5)

S i l i c a g e l GF254(Merck)

0.1 N N a 2 C 0 3 impregnated

d

u1 P

27. Chloroform-ethanol (95%) (9: 1)

Silica gel G

0.29

S e p a r a t i o n f r o m degradat i o n products

61

28. Chloroform-ethanol (95%) (9:l)

S i l i c a g e l G , 1% KOH impregnated

0.16

S e p a r a t i o n from degradat i o n products

134

29. Benzene-chloroforme t h a n o l ( 2 : 4: 1)

Silica gel G

0.43

T e s t i n g of p u r i t y o f ergot alkaloids

135

3 0. Hep t a n e - c a r b o n t e trachloride-pyridine (1:3: 2 )

Silica g e l G

0.16

T e s t i n g of p u r i t y o f ergot alkaloids

135

31. Chlorofonn-acetoned i e t h y l a m i n e (5:4:1)

Silica gel

0.24

Quantitative analysis

163

S o l v e n t system 32. Chlorof om-me t h a n o l ( 9 : 1) 33. Chloroform-methanol

(7:l)

34. Chloroform-ethanol ( 9 5 % ) (9: 1)

Sorbent

Rf -

A p p l i c a t i o n and comment

Reference

Silica gel

0.50

Quantitative analysis

136,137

S i l i c a g e l GF254

0.52

Quantitative analysis

8

0.36

Quantitative analysis

8

S i l i c a g e l GF254

35. Chloroform-methanol

(17: 3)

Silica gel G

0.64

Quantitative analysis

112

36. Chloroform-methanol

( 4 : 1)

Silica gel G

0.75

Quantitative analysis

112

Silica gel

0.22

S e p a r a t i o n from o t h e r e r g o t a l k a l o i d type compounds

138

S i l i c a gel

0.58

S e p a r a t i o n from o t h e r e r g o t a l k a l o i d type compounds

138

S i l i c a g e l F254 (Merck , precoated)

0.32

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

S i l i c a g e l F254 (Merck, precoated)

0.39

I d e n t i f i c a t i o n , separ a t i o n from L S D

139

S i l i c a g e l F254(Merck, p r e c o at e d )

0.63

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

(1:9)

37. Morpholine-toluene

38. Chloroform-methanol

(9: 1)

39. Acetone

40. Acetone-chloroform

41. Acetone-methanol

(4:l) ( 4 : 1)

S o l v e n t system 42. C h l o r o f o r m

43. C h l o r o f o r m - a c e t o n e

(6:l)

So rbe n t

Rf

A p p l i c a t i o n a n d comment

S i l i c a g e l F254 (Merck, precoated)

0.00

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

S i l i c a g e l F254 (Merck, p r e c o ated)

0.02

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

Reference

44. Chloroform-methanol

(4: 1)

S i l i c a g e l F254(Merck, precoated)

0.62

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

45. Chloroform-me t h a n o l

( 9 :1)

S i l i c a g e l F254(Merck, precoated)

0.35

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

S i l i c a g e l F?,, (Merck, precoated)

0.69

I d e n t i f i c a t i o n , separ a t i o n f r o m LSD

78,139

S i l i c a g e l F254 (Merck, precoated)

0.73

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

48. Chloroform-cyclohexaneisopropylamine (5:5:1)

Silica gel F (Merck, 254 precoa ted)

0.11

I d e n t i f i c a t i o n , separ a t i o n f r o m LSD

139

49. Chloroform-cyclohexaned i e t h y l a m i n e ( 5 : 5: 1)

S i l i c a g e l F254 (Merck, pr e coa t e d )

0.02

I d e n t i f i c a t i o n , separ a t i o n f r o m LSD

139

50. l , l , l - t r i c h l o r e t h a n e methanol ( 9 : l )

S i l i c a g e l F254 (Merck , precoa t e d )

0.20

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

-2-

47. M e t h a n o l - a c e t a t e (pH 4.5) ( 9 : l )

buffer

Sorbent

Solvent system

Aluminium oxide F

51. Acetone

(Merck ,

A

254 precoated)

Rf

A p p l i c a t i o n and comment

0.48

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

Refereme

52. 1 , 1 , l - t r i c h l o r o e t h a n e methanol (9: 1)

Aluminium oxide F

0.52

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

53. l , l , l - t r i c h l o r o e t h a n e methanol (96:4)

A l u m i n i u m oxide F254

0.20,

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

54. 1,1,l - t r i c h l o r o e t h a n e methanol (98:2)

A l u m i n i u m oxide F254

0.04

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

55. l,l,l-trichloroethananolmethanol (99: 1)

Aluminium oxide F

0.00

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

56. n-Butanol-citric water

C e l l u l o s e (Merck) sprayed with 5% sodium dihydrogen c i t r a t e and dried

0.77

I d e n t i f i c a t i o n , separ a t i o n from LSD

139

Silica gel G

0.65

Identification

140

P

254 (Merck, p r e c o a t e d )

(Merck, p r e c o a t e d )

(Merck , p r e c o a t e d )

m

acid-

(870 n1:b.B g:130 ml) 57. Benzene-acetone-diethylether-ammonium hydroxide ( 2 5 % ) (4:6:1:0.3)

254 (Merck, p r e c o a t e d )

Sorbent

Rf

A p p l i c a t i o n a n d comment

58. Benzene-chloroform ( 4 : 5 ) s a t u r a t e d w i t h formamide a n d mixed w i t h 10% methanol

Silica gel G

0.41

Identification

140

59. Benzene-n-heptanec h l o r o f o m - d i e t h y 1m i n e (40:20:30:10)

Silica gel G

0.05

Identification

140

Aluminium o x i d e

0.82

Identification

141

6 1. D i i sopropy 1- e t h e r -t e trahydrofuran-toluene-die t h y l a m i n e (70: 1 5 :15: 0 . 1 )

S i l i c a g e l , formamide impregnated

0.11

S e p a r a t i o n of e r g o t alkaloids

142

62. Dioxane-cyclohexaned i e t h y l a m i n e (10: 20 :0 . 5 )

Polyamide

0.05

S e p a r a t i o n of a l k a loids

143

63. Chloroform-cyclohexanediethylamine (10:20:0.5)

Po 1yamide

0.15

S e p a r a t i o n of a l k a loids

143

64. 2-Butanon-cyclohexanediethylamine (20:30:0.5)

Polyamide

0.11

S e p a r a t i o n of a l k a loids

143

6 5. E t h a n o l - c h l o r o forma c e t i c a c i d (20:200:0.5)

Polyam ide

0.97

S e p a r a t i o n of a l k a loids

143

Solvent system

60. C h l o r o f o r m - e t h a n o l

d

I P D

(9:l)

-

Reference

Sorbent

Rf -

66. Water-ethanol-pyridine (10:0.5:0.3)

Polyamide

0.10 Separation of alkaloids

143

6 7 . Cyclohexane-ethylacetate

Polyamide

0.01

Separation of alkaloids

143

68. Water-ethanol-dimethylm i n e (88:12:0.1)

Polyamide

0.03

Separation of alkaloids

143

69. Chloroform-ethanol (10:1)

Silica gel

0.51

Quantitative analysis

113

70. Chloroform-methanol ( 9 : l )

Silica gel G, 0.1 N NaOH impregnated

0.51

Identification

144

71. Benzene-heptane-chloroform (6:5:3) followed by benzene-heptane (6:5)

Cellulose, formamide impregnated

0.06

Identification

145

Solvent system

Application and comment

Reference

n-propanol-dimethylamine ( 30: 2.5: 0.9: 0.1)

A

m 0

ERGOTAMINE TARTRATE

151

Table 8 V i s u a l i z a t i o n o f Ergotamine on Thin Layer P l a t e s No. -

Treatment

Result

Reference

1. p-dimethylaminobenzaldehyde Reagent ( v a r i o u s modifications)

B h e - v i o l et

58,78,113, 120,132, 1 3 5 ,1 37 ,1 3 8 139 ,1 4 4

2. U l t r a v i o l e t l i g h t ( A = 254 o r 366 nm)

Blue

8,78,120 124,128, 132,138, 139

3. I n s i t u scanning

62, 115, 116 ,117 , 119

4. I o d i n e v a p o r

143

5. Dragendorff Reagent

58

,

6. Ammoniated copper sulphate

Violet-brown s p o t s on l i g h t g r e e n background

58,140,141

7. Xanthydrol-hydrogen peroxide

Blue-violet

140

8. Ninhydrin-cadmium acetate

Red-violet s p o t s on p i n k background

140

9 . Iodo p l a t i n a t e Reagent

Grayish v i o l e t

58,143,146

10. Potassium permanganate 11. F e r r i c c h l o r i d e glyoxylic acid

78 Blue

124

152

BO KREILGKRD

p a r a t e C e l i t e 545 columns ( 1 4 7 , 1 4 8 ) . The f i r s t column i s i m p r e g n a t e d w i t h sodium b i c a r b o n a t e a n d t h e a l k o l o i d bases a r e e l u t e d w i t h c h l o r o f o r m . The s e c o n d column i s i m p r e g n a t e d w i t h a 20% c i t r i c a c i d s o l u t i o n and ergotaminine i s e l u t e d with a p o r t i o n of chloroform. E r g o t a m i n e i s e x t r a c t e d w i t h c h l o r o f o r m from t h e e x t r u d e d s u p p o r t which i s s u s p e n d e d i n a n aqueous b i c a r b o n a t e s o l u t i o n . The a l k o l o i d content i n each f r a c t i o n i s determined using T h i s method h a s b e e n t h e van U r k r e a c t i o n . a d a p t e d by t h e USP X I X i n t h e a s s a y o f e r g o t a mine t a r t r a t e i n j e c t i o n (1) a n d i n a n a l y s i s o f t a b l e t s containing ergotamine t a r t r a t e , tropane a l k a l o i d s and p h e n o b a r b i t a l ( 6 4 ) . The met h o d , however, d o e s n o t t a k e t h e p r e s e n c e o f o t h e r d e g r a d a t i o n p r o d u c t s , s u c h as a c i - d e r i v a t i v e s , l y s e r g i c and i s o l y s e r g i c a c i d a i d e i n t o account. E r g o t a m i n e and e r g o t a m i n i n e i n d r u g s h a v e b e e n d e t e r m i n e d u s i n g C e l i t e 545 i m p r e g n a t e d w i t h formamide a s t h e s t a t i o n a r y p h a s e a n d benz e n e - p e t r o l e u m e t h e r ( 9 : 1) a s t h e m o b i l e p h a s e (149). Ergotamine can be q u a n t i t a t i v e l y s e p a r a t e d from o t h e r e r g o t a l k a l o i d s u s i n g a n aluminium o x i d e column and m e t h y l e n e c h l o r i d e add i n g i n c r e a s i n g amounts o f m e t h a n o l a s e l u t i n g s o l v e n t ( 1 2 , 1 5 1 . C a r l e s s ( 1 5 0 ) u s e d columns of c e l l u l o s e i m p r e g n a t e d w i t h a pH 3 . 0 McIlvane c i t r a t e - p h o s p h a t e b u f f e r and e t h e r , adding 0 . 1 % p y r i d i n e as m o b i l e p h a s e f o r s e p a r a t i o n of e r g o t a l k a l o i d s . Only a b o u t 8 0 % e r g o t a m i n e i s recovered (150)

.

6.5.4

High P r e s s u r e L i q u i d Chromatography (HPLC 1

W i t h i n t h e l a s t few y e a r s h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h i c methods f o r t h e a n a l y s i s o f e r g o t a m i n e h a v e b e e n d e v e l o p e d . Ads o r p t i o n c h r o m a t o g r a p h y i s c a r r i e d o u t on s i l i ca g e l w i t h s e v e r a l d i f f e r e n t o r g a n i c s o l v e n t s a s m o b i l e p h a s e s ( 1 9 , 1 0 3 , 1 5 3 ) . A reverse phase Bondapak p h e n y l / C o r a s i l o r pBondapak c 1 8 column w i t h a c e t o n i t r i l e - a q u e o u s ammonium carb o n a t e b u f f e r as mobile p h a s e , p e r m i t s s e p a r a t i o n of e r g o t a m i n e from i t s d e g r a d a t i o n products (154,155). Increased s e n s i t i v i t y i n t h e

ERGOTAMINE TARTRATE

153

a n a l y s i s of e r g o t a m i n e c o u l d b e a c h i e v e d by u s i n g p i c r i c a c i d as c o u n t e r i o n i n f o r m i n g a n i o n - p a i r which o n t h e s i l i c a g e l column i s d i s t r i b u t e d between a s t a t i o n a r y aqueous phase and a m o b i l e o r g a n i c p h a s e ( 1 5 2 ) . An i n c r e a s e d s e n s i t i v i t y r e l a t i v e t o common f l u o r i m e t r i c d e t e c t o r s ( 1 9 , 1 5 1 ) c o u l d b e a c h i e v e d by u s i n g a h i g h - p r e s s u r e Xenon a r c lamp w i t h a n i n t e g r a l c o l l i m a t i n g mirror a s e x c i t a t i o n s o u r c e ( 1 0 3 ) . A l s o U V - d e t e c t o r s a t 254 nm h a v e been u s e d (153-155) t o d e t e c t e r g o t a m i n e . R e c e n t l y Bethke e t a1 ( 1 5 6 ) d e s c r i b e d a r e v e r se p h a s e HPLC method w i t h s o l v e n t g r a d i e n t which i n l e s s t h a n 20 m i n u t e s e n a b l e d them t o d e t e r m i n e t h e c o n t e n t of e r g o t a m i n e a s w e l l a s a l l e p i m e r i z a t i o n and h y d r o l y s i s p r o d u c t s i n pharmaceutical preparations. 7.

D e t e r m i n a t i o n i n B i o l o g i c a l Systems

E r g o t a m i n e h a v e b e e n a n a l y z e d i n plasma by Kopet & D i l l e (69) used t h e van Urk reaction t o determin e e r g o t a m i n e i n b l o o d and t i s s u e s , w h i l e a n a l y s i s of e r g o t a m i n i n e i n plasma i s done u s i n g HPLC equipped w i t h a f l u o r e s c e n c e d e t e c t o r ( 1 0 3 ) . B i o a v a i l a b i l i t y s t u d i e s on e r g o t a m i n e t a r t r a t e h a v e b e e n done m o n i t o r i n g plasma and u r i n a r y r a d i o a c t i v i t y a f t e r i n g e s t i o n of 3 H - l a b e l l e d e r g o t a m i n e tartrate (32). TLC f o l l o w e d by i n s i t u f l u o r i m e t r y ( 1 1 5 ) .

8.

Determination i n Pharmaceutical Preparations

The f o l l o w i n g methods h a v e b e e n a p p l i e d t o a n a l y s i s of e r g o t a m i n e t a r t r a t e i n p h a r m a c e u t i cals: References Colorimetry

81,91,92,99,157

Fluorimetry

17

P a p e r chromatography

47

Column chromatography

64,148,149

T h i n l a y e r chromatography

8,6l,62,113,115,117,158

High p r e s s u r e l i q u i d chromatography

152,156

154

BO

KREILGWRD

Re ferences

1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

The United States Pharmacopoeia XIX, Mack Printing Co., Easton, Pa., 1975, pp.173-175. Pharmacopea Nordica 1963, Editio Danica, Nyt Nordisk Forlaq, A.Busck, Copenhagen 1963. European Pharmacopoeia Volume 111 , Maisonneuve S.A. , France, 1975, pp. 17-19. C.C.Cromp and F.G.Turney, J.Forensic Sci. 12, 538 (1967). A.Hofmann, Die Mutterkornalkaloide, F.Enke V e e lag, Stuttgart (1964). R.G.Mrtek, H.L.Crespi, G.Norman, M.I.Blake and J.J.Katz, Phytochemistry 7, 1535 (1968). N.J.Bach, H.E.Boaz, E.C.KErnfeld, C.-J.Chang, H.G.Floss, E.W.Hagaman and E.Wenkert, J.Org. Chem. - 39, 1272 (1974). B.Kreilg5rd and J.Kisbye, Arch.Pharm.Chemi Sci. Ed. 2, 1 (1974). J.Bayer, Magy.Kem.Foly 63, 197 (1957). Z.Gawrych and I.Wilczynza, Acta Pol.Pharm. 22, 1 (1965). 38, A.Stoll. and W-Schlientz, Helv.Chim.Acta 585 (1955). A.L.Kapoor, H.Schumacher and J.BCchi, Pharm. Acta Helv. 32, 411 (1957). A.Bowd, J.B=udson and J.H.Turnbul1, J.Chem. Soc.Perkin 11, 1312 (1973). A.Stol1 and A.Hofmann, Helv.Chim.Acta 26, 2070 (1943). 101, W.Soffe1 and H.Rochelmeyer, Pharm.Ztg. 1059 (1956). A.Stoil and A.Riiegger, Helv.Chim.Acta 37, 1725 (1954). W.D.Hooper, J.M.Sutherland, M.J.Eadie and J.H.Tyrer, Anal.Chim.Acta 69, 11 (1974). A.C.Metha and R.A.Chalmers,Chem.Anal.(Warsaw) 17, 565 (1972). R.A.Heacock, K.R.Langille, J.D.MacNei1 and R.W.Frei, J.Chromatogr. 77,425 (1973). M.Barber, J.A.Weisbach, B.Douglas and G.O.Dudek, Chem.Ind. (London) 1072 (1965). D.Voigt, S.Johne and D.GrGger, Pharmazie 2 , 697 (1974). J.Vokoun and Z.Rehacek, Coll.Czech.Chem.Com. 40, 1731 (1975). J.Vokoun, P.Sajd1 and Z.Rehacek, 2bl.Bakt.Abt. I1 129, 499 (1974).

ERGOTAMINE TARTRATE

24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.

155

T.Inoue, Y.Nakahara and T.Niwaguchi, Chem. Pharm.Bul1. 20, 409 (1972). L.C .CraigI Proc .Nat.Acad.Sci.U. S. 61, 152 (1968). G.H.Svoboda and G.S.Shahovskoy, J.Amer.Pharm. Ass.Sci.Ed. 42, 729 (1953). S.Smith and G.M.Timmis, J.Chem.Soc. 1440 (1936). A.E.Beesley and G.E.Foster, Analyst 70,374 (1945). A. Stoil I Helv.Chim.Acta 28, 1283 (1945). A.Stol1 I Schweiz.Apoth.Ztg. 60, 341 (1922). J. Sage1 I Pharm.Weekb1. 107,119 (1972). R. Schmidt and A.Fanchamp, Europ.J.Clin.Pharma-col. - 7, 213 (1974). M .A.Zoslio, H.V.Mauldinq and J.J.Windheuser, 58, 222 (1969). J.Pharm.Sci. Merck Index, 8th Ed., Merck & Co.Inc.Rahway, New Jersey, 1968. C.Lorincz, Herba Hung. 5, 211 (1966). H.Hellberg, Farm.Revy 50, 17 (1951). B.Berde, A.Cerletti, H2.Dengler and M.A.Zoglio, Third Migraine Symposium 24.-25.April (1969). Ed. by A.L.Cochrane. Heinemann, London 1970. M.Beran and M.Sermonsky, Cesk.Farm. 11, 440 (1962). H.V.Maulding and M.A.Zoglio, J.Pharm.Sci. 2, 700 (1970). Swiss Patent No. 79879 (1918). F.Gstirner and H.O.MGller, Arch.Pharm. 589 (1955). V.Mascov, E.Nichiforescu, L.Rosca, C.Rizescu and I .Veiea, Farmacia (Bucharest)-21, 557 (1973). A.Hofmann, A.J.Frey and H.Ott, Experientia 17, 206 (1961). E .C .Kornfeld I E J .Fornefeld , G B. Kline I M.J.Mann, R.G.Jones and R.B.Woodward, J.Amer. 76, 5256 (1954). Chem.Soc. A.Hofmann, H .Ott, R.Griot, P.A .Stadler and A.J.Frey, Helv.Chim.Acta 46, 2306 (1963). A.Stol1, A.Hofmann and F.Troxler, Helv-Chim. Acta -32, 506 (1949). W.Schlientz, R.Brcnner, A.Hofmann, B.Berde and E.StGrmer, Pharm.Acta Helv. 36, 472 (1961). B.Kreilg5rd and J.Kisbye, Arch.Pharm.Chemi S c i Ed. - 2, 38 (1974). H.Ott, A.Hofmann and A.J.Frey, J.Amer.Chem.Soc. 88, 1251 (1966).

z,

.

.

156

50.

51.

52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80.

BO KREILGKRD

W.A.Jacobs and L.C.Craig, J.Biol.Chem. 104, 547 (1934). 106, W.A.Jacobs and L.C.Craig, J.Biol.Chem. 393 (1934). S.Smith and G.M.Timmis, J.Chem.Soc. 763 (1932). S.Smith and G.M.Timmis, J.Chem.Soc. 1543(1932). J.Kolsek, Mikrochim.Acta 1377 (1956). H.Hellberg, Acta Chem.Scand. 11, 219 (1957). F.Troxler and A.Hofmann, HelvThim.Acta 42, 793 (1959). R.Adamski, J.Lutomski, A.Socha and H.Speichert, Farm.Po1. 24, 43 (1968). K.C.Guven G d T.Guneri, Eczacilik Bul. 13, 57 (1971). M. Sprung , Pharmazie 16 , 515 (1961) J.Trzebinski and T.WGcko, Acta Pol.Pharm. 24, 579 (1967). 40, 25 M.Sahli and M.Oesch, Pharm.Acta Helv. (1965). 106, 865 (1971). J.M.G.J.Frijns, Pharm.Weekb1. British Pharmacopoeia, The Pharmaceutical Press, London 1973. I.Juhl, Arch.Pharm.Chemi 2,667 (1966). A.E.H.A. El-Shamy, F.M. El-Anwar and A.A.Kassem, J.Drug Res. 2, 159 (1973). L.S.Goodman and A.Gilman, The Pharmacological Basis of Therapeutics, Fourth Ed. The MacMillan Compagny, London 1971 , p. 902. E.Rothlin, Bull.Schweiz Akad.Med.Wiss. 2, 249 (1946/47). E.Rothlin, Helv.Chim.Acta 29 , 1290 (1946) J.C.Kopet and J.M.Dille, J%er.Pharm.Ass. 31, 109 (1942). K.Macek, M.Semonsky, S.Vanecek, V.Zikan and A.Cerny, Pharrnazie-2, 752 (1954). -narm.Weekb1. 66, 473 (1929) H.W. van Urk, Pharm.Weekb1. 286, 509719531. M.P6hm, =h.Pharm. 286 , 50971953) of E.G.C.Clarke, Isolation and Identification, The Pharmaceutical Press. Press , London 1969. Druss , -Drugs D.L.Andersen, J.Chromatogr. 41, 491 (1969) G.Dusinsky and L.Faith, ith, Pharzzie - -22, 475 (1967). J.Bayer, Acta Pharm.Hung. 28, 35 (1958). A.Harmsma, Pharm.Weekb1. 65, 1121 (1928). E.G.C.Clarke, J.Forensic Zi.Soc. 2, 46 (1967). F.Adamanis, E.Pawelczvk and Z.Plotkowiakowa, 513 (1961). Farm.Po1. N.L.Allport and T.T.Cocking, Quart.J.Pharm. 5, 341 (1932). Pharmacol. -

.

.

.

&,

EflGOTAMlNE TARTRATE

157

E.Ermer, Pharm.Ztg. 120, 149 (1975). G.E.Foster, J.Pharm.Pharmaco1. 7, 1 (1955). W.N.French, J.Pharm.Sci. 54, 1726 (1965). 1.Gyenes and J.Bayer, Pharmazie 16, 211 (1961). 85. P.Horak, Cesk.Farm. 17, 37 (1968). 86. Y.Kazutaka. T.KawataS. T.Tabata, S.Fukushima 73, 268 and M.Ito,.J.Pharm.Soc: (Japan) (1953). 87. V.Pedersen, Arch.Pharm.Chemi 62, 675 (1955). 88. F.Schlemmer, P.H.A.Wirth and TPeters, Arch. Pharm. 274, 16 (1936). 89. M.I.Smith, Pub.Health Rep. 45, 1466 (1930). 90. J.W.Strong and F.A.Maurina, J.Amer.Pharm. Ass.Sci.Ed. 42, 414 (1953). 91. F.D.Snel1 anTC.T.Snel1, Colorimetric Methods of Analysis. Including Photometric Methods. Volume IV AA, Van Nostrand Reinhold Comp. 1970. 92. R.Voigt and F.Weiss, Pharmazie 13, 319 (1958). 93. R.Voigt, Mikrochlm.Acta, 619 (1959). 94. F.Wokes and H.Crocker, Quart.J.Pharm.Pharmacol. - 4 , 420 (1931). 95. L.E.Michelon and W.J.Kelleher, Lloydia 26, 192 (1963). 96. E.Schulek and G.Vastagh, Dan.Tidsskr.Farm. 13, 101 (1939). 37, 6 7 97. L.Vida and G.Vastagh, Acta Pharm.Hung. (1967). 98. E.Graf and E.Neuhoff, Arzneimittel-Forsch. 4, 397 (1954). 1 0 6 , 515 99. H.J. van der Pol, Pharm.Weekb1. (1971). 100. H-Wachsmuth and L. van Koeckhoven, J.Pharm. Belg. 2 , 378 (1963). 101. E.M.Karacsony and B.Szarvady, Planta Medica 11, 169 (1963). 102. 1.Gyenes and K.Szasz, Magy.Kem.Foli. 61, 393 (1955). 103. R.J.Perchalski, J.D.Winefordne !r and B.J.Wi 1der, Anal.Chem. 47, 1993 (19751 . 104. L.Reio, J.ChromaGgr. 68, 183 (1972). 105. L.Wichlinski and J.Trzebinski, Acta Pol. Pharm...-20, 32 (1963). 106. P.Heinanen, L.Tuderman and E.N 'issilz, Suom Apt.Lethi 46, 133 (1957). 107. K.Macek, A.Cerny and M.Semonsk y , Pharmazie 388 (1954). 108. J.Kolsek, Mikrochim.Acta, 1500 (1956).

81. 82. 83. 84.

158

109. 110.

111. 112. 113.

114. 115. 116. 117.

118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135.

BO KREILGARD

K.Macek and S.Vanecek, Pharmazie 10, 422 (1955). P.Heinanen, M.Jarvi and M.LahteenmZki, Farm. Notisbl. 68, 155 (1959). R.Adamski, J.Lutomski, A.Socha and H.Speichert, Farm.Po1. 23, 571 (1967). S.Keipert and R.V=gt, J.Chromatogr. 64, 327 (1972). M.Klavehn, H.Rochelmeyer and J.Seyfried, Deut.Apoth.Ztg. 101, 7 5 (1961). J.L.McLaughlin, J.E.Goyan and A.G.Pau1 i J.Pharm.Sci. 53, 306 (1964). M.Amin and W.Spp, J.Chromatogr. 118 , 225 (1976). H.Bethke and R.W.Frei, J.Chromatogr. 91, 433 (1974). E.Eich and W.Schunack, Planta Med. 2 , 58 (1975). K.Genest, J.Chromatogr. 19, 531 (1965). M.Vanhaelen and R.Vanhaelen-Fastre I J-Chromatog:. 72, 139 (1972). K.Roder, E.Mutschler and H.Rochelmeyer, Pharm. Acta Helv. 42, 407 (1967). P.Horbk and S.Kudrndc, Cesk.Farm. 15, 483 (1966). ;.A. Dal Cortivo, S.R.Broich, A.Dihrberg and B.Newman, Anal.Chem. 38, 1959 (1966). 1.Zarebska and A.Ozarowski, Farm.Po1. 22, 518 (1966). 29, A.Peuch, C.Duru and M.Jaaob, J.Pharm.Belg. 126 (1974). M.PEhm, Arch.Pharm. 289, 324 (1956). D.GrEger and D.Erge, Pharmazie 18, 346 (1963). L.Wichlinski, Acta Pol.Pharm. 26, 617 (1969). V.Mascov, L.Rosca and E.NichifGescu, Farmacia (Bucharest) 21, 499 (1973). J.Reichelt and S.Kudrnac, Cesk.Farm. 23, 13 (1974). A.R.Sperling, J.Chromatogr.Sci. 12, 265 (1974). L . Wichlinski and Z .Skibinski I Farm.Pol. 22, 194 (1966). S.Agurel1, Acta Pharm.Suecica 2, 357 (1965). E.R6der, E.Mutschler and H.Rochelmeyer, Z.Anal.Chem. 244, 46 (1969). J.Tyfczyfiska, Diss.Pharm.Pharmaco1. 18,491 (19661 M.Zinser and C.Baumggrte1, Arch.Pharm. 297, 158 (1964).

.

ERGOTAMINE TARTRATE

159

Y.Petrova. T.Tomova and L.Fili~ova. Farmatsiya (Sofia) 22, 9 (1972). 137. E.Stah1, D i i n n z h i c h t - C h r o m a t o g r a p h i e , Ein Laboratoriumhandbuch, 2.Ed. Springer, Berlin 1967. 138. G.V.Alliston and M. J. de Faubert Maunder, J.Pharm.Pharmaco1. 23, 555 (1971). 139. R.Fowler, P.J.Gomm and D.A.Patterson, J.Chromatogr. 72, 351 (1972). - -17, 46 140. K.C.Guven and TGuneri, Eczacilik Bul. (1975). 141. K.C.Guven and L.Eroglu, Eczacilik Bul. -10, 53 (1968). 142. J.Reichelt and S.Kudrnac, J.Chromatogr. 87, 433 (1973). 143. H.-C.Hsiu, J.-T.Huang, T.-B.Shih, K.-L.Yang, 14, K.T.Wang and A.L.Lin, J.Chin.Chem.Soc. 161 (1967). 144. W.N.French and A.Wehrli, J.Pharm.Sci. 54, 1515 (1965). 145. K.Teichert, E-Mutschler and H.Rochelmeyer, Deut.Apoth.Ztg. 100, 283 (1960). 146. F.Sita, V.Chmelova and K.Chme1, Cesk.Farm. 22, 234 (1973). 43, 224 147. T.G.Alexander, J.Ass.Offic.Agr.Chem. (1960). 148. T.G.Alexander, J.Pharm.Sci. 52, 910 (1963). 95, 149. J.J.A.M. van de Langerijt, Pharm.Weekb1. 133 (1960). 150. J.E.Carless, J.Pharm.Pharmaco1. 5, 883 (1953). 84, 181 151. I.Jane and B.B.Wheals, J.ChromatEgr. (1973). J.Chromatogr. 152. W.Santi, J.M.Huen and R.W.Frei, 115, 423 (1975). 153. J.D.Witter,Jr. and J.H.Kluckhoh .n, J .Chromatogr.Sci. 11, 1 (1973). 154. ApplicationSheet No DS 042, Wa ters Associates,Mass., U.S.A. 155. Application Sheet No.AN 118, Waters Associates,Mass., U.S.A. 156. H-Bethke, B.Delz and K.Stich, J.Chromatogr. 123. 193 (1976). 157. S.Czyszewska, F.Kaczmarek, L.Lutomski and H.Speichert, Herba Pol. 12, 87 (1966). 158. V.Prochazka, F.Kavda, M.Eucha and J.Pitra, Cesk.Farm. 2 , 493 (1964). 159. M.P&m and L.Fuchs, Naturwissenschaften 41, 63 (1954). This profile attempts to cover the literature on ergotamine tartrate published up to June 1975. 136.

FENOPROFEN CALCIUM

Christine K , Ward and Roger E. Schimer

162

CHRISTINE K. WARD AND ROGER E. SCHIRMER

CONTENTS

1. 2.

3. 4. 5. 6.

7.

8.

9. 10.

Description Phys ica 1 P r o p e r t ies 2.1 C r y s t a l C h a r a c t e r i s t i c s 2.1.1 C r y s t a l . Forms a n d H y d r a t e s 2.1.2 M e l t i n g Range a n d D i f f e r e n t i a l Therma 1 Ana l y s is 2.2 S o l u b i l i t y 2.3 pKa 2.4 E l e c t r o n i c S p e c t r a 2.4.1 U l t r a v i o l e t A b s o r p t i o n S p e c t r u m 2.4.2 O p t i c a l R o t a t i o n 2 . 5 I n f r a r e d Spectrum 2.6 N u c l e a r M a g n e t i c Resonance S p e c t r u m 2.7 Mass S p e c t r u m Synthesis D e g r a d a t i o n of F e n o p r o f e n C a l c ium Me t a bol i s m of F e n o p r of e n 5 . 1 M e t a b o l i t e s of F e n o p r o f e n 5.2 P h a r m a c o k i n e t i c s E lementa 1 A n a l y s i s C h r o m a t o g r a p h i c Methods of A n a l y s i s 7 . 1 T h i n Layer Chromatography 7.2 Gas Chromatography 7.3 High P r e s s u r e L i q u i d Chromatography T i t r i m e t r i c D e t e r m i n a t i o n s ( f e n o p r o f e n and ca l c ium) S p e c t r o p h o t o m e t r ic A n a l y s i s A n a l y s i s of F e n o p r o f e n i n B i o l o g i c a l S a m p l e s

FENOPROFEN CALCIUM

1.

163

Descr i p t i o n -

F e n o p r o f e n C a l c i u m is c a l c i u m 2 - (3-phenoxyphenyllpropionate dihydrate (I).

ii

[

‘I

coo- 1

2

I.

-2H$

2

E m p i r i c a l Formula ( C 1 5 H 1 3 0 3 ),Ca*2H20 558.60 M o l e c u l a r Weight I t is a n odorless, w h i t e , c r y s t a l l i n e powder. 2.

C r y st a l Forms a n d H y d r a t e s F e n o p r o f e n c i u m occurs a s

Physical Properties 2.1.1

a

c r y s t a l l i n e d i h y d r a t e w h i c h is s t a b l e from 94% t o less t h a n 1%r e l a t i v e h u m i d i t y a t room t e m p e r a t u r e . Only o n e c r y s t a l f o r m has b e e n o b s e r v e d f o r t h e d ih y d r a t e

.

2.1.2

M e l t i n g Range a n d D i f f e r e n t i a l Therma 1 Ana l y s is When r u n i n a n o p e n pan the thermcgram of F e n o p r o f e n C a l c i u m (!ee f i g u r e 1) e x h i b i t s a large e n d o t h e r m n e a r 94 C c o r r e s p o n d i n g t o a l o s s of water accompanied by c o l l a p s e o f t h e c r y s t a l s t r u c t u r e t o a g l a s s . When t h e loss of v o l a t i l e s is r e s t r i c t e d , a s i n a m e l t i n g p o i n t t u b e , t h $ e n d o t h e r m a p p e a r s a t h i g h e r temperature (118-123 C ) a n d is accompanied by p a r t i a l l i q u i f i c a t i o n of t h e s a m p l e . T h i s does n o t a p p e a r t o be a t r u e m e l t .

THERMAL ANALYSIS OF FENOPROFEN CALCIUM

r

loo -90

TGA

-80 -70 -60 DTA

-50 -40 94

-30 Figure 1

FENOPROFEN CALCIUM

2.1.3

X-ray Powder P a t t e r n of Fenoprofen f!a lc i u m

d -

1/11

d

13.40 9.70 7.31 6.70 6.06 5.79

100 10 60 60 50 50 80 90 90 70 10 70 05 05 30

4.83

4.47 4.27 4.07 3.89 3.75 3.53 3.40 3.27 2.2

165

05 10 10 10 20 15 05 05 02 10 20 05 15 02

3.12 3.06 2.96 2.85 2.75 2.55 2.42 2.35 2.22 2.15 1.99 1.91 1.85 1.76

Solubility Solubility <mg/m11

Solvent Methanol 1-Hexano 1 Chloroform Cyc lohexane Water Buffer pH 1.2 4.0 6.0

8 11 0.01 -0.01 2.5 0.12 0.28 3.30

Temperature 3 7O 3 7O 3 7O 3 7O 25OC 25 O C 25 O C 25OC

2.3 pKa

Water 66% D i m e t hylf ormamide/34$ water

4.5 7.6

-

2.4 E l e c t r o n i c Spectra 2.4.1

U l t r a v i o l e t Absorption Spectrum The u l t r a v i o l e t spectrum of Fenoprofen C a l c i u m i n methanol is shown i n F i g u r e 2 . The s p e c t r e x h i b i t s maxima a t 266, 272, and 278 nm w i t h E @ v a l u e s of 61.3, 70.0, and A bill 63.2, r e s p e c t i v e l y .

,

_I

166

CHRISTINE K. WARD AND ROGER E. SCHIRMER

0.5

0 I

250 Figure 2.

I

300

I

350

i

380

U l t r a v i o l e t Spectrum of F e n o p r o f e n Calcium

FENOPROFEN CALCIUM

167

O p t i c a l Rotation Althousch F e n o p r o f e n Calcium is used a s t h e racemic mixture, o p t i c a l r o t a t i o n s have been r e p o r t e d f o r t h e c o r r e s p o n d i n g e n a n t i o mer i c a c i d s . 1 ta12i C = 1 - i n CHC1, d - (+I-Fenoprofen Acid +46.0’ -45.7O 1- (- -Fenopr of e n A c i d 2.4.2

2.5

I n f r a r e d Spectrum The i n f r a r e d sDectrum of F e n o m o f e n Calcium i n a KBr d i s k is g i v e n i n F i g i r e 3. The s p e c t r u m w a s o b t a i n e d u s i n g a Beckman IR12 I n f r a r e d S p e c t r o p h o t o m e t e r . Major band a s s i g n ments a r e as f o l l o w s : Band -

P o s i t i o n , CM-l

Assignment

3660, 3600 and 3300

-OH s t r e t c h i n g of hydrate

1560 ( v e r y s t r o n g , b r o a d ) and 1420

CO; a s y m m e t r i c and symmetric s t r e t c h i n g

1490, 1440 and 1450

aromat i c r i n g st r e t c hing

1260 t o 1210 (several bands)

C-0-C asymmetric e t h e r stretching

930 t o 695 ( s e v e r a l bands)

p r i m a r i l y aromat i c out of p l a n e bend i n g

.

-

N u c l e a r Magnetic Resonance Spectrum T h e y nmr s p e c t r u m of Fenoprofen C a l c i u m i n d e u t e r a t e d d i m e t h y l s u l f o x i d e a c i d i f i e d w i t h t r i f l u o r o a c e t i c a c i d is g i v e n i n F i g u r e 4. Assignments of t h e bands a r e as f o l l o w s : 2.6

--

Band P o s i t i o n , ppm

Assignment

8 . 4 - 6 . 0 (complex mult i p l e t )

aroma t i c p r o t o n s

3 . 7 0 ( q u a r t e t , J = 7Hz)

-CH-

1.35 ( d o u b l e t , J = 7Hz)

-CH3 -

-

168

E a,

0

Enl E a, E

a,

a

k rd k

H

w c

m

a, k

169

170

CHRISTINE K. WARD AND ROGER E. SCHIRMER

2.7

Mass Spectrum The mass s p e c t r u m of F e n o p r o f e n is presented in Figure 5 . 3.

Synthesis The s y n t h e s i s of F e n o p r o f e n Calcium is presented i n Figure 6 .

4.

Stability

F e n o p r o f e n is q u i t e s t a b l e t o a c i d % b a s e , and heat. For example, s t o r a g e a t 135 C f o r s i x d a y s r e s u l t s o n l y i n t h e loss of t h e waters of hydratio!. Samples stored f o r o v e r three y e a r s a t 37 C showed no d e g r a d a t i o n a t a l l . However, d e g r a d a t i o n of F e n o p r o f e n c a n be induced by e x p o s i n g a q u e o u s s o l u t i o n s of t h e d r u g t o i n t e n s e u l t r a v i o l e t l i g h t . Under these c o n d i t i o n s p h o t o - f r ies r e a r r a n g e m e n t s o c c u r l e a d i n g t o a m i x t u r e of t h e f o l l o w i n g isomeric biphenyls2 :

y COOH

ycoon

No d e g r a d a t i o n of Fenoprof e n C a l c ium has been o b s e r v e d i n a n y f o r m u l a t i o n .

I

I

,yY'

.'I

200 F i g u r e 5.

1

It.

II

I,

1 1 1 1 1 ~ 1 ' 1 1 1 1 1 1 1 ' 1 ' 1 1 1 ' 1 ' 1 ' 1

220

240

260

280

Mass S p e c t r u m of F e n o p r o f e n Calcium

300

320

340

/

172

w

0 k

c

0

a (u

b4

h

c

rn

a

r.4

-4

F ENOPRO F EN CALCIUM

173

Table 1 Urinary M e t a b o l i t e s of Fenoprofen3'4

H3c\c00H

3%

(unchanged Fenopr of e n )

455

d

COOH

I1 0

2%

42%

F i r s t unidentified acid l a b i l e conjugate Second u n i d e n t i f i e d a c i d l a b i l e c o n j u g a t e

35 5%

I74

5.

CHRISTINE K. WARD AND ROGER

E. SCHIRMER

Metabolism 5.1

Metabolites The p r i n c i p l e r o u t e s of metabolism of F e n o p r o f e n i n v o l v e h y d r o x y l a t i o n of t h e t e r m i n a l p h e n y l g r o u p and c o n j u g a t i o n w i t h g l u c u r o n i c a c i d . 3 ) 4 The s t r u c t u r e s and t y p i c a l percentages of t h e m e t a b o l i t e s i n human u r i n e a r e p r e s e n t e d i n T a b l e 1. 5.2

Pharmacokinetics -*compartment open mode 1 p r o v i d e s a r e a s o n a b l y a c c u r a t e d e s c r i p t i o n of F e n o p r o f e n c o n c e n t r a t i o n s i n plasma f o l l o w i n g o r a l d o s e s . 596 R e p r e s e n t a t i v e v a l u e s of t h e k i n e t i c p a r a m e t e r s f o r t h e one compartment model a r e g i v e n i n F i g u r e 7.5 K i n e t i c p a r a m e t e r s have a l s o been r e p o r t e d f o r t h e t w o compartment open model. 59 6 Rena 1 c l e a r a n c e v a l u e s f o r Fenoprof e n range from 3 8 . 6 t o 4 7 . 8 ml/min a n d suggest that t u b u l a r r e s o r p t i o n of F e n o p r o f e n o c c u r s . 6.

E lement a 1 Ana l y s is

Element Ca C H 0 7.

Calcium Fenoprof e n Anhydrous D i hydra t e

--

7.67 68.94 5.01 18.37

7.17 64.50 5.41 22.91

Chromatographic Methods of A n a l y s i s 7.1

T h i n Layer Chromatography S e v e r a l t h i n l a y e r s y s t e m s have been r e p o r t e d f o r s e p a r a t i o n o f F e n o p r o f e n from its s y n t h e t i c p r e c u r s o r s and metabolites. T h e s e s y s t e m s are summarized i n T a b l e 2 . S i l i c a g e l p l a t e s were u s e d in a l l cases. The Roman numerals r e f e r t o t h e structures given in

Figure 6 .

-

PLASMA COMPARTMENT rn

I

f D

kab=0.15 min-1

f/V = 0.14 Q-1

kd=0.005 min-1

1

kab= absorption rate constant kd= elimination rate constant f D = fraction of dose absorbed x dose V = volume of the plasma compartment Figure 7.

Pharmacokinetics of Fenoprofen Calcium

3

I: *I

O

O O

4O

rl

176

Ic

ID

m 00 In

10

(0

cv

cv 0

Q

E

N

Q

: In

21 8 Q,

In

(D

In

(0

Q,

(0

In

m W

Q,

z (D

El

H

: In

3

El 3 w

v)

m

b m

0

W

m

alc

+o

v

drl

.rO al4

0

dn

W

m

>I

Iy"

u ( 0

"I

5

dl

b

b

C U Q , ( D C D

In

rl

CUN

cvcv

(D

N

I n Q ,

W W

b

0

w

d(

t-

0

A

CO -I

IX

Ref. -

66

62

7

56

96

91

7

89

87

98

91

7

92

91

98

92

7

S o l v e n t System

V VI VII VIII -I -

17.

Ethyl ether

63

49

60

54

18.

Ethyl etheracetic a c i d (98-2)

86

82

89

19.

Ethyl etheracetic a c i d (95-5 1

87

82

20.

Ethyl etheracetic a c i d (90- 10)

92

85

FENOPROFEN CALCIUM

179

7.2

G a s Chromatography C a l c i u m F e n o p r o f e n r a w materials a n d f o r m u l a t i o n h a v e b e e n a n a l y z e d b y gas chromatog r a p h y . The s a m p l e is p r e p a r e d by s u s p e n d i n g t h e d r u g or c r u s h e d f o r m u l a t i o n i n a q u e o u s h y d r o c h l o r i c acid and e x t r a c t i n g w i t h chlorof o r m , d r y i n g t h e chloroform o v e r a n h y d r o u s sodium s u l f a t e a n d e v a p o r a t i n g t h e chloroform a s necessary t o c o n c e n t r a t e t h e s a m p l e . The F e n o p r o f e n a c i d is s i l y l a t e d by warming a t 6OoC f o r 15 m i n u t e s w i t h N,O-bis- ( t r i m e t h y l s i l y l ) trif luoroacetamide and then i n j e c t e d o n t o t h e column. S e v e r a l sets of c h r o m a t o g r a p h i c c o n d i t i o n s s u i t a b l e for t h e a n a l y s i s are summarized i n T a b l e 3. Diphenamid a n d m - d i p h e n y l b e n z e n e have been used as i n t e r n a l s t a n d a r d s .

7.3

High P r e s s u r e L i q u i d Chromato r a p h y a d by Fenoprof e n Calcium c high p r e s s u r e l i q u i d chromatography u s i n g t h e following condit ions :

Column :

30 c m x 4 mm s t a i n l e s s s t e e l column packed w i t h p-Bondapak C 18.

Temperature :

Ambient ( a p p r o x i m a t e l y 25OC )

S o l v e n t Flow Rate :

100 ml/hr

Detector:

U l t r a v i o l e t , 2 8 0 nm

Sample S i z e :

A p p r o x i m a t e l y 22 mcg o n c o lumn

E l u t ing Solvent :

(-1100 p s i )

600 m l d e i o n i z e d water 4 0 0 m l a c e t o n i t r i l e and

20 ml g l a c i a l acetic a c i d

Internal Standard :

p - c h l o r o b e n z o i c acid

Table 3 C o n d i t i o n s for G a s Chromatography of S i l y l a t e d Fenoprofen A rox. Solid Support Length ID Been Temp.

Tfme

3.8$, W 9 8

D iat opor t S

1.0$, W98

Gas Chrom

Q

0.5%,OV17

Liquid Phase L

1.%,

oV17

-

- w p ~ -~ ~ ~

3 ft.

3 mm

175OC

4 min

2 ft.

3 mm

15OoC

4 min

Chromosorb GHP

4 ft.

3 mm

175OC

2 min

Chromosorb G-

3 ft.

3 mm

14OoC

6 . 6 min.

A W DMCS

ion

Ref 8

FENOPROFEN CALCIUM

8.

181

T i t r i m e t r i c Determination

The c a r b o x y l a t e f u n c t i o n may be d e t e r m i n e d by p o t e n t i o m e t r i c t i t r a t i o n w i t h p e r c h l o r i c a c i d using glacial acetic acid as t h e s o l v e n t . Calcium c a n be d e t e r m i n e d by t i t r a t i o n w i t h 0.05 M EDTA u s i n g C a l c o n i n d i c a t o r . About 1.5 g of F e n o p r o f e n Calcium is d i s s o l v e d i n e t h a n o l and d i l u t e d t o 100 m l w i t h e t h a n o l . 10 m l of t h i s s o l u t i o n a r e t h e n t i t r a t e d t o t h e blue endp o i n t i n a s o l u t i o n c o n t a i n i n g 7 0 m l of water, 2 m l of 10%sodium h y d r o x i d e , 1 d r o p of 1% g e l a t i n , 3 d r o p s of 10% KCN a n d 2 drops of C a l c o n i n d i c a t o r s o l u t ion.

9.

Spectrophotometric

Determinations

F e n o p r o f e n acid has b e e n d e t e r m i n e d by m e a s u r i n g t h e a b s o r b a n c e a t 272 nm i n m e t h a n o l s o l u t i o n s ac i d i f i e d w i t h acet i c a c i d . F e n o p r o f e n Calcium has been d e t e r m i n e d by measuring t h e a b s o r b a n c e a t 2 7 0 nm i n a pH 7.5 phosphate b u f f e r s o l u t i o n . 10,

A n a l y s i s of F e n o p r o f e n i n B i o l o g i c a l Samples

F e n o p r o f e n has b e e n d e t e r m i n e d i n blood plasma s a m p l e s 8 by gas chromatography f o l l o w i n g e x t r a c t i o n . F e n o p r o f e n was first e x t r a c t e d i n t o hexane from t h e a c i d i f i e d plasma s a m p l e , t h e n e x t r a c t e d o u t of t h e hexane i n t o 0 . 1 N s o d i u m h y d r o x i d e s o l u t i o n , and f i n a l l y e x t r a c t e d back i n t o hexane a f t e r a d j u s t i n g t h e pH of t h e a q u e o u s s o l u t i o n t o a b o u t 3. The hexane w a s evaporated and t h e fenoprofen s i l y l a t e d u s i n g hexamethyl d i s i l i z a n e i n c a r b o n d i s u l f i d e . T h e c a r b o n d i s u l f i d e s o l u t i o n is t h e n i n j e c t e d o n t o a 3 f t . 3.8% W98 on D i a t a p o r t S operated a t 175O C .

182

CHRISTINE K. WARD AND ROGER E. SCHIRMER

References ---

1. 2.

3. 4.

5.

W.S. M a r s h a l l , U.S. P a t e n t 3 , 6 0 0 , 4 3 7 (1971 t o E l i L i l l y and Company). A . Dinner, Unpublished r e s u l t s . A . Rubin, P. Warrick, R.L. Wolen, S.M. C h e r n i s h A.S. R i d o l f o , C.M. Gruber, J r . , J . Pharmacol, E X ~ .Ther. 183, 449(1972) A . Rubin, S.M. C h e r n i s h , R. C r a b t r e e , C.M. Gruber, Jr., L. Helleberg,B.E. Rodda, P. Warrick, R.L. Wolen, and A.S. R i d o l f o , C u r r . Med. R e s . Opinion 2, 529(1974). A . Rubin, B.E. Rodda, P. Warrick, A.S. R i d o l f o , and C.M. Gruber, Jr., J. Pharm. S c . 6 0 , 1797 (1971) A . Rubin, B.E. Rodda, P. Warrick, A.S. R i d o l f o , and C.M. Gruber, J r . , J . Pharm S c . 61, 739 (1972 ) R.H. B i s h a r a , J. Ass. Off. Anal Chemists 5 6 , 657 (1973)J . F . Nash, R. J . Bopp and A . Rubin, J . -Pharm Sc. 60, 1062(1971).

- -

---

---

-

6. 7. 8.

-

A

-

- --

-

- -

- -

-

ISONlAZID

Glenn A. Brewer

184

GLENN A. BREWER

CONTENTS

1.

2.

3. 4. 5. 6.

Description 1.1 N a m e , F o r m u l a , Molecular W e i g h t 1 . 2 A p p e a r a n c e , C o l o r , Odor, T a s t e P h y s i c a l and Chemical P r o p e r t i e s 2 . 1 Spectra 2.11 I n f r a r e d Spectrum 2.12 U l t r a v i o l e t Spectrum 2.13 Chemiluminescence 2.14 Fluorescence Spectrum 2 . 1 5 N. M. R. Spectrum 2 . 1 6 E. S. R. S p e c t r u m 2 . 1 7 Mass S p e c t r o m e t r y 2.2 P h y s i c a l P r o p e r t i e s of t h e S o l i d 2.21 Melting C h a r a c t e r i s t i c s 2 . 2 2 D.T.A. a n d D.S.C. 2.23 T.G.A. E l e c t r i c a l Moment 2.24 2.25 E l e c t r i c a l Conductivity 2.26 Crystal characteristics 2.27 X-Ray D i f f r a c t i o n 2.3 S o l u b i l i t y 2 . 3 1 Water S o l u b i l i t y 2.32 Solubility i n Solvents 2.4 Physical Properties of Solution 2 . 4 1 PH 2.42 D i s s o c i a t i o n Constant 2.43 Photolysis Constant 2.44 Oxidation P o t e n t i a l Metal Complexes History, S y n t h e s i s and Manufacturing Stability A n a l y t i c a l Chemistry 6.1 Identity T e s t s 6.2 Methods o f A n a l y s i s 6 . 2 1 General Reviews 6 . 2 2 Colorimetric Methods 6 . 2 3 S p e c t r o p h o t o m e t r i c Methods 6 . 2 4 F l u o r i m e t r i c Methods 6 . 2 5 T i t r i m e t r i c Methods 6 . 2 6 E l e c t r o c h e m i c a l Methods

ISON IAZlD

7. 8. 9.

185

6 . 2 7 G r a v i m e t r i c Methods 6 . 2 8 M i c r o b i o l o g i c a l a n d E n z y m a t i c Methods 6 . 2 9 M i s c e l l a n e o u s Methods 6 . 3 C h r o m a t o g r a p h i c Methods 6 . 3 1 P a p e r Chromatography 6 . 3 2 Thin-Layer Chromatography 6 . 3 3 I o n Exchange c h r o m a t o g r a p h y 6 . 3 4 O t h e r C h r o m a t o g r a p h i c Methods 6 . 4 D e t e r m i n a t i o n of I s o n i a z i d and i t s Metabolites i n Body F l u i d s and T i s s u e s 6 . 4 1 G e n e r a l Reviews 6.42 C o l o r i m e t r i c Methods 6 . 4 3 T u r b i d i m e t r i c Method 6 . 4 4 F l u o r i m e t r i c Methods 6 . 4 5 E l e c t r o c h e m i c a l Methods 6 . 4 6 G a s o m e t r i c Methods 6 . 4 7 M i s c e l l a n e o u s Chemical A s s a y s 6.48 M i c r o b i o l o g i c a l Assays 6.49 Chromatographic Assays Drug Metabolism Biopharmaceutics References

186

GLENN A. BREWER

1. Description 1.1 Name, Formula, Molecular Weiqht Generic names - Isoniazidl, Isonicotinic Acid Hydrazide, INH, Isonicotinoylhydrazine, Isonicotinyl hydrazide, Isonicotinylhydrazine, Tubazid, ISoniazidum Chemical names - 4-Pyridinecarboxylic acid hydrazide, pyridine-4-carboxyhydrazide, pyridine- y-carboxylic acid hydrazide. Chemical Abstracts Registry No. 54-85-3 2

.

2HNm0c-)+( C6H7N30

Mol. Wt. 137.14

1.2

Appearance, Color, Odor, Taste Colorless or white crystalline powder which is odorless and has at first a slightly sweet and then bitter taste3. Physical and Chemical Properties 2.1 Spectra 2.11 Infrared Spectrum The infrared spectrum of isoniazid and other hydrazides of carboxylic acid have been recorded and band assignments were made4 , Nagano et a15 in a later paper made band assignments for isoniazid, metal complexes of isoniazid and related compounds The infrared spectra of isoniazid as a solid in a KBr pellet and as a mull in mineral oil are shown in Figures 1 and 2 . The following assignments have been made by Mrs. Toeplitz6. Frequency(cm-l) A s s iqnment 3300-3000 Bonded NH and C-H 1670 c=o 1560 Amide I1 1640 NH2 deformation 1610t ring C=C and C=N 1500/ 2.

.

WAVELENGTH (MICRONS)

FRMUENCY (W')

F i g u r e 1:Infrared

spectrum of isoniazid as a KBr p e l l e t .

WAVELENGTH (MICRONS)

A

8

FREQUENCY (W')

Figure 2:Infrared

s p e c t r u m of i s o n i a z i d i n m i n e r a l o i l m u l l .

ISONIAZID

2.12

189

U l t r a v i o l e t Spectrum Numerous a u t h o r s h a v e r e c o r d e d t h e u l t r a v i o l e t s p e c t r u m o f i s o n i a z i d i n a number o f s o l v e n t s 7 , 8 ~ 9 ~ 1 0 , 1 1 ~ 1 2 . The e f f e c t of t h e p H o f t h e s o l u t i o n on t h e r e s u l t i n g s p e c t r u m h a s been noted. Zommer13 h a s r e c o r d e d t h e s p e c t r a of t h e h y d r a z o n e s of i s o n i a z i d and a c e t o n e o r p-hydroxybenza ldehyde. The u l t r a v i o l e t s p e c t r u m o f i s o n i a z i d i n d i l u t e a c i d (0.01N aqueous HC1) shows t w o a p p r o x i m a t e l y e q u a l maximima a t 213 nm ( E Y i m 437) a n d 265 nm ( E l % 4 1 7 ) . The minimum 1c m o c c u r s a t 233 nm. The s p e c t r u m i n d i s t i l l e d w a t e r shows a b r o a d peak a t 2 6 1 nm (EFgm 306) w i t h o u t a d e f i n e d minimum. T h e r e i s a s h o u l d e r a t 208 nm. I n d i l u t e a l k a l i (0.01N a q u e o u s a l k a l i ) t h e s p e c t r u m t a k e n i m m e d i a t e l y shows a s h o u l d e r a t 266 nm ( E F i m 293 a n d p e a k s a t 272 nm (EFgm 298) a n d 2 9 5 nm (Elcm % ! 2 8 4 ) . On s t a n d i n g t h e s e p e a k s s h i f t so t h a t a t 2 h o u r s t h e r e a r e p e a k s a t 256 nm ( E l % 1 7 3 ) , 262 nm ( E E m 1 7 0 ) a n d 325 nm ( E r g m 7 6 ) . 1c m A t 24 h o u r s t h e 325 nm peak d i s a p p e a r s . The same s h i f t t a k e s p l a c e w i t h h i g h e r c o n c e n t r a t i o n s of a l k a l i e x c e p t t h a t i t occurs more r a p i d l y . The u l t r a v i o l e t s p e c t r u m t a k e n i n m e t h a n o l i c r a t h e r than aqueous s o l v e n t s a r e s i m i l a r t o those i n water except t h a t the absorption maximima g e n e r a l l y o c c u r a t s l i g h t l y lower wavelengths. 2.13 Chemiluminescence C a e n l 5 h a s o b s e r v e d a weak c h e m i l u m i n e s c e n c e o f i s o n i a z i d when s o l u t i o n s a r e o x i d i z e d w i t h sodium h y p o c h l o r i t e . The lumj n e s cence i n c r e a s e s w i t h pH from 1 0 . 2 t o 13. The maximum o f t h e e m i s s i o n c u r v e i s a t 0.552 p c o r r e s p o n d i n g t o a n e n e r g y o f 5 1 K c a l , Two t h e o r i e s f o r t h e o b s e r v e d l u m i n e s c e n c e a r e o f f e r e d , b o t h of which depend on t h e p r e s e n c e o f f r e e OH a n d H 0 2 r a d i ca 1s

.

190

GLENN A. BREWER

2.14

Fluorescence Spectrum I s o n i a z i d shows a n i n t e n s e f l u o r e s c e n c e s p e c t r u m when o x i d i z e d w i t h p e r o x i d e o r a f t e r cleavage of the p y r i d i n e r i n g with This fluorescence is t h e basis cyanogen bromide. o f s e v e r a l s e n s i t i v e methods t o d e t e r m i n e i s o n i a z i d i n b i o l o g i c a l m a t e r i a l s ( S e e S e c t i o n 6 . 4 ) . When a s o l u t i o n o f i s o n i a z i d a t pH 6 . 5 t o 7 . 5 w a s t r e a t e d w i t h d i l u t e p e r o x i d e a t 100°C f o r 30 m i n u t e s w e found t h e e x c i t a t i o n maximum a t 333 nm and t h e e m i s s i o n p e a k a t 415 nm 14 A f t e r i s o n i a z i d i s rea c t e d w i t h cyanogen b r o m i d e r e a g e n t i n 1 . 8 N a l k a l i n e s o l u t i o n a t room temperature w e f o u n d a n a c t i v a t i o n maximum a t 312 nm a n d a f l u o r e s c e n c e maximum a t 392 nmI4. I s o n i a z i d a l s o f l u o r e s c e s when r e a c t e d w i t h c e r t a i n a r o m a t i c c a r b o n y l compounds (Section 6.24). 2.15 N. M. R. S p e c t r u m Several authors have studied t h e n u c l e a r magnetic resonance spectrum o f t h e h draz i d e s o f c a r b o x y l i c a c i d i n c l u d i n g i s o n i a z i d y 6 9 l7 9 18. H i l l e r b r a n d and c o - w ~ r k e r s ~ gt us d i e d t h e N. M. R. spectrum o f t h e copper s a l t . The N.M.R. spectra o f i s o n i a z i d a n d *zO exchanged i s o n i a z i d a r e shown i n F i g u r e s 3 a n d 420. The 60 MHz NMR s p e c t r u m of i s o n i a z i d , i n dimethyl sulfoxide-d6 containing tetramethyl s i l a n e a s i n t e r n a l r e f e r e n c e shows t h e p r e s e n c e o f hydrazino protons resonances a t (ppm) 4 . 6 0 ( b r o a d , 2 H , e x c h a n g e d ) and 10.15 ( b r o a d , l H , exc h a n g e d ) . The a r o m a t i c p r o t o n s r e s o n a n c e s appear a s m u l t i p l e t s a t 7 . 7 3 ( 2 H ) and 8 . 7 0 ( 2 H ) . ( F i g u r e s 3 a n d 4 ) . The complex p a t t e r n of t h e resonances, o t h e r than t h e expected doublets, suggests charge d i s t r i b u t i o n i n t h e p y r i d i n e ring. However, t h e h i n d e r e d r o t a t i o n a r o u n d t h e N-C=O a s w e l l as C - a r y l b o n d s c a n n o t be r u l e d o u t . The NMR s p e c t r u m i n m e t h a n o l -d4 w a s s i m i l a r t o F i g u r e 4 , t h e h y d r a z i n o p r o t o n s h a v i n g b e e n exchanged. The a d d i t i o n of d e u t e r a t e d H C 1 d i d n o t a l t e r t h e spectrum except a downfield s h i f t of t h e aromatic

.

191

a,

a

.d

3

m

5

W d

h

d

E

5a, .d

0 k

a al

5

a, U

a

c 0 m .d

w 0

192

a,

a m

al

EX a,

a X 0

-rl

ISON IAZ I D

193

p r o t o n s r e s o n a n c e b y 0 . 1 ppm. 2.16 E. S. R. Spectrum H i l l e r b r a n d a n d co-workers19 u s e d E l e c t r o n S p i n Resonance t o s t u d y c h a r g e t r a n s f e r i n t e r a c t i o n s between i s o n i a z i d and c o p p e r i o n s . 2.17 Mass S p e c t r o m e t r y G i l l i s 2 1 h a s d i s c u s s e d t h e fragment a t i o n p a t t e r n f o r i s o n i a z i d a n d s i m i l a r compounds. F i g u r e 5 shows t h e e l e c t r o n - i m p a c t mass s p e c t r u m o b t a i n e d on an A E I MS902 mass s p e c t r o m e t e r e q u i p p e d w i t h a d a t a a c q u i s i t i o n s y s t e m . The M+ o c c u r s a t m / e 137 and t h e f r a g m e n t i o n s r e s u l t from e i t h e r d i r e c t bond c l e a v a g e ( m / e 106, 78) o r t h r o u g h t h e e l i m i n a t i o n o f HCN from t h e p y r i d y l r i n g ( m / e 5 1 ) .

m/e

m/e

106

]

51

m/e

78

Physical Properties of t h e S o l i d 2.21 Melting C h a r a c t e r i s t i c s The m e l t i n g p o i n t o f i s o n i a z i d i s u s e d a s s p e c i f i c a t i o n i n t h e U n i t e d S t a t e s Pharma~ o p o e i aand ~ ~European Pharmacopoeia3. The m e l t i n g p o i n t o c c u r s between 1 7 0 a n d 174OC. 2.22 D.T.A. and D.S.C. D i f f e r e n t i a l thermal a n a l y s i s w a s used t o s t u d y i s o n i a z i d b e f o r e t h e t e c h n i q u e gained i t s c u r r e n t p o p u l a r i t y 2 4 3 25. P i r i s i 2 6 showed t h a t i s o n i a z i d i n t h e p r e s e n c e o f z i n c , c o p p e r and ' i r o n s a l t s and m e r c u r i c o x i d e g i v e s an a b n o r m a l D.T.A. pattern. D r . J a c o b s o n 2 7 h a s shown t h a t t h e 2.2

194

GLENN A. BREWER

3793

ISONIHZID LOT 866434 1

1O O t

I

90-

I

WR

I

-t-a

l

-

80-

1 :

70.60-

58--

_I

a 10 o t-t

48-

--

t-

30-

Z

W

20-

--

6 .

INTENSITY

0

w

J0 3 (2,

-Fl+T-r

3

5

-

-

i

4

d

+

MFISS/CHRRGE SUM = 4 4 6 7 2 B R S E PERK 2 =24.36

Figure 5:Low-resolution mass spectrum of isoniazid.

ISON IAZlD

195

S q u i b b House S t a n d a r d of i s o n i a z i d shows a s h a r p endotherm a t 17OoC u s i n g DuPont t h e r m a l a n a l y s i s

equipment. The p u r i t y of t h i s s t a n d a r d w a s d e t e r m i n e d t o be 99.95 mole p e r c e n t u s i n g a P e r k i n E l m e r DSC-1B d i f f e r e n t i a l s c a n n i n g colorimeter27. 2.23 T.G.A. Thermogravimetry c a n be u s e d t o d e t e r m i n e m o i s t u r e or r e s i d u a l s o l v e n t s i n i s o n i a z i d . When t h e S q u i b b House S t a n d a r d w a s t e s t e d no loss on d r y i n g was r e c o r d e d 2 7 . 2.24 E l e c t r i c a l Moment Lumbroso and Barassin’* d e t e r m i n e d t h a t t h e e l e c t r i c a l moment of i s o n i a z i d w a s 2.92 I.L. 2.25 E l e c t r i c a l C o n d u c t i v i t y The e l e c t r i c a l c o n d u c t i v i t y of a compressed t a b l e t of i s o n i a z i d w a s d e t e r m i n e d a t t e m p e r a t u r e s between 50 and 1500C29. 2.26 C r y s t a l Characteristics B h a t a n d co-workers30 h a v e r e p o r t e d t h a t i s o n i a z i d c r y s t a l s a r e o r t h o r h o m b i c , space g r o u p P 2 1 2 1 8 1 , w i t h a , 14.915 ( 1 5 ) b, 11.400 ( 1 0 ) c, 3.835 ( 5 ) ~ d , ( m e a s u r e d ) = 1.417 ( 7 ) d ( c a l c u l a t e d ) = 1.395 and 2 = 4.

196

GLENN A. BREWER

2.27

X-Ray D i f f r a c t i o n The powder x-ray d i f r a c t i o n c u r v e f o r i s o n i a z i d i s shown i n F i g u r e 6 3 f The r e l a t i v e i n t e n s i t i e s f o r t h e v a r i o u s peaks a r e g i v e n below:

.

Interplanar Distances Relative I n t e n s i t i e s d (ANGSTROMS ) 0.098 8.84 0.408 7.30 0.398 6.10 0.451 5.64 1.000 5.25 0.502 4.49 0.296 3.69 0.398 3.51 0.102 3.42 0.068 3.36 0.197 3.27 0.235 3.10 0.060 3.04 0.058 3.01 2.80 0.170 0.076 2.63 0.168 2.47 0.115 2.42 0.187 2.33 2.3 S o l u b i l i t y 2 . 3 1 Water S o l u b i l i t y32 F o u r t e e n g r a m s of i s n i a z i d -re s o l u b l e i n 100 m l o f water a t 25OC. T w e n t y - s i x grams a r e s o l u b l e i n 100 m l of water a t 4OoC. 2.32 Solubility i n Solvents32~33 Solvent S o l u b i l it ethanol(25OC) 2 g/100 e t h a n o l ( b o i l i n g ) 10 g/100 m l ch l o r o form 0 . 1 g/100 m l ethyl ether very s l i g h t l y soluble benzene i ns o l u b l e

mf

104

,

100

I.,

h M

I

h

co

70

60

M

re

30

M

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ut

- 0

Figure 6:X-ray powder-diffraction pattern of isoniazid.

4

c

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198

G L E N N A. BREWER

2.4

Physical P r o p e r t i e s of Solution 2.41 pH The pH o f a s o l u t i o n ( 1 i n 10) should b e between 6.0 and 7.523. 2.42 D i s s o c i a t i o n Constant There i s a d i s c r e p a n c y i n t h e l i t e r a t u r e o n t h e d i s s o c i a t i o n c o n s t a n t s of isoniazid. T h i s i s i n p a r t due t o t h e d i f f e r e n t methods of measurement employed. F a l l a b 3 4 determined t h e b a s i c d i s s o c i a t i o n c o n s t a n t a s 3 x 10-11 measured conductC a n i 6 and D j o r d j evi635 e s t a b l i s h e d ometrically. t h a t t h e 1st b a s i c c o n s t a n t s h o u l d be a s c r i b e d t o t h e p y r i d i n e n i t r o g e n and t h e 2nd t o t h e h y d r a z i n e group. T h i s i s c o n t r a r y t o p r e v i o u s work by Cingolani and G a ~ d i a n o ~ ~ . Nagano and c o - ~ o r k e r sdetermined ~~ t h e dissociation constants potentiometrically as PK1 = 2.13, PK2 = 3.81, PK3 = 11.03. S a l v e s e n and G l e n d r a n c ~ ed~e ~ termined t h e d i s s o c i a t i o n c o n s t a n t s i n 1 . O M sodium and K2 = c h l o r i d e s o l u t i o n a s K1 = 9.80 x 1.42 10-4. Zommer and Szuszkiewicz'l have e s t a b l i s h e d pK1 = 1 0 . 7 5 and pK2 = 11.15 and prot o n a t i o n c o n s t a n t s cf 3.57 f o r t h e p y r i d i n e N and 1.75 f o r t h e h y d r a z i d e N. Rekker and N a ~ t found a ~ ~ t h a t solu t i o n s o f i s o n i a z i d became yellow a t p H 10 and 2.7. The c o l o r i s r e v e r s i b l e on changing t h e pH. They e x p l a i n e d t h i s b e h a v i o r on t h e b a s i s o f t h e e x i s t ance o f two p o s i t i v e i o n s , a monovalent yellow p o s i t i v e i o n and a d i v a l e n t c o l o r l e s s p o s i t i v e ion. The pK v a l u e s a r e pK' = 2 . 0 0 , pK" = 3.6 and pK"' = 10.8. 2.43 Photolysis Constant S a l v e s e n and Eiki113' e s t a b l i s h e d t h e p h o t o l y s i s c o n s t a n t s f o r i s o n i a z i d a t 20°C and o ' * and 370 nm i n M NaCl s o l u t i o n a s kl = 1 . 0 0 x l k2 = 1.45 x 10-4.

ISONI AZ I D

199

2.44

Oxidation P o t e n t i a l The o x i d a t i o n p o t e n t i a l s f o r i s o n i a z i d a t v a r i o u s pH v a l u e s w e r e d e t e r m i n e d b y vu 1t e r i n 4 0 . Solution i n Ef 1 N HC1 0.70 0.025M Na2B407 0.25 3N NaOH -0.22 3.

Metal Complexes I s o n i a z i d forms metal complexes w i t h many d i v a l e n t i o n s . T h e s e complexes h a v e b e e n used i n t h e d e t e r m i n a t i o n o f i s o n i a z i d (see S e c t i o n s 6.22, 6.25 and 6.29). T a m u r a a n d Nagano4I h a v e d e t e r m i n e d t h e c o n s e c u t i v e f o r m a t i o n c o n s t a n t s f o r t h e complex formed b e t w e e n i s o n i a z i d a n d C d ( I 1 ) . The e x p e r i ments were c a r r i e d o u t a t pH 7.2 ( a d j u s t e d w i t h NaOH) i n M NaN03 u s i n g 0.001M Cd(N03)2 a t 25OC. The d e t e r m i n a t i o n was made p o l a r o g r a p h i c a l l y . The v a l u e s d e t e r m i n e d were k l = 35, k2 = 0 . 5 7 , k3=52.5. A t high concentrations o f i s o n i a z i d yellow c r y s t a l s o f Cd ( I N H ) 2 (NO3)2-H20 p r e c i p a t e d from s o l u t i o n indicating t h a t contrary t o t h e polarographic data t h a t t h e 2 : l complex is more s t a b l e t h a n t h e 3 : l complex. By p H t i t r a t i o n t h e s t e p w i s e f o r m a t i o n c o n s t a n t s w e r e k l = 1 2 . 2 , k2 = 1 2 . 6 , a n d k 3 = 3.4. The Same a u t h o r s 4 2 s t u d i e d t h e f o r m a t i o n c o n s t a n t s o f i s o n i a z i d a n d C u ( I I ) , Zn, N i ( I I ) , C o ( I 1 ) a n d Mn (11). The complexes o f c o p p e r a n d i s o n i a z i d h a v e been e x t e n s i v e l y s t u d i e d by I ~ h i d a t e ~ ~ . 4.

History, Synthesis and Manufacturinq I s o n i a z i d w a s f i r s t p r e p a r e d b y Meyer a n d M a l 1 Y s o 0 i n 1912 b y h e a t i n g a m i x t u r e o f ison i c o t i n i c a c i d a n d h y d r a z i n e above 30OoC. The a c t i v i t y of t h e compound a g a i n s t Mycobacterium SJ. was f i r s t r e c o g n i z e d b y C h o r i n e 5 0 1 a n d b y Huant502 i n 1945. The d r u g w a s r e p o r t e d a s a u s e f u l t u b e r c u l o s t a t i c a g e n t b y F a r b e n f a b r i k e n - B a y e r , A. G., Hoffmann-LaRoche, I n c . a n d E. R. S q u i b b & S o n s , I n c .

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i n 1952503. The b a s i c method of m a n u f a c t u r e o f i s o n i a z i d i s t h e condensation of hydr azine with a y - s u b s t i t u t e d pyri d i ne H y d r a z i n e c a n be d i r e c t l y c o n d e n s e d w i t h i s o n i c o t i n i c a c i d , The w a t e r formed i n t h e rea c t i o n i s u s u a l l y removed b y a z e o t r o p i c d i s t i l l a tion44945946947, E s t e r s o f i s o n i c o t i n y l a c i d c a n be h y d r o l y z e d and t h e r e s u l t i n g a c i d condensed w i t h h y d r a z i n e . Ammonia i s u s u a l l y employed f o r t h e h y d r o l y s i s 4 8 . Y - P i c o l i n e c a n be o x i d i z e d i n 70% s u l f u r i c a c i d w i t h manganous d i o x i d e t o form i s o n i c o t i n i c acid. The c o r r e s p o n d i n g a c i d c h l o r i d e i s made with thionyl chloride. The a c i d c h l o r i d e i s t h e n r e a c t e d w i t h h y d r a z i n e i n anhydrous benzene t o y i e l d isoniazid49. I n a modification of this procedure t h e a c i d c h l o r i d e i s r e a c t e d w i t h e t h a n o l t o form t h e e t h y l ester which i s t h e n r e a c t e d w i t h h y d r a z i n e i n e t h a n o l t o form i s o n i a z i d 5 0 . I n a s i m i l a r manner o n e c a n o x i d i z e 2 , 4 d i m e t h y l p y r i d i n e w i t h s e l e n i u m and s u l f u r i c a c i d . T h e m i x t u r e i s n e u t r a l i z e d w i t h ammonia. A m i x t u r e o f i s o n i c o t i n i c a c i d , i s o n i c o t i n a m i d e and i s o n i c o t i n i c h y d r a z i d e i s forrned51.

.

5.

Stability The s t a b i l i t y o f i s o n i a z i d h a s b e e n s t u d i e d e x t e n s i v e l y i n s o l u t i o n a n d i n v a r i o u s pharmaceut i c a l p r e p a r a t i o n s . Of p a r t i c u l a r i n t e r e s t i s t h e r e a c t i o n of t h e h y d r a z i n e g r o u p w i t h n a t u r a l l y o c c u r i n g a l d e h y d e s and k e t o n e s such a s s u g a r s o r k e t o a c i d s and the complexation o f i s o n i a z i d w i t h metal i o n s . Lewin a n d H i r ~ c h a~v e~ shown t h a t n o n - i o n i c c h e l a t i n g material can l a r g e l y p r e v e n t t h e degrad a t i o n o f i s o n i a z i d when n e u t r a l a n d a l k a l i n e s o l u t i o n s a r e a u t o c l a v e d . They n o t e d t h a t C u ( I 1 ) and Mn(I1) i o n s a c c e l e r a t e d t h e d e g r a d a t i o n o f i s o n i a z i d i n t h e p r e s e n c e o f hydrogen peroxide. P o o l e a n d M e ~ e r e~p o~r t e d t h a t i s o n i a z i d i s u n s t a b l e i n human o r r a b b i t plasma w h i l e i t i s

ISON I A2 ID

201

s t a b l e f o r s e v e r a l weeks i n b u f f e r e d aqueous s o l u t i o n s a t pH v a l u e s below 8 . The i n s t a b i l i t y i n plasma i s q u i t e marked even a t r e f r i g e r a t o r temperatures. Kakemi and c o - ~ o r k e r shave ~ ~ studied t h e d e g r a d a t i o n o f i s o n i a z i d i n aqueous s o l u t i o n under a n a e r o b i c c o n d i t i o n s . A l k a l i n e h y d r o l y s i s under a e r o b i c c o n d i t i o n s y i e l d s a m i x t u r e of i s o n i c o t i n i c a c i d , i s o n i c o t i n a m i d e and 1 , 2 d i i s o n i c o t i n o y l h y d r a z i n e p l u s s m a l l amounts of u n i d e n t i f i e d products. Under a n a e r o b i c c o n d i t i o n s i s o n i c o t i n i c a c i d and 1 , 2 d i i s o n i c o t i n o y l h y d r a z i n e were t h e p r i n c i p a l p r o d u c t s . When EDTA was added t o t h e r e a c t i o n m i x t u r e only i s o n i c o t i n i c a c i d was formed. F i r s t o r d e r k i n e t i c s were followed. Inoue55 found t h a t a t pH 3 . 1 u n d e r a n a e r o b i c c o n d i t i o n s i s o n i a z i d h y d r o l y z e s t o form i s o n i c o t i n i c acid. Pseudo f i r s t o r d e r k i n e t i c s a r e followed. A t lower pH v a l u e s t h e e f f e c t of b u f f e r t y p e can be s e e n . A c t i v a t i o n e n e r g i e s were c a l c u l a t e d for t h e h y d r o l y s i s by d i f f e r e n t i o n i c s p e c i e s . Horioka and c o - ~ o r k e r sfound ~~ t h a t losses o f i s o n i a z i d were encountered when t h e drug was blended w i t h v a r i o u s a n t i a c i d p r e p a r a t i o n s . The e f f e c t of t e m p e r a t u r e , humidity and pH on t h e s t a b i l i t was determined. Haldg7 found t h a t i s o n i a z i d underwent slow o x i d a t i o n i n aqueous s o l u t i o n , b u t i n t h e p r e s e n c e of s u c r o s e t h e i s o n i a z i d r e a c t e d w i t h t h e a l d o hexoses formed on i n v e r s i o n . The r e a c t i o n w i t h s u c r o s e could b e i n h i b i t e d by t h e a d d i t i o n o f 0.3% sodium c i t r a t e . Pawelczyk and c o - ~ o r k e r sfound ~~ t h a t a s long a s c o n d i t i o n s were k e p t a n a e r o b i c t h a t t h e decompos i t i o n of i s o n i a z i d i n t h e pH r a n g e 3 t o 7 followed f i r s t order kinetics. They r e p o r t e d t h a t a 1% s o l u t i o n of t h e drug was 37 times more s t a b l e a t PH 6 t h a n a t pH 3.The e f f e c t of d i f f e r e n t b u f f e r s p e c i e s on t h e r a t e o f t h e r e a c t i o n was noted. Wu and co-workers59 i n v e s t i g a t e d t h e browning r e a c t i o n between l a c t o s e and i s o n i a z i d i n t h e

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G L E N N A. BREWER

s o l i d s t a t e with d i f f u s e r e f l e c t a n c e spectrophotometry. T h i n - l a y e r chromatography was u s e d t o demonstrate t h e p r e s e n c e of i s o n i c o t i n o y l hydrazones of l a c t o s e and h y d r o x y m e t h y l f u r f u r a l . InoueG0 h a s e s t a b l i s h e d t h e e f f e c t of t h e p r e s e n c e of copper (11) i o n s on t h e r a t e of oxidat i o n of i s o n i a z i d i n s o l u t i o n . The r e a c t i o n p r o d u c t s were i s o n i c o t i n i c a c i d , i s o n i c o t i n a m i d e , 1,2-diisonicotinoylhydrazine, i s o n i c o t i n e carboxaldehyde and i s o n i c o t i n o y l hydrazone. The copper c h e l a t e s of i s o n i a z i d a r e degraded by a f i r s t o r d e r r e a c t i o n and t h e r a t e i s determined b y t h e r a t i o of t h e c o n c e n t r a t i o n of c h e l a t e d s p e c i e s p r e se n t Inoue and Ono61 have e s t a b l i s h e d t h e k i n e t i c s of t h e d e g r a d a t i o n of i s o n i a z i d i n t h e p r e s e n c e o f Managenese (11). Shchukin62 h a s s t u d i e d t h e r e a c t i o n of copper (11) w i t h i s o n i a z i d . Kakemi and co-workers63 s t u d i e d t h e s t a b i l i t y of t h e sodium m e t h a n e s u l f o n a t e salt of i s o n i a z i d from p H 3 t o 9. Rao and c o - ~ o r k e r shave ~ ~ demonstrated t h a t i s o n i a z i d i n s y r u p f o r m u l a t i o n s undergoes hydrazone formation w i t h t h e f r e e g l u c o s e t h a t i s p r e s e n t . Absorption of t h i s hydrazone i s r e p o r t e d t o b e impaired. The a u t h o r s s u g g e s t t h e u s e of s o r b i t o l a s a replacement f o r s u c r o s e .

.

6.

A n a l y t i c a l Chemistry 6.1 Identity Tests A l a r g- e number of i d e n t i t y t e s t s have b e e n e s t a b l i s h e d f o r i s o n i a z i d . Most of t h e s e a r e colorimetric and a r e r e p o r t e d below i n t a b u l a r form.

Reaqen t

8 w

p- Dimethylaminobenza l d e h y d e A l k a l i n e Na2Fe ( C N ) 5NO Q C F e (CN) 6J + light Dini t r o c h l o r o b e n z e n e o-ninitrobenzene R e d u c t i o n w i t h Zn/HCl and ph e n y l h y d r a z i n e 1 , 2 Naphthoquinone4 - S u l f o n i c a c i d + NaOH 1,2,4-Aminonaph t h o 1 s u l f o n i c a c i d SbC13, SbC15 o r AsC13 E p i ch l o r o h y d r i n Dimethylglyoxime E t h y 1en i c d i ca rbox y 1i c a c i d s (fumaric, maleic a c i d s , e t c . ) Naphthoqu inone-HgC12 3,5-Dini t r o s a l i c y l i c a c i d N i n h y d r in BrCN a n d NaOH Benzyl c h l o r i d e NaOH D r a g e n d o r f f ' s Reagent

rnlnr

Reference

i n t e n s e yellow i n t e n s e orange pink purple violet

66,67,68,74,85,86 69,74 70 71,74,86 72

y e 1low

73

bright red orange t o red

74,75,76 77 78 79 80 81

--

red red ye 1low

-brown r e d r e d orange green-blue f l u o r . b l u e fluorescence red

82 83 84,85 85 85 86,94

I n a d d i t i o n t o t h e s e c o l o r r e a c t i o n s a number o f c o l o r e d p r e c i p i t a t e s can be formed o n t h e a d d i t i o n of m e t a l s a l t s o r a c i d s t o i s o n i a z i d .

Reagent Am03 S e02 HgC12 cuso4 Hg2C12 KI AuI Lead a c e t a t e + K I KBr

N

e 0

~205.12~03 Picrolonic acid Tannic a c i d V i t a l i l s reagent Mecke' s r e a g e n t Frghde' s r e a g e n t Mandelin' s r e a g e n t Alloxan D i s u I f imides M e t h y 1i o d i d e K2 C r 2 0 7 Pho sphomo 1yb d i c a c id P i c r i c Acid Reineckel s s a l t Styphnic a c i d

Color of P r e c i p i t a t e White Red White Blue White Amorphous mass Dark c r y s t a l s Yellow a c i c u l a r c r y s t a l s E f f e r v e s c e n c e followed by b l a c k and c o l o r l e s s crystals Precipitate Green-ye1 low PPt Yellow mass Rose-sienna Blue Red White p p t

-Yellow n e e d l e s

--

--

Reference 74,94 78,87,88 74 86 86 86,93,95 86 86 86

86 86,93 86 86 86 86 86 89 90 91 92 92 93,94 94 94

94 94 94

Kay's reagent Vaillef s reagent Na 2 P t B r 6

F e i g l a n d c o - w o r k e r s g 6 h a v e r e p o r t e d a s p o t t e s t i n which i s o n i a z i d i s ( C N ) 6-7 q u a t e r n i z e d a n d p y r o l y z e d w i t h Na2S203 a t 180OC. A c i d i f i e d Fe i s used f o r d e t e c t i o n .

Be

97 98 Popkov and Amelink h a v e r e p o r t e d on m i c r o c r y s t a l l i n e t e c h n i q u e s f o r t h e detection of isoniazid. 6 . 2 Methods of A n a l y s i s 6 . 2 1 G e n e r a l Reviews Deltombe99, S l o u f l O O , Robles a n d Unzueta'Ol, Yalcindaglo2, B r a n d y s l 0 3 a n d G a r c i a a n d c o - w o r k e r s l o 4 h a v e a l l p u b l i s h e d r e v i e w s on t h e q u a l i t a t i v e and 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 i s o n i a z i d . 6 . 2 2 C o l o r i m e t r i c methods A number of a u t h o r s h a v e formed h y d r a z o n e s of i s o n i a z i d w i t h v a r i o u s aldehydes and k e t o n e s and used t h e h i q h l y c o l o r e d p r o d u c t s t o determine t h e d r u g . O f t h e v a r i o u s a ldehydcs u s e d , p-dimei?hylaminobenza l d e h y d e lo8, log, I l o ,lll. Benza1dehydes7, I 6 O , e a r s t o be t h e m o s t 0 ular105,106, lo7; a n d v a n i l l i n 1 1 3 ' 197yPgghave a l s o been u s e d . The o f f i c i a l method o f t h e AOAC i s t h e r e a c t i o n of i s o n i a z i d w i t h b e n z a l d e h y d e i n sodium b i c a r b o n a t e solution. The a b s o r b a n c e o f t h e h y d r a z o n e i s m e a s u r e d a t 302 nm. The absorbance a t 375 nm ( b a c k g r o u n d ) i s s u b s t r a c t e d a s a c o r r e c t i o n 1 6 9 . Sodium 1,2-naphthoquinone-4-sulfonate r e a c t s w i t h t h e h y d r a z i d e p o r t i o n o f i s o n i a z i d i n a l k a l i n e s o l u t i o n t o p r o d u c e a n orange-red c o l o r w i t h a maximum a t 480 nm1149115. 2-3-Dichloro-1,4-naphthoquinone r e a c t s

?R

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GLENN A. BREWER

with isonia zid t o give a b lu e co lo r i n a l k a l i n e ~ o l u t i o n ~ l ~The , ~ r~e a~c t. i o n i s u s e f u l w i t h pharmaceutical p r e p a r a t i o n s which a l s o c o n t a i n sodium a m i n o s a l i c y l a t e l l 8 . An a s s a y u t i l i z i n g 1, 4-naphthoquinone h a s a l s o been regorted1l9. I s o n i a z i d r e d u c e s phosphomol b d a t e i n a l k a l i n e s o l u t i o n t o molybdenum b l u e 1 2 0 , l Y l . In a s i m i l a r r e a c t i o n molybdophosphotungstate g i v e s a b l u e color122. An a s s a y u t i l i z i n g molybdic a c i d i n a l k a l i n e a c e t o n e s o l u t i o n h a s also been r e p o r t e d l 2 ? I s o n i a z i d r e a c t s w i t h cvano en c h l o r i d e 1 2 4 ~ 1 2 5 ~ 1 2 6 c, h l o r o r h o d a n a m i n e l 2 7 ~ 18 o r

2

cyanogen bromide129 t o form g l u t a c o n i c d i a l d e h y d e which can t h e n be condensed w i t h b a r b i t u r i c o r 2t h i o b a r b i t u r i c a c i d s t o y i e l d c o l o r e d polymethine dyes. I s o n i a z i d reduces f e r r i c y a n i d e t o f e r r o c y a n i d e . The amount o f f e r r o c y a n i d e can be determined by t h e a d d i t i o n of f e r r i c i o n t o y i e l d a b l u e colorl30,131. Sodium pentacyanoaminoferroate r e a c t s w i t h i s o n i a z i d t o g i v e a yellow chromogen 132 I s o n i a z i d r e a c t s w i t h 1-chloro-2, 4-dinitrobenzene i n a l k a l i n e s o l u t i o n t o g i v e a p u r p l e color133,74,134. l-Fluoro-2,4 d i n i t r o b e n z e n e a l s o r e a c t s i n a s i m i l a r manner 135 I s o n i a z i d forms c o l o r e d complexes w i t h many m e t a l s which can be used i n a n a l y t i c a l c a n be formed w i t h ammonium methods. vanadate f e r r i c c h l o r i d e and 2 , 2 1 b i ~ y r i d i n e l ~copper139 ~, and N i c k e l (11) and f e r r i c i0nl40. Reineckels s a l t forms a w a t e r i n s o l u b l e p r e c i p i t a t e with i s o n i a z i d . This p r e c i p i t a t e d i s s o l v e s i n a c e t o n e and t h e c o n c e n t r a t i o n of i s o n i a z i d can b e determined c o l o r i m e t r i c aiiy141, 142. The f o l l o w i n g compounds have a l s o been used i n c o l o r i m e t r i c assays for i a o n i a z i d .

.

13g~y9’~358,

Reagent Reference ch loropicrin 504 epichlorohydrin 143 ninhydrin 144 145 tripheny1tetrazolium ch loride 9-chloroacridine 146 dinitrobenzoic acid 147 p-aminosalicylate-HVO~ 148 1,2,4-aminonaphtholsulfonic acid 77 p-ni trophenyldiazonium fluoroborate 149 7-chloro-4 nitrobenzo-2-oxa-1,3-diazole 150 acid chrome dark blue 151 2-bromo-1-acetonaphthone 112 N- (4-pyridyl)pyridinium chloride 152 174 picryl chloride 6.23 Spectrophotometric Methods A number of authors have utilized the strong absorbance of isoniazid in the ultraviolet as a means of determining the concentration of the drug. In many methods the a @ : f ! 7 4 IPS in alkaline and acid solution as an identity test 1659237. Isoniazid can be determined in the presence of p-aminosalicylate by an ultraviolet assay162,163,164. 6.24 Fluorimetric Methods Although isoniazid does not have any native fluorescence several sensitive fluorometric assays have been reported for the drug. Isoniazid is coupled with 2-hydroxy-1-naphthaldehyde to give a yellowgreen fluorescence. The compound has an excitation maximum at 495 m and an

Y

y3,iQ%, ar39:'iB;

Fh

208

GLENN A. BREWER

e m i s s i o n maximum a t 534 nrn166~167. I n a n o t h e r method t h e p y r i d i n e r i n g i s c l e a v e d w i t h cyanogen b r o m i d e t o form A S c h i f f ' s base i s t h e n formed glutacondialdehyde. w i t h 4 - a m i n o b e n z o i c a c i d which has a n e x c i t a t i o n maximum a t 336 6 . 2 5 T i t r i m e t r i c Methods A large variety of titrimetric methods h a v e b e e n employed f o r the d e t e r m i n a t i o n of i s o n i a z i d i n b u l k and i n formulated products. A series of reviews have been w r i t t e n on t i t r i m e t r i c methods170, 1 7 1 9 1 7 2 3 1 7 3 ~ 202. The o f f i c i a l m e t h o d s of a n a l y s i s i n t h e U . S. P. 2 3 , B. P. 174 a n d E u r o p e a n P h a r m a c o p o e i a 3 a r e t i t r i m e t r i c methods. I n t h e U.S.P,23 a n i t r i t e titration is utilized. I n t h e B.P. 174 t h e i s o n i a z i d i s r e a c t e d w i t h bromine and t h e e x c e s s bromine i s t i t r a t e d w i t h t h i o s u l f a t e a f t e r t h e l i b e r a t i o n o f i o d i n e by t h e a d d i t i o n o f potassium iodide. I n t h e European P h a r m a c o p o e i a 3 a d i r e c t t i t r a t i o n w i t h bromate i s u t i l i z e d w i t h t h e a d d i t i o n o f ethoxychysoidine a s an i n d i c a t o r . The v a r i o u s t i t r i m e t r i c methods a r e summarized i n t h e Table.

I C 1, K I

Titrant t h i o s u 1f a t e KBr03 alkali KBrO3 mr03 t h i o s u lf a t e KIO~ thiosulfate t h i o s u 1f a t e t h i o s u l f at e t h i o s u l f at e

non-aqueous

HC lo4

non-aqueous non-aqueous

NaN02 HC 104 HClOq

Reaq e n t

mr ,m r 0 3 ,KI m r Br2

-

~ 1 0 3KI , H I , K2Cr207, K I

~ ~ 1 0 4 , N

8

non-aqueous Cd++ Cd++ cU++,

NH4SCN

CU++, N H ~ S C N

I2

Indicator starch ethoxychrysoidine pheno lphtha l ei n methyl orange p o t e n t iome t r i c starch ethoxychrysoidine starch starch starch starch thermome t r i c crystal v i o l e t or methyl v i o l e t

Sb e l e c t r o d e

Complexon 111 CaC12

g l a s s electrode potentiome tric thymol b l u e eriochrome B l a c k T methylthymol B l u e

Am03 EDTA

mu r e x i de

NaClO4 NaOMe

Reference 1 7 5 ,1 7 7 , 1 7 9 , 1 8 2 176,106,180,184 178 181,186 1 8 1 , 1 8 3 ,1 8 5 ,1 8 7 74,188,192,196 189 1 9 0 , 1 9 3 ,1 9 5 191 106,194 198 200 217,211,210,209,208, 207,206,204,205,216, 213,201,74,57,203, 2 14 233 212,276 215 2 18 2 02 219,221 220 222 2 2 3 ,224

E

% m

d

~m m a ,

d

0 0 0

a E E

U

H H

C 0

3

dd

C O D

0 a 0 mEcn NUf:

h

d

9

a,

a 0 k c,

u

a,

c

a,

k

a,

a m a -ti

I

I

m

4J

m

k

E

0

.ti

5

a .ti a

I

rl

h C

a,

a,

5k

4

tn

0-

a

a,

d

nl

a,

m

.d

m

4J

z

c,

-

Ill

m

4J

o"+ cn+

C cr a, m m rn W

I

3 3

u u

E 7

m

d a, Ik

210

I

H

h

d

m

'

Nesslerl s reagent I2

-

C e (So4) 2

-

K2Cr207 isopropenyl t r i chloroacetate

Y

sodium d i e t h y l d i thiocarbama t e hydraz i n e ammonium hexani t r o c e r a t e (IV) Mohrrs s a l t C e (NO314 Mohrfs s a l t

cuso4

2 56

starch a-naphthof lavone P t electrode P t electrode diphenylamine

2 57 2 58

259 2 60 261

NaOH bromphenol b l u e 262 KOH K3Fe (CN) 6 P t electrode 263,264 K 3Fe (CN) 6, KOH, H2S04, K I t h i o s u I fa t e starch 265 6.26 E l e c t r o c h e m i c a l Methods A number of a u t h o r s have d e t a i l e d p o l a r o g r a p h i c methods f o r i s o n i a z i d . The r e d u c t i o n a p p a r e n t l y o c c u r s i n two s t e p s ( t o t a l o f 4 e l e c t r o n s ) b u t t h e s t e p s a r e n o t s u f f i c i e n t l y w e l l s e p a r a t e d t o be u t i l i z e d The h a l f wave p o t e n t i a l bea n a l y t i c a l l y , s o t h a t t h e s i n g l e wave i s used. b u t t h e h e i g h t d i d n o t change r e a t l y comes more n e g a t i v e a t h i g h e r pH v a l u e s H r a n g e s t u d i e d 266,153,267,266,269,270,271,272,273,274,275,27 ,278, over t h e 279,280,2Kl

4

A.C.Polarography h a s been used by Sato282 and Vallon and c o - ~ o r k e r s ~ ~t h~ei n a s s a y o f i s o n i a z i d . Okuda and co-workers284 rea c t e d i s o n i a z i d w i t h 1,2-naphthoquinone-4-sulfonic a c i d and have t h e n used p o l a r o g r a p h y t o measure t h e r e a c t i o n p r o d u c t . se e r p l au h o r s h ve r e r t e d c o u l o e t ' c g e t h o d s for t h e a n a l y s i s o f i s o n i a z i x w i t h ekectroccemica??y g e n e r a t e 8 c 6 i o r i n e 285,286 o r bromine286,287,288,289.290.

212

GLENN A. BREWER

6.27

Gravimetric Methods R e l a t i v e l y few g r a v i m e t r i c a s s a y s have been r e p o r t e d f o r i s o n i a z i d . T h i s i s probably because of t h e l a r g e number of c o l o r i m e t r i c , t i t r i m e t r i c and e l e c t r o c h e m i c a l methods a v a i l a b l e which a r e f a s t e r and more convenient than t h e g r a v i m e t r i c methods. Leal and Alves 234 have r e p o r t e d an a s s a y using p i c r i c a c i d t o form a w a t e r i n s o l u b l e salt. Akiyama and co-workers291 prec i p i t a t e i s o n i a z i d e a s t h e C u ( I 1 ) o r Hg(I1) s a l t s . The s a l t s a r e r e d i s s o l v e d i n h y d r o c h l o r i c a c i d and t h e metal i s then r e p r e c i p i t a t e d a s t h e s u l f i d e which i s determined g r a v i m e t r i c a l l y . The z i n c 2 9 2 and cadmium293 s a l t s can be measured by d i r e c t gravimetry. The benzyli d e n e d e r i v a t i v e can be determined e i t h e r gravimetrically o r v o l ~ m e t r i c a l l y ~ ~ I s~o .n i a z i d can b e q u a t e r n i z e d and t h e s a l t can b e then measured v o l u m e t r i c a l l y o r g r a v i m e t r i c a l l y 2 95. The phosphotungstate of i s o n i a z i d can b e determined gravime t r i c a 1 1 ~ 1 6 4 . 6.28 M i c r o b i o l o q i c a l and Enzymatic Methods S e v e r a l a g a r d i f f u s i o n microbiol o g i c a l a s s a y s u t i l i z i n g s t r a i n s of a c te rium have been r e p o r t e d f o r i s o n i a ~ i d ~ ~ ~ I s o n i a z i d i n h i b i t s many enzyme systems and a number of t h e s e might b e s e l e c t e d a s t h e b a s i s of enzymatic a s s a y s . Examples of enzyme systems which a r e i n h i b i t e d a r e pea cotyledon amine o x i d a s e , c a r r o t r o o t L-glutamic decarboxylase and wheat s e e d l i n g t r a n s a m i n a ~ e ~ The ~ ~ . inhibition i s r e v e r s e d by t h e presence of k e t o a c i d s . 6 . 2 9 Miscellaneous Methods O s c i l l o m e t r i c t i t r a t i o n s have been used t o determine i s o n i a ~ i d301. ~ ~ ~ Ijs o n i a z i d can be assayed g a s o m e t r i c a l l y a f t e r o x i d a t i o n w i t h iodate3O2 o r f e r r i c y a n i d e 3 0 3 304. Conductometric t i t r a t i o n s with sodium hydroxide o r h y d r o c h l o r i c a c i d have been J

~

~

~

u s e d t o measure i s o n i a z i d c o n t e n t 3 0 5 , 3 0 6 . The c o p p e r c h e l a t e of i s o n i a z i d i s s o l u b l e i n m e t h y l i s o b u t y l ketone. The c o p p e r c o n t e n t of t h e c h e l a t e i s d e t e r m i n e d i n t h e o r g a n i c p h a s e by a t o m i c a b s o r p t i o n s p e c t r o m e t r y 3 0 7 . I s o n i a z i d i n p u r e s o l u t i o n s can b e d e t e r m i n e d by r e f r a c t o m e t r y 3 08 6.3

5 w

Chromatographic Methods 6 . 3 1 Paper Chromatoqraphy Numerous p a p e r c h r o m a t o g r a p h i c s y s t e m s h a v e b e e n u s e d t o s e p a r a t e i s o n i a z i d from i n t e r m e d i a t e s u s e d i n t h e s y n t h e s i s , d e g r a d a t i o n p r o d u c t s and m e t a b o l i c p r o d u c t s . Since isoniazid absorbs strongly i n t h e u l t r a v i o l e t and g i v e s a number of c o l o r r e a c t i o n s 3 0 9 t h e r e i s no problem i n d e t e c t i n g o r q u a n t i t a t i n g t h e d r u g a f t e r t h e s e p a r a t i o n h a s been completed. A t a b l e of some p a p e r c h r o m a t o g r a p h i c s y s t e m s i s g i v e n below: Solvent Syst e m Detection Use Ref. Water s a t u r a t e d b u t a n o l C14 l a b e l l e d Urine metabolites 310 Isoamyl alcohol-waterCNBr ,Microb i o 1. Urine metabolites 311 a c e t i c a c i d ( 5 0 :50 :1 . 5 ) I s o p r o p a n o l - w a t e r (85 :1 5 ) Urine metabolites 312,313 Butano 1-ammonia -Derivatives 31 4 1st Dimension sec. b u t a n o l w a t e r (saturated)

--

2nd Dimension i s o a m y l alcohol-acetone-acetic a c i d - w a t e r (56:24:6: 1 4 )

CNBr- o-phenyl-

enediamine dimethylbenza l d e h y d e

Urine

Butanol-10% NH4OH(lO:2) circular Butanol-water(4:l) ascending 2,4-lutidine-isoamyl alcohol-water (5: 100: 9) Butanol-HC1-pet. ether or Butanol-HCl-H20(paper sat. with KC1 solution) (a)Butanol-ethanol-water (2:2:1) (b)Butanol-pyridine-water (16:4: 3 )

Butanol sat.ammoniaca1 Impurities with silver nitrate dimethylaminobenzaldehyde Dosage forms

3 16

methanolic dinitrochlorobenzene iodine-platinic iodide

Impurities

318

other basic substances

319

metabolites

320

metabolites metabo 1ites in urine

322,323

I

(c)Ethanol-l.5N NHqOH-water ultraviolet (17:1:2) (d)Phenol-isopropanol-water (16:1:5) 0.5 ammonium chloride u 1traviole t (a)Butanol saturated with water -(b)Propanol-water( 8 0 : 2 0 )

1

317

321

(a) 1sopropanol-25% NH20H(85: 15) (b) Isopropanol-water(85:15) (c) Isopropanol-formic acidwater (80:1O:lO) Pyridine-Water(65:35) Isopropanol-NH40H-water (7:1:2) Butanol-acetic acid-water ( 5 :1:4) ( a ) E thyl me thyl ketone-acetoneformic acid water(40:2:1:6) (b)E thy 1 me thy1 ketone-diethy lam inewater (921:2 :7 7 ) (c)Methyl isobutylketone-formic acid-water(ketone sat.with 4% formic acid) (d)Chloroform-methanol-formic acidwater(CHC13 sat.with 1 part H20 and 1 part 4% formic acid) (e)Benzene-ethylmethyl ketoneformic acid-water(9 parts benzene plus 1 part ketone sat.with 2% formic acid) (f)Benzene-formic acid-water (benzene sat. with 2% formic acid)

1 1

2 Ln

CI4 and spray reagents

-FeC13 and

K 3 F e (CN)6

1

Me tab01ites

324

Metabolites Metabolites

325 326

327

I

2,4,6 trinitrobenzene-sulfonic acid chlorani1ic acid

1

copper sulfate in ethanol then 0.1% benzidine in 50% aqueous ethanol

la)ISO-propanol-water (17:3) (b)Butanol-acetic acid-water(4:1:5) (c)1.4M potassium phosphate buffer pH 7.0 Butanol-acetone-water(45:5:50)

Butanol-phosphoric acid-water(3:1:3) (a)Butanol S a t . with water in atmosphere of NH3 (b)95% ethanol-M ammonium acetate(7:3) adjusted to pH 5 6.32

N m

--

3 28

329 330 33 1

Thin-layer Chromatrogaphy In recent years several authors have developed thin-layer chromatographic system for isoniazid. These are presented in tabular form. System Chloroform-methanol(8:2) Chloroform-acetone-diethylamine (5:4:1) Cyclohexane-chloroform-diethylamine (4:5:1) Butanol-phosphoric acid-water(3:1:3) Acetone-methanol-NJQOH (50: 50: 1) (a)Pkthanol (b)Chloroform (c)Ethanol

\

Detection Folin-Ciocalteu or Phosphomo 1ybdate

-dimethylaminobenzaldehyde 5:l mixture 10% cuso4 and 10% NH~OH

Use separation from other drugs

Ref 332

derivatives 330 identification 333 identity test

334

ISopropanol-acetone(6:4 )

--

chloroform-methanol (6:4)

--

Chloroform-methanol(125:60) (a!Ethyl acetate-cyclohexanedioxane-methanol-waterNH40H(50:50:10:10:1.5:0.5)

UV iodine

I

separation of hydrazine separation of isonicotinic acid hydrazone with lactose

(b)same solvent but (50:50: 10: 1O:O. 5: 1.5) Ninhydrin or separation from (c)Ethyl acetate-cyclohexane0.5% H2S04 drugs of abuse NHqOH-methanol-water (70:15:2:8:0.5) (d)E thyl acetate-cyclohexaneNHqOH-methanol(56 :40:0.4:0.8) (e) same but (70:15 :5 :10) (f)E thyl acetate-cyclohexaneNH40H ( 5 0:40:0.1) Methanol-NH4OH-H20(100:1:4) KMnO4 bromothymol other drugs blue

335 335 59

336

\

337

rn 3

338 254 nm U.V. iron chloride(a)Chloroform-methanolhexacyanoferrate,molybdo13N ammonia (90:10: 1) phosphoric acid. Folin(b) Benzene-;oethano1Ciocalteu,potassium diethylamine (90:10:1) permanganate,ammoniacal (c)Chloroform-hexanol13N ammonia(90:10:0.2) silver nitrate,amminepenta(d)Chloroform-ethyl acetate cyanoferrate,iodoplatinate, iodine,Dragendorff, cinna13N ammonia ( 50 :50 :1) maldehyde triphenyltetra(e) chloroform-acetonezolium,dithiocarbamate acetic acid (90:10: 1) or ammonium molybdate (f)Benzene-acetonediethylamine (50:50 :1) (g) Chloroform-acetoneacetic acid(50:50:1) Nishimoto and T ~ y o s h i m a ~ ~found ' that isoniazid showed tailing on thin-layer chromatography due to trace metals in the silica gel. When the adsorbent was treated with EDTA the tailing was eliminated. Wijnne and c o - w o r k e r ~found ~ ~ ~ that isoniazid could be quantitated after thin-layer chromatography by coulometric titration. Schmidt341 showed that isoniazid could be revealed on a thin-layer plate by exposure to iodine vapor. Kawale and c o - w o r k e r ~sprayed ~~~ thin-layer plates with 1% mercurous nitrate to reveal isoniazid as black spots. 6.33 Ion exchanqe Chromatoqraphy Tsuji and Sekiguchij4j have shown that isoniazid is quantitatively adsorbed on Dowex 50 cation exchange resin in various metal forms. The strength of adsorption decreases in the following order:Cu++k Ni'+2 Hg++> H+ > Co++ > cd++k En++>Fe++ > Pb++> m++> ~ l + + + .

1

ISONIAZID

219

No a d s o r p t i o n o c c u r s on r e s i n i n t h e Ba++, Mg++, Ca++ o r Na+ forms. H e l l e r and c o - ~ o r k e r ss ~ e p~a ~ rated a c e t y l i s o n i a z i d from i s o n i a z i d on a column o f Dowex 1-X8 i n t h e p y r u v a t e form. 9 developed a Kakemi e t -a~~~~ c h r o m a t o g r a p h i c method f o r t h e s e p a r a t i o n o f i s o n i a z i d from some d e g r a d a t i o n p r o d u c t s . I s o n i a z i d i s a d s o r b e d on a weak c a t i o n e x c h a n g e r s u c h a s A m b e r l i t e CG-50 i n t h e hydrogen form. I s o n i c o t i n i c a c i d i s n o t a d s o r b e d and i s d e t e r m i n e d c o l o r i m e t r i c a l l y u s i n g cyanogen bromide. To determine i s o n i c o t i n a m i d e t h e sample s o l u t i o n i s o x i d i z e d w i t h a l k a l i n e f e r r i c y a n i d e and t h e n p a s s e s t h r o u g h a column o f s t r o n g a n i o n e x c h a n g e r s u c h a s Dowex 1-X8 i n t h e c h l o r i d e form. The amide i s unchanged Another degradaand i s n o t a d s o r b e d on t h e r e s i n . t i o n product,1,2-diisonicotinoyl h y d r a z i d e i s d e t e r m i n e d b y a d j u s t i n g t h e s a m p l e t o pH 8 . 9 w i t h b o r a t e b u f f e r and d e t e r m i n i n g t h e a b s o r b a n c e a t 329 nm. P e t e r s a n d c o - ~ o r k e r s 347 ~ ~ ~w ,e r e a b l e t o s e p a r a t e and q u a n t i t a t e a l a r g e number o f m e t a b o l i t e s of i s o n i a z i d u s i n g Dowex AG-50-X4 r e s i n i n t h e hydrogen and ammonium forms. S e l e c t i v e c o l o r r e a c t i o n s were u s e d t o d i f f e r e n t i a t e t h e g r o u p s of m e t a b o l i t e s . Fan and Wald348 s e p a r a t e d p - a m i n o s a l i c y l i c a c i d from i s o n i a z i d u s i n g a Dowex 2 - X 8 column. I n o u e and c o - w ~ r k e r su~s e~d ~ a s y s t e m s i m i l a r t o t h a t o f Kakemi e t a 1 3 4 3 t o s e p a r a t e i s o n i a z i d from i t s d e g r a d a t i o n p r o d u c t s . Lewandowski and S y b i r ~ k a ~ ~ O s e p a r a t e d i s o n i a z i d from i s o n i c o t i n i c a c i d b y p a p e r chromatography u s i n g b u t a n o l s a t u r a t e d w i t h water. The p a p e r was c o n n e c t e d w i t h an i o n exchange p a p e r i n t h e a c i d form. The s p o t s were e l u t e d w i t h d i o x a n e . The s h a r p z o n e s on the i o n exchange p a p e r were v i s u a l i z e d w i t h i c r y l c h l o r i d e Darawy a n d Mobarak3" chromatog r a p h e d s e v e r a l d r u g s on CM-82 c a r b o x y m e t h y l c e l l u l o s e c a t i o n exchange p a p e r u s i n g a w a t e r -

-

220

GLENN A. BREWER

acetone-foramide(l0:l:l) solvent system. 6.34 Other Chromatoqraphic Methods Barreto and Sabin0352 used a anhydrous sodium sulfate column eluted with chloroform-diethylamine(9:1) to concentrate metabolites of isoniazid from serum or urine. Smolarek and Dlugosch353 separated isoniazid and p-aminosalicylic acid by paper electrophoresis in barbital buffer, pH 8.5. B a r r e t ~used ~ ~ ~ two dimensional electrophoresis to separate the metabolites of isoniazid. also used paper electrophoresis to separate several acyl hydrazides. A pH 2.0 acetate buffer was used. Isoniazid was separated from several antituberculosis drugs by gas chromatography356,357, A silanized chromosorb G coated with 6% QF1 was used. Gas chromatography was used to separate the products of oxidation of hydrazides with Fehling’s solution358. 6.4 Determination of Isoniazid and its Metabolites in Body Fluids and Tissues The methods described in this section were specifically developed for the determination of isoniazid in body fluids and tissues. Many of the methods are similar to other general analytical methods described in Section 6.2 perhaps differing only in the extraction procedure. 6.41 General Reviews Terze and D a d i ~ t o u ~studied ~’ a number of color reactions to determine their application to blood level assays. Ginoulhiac360 also made a literature review of blood level methods, A critical review of methods for isoniazid determination has been written364 6.42 Colorimetric Methods Colorimetric methods are most popular for the determination of isoniazid in biological samples. The methods are listed in tabular form.

.

Reaqent Dimethylaminobenzaldehyde

Pretreatment of sample acid hydrolysis

Dimethylaminobenzaldehyde Dimethylaminobenzaldehyde

none extraction into isoamyl alcoholether from alkaline solution deproteinization with ~ ~ 1 0 ~ deproteinization with trichloroacetic acid none

Dimethylaminobenzaldehyde Dimethylaminobenzaldehyde N

5

Vanillin Vanillin Vani 11in Van i11in Cinnama ldehyde Cinnamaldehyde

Type of specimen Ref. serum & urine 361,370, 375 urine 362,363 plasma and 365,366, urine 367,3 68, 369. serum

37 1

serum and tissues

372

serum

373,376, 377 374,432, 433,434 375

deproteinization serum with trichloroacetic acid extraction with serum organic solvent extraction with milk propanol deproteinization serum with trichloroacetic acid extraction with serum butanol-chloroform

3 78 379,380, 429,430 38 1

o-Nit robenza ldehyde Sa 1icyla 1dehyde- FeC1 S a licy la ldehyde

Salicylaldehyde Glutaconic aldehyde @-diketone N

E

Catecho1 Catecho1 H2 0 2 CNBr

-

CNBr A Ikaline

hydrolysisCNBr NHqV03-H2S04 KCN, Chloramine Tbarbituric acid

deproteinization with trichloroacetic acid extraction into isoamyl alcohol-ether from alkaline solution none extraction with acetone deproteinization with trichloroacetic acid none deproteinization with trichloroacetic acid automated method deproteinized serum deproteinized trichloroacetic acid deproteinized trichloroacetic acid acid hydrolysis

serum

382

serum

383

bio logica1 fluids cadavers

3 84

plasma

386

biological ma teria Is citrated blood serum serum & urine biological fluids urine

387

urine

385

388 389 390 391,404, 411,412 392

393,394,395, 396,397,398, 399,370,435 plasma,urine 400,401 tissues,serum

1-amino-2-naphthol-4sulfonic acid Naphthoquinone-4sulfonic acid Naphthoquinone-4sulfonic acid 2,4,6-trinitrobenzenesulfonic acid Dinitrochlorobenzene Dinitrochlorobenzene

rJ .

K3Fe (CN)6 Sodium pentacyanoaminoferroate K3Fe (CN)6 Ni tropentacyanoferroate Na ph thoquinone Na ph thoq inone H202,CNBr, aniline

Picryl chloride KMn04,BrCN,NH3

urine biol. fluids urine deproteinization Zn (OH) 2 extraction methyl isobutyl ketone deproteinized serum deproteinized tissue deproteinized with sodium tungstate deproteinized with phosphoric acid

--

trich loracetic acid tungstic acid extraction BuOH,Et20 Py tein-free fiPtrate

402

urine

403,404,405, 406,407 408

whole blood

409,410

urine serum

411 412,413

serum tissue, urine spina1 fluid serum

414 415,416,417

spinal fluid urine blood blood urine plasma urine spinal fluid plasma

418

419 420 421 422 423,424 425

3 P

4-pyridylpyridinium trichloracetic acid plasma 426 dichloride,NaOH,HCl filtrate KBrO3+ methyl o r a n g e acid tungstate blood 427 Zn powder + h e a t -urine 428 6 . 4 3 T u r b i d i m e t r i c Method I s o n i a z i d r e d u c e s K2Hg14 t o form HgI w h i c h i s i n s o l u b l e . The r e s u l t i n g t u r b i d i m e t r y c a n be measuEed t o d e t e r m i n e t h e amount o f i s o n i a z i d Wagner a n d co-worker-431 h a v e a p p l i e d t h i s method t o b l o o d f o l l o w i n g present. d e p r o t e i n i z a t i o n w i t h b a r i u m h y d r o x i d e and z i n c s u l f a t e . 6 . 4 4 F l u o r i m e t r i c Methods A number of f l u o r i q e t r i c m e t h o d s € o r i s o n i a z i d h a v e b e e n reported. H e d r i c k a n d c o - ~ o r k e r sa~b s~o ~ r b e d a p r o t e i n f r e e f i l t r a t e of s e r u m on p H 6 . 5 A m b e r l i t e XE-64 i o n e x c h a n g e r e s i n . T h e i s o n i a z i d was e l u t e d w i t h d i l u t e a c i d and t h e n r e a c t e d w i t h hydrogen p e r o x i d e i n pH 8.7 b u f f e r . The o x i d a t i o n p r o d u c t f l u o r e s c e s a t 415 nm when a c t i v a t e d by u l t r a v i o l e t l i g h t a t A s l i t t l e a s 0.05 y/m1 o f s e r u m c a n be d e t e r m i n e d . 320 nm. S c o t t and Wright437 r e a c t e d s a l i c y l a l d e h y d e w i t h i s o n i a z i d and reduced t h e r e s u l t i n g hydrazone. The r e s u l t i n g compound w a s h i g h l y f l u o r e s c e n t . R e i s s , Morse a n d P u t s c h 4 3 8 a s s a y e d i s o n i a z i d f l u o r i m e t r i c a l l y a f t e r a b s o r p t i o n a n d e l u t i o n from i o n e x c h a n g e r e s i n and t r e a t m e n t w i t h a l k a l i n e c y a n o g e n bromide. Wilson, Lever and u t i l i z e d t h e f l u o r e s c e n c e of t h e z i n c c h e l a t e of t h e hydrazone of i s o n i a z i d w i t h pentane-2,4-dione i n an a s s a y i n serum. E l l a r d , Gammon a n d W a l l a c e 4 4 0 h a v e d e v e l o p e d s p e c i f i c f l u o r i m e t r i c a s s a y s f o r i s o n i a z i d , a c e t y l i s o n i a z i d , mono-and d i a c e t y l h y d r a z i n e , i s o n i c o t i n i c a c i d a n d i s o n i c o t i n y l g l y c i n e i n serum a n d u r i n e . Boxenbaum a n d Riegelman441 h a v e a l s o d e v e l o p e d a s s a y s f o r i s o n i a z i d and i t s m e t a b o l i t e s i n whole blood. M i c e l i , O l s o n a n d Weber442 h a v e e s t a b l i s h e d a micro method f o r

ISONlAZlD

225

t h e f l u o r i m e t r i c d e t e r m i n a t i o n of i s o n i a z i d i n serum. A s l i t t l e a s 2 5 p1 o f s e r u m can be u s e d i n t h e assay. P e t e r s , Morse a n d Schmidt 443 and 0' B a r r , K e i t h and B l a i r 4 4 4 h a v e compared f 1 U O r i m e t r i c and m i c r o b i o l o g i c a l a s s a y s f o r i s o n i a z i d i n serum. 6.45 E l e c t r o c h e m i c a l Methods Lauermann a n d O t t o 4 4 5 h y d r o l y z e d i s o n i c o t i n i c a c i d h y d r a z i d e and i t s m e t a b o l i t e s t o i s o n i c o t i n i c a c i d w i t h a l k a l i . The h y d r o l y s i s product w a s determined p o l a r o g r a p h i c a l l y . The a u t h o r s found t h a t t h e r e s u l t s o b t a i n e d b y t h i s method i n t h e a n a l y s i s o f c a d a v e r i c f r a c t i o n s was comparable t o t h o s e o b t a i n e d when t h e method o f N i e l s c h a n d G i e f e r 4 0 1 was u s e d . The p o l a r o g r a p h i c method was l e s s t i m e consuming. Kane 446 d e t e r m i n e d i s o n i a z i d i n biological f l u i d s without p r i o r separation. 6.46 G a s o m e t r i c Methods The h y d r a z i n e g r o u p i n i s o n i a z i d c a n b e r e a d i l y decomposed i n t o n i t r o g e n g a s . Several a u t h o r s have u t i l i z e d t h i s r e l a t i v e l y s e l e c t i v e f i n i s h f o r b l o o d and u r i n e l e v e l a s s a s . S t r i c k l a n d a n d H e n t e l 1;47 r e a c t e d i s o n i a z i d w i t h sodium i o d a t e i n a l k a l i n e s o l u t i o n . The a s s a y i s n o t e f f e c t e d b y t h e p r e s e n c e o f pa m i n o s a l i c y l i c a c i d which i s o f t e n g i v e n i n conjunction with isoniazid. H a r t i n g and G e r z a n i t s 448used a l k a l i n e f e r r i c y a n i d e t o l i b e r a t e t h e nitrogen gas. I t o and c o - w ~ r k e r 7s450 ~ ~ were ~ able t o s e l e c t i v e l y u s e c o p p e r , i r o n a n d chromium azometry t o determine i s o n i a z i d and i t s v a r i o u s metabolites i n urine. 6.47 M i s c e l l a n e o u s Chemical A s s a y s V e r r o t t i and B a r d e l l i 4 5 1 d e t e r m i n e d i s o n i z i d i n cerebrospinal f l u i d by iodometric ti t r a t i o n . Schwenk a n d c o - ~ o r k e r s ~employed ~* a radioimmunoassay f o r t h e d e t e r m i n a t i o n o f isoniazid i n biological fluids.

Microbioloqical Assays Although isoniazid is readily measured in biological fluids and tissues by chemical assays, as with many antibacterial substances a number of microbiological assays for this substance have been proposed. Microorganism Type of assay S ens itiwi ty Ref. 2.5-30 y/ml 453 Mycobacterium phlei agar diffusion Koch bacilli turbidimetric -454 tubercle bacteria cord formation -455 bacilli vertical diffusion -456 Mycobacterium vertical diffusion -457 tuberculosis HV37 Mycobacterium agar diffusion -458 tubercu10s is 0.49 y/m1 459 vertical diffusion Mycobacterium tuberculosis H37Rv and H 3 7 R a assay of isoniazid 378 BGG in milk-agar diffusion 6.48

N

N m

>

Mycobacterium tuberculosis

--

vertical diffusion vertical diffusion

460 46 1

vert ica 1 di f€usion tube dilution vertical diffusion for urine

462 463,464

ISON IAZlD

227

Bartmann and F r e i s e 4 6 5 s t u d i e d t h e t i s s u e b i n d i n g of i s o n i a z i d w i t h t h e m i c r o b i o l o g i c a l assay. They found 40% b i n d i n g w i t h human t i s s u e w h i l e mice and g u i n e a p i g t i s s u e g a v e 80% binding. Incubating the t i s s u e a t an e l e v a t e d temperature d i d not r a i s e t h e recovery. N i ~ h i P~o o~l e~ and , Meyer467, T a n s i n i and c o - w o r k e r ~ ~P~e t~e ,r s and c o - w o r k e r s 433 and O ’ B a r r & a4!*a l l compared v a r i o u s c h e m i c a l a s s a y s and m i c r o b i o l o g i c a l a s s a y s . A l l w o r k e r s c o n c l u d e t h a t t h e two methods g a v e c o m p a r a b l e results 6 . 4 9 Chromatographic A s s a y s The m e t a b o l i s m o f i s o n i a z i d i s complex and many w o r k e r s h a v e s e l e c t e d chromatog r a p h i c a s s a y s t o measure t h e d r u g i n t i s s u e and biological fluids. T h e s e methods p r o v i d e t h e s p e c i f i c i t y t h a t a r e n o t g i v e n b y many c h e m i c a l methods. A l a r g e number of c h r o m a t o g r a p h i c s y s t e m s a r e g i v e n i n s e c t i o n 6.3. Many of t h e s e methods c o u l d p r o b a b l y be u s e d t o measure i s o n i a z i d i n t i s s u e s and b i o l o g i c a l f l u i d s . The methods g i v e n i n t h i s s e c t i o n h a v e been d e v e l o p e d j u s t f o r t h i s purpose. Makino and c o - ~ o r k e r sf o ~ l~ l o~w e d t h e m e t a b o l i s m of i s o n i a z i d i n l i v e r and i n u r i n e by p a p e r c h r o m a t o g r a p h y ( w a t e r s a t u r a t e d b u t a n o l , 1% ammonia-isopropanol(3:20), b u t a n o l s a t u r a t e d w i t h 0.02M p h o s p h a t e b u f f e r p H 7 . 4 , 1%ammonia s a t u r a t e d b u t a n o l and b u t a n o l - a c e t i c a c i d - w a t e r

.

(4:1:5)).

L e ~ s c h n e r ~u ~ s egd s e c - b u t a n o l s a t u r a t e d w i t h w a t e r and i s o a m y l a l c o h o l a s developing solvents. I ~ a i n s k ys e~p a~r a~t e d t h e h y d r a z o n e s o f i s o n i a z i d and p y r u v i c and a - k e t o g l u t a r i c a c i d from i s o n i a z i d w i t h p a p e r chromatography. S e z a k i 470 s e p a r a t e d i s o n i a z i d from p y r a z i n a m i d e i n u r i n e b y means of A m b e r l i t e IRA-400. B e l l e s and L i t t l e m a n 4 7 1 u s e d Dowex 50-X8 t o s e p a r a t e i s o n i a z i d from a c e t y l i s o n i a z i d . Abiko

228

GLENN A. BREWER

and c o - ~ o r k e r suse ~~~ Dowex 1-XlO to separate these as well as the hydrazone of glucuronic acid. Peters, Miller and Brown346 utilized ion exclusion chromatography to separate metabolites of isoniazid into ionized, slightly ionized and unionized groups of compounds. The individual metabolites were measured with specific colorimetric assays. Okudaira and c o - ~ o r k e r s ~ used ’~ Dowex 1 and Dowex 50 columns in tandem to separate the various metabolites of isoniazid. Paper chromatographic systems have been used to isolate the various metabolites of isoniazid474,475,352. Barreto and S a b i n have ~ ~ ~described ~ a two dimension separation of isoniazid metabolites using paper chromatography and paper electrophoresis The same authors352 have also used a sodium sulfate column developed with chloroform-diethylamine(9O:lO) to separate the metabolites of isoniazid. Fartushnyi and S ~ k h i n *have ~ ~ used TLC to determine isoniazid and other drugs in cadavers. Cattaneo, Fantoli and Ferrari478 claim that their chromatographic studies indicate that the tumorogenic effect of isoniazid in mouse lung is due to the large amount of isonicotinic acid produced in that organ. Hughes479 separated acetylisoniazid from isoniazid by counter-current distribution (butanol-ethylene dichloride- 9:1 - 2M phosphate buffer pH 5.1). Ozawa and Kiyomoto480 isolated three conjugated metabolites of isoniazid by paper used paper chromatography. Cuthbertson et chromatography to determine isonicotinoylglycine. They used the following systems: Water saturated butanol Methylethy1ketone:acetic acid:water(49:1:50) Propano1:water (4:1) Zamboni and D e f r a n ~ e s c h iused ~~~ a isopropanol:water(85:15) system to separate the hydrazones of pyruvic and a -ketoglutaric acid from

i son i a z id. 7. Druq Metabolism The drug metabolism of isoniazid is unusually complicated in that it is a very reactive molecule and can undergo non-enzymatic reactions in the body. A general metabolic pattern is indicated in diagram. Non-enzymatic

Enzymatic 0

II

isonicotinoylhydrazones of glucose, a-ketoglutaric acid, aldehyde pyruvic acid etc. N ID

acetylisoniazid

or ketone

0

isonicotinamide N , N * diisonicotinoylhydrazide

230

GLENN A. BREWER

The major metabolite of isoniazid is N-acetylisoniazid. The rate of acetylation is has genetically ~ o n t r o l l e d 485. ~ ~ ~ ,It ~ ~ ~ been established that the slow acetylation is a autosoma1 recessive trait. The acylation occurs by N-acetyl transferase. Six hours after the oral administration of 4 mg/Kg of isoniazid fast acetylators have plasma concentrations of 0.2 pg/ml or less while slow acet lators have plasma levels higher that 0.4 pg/ml 48Yj In a metabolic scheme,such as the one indicated earlier,relative amounts of the various metabolites found in the urine will differ for each individual and will depend on genetic factors, previous drug history (enzyme induction) and general nutrition (availability of ketoacids). Reviews on the drug metabolism of isoniazid have been re ared b a number of authors487,488, 489,490,49!?, 462,493,i94,495,496,497

.

Toth and Shimizu have reported that the continuous administration of N-acetylisoniazid in rats has markedly increased the incidence of lung tumors in this species. Since the N-acetyl derivative is a major metabolite in man this poses some questions on the long term administration of the compound497. 8. Biopharmaceutics Kakemi and co-workers498 determined the rate of absorption of derivatives of isoniazid in the stomach and intestine. The authors report a rough correlation between degree of absorption and lipidwater partition coefficient.

ISONIAZID

231

9.

References

1.

G r i f f i t h s , M. C. ; Dickerman, M. J. a n d M i l l e r , L.C. ; USAN and t h e USP D i c t i o n a r y of Druq Names page 152 ( 1 9 7 5 ) . Anon; Chemical Abstracts S e r v i c e , R e g i s t r y Handbook-Number S e c t i o n ( 1 9 7 4 ) . Anon; European Pharmacopoeia Volume 1 , p a g e 310 (1969). P r e v o r x e k , D. ; B u l l . SOC. chim. F r a n c e , 795-801 (1958) ;C.A. 53 2 1 1 6 0 b ( 1 9 5 9 ) . Nagano, K. ; K i n o s h i t a , H. a n d Hirakawa,A. ; Chem. Pharm.Bul1. l2,1198-1206 ( 1 9 6 4 ) ;C.A. 62 2759a ( 1 9 6 5 ) . T o e p l i t z , B . ; E. R. S q u i b b and Sons: P e r s o n a l Communication. Lapp, C. ; B u l l , SOC. pharm. Nancy 3 4 , 14-17 ( 1 9 5 7 ) ; C.A. 53 21145d ( 1 9 5 9 ) . G r e b e n n i k , L. I. ;Farmakol. i T o k s i k o l . 24, 233-7 ( 1 9 6 1 ) ; C . A . 55 26263a ( 1 9 6 1 ) . Pinyazhko, R. M. ;F a r m a t s e v t . Zh. 2 0 , 18-22 ( 1 9 6 5 ) ; C.A. 64 7969e ( 1 9 6 6 ) . Kracmar, J . ; Kracmarova, J. and Zyka, J . ; Pharmazie 2 3 , 567-73 ( 1 9 6 8 ) ; C . A . a 3 1 7 1 9 r (1969). Zommer, S. and S z u s z k i e w i c z , J. ;Chem.Anal. 14, 1075-83 (1969) : C.A. 2 89616n ( 1 9 7 0 ) . C e l e c h o v s k y , J . ; Greksakova, 0. and P o l a s e k , E . ; A c t a Fac. Pharm. l 7 , 5 1 - 5 ( 1 9 6 9 ) ; C . A . B 112880a (1970). Zommer, S. ; A c t a Pol. Pharm. 29,533-5 (1972) ; C.A. 78 158502k ( 1 9 7 3 ) . Dunham, J. ; E. R. S q u i b b a n d S o n s ; P e r s o n a l Communication. Caen, G. ; J . Chim.phys. 51,60-3(1954);C.A. 48 10438e ( 1 9 5 4 ) . T i t o v , E. V. ;Kapkan, L. M. ;Rybachenko,V. I. and Korzhenevskaya, N . G . ; R e a k t s S p o s o b n o s t O r q . S o e d i n 2, 673-81 (1968) ; C.A. 70 6 7 4 4 8 r ( 1 9 6 9 ) . T i t o v , E.V. and Kapkan, L.M.; Dokl.akad.Nauk -SSSR 184,1342-5 ( 1 9 6 9 ) ;C.A. 70 114411k ( 1 9 6 9 ) .

2. 3. 4. 5.

6. 7. 8.

9. 10.

11.

12.

13. 14. 15.

16.

17.

232

GLENN A. BREWER

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233

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36. 37.

38. 39.

40. 41. 42. 43.

44. 45.

46. 47. 48. 49. 50. 51. 52. 53. 54.

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-

-

234

55. 56. 57. 58. 59.

60. 61. 62. 63.

64.

65. 66. 67. 68. 69.

70.

71.

GLENN A. BREWER

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ISONIAZID

72. 73. 74. 75. 76.

77. 78.

79.

80.

81.

82. 83. 84. 85. 86. 87.

88. 89. 90.

235

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a.

:=.

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144. 145.

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239

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16,696-9

-

-

-

.

171. 172. 173.

174. 175. 176. 177.

178. 179. 180.

-

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24 1

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GLENN A. BREWER

Mitchell,J. R. and Jollow,D.J. ;Druq Interact. 65-79(1974) :C.A.U 92730g(1975). 495. Iwamoto,T. :J. Pharm. SOC.Japan 74 36-9 (1954)i C.A. & 5362b(1954). 496. Nakazono,I. and Hirata,H. ;Kekkaku 3 4 101-6(1959) ;C.A.S 25288e(1960). 497. Toth,B. and Shimizu,H. ;Eur.J. Cancer 9, 285-9(1973) :C.A.B 101439m(1973). 498. Kakemi,K. ;Arita,T. ;Sezaki,H. and Takasugi, N. :Chem. Pharm.Bul1. JJ 551-7 (1965);C.A.Q 7511c(1965). 499. Juchau,M.R. and Horita,A.;Drug Metabolism Reviews, DiCarlo F.J. Editor, Vol. 1 pages 71-86, Marcel Dekker Inc. New York, 1973. Meyer,H. and Mally,J. ;Monatsh.Chem 2 3 . 9 500. 393(1912) ;C.A.6_ 2073(1912). 501. Chorine,V. : Compt. rend,acad. sci. 220,150 (1945). 502. Huant,E.:Gazette des Hopitaux Aug.15(1945). 503. Long,E.S.:The Chemistry and Chemotheraw of Tuberculosis.3rd ed.,The Williams & Wilkins C Co. ,Baltimore,1958. 504. Kirschbaum,A. ;Pharm.Acta Helv. 27 229-33 (1952):C.A. 47 2429b (1953). 494.

KANAMYCIN SULFATE

Paul J. Clues, Maurice Dubost and Hubert Vanderhaeghe

260

PAUL J. CLAESetel.

TABLE OF CONTENTS 1 . Description 1.1.

Name, Formula, Molecular Weight

1.2. Appearance, Color, Odour 1.3. Definition of International Unit 2 . Physical Properties 2 . 1 . Spectra 2.11.

Infrared Spectra

2.12. Ultraviolet Spectra 2.13. Nuclear Magnetic Resonance Spectra 2.14. Mass Spectra 2.2. Optical Rotation 2.3. Electrometric Titration Curve-pK Values 2 . 4 . Crystal Properties 2 . 5 . Melting Range 2 . 6 . Thermal Analysis 2 . 7 . Solubility

3 . Synthesis 3.1.

Fermentation-Biosynthesis

3.2.

Chemical Synthesis

4 . Stability-Degradation

5 . Inactivation by Enzymes 6 . Mode of Action 7 . Pharmacokinetics

8. Methods of Analysis

Identification 8 . 2 . Determination of Sulfate

8.1.

8 . 3 . Loss on Drying 8.4. Microbiological Assay 8 . 5 . Assay of Kanamycin B

KANAMYCIN SULFATE

8.6. Chromatographic Analysis

8.61. Paper 8.62. Thin Layer 8.63. Ion Exchange 8.64. Gas Liquid 8.7. Electrophoretic Analysis

9. Determination in Body Fluids and Tissues 10. References Cited

26 1

PAUL J. CLAES et a / .

262

1 . Description 1.1.

Name, Formula, Molecular Weight Kanamycin or kanamycin A (I) is the major component

of the antibiotic complex produced by certain strains of Streptomyces kanamyceticus'

. Its structure was established as

0-(6-amino-6-deoxy-CK-D-glucopyranosyl)-( deoxy-CY -D-glucopyranosyl-( 1

-

1 --4)-0-[3-amino-3-

6)] -1,3-diamino-l,2,3-trideo-

xy-scyllo-inositol. Since 1972 the compound has been listed

-

in Chemical Abstracts under the heading D-streptamine,

O-3-amino-3-deoxy-CY-D-glucopyranosyl- ( 1 6)-0-[ 6-amino-6deoxy-CY-D-glucopyranosyl-( 1 411 -2-deoxy-. The numbering is

-

given in the formula below. The carbon atoms of the 2-deoxy-

6'

1' I

0

OH

,2

\I 0

Kariadlycin A free base : C,8H36N4011

M.W. 484.50

Kanatnycin A monosulfate monohydrate : C18H36N401 1 0H2S04'H20

M.W. 600.59

KANAMYCIN SULFATE

263

..., those of the

streptamine ring are numbered as 1,2,3,

amino sugar moieties linked at C-4 and C-6 of 2-deoxystreptamine respectively as 1 ' ,2',3',

... and

1",2",3",.

..

Kanamycin A is supplied in two forms, a crystalline monosulfate monohydrate and a salt with a higher sulfate content. The latter is more readily soluble in water and is 2 designated in the Brit. Ph. Add. 1975 as kanamycin acid sulfate. The sulfate (SO ) content calculated for the monosul4 fate monohydrate is 15.99 2 . In most commercial samples the sulfate content varies from 16 to 16.4 %3'4. The monosulfate 5 monohydrate is reported in the U.S. Ph. XIX and in the Brit. 6 Ph. 1973 under the heading kanamycin sulfate. To avoid confusion the designation kanamycin monosulfate should be preferred. The limits of the pH ( 1 2 aqueous solution) given in 8 the Code of Federal Regulations7 and in the Eur. Ph. are from 6.5 to 8.5. Kanamycin acid sulfate, the name used in Brit. Ph. Add. 1975, which is sometimes referred to as kanamycin bisulfate, is obtained by adding sulfuric acid to a solution of the monosulfate and drying by a suitable procedure. Its sulfate content (dry basis) may vary from 24 to 26 %. Percentages sulfate calculated for C H N 0 1.6 H2S04 and C18H36N4011. 18 36 4 1 1 ' 1.8 H SO are respectively 23.95 and 26.14 X . It is obvious 2 4 from these figures, that the name kanamycin bisulfate is not a correct designation. The limits of pH given by the Eur. Ph.8 are from 5.5 to 7.5. Kanamycin B (11) and kanamycin C (111) are two minor components of the antibiotic complex. They differ from kanamycin A in the nature of the amino sugar linked to the 4-position of the 2-deoxystreptamine moiety (2,6-diamino2,6-dideoxy-D-glucose for I1 and 2-amino-2-deoxy-D-glucose

PAUL J. CLAES et a / .

264

for 111). Kanamycin B, also referred to as bekanamycin, is R available as its sulfate salt under the name Kanendomycin (Meiji)

.

6

"

CH20H I

I

' 1" HO

I1

kanamycin B : R 1 = R

I11 kanamycin C : R IV

amikacin : R

1

1

-

2

NH2, R3 = H, R4 = OH

NH2, R2 = OH, R3 = H, R4 = OH

= H, R

2

R4 = OH

V

=

tobramycin : R 1 = R

2

-

-

NH,, R3 = L(-)-CO-CH-(CH,),-NH,, ..1 OH

NH2, R3

-

H , R4 = H

-

KANAMYCIN SULFATE

265

The two antibiotics amikacin (or BB-8) (IV) and tobramycin (V) are structurally related to the kanamycins. The former is obtained by selective !-acylation

of kanamycin A at

the I-amino group with L(-)y-amino-CY-hydroxybutyric

acid

9

.

The latter is a 3’-deoxykanamycin B produced by Streptomyces 10

tenebrarius 1.2.

.

Appearance, Color, Odour The monosulfate monohydrate is a white or almost

white, odourless or almost odourless crystalline powder. The acid sulfate is amorphous. 1.3. Definition of International Unit

11

The International Reference Preparation is a sample of kanamycin monosulfate ( 1 7 . 2 % SO4) established in 1959. The International Unit was defined in 1962 as the activity contained in 0.001231 mg of the International Reference Preparation, corresponding to a potency of 812 U/mg

.

2 . Physical Properties 2.1.

Spectra

2 . 1 1 . Infrared Spectra

Infrared spectra of kanamycin monosulfate monohydrate and of the free base have been published by Maeda

12

.

These spectra are typical for polyhydroxy polyamino compounds. However, no characteristic bands, which would permit differentiation from related aminoglycosidic antibiotics, are present.

PAUL J. CLAESetal.

266

2.12. Ultraviolet Spectrum Kanamycin free base and its sulfate salts show end 12 absorption only

.

2.13. Nuclear Magnetic Resonance Spectra The PMR spectrum of kanamycin free base, determined on a Varian XL-100 instrument at ambient temperature, is presented in the figure I . The spectrum was obtained by dissolving 60 mg crystalline free base in 0.5 ml D20, containing sodium 3-(trimethylsilyl)propane-l-sulfonate as internal standard. In the spectrum, which is in agreement with that publishedI3, signals appear in three separate regions. The lowest field contains two one-protor; doublets, due to anomeric protons. The highest field shows two one-proton signals, due to the methylene group of the deoxystreptamine moiety. Signals from the remaining protons, attached to carbon atoms Table I. PMR Spectral Assignments of Kanamycin A free base Assignment

I

Chemical Shift*

Coupling Constant = 13 Hz, Jaa= 12 Hz gem J~ = 13 Hz, J = 4 HZ gem ea

2-Ha

1.22 (m)

2-He

1.96

3"-H

2"-H

2.90 (m) 3.48 (m)

2'-H

3.55

(m)

3'-H

3.77

(m)

I "-H

5.03

(d)

J = 3.8 Hz

1 '-H

5.79

(d)

J = 3.3 Hz

(m)

J

J = 3.8 Hz, J = 10 Hz J = 3.3 Hz, J

= 9.5

Hz

d = doublet; m = multiplet; gem = geminal; a = axial; e = equatorial. The 6-values, given in this table, refer to sodium 3-(trimethylsilyl)propane-l-sulfonate as internal standard. They can be converted into b-values, referring to TMS, by adding 0.48 ppm.

3'" 4

h)

m

U

Fig. 1. PUR spectrum (lo0 Mc) of kanamycin A free base, taken in D20 with sodium 3-(trimethylsilyl)propane-l-sulfonate as internal standard.

PAUL J. C U E S et a / .

268

bearing "H2,

-OH or -0-,are found in the central region.

The spectral assignments, given in the figure and summarized in Table I, have been discussed in detail by Naganawa et 13 al. -

.

L

Spectral data, observed for a solution of the monosulfate of kanamycin A in D20 solution and for its tetrahydrochloride, are given in Table 11. It can be seen that protonation of the amino groups causes a downfield shift of some of the protons. This effect is less pronounced for the monosulfate. The PMR spectrum of kanamycin B has been reported by Koch et a1.I5. Assignment of the signals in carbon-I3 NMR spectra of kanamycin AI6 and BI7 have been reported recently.

*

Table 11. Chemical Shift Values observed for Kanamycin A

14

Salts Kanamycin mono sulfate

***

L_

Protons

Kanamycin 4 DC1

2-Ha

1.48 (m)

1.98 (m)

2-He CHO, - Cfi20

2.16 (m)

2.6 (m)

- CE2N CHN, anomeric protons

* ** ***

2.95

- 4.2

(m)

5.09 (d) and 5.52 (d)

2.95

- 4.2

(m)

5.18 (d) and 5.58 (d)

6-Values relative to sodium 3-(trimethylsily1)propaneI-sulfonate as internal standard. Saturated solution in D20 35 mg kanamycin A free base in 0.35 ml IN - DC1.

2.14. Mass Spectra The high- and low resolution mass spectra of volatile -and N-acetylderivatives of kanamycin A (N-acetyl-N,O-methyl0-trimethylsilylkanamycin) have been determined and interpreted by De Jongh et a1.I8. The electron impact spectra of both

KANAMYCIN SULFATE

269

derivatives show a very small molecular ion peak, which may be obscured by background or noise. More intense are the (M+1) peak in the spectrum of the N-acetyl-N,O-methyl -deriva-

tive and the (M-15) peak in that of the IJ-acetyl-0-trimethylsilyl derivative. Other diagnostic peaks, observed in the spectra of both derivatives, result from a cleavage of glycosidic bonds or C-0 bonds connecting a hexose to the deoxystreptamine unit. The m/e values of these peaks reveal the sequential arrangement and the gross structure of the saccharide- or the aminocyclitol units, of which kanamycin is composed. Mass spectra of deuterated analogs and the chemical inonization mass spectrum of IJ-acetyl-E,g-methylkanamycin A 18 are also described in the paper of De Jongh et al.

.

Mass spectra of the underivatized free bases of kanamycin A and B and of other aminoglycoside antibiotics (up to the pseudotrisaccharide level) have been reported by Daniels

-et al. 19y20.The electron impact spectrum of underivatized kanamycin A shows a MH

+

peak at the highest mass ion. Other

diagnostic fragment ions arise from glycosidic cleavage and from a cleavage of one of the sugar units. Some of the diagnostic peaks observed in the spectra of derivatized and underivatized kanamycin A are given in the following scheme :

moiety

geox;yk;r;taminefi m/e 3 0 6 ( a ) , A 530 (b) ,720(c)

1

f-aminog moiety lucose k m / e 162(a), 260(b) ,420(c)

+

(a) Kanamycin A underivatized : m/e 485 ( M + l ) (b) N-Acetyl-N,O-methylkanamycin A : m/e 807 (M+l) , m/e 806 (M)+ + (c) N-Acetyl-0-trimethylsilylkanamycin A : m/e 1156 (M) , m/e 1141 TM-15)' +

270

PAUL J. CLAESetal.

2 . 2 . Optical Rotation

The following specific rotations have been reported; for the free base _-_-----------

[a];'+

1400 (c I , H ~ o ) ,% + 67.830

[a]:+

146' (c 1 , 0.1N- H 2 S 0 4 ) ,

[

150.5'

(c 1 , 0.2E

Maeda

% + 70.737 H 2 S 0 4 ) , % + 72.917

12

--

Cron et al. 21

--

Claes et al.

---------------

for the monosulfate Maeda

mmoles

HCI a d d e d

12

-

Fig. 2. Electrometric titration curve of kanamycin A free base.

22

KANAMYCIN SULFATE

For each of these[(XID were calculated. The

27 1

values the mole@tilar rotations (%)

%,

calculated froin the specific rota-

tiori of the free base measured in the authors' laboratory in 0.2l H2S04, is almost identical to that of the monosulfated The

%

calculated from the value of Cron et a1.*' is somewhat

lower. 2.3. Electrometric Titration Curve-pK Values Apparent pK values of 6.40, 7.55, 8.40, and 9.40 a were derived from the electrometric titration curve of kanamycin A given in figure 2 . The curve was determined23 with an automatic Radiometer titration assembly (TTT 1 and SBR 2) for an aqueous solution (5 ml) of 0.1 nnnol kanamycin A free base

and 0.5 mmol KOH. Titration was carried out with HC1 0.5N. 2.4. Crystal Properties The X-ray powder diffraction pattern obtained24 for a commerc a1 sample of kanamycin monosulfate monohydrate containing 2 to 3 % of the B component is presented in Table I11 Experimental conditions Philips PW 1050/25 vertical goniometer, supplied with flat rotation specimen holder PW 1064/20 Generator : PW 1130/00 60 kV-3kW 2 kW normal focus Cu tube : 40 kV-40 mA

Divergence slit : 1 " Receiving slit : 0.1" No beta filter

Focusing monochromator : PW 1966/40 Proportional counter PHS employed; F.S.D. 4 x 103 cps; time constant 1 s Scanning speed : 0.5" 20 per minute

272

PAUL J. CLAES et a / .

Chart speed : 10 mm/min. Table 111. X-Ray Powder Diffraction Data : 1 -

12.450 7.242 7. I26 6.317 6.215 5.965 5.090 5.039 4.882 4.783 4.658 4.599 4.506 4.142 4.101 4.004 3.842 3.793 3.725 3.640 3.455 3.484 3.466

* **

10

21 12 24 28 24 22 16 100

41 9 7 10

47 58 6 6 2 7 41 16 26 20

d =

d* (A)

**

I/Io

d* (A)

1/17

2.362 2.329 2.304 2.290 2.273 2.232 2. I83 2.141 2.116 2.100 2.077 2.052 2.029 1.993 1.973 1.899 1.848 1.805 1.756 1.734 1.678

8 4 5 7 6 4 5 2

L_

3.345 3.264 3.232 3. I98 3.164 3.110 3.038 2.983 2.943 2.898 2.87 1 2.834 2.805 2.759 2.694 2.644 2.599 2.556 2.522 2.488 2.436 2.414 2.374

10 6 8 11

12 31 4 3 7 2 2 7 2 11

2 2 5 8 6 3 6 5 7

1 1

2 3 2 5 3 4 3 2 2 2 2

n h = interplanar distance 2 sine

I/Io = relative intensity (based on highest intensity of 1.00). The crystal structure of kanamycin monosulfate monohy-

drate and of the isomorphous kanamycin monoselenate monohy25 drate has been determined by X-ray analysis

.

2.5. Melting Range The following melting (decomposition) temperatures have been reported :

273

KANAMYCIN SULFATE

for the free base of kanamycin A ______--______----__------__-12

250'

Maeda

255" (decomp.)

Claes et al.

--

for the monosulfate __----------------268-276" (decomp.)

Maeda

22

12

2.6. Thermal Analysis The differential scanning calorimetry (DSC) curve shows26 two endotherms (respectively at 120' and 170')

for

kanamycin monosulfate. This is in agreement with the results of loss on drying given in section 8.3. However, no transition was noted27 below 250' in the differential thermal analysis (DTA) curve of the monosulfate. This is in apparent contradiction with DSC measurements and with the results of loss on drying. 2.7. Solubility The free base, the monosulfate and the sulfate of kanamycin A are soluble in water and almost insoluble in organic solvents such as alcohol, acetone, ether ethyl acetate and benzene. The free base is slightly soluble in formamide12. The following solubilities have been reported. Kanamyc in

Solvent

Solubility

References

monosulfate

water 5 0 Z aq.MeOH water

12 6,8,23

acid sulfate

350 m g / m l 1 part in 8 parts 5 mg/ml 1 part in 1 part

12

6,8

The solubility of kanamycin sulfate in water at various pH values is given in a paper by Granatek et a1.28. Solubilities in various solvents are also given in this paper.

274

PAUL J. CLAES et a / .

3 . Synthesis

3.1. Fermentation - Biosynthesis Kanamycin is produced commercially by fermentation. The isolation of crystalline kanamycin A monosulfate has been described by Maeda12. In this procedure the antibiotic is extracted from the culture filtrate by adsorption on a cation exchange resin (Amberlite IRC-50) in the sodium form and eluted from the resin with 2N NH OH. The eluate is concentra-

-

4

ted, adjusted to pH 9 with H2S04, decolored over active with NH OH. Addition of 4 methanol affords a precipitate of the crude crystalline monocarbon and adjusted to pH 8.0-8.2

sulfate monohydrate of kanamycin A, which is recrystallized from water-methanol or water-methylcellosolve. The biosynthesis of kanamycins has been studied by Kojima et al. 29,30. A review of the biosynthesis of aminocyclitol antibiotics is given by Rinehart et al.31 -0

-

.

3.2. Chemical Synthesis Total synthesis of the kanamycins A, B and C was achieved in 1968 by

s.

Umezawa and coworkers3 2 - 3 4 . An alter-

native independent synthesis of kanamycin A has been reported 35 in a paper by Nakajima et al.

.

4 . Stability

-

Degradation

The stability of kanamycin A free base and sulfate has

--

been investigated by Granatek et al. 28. Unfortunately the authors did not mention whether the monosulfate or the sulfate with another composition was used in their experiments. The following results, taken from their paper, illustrate that both forms are extreqely stable as powders. After storage for 4 months at 56' on qverage no loss of activity

KANAMYCIN SULFATE

275

was observed for the free base. That of the sulfate, stored in identical conditions was 4.3 %. In a pH range of 2.6 to 7.9, aqueous solutions of kanamycin showed an average loss of only 3.5 % after storage for 4 months at 56". These authors observed that solutions are subject to darkening, due to air oxidation. The color change has no effect on the potency. The crystalline monosulfate can be heated as a powder 4 for 6 hr at 150" without loss of activity . During determination of the structure of kanamycin A acid degradation has been investigatedI2. It was found that the antibiotic is almost unaffected by refluxing with IN - HC1 in methanol. In 6N - aqueous HC1 at l o o " , 97 Z of the biological activity was destroyed after 45 min and kanamycin was almost completely hydrolysed into its three components : 2-deoxystreptamine, 6-amino-6-deoxy-D-glucose and 3-amino3-deoxy-D-glucose. Kanamycin, like the related antibiotics neomycin, paraneomycin and gentamicin, is very stable in alkaline medium. No loss in activity was found when these antibiotics were

refluxed for 48 hr in 1.9N - aqueous NaOH36

.

5. Inactivation by Enzymes Aminoglycoside-modifying enzymes can be found in a wide

variety of resistant bacteria and are known to be coded by plasmids. In most cases the enzymatic modification of the antibiotic results in complete inactivation. The three known modifications induced by these enzymes are : N-acetylation,

0-phosphorylation, and 0-adenylylation.

These mechanisms of inactivation have been reviewed by Benveniste and Davies37

.

A recent article by Haas and D ~ w i n gdescribes ~~ the isolation

and assay of these enzymes. The kanamycin A-modifying enzymes,

Table IV. Kanamycin A Modifying Enzymes

2

Enzyme

Cof actor

Modification induced in kanamycin A

Other Substrates

Kanamycin acetyltransferase (KAT)

Acetyl coenzyme A

Acetylation of the 6'-amino group

Neomycins, kanamycin B, gentamicin Cia, gentamicin C2, tobramycin, butirosins, ribostamycin, sisomicin, BB-K8 (amikacin)

Gentamicin acetyltransferase I11 (GATIII)

Acetyl coenzyme A

Acetylation of the +amino group (of deoxystreptamine)

Kanamycins B & C, gentamicins, sisomicin, ribostamycin, tobramycin, lividomycins

Gentamicin adenyltransferase (GAdT)

ATP

Adenylylation of the 3'-hydroxyl group

Kanamycins B & C, gentamicins, t obramycin

Neomycin phosphotransferase I WTI)

ATP

Phosphorylation of the 3 '-hydroxyl group

Kanamycins B & C, neomycins, lividomycins, ribostamycin, gentamicins A & B

Neomycin phosphotransferase I1 (NPTII)

ATP

Phosphorylation of the 3 '-hydroxyl group

Kanamycins B & C, neomycins, butirosins, ribostamycins, gentamicins A & B

KANAMYCIN SULFATE

277

their substrates, co-factors and the modifications induced in the kanamycin molecule are summarized in Table IV. The data presented in this table are taken from references 37 and 38. The application of aminoglycoside-modifying enzymes in the assay of kanamycin and related antibiotics will be discussed in section 9. 6. Mode of Action

The mode of action of kanamycins is similar to that of other aminoglycoside-aminocyclitol antibiotics and has been reviewed by Weisblum and Davies3’ and by Gale et al.40. These L

L

drugs inhibit protein synthesis through an interaction with the 30s ribosomal subunit. They also induce a misreading of the codon. The significance of the latter effect for the lethal action of the antibiotic is not clear. A structure-activity relationship among the aminoglyco-

41

side antibiotics is reported by Benveniste and Davies

.

7. Pharmacokinetics Earlier work on absorption, distribution and excretion of kanamycin in humans was reviewed by K ~ n i nin~ 1966. ~ A comparative pharmacokinetic study of kanamycin and amikacin (a semisynthetic aminoglycoside antibiotic derived from kanamycin A) in dogs and human has been reported recently by Cabana and Taggart43. The kinetic profiles of both antibiotics are almost identical. The results presented in this paper are similar to those obtained in a previous study44. In humans, serum concentrations of about 20 pg/ml were observed at 1 hr after a 500 mg intramuscular dose. The plasma half-life of kanamycin is approximately 2.3 hr. Clearance in man was primarily by glomular filtration, and urinary excretion of the

278

PAUL J. CLAESeral.

unchanged antibiotic accounted for 83 % of the dose. No

45-47 protein binding of kanamycin by human serum was observed Kanamycin sulfate is poorly absorbed from the gastrointestinal tract and large amounts of kanamycin are recovered in the 42 stools of patients given the drug by mouth

.

The distribution of kanamycin in tissues, after parenteral administration, has been studied by several 48-50 authors 8 . Methods of Analysis

8.1. Identification Kanamycin generates a violet color when heated with ninhydrin. This color reaction, which is not specific since it is due to the presence of primary amino function, is given

as identification test in the Eur. Ph.8, Brit. Pharm. Codex

1968’’ and in the Code of Federal Regulations7. The characteristic melting point (about 235’ with decomp.) of the crystalline picrate salt of kanamycin is also useful as identification test. The procedure is described in the Brit. Pharm. 8

Codex5’ and in the Eur. Ph.

.

Thin layer chromatography (TLC) on silica gel H with a solvent system consisting of 3.85 % aqueous amonium acetate 6 has been described as identification in the Brit. Ph. 1973 (cf. section 8.62, solvent system V). A ninhydrin reagent (solution in butanol) is used for detection. The TLC system described by Dubost et al.52 for the semiquantitative determination of the B-component in commercial samples of kanamycin (section 8.62, solvent system VI) is also a specific method for the identification of kanamycin A23. The chromatography is carried out on Merck pre-

KANAMYCIN SULFATE

279

coated silica gel plates with a 15 2 aqueous solution of KH PO

as a solvent system. Spots are visualized by the color 2 4 reaction with ninhydrin or by a spray consisting of a 0.2 2

alcoholic solution of 1,2-dihydronaphthalene and sulfuric

- in a ratio of 1:1, acid 9N

followed by heating f o r 5 to 10

min at 150". Differentiation of kanamycin from related aminoglycoside antibiotics is based on Rf values and the color observed after visualization with the 1,2-dihydroxynaphthalene reagent. Kanamycin gives a brown colored spot, whilst blue spots are obtained for paramomycin and neomycin. Merck precoated silica gel plates may be replaced by plates coated with silica gel H containing 1 2 carbomer. In the latter case a 7 2 aqueous solution of KH PO is used as a solvent 2 4 system8'23 (section 8.62, system VII). 8.2. Determination of Sulfate The limits for the sulfate content (SO ) are for 4 6 kanamycin monosulfate, from 15.7 to 17.3 2 (Brit. Ph. 1973 ) 8 and from 15.0 to 17.0 2 (Eur. Ph. ) , for the acid sulfate from 23.0 to 26.0 2 (both Pharmacopoeias). A gravimetric assay method has been described in the Brit. Ph. 19736. A facile method for the determination of the sulfate in kanamycin and in related aminoglycoside antibiotics has been reported by Roets and Vanderhaeghe53. In this method the sulfate ion is titrated with BaCl 0.1M, using thorin as indica2 tor, after fixation of the kanamycin free base by ion exchange on a column filled with a suitable strongly acidic resin

+

in the H form (e.g. Dowex 50W-X8, 200-400 mesh).

8 The most convenient method is described in the Eur. Ph.

and consists in the precipitation of the sulfate with a known amount of BaCl in the presence of ammonia, followed by a 2

280

PAUL J. CLAES er a/

titration of the excess of barium ions with sodium edetate. This procedure, which is given below, has been adapted3Y4 from a complexometric titration described by Anderegg

al.54.

et

Kanamycin sulfate (0.250 g) is dissolved in 100 ml

water and sufficient concentrated ammonia is added to adjust the pH to 1 1 . After addition of barium chloride 0.1M - (10 ml) and of phthaleinpurple (0.5 mg), the solution is titrated with 0.1M - sodium edetate, adding 50 ml of ethyl alcohol when the color of the solution begins to change. Titration is continued until the violet-blue color completely disappears. 8.3. Loss on Drying The water present in kanamycin monosulfate monohydrate can only be removed after heating at high temperature. A loss on drying of 2 to 3.5 2 was noticed after heating samples for 6 hr at 1 5 0 ' ~ ~ (the ~ calculated amount of water is 3.0 2 ) . X-Ray powder diffraction patterns of samples heated for 6 hr at 150' revealed a transformation into another crystalline form24. Heating for 4 hr at 150' or 6 hr at 120' is not sufficient for the removal of water present. According to the Brit. Ph.6 and the Eur. Ph.8 the loss on drying for kanamycin monosulfate is determined after heating for 3 hr at 60" in vacuo (5 mn Hg or less) over phosphorus pentoxide. This treatment does not alter the X-ray powder

6

The limits for this loss on drying is 3 % 8

(Brit. Ph. ) and 1.5 Z (Eur. Ph. ) . The values actually observed under these conditions vary from 0.2 to 0.7 2. For kanamycin acid sulfate the same procedure (3 hr at

in vacuo over P 0 ) is recommended in the Brit. Ph. (Add. 2 5 2 1975) and in the Eur. Ph.7. The limit given in both Pharmacopoeias is 5 2 .

60'

KANAMYCIN SULFATE

281

The water content of kanamycin sulfate has also been determined by the K. Fischer mthod. Results obtained in different laboratories are not always in agreement with each other. This may be due to the fact that the kanamycin sulfates are almost insoluble in methanol. Methanol may be replaced as a solvent by pyridine or formamide. In these cases the solvents must be strictly anhydrous.

8.4. Microbiological Assay

6 The minimum potency required by the Brit. Ph. 1973

is 735 I . U . per mg for kanamycin (mono) sulfate and 670 I . U . per mg for the acid sulfate. The requirements of the Eur. Ph.8 will be respectively 750 and 670 I.U. per mg. The minimum potency requirements of the FDA7 for kanamycin (mono) sulfate is 750 mcg per mg. Prescriptions for the microbiological assay using the diffusion procedure can be found in different compendia. The Brit. Ph.6 recommends as test organisms Bacillus pumilus NCTC 8241, whereas the Eur. Ph.8 --

suggests the use of Bacillus

subtilis ATCC 6633 or NCIB 8054, or Staphylococcus aureus 7 ATCC 6538P or NCTC 6571. The FDA prescribes Staphylococcus

--

aureus ATCC 6538P. Details of the FDA procedure can also be

--L_

found in refs. 55 and 56. No detailed description of the turbidimetric assay of kanamycin has been published although it is used in some laboratories. For general infomation about this method see ref. 57. 8.5. Assay of Kanamycin B The Code of Federal Regulations7 described the determination of the B-component in commercial kanamycin samples. The method, which is similar t o the procedure origi-

282

PAUL J. CLAESet a/.

nally reported by Wakazawa et al.58, is based on the fact that kanamycin B is more resistant to acidic hydrolysis than kanamycin A. Thus the commercial sample is heated for 1 hr at 100" in HC1 6N - and the residual antibacterial activity is

assayed using Bacillus subtilis ATCC 6633. The limit for the 7

B-component given in the Code of Federal Regulations is 5 2 . A method using column chromatography on Dowex 1-X2 ionexchange resin in the OH- form (section 8.63), using the reaction with ninhydrin as detection method, is described in the Brit. Ph. 19736. The limit for kanamycin B in commercial samples given in this Pharmacopoeia is 3

9,.

A limit test for kanamycin B by thin layer chromatogra-

phy on Merck precoated silica gel plates has been reported by Dubost -et al.52 (section 8.62). The precoated plates can be replaced23 by silica gel H layers containing 1 2 carbomer (Carbopol 934). In this case the percentage of KH PO must be 2 4 lowered from 15 to 7 %.Ninhydrin is used for detection in both systems. The procedure using the carbomer-containing layers is recommended in the Eur. Ph.8 as a limit test for the B-component. The intensity of the secondary spot must be lower than that observed for a reference solution consisting of the kanamycins A and B in a ratio 25:l.

Commercial samples show in these systems a third spot with a higher Rf value than that of either kanamycin A or B. The minor components responsible for this spot were identified22 by degradation and mass spectral studies as paromamine and as 6-0-(3-amino-3-deoxy-CY-D-glucopyranosyl)deoxystreptamine.

KANAMYCIN SULFATE

283

The solvent system of Peterson and Reinecke5',

which

8.6. Chromatographic Analysis 8.61. Paper Chromatography consists of water-saturated butanol containing 2 Z p-toluene 1,21 ,60-63 sulfonic acid, has been used by a number of authors for differentiation of kanamycin A from the B- and C-components. The Rf values 0.12-0.18 for kanamycin A, 0.26-0.28 for kanamycin B and 0.20-0.24 for kanamycin C have been reported by Rothrock _ et -a1.61 (descending chromatography of 40 to

48 hr on Whatman no. I paper with the Peterson and Reinecke solvent system). Kanamycins were visualized by bioautographyl y60, ninhydrin reaction62'63 and "chromato red" staining6*. Another system used for differentiation of the three kanamycins was reported by Kojima et al.30. It consists of n-butanol-pyridine-acetic

acid-water (6:4:1:3) (v/v)

(descending chromatography for 5 days at 20-25'). Differentiation of kanamycin from related antibiotics by paper chromatography using a combination of several solvent 64-67. A systems has been described in a number of papers review article on paper chromatography of antibiotics has 68 been published recently

.

8.62. Thin Layer Various TLC systems for separation of kanamycin A from its congeners (kanamycin A and B) and from other watersoluble basic antibiotics have been reported. Details are given in Table V. Separation of kanamycin A from tuberculostatic antibiotics such as rifamycin SV, capreomycin, viomycin, cycloserine, and streptomycin (or dihydrostreptomycin) 69 has been reported by Voigt and Maa Bared

.

N

O- ,

~

W N

R W L l

-

(0

w u m W e

W

u W

~

N

W

U

W

N

~ W

-

W

C

W

0

N

0

Ln

Ln

N

R

c.

W

W R

0

-

ocl

m

-o w

-

w

m

v1

W

uc: w

W

0 N

-

c"

m

u

Ll

N

0

u o o

VI

W L n W W

O

-

O

P v1

0

Ln

w U

f - W

C

l

P

w

m N

h

O

L

-

W lL wl L

W W

W P

0

U

N u u W N W N

> u C . W N V I W L - L n Q I

W N

N w

u U

P o

V8Z

-

~u

0

lu

o

m

Ll

0 m

I>

VI N

a

* P o .

-

U

W

:I m h 0..

W V I V t W L n

0

u

~

~

Eacitracin

Polyniyxin B

Capreomycin

Viomycin

Dihydrostrept.

Streptomycin

Spectinornycin

Centarnicin C,

Paromomycin,,

Paromamine

Neoaycin,,

Neamine

Kanamycin C

Kanamycin B

Kanamycin A

u Reference

Revelation

Plate

System

KANAMYCIN SULFATE

285

visualinarios-ol-tpe,astibiotics_.sed_is~-~~~~e~~~~~ L------------iven in Table V P = spray of 10 % potassium permanganate followed by a spray 70 of a 0.2 2 bromophenol blue solution N = ninhydrin reagent C1 = spray of a NaOCl solution containing 0.5 2 active chlorine followed, after evaporation of the chlorine, by a spray of a 0.5 2 KI solution containing 1 % starch NR = spray of a 0.2 2 naphthoresorcinol (1,3-dihydroxynaphtalene) solution in ethanol, followed by a spray of H2S04 9N - and heating for 5 to 10 min at 150' PG = as NR, but with phloroglucinol instead of naphthoresorcinol OR = as NR, but with orcinol instead of naphthoresorcinol DN = p-dimethylaminobenzaldehyde-ninhydrin reagent69 NP = sodiumnitroprusside-permanganate reagent69 ON = oxidized nitroprusside reagent79 80 CT = chlorine-tolidine reagent MS = Mathis-Schmitt reagent81 Solvent systems for the TLC procedures in Table V -----------------------------------given --------------_ I

: Upper layer of CHCl -MeOH-17 % ammonium hydroxide

( 2 : 1 : 1)

70

3

70

I1

: n-Propanol-pyridine-HOAc-water ( I5 : 10:3 : 10)

I11

: CHCl -MeOH-28 2 ammonium hydroxide-water (1:4:2:1)

IV

: 10 %

V

: NH40Ac (3.85 g) in water (100 ml) (tank saturated

72

3 tank saturated overnight

aqueous solution of NaH PO 2H20-MeOH-EtOAc 2 4' 52 (8:7 :3)

overnight)78

VI

:

Aqueous solution of KH PO 15 2 (tank saturated over2 4 52 night)

286

PAUL J. CLAES et a/

Results given for this system can only be obtained on Merck precoated plates. It was found that the separating power is mainly due to the presence of a polycarboxylic resin which is used as a hinder in these Merck plates. Similar results can be obtained with system VII in which a small amount of a polycarboxylate resin (carbomer) is added to the silica gel8,23 VII

: Aqueous solution of KH PO

night)

8,23

2 4

.

7 % (tank saturated over-

VIII : n-Propanol-EtOAc-water-25 % ammonium hydroxidepyridine-3.85 % in water (100:20:60:20:10:200) 73

IX

:

X

: MeOH-EtOAc-water-25 % ammonium hydroxide-pyridine-

EtOH-EtOAc-water-25 % ammonium hydroxide-pyridine73 3.85 % NH40Ac in water (100:20:60:10:200) 3.85 % NH40Ac in water (100:20:60:20:10:200)

73

25 % Ammonium hydroxide-water-Me2C0 (16:144:40)73 74 XI1 : Water-sodium citrate-citric acid (100:20:5) 75 XI11 : n-Propanol-pyridine-HOAc-water (15:10:3:12)

XI

:

XIV

: MeCOEt-MeOH-isopropanol-7.9N - ammonium hydroxide

(10:8:5:3:7) (tank and plate saturated deve1opment) 76 XV

:

-

double

1.5M NaOAc (adjusted to pH 8.5) containing 1.OM- NaCl 77 and 10 % s - b u t a n o l

Plates_for_the-T4c_Erocedures_given_iq_T~~~e-~ SG-I : Silica gel G thickness of layer and mode of activation not specified SG-2 : Silica gel G (0.25 mm) activated for 1 hr at 110' SG-3 : Silica gel G (0.75 mm) not activated SG-4 : Silica gel (0.5 mm) activated for 1 hr at 110' SH

:

Silica gel H (0.25 mm) activated for 1 hr at 110'

MP

:

Merck precoated silica gel F-254 plates activated for 1 hr at 110'

KANAMYCIN SULFATE

287

SH-C : Silica gel H containing 1 % carbomer (adjusted to pH 7) activated for 1 hr at l l O o SK

:

Silica gel G 1

-

kieselguhr G (1:2) activated for 1 hr at

lo0

KG

:

Kieselguhr G (0.25 mm) activated for 1 hr at 120'

C-I

:

Machery Nagel cellulose powder 300 (0.25 mm) dried for 20 min at 100'

C-2

:

Idem, dried €or 2-3 hr at 100-105°

IE

:

Dowex 50 x 8 type resin-coated TLC plates in the sodium cycle (Machery Nagel Ionex 25 SA)

8.63. Ion Exchange Column chromatography of kanamycins and related antibiotics on both acidic and basic ion exchange resins has been reported. Separation on acidic resins is by classical ion exchange chromatography. The separating capacity of strongly basic resins is based on non-ionic adsorption of the antibiotic by the quarternary ammonium groups of the resin. This chromatographic system, which is now referred to an ion exclusion chromatography, was introduced by Rothrock et al. 61 for the separation of the kanamycins A , B and C (the order of elution is B, C, A ) . The procedure permitted isolation of crystalline kanamycin C. Improvements of the original procedure have been reported82. Other applications in the field of aminoglycoside antibiotics have been reviewed recently by Umezawa and Kondo83 . Experimental details of ion exclusion chromatography can be found in several papers6 1 y72y82-84.Most of the separations were carried out on Dowex 1-X2

(100-200 mesh) resin

(Dow Chemical Co., Midland, Michigan) or on Biorad AG 1-X2 (100-200 mesh) resin (Bio-Rad Laboratories, California) both

PAUL J. CLAESet a / .

288

in the OH- form. The resins contain trimethylammonium groups on a polystyrene backbone with a low degree of cross-linking. After application of the antibiotic, the column is developed -free water. Detection systems based on a continuous 2 measurement of electric conductivity, optical rotation and with

CO

colorimetry after reaction with ninhydrin have been used. High performance liquid chromatography (HPLC) of kanamycin A and B based on ion exclusion has been reported recently on Aminex A-2J85 and Biorex 9 resins86 (Bio-Rad Laboratories, California). Weakly acidic carboxylate resins, such as Amberlite IRC-50 (Rohm and Haas Co.,

Philadelphia), are widely used in

industry for the isolation of kanamycin and other aminoglycoside antibiotics from culture filtrates12. The antibiotic

+

+

is adsorbed on the carboxylic resin in the Na or NH4 and eluted with IN - aqueous ammonium hydroxide. Separation of the three kanamycins and of other minor components present in commercial samples was achieved on the chromatographic grade resin by elution with 0.2N NH OH22. Gradient elution has been - 4 83 used for separation of other aminoglycoside antibiotics

.

References for applications of carboxylic-, sulfonic- and phosphonic acid resins, and of cellulose- and sephadex-ion exchangers in extraction and purification of aminoglycoside antibiotics can be found in a review article by Umezawa and K ~ n d o ~High ~ . performance liquid Chromatographic (HPLC) determination of kanamycins A and B on a pellicular cation exchanger such as Zipax SCX (Dupont) has been reported 87

recently

.

KANAMYCIN SULFATE

289

8.64. Gas Liquid Tsuji and RobertsonS8 reported gas chromatographic separation of the 0,N-trimethylsilyl derivatives of kanamy-

tins A , B and C on a 3 x 1830 nun glass column packed with 3 Z OV-1 on Gas Chrom Q at a temperature of 300' using a

flame ionization detector. The volatile derivatives were prepared by silylation (45 min at 75')

of a freeze-dried

sample of kanamycin sulfate with 1-trimethylsilylimidazole in pyridine (Tri-Sil 2, Pierce, Rockford, Illinois) and

N-trimethylsilyldiethylamine. Addition of trilaurin as an internal standard permits quantitative analysis. Similar conditions were described for TMS derivatives of neomycin and paromomycin and other aminoglycoside antibio88 ticsg9. The order of elution given in the original paper is kanamycin B, kanamycin A and kanamycin C. Japanese authors''

found that kanamycin C was eluted before kanamycins

A and B y under GLC conditions similar to those employed by

Tsuji and Robertson. Numerous factors may easily interfere with the GLC determination of neomycin and of other aminoglycoside antibiotics. These have been discussed by Margosis and Tsujigl.The solution to some of the common problems encountered during GLC analysis of neomycin is given by these authors, and also by Tsuji and Robertson8'

in a review arti-

cle on GLC of antibiotics. GLC of N-trifluoroacetyl-0-trimethylsilyl derivatives of a number of aminoglycoside antibiotics (including the three kanamycins) has been reported by Omoto et al. 90

.

8.7. Electrophoresis High-voltage paper electrophoresis of kanamycins and other water-soluble basic and amphoteric antibiotics has

290

PAUL J. CLAES et a / .

been described by Maeda et a1 .92. The spots of the kanamycins L-

were visualized with ninhydrin. Mobilities relative to alanine (Rm values) are 1.82 for kanamycin A , 1.92 for kanamycin B and 1.85 for kanamycin C. In a review articleg3 on electrophoresis of antibiotics by two of the authors of the original paper somewhat different Rm values are given ( 1 . 7 4 ,

1.89 and 1.70, respectively for the kanamycins A , B and C). Electrophoretic separation of aminoglycoside antibiotics including kanamycin A has been reported by Ochabg4. Resolution of antibiotic mixtures in serum samples by high-voltage 95 electrophoresis on agarose is described by Reeves and Holt

.

9. Determination in Body Fluids Since kanamycin acid, like other aminoglycoside antibiotics, may cause ototoxicity and renal impairment, it is advisable to monitor the antibiotic level in the serum of patients receiving these drugs. Special and rapid assay procedures have been worked out for this purpose. Sabath % al.96'97 described a microbiological assay method. Interfe-

L

rence by penicillins and cephalosporins can be eliminated by a treatment of the serum with a "broad-spectrum" /j-lactamase96'98. A method based on the inhibition by aminoglycoside antibiotics of the urease activity of Proteus s p . has been reported by Noon et al. ". A semiquantitative determination of kanamycin in serum and urine, based on a visual comparison of fluorescent intensity with that of reference samples on TLC plates after reaction with 7-chloro-4-nitrobenzo-2oxa-l,3-di'azole, has been developed by Benjamin et al. -

L

100

.

Enzymatic assays employing aminoglycoside-modifying enzymes (section 5) have been introduced recently1 0 1 y 1 0 2 . In these procedures the antibiotic is enzymatically modified in the

KANAMYCIN SULFATE

29 1

presence of a radiolabeled cofactor. Kanamycin acetyltransferase (KAT) l o ' and gentamicin acetyltransferaselo2 have been used in the assay of kanamycin. 10. Reference Cited 1.

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4.

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5.

United States Pharmacopoeia, XIX, --

6.

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Code of Federal Regulations, 3, § --

8.

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-

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--

e

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--

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-

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_

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91.

M. Margosis and K. Tsuji, J. -Pharm. Sci. , 62, 2946

-

( 1 973).

92.

K. Maeda, A. Yagi, H. Naganawa, S. Kondo and H. Umezawa, -J. Antibiotics, 22, 635 (1969).

93.

H. Umezawa and S. Kondo, Methods in Enzymology, 43, 279 ( 1 975)

94.

S.

-

.

-.,

Ochab, Pol. - J. Pharmacol. Pharm

25, -

105 (1973).

296

PAUL J. CLAES et a / .

95. D.S. Reeves and H.A. Holt, 2. Clin. Pathol., 28, 435 (1975). 96. L.D. Sabath, J.I. Casey, P.A. Ruch, L.L. Stumpf and M. Finland, Antimicrob. &. Chemother.-1970, 83 (1971). 97. L.D. Sabath, "Analytical Microbiology", Vol. I1 , p. 235, Academic Press, New York (1972).

a.

Microbiol., 21, 1002 98. S.A. Stroy and D.A. Preston, (1971). 99. P. Noone, J.R. Patton and D. Samson, Lancet, 2, 16 (1971). 100. D.M. Benjamin, J.J. McCormack and D.W. Gump, Anal. Chem., 45, 1531 (1973). 101. M.J. Haas and J. Davies, Antimicrob. &. Chemother., 4, 497 (1973). 102. J.M. Broughall and D.S. Reeves, Antimicrob. &. Chemother., 8, 222 (1973).

- -

KETAMINE

William C. Sass and Salvatore A . Fusan

298

WILLIAM C. SASS AND SALVATORE A. FUSARI

Contents

1. Description

1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color, Odor 2.

Physical Properties 2.1

Spectral 2.11 2.12 2.13

2.2 2.3 2.4 2.5 2.6 2.7

Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Absorption Spectrum

Mass Spectrum Differential Thermal Analysis and Melting Point Solubility Optical Rotation Ionization Constant Crystal Properties 2.71 2.72

Derivative Crystallinity X-Ray Diffraction

3.

Synthesis

4.

Decomposition

4.1 Metabolic Decomposition 4.2 5.

Chemical Decomposition

Methods of Analysis 5.1 5.2 5.3 5.4 5.5 5.6 5.7

Elemental Analysis of the Hydrochloride Ion-Pairing Colorimetric and Fluorescence Ultraviolet Differential Thermal Analysis Non Aqueous Titration Tritium Labeling Chromatography 5.71 5.72 5.73

Paper Chromatography Thin Layer Chromatography Gas Chromatography

KETAMINE

5.74

Liquid Chromatography

6.

Determination in Body Fluids

7.

References

299

300

WILLIAM C. SASS AND SALVATORE A. FUSARl

1. Description

1.1 Name, Formula, Molecular Weight' Ketamine is 2-(2-~hlorophenyl)-2-(methylamino)cyclohexanone. The hydrochloride bears the clinical investigation number CI-581. 0

CH 2

Molecular formula: C13H16ClNO Molecular weight: 237.74 Molecular formula of the hydrochloride: C13H16C1NO.HC1 1.2 Appearance, Color, Odor Ketamine and the hydrochlor'de are both odorless, white crystalline powders.

i

2. Physical Properties 2.1

Spectral 2.11

Infrared Spectrum

Infrared spectra of the base in chloroform (Figure 1) and of the hydrochloride as a 0.5% disper ion in potassium bromide (Figure 2) were obtained with a Perkin-Elmer Model 621 grating infrared spectrophotometer. The high energy absorption between 2600 and 3000 cm.-l of the hydrochloride has been ascribed to the amine hydrochloride while that at 1730 and 780 cm.-1 result from carbon-oxygen stretching and phenylhydrogen bending respectively. (1200 cm-1 is CHC13) 2.12 Nuclear Magnetic Resonance Spectrum Figure 3 shows the proton magnetic

2.5

4

3

WAVELENGTH 5 6

7

MICRONS 8 9 10

18 22

12 14

3550

t---

-4-

4000

3500

300C

-

- -

I

2500

2WC

17CO -i40C l!i)O FREQIJENCY 'CM ' )

800

Figure 1. infrared Spectrum of Ketamine Base in Chloroform. (1601.0 cm- is polystyrene reference peak)

500

200

302

k 0

k

a

a c

H

N

SOLVENT TEMPERATURE FILTER BANDWIDTH R F FIELD SWEEP TIME SWEEP WIDTH SWEEP OFFSET SPECTRUM AMP. INTEGRAL AMP.

. .

D20 25

OC

4

cps

0.2 250 500

mG

0

sec

cps cps

16 2.5

w 0 w

,/--

1

I

8.0

Figure 3.

7.0

6.0

I

5.0

1

PPM(6)

4.0

I

3.0

60 MC NMR Spectrum of Ketamine Hydrochloride in D20.

I

I

2.0

1.0

* 0

304

WILLIAM C. SASS AND SALVATORE A. FUSARI

W a v o Ionurh (nm)

1% a 1 cm

2 76

20 4

269

23.2

J’

lox

W a v o I o n g t h (nm)

Figure 4 . Ultraviolet Spectrum of Ketamine Hydrochloride in 0.1 N Hydrochloric Acid.

KETAMINE

305

resonance spectrum of ketamine hydrochloride in D20 at 60 y g . Hz. The following assignments have been made: Structural Assignments LPF!

# of Protons and Description

2.0

5 - protons of cyclohexanone ring. Shape of absorption peak is typical of cyclohexyl ring protons

2.6

5

-

Sharp peak is N-CH3, - rounded peak at 2.7 ppm represents 2 protons of cyclohexanone ring

3.5

1

-

One of protons on cyclohexanone ring. This proton is most probably on the carbon ci to the carbon bearing -N-CH3 Hydrogen bonding of m s proton to N would lower its chemical shift

4.8

2 - Two protons. Total integration is 19 spaces; subtract 5 spaces for D20 blank to give 14 spaces or two protons. These are exchangeable protons so that they are protons -NH - and H-C1 -

7.7

4

-

Aromatic ring protons

2.13 Ultraviolet Absorption Spectrum Figure 4 is the ultraviolet spectrum5 of ketamine hydrochloride in 0.1N hydrochloric acid obtained on a Cary 15. The two maxima at 276 and 269 nm. represent a(l%, 1 cm.) values o f 20.4 and 23.2 respectively.

In 0.1N sulfuric acid, maxima at 264 nm. (a 1%, 1 cm. = 16.6), 269 nm. (a 1%,

306

WILLIAM C. SASS AND SALVATORE A. FUSARI

Wove length

(nm)

301

5.0 7.0

2 74 268

98

261

10.5

\

25X 0

m

O N 0

R

ti

S

N

8

N

W o v e length

1% a l cm

\

4

8

5

3

(nm)

Figure 5 . Ultraviolet Spectrum of Ketamine Base in 95X methanol - 0.01N sodium hydroxide.

3

K ETAM IN E

1 cm. = 23.2), and 2 6 nm. (a 1%, 1 cm. have been reported.2i:

=

307

20.3)

Figure 5 represents the alkaline spectrum (0.01N sodium hydroxide in 95% methanol) with the following a(l%, 1 cm.) values: 301 nm. (5.03); 276 nm. (7.07); 268 nm. (9.80) ; and 261 nm. (10.58). 2.2 Mass Spectrum Although the mass spectrum of the hydrochloride cannot be easily obtained because of its low volatility, the normalized fragmentation pattern of the base6~30is shown in Figure 6. Tabulated values are f0.5 mass unit. The pattern is consistent with a progressive l o s s of C2H4 (209), CO (181), and CH2NH (152). Fragments at 211, 183, and 154 would result from the chlorine isotope. 2.3 Differential Thermal Analysis and Melting Point A Ketamine reportedly melts at 92-930'. differential thermal analysis thermogram6 of the base run on a Mettler DTA (Figure 7) displays only a single melting endotherm at 92.25OC. The heat of fusion was found to be -25.81 m cal/mg. The observed specific heat at 90° is 1.9 m cal/mg. OC. Decomposition of the hydrochloride precludes a precise determination of thermodynamic properties. 2.4 Solubility5 One gram of the hydrochloride will dissolve in:

6 14 60 60

ml. ml. ml. ml.

of of of of

methanol 95% ethanol (USP) chloroform absolute ethanol

One gram of the hydrochloride is incompletely dissolved in 60 ml. of acetone, ether, benzene, DMF, or dioxane.

RRW DRTR

DR. SRSS. P A R K E D F I V I S : B R S E FORM 16-JUN-76 FI 52 T I C = 8118 3.96 M I H RGNGE so THRIJ 250 THRESHOLU = 0 . 0 0 fl/E

I? I

fl/E

R

I

s e ) . ~ 5 m 0.16

7 6 . 3 ~ 0.31

53.258

7i.117 32 l? 69

5 9 . 177

62.89R 64.E18 67.314 78.302 72.537 i>.995

0.?G

0. 16 0. 1 9 0.19 0.14 17.18 3.50

n.52

Figure 6.

89

.375 91.548 9H.173 101.466 li32.232

0.35: 0.18

0.16 0.21: 0.25 0.22

M/E 183.201 110.484 112:?02 214.900 116.19?

R I 0.52 0.50 0.28 8.R6 0.64

117.302

n.csr

123.972

0.16 1.06 0.36

i3.18

125.630

0.34 0.42

127.4'34

1.28

123.44%

128.392

R.28 0.20

M/E 130.949 171.5rlc 132.378 137.833 i:?.-e2 138.982 148.183 141.351 143.93Z 144.OIJtl 14F.355

Mass Spectrum of Ketamine Base

R I B.36 8.38 8.52 R.74 1.74 8.70

1.82 0.24 0.92 1 . 12

1.66

MYE

R I

146.875 0 . 2 6 148.333 0. 18 1 ~ 0 . ~ 3 98.82 151.681 3.62 352.718 0.80 1.94 154.312 356.858 6.24 164.637 8 . 2 4 1.30 165.8 14 1.72 i 66.8 2 0 168.130 I .sa

fl/E

P I

169.ias 0.80 171.283 8.16 174.244 1.94 0.36 175.468 178.322 @.IS 179.666 m . 7 0 188.F14 2". 10 ltt1.413 ?.8C 188 1 0 1 9.40 1 8 4 . ~ 8 9 1.:-

184.HRZ 19R. 154

KETAM IN€

Figure 7.

Differential Thermal Analysis Melting Curve.

309

310

WILLIAM C. SASS AND SALVATORE A. FUSARI

2.5 Optical Rotation Isolation of the d(+) isomer of the hydrochloride from a-racemzc mixture using dcamphorsulfonic acid' resulted in a com o k d with a s ecific rotation o ( a ) g 0 = +gog (0.98% in methanol!. Other physical6 and physiological properties were similar to unresolved commercially available material. 2.6 Ionization Constant The pKa of ketamine and the N-dealkylated metabolite are3I 7.5 and 8.65. The pH of 10, 50, and 100 mg./ml. solutions of the hydrochloride are 4.63, 4.16, and 3.92 respectively. 2.7 Crystal Properties 2.71 Derivative Crystallinity24

In latinic iodide solution, rhomboidal plates are gormed (sensitivity to 1 in 1000 solution). With potassium bismuth iodide solution, small plates are formed (also sensitivity to 1 in 1000). 2.72 X-Ray Diffraction X-Ray Diffraction values on the hydrochloride obtained on a Norelco Diffractometer6929 using Copper K2 radiation ( A = 1.5418) and a crystal monochrometer are listed in Table I. Variations in the X-Ray pattern of the base suggest that polymorphism may occur. TABLE I X-Ray Diffraction of Ketamine Hydrochloride

9.70 7.43 6.92

6.44 6.14 5.90

5.8 100.0

3 . 0 15.6 11.6 1.8

5.30 4.87 4.63 4.55 4.32 4.14

1.4

42.1 5.9 12.1 1.5 7.9

KETAM INE

31 1

100 WI1)

20.4 81.7 2.4 5.9 3.1 7.6 3.7 5.7 3.6 1.6 7.0 13.2

4.13 3.72 3.57 3.45 3.35 3.25 3.22 3.17 3.15 3.05 2.92 2.90

2.70 2.63 2.44 2.11 2.02 1.83 1.79 1.75 1.64 1.44

35.2 2.7 3.4 1.8

4.0 1.8 2.6 2.6 2.6 1.5

3. Synthesis 3.1 Ketamine hydrochloride may be pre aredl' from o-chlorobenzaldehyde by the procedure3 shown in Figure 8.

4. Decomposition 4.1 Metabolic Decomposition An initial rapidlfrop in h 11 min. 17 rnin.l2, levels (half-life 10 min. and 25 min.13) due to distribution of drug to the tissues is followed by a first order decrease in plasma lev ith a half-life of about 2.5 hours.f P 2 X 2

Describing the absorption pharmacokinetic behavior of ketamine following intravenous injection by a two-compartment mode the half-life of the 6-phase has been reportedh5 as 2.52 hr., 3.99 f1.23 hr., and 6.84 f2.97 hr. for ketamine, N-dealkylated amine, and the dehydro-N-dealkylated metabolites respectively. In addition to small amounts of the intact drug excreted, the decomposition scheme shown in Figure 9 has been suggested.llp15,25 No indication of otein binding was observed.11 Another report33 suggests that if present, protein binding does not exceed 12%.

WILLIAM C. SASS AND SALVATORE A. FUSARI

312

1. aq. C H 3 0 H , NaOH

*

Ar*-CH=NOH

2 . HC1 i1

1. 1. A1220

Ar-CN

P

2.

2 . NaOH

BrMgcl 0 11

CUCl H20, HC1

Ar/'>C7 H

I11

-

IV

0ch3

F1

*=2

A,/'=

cc14

1

CH30Na CH30H, A f

Br

v

VI

VIII

VII

/cm NCH 3

Ar

*HC1

HO /'

IX

*

pc. /

X k e t a m i n e hydrochloride

=

a

c1

F i g u r e 8.

Synthetic Procedure

4 !-l

H

H

a aJ

U

3

rd M 'r)

U

I a,

9

3

C0P

H H

8 *

m

U

H H H

PI

a bo

rd

+J

0 0 313

H W

*

4 U

U

6-

314

W I L L I A M C. SASS A N D S A L V A T O R E A. F U S A R I

4.2 Chemical Decomposition Ketamine in aqueous solution has been shown14 to react under accelerated conditions of high temperature and pH by a process which involves initial formation of 1-[ (2-chlorophenyl) (methylimino)methyl]cyclopentanol (I)(Figure 1 0 ) . This intermediate, depending on temperature and pH, may then isomerize back to Ketamine or hydrolyze to (2-chlorophenyl)(l-hydroxycyclopentyl) methanone (111), the primary product of this reaction. 2-(2-chlorophenyl)-2-hydroxycyclohexanone (IV) which may be a major, although not primary, product results from isomerization of the cyclopentyl hydroxyketone (111). When the accelerating conditions are avoided, aqueous solutio and the powder exhibit extraordinary stability.

YE

5. Methods of Analysis 5.1 Elemental Analysis of the Hydrochloride E1ement %C % H % N % C1 (total) % C1 (ionic)

Found3

Theory

57.05-57.29 6.49-6.61 4.95 25.88-26.02 13.06

56.94 6.25 5.11 25.86 12.93

5.2 Ion-Pairing Colorimetric and Fluorescence Ion pair extraction into an organic phase using methyl orangel5 is reported to be a less sensitive method than extraction with xylene red B into 1,2- ichloroethane followed by fluorescence analysis.f 6 Excitation and e ssion wavelengths of 562 and 578 nm. were used. With a modification of the xylene red B procedurel7, atropine, diazepam, pentobarbital, fluothane, oxytocin, and ergometrin have been shown not to interfere with the assay, although two of the ketamine metabolites do.

Ti

315

KETAM INE

Ar

Ar*

Ketamine Hydrochloride

Ketamine Base

9 0: -

Ar

c1

IV

Figure 10.

I11

Chemical Decomposition

1

I

316

WILLIAM C. SASS AND SALVATORE A. FUSARI

5.3 Ultraviolet In the absence of interfering substances, ketamine may e analyzed directly by ultraviolet spectroscopy.

b

5.4 Differential Thermal Analysis Pure base may be analyzed by thermal analysis.6 Figure 7 is a thermogram of a recrystallized sample which contains less than 1 x 10-3 mole % impurity. 5.5 Non Aaueous Titration A sample dissolved in glacial acetic acid containing mercury (11) acetate may be titrated with 0.1N perchloric acid in glacial acetic acid to the blue-green end point of crystal violet.5 5.6 Tritium Labeling Heating of ketamine hydrochloride to 100°C. with trifluoroacetic acid and tritiated water in the presence of pre-reduced platinum catalyst for 18 hours formed the labeled product with at least 7% tritium incorporation alpha to the carbonyl.16 Labile tritium hould be removed by treatment with strong alkalil8 to avoid tritium incorporation in body water. Labeled ketamine hydrochloride has been used to study metabolic decomposition.11 15 9

5.7 Chromatography 5.71 Paper Chr~matography~~ A 2.5 pl. spot of a 1% solution in 2N acetic acid is applied to Whatman No. 1 paper previously dipped in a 5% sodium dihydrogen citrate solution, blotted, and dried. Development in an unequilibrated chamber with a solution of 4.8 grams of citric acid in 130 ml. of water plus 870 ml. of n-butanol resulted in a zone at Rf 0.55 which was visible under ultraviolet light after spraying with iodoplatinate or bromocresol green solution.

KETAM IN E

317

5.72 Thin Layer Chromatography Two of the four tritium labeled metabolites and intact ketamine hydrochloride have been separated on Silica Gel GF using chloroform: ethyl acetate:methanol:ammonium hydroxide (60:35: 5:l). The intact molecule at Rf = .65 and metabolites were detected by their radioactivity.11 Separation of the unresolved metabolites19 was accomplished on Aluminum Oxide HF using chloroform: cyc1ohexane:diethylamine (60:40:2). Chloroform: cyc1ohexane:ethyl acetate:ammonia (25:50:25:5) has been used25~27to separate ketamine (Rf = 0.58) and the N-dealkylated metabolite (Rf = 0 . 4 1 ) on a LQ6D plate. The other major metabolite is separated but exists as a diffuse zone. All were visualized by exposure to iodine.

A system5 used to separate ketamine hydrochloride and (2-chlorophenyl)(l-hydroxycyclopenty1)methanone is Kieselgel DF-5 using benzene: methano1:ammonium hydroxide (9O:lO:l). Rf values of 0.7 and 0.6 respectively are observed for the compounds under 254 and 366 nm. ultraviolet light. Concentrated ammonium h droxide in methanol (1.5:lOO) has also been used2Z to develop samples on activated silica gel G. The main zone at Rf 0.72 was made visible with acidified iodoplatinate spray. 5.73 Gas Chromatography Since gas chromatography allows rapid, quantitative analysis of ketamine and its degradation roducts, numerous systems have been utilized.20,51 The use of all glass systems22 and the avoidance of evaporation to dryness13 have been suggested to avoid degradation. Chromatographic conditions employed are summarized in Table 11. 5.74 Liquid Chromatography Reverse phase chromatography on C18 Microbondapak columns using water:acetonitrile (1 : 1) has been employed28 to separate the p-nitro-

TABLE I1 Conditions Used In Gas Chromatographic Separations of Ketamine Ref. Column

Column Temp.

Detector

Internal Standard

11

1% ECNSS-M

155O

FID, EC

o-trifluoromethyl and o-Bromo analogs*

12

3% OV-17 3%

195O

E.C. o f heptafluorobutyryl derivative

o-Bromo analog"

13

1% OV-101 and 3% succinamine polymer on (100/120 Gas Chrom Q)

158O

FID

CL-392

15

1% ECNSS-M (80/100 Gas Chrom P)

170°

20

2.5% SE-30 (80/100 Chromasorb G)

200°

FID

21

1% DDTS Gas Chrom Q

180'

FID

(100/120 Gas Chrom Q)

o-trifluoromethyl analog* Pentob arbita1

TABLE I1 (Continued) Column Temp.

Detector

Internal Standard

98-180O

FID

methyldiphenylamine

2YL SE-30 (80/100 Chromasorb G)

zooo

FID

Pentobarbita1

26

0.5% PEG 20000 M (80/100 Chromasorb G- DMCS )

90-2oooc.

FID

Carbothesin

28

10% UCW-982 (80/100 CWAW-DMCS)

27OoC.

FID

- (all separated as

1% Carbowax 20-M ( 6 0 / 8 0 Gas Chrom G AW - DMCS )

21oOc.

FID

Ref.

Column

22

0.5% polyethyleneglycol (20,000 M) (80/100 Chromasorb G) silinized

23

w, W

25 27

9

*analogs of ketamine

@ 3O/min.

p-nitrobenzamides)

320

WILLIAM C. SASS AND SALVATORE A. FUSARI

benzamide derivatives of ketamine and its metabolites. Derivatization is required to enhance the otherwise low absorbance at 254 nm.

6.

Determination in Bodv Fluids

Ion-pairing l5 thin layer chr chromatography

tritium labeling18 5 J 1 1 J 1 9 , 2 5 , 2 7 gas J 22 25-28,31 and liquid techniques have been applied to the determination of ketamine and its metabolites from body fluids. J

s

?f'€ES~fT~!!B,

KETAMINE

321

References (Current to June, 1 9 7 6 )

7.

1. The Merck Index, Eighth Edition, 599 ( 1 9 6 8 ) . 2 . RX Bull, 3 , 5-10 ( 1 9 7 2 ) . 3 . Wheeler, L.M., Parke, Davis & Co., Personal

Communication.

4 . Fusari, S.A., Parke, Davis & Co., Personal

Communication.

5 . Chang, J.H., Parke, Davis & Co., Personal

Communication.

6 . Sass, W.C., Parke, Davis & C o . , Personal

Communication.

7 . Nordin, I.C., Parke, Davis & Co., Personal

Communication.

8 . O’Connor, R.E., Parke, Davis & Co., Personal

Communication.

9 . McCarthy, D.A., Parke, Davis & Co., Personal

Communication,

1 0 . Chem. Abs. 6 5 , 5414h ( 1 9 6 6 ) .

11. Chang, T., uazko, A.J., Int. Anesthesiol. Clin. 1 2 , 157-77 ( 1 9 7 4 ) . 1 2 . Chang,T.,Glazko, A.J., Anesthesiology 36, 401-4 ( 1 9 7 2 ) . 1 3 . Hodshon, B.J., Ferrer-Allado, T . , Brechner, V.L., et. al., Anesthesiology 3 6 , 506-8 ( 1 9 7 2 ) . 1 4 . Philip, J., Parke, Davis & C o . , Personal

Communication.

1 5 . Chang, T., Dill, W.A., Glazko, A.J., Fed. Proc. 2 4 , 268 ( 1 9 6 5 ) . 1 6 Dill, W.A.,Chucot, L . , Chang, T . , Glazko, 3 4 , 73-6 ( 1 9 7 1 ) . A.J., Anesthesiology 1 7 . Nishijima, M., Fujii, A . , Kojima, T., et. al., Jap. J. Anesthesiol. 2 1 , 8 8 1 - 5 ( 1 9 7 2 ) . 18 Blackburn, C.E., Ober,R.E., J. Labelled Compounds 2, 38 ( 1 9 6 7 ) . 1 9 . Glazko, A.J., Parke, Davis & C o . , Personal

Communication.

2 0 . Finkle, B.S., Cherry, E.J., Taylor, D.M., J. Chromatogr. Sci. 9 , 393-419 ( 1 9 7 1 ) . 2 1 Jenden, D.J., Roch, R . , Booth, R., J. Chromatogr. Sci. 10, 1 5 1 - 3 ( 1 9 7 2 ) . 22 Wieber, J., HengstmaE, J., In: Ketamin, Neue 9

Ergebnisse In Forschung Und Klinik, Report of the 2nd Ketamine Symposium, Mainz, Apr. 7 2 , Edited by M. Gemperle et. al., Berlin, Springer-Verlag; Anaesthesiol. Resuscitation 6 9 , 146-50 (1973).

322

WILLIAM C. SASS AND SALVATORE A. FUSARI

113, 69-95 ( 1 9 7 5 ) . 2 3 . Moffat, A.C., J. Chromatogr. 24. Clarke, E.G.C., Isolation and Identification of Drugs, 1 9 6 9 , The Pharmaceutical Press, 1 7 Bloomsbury Square WC1, London, England.

2 5 . Kochhar, M.M., et. al., Res. Commun. Chem. 1 4 , 3 6 7 - 7 6 , June 7 6 . Pathol. Pharmacol. 26. Wieber, J . , et. al., Anaesthesist 2 4 , 260-3, June 75. 9(1), 2 7 . Kochhar, M.M., et. al., Clin. Toxicol. 2 0 - 1 . 1976. 114, 2 8 . Needham, L.L.,et. al., J. Chromatogr. 220-2, 1 2 NOV. 75. 2 9 . Krc, J., Parke, Davis & Co., Personal

-

Communication.

3 0 . Leavett, R . , Michigan State University,

Personal Communication. 31. Cohen, M.L., Trevor, A.J., J. Pharmacol. Exp. Ther., 1 8 9 , 3 5 1 - 8 , May 1 9 7 4 .

MINOCYCLINE

V M i m i r Zbinovsky and George P. Chrekian

VLADlMlR ZEINOVSKY AND GEORGE P. CHREKIAN

324

CONTENTS

1.

Description

1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color, Odor 2.

Physical Properties 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10

Infrared Analysis Nuclear Magnetic Resonance Spectrum Ultraviolet Spectra Mass Spectra Optical Rotation Thermogravimetric Analysis Differential Thermal Analysis Solubility Solvent Partitioning Data Crystal Properties

3.

Synthesis

4.

Stability, Isomerization, Degradation

5.

Pharmacodynamic Studies

6.

Methods of Analysis 6.1 6.2

Elemental Analysis Chromatographic Analysis

6.21 Thin Layer 6.22 Column 6.3

Direct Spectrophotometric Analysis

MINOCYCLINE

325

MINOCYCLINE HYDROCHLORIDE

1.

Description

1.1 Name, Formula, Molecular Weight Minocycline hydrochloride is known chemically as 4,7-bis (dimethylamino)l,4-4a,5,5a, 6,11,12a-octahydro-3,10, 12,-12a-tetrahydroxy-l,ll-dioxo-2-naphthacenecarboxamide monohydrochloride and by the trivial name 7-dimethylamino-6demethyl-6-deoxytetracycline hydrochloride.

OH .HCL CONHz OH

0

C23H2,NJO,.HCL

1.2

0

OH

MOL. Wt.:

493.94

Appearance, Color, Odor

Minocycline hydrochloride occurs as a yellow crystalline powder, It is essentially odorless and has a somewhat bitter taste. 2.

Physical Properties 2.1 Infrared Analysis1

The infrared spectrum of Minocycline HC1 (Lederle House Standard No, 7516B-172) is presented in Figure 1, In a multi-functional molecule like Minocycline HC1, most maxima represent a composite envelope of overlapping absorption peaks. In these cases it is not possible to uniquely

FIGURE 1 I n f r a r e d Spectrum of Minocycline HC1.2H20 i n KBr P e l l e t : Instrument: Ferkin-Elmer

FREQUENCY (CM-’)

WAVELENGTH (MICRONS)

21

MINOCYCLINE

327

a s s i g n maxima. Thus, t h e maximum a t a b o u t 2.9 IJ r e p r e s e n t s t h e NH2 s t r e t c h i n g of t h e 2-carboxamido, t o g e t h e r w i t h 1 2 hydroxy. The remainder of t h e broad a b s o r p t i o n up t o 5.0 !-I i s composed of t h e hydrogen bonded p h e n o l i c and e n o l i c hydroxy groups p l u s t h e hydrogen atom on t h e p r o t o n a t e d dimethylamino group. The maxima a t 6.07 1.1 i s t h e c a r b o n y l of t h e 2-carboxamido group, b u t t h e broad maxima c e n t e r e d a t about 6.25 1.1 i s a composite of conjugated hydrogen bonded k e t o n e s , p l u s t h e conjugated double bond systems p r e s e n t i n t h i s molecule. The maxima a t about 7 . 7 is a composite of t h e s t r o n g l y hydrogen bonded p h e n o l i c and e n o l i c hydroxyl groups p l u s a c o n t r i b u t i o n from t h e 2-carboxamido group and t h e maxima a t a b o u t 8.2 1.1 i s composed of r e l a t i v e l y unbonded p h e n o l i c hydroxy groups. 2.2

Nuclear Magnetic Resonance Spectrun’

The M.IR spectrum, F i g u r e 2 , i n hexadeuterodimethyls u l f o x i d e c o n t a i n i n g t e t r a m e t h y l s i l a n e as i n t e r n a l s t a n d a r d i s a s i n g l e s c a n on a HA-100D Varian Spectrometer. The s p e c t r a l assignments of Minocycline h y d r o c h l o r i d e are shown i n T a b l e I. TABLE I

NMR S p e c t r a l Assignments of Minocycline Hydrochloride Chemical S h i f t s ( A )

c-

NH2

2.60

S

2.94

S

4.34

S

7.41

d; J8,q = 8

6.83

d; J8,9 = 8

9.05

9.53

(2 broad s i n g l e t s )

It 0 C1o

-

OH

11.30

s = s i n g l e t ; d = d o u b l e t ; J = coupling c o n s t a n t i n Hz

MINOCYCLINE

329

2.3 Ultraviolet Spectrum Martell et a12 in 1967 determined the ultra-violet properties of Minocycline. They reported -

X max

in 0.1N HCL

X max

in 0 . 1 N NaOH 2.4

352 nm (log 263 nm (log 380 ~1 (log 243 nm (log

E

6

E E

4.16) 4.23) 4-30] 4.38)

Mass Spectrum1

The mass spectrum of Minocycline hydrochloride was run on an AEI MS-9 mass spectrometer and is shown in Figure 3 . At temperatures close to the melting point the salt decomposes to the free base and HC1, and the mass spectrum is a composite of both compounds. The molecular ion of Minocycline is fairly strong and is observed at m/e 457, consistent with the elemental composition C23H27N307. Loss of NH3,NH3 and (CH3)2NH, and C4H3N03 from the molecular ion affords ions at m/e 440, 395 and 344 respectively. A complete listing of the elemental composition of the major ions in the mass spectrum of Minocycline is available from Dr. R. T. Hargreaves, Lederle Laboratories. 2.5 Optical Rotation The following rotation was determinedl for Minocycline HC1.2H20 in 0.1N HC1: Cal

25

-

166',

conc. = 0.524

2.6 Thermogravimetric Analysis7 indicates that Minocyclineohydrochloride loses its water of hydrgtion between 75' and 150 and begins to decompose at about 175

.

2.7 Differential Thermal Analysis7 curves for Minocycline hydrochloride exhibit one melting and/or decomposition endotherm at 217

.

2.8

Solubility

Barringer et a13 in a monograph on Minocycline accumulated data related to unusual in vitro and in vivo properties of Minocycline and compared them to other tetracyclines antibiotics. The solubility of tetracyclines is a complex

2

w

mC

H

2

0 hl

V

Ti

X

..

U L)

a

a

rn

rl

l-l

330

0

"

v)

r

W

a

E

0 7 o a " m

0

0

N Ln

.. m

h

(u

0 . -.

u

0 I J

(I

w

P

W

h

nlln

o

"

L u

J

Y

I

w

I >

D

I na 0

0

h

m

" m

J

E

z CI

0 ..I 0

0

MlNOCYCLlNE

33 1

phenomenon. There are 16 p o s s i b l e i o n i c m i c r o s t r u c t u r e s f o r Minocycline. Thus, t h e observed s o l u b i l i t y is g e n e r a l l y n o t t h a t of a s i n g l e e n t i t y b u t r e p r e s e n t s t h e sum of t h e t o t a l of two o r mcre s p e c i e s i n a s o l u t i o n a t a given pH v a l u e . Minocycline, u n l i k e o t h e r a n t i b i o t i c s , c o n t a i n s two amino groups which a r e r e s p o n s i b l e f o r hundred-fold s o l u b i l i t y of Minocycline n e u t r a l i n water over t h a t of t e t r a c y c l i n e . The s o l u b i l i t y of Minocycline monohydrochloride d i h y d r a t e i n v a r i o u s s o l v e n t s and of Minocycline n e u t r a l i n water are given i n T a b l e I1 and Table I11 r e s p e c t i v e l y . TABLE I1

Aqueous S o l u b i l i t y of Minocycline a t 25OC.

mdml Neutral

pH 6.7

52

Hydrochloride

pH 3.9

15

Dihydrochloride

pH 0.8

>500

TABLE I11

S o l u b i l i t y of Minocycline Hydrochloride .2H7O i n Various S o l v e n t s a t 25'Cj %

Solvent

mdml

w/v

Hexane

0.004

0.0004

Benzene

0.02

0.002

Chloroform

0.13

0.013

Ethyl Acetate

0.3

0.03

Methyl E t h y l Ketone

0.4

0.04

1-Oc t a n o l

0.5

0.05

Ace t o n e

0.6

0.06

Dioxane

0.7

0.07

1-But ano 1

4.4

0.44

2-Pr opano 1

7

0.7

Methanol

14

1.4

Water

16

1.6

Abs. Ethanol

42

4.2

332

VLADlMlR ZBINOVSKY AND GEORGE P. CHREKIAN

2.9

P a r t i t i o n i n g Data

L i t e r a t u r e v a l u e s a c c o r d i n g t o C o l a i z z i and Klink4 f o r t h e a p p a r e n t p a r t i t i o n c o e f f i c i e n t s of Minocycline i n a w a t e r : n-octanol system a t v a r i o u s pH v a l u e s are r e p o r t e d i n T a b l e I V . The optimum pH v a l u e f o r t r a n s f e r i n t o t h e o r g a n i c phase is about 6.6 a t which pH t h e n e u t r a l z w i t t e r i o n i c form is predominant and a l s o c o i n c i d e s w i t h t h e i s o e l e c t r i c p o i n t of Minocycline. TABLE I V

Apparent P a r t i t i o n C o e f f i c i e n t s (Octanol/Aqueous B u f f e r ) of Minocycline Hydrochloride

2.1 0

3.9

5.6

6.6

8.5

0.051

1.11

1.48

0.36

2.10 C r y s t a l P r o p e r t i e s The X-Ray powder d i f f r a c t i o n p a t t e r n of Minocycline h y d r o c h l o r i d e is shown i n Table V.

M INOCYCLI N E

333

TABLE V Powder X-Ray Diffraction Pattern of Minocycline HC15 d (Ao)*

I/IO**

12.0 7.05 6.60 5.70 5.20 4.95 4.73 4.45 4.28 4.00 3.82 3.68 3.56 3.43 3.26 3.03 2.86 2.73 2.67 2.60 2.44 2.31 2.25 2.13 2.06 1.96 1.91 1.85 1.72 1.52 1.20

* **

d = (interplanar distance)

0.15 1.00

0.04 0.08 0.07 0.09 0.09 0.01

0.06 0.04 0.15 0.50 0.45 0.02 0.40 0.04 0.05 0.02 0.02 0.01 0.06 0.02 0.02 0.02 0.01 0.01 0.01

0.03 0.02 0.01

0.02 n X 2 sin 0, X = 1.539A0

Based on highest intensity of 1.00 Radiation: Kal, and Ka2 Copper

334

3.

VLADlMlR ZBINOVSKY AND GEORGE P. CHREKIAN

Synthesis

P r e v i o u s s y n t h e s i s of Minocycline was achieved by a sequence of r e a c t i o n s based on n i t r a t i o n of 6-demethyl-6-deox y t e t r a c y c l i n e 2 . I n t h i s s y n t h e s i s two isomers (7 and 9 n i t r o ) were formed. Removal of u n d e s i r a b l e 9 - n i t r o isomer involved t e d i o u s procedures. L a t e l y , L. B e r n a r d i and a s s o c i a t e s 6 were a b l e t o b l o c k p o s i t i o n 9 w i t h a t e r i a r y b u t y l group and t h u s s i m p l i f y t h e r e a c t i o n and improve t h e y i e l d s . The r e a c t i o n scheme of t h i s new s y n t h e s i s is g i v e n i n F i g u r e

4. 6-demethyl-6-deoxytetracycline (I) was a l k y l a t e d t o g i v e (11) w i t h excess of t e r t i a r y b u t y l a l c o h o l and methane s u l f o n i c a c i d . By adding f o u r e q u i v a l e n t s of m02, compound (111) w a s o b t a i n e d i n 76% y i e l d based on ( I ) . I n t e r m e d i a t e compound (111) was c a t a l y t i c a l l y reduced over Pt02 t o g i v e 7-amino- 9- t e r t i a r y butyl-6-demethyl-6-deoxyt e t r a c y c l i n e (IV) which was t h e n r e d u c t i v e l y methylated t o (V). The l a s t s t e p involved t h e removal of t h e t e r t i a r y b u t y l group from p o s i t i o n 9. T h i s was accomplished by u s i n g t r i f l u o r o m e t h a n e s u l f o n i c a c i d w i t h 63% y i e l d . 4.

S t a b i l i t y , I s o m e r i z a t i o n , Degradation

I n t h e dry-powder s t a t e t h e Minocycline, l i k e o t h e r t e t r a c y c l i n e s , i s s t a b l e a t l e a s t 3-4 y e a r s when s t o r e d a t 0 room t e m p e r a t u r e ( 2 5 C). Minocycline, l a c k i n g hydroxyl groups a t both C5 and c6 does n o t form t h e anhydro, iso, o r e p i compounds, which a r e t h e common d e g r a d a t i o n compounds formed from o t h e r t e t r a c y c l i n e a n t i b i o t i c s . However, i t r e a d i l y undergoes b o t h 4-epimerization and o x i d a t i v e d e g r a d a t i o n . S i n c e t h e D r i n g of Minocycline i s a s u b s t i t u t e d p-aminophenol, i t i s more s u s c e p t i b l e t o o x i d a t i o n t h a n o t h e r t e t r a cyclines. S t a b i l i t y d a t a f o r s o l u t i o n s of Minocycline a t v a r i o u s pH v a l u e s a r e summarized i n T a b l e V I . Minocycline s o l u t i o n s a t pH 4 . 2 and 5.2 r e t a i n e d 90% of t h e i r i n i t i a l potency f o r 1 week a t room temperature. These s o l u t i o n s were more s t a b l e t h a n any o t h e r t e t r a c y c l i n e a n t i b i o t i c s o l u t i o n s t u d i e d . However, none of t h e t e t r a c y c l i n e a n t i b i o t i c s are s t a b l e enough t o p e r m i t t h e p r e p a r a t i o n of a p r e c o n s t i t u t e d aqueous s o l u t i o n a s a p r a c t i c a l dosage form. The a d d i t i o n a l amino group i n Minocycline, b e s i d e s cont r i b u t i n g t o i n c r e a s e d s o l u b i l i t y of Minocycline n e u t r a l i n w a t e r , i s a l s o r e s p o n s i b l e f o r d i f f e r e n c e s i n physico-chemical

d w

335

t

t

336

VLADlMlR ZBINOVSKY AND GEORGE P. CHREKIAN

and p h y s i o l o g i c a l p r o p e r t i e s . The i s o e l e c t r i c p o i n t of Minocycline is a f u l l pH u n i t h i g h e r (pH 6 . 4 ) t h a n t h a t of most o t h e r t e t r a c y c l i n e a n t i b i o t i c s (pH ca. 5 . 5 ) and consequently has a p o t e n t i a l therapeutic significance. This property accounts f o r i t s g r e a t e r p a r t i t i o n i n g c h a r a c t e r i n t o l i p o i d material a t e s s e n t i a l l y n e u t r a l pH, i n c l u d i n g t h y r o i d , b r a i n and f a t t i s s u e . TABLE V I

Minocycline S o l u t i o n S t a b i l i t y Data % I n i t i a l A c t i v i t y Retained Days S t o r e d a t 2SoC

PH 0.5

1

1.5

2

1.85

96

94

91

22

2.5

97

95

93

81

3

4

7

8

9

11

14

4.2

99

96

98

95

90

91

90

87

84

5.2

98

98

98

96

92

89

85

81

72

6.2

98

95

93

89

76

72

64

53

37

5.

Pharmacodynamic S t u d i e s

R. C. K e l l y and A s s o c i a t e s 8 found t h a t t h e maximum serum c o n c e n t r a t i o n of Minocycline was a t t a i n e d by t h e f i r s t sampli n g a t 1 hour and t h a t serum h a l f l i f e a f t e r o r a l a d m l n l s t r a t i o n of Minocycline w a s 1 6 hours.

Minocycline showed e x c e l l e n t t i s s u e p e n e t r a t i o n due t o i t s h i g h e r z w i t t e r i o n i c form which is predominant a t pH 6 . 6 , a p p r o x i m a t e l y one pH u n i t h i g h e r t h a n f o r o t h e r t e t r a c y c l i n e s . An advantage f o r t h i s h i g h l y l i p o p h y l i c t e t r a c y c l i n e h a s been p o s t u l a t e d i n terms of t h e r a p u e t i c e f f i c a c y , i . e . a r a p i d and h i g h c o n c e n t r a t i o n of a n t i b i o t i c where r e c o r d e d . Okubo and a s s o c i a t e s 9 e s t a b l i s h e d t h a t i n r a t s a f t e r a s i n g l e o r a l dose, c o n c e n t r a t i o n s i n a l l t i s s u e d s t u d i e s were h i g h e r t h a n i n blood. When t h e Minocycline was a d m i n i s t e r e d t o p a t i e n t s b e f o r e s u r g e r y , a similar h i g h t i s s u e - b l o o d r a t i o was found a f t e r

MINOCYCLINE

337

t h e organ was removed. The h i g h e s t accumulation of Minocyc l i n e w a s found i n g a l l b l a d d e r , t h y r o i d , duodenum and l i v e r . Minocycline i s metabolized t o i n a c t i v e s u b s t a n c e s t o a g r e a t e r e x t e n t t h a n o t h e r known t e t r a c y c l i n e s . 6.

Methods of A n a l y s i s 6.1

Elemental A n a l y s i s f o r C23H27N307HC1.2H20

Element

% Theory

Reported Ref.

C

52.12

52.12

H

6.09

6.19

N

7.93

7.79

c1

6.69

6.72

6.2

Chromatographic A n a l y s i s 6.21 Thin Layer Chromatographic A n a l y s i s

S e p a r a t i o n and 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 of Minocycline i n t h e p r e s e n c e of r e l a t e d minor components w a s achieved on diatomaceous e a r t h , used as s u p p o r t i n g phase. P l a t e s were p r e p a r e d by s p r e a d i n g i n t o a t h i n l a y e r a m i x t u r e of diatomaceous e a r t h , pH 6 EDTA b u f f e r , p o l y e t h y l e n e g l y c o l 400 and g l y c e r i n . P l a t e s were developed w i t h a s o l v e n t cons i s t i n g of a m i x t u r e of pH 6 EDTA b u f f e r and e t h y l a c e t a t e cyclohexane ( 9 : 2 ) . T h i s system was p r e v i o u s l y used by P . P. AscionelO i n a s e p a r a t i o n of o t h e r t e t r a c y c l i n e s by t h i n l a y e r chromatography. The Rf of Minocycline i n t h i s system was approximately 0.2. By rechromatography i n t h e same system t h e Minocycline s p o t can be moved h a l f way on t h e p l a t e , t h u s g i v i n g complete s e p a r a t i o n from t h e r e l a t e d compounds. 6.22 Column Chromatographic A n a l y s i s Minocycline and r e l a t e d i m p u r i t i e s were s e p a r a t e d on an a c i d - s o l v e n t washed diatomaceous e a r t h column.11,12 Supporting phase was p r e p a r e d by mixing t h e d i a tomaceous e a r t h w i t h 5% v / v p o l y e t h y l e n e g l y c o l 400 (PEG-400)g l y c e r i n e m i x t u r e i n 0.lM EDTA pH 6 b u f f e r . Minocycline and r e l a t e d compounds were e l u t e d w i t h s t e p w i s e i n c r e a s i n g p o l a r i t y of t h e chloroform-cyclohexane m i x t u r e and determined s p e c t r o p h o t o m e t r i c a l l y a t 358 tun. 98-102% r e c o v e r y of t h e t o t a l s p e c t r a l v a l u e of t h e charge was o b t a i n e d .

338

VLADlMlR ZBINOVSKY AND GEORGE P. CHREKIAN

6.3

Direct Spectrophotometric Analysis

U. V. Absorption maximum of Minocycline a t 358 nm has been e x t e n s i v e l y used f o r a s s a y p u r p o s e s , e s p e c i a l l y f o r r e a d i n g of column e l u a t e s . The c o n c e n t r a t i o n of 1 6 micrograms p e r m l w a s used i n a c i d i f i e d methanol-chloroform s o l u t i o n .

Minocycline HC1 h a s a d i s t i n c t i n f r a r e d spectrum which can be used i n q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s i s .

-

A l i n e a r concentration a b s o r p t i o n r e l a t i o n s h i p was achieved by Ace and J a f f e , 1 3 u s i n g pH 6.5 b u f f e r i n an e x t r a c t i o n of Minocycline. The f l u o r e s c e n c e of t h e f i n a l p r o d u c t was r e a d a t a n e x c i t a t i o n wavelength of 380 run and a n emission wavelength of 480 nm u s i n g a f i l t e r c o l o r i m e t e r .

MI NOCYCLI N E

339

REFERENCES 1. W. Fulmor, L e d e r l e L a b o r a t o r i e s , p e r s o n a l communication.

2.

M. J. M a r t e l l , J . H. Boothe, J . Med. C h e m . , x ,

44 (1967).

3.

W. C. B a r r i n g e r , W. S h u l t z , C. M. S i e g e r and R. A. Nash, Am. J . of Pharmacy, 179 (1974).

4.

J . L. C o l a i z z i , P . R. Klink, J . Pharm. S c i . ,

146,

58,

1184

(1969).

5.

P . Monnikendam, L e d e r l e L a b o r a t o r i e s , p e r s o n a l communication.

6.

L. B e r n a r d i , R. D e C a s t i g l i o n e , V. Colonna, P. Masi, I1 Farmaco, Ed. Sc., 30 736 (1975).

7.

L. M.

8.

Brancone, L e d e r l e L a b o r a t o r i e s , p e r s o n a l communication.

R. G. K e l l y , L. A. Kanegis, T o x i c o l , Appl. Pharmacol.,

11, 1 7 1 (1967). 9.

10.

H. Okubo, Y. Fujimoto, Y . Okamoto, J . Tsukada, J a p . J. A n t i b i o t . , 22, 430 (1969).

P. P. Ascione, J . B. Zagar, and G. P. Chreklan, J. Pharm. S c i . , 56, 1393 (1967).

11. P. P. Ascione, L e d e r l e L a b o r a t o r i e s , p e r s o n a l communlcat ion. 12.

P. P. Ascione, J. B . Zagar and G . P . Chrekian, J . Pharm. S c i . , 56, 1396 (1967).

13.

L. N. Ace and J . N. J a f f e , Bioch. Medicine, (1975)

12, 401

NYSTATIN

Gerd W.Michel

GERD W. MlCHEL

342

TABLE OF CONTENTS 1.

DESCRIPTION 1.1 1.2

1.3 2.

Name, Formula, Molecular Weight, Elemental Composition Appearance, Color, Odor Standards and Regulatory Status

PHYSICAL PROPERTIES 2.1

Crystal Properties 2.1.1 2.1.2

2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16

Optical Crystallographic Properties X-Ray Powder Diffraction

Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Fluorescence Spectrum Mass Spectrum Optical Rotation Optical Rotatory Dispersion Melting Range Differential Thermal Analysis Thermogravimetric Analysis Solubility Countercurrent Distribution Ionization Constants Aggregation Polarography

3.

BIOSYNTHESIS

4.

METHODS OF MANUFACTURE 4.1 4.2 4.3

Historical Microbiological Processes Isolation and Purification Processes

NYSTATIN

TABLE OF CONTENTS (Cont'd) 5.

STABILITY - DEGRADATION 5.1

Dry Thermal Degradation

5.1.1 5.1.2 5.1.3 5.1.4 5.2 5.3 5.4 5.5

Stability Stability Stability Stability

of of of of

Amorphous Product Crystalline Product Solid Dosage Forms Ointment Formulations

Stability in Solution Stability under Radiation Microbial Degradation Stabilization

6. METHODS OF ANALYSIS 6.1 6.2 6.3 6.4 6.5

Elemental Analysis Neutralization Equivalents Identification Tests color Reactions Direct Spectrophotometric Analysis 6.5.1 6.5.2 6.5.3

6.6 6.7

Fermentation Liquids and Products Pharmaceutical Preparations Other Applications

Colorimetric Analysis Chromatographic Analysis 6.7.1 6.7.2 6.7.3 6.7.4

Paper Chromatography Thin-Layer Chromatography Gas-Liquid Chromatography High Performance Liquid Chromatography

Electrophoretic Analysis Polarographic Analysis 6.9 6.10 Titrimetric Analysis 6.11 Microbiological Methods

6.8

7. REFERENCES 8.

ACKNOWLEDGMENT

343

344

GERD W. MICHEL

1. DESCRIPTION

1.1 Name, Formula, Molecular Weight, Elemental Composition Nystatin is a prominent member of a relatively large and varied group of structurally related, highly unsaturated antifungal antibiotics produced by various strains of streptomycete species of microorganism^^-^. Based on their chemical structure - and to distinguish them from numerous other antibiotics which also have antifungal properties8 , - this group of important therapeutic agents is commonly referred to as the polyene macrolide antifungal antibiotics. All members within this class of antibiotic agents have in common (a) a macrocyclic ring of carbon atoms closed by lactonization, and (b) the presence of a series of conjugated carbon double bonds. The latter grouping represents the chemically most characteristic feature of polyene macrolides and serves to further classify this group of natural products into tri-, tetra-, penta-, hexa- and heptaenes, according to the type of conjugated chromophore present in the molecule2 110-15. Attempts at complete tabulation of all presently known polyene antibiotics within this class have been published in several comprehensive review articles4rl2r 13 I i6-27. Following the above nomenclature, nystatin may be chemically classified as a tetraene macrolide antibiotic. ~ lthe Division of Isolated in 1950 by Hazen and B r ~ w n ~ * -of Laboratories and Research, New York State Department of Health, Albany, N.Y., it was the first of the polyene macrolides to be discovered and is since produced biosynthetically on large scale by fermentation with strains of Streptomyces n ~ u r s e i ~ ~ 33, S. albulus34-36 and S. aureus3r6,32r34. Initially called fung~cidin28r29132,it was later given the name nystatin (N.Y. State-in)4~32, but is also listed under several other proprietary synonyms3 37-40: Moronal, Mycomycin, Mycostatin, Nilstat, Nitacin, Nystan and Stamicin. The designation most commonly used in the chemical, pharmaceutical and medical reference l i t e r a t ~ r e ~ ,~ including , ~ ~ - ~ ~ Chemical Abstracts , is nystatin. As is true for many polyene macrolide antibiotics, a complete and satisfactory chemical characterization of nystatin with respect to its precise molecular structure, stereochemistry and absolute configuration is still outstanding,

345

NYSTATIN

despite extensive efforts in a number of l a b o r a t o r i e ~43-60. ~~ Early degradation studies by several investigator^^^-^' established the antibiotic to be a macrocyclic C41-polyene lactone linked glycosidically to the pyranose form of the amino sugar mycosamine (3-amino-3,6-dideoxy-g-mannose)43-48. While the structure of the aglycone portion of the molecule (nystatinolide)46, containing a diene and tetraene chromophore, has been deduced from the isolation of degradation products, Chong and R i ~ k a r d shave ~ ~ only recently provided experimental evidence, subsequently confirmed by Borowski et a1.59, for a glycosidic linkage of the sugar moiety to the C-19 position of the aglycone. Present knowledge therefore suggests the nystatin molecule to be identical with structure 158-61, without regard to its stereochemistry. I

I Molecular Weight:

926.13

Very recent work58i60 has indicated that nystatin, in its crystalline state and in neutral hydroxylic solutions at ambient temperatures, may exist in the hemiketal form rather than the hydroxy-ketone structure (I) depicted above. In analogy to amphotericin B6* 63, a structurally related polyene macrolide whose crystalline N-iodo-acetyl derivative was found to exist as a cyclic hemiketal, a pyranoid hemiketal linkage (111) in nystatin could arise from the formation of an oxygen bridge between carbon atoms 13 and 17 of the hydroxy-ketone moiety (II), according to the following scheme:

346

GERD W. MlCHEL

OH

7

COOH

17

C OOH

OH

While the available chemical evidence supports the structural characteristics of nystatin as outlined above, it should also be noted, however, that commercial nystatin products are not necessarily homogeneous compounds, but may reresent mixtures of chemically closely related components56, 8159. Shenin et al.561 for instance, examined several lots of pharmaceutical grade nystatin (including the International Standard) by countercurrent distribution in a suitable solvent system and found all products to contain two Chemically distinct components, A1 and A2, in varying proportions. In a more recent study, Porowska et a1.64165 adopted the same technique under modified conditions to demonstrate that some commercial nystatin products may, in fact, be separated into three distinctly different constituents (designated nystatin A l r A 2 and A3), two of which (A1 and A2) are apparently identical with those characterized by Shenin et a1.56, while the third constituent (A3) represents another tetraene component, also shown to be part of the polifungin-A complex produced by Streptomyces noursei var polifungini66-69.

--

.

The lack of uniformity between individual nystatin products generated under a wide variety of possible fermentation conditions16127 ,70:71 , combined with the exceptional difficulties normally encountered in the isolation of strictly pure materials, poses unique problems in a satisfactory analytical characterization of this widely produced chemotherapeutic agent, at present. As a result, depending on the source, purity and uniformity of the examined sample, reported physico-chemical property data on nystatin can be expected to vary over a wide range and are not necessarily characteristic for the uniform, highly purified compound. Thus, for the purpose of this profile and in an attempt to overcome some of the obvious discrepancies between various literature data, a typi-

NYSTATI N

347

cal production lot (Squibb Research Standard #MYNM-lSO-RP) has been selected for characterization by the more common analytical methods, and reference is made to it whenever possible. 1.2 Appearance, Color, Odor

Nystatin is a light yellow to yellow crystalline powder with a faint, characteristically musty odor; slightly hygroscopic and light-sensitive. 1.3

Standards and Regulatory Status

The biological activity of commercial preparations of nystatin is expressed in units per mg, based on a potency of 1000 units per mg originally assigned to a batch of nystatin set aside by the FDA for reference purposes as the first primary standard. Since then, improved isolation techniques have led to the production of materials with substantially increased potencies. However, the first primary reference is still in use as a reference point in the assignment of potency values to later working standards40a. A.

FDA and USP Standards

The most recently adopted FDA standard material, after collaborative assay by the National Center for Antibiotic Analysis (NCAA) and other laboratories, has been defined to contain 6088 units per mg72; this material is identical with the current USP Reference Preparation of Nystatin. B.

International Standard

An international collaborative study of nine laboratories in six countries resulted in the adoption of a first International Standard (WHO Standard) for Nystatin by the World Health Organization Expert Committee on Biological Standardization in 196373. The reference material selected f o r this study was assayed against the USP Reference Preparation of Nystatin available at that time and was established to contain 3000 International Units (IU) per mg. Accordingly, the International Unit of Nystatin is defined as the activity in 0.000333 mg of the International 73. The methodology associated with standardization and revised outlines of the recommended standard microbiological assay procedures have been reported recently74 and are recorded in the Code of Federal Regulations75.

GERD W. MlCHEL

348

The minimum allowable potency for commercial nystatin products was reviewed by the Food and Drug Administration during 1973 and raised from 2000 units to 4400 units per mg, effective 197576. Official monographs for nystatin are listed in the United States Pharmacopeia XIX41 and British Pharmacopeia 197342. 2.

PHYSICAL PROPERTIES 2.1

Crystal Properties 2.1.1

Optical Crystallographic Properties

The following optical crystallographic constants of nystatin (without reference to crystal system and habit) have been reported7’, 78: Optic Sign: + Elongation: Extinction: para1le1 Refractive Indices: na = 1.512 nB = 1.583 n = 1.682

Y

2.1.2

X-Ray Powder Diffraction

To date, three distinctly different crystal forms of nystatin, referred to as Types A , B and C, have been observed79. A l l three forms are readily identified by their characteristic X-ray powder diffraction patterns80 (Section 2.1.2) , solid-state infrared spectrael (Section 2.2) and thermal behaviouraO (Section 2.10) The more commonly occurring forms, Types A and B, are known to be interconvertible82 on changes in environmental moisture content and apparently represent crystal modifications with different degrees of hydration.

.

The X-ray powder diffraction data80 for crystal forms A, B and C are given in Tables I and 11, respectively, and their corresponding diffraction patterns are presented in Figure 1 (Squibb Res. Std. #MYNM-150-RP, Type A), Figure 2 (Squibb Res. Std. #MYNM-150-RP/HI Type B), and Figure 3 (Squibb Res. Std. #WSC-08982-FPI Type C), respectively.

NYSTATI N

349

TABLE I X-Ray Powder Diffraction Patterns of Nystatin Type B

Type A

Squibb Res. Std. #MYNM-150-RP Squibb Res. Std. #MYNM-150-RP/H (Figure 1) (Figure 2) d

(8)*

29.0 10.5 10.1 8.70 7.80 7.10 6.34 6.0 5.31 4.76 4.45 4.32 4.08 3.79 3.23

I/I,** v

0.34 0.32 0.15 0.22 0.11 0.22 0.85 0.29 0.37 0.17 0.85 1.00 0.78 0.39 0.16

25.0 12.6 10.8 8.60 8.00 6.90 6.43 5.90

4.98 4.52 4.20 4.00 3.77 3.13 0

*d = Interplanar distance (A),

nh

0.27 0.40 0.15 0.26 0.17 0.46 0.36 0.47 0.48 0.92 0.70 0.69 1.00

0.17

2 sin 0 **I/Io = Relative intensity (based on highest intensity of 1.00) Radiation:

Koll

and Ka2 Copper

350

GERD W. MICHEL

TABLE I1 X-Ray Powder Diffraction Pattern of Nystatin Type C Squibb Res. Std. #WSC-08982-FP (Figure 3) d

(g)*

1/1

-0-

**

0.19

25.0 20.0 9.30 7.15 6.28 5.90 5.60 5.26 5.15 4.67 4.51 4.27 4.19 4.10 4.00 3.68 3.60

0.80

0.26 0.20 0.93 0.20 0.64 0.59 0.30

0.60 0.55 0.59 0.46 1.00 0.27 0.21 0.47 0

*d = Interplanar distance ( A ) ,

nX sin

**I/Io = Relative intensity (based on highest intensity of 1.00) Radiation:

K

Crl

and K

a2

Copper

Figure 1.

X-Ray Powder Diffraction Pattern of Nystatin, Type A

(Squibb Res. Std. #MYNM-150-RP) Instrument: Philips Norelco Diffractometer

1

I

4:Il

I

I

w

N UI

Figure 2.

X-Ray Powder Diffraction Pattern of Nystatin, Type

(Squibb Res. Std. #MYNM-150-RP/H) Instrument: Philips Norelco Diffractometer

.o

0

d

N

w m w

Figure 3.

X-Ray Powder Diffraction Pattern of Nystatin, Type c (Squibb Res. Std. #WSC-08982-W) Instrument: Philips Norelco Diffractometer

354

GERD W. MICHEL

2.2

Infrared Spectrum (IR)

The infrared absorption spectrum” of nystatin (Squibb Res. Std. #MYNM-150-RPI Type A ) as a mineral oil mull is presented in Figure 4 . A spectrum of the same standard taken as a potassium bromide pellet (1.5 mg/300 mg KBr) was essentially identical to the one presented. Tentative assignments for some characteristic infrared absorption bands18153183-85are listed in Table 111. Table I11 Infrared Spectral Assignments for Nystatin (Squibb Res. Std. #MYNM-150-RPI Type A) Frequency (cm-’) 998

1065 1375 1448 1572 1705 3300-3500

Vibrational ModeE6‘ 87 CH Deformation (out-of-plane) in -CH=CH- (trans) C-OH Stretching CH3 Deformation (sym. CH3 Deformation (aSym.1 CH2 Deformation Carboxylate Ion18 1 83 Lactone (unstrained)l8rE3 NH, OH Stretching83

The IR spectrum shown in Figure 4 is in substantial agreement with spectra previously published by J.D. Dutcher et ~ ~ A.O.3 Hayden , et al.5t88 (Spectrum #85 in Hayden’s compendium of spectra measured on a Perkin-Elmer Model 21 spectrophotometer with sodium chloride prism) and H. Umezawa8’. Examination of the solid-state IR spectra (mineral oil mull) of crystal forms Type B and Type C, presented in Figures 5 and 6, resp., reveals distinct absorbance differences both between these two modifications and in their relation to the Type A form (Figure 4): In the Qpe B modification, for instance, the absorption band assigned to the carboxylate ion is shifted to 1560 cm-l, while the comparatively sharp band associated with the lactone carbonyl stretching vibration is observed near 1745 cm-l. In addition to several other absorption changes, relative to the Type A form, in the 900-1000 cm-l and 1350-1420 cm-1 regions, this form also displays a band of medium intensity near 1640 cm-1.

355

4

a,

Infrared Spectrum of Nystatin, Type A (Squibb Res. Std. #MYNM-150-W) Mineral Oil Mull Instrument: Perkin-Elmer Model 621 rl

cv

WAVELENGTH (MICRONS)

3500

2500

ZOO0

1800

1600 FREQUENCY

Figure 5.

la00

(CM’)

1200

1OO0

Infrared Spectrum of Nystatin, Type B (Squibb Res. Std. #MYNM-150-RP/H) Mineral Oil Mull Instrument: Perkin-Elmer Model 621

800

600

200

357

7

a, &

I n f r a r e d Spectrum of N y s t a t i n , Type C (Squibb R e s . S t d . #WSC-08982-FP) Mineral O i l M u l l Instrument: Perkin-Elmer Model 621

358

GERD W. MICHEL

The Type C form, in contrast, is characterized by two neighboring, sharply resolved absorption bands near 990 and 1005 cm-1, not present in either Type A or Type B crystal form. An additional band appears in the C-0-C stretching region near 1040 cm-l, while the relatively strong, broad absorption at 1540 cm-1, assigned to the ionized carboxyl group, is complemented by two weak, but definite bands at 2630 and 2700 cm-l and the presence of a broad absorption near 2090 cm-1, both typical for the zwitterionic structure of amino acids86. Another strong, symmetrical band in the functional group region at 1695 cm-1 can be attributed to the lactone carbonyl stretching frequency. Of special diagnostic value in the identification of the Type C crystal form, however, is a sharp absorption band at 3600 cm-l, absent in both Type A and Type B modifications and tentatively assigned to the "free" OH stretching mode of a cyclic hemiketal linkage (between C-13 and C-17)90. 2.3

Nuclear Magnetic Resonance Spectrum (NMR)

The 100 MHz NMR spectrumg1 of nystatin is shown in Figure 7. Proton assignments for the observed chemical shifts are tabulated below. Table IV NMR Spectral Assignments for Nystatin (Squibb Lot #88645) Chemical Shift (ppm) 0.87 (6.0 Hz) 0.97 (6.0 Hz) 1.10 1.16 1.44 1.83 2.26 2.78 3.18 5.06 5.58 5.98 6.21

Mu 1tiplicity Doublet Doublet Mu1tip1et

Assignment Secondary Methyl Group I,

11

,I

I,

,I I,

I,

II

,I

Methylene Proton I,

II

I1

11

0,

I,

Methine Proton (-CEO-) I1

"

(-CFOC=O)

Olefinic Proton Mu1tiplet

I,

,I

II

I,

In addition, broad resonance occurs at 6 3.92 (NH2, OH, H20) which is exchanged with D2O91.

2 )1

1

'

1

1

.

1

'

" "

I

" 4

1

1

' ' I

1

" 1 ;

1 ' '

1,'

'

Figure 7.

1

' ','"';"

1 ' ;

'

I

";'

I 1

' ' , I

' I '

j

"

'

'11'

I " , '

"

I/,'

NMR Spectrum of Nystatin (Squibb Lot #88645) Solvent : DMS0-d 6 Instrument: Varian Model XL-100-15

'

' I : "

' 1 ; ' '

I1,""

L

360

GERD W. MICHEL

2.4

Ultraviolet Spectrum ( W )

In agreement with the classification of nystatin as a polyene macrolide containing a conjugated tetraene and a diene chromophore, its ultraviolet spectrum exhibits three intense, very sharp absorption bands, separated by narrow valleys, in the region between 280 and 340 nm, typical for the tetraene chromophore and characteristic for several other polyene macrolide antibiotics in the same chemical categoryllil2113, 15,18a,92,93. The ultraviolet absorption spectrumg4 of nystatin reproduced in Figure 8 was obtained from a methanol solution of Squibb Res. Std. #MYNM-150-RP at a concentration of 1.076 mg per 100 ml of methanol. Since methanolic solutions of nystatin are known to have a limited stability, the spectrum was recorded within 10 min. after sample preparation. Under these conditions, the following three principal absorption bands were obtained: Xmax

nm 280 (sh) 291 304 318

E

(l%, 1 cm) 298 567 866 789

These three distinct, regularly spaced peaks characteristic for unhindered, coplanar systems of conjugation - form the main absorption bands for nystatin and are assigned to the tetraene chromophore (possibly an all-trans configuration)12,18a,83,95. A minor inflection5r 32 8 3 f is noted at 280 nm (El' = 298), and an additional band at 231 nm of lower absorptit??y (Ei:m= 290) has been attributed to the diene linkage (trans,trans-1,4-disubstituted)lea,83.

The spectrum is in good agreement with the absorbances originally recorded for nystatin by Brown and Hazen3', by Dutcher -et a1.32,83,95 and those documented by other investigators, as listed in Table V. Two similar spectra of nystatin, measured as methanol solutions in the presence of 0.1% of glacial acetic acid and 0.1% of 0.1N sodium hydroxide, respectively, are listed in the collection of USP and NF reference standards compiled by

TABLE V

U l t r a v i o l e t A b s o r p t i o n of N y s t a t i n

Source Bolshakova e t a l . Brown a n d Hazen Doskochilova and G e s s Dutcher Dutcher e t a l . Dutcher e t a l . H a m i 1t o n - M i l l e r O r o s h n i k a n d Mebane Oroshnik e t a l . Shenin e t a l .

Umezawa Vining e t a l .

Reference

Xhmax (nm)

52 30 96 95 32 83 97 18a 12 56 89 13

291,304,318 291,305,319 230,292,304.5,318 230,290,305,320 292,304.5,318 231,292,305,320 292,306,321 230,291,304,318.5 292,304.5,318 230,291,304,319 235,291,304,319 292,304.5,318

c

c 2

362 -4J

Ultraviolet Spectrum of Nystatin (Squibb Res. Std. #MYNM-150-=) Solvent: Methanol (1.076 mg/100 ml) Instrument: Cary 11 Spectrophotometer

k

a,

NYSTATI N

363

.

Hayden et al 5, 88. The corresponding absorbance maxima (#85 of Hayden’s compendium) are quoted as follows: 280,290,304 and 318 nm (in acidic medium), and 230,280,290 and 317 nm (in alkaline medium). The special nature of the ultraviolet absorption spectra of polyene macrolide antibiotics and their significance in the interpretation of structural differences between closely related Streptomyces antifungal polyenes are thoroughly discussed in a review article by Oroshnik and Mebanelaa. 2.5

Fluorescence Spectrum

Schroeder et al. utilizing a computer-centered combination spectrophotometer-spectrofluorometer system, examined the fluorescence properties of freshly prepared aqueous nystatin solutions (8.39 pM in 0.05M citrate-phosphate buffer, pH 4, containing 0.3% dimethylsulfoxide) and observed corrected maxima for excitation and fluorescence, respectively, at 323 and 402 nm. Similar activation and emission data are reported by Kading9 for dilute solutions of nystatin in a 1:l (by vol.) methanol/water system containing approximately 5 micrograms of substrate per nl of solvent. under these conditions, using a Perkin-Elmer Model 204 fluorescence spectrometer, excitation maxima were observed at 310 and 321 nm, with corresponding maximum fluorescence emission at 429 and 409 nm, respectively. The excitation and emission s ectra of nystatin (Squibb Lot #88645), recorded by NooneYo0 and obtained from a methanol solution at a concentration of 10 ppm, are presented in Figure 9. Excitation at 325 nm produced emission with a maximum at 422 MI. 2.6

Mass Spectrum

The use of mass spectrometry with respect to nystatin has been limited to the determination of molecular weights and the identification of cleavage products in early structure elucidation studies5*157-591 but has not been extended to investigations of the intact, underivatized molecule, most likely because of inherent problems associated with its high molecular weight and the complex, polyfunctional nature of the molecule. Recently , however , Haegele and DesideriolOl examined the pertrimethylsilylated (per-TMS) derivative of nystatin and

100

-

90-

200 Figure 9.

440 520 Wavelength - nanometers

280

360

Fluorescence Spectra of Nystatin (Squibb Lot #88645) Solvent: Methanol Instrument: Aminco-Bowman SPF

600

NYSTAT I N

365

reported its complete low resolution mass spectrum, including a detailed rationalization for the genesis of the observed ion species and a proposal for the respective fragmentation pathways. The mass spectral fragmentation pattern of per-TMS nystatin is characterized by consecutive losses of MTS, TMSOH and the mycosamine moiety, with the most abundant ions in the low mass range of the spectrum arising from the amino sugar portion of the molecule. The authors101 conclude that the apparent driving force behind most of the fragmentation processes is to be sought in the energetically favored extension of the conjugated polyene system to a highly conjugated ion species (m/e 870) and the production of neutral molecules, facilitated by the stability of the leaving groups - TMSOH and the amino sugar moiety. Other important features of the mass spectrum of perTMS nystatin include: Loss of a TMS group produces an ion cluster at m/e 1716; elimination of three molecules of TMSOH from m/e 1716 leads to the formation of ions at m/e 1626, 1536 and 1446.

Elimination of the amino sugar portion - with retention of the glycosidic oxygen by the aglycone - produces the [M-362]+ ion at m/e 1427; it loses in succession eight molecules of TMSOH to form the respective ion species. Expulsion of the neutral sugar moiety forms the iM-3791' ion at m/e 1410; the required hydrogen atom for this process is postulated to arise from C-18 to produce an ion in which the conjugation is extended. Up to six molecules of TMSOH are then eliminated from this ion to form a series of ions (m/e 1320, 1230, 1140, 1050, 960) and to produce finally the highly conjugated ion at m/e 870.

Loss of one and two molecules of TMSOH from [MI+ generates ions at m/e 1699 and 1609. The proposed fragmentation mechanisms have been corroborated by stable deuterium isotope (dg) derivatives and were confirmed by accurate mass measurements. For the formation of the TMS derivative, standard published procedures were followed by the authorslol without

366

GERD W . MICHEL

modification. Low resolution mass spectra were obtained with an Atlas/Varian CH-7 mass spectrometer and high resolution spectra on a DuPont/CEC 21-llOB instrument. Detailed instrumental conditions are givenlo'. 2.7

Optical Rotation

Early investigators determined the specific rotation of nystatin in several solvents; their data, and those characteristic for Squibb Lot #a8645 are as follows: T [ a ]D

T,OC

-100

25

-ao

25

-8O

-

+21°

25

+120

25

-70

25

Solvent

Reference

AcOH (C, not specified) AcOH (C, not specified) AcOH (C, not specified) Pyridine (C, not specified) DMF (C, not specified) 0.1N HC1 in MeOH (C, not specified)

18a, 32 83

95 18a, 32, 83 18a, 32 18a, 32

Squibb Lot #a8645

+

8.05

22.5

DMF

94

(C = 1)

+21.04O 2.8

22.5

Pyridine (C = 1)

94

Optical Rotatory Disperson ( O m )

The optical rotatory dispersion curve of nystatin (methanol solution) in the 250-450 nm region has been presented by Chong and RickardsbO; from a comparison of the ORD characteristics of the parent antibiotic with those of its dihydro- and perhydro-derivatives, the authors conclude that nystatin - in neutral hydroxylic solution at ambient temperatures - is likely to exist as a cyclic hemiketal (in analogy to amphotericin B)62. 2.9

Melting Range

Nystatin does not exhibit a sharp melting point. Dutcher et al. report gradual decomposition above 160°C32 and

NYSTATIN

367

1650Ce3, respectively, without melting by 25OoC. Squibb Res. Std. #MYNM-150-RPI when heated on a Mettler Model FP52 microscope hot stage at a rate of 10°C/min and viewed in polarized light, shows a distinct phase transition at 165.5-168.5OC with concurrent loss of birefringence. 2.10 Differential Thermal Analysis (DTA) The thermal properties of nystatin vary markedly with the nature of the crystal modification (Types A , €3 and C; see Sections 2.1.2 and 2.2), and their specific characteristics represent a useful supplementary tool in the identification of each of the three observed forms. A differential thermal analysis (DTA) study of the recognized modifications was performed by Jacobson and Valentil02 between room temperature and 25OoC using a DuPont Model 900 Differential Thermal Analyzer under the following operating conditions: Sample : Reference: Heating Rate : Temperature Scale: AT :

Microtube (1.6-1.8mm) / Air Atmosphere Glass Beads 15O~/min 50°C/in. 1°C/in.

The respective thermogramsg4 , reproduced in Figure

10, show the following prominent features:

Type A (Squibb Res. Std. #MYNM-150-W) Single , well-defined endotherm at 169OC (corr.) , corresponding to the sharp phase transition discernible under polarized light on heating of the sample on a microscope hot stage (Section 2.9). Above this temperature, rapid decomposition takes place. Type B (Squibb Res. Std. #MYNM-150-RP/H) Two sharp endotherms at 115OC and 171OC (both corr. ) . Type C (Squibb Res. Std. #WSC-08982-FP) Single sharp endotherm at 153OC (corr.), followed by a broad endothermal band in the 160-185OC range. 2.11 Thermogravimetric Analysis - (TGA) A thermogravimetric analysis (TGA) of samples of the

368

GERD W. MICHEL

0 X W

1

TYPE A

%TV

l-

a

TYPE B

1 0

0

z

153 'C

1

50 F i g u r e 10.

7

TYPE C

W

I

I

1

100 150 200 Temperature, "C

1

250

DTA and TGA Thermograms of N y s t a t i n

(Types A , B and C) Instruments ; DuPont Model 900 D i f f e r e n t i a l Thermal Analyzer DuPont Model 950 Thennogravimetric Analyzer

NYSTATIN

369

three identified crystal modifications of nystatin (Types A, B and C; see Sections 2.1.2 and 2.2) under a nitrogen atmosphere has been conductedlo2 using a DuPont Model 950 Thermogravimetric Analyzer under the following operating conditions: Sample Atmosphere: Nitrogen Sweep (30-40 ml/min) Heating Rate: 15O~/min Temperature Scale: 50°C/in. Sensitivity: 2 mg/in. The corresponding TGA curvesg4 , superimposed on Figure 10, indicate the following continuous weight losses for the three crystal forms: Weight Loss %

Temperature

2.5 -15

up to 13OoC up to 2oooc

12.5 -20

up to 13OoC up to 185OC

4.0 -12

up to 13OoC up to 20oOc

2.12 Solubility Nystatin is practically insoluble at room temperature in water and common non-polar solvents, sparingly soluble in lower aliphatic alcohols, and readily soluble in formamide, N,N-dimethylformamide, dimethylsulfoxide, pyridine, ethylene glycol and propylene 32, 37 8 3 . Its solubility in polar solvents is reported to be substantially increased in the presence of 10 to 20% water32. Solutions and suspension of nystatin in water 37 lower alcohols, highly alkaline and acid media (e.g., glacial acetic acid, 0.05N methanolic HC1 or NaOH)32r83,95 are rapidlv inactivated soon after preparation. As part of a comprehensive study of 18 different antibiotics completed in 1957, Weiss et al. 5t103 reported the solubility of pooled commercial nystatin samples in 24 solvents at room temperature (28 4OC). These data, together with the results of solubility determinations for Squibb Lot #88645 in several selected solvents at 24 + loCio4, are summarized in Table VI. The discrepancies between the results of

370

GERD W. MlCHEL

both determinations are noted and evidently result from differences in the purity and/or homogeneity of the examined samples. Table VI Solubility of Nystatin

Solvent Water Methano1 Ethanol 2-Propanol Isoamyl Alcohol Cyclohexane Benzene Toluene Petroleum Ether 2,2,4-Trimethylpentane Carbon Tetrachloride Ethyl Acetate Isoamyl Acetate Acetone Methyl Ethyl Ketone Diethyl Ether lI2-Dichloroethane 1,4-Dioxane Chloroform Carbon Disulfide Pyridine Formamide Ethylene Glycol Benzyl Alcohol

Solubility, mg/ml Weiss et a d o 3 Squibb Lot #886451°4 [ 28+4OC] [ 24~1% I 4.0 11.2 1.2 1.2 2.4 0.505 0.28 0.285 0.16 0.03 1.23 0.75 0.55 0.39 0.75 0.30 0.45 2.1 0.48 0.40 >20 >20 8.75 2.65

0.36 10.23 0.83 0.23 <0.1 <0.1

<0.1 0.10

<0.1

16.63

As part of a general study of the physical properties of nystatin intermediates isolated by mycelium extraction with lower alcohols and vacuum concentration of the resulting aqueous alcoholic extracts, Trakhtenberg et al.105 examined the effect of changes in the water content of several solvents (acetone, methanol, ethanol and 2-propanol) on the solubility of the isolated products. While methanol-water mixtures provided maximum solubility for the antibiotic intermediates at water levels below 10 vol. %, the authorslo5 found substantial increases in the solubility of the test products in the binary systems ethanol/water, isopropanol/water and acetone/water

NYSTAT I N

37 1

with increasing water concentrations (ranging up to 50 vol.% in isopropanol). In contrast, the solubility of the examined materials in 70% aqueous methanol was determined to be only one-half of their solubility in neat methanol (8.26 mg/ml). In a similar, later study conducted with a slightly purer, crystalline product (activity 4500 units/mg), Kleiner and Ionoval06 examined the solubility of nystatin in binary mixtures of methanol, ethanol and isopropanol containing up to 50 vol.% of water and, in substance,confirmed the general observations made by Trakhtenberg et a1.1°5 with less pure preparations. While the solubility of nystatin in methanol was again found to have its maximum (9.2 mg/ml, 24 + l0C) in the absence of water, solubilities were shown to begreatly enhanced in ethanol and isopropanol with increases in water content in both solvents. Maximum solubilities for nystatin were reported to reach 4.0 mg/ml in 75 vol.% aqueous ethanol (0.55 mg/ml in anhydrous ethanol) and 2.2 mg/ml in 70 vol.% aqueous isopropanol (0.68 mg/ml in anhydrous isopropanol) at 24 2 l0C, as compared to 9.2 mg/ml in anhydrous methanol at the same temperature. Solubility profiles of nystatin for the solvent systems methanol/water and ethanol/water ( 2 3 2 l0C) have been determined82 with Squibb Res. Std. #MYNM-150-RPI following (a) the gravimetric procedure outlined by Weiss et al.103, and (b) a spectrophotometric method referred to in Section 6.5. Individual solubility data are summarized in Table VII, and the corresponding solubility curves are presented in Figures 11 and 1 2 . 2.13

Countercurrent Distribution

In 1968, Shenin et al.56 described a method for the separation of commercial nystatin preparations into two closely related components, designated A1 and A by countercurrent 2' distribution in an n-amyl alcohol/isoamyl alcohol/pH 5 citrate phosphate buffer system. In particular, the selected method involved 200-transfer distributions and the isolation of the pure constituents by subsequent extraction of the upper phase with a three-fold volume of petroleum ether, followed by washing of the organic phase with water and acetone, removal of the solvent and drying of the resulting residue. Subsequent redistribution of the individually isolated components A1 and A2 in the same solvent system showed the complete absence of the companion fraction originally present in the starting material, thus evidently excluding the possi-

372

GERD W. MlCHEL

bility that either component may be the product of partial degradation during experimentation.

Table VII Solubility of Nystatin in MeOH/H20 and EtOH/H30 Systems at 23

+

l0C

(Squibb Res. Std. #MYNM-150-RP)

% Solvent (vol /vol . I

.

100 98 96 94 93 92 90 80 75 70 65 60 50 40 30 25 20 10 *)

MeOH/H20 GraviSpectrometric photom. Method Method mg/ml u/ml* ) 9.0440 9.6530 9.8440

52 ,1 9 3 54,627 55,428

9.4520

EtOH/H20 GraviSpectrometric photom. Method Method u/ml * 1 mg/ml 1.1240 1.0880 1.5160 2.0520

-

53,493

-

3800 4175 5910 9045

8.8080

47,957

11,713 10,972 7284

2.4333

11,019

2.3680 2.3920 1.8560 1.7240 1.5960 1.5560 1.1480 0.7920 0.5720

6403 6463 5956 4322 2528 1191

0.3960 0.2480

450 356

-

-

-

0.6520

-

0.3240

-

-

-

-

1961

-

-

-

-

-

-

-

Based on a potency for Squibb Res. Std. #MYNM-150-W of 6190 u/mg (spectrophotometric assay, Section 6 . 5 ) .

0. 0. 0. 0. 0. 0. 0. 0. 0. 0.

373

40

u o u

+I

rl

+I

m

S o l u b i l i t y P r o f i l e of N y s t a t i n ( S q u i b b Res. S t d . #MYNM-150-RP) S o l v e n t S y s t e m s : Methanol/Mater, 2 3 + l0C Ethanol/Water , 2 3 +-l°C

AW .

P

Figure 12.

Solubility Profile of Nystatin (Squibb Res. Std. #MYNM-150-RP) Solvent Systems: Methanol/Water, 23 + l0C Ethanol/Water I 2 3 L-l°C

NYSTATI N

375

The authors56 examined several pharmaceutical grade products and found all of them to contain both components, although in varying ratios, depending on their origin and/or their degree of purity, but generally established the A1-component (distrib. coefficient 4.6) to be present in much larger quantities than the A2-component (distrib. coefficient 16.8). Although both fractions apparently represent distinct chemical species, they nevertheless exhibit a number of closely related features, including essentially identical IR spectra, similar W spectra characteristic of a tetraene chromophore and, when subjected to acid hydrolysis, both constituents yield mycosamine as one of the reaction products. Moreover, as freshly generated products, both components are said to exhibit effectively equal bioactivities. However, a marked difference between both components was found in their relative stabilities as determined by an "express" method not further described in detail. While, under these conditions, the A2component was found to remain stable, component A1 lost approx 50% of its initial bioactivity. Other investigators1071108 have reexamined the findings reported by Shenin et al. with various samples of pharmaceutical grade nystatin and - despite the lack of adequate experimental details in the original publication56 - were able to confirm the presence of two or more constitutents in all examined nystatin products. Recently, Porowska et al. 6 4 r 6 5 adopted a countercurrent distribution technique to establish the close chemical relationship between nystatin and polifungin (produced by Streptomyces noursei var. polifungini, ATCC 21581), while also being able to demonstrate that both tetraene antibiotics are not homogeneous entities but, in fact, represent complexes of up to four biologically active main components. During the course of this investigation, samples from several lots of pharmaceutical grade nystatin were shown to be separable by consecutive countercurrent distribution from two different solvent systems (methanol/chloroform/pH 8.2 borate buffer and methanol/chloroform/l% aq. NaCl soh.; 400 transfers) into three closely but chemically distinct constituents designated as nystatin A1 (main component), A2 and Aj. On comparison to similar fractions isolated concurrently from the polifungin complex, all three pure components separated from nystatin were also found to be common to polifungin. Moreover, the evidence presented suggests that two of the constituents derived from the nystatin complex, namely A 1 and A2, are evidently identical with those characterized by Shenin et a1.56,

376

GERD W. MlCHEL

while the third component ( A 3 ) represents a newly isolated bioactive constitutent. Based on the evidence at hand, as supported by TLC and bioautographic comparisons, the a u t h o r ~ ~ conclude ~ l ~ ~ that all three nystatin constituents are identical with those contained in the polifungin-A complex, while the fourth tetraene component separated from the polifungin complex, designated polifungin B, is apparently the o n l y main constituent differentiating both nystatin and polifungin complexes from each other. 2.14 Ionization Constants Nystatin is an amphoteric compound with two ionizing groups, namely a carboxyl and an amino function. Ray-Johnson log determined the ionization constants of nystatin in a mixture of N,N-dimethylformamide/water (50:50) by direct titration and - following the general procedure of Albert and SerjeantllO - calculated the following pKa values from the titration curves: pK1 (proton gained) = 5.12 = 8.89 pK2 (proton lost) Recently, Valentilg5 determined the ionization constants and the isoelectric point of nystatin in a ternary s o l vent system composed of methanol, 2-methoxyethanol and water by potentiometric titration and established the following apparent pK, values from the respective equilibrium constants:

The isoelectric point for nystatin in this system, calculated from the average of pK1 and pK2, was found to be at pH 7.18. There is, as yet, no experimental evidence to establish whether nystatin exists at the isoelectric point as a zwitterion or as an un-ionized molecule. Resolution of this question requires the examination of singly charged derivatives of the antibiotic, such as an ester and/or suitable salt. The zwitterionic nature of a closely related polyene macrolide antibiotic, amphotericin €3, was lately confirmed by such techniqueslg6-

NYSTATI N

377

2.15 Aggregation Molecular weight determinations with the aid of a Beckman Model E Analytical Ultracentrifuge have been performed by Kirschbaumlll on the clear supernatant of saturated nystatin-Type A and -Type C solutions in 90% methanol/lO% water at 4O, 20° and 37OC without equilibration between removal of the undissolved nystatin (by low-speed centrifugation) and the start of the MW analysis. Under these conditions, nystatinType A was found to exist in solution predominantly as a dimer, while nystatin-Type C is mainly a tetramer. This relationship, as established in one experiment, was maintained for solutions in equilibrium with undissolved nystatin for up to 98 hours prior to the low-speed removal of the undissolved product and subsequent MW determination on the supernatant. From a comparison of the UV-absorption spectra of nystatin solutions in methanol/0.05% acetic acid and various aqueous buffer systems (pH 4.5, 6.8 and 9.01, Lampen et al.ll* concluded that the low extinction values typical for the aqueous media are likely to reflect that nystatin is present as micelles and is not in true solution. This inference was supported by the observation that nystatin is not dialyzable under these conditions (pH 4.5 and 6.8, 10-30 ug of nystatin per ml of 0.1% aq. dimethylsulfoxide solution), and the product could be recovered unaltered at the end of the dialysis experiment. 2.16 Polarography A solution of nystatin in 25% aqueous ethanol, containing tetrabutylammonium hydroxide (0.15)! as basic electrolyte, has been reported by Kramarczyk and Berg113 to be irreversibly reduced with a half-wave potential of -1.65 volts, as measured against a normal calomel electrode. 3.

BIOSYNTHESIS

The structure of nystatin (see Section 1.1) is generally consistent with the biosynthetic pathway postulated for the entire class of biogenetically related macrolide antibiotics114 , including the polyene and erythromycin sub-groups (polyketide pathway) . Isotopic tracer studies by Birch -et a1.49,50,115 with fermentation cultures of Streptomyces noursei and degradation of the resulting labelled nystatin provided evidence in support of the polyketide pathway, and also allowed for the

378

GERD W. MICHEL

tentative assignment of partial structures for the nystatin molecule. 4. METHODS OF MANUFACTURE 4.1

Historical

Nystatin was first isolated by Hazen and B r ~ w n ~ ~ , ~ ’ in 1950 from the surface growth of a liquid glucose-tryptone culture of a natural soil actinomycete (strain No. 48240) later designated Streptomyces noursei33r40ar116 - originatin from a farm soil specimen recovered in Fauquier County, Va. 28

.

4.2

Microbiological Processes

While Hazen and Brown, in their original experiments leading to the discovery of nystatin, employed conventional surface culture techniques for the growth of the Stre tomyces organism under static condition^^^, Dutcher et a 1 * later succeeded in cultivating the organism by the method of deep fermentation (i.e., submerged culture, under aerobic conditions), thus providing the basis for an economical large-scale industrial production of the antibiotic. In efforts to further improve the productivity of commercial fermentations, a large variety of yield-influencing factors - including the selection of high-productivity strains and mutants341 36,117-127 , modifications in media and cultural c o n d i t i ~ n s l ~most ~ - ~suitable ~~ for the growth of the antibiotic-producing organism, etc. - have since been explored and recorded, predominantly in the patent literature2. The original 5. noursei strain (No. 48240)29t33i116, several subsequently isolated mutants (generated by exposure to X-ray and UV-irradiation or after treatment with nitrogen m ~ s t a r d ) ~ ~ *as , ~well ~ ~ ,as s ecific strains of Streptomyces albulus (e.g. , ATTC-12757) 34,p6 and other nystatin-producing Actinomyces o r g a n i s r n ~ l 70,126r140 ~,~~~ are known to co-produce secondary metabolites - e . g . , cyc1oheximi.de (actidione)29r 34-361125~antitumor antibiotic E7335 - in substantial quantities under particular culture conditions. 4.3

Isolation and Purification Processes

The isolation of nystatin from culture broth141-147 on industrial scale is most commonly based on extractive recovery procedures, involving (a) the admixture of an appropriate, water-miscible organic solvent to the whole fermenta-

NYSTATIN

379

tion broth (with or without pH adjustment), followed by (b) the removal of insoluble broth constituents via filtration, and (c) the separation of the antibiotic by either fractional precipitation or extract concentration, or suitable combinations thereof. A substantial number of reported p r o c e ~ s e s ~ ~ ~ 148-154 avoid the use of large solvent quantities usually required in whole broth extraction methods by first providing for the separation of a nystatin-rich mycelium cake intermediate (moist or dried) from which the antibiotic may then be extracted by any one of several suitable solvents or solvent combinations, following procedures similar to those adopted for whole broth extraction methods. The solvents and solvent combinations most widely used in the large-scale isolation of nystatin from fermentation broths or mycelial cakes include methano133,116r131,149, 151-153, ethano1116,153, n-but~no~33136,114,116,145~147, n-propanolll6 , i ~ o p r o p a n o l l l 6 , I~153 ~ ~,- methanol/ethanol ~~~ (1:1)116 , acetone33 I 1491151 , 80% acetic acid/xylene150 and pyr idine154. Other, more unique recovery methods take advantage of the known ability of nystatin to form a variety of soluble complexes with inorganic salts in organic solvents - e.g., with CaC12 in m e t h a n 0 1 ~ ~ , 1 4 2 , 1or ~ ~ with , NaI, NaSCN, KSCN and NH4SCN in acetone146 - which readily dissociate into the free antibiotic and the corresponding salt component on addition of water to the respective solution. Alternate isolation methods for nystatin are based on a property peculiar to its chemical nature, namely the pronounced tendency to form relatively stable aqueous emulsions with a number of water-immiscible organic solvents (alcohols, esters and ketones)144,145, thus permitting a direct separation of the antibiotic from nystatin-containing broths by flotation. A majority of the present recovery methods, however, produces relatively impure, low-potency intermediates requiring further purification, generally by procedures adapted from established broth or mycelial cake extraction techniques32r 117,141-146,149-152,155-160~

5. STABILITY - DEGRADATION Nystatin shares with many other complex polyene macrolide antibiotics a high degree of sensitivity to heat, light, oxygen, and extremes of pH, both as pharmaceutical grade bulk material in the solid state and in solution or suspension. However, very few reliable quantitative data are at

380

GERD W. MlCHEL

hand on the chemistry of various possible degradation processes and on the nature of the degradation product, resulting from exposure of the antibiotic to a variety of environmental conditions. Results of the few published experimental studies, listed below, often appear contradictory and are not readily interrelated, as they commonly reflect significant differences in experimental conditions (including assay methods), as well as wide variations in the origin, purity and homogeneity of the examined products (e.g., crystalline E. amorphous product, and/or mixtures thereof). In general terms, both the highly unsaturated nature of the molecule and the presence of a pH-sensitive lactone ring linkage undoubtedly contribute to the inherent susceptibility of nystatin to deactivation.

5.1

Dry Thermal Degradation

Among several general statements in the literature3' 4,7,39,83,95, it is r e p ~ r t e d that ~ ~ ,nystatin ~ ~ ~ - in the dry solid state-has been stored under refrigeration for up to 4# years without appreciable loss of activity, but approx. 25% of its activity was lost in 6 months at 4OoC under non-specified storage conditions. 5.1.1

Stability of Amorphous Product

Bashkovich and coworkers161 report that inactivation of amorphous nystatin, when exposed to atmospheric oxygen, is greatly enhanced by the presence of & 9% moisture, and suggest that loss of activity is the result of oxidative polymerization. Inactivation was also found to be increased by the presence of polyvalent metal ions (Ca2+, Fe3+, Cu2+, Mn2+, A 1 3 + , Co2+ and Ni2+) , but this effect is said to be minimized by the addition of a suitable complexing agent, such as Na-hexametaphosphate. Results of accelerated stability studies carried out by shaking nystatin powders at room temperature for 17 days in a sealed tube containing an oxygen atmosphere and exposed to W light are reported to correlate well with the extent of deactivation after normal storage for one year at 4OC. 5.1.2

Stability of Crystalline Product

Accelerated heat stability tests conducted by Trakhtenberg et al .lo5 with dry nystatin materials (isolated by extraction of mycelium cake with primary alcohols) showed that samples which were practically stable on storage under refrigeration nevertheless rapidly degraded at elevated

NYSTATI N

381

temperatures as evidenced by an activity loss of approximately 75% on storage for 3 hours at 100°C; the presence of moisture was found to enhance thermal decomposition, as was also noted by other investigatorsl6211641165. Kleiner and 1 0 n o v a l ~examined ~ the stability of crystalline commercial nystatin samples on heating in sealed tubes at 80° and 100°C and established first-order kinetics for the degradation under these conditions, with half-life periods of 1.33 x lo3 min at 80°C, and 0.88 x lo2 min at 100°C. The addition of antioxidants (e.g.I thiourea and Nametabisulfite) was found not to protect the antibiotic from thermal decomposition. As part of an investigation to explore potential methods other than a heat-resistance test for the determination of nystatin stability, Kuzovkov et al.164 studied the effect of storage under controlled humidity conditions and developed an expedient, qualitative ("express") method for stability studies. Nystatin samples were stored in open vessels over 10% H2SO4 in a hermetically sealed chamber at 2OoC and 98% rel. humidity for a 30-day period. The authors164 found that preparations oliherwise shown to be unstable under normal ambient conditions lost 30-70% of their initial activity after 30 days in the high-humidity environment, while samples which were considered stable at room temperature also appeared to be stable for longer periods in the humid atmosphere. No quantitative relationship was established between the activity loss in the high-humidity environment and storage under ambient conditions. The method described appears, therefore, only useful as a qualitative test for the estimation of nystatin stability.

Lokshin et al. 165 provided evidence that the enhanced stability of well-dried nystatin is best preserved by storage over P2O5, in the absence of atmospheric oxygen. Benzoylperoxide, polyvalent transition metal ions (Fe3+, Co2+ and Cu2+) and high ambient humidity are reported to greatly reduce the stability and biological activity of dried products on storage at room temperature. Unidentified polymerization products, insoluble in organic solvents and in inorganic acids and bases, were shown to accumulate on prolonged storage under unprotected conditions as a consequence of aerial oxidation; mycosamine has been identified as one of the reaction products from the acid hydrolysis of the isolated polymeric constituents. Crystalline nystatin, as opposed to the amorphous

382

GERD W. MICHEL

product, and nystatin purified by treatment with Na-hexametaphosphate in aqueous isopropanol solution165 1 166 were reported to have superior stability, while being less susceptible to the deteriorating effect of humidity. The authors165 suggest that aerial oxidation is the prominent cause of nystatin deactivation and also postulate that conditions of high relative humidity promote the decomposition of peroxide compounds formed during air oxidation. Among several antioxidants examined, butoxytoluene and butoxyanisole proved to be the most effective stabilizing agents. 5.1.3

Stability of Solid Dosage Forms

Thermostability tests conducted by Tebyakina et al. 167 on pharmaceutical grade samples of nystatin - as dry bulk powders and in solid dosage forms (tablets, pills) - revealed marked differences between various products after storage for up to two years at 5OC and at room temperature; while the forz mulated products effectively retained their original activity at both temperatures, bulk powders were subject to substantial degradation on storage, with activity losses for some samples ranging in the order of 20-30% over a 2-year storage period at 5OC. Addition of tetracycline to the dry dosage forms was reported to improve their thermal stability. S. Boteanu and coworkers162 investigated a variety of dragee formulations under long-term storage conditions to establish a semi-quantitative relationship between excipient composition and the effects of heat exposure, relative humidity, UV- and IR-irradiation and pH on the rate of product degradation over periods of up to 720 days.

More recently, Elkouly et a1.l6' compared the stability of nystatin in five different suppository bases against dry nystatin powder when stored at '5 and 25OC. At either temperature, the dry powder was found to decompose on storage but, as expected, with a markedly lower rate at 5OC than at 25OC, in general agreement with the findings of other investig a t o r 1~ ~ 1~167. ~ At both temperatures, however , it was established that Siopotencies of the dry powder decreased at an appreciably faster rate during the first three months of storage (approx. 30% and 50% activity l o s s at 5O and 25OC, respectively) than during the following period, consistent with early observations by Dutcher et al.83 on lyophilized nystatin powders. The storage stability characteristics of the antibiotic in the selected suppository bases followed a similar pattern over the first 3-month period, with slightly higher initial decomposition rates at both temperatures, but near-

NYSTAT IN

383

equal residual biopotencies after 6-month storage at either temperature (approx. 65% activity l o s s ) . A s part of this study, Elkouly and coworkers168 employed, in parallel, two of the most commonly adopted quantitative procedures for the determination of potency changes during long-term storage of nystatin and its formulated products - namely a microbiological (cup-plate agar diffusion assay169) and a direct spectrophotometric method (see Section 6.5) - and found very poor agreement between both procedures. In fact, the microbiological assay data provided evidence for substantial, progressive biopotency losses over the entire 6month test period; concurrent monitoring of the UV-absorbance of nystatin at one of its three prominent absorption bands (319 nm) during the same test interval indicated effectively no absorbance changes for the storage samples at both test temperatures, thus evidently precluding the use of the direct spectrophotometric method as a reliable tool in stability studies. A similar conclusion was reached by Dutcher at a1.83 during early studies of the chemical and biological properties of nystatin, and has found further support in the recent findings generated by Hamilton-Millerg7 during the examination of pH and temperature effects on the stability of nystatin solutions; in addition, several other i n v e s t i g a t o r s 2 7 ~ 9 6 ~ 1 ~ ~ ~ ~ ~ ~ ~ 171 have commented on the lack of a meaningful correlation between biological and spectrophotometric assays of polyene antibiotics.

5.1.4

Stability of Ointment Formulations

The stability of nystatin in twelve different ointment bases held at 37OC for various time periods (up to 75 days) was examined by Trivedi and Shah172 by the agar cupplate method using Saccharomyces cerevisiae as the test organism. The degradation reaction was found to follow first-order kinetics, and half-life times are listed. Among the examined ointment bases, a composition of polyethylene glycol 400 and 4000, Span 60 and water showed maximum stability, optimum diffusion through agar and release through parchment paper. 5.2

Stability in Solution

Studies by Trakhtenberg et a1.1°5 have shown that solutions of nystatin in methanol, both under conditions of acid (0.05N HC1) and alkaline pH (0.05N NaOH), are highly unstable and lead to a near-complete loss of bioactivity within a matter of hours, without appreciable changes in the extinction attributed to the polyene chromophore.

384

GERD W. MICHEL

Lokshin et al. examined the kinetics of degradation for highly purified nystatin samples in anhydrous dimethylformamide solutions at several temperatures ranging from 32O to 56OC, both in the presence and absence of atmospheric oxygen. While, under these conditions, essentially no loss in biological activity was observed in the absence of aerial oxygen even after storage of the solutions at 56OC for 120 hours, rapid inactivation took place in the presence of air. Although the formation of peroxide derivatives was found to be related to the degree of deactivation, l o s s of bioactivity (e.g., 90% at 56OC/120 hours) showed no correlation with a concurrent decrease in W-absorbance (e.q., only 50-60%). The rate of autoxidation of nystatin in dimethylformamide solutions was further studied by Zhdanovich et al. 174 and shown to be accelerated in the presence of heavy metal ions (Fe3+, Co2+ and, esp., Cu2+), but retarded by the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT) at concentrations of 1-1.5% of the antibiotic weight. Hamilton-Millerg7 recently investigated the effects of temperature and pH on the stability of nystatin (and amphotericin B) solutions in phosphate-citrate buffers of different pH values (range pH 3 to 8 ) and concluded that nystatin solutions, when held at 37OC, are optimally stable between pH 5 and 7 , while rapid breakdown was observed at pH 3 and 4 (approx. 90% destruction in about 3 and 6 hours, resp.). Periodic examination by both microbiological and spectrophotometric assay methods of test solutions incubated at pH 5, 6.5 and 7 showed that the loss of biological activity proceeded at a faster rate (4 to 8 times as rapid) than did the loss of extinction characteristic of the tetraene chromophore (321, 306 and 292 nm). The authorg7 suggests that the mechanism of deactivation under the selected test conditions is not determined by an epoxidation of the type established for the aerial autoxidation of other polyene macrolide antibioticsg2. In general, loss of bioactivity followed first-order kinetics at temperatures between 37O and 100°C, except under acid conditions. Thermodynamic parameters have been calculated from the Arrhenius plots of the respective thermal stability data, and values for the apparent activation energy, entropy, enthalpy and free energy of activation characteristic for the loss of bioactivity are given. Boudru and B ~ u i l l e t lexamined ~~ the stability characteristics of nystatin powders dissolved in a pH 1.6 artificial gastric medium and observed bioactivity losses at 25OC in the order of 35% after 15 min and 84% after 90 mint while an

NYSTAT I N

385

increase of the temperature to 37OC resulted in a total loss of biological activity within 60 min. Inoculation of the same medium (pH 1.6) at 37OC with Candida albicans, however, showed a near-complete growth inhibition for the microorganism under these conditions. Identical experiments with nystatin in the form of sugar-coated tablets and powders in suspension produced similar results, leading to a complete destruction of the microorganism after 30 min and 60 min, resp., of incubation in the gastric medium. 5.3

Stability under Radiation

The use of Y-radiation to sterilize nystatin (and other polyene antibiotics and their salts) was examined by Tsyganov and V a ~ i l e v a l ~ ~Exposure . of the antibiotic to radiation doses in the order of l o 6 rads produced satisfactory sterilization effects, but decreased the biological potency of the product by approx. 10% without, however, leading to detectable differences in the toxicity between irradiated and nonirradiated samples, neither as freshly treated specimens, nor after 1-year storage at room temperature. 5.4 Microbial Degradation The microbial degradation of nystatin by various strains of lower pathogenic fungi has been examined177, and no significant differences were found in the rate or degree of its degradation by various species of dermatophytes. However, marked differences were found in the rate of enzymatic degradation by microorganisms which were adapted and not adapted to nystatin. After a 4-hour exposure of nystatin to non-adapted strains in a suitable culture medium, approx. 70% of the antibiotic was still intact after inoculation, whereas the antibiotic was completely degraded by adapted strains during the same time period. 5.5

Stabilization

In addition to the examined stabilization methods161, 163,165-1671174 for nystatin quoted above (Sections 5.1.1, 5.1.3 and 5 . 2 ) , suppository formulations of the antibiotic are reported178 to be stabilized by the incorporation of mixtures , of antioxidants - i.e., butylated hydroxytoluene (0.01%) butylated hydroxyanisole (0.005%) and citric acid (0.005%)into a base consisting of lanolin/paraffin/hydrous fat (8:l:lL Similar stabilizing effects have been attributed by Hermansky and V o n d r a ~ e k ’to ~ ~ several other antioxidants, in-

386

GERD W . MICHEL

cluding hydroquinone, 8-naphthol, propylgallate and 2,6-ditert-butyl-o-cresol.

-

6. METHODS OF ANALYSIS

6.1

Elemental Analysis

Based on the results of various structure elucidation studies of the past, several conflicting proposals for the molecular com osition of nystatin have been made in the literaturel8a I 31I 33 I 46 49 50I 54 83 95 105 i 115. Among these , latest experimental e v i d e n ~ elo’ ~ ~supports , ~ ~ ~ an elemental composition corresponding to the empirical formula C47H75N017 (MW 926.13) for the unresolved antibiotic complex; the same formula is also postulated for nystatin A159, the pure main component of the nystatin complex, isolated by countercurrent distribution. I

I

I

I

I

In light of the finding that nystatin is not an individual compound but rather a variable mixture of several chemically related, active constituent^^^^^^^^^ , present assignments for the antibiotic complex should be viewed with reserve, as illustrated by the general lack of agreement between experimental microanalytical data and the theoretical elemental composition for the proposed empirical formula (see Table VIII for a listing of elemental analyses quoted in the literature). TABLE VIII Elemental Analysis of Nystatin

C

Theory 60.95 (Calculated for C47H75N017)

%

%

*)

Found

58.86 58.50 58.42 58.58 58.42 58.22 58.21 58.86

Squibb Res. Std. #MYNM-150-RP

Element H -

N -

8.17

1.51

8.97 8.57 8.18 8.28 8.18 8.21 8.26 8.21

1.7 1.6

Ref. -

32 32 1.66 83 1.62 83 1.6 95 1.51 105 1.75 105 1.64 180*)

NYSTATI N

6.2

387

Neutralization Equivalents

Nystatin has been titrated, both as a base (with perchloric acid in glacial acetic acid32,83,95) and as an acid (with sodium methoxide in ~ y r i d i n eand ~ ~ methanolg5). The following neutralization equivalents were determined: Neutralization Equivalents (NE) As Base

As acid

Ref. -

956 955,956 955

922

32 83 95 181*)

-

*)

6.3

-

950 950

Squibb Res. Std. #MYNM-150-W

Identification Tests

Nystatin may best be identified by its characteristic IR and W absorption spectra, as well as its X-ray diffraction pattern (see Sections 2.1, 2.2 and 2.4). The Federal Register75c describes an identity test for nystatin involving the recording of the W spectrum of nystatin in the 220-320 nm range and the determination of the absorbances at five selected absorption maximala2 leg. A series of qualitative, non-specific chemical identification tests quoted in the literature2 are listed below. Test -

Response

Ref. -

Benedict Carbazole Mo 1isch Schiff

Positive Positive Positive (Faint) Positive (Atypical)

2 2 ,32,126 2,31,32,83,126 2,32,83,126

In addition2, nystatin decolorizes solutions of bromine-waterle3 , bromine-carbon tetra~hloride~~ ,126,184, iodine-potassium iodide183 , and potassium per~nanganate~~ ,126 le4. However, it does not give positive tests with biuretle3, Fehling32,83,126f ferric chloride32r126t183, Millon32183t126, ninhydrinle3 ,T 0 1 l e n s ~r 83 ~ 126 , and 2 ,4-dinitrophenylhydrazine 83 reagents. I

388

GERD W . MICHEL

6.4 Color Reactions

Seve a1 color reactions typical for nystatin have been reported’ (see tabulation below). Reagent

Color

Hydrochloric Acid Yellow Pink Phosphoric Acid Sulfuric Acid, Conc. Violet to Blue to Black Strong Blue FeC13-K3Fe (CN) SbC13 in Chloroform Pink (Carr-Price)

Ref. 183 183 32,126,183,184 32,184 184

Other tests suitable for the identification of nystatin involve color reactions which are common to a large number of polyene macrolides. Into this category belong the characteristic formation of a chloroform-extractable, dark yellow color constituent on heating of nystatin in sodium hydroxide solution, the transient appearance of a red-violet color with concentrated sulfuric acid, and the formation of a blue coloration on addition of concentrated hydrochloric acid or trichloroacetic acid to an alcoholic solution of n y ~ t a t i n l ~ ~ . Laubielg7 noted that a pink color is formed by heating an alcoholic solution of nystatin in the presence of resorcinol and concentrated hydrochloric acid (Selivanof reaction) to reflux temperature; on dilution of the mixture, the color component may be extracted into isoamyl alcohol. A l though this reaction was shown to be very sensitive and may be suitable for the detection of nystatin at levels of approx. 50 pg, the method is non-specific as several other antibiotics produce similar color reactions. A related procedure, described by the same author197 and claimed to be more specific for nystatin, involves the reaction of an alcoholic nystatin solution with a mixture of concentrated hydrochloric acid and dilute aqueous ferric chloride; the intensity of the green color component formed in this reaction is reported to allow the detection of nystatin at levels identical to those quoted above. This procedure has been evaluated by Szucslg8 as an identity test for the determination of nystatin in the presence of a series of excipient materials commonly found in tablet formulations. Color reactions adapted for use in the quantitative analysis of nystatin by colorimetric assay methods are covered

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in Section 6.6. 6.5

Direct Spectrophotometric Analysis

The ultraviolet absorption properties of nystatin are discussed in Section 2.4. Because of the distinct spectral fine structure of polyene macrolide antibiotics11,12,13,15i18a,g2,g3,ultraviolet absorbance measurements are widely accepted as the most expedient tools in analytical methodology. Quantitative spectrophotometric methods for the determination of nystatin, utilizing the characteristic absorption of the conjugated tetraene chromophore with intense absorption bands near 291, 304 and 318 nm, have been employed in a variety of investigations, including the rapid differentiation of nystatin from other olyene macrolides derived from Stre tom ces species11,15 ,18a, ", in stability studies97 I 1051 47-1 and in chemical transformations52 i 190-192. Although these methods were found by some authorsg6' 185 to correlate acceptably with the biological activit of the antibiotic , the majority of ~ t u d i e s l l ,83,96,97,185,168, ~~ 170,1711 however, have established either unsatisfactory or only marginal relationships between spectrophotometric and biological assays, most likely as the result of substantial variations in the state of purity and homogeneity of the examined products, specifically with respect to differences in the ratio of active components. The lack of an adequate agreement between both analytical methods has greatly reduced the usefulness of ultraviolet spectrophotometric procedures as tools for the assessment of product purity. Nevertheless, spectrophotometric methods are being utilized, for convenience reasons, in many process control applications5'96r185-187, particularly in the measurement of nystatin concentration in fermentation broths, unpurified products and various recovery samples. 6.5.1

Fermentation Liquids and Products

The absorbance of nystatin at 304 nm has been used to 5 determine the concentration of nystatin in fermentation broth. The assay does not reflect the stability of nystatin to acid and heat, but is suitable for process control uses. Another direct spectrophotometric assay for the determination of nystatin in fermentation broth, based on the

390

GERD W. MICHEL

measurement of t h e d i f f e r e n c e i n e x t i n c t i o n s a t 304.5 and 312 nm, i s reported by Doskochilova and Gessg6. The method dess c r i b e d i s claimed t o give r e s u l t s comparable t o t h o s e obt a i n e d by t h e b i o l o g i c a l a s s a y , using an a c t i d i o n e - r e s i s t a n t s t r a i n of Candida a l b i c a n s (BUCAV 4 4 ) as t e s t organism i n a p l a t e method of c u l t i v a t i o n . S a t i s f a c t o r y agreement between both spectrophotometric and b i o l o g i c a l methods w a s r e p o r t e d t o be maintained during t h e e n t i r e course of a fermentation. However, on prolonged fermentation beyond t h e a t t a i n m e n t of maximum a n t i b i o t i c a c t i v i t y , both methods begin t o d e v i a t e from each o t h e r , with t h e b i o l o g i c a l assay i n d i c a t i n g a sharper d e c l i n e i n a c t i v i t y of t h e c u l t u r e f l u i d than r e f l e c t ed by t h e spectrophotometric method. The authors96 e x p l a i n t h i s discrepancy with t h e l i k e l y decomposition of t h e a n t i b i o t i c on extended fermentation, concurrent l o s s of bioa c t i v i t y , but r e t e n t i o n during decomposition of t h e polyene chromophore responsible f o r t h e u l t r a v i o l e t absorption of nystatin. A l t e r n a t e spectrophotometric assay procedures f o r t h e determination of n y s t a t i n , developed by Sherman e t al.186,187 , attempt t o account f o r t h e presence of u l t r a v i o l e t - a b s o r b i n g b a l l a s t substances i n fermentation l i q u i d s and u n p u r i f i e d i n termediates which otherwise tend t o a f f e c t t h e d e s i r e d accuracy of q u a n t i t a t i v e assay methods based on e x t i n c t i o n measurements. The proposed d i f f e r e n t i a l methods, a p p l i c a b l e t o both broth and i s o l a t e d product samples, involve t h e e x t i n c t i o n measurement of n y s t a t i n s o l u t i o n s ( i n methanol/dimethyls u l f o x i d e mixtures) a t t h e absorption m a x i m u m i n t h e 302-306 nm range, p l u s t h e determination of t h e e x t i n c t i o n f o r t h e m i n i m a on e i t h e r s i d e of t h e peak a b s o r p t i o n , i . e . , near 295 and 312 nm, r e s p e c t i v e l y . D e t a i l s of t h e q u a n t i t a t i v e procedures developed f o r t h e determination of n y s t a t i n b r o t h and t h e p u r i t y of bulk product i n r e l a t i o n t o a s t a n d a r d sample a r e o u t l i n e d below: (a)

Nystatin i n Broth Procedure186 Measure 20 m l of well-mixed whole b r o t h and t r a n s f e r i n t o a 6" x 1" screw-cap t e s t tube. To d e a e r a t e t h e b r o t h sample, s p i n f o r 5 min a t 2000 rpm i n a s u i t a b l e c e n t r i f u g e , and again mix t h e t e s t tube c o n t e n t s on a Vortex Mixer f o r 15-30 s e c . P i p e t t e 2 m l of t h e well-mixed sample i n t o a 100-ml volumetric f l a s k , add 75 m l of dimethyl-

NYSTATI N

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sulfoxide and agitate on a rotary shaker at moderate speed for 15 min. Bring up to volume with dimethylsulfoxide, shake up by hand to mix and filter the mixture by gravity through Whatman #4 filter paper. Pipette 2 ml of the clear filtrate into a 100-ml volumetric flask, bring up to volume with absolute methanol and mix well. Read the sample against a reagent blank ( 2 ml of dimethylsulfoxide, brought up to 100 ml with absolute methanol) on a suitable spectrophotometer in 1 cm silica cells. Determine the maximum absorbance for nystatin in the 302-306 nm region, and determine the absorbance at the minima on either side of this peak (in the range of 296 and 312

nm). Calculation:

K

=

Nystatin units/ml

Absorbance at about 304 nm Absorbance at about 296 nm = Absorbance at about 312 nm = Dilution factor (2500) = Potency of nystatin reference standard (units/mg ) K = Standardization factor determined with nystatin reference standard by the procedure outlined below. A B C D E

= =

Standardization Weigh accurately about 5 mg of standard nystatin powder and transfer into a 500-ml volumetric flask. Add 5 ml of dimethylsulfoxide and dissolve the powder. Bring up to volume with absolute methanol and mix well. Read the standard solution against a reagent blank in 1 cm silica cells on a suitable spectrophotometer. Keep the slit width constant and maintain the same setting for sample assay. Determine the maximum absorbance of nystatin in the 302-306 tun range and the minima on each side of this peak.

392

G E R D W . MICHEL

Calculation: Standardization factor K =

(A

B + C - 7) x D

Weight of standard in mg

Absorbance at about 304 nm Absorbance at about 296 nm C = Absorbance at about 312 nm D = Dilution factor (500)

A = B =

(b) Nystatin Products Procedure187 Weigh accurately 85-105 mg of nystatin into a 100-ml volumetric flask. Add 10 ml of dimethylsulfoxide and shake to dissolve the powder. Bring up to volume with absolute methanol and mix well. Pipette 1 ml of the clear solution into a 100-ml volumetric flask, bring up to volume with absolute methanol and mix well. Read the sample against absolute methanol as a reagent blank on a suitable spectrophotometer in 1 cm silica cells. Determine the maximum absorbance for nystatin in the 302-306 nm region, and determine the absorbance at the minima on either side of this peak (in the range of 296 and 3 1 2 nm). Calculation : ( A - -B)+x DC x E 2

K x Weight of sample in mg

=

Nystatin units/mg

A = Absorbance at about 304 nm B = Absorbance at about 296 run C = Absorbance at about 312 nm D = Dilution factor (l0,oOo) E = Potency of nystatin reference standard (units/mg) K = Standardization factor determined with nystatin reference standard by the same procedure as outlined above under (a) for nystatin in broth.

NYSTATIN

6.5.2

393

Pharmaceutical Preparations

Aiteanu and Medianu188 examined the stability of nystatin in N,N-dimethylformamide (DMF)/ethanol mixtures and found such solutions to be stable for 24 hours, as concluded from the measurement of extinction coefficients for the absorption maxima at 291,304, and 318 nm. 6.5.3

Other Applications

Special applications of ultraviolet spectrophotometric techniques to the examination of chemical transformations of nystatin have been reported by Bolshakova et a1.52, lgo, Korchagin et a1.lg1 and, more recently, by Udvardy et al. lg2. The latter authors examined the addition of iodine monochloride and bromine to nystatin by a combination of spectrophotometric, titrimetric and thin-layer chromatographic methods in an attempt to correlate biological activity with the tetraene content of a large number of nystatin production batches. Further details are discussed in Section 6.10. Wayland and Weisslg3 developed a system of chemical identity tests for the specific, positive characterization of antibiotics in sensitivity disks to supplement the quantitative information obtained by microbiological assay techniques. The system is suitable for the microquantities involved in antibiotic disks, positively identifies the chemical nature of the antibiotic in an unknown disk sample and was screened for interference from other disk antibiotics. Within this scheme of chemical test procedures - involving a sequence of colorimetric, TLC and paper chromatographic tests, in combination with microbiological response and potency data - nystatin is identified by its characteristic absorption peaks at 291, 304, and 318 nm. A general survey of spectrophotometric methods for antibiotic determination in the ultraviolet and infrared regions was published by Untermanlg4 in 1965.

6.6 Colorimetric Analysis

Several colorimetric methods have been published for the determination of nystatin as bulk material and in pharmaceutical formulations. The earliest methods described by Laubielg7 and Szucs lg8 are semi-quantitative procedures based on the formation of distinct color components (see also Section 6.4).

394

GERD W. MlCHEL

Characteristic colorations are also formed upon treatment of dimethylformamide solutions of nystatin with either dilute aqueous sodium hydroxide or concentrated hydrochloric acid1''. The latter reaction, described by O Z S O Z ,~ ~ ~ producing a light-blue color on addition of 0.25 ml of concentrated HC1 to a solution of 25-100 units of nystatin in 0.1 ml of DMF, has only found use as a qualitative test in the identification of nystatin, specifically in ointment formulations. The color reaction resulting from the admixture of dilute sodium hydroxide to a DMF solution of nystatin reported by Unterman200 , however, has been developed as a quantitative procedure suitable for the analysis of the antibiotic in tablet formulations. Unterman201 also found that nystatin produces a reddish-yellow color when reacted in DMF solution with AlC13, and proposed that the reaction be used as a quantitative method for the determination of the antibiotic. Ochab202 later worked out optimum reaction conditions, established a linear relationship between the concentration of nystatin and the absorbance of the color component at 435 nm and - based on good agreement between colorimetric and biological assay data - adapted this method for the quantitative assay of the antibiotic in pharmaceutical dosage forms. A different color reaction, also reported by Unterman 203, involves the formation of a yellow-brown colored complex on treatment of nystatin with 6% anhydrous methanolic titanium tetrachloride solution. The absorption spectrum characteristic for this complex is different from the parent antibiotic, but retains the unique absorption maximum at 318 nmg6; the reaction has not been adapted for quantitative use. However, an apparently related color reaction described by Mazor and Papay 204, based on the generation of a reddish-brown color complex on addition of a TiC14 solution in DMF to a nystatin solution in the same solvent, has been proposed as a method for the colorimetric determination of nystatin. The resulting complex with a molar ratio of nystatin : titanium of 1:3 exhibits strong absorbance at 450 nm and its formation obeys LambertBeer's law. As the colored complex no longer shows significant absorption in the ultraviolet range, the authors presume that nystatin decomposes under the conditions of the reaction and the resulting complex is, in fact, formed with one of the decomposition products.

.

Chang et a1 205 have proposed a colorimetric method for the assay of nystatin, both as bulk material and in phar-

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395

maceutical formulations, which utilizes the formation of a yellow color produced on heating DMF solutions of nystatin with aqueous sodium hydroxide. Although good agreement between colorimetric and microbiological assay results is reported, the presence of sugars is known to interfere with this method5. The procedure was applied to the measurement of nystatin activity in creams, ointments and capsules, and was also employed in stability studies. A more recent colorimetric method for the determination of nystatin reported by Amer and Habib2O6l2O7 is based on the reaction of the alkaline hydrolysis products of nystatin with p-aminoacetophenone in the presence of concentrated hydrochloric acid. A general colorimetric procedure proposed by D r y ~ n l ~ ~ for the determination of several natural antifungal compounds (incl. nystatin, amphotericin B, and pimaricin) involves the dissolution of the polyene antibiotic in MeOH/CHC13 (2:l) mixtures, addition of 37% hydrochloric acid containing 20 vol.% of ethanol under cooling, formation of a blue color within -8 min at room temperature, and photometric measurement of the extinction at 620 nm against a blank. Korchagin et a1.lg1 have suggested a colorimetric determination of nystatin based on the absorbance measurement of DMF-EtOH solutions following treatment with concentrated phosphoric acid for 6 min at 100°C. Photometric measurements of the stable color formed under these conditions are claimed to correlate well with direct spectrophotometric determinations and microbiological assays generated by the agar-diffusion method. The procedure has also been applied to the determination of degradation products formed on storage of methanolic nystatin solutions in the presence of acid (pH 4 ) and alkali (pH 9).

6.7 Chromatographic Analysis Chromatographic methods have been widely employed in the detection and identification of nystatin, mainly as qualitative tools to differentiate the antibiotic from other known and unknown polyene antifungal agents generated by a wide variety of antibiotic-producing microorganisms, predominantly those isolated from Streptomyces species16 170 ,208r 221. As many of the polyene antibiotics which have been isolated are known to be actually mixtures of two or more active constituents, chromatographic comparisons with previously

396

GERD W. MlCHEL

identified products are the most expedient means of establishing uniqueness of a newly isolated antibiotic and providing criteria for its classification. Frequently, available chromatographic separation methods are combined with the detection of the active component on the developed chromatogram by bioautography. The application of this special detection method in paper and thin-layer chromatographic studies of antimicrobial substances as well as its general scope in the antibiotic field have been critically reviewed by Betina214 in a recent comprehensive publication. 6.7.1

Paper Chromatography

A variety of paper chromatographic systems have been developed for nystatin, and a number of these are summarized in Tables IX and X.

The general utility of paper chromatographic methods in the differentiation of nystatin from chemically closely related polyene macrolide antibiotics produced by a large number of organisms and in their separation into individual, biologically active components from complex mixtures of similar olyenes is illustrated in several reviews16 70,208-210 1 212 12y3 and individual studiesla31211I 215-227. A simple paper chromatographic procedure for the qualitative determination of nystatin in pharmaceutical dosage forms and in admixtures with other antibiotics has been developed by Ritschel and Lercher2I7.

A n-butanol/ethanol/water (5:1:4) system together with Whatman No. 1 paper has been utilized by Struyk et a1.211 in a descending method (17-hour development) to separate nystatin from pimaricin and amphotericin A , all closely related tetraene macrolides with similar physical and biochemical characteristics. In a related application, paper chromato raphy was the method of choice selected by Rao and Cullen2” to establish the identity of one among five different active metabolic products (including antitumor antibiotic E-73) isolated from a culture broth of Streptomyces albulus. A special paper chromatographic technique developed by Betina and Nemec2241225, termed “pH-chromatography”, has been applied to nystatin. This method, specifically designed

TABLE IX Paper Chromatography Systems for Nystatin Solvent System (See Table X)

w

U (D

H I J

K L

M

Paper

Development Time (hrs)

Whatman No. 1 Not reported Whatman No. 1 Whatman No. 1 Arches No. 302 Whatman No. 1 Whatman No. 1 Arches No. 302 Schleicher & S c h h l 2043b, "hydrophobed" Schleicher & S c h h l 2043b, " hydrophobed" Whatman No. 1 Whatman No. 2 Whatman No. 1 Not reported Whatman No. 4

Method of Detection (See Table X )

15-16

1 2

18 15-16 16

3

-

-

15-16 18 15 21 17 18-24 6-7

-

3-

Reference 126 ,215

6,7,8

0.25,O.32 Not reported 0.22 0.76,O. 9 0.58 0.56 0.73,O.63 0.44 Not reported

221,222 126 ,215 218 177 126,215 218 217

617,8

Not reported

217

2 2 1

Not reported 0.40 0.82 ,O. 78 Not reported Not reported

211 220,221 126,215 219 65,67

1 4 5 1

4

-

2

65

TABLE X Paper Chromatography Systems €or Nystatin Solvent Systems

A B C

D E F G

H I J K L

M

Methods of Detection

1 2 3 4 5

6 7 8

n-Butanol, Water Saturated n-Butanol/Acetic Acid/Water (2:l:l) n-Butanol/Acetic Acidmater (4:1:5) n-Butanol/Acetic Acid/Water (4:l:l) n-Butanol/Pyridine/Water (1:0.6:1) n-Butanol/Pyridine/Water (2:1:2) n-Butanol/Pyridine/Acetic Acid/Water (15:10:3:12) n-Butanol (Water Satd.)/Ethyl Ether (Water Satd.)/Acetic Acid (5:l:l) n-Butanol/Ethanol/Water (5:1:4) n-Butanol/Ethanol/Water (5:1:5) Acetone/Water (1:1) 70% Aqueous Isopropanol Methanol/Chloroform/l2.5% Ammonia (1:2:1) , Lower Phase Bioautography vs. Penicillium oxalicum 99 Bioautography vs. Saccharomyces cerevisiae ATCC 9367 Bioautography vs. Saccharomyces carlsbergensis K-20 0.02N Potassium Permanganate Spray Reagent Ultraviolet Light 9% Ferric Chloride Spray Reagent 0.25% or 0.5% p-Dimethylaminobenzaldehyde Spray Reagent Ninhydrin-Stannous Chloride Spray Reagent

NY STAT I N

399

for the analysis of substances of biological origin, involves the chromatography of a selected antibiotic on a series of chromatographic paper strips buffered to pH values ranging from 2 to 10. Suitable organic solvents (e.g., n-butanol) saturated with water are used in the development of the strips by the ascending method, and the developed spots are visualized by microbiological detection. The authors2241225 propose this technique as a convenient means for the simultaneous determination of the ionic character of a given antibiotic and of the optimal pH values for its extraction into a suitable organic solvent (pH equal to the highest Rf value on the pHchromatogram) and, conversely, its re-extraction from the solvent into water (pH corresponding to lowest Rf value). When applied to nystatin, the resulting pH chromatogram - generated on Whatman No. 1 paper with water-saturated n-butanbl as development solvent, and covering the range of pH 2-10 - manifests the expected variations of Rf values with pH changes as anticipated for an amphoteric antibiotic, with two Rf maxima near pH 4 and pH 8, and an Rf minimum in the range pH 5-6. In addition to the methods listed in Table X for the visualization of nystatin after development by paper chromatography either through bioautography or the use of appropriate chemical detection reagents, L i t ~ i n e n k orecorded ~~~ a series of color reactions adaptable to the localization of several common antibiotics on paper chromatograms, including nystatin, and reportedly suitable for the monitoring of antibiotic concentration and purity during production. 6.7.2

Thin-Layer Chromatography

Several thin-layer chromatographic systems have been developed for the separation and identification of nystatin, primarily for use in qualitative procedures to differentiate the antibiotic from other related polyene antifungals. Some of the systems reported in the literature are summarized in Tables XI and XII. Although the thin-layer chromatographic systems listed in Tables XI and XI1 have thus far only found use as qualitative methods for the separation and identification of nystatin, their generally improved resolution - in comparison to paper chromatographic techniques - has greatly enhanced the possibility to rapidly separate individual components within a complex of closely related polyene antibiotics, as recently demonstrated by Porowska and c o - ~ o r k e r Is65 ~ ~with the isolation of three different constituents from the nystatin complex, utilizing both thin-layer and paper chromatographic techniques

TABLE XI Thin-Layer Chromatography Systems f o r Nystatin Solvent System (See Table XII) A A A €3

C

D E F G H I

I J

K L M N 0

P

Q R

Adsorbent

Method of Detection (See Table XII)

Silica Gel 6060 (Eastman) Silica Gel 6060 (Eastman) pH 2 Silica Gel 6060 (Eastman), pH 11 Silica Gel G (Merck) Silica Gel G (Merck) Silica Gel G (Merck), pH 8 Silica Gel G (Merck) Silica Gel 6060 (Eastman) Silica Gel GF (Analtech) Silica Gel G (Merck), pH 3 Silica Gel G (Merck) Silica Gel G (Merck) Silica Gel G (Merck) Silica Gel G (Merck) Silica Gel G (Merck) Silica Gel G (Merck) Silica Gel G (Merck) Silica Gel 60 F-254 (Merck) Silica Gel G F (Analtech) Kieselgur G, impregn-with 0.15 EDTA Sephadex G-15, pH 6

*Migration of nystatin relative to penicillin-G (1.0)

3-

Reference

0.5 0.5 0.45 0.66 0.54

228 228 228 184 184 229-231 232 228 233 235 232 229-231 221,236 221,236 232 232 232 237 234 238 239

0.18 0.28 0.22 0.45,O.51 0.55 0.53 0.18 0.45 0.45 0.65 0.76 0.63 0.38,0.40,0.43 0.25,0.27,0.32 0.0 0.2 (+el)*

TABLE X I 1 Thin-Layer Chromatography Systems for Nystatin Solvent Systems

A B C

D E F G

H I

Methanol Methanol/Acetone/Acetic Acid (8:l:l) Methanol/Isopropanol/Acetic Acid ( 9 : l : O . l ) Ethanol/Ammonia/Water (8:l:l) Ethanol/Ammonia/Water/Dioxane (8:l:l:l) n-Butanol/Methanol (1:l) n-Butanol/Methanol/Water (5:3:2) n-Butanol/Acetic Acid/Water (2:l:l) n-Butanol/Acetic Acid/Water (3:l:l) n-Butanol/Acetic Acid/Water (4:1:2) n-Butanol/Pyridine/Water (2:1:2) n-Butanol/Pyridine/Water (3:2:1) n-Butanol/Pyridine/Acetic Acid/Water ( 5: 10:3 : 2 ) n-Butanol/Dioxane/Acetic Acid/Water (6:1:2:2) n-Amy1 Alcohol/Acetic Acid/Water (2:l:l) Ethyl Acetate/Isopropanol/Water (5:5:3) Methyl Ethyl Ketone/McIlvaine Buffer, pH 4.7/Ethanol (100:6.4:22) - Phosphate Buffer (KH2P04-NaOH, pH 6.0), 0.5M- NaCl 0.025M

TABLE XI1 (Cont'd.) Thin-Layer Chromatography Systems for Nystatin Methods of Detection

1 2 3

P h) 0

8 9 10

11 12 13

Ultraviolet Light228,235i236 Bioautography vs. Candida albicans228 0.2% p-Dimethylaminobenzaldehyde Spray Reagent (in H2SO4, contg. trace FeC13)218,235 0.5% Potassium Permanganate/O. 2% Bromophenol Blue Spray Reagent231 5% Potassium Permanganate Spray Rea ent (or H3P04)242 Charring with mineral acid (H2SO4)233 1% p-Dimethylaminobenzaldeh de/20% SbC13 Spray Reagent (in EtOH, contg. HC1)235 Ultraviolet Light (Fluorescence @ 350 nm)235 0.02N Potassium Permanganate Spray Reagent221r236 Iodine/2,7-Dichlorofluorescein Spray Reagent237 Bioautography vs. Candida tropicalis SC 1674237, or Saccharomyces cerevisiae SC 160c123~2 3 9 p 240 Chlorine/o-Toluidine Spray Reagent243 Bioautography vs. Saccharomyces cerevisiae ATCC 9763237 239 1 240

NYSTAT IN

403

as complementary tools. Similar separations of the nystatin complex into its components have been achieved by Targos and M e t ~ g e rand ~~~ Kocy and Cole237. N ~ s s b a u m e r *proposed ~~ a TLC procedure to establish the degree of nystatin degradation in the formulated drug by monitoring the appearance of a primary oxidation product with an Rf value of 0.73-0.75, compared to an Rf value of 0 . 4 5 for the intact antibiotic. A unique application of thin-layer chromatography has been reported by Zuidweg et a1.239 with the use of Sephadex G-15 as adsorbent medium. Instead of organic solvent mixtures, this medium utilizes an aqueous buffer solution as the developing agent, thus avoiding the possible formation of false inhibition zones during bioautographic development due to incomplete removal of solvent. By combining Sephadex TLC with bioautography (against 2. cerevisiae ATCC 9763 for nystatin), the authors239 accomplished the often problematic separation and qualitative analysis of antibiotics mixtures, including nystatin, amphotericin B, and various penicillins and tetracyclines.

--

Combinations of paper and thin-layer chromatographic methods have been applied by Zhdanovich et a1.226 to the separation and identification of decomposition products of nystatin arising from the partial and total oxidation of the antibiotic with KMnO4 in acidic media. Some of the identified products - including succinic, formic, malic and lactic acid are also claimed to be formed as secondary decomposition products during the natural degradation of nystatin on storage.

-

In an effort to overcome the mechanical problems associated with the need to provide a proper surface contact between the inoculated agar layer and the rigid, glass-backed TLC plate in bioautographic detection methods214 for antimicrobial substances, Meyers and Smith240 introduced the use of spread-layer chromatograms and developed a now commonly adopted transfer technique which consists of inserting a sheet of filter paper between the TLC plate and agar surface. The resulting sandwich is incubated overnight at 37OC with the chromatographic plate and filter paper contacting the agar layer. This method produces sharp, well defined antibiotic zones of inhibition, with sensitivities comparable to those realized with paper chromatograms. Basic alumina, neutral alumina, and silica gel H were found to be suitable adsorbent media for this technique. In the bioautography of nystatin,

404

GERD W. MICHEL

S . cerevisiae served as a useful indicator organism. Several later modifications of this detection method are reported2*I. For the determination of the antioxidant 2 , 6 - d i - Z butyl-4-methylphenol (BHT) in admixture with commercial nystatin in pharmaceutical bulk materials, a TLC procedure has been proposed by Korchagin et al.241. 6.7.3

Gas-Liquid Chromatography

As part of a comprehensive study to establish a general analytical screening scheme for a wide range of materials encountered in forensic toxicology (common poisons, drugs, and human metabolites) , Finkle et al.244 developed a simple GLC system, utilizing four different columns and three liquid phases, to detect any one of almost 600 different substances, including nystatin, to a sensitivity limit of 2 pg/ml in blood, urine and tissue specimens.

During the examination of several polyene antifungal antibiotics by pyrolysis-gas chromatography, Burrows and Calam 245 have shown that nystatin and amphotericin B can be distinguished from each other and from three other polyene macrolides (candicidin, levorin and trichomycin) by the gas chromatograms of their pyrolysis products. 6.7.4

High Performance Liquid Chromatography

Lately, high performance liquid chromatography has been employed in several instances to separate and characterize the individual components of macrolide antibiotic complexes with similar chemical structure246-248. In efforts specifically aimed at the development of a rapid separation method applicable to all chromophore classes of the polyene macrolide antifungal antibiotics, Mechlinski and Schaffner2471248 recently applied a high-speed liquid chromatography (HSLC) technique to the analysis of several prominent polyene antibiotics, including nystatin. In brief, the reported procedure involves the use of a non-commercial liquid chromatograph composed of a Milton Roy high-pressure reciprocating pump with pulse dampener connected to a septum injector, followed by a chromatographic column, a 350 nm W monitor and waste reservoir. The separation of the nystatin complex was achieved in a reverse-phase mode with a mixture of water/methanol/THF (420:90:60 or 420:90:50) as the mobile phase, resulting in the

NYSTATI N

405

isolation of three distinct polyene components, two of which including the main component - were identified as tetraenes, while the third constituent proved to be a heptaene macrolide by spectrophotometric examination. The entire analysis was completed within approx. 15 min, with a retention time for the main component of approx. 4-5 min with both mobile phase solvent mixtures. Possible adaptation of the procedure for use in the quantitative analysis of the individual components is indicated and may require an adjustment in detector response, possibly by increasing the sensitivity of the instrumentation through the use of a continuously variable wavelength UV detector which would allow each chromophore to be monitored at its respective absorption maximum. 6.8 Electrophoretic Analysis

Paris and Theallet2l8 separated a number of antibiotics, including nystatin, by high-voltage paper electrophoresis on Arches 302 paper at a potential gradient of 15.3 volts/ cm over a 2-hour period. With 5% aqueous formic acid solution (pH 2) as electrolyte, nystatin showed a displacement toward the cathode of 13 mm in 2 hours and, over the same timeperiod, a migration of 17 mm toward the anode in an alkaline Verona1 buffer solution (pH 8.6). In the separation of complex antibiotics mixtures, the use of paper electrophoresis at different pH ranges is suggested as a supplemental technique to ordinary chromatographic methods. Electrophoretic mobilities of nystatin, amphotericin A, amphotericin B and several other antibiotics in various different electrolyte systems (salt solutions and solvents) are also reported249. 6.9

Polarographic Analysis

The use of polarography in the determination of antibiotics has been discussed in a recent review by Unterman and WeissbuchZ50. As outlined in Section 2.16, the polarographic behaviour of nystatin has been examined113. Icha and S t r o ~ o v ahave ~ ~ ~reported the determination of nystatin content in the fermentation medium, mycelium and bulk product by oscillopolarographic evaluation of its degradation products resulting from alkali treatment.

406

GERD W. MlCHEL

6.10 Titrimetric Analysis

From a series of potentiometric titrations of nystatin with either glacial acetic acid or mixtures of glacial acetic acid and benzene, dioxane or chloroform as solvent media, and perchloric acid in acetic acid or dioxane as titrants, Mazor and Papay252 evolved an optimum set of conditions for the titration of nystatin in non-aqueous media. The best results for the determination of the antibiotic by both potentiometric and visual endpoint titrations have been obtained with a solution of 5-50 mg of nystatin in 15 ml of a 1:14 (v/v) mixture of glacial acetic acid/dioxane and titration with standard 0.01N perchloric acid in dioxane, using either a glass-calomel electrode combination in a potentiometric procedure, or a visual endpoint determination with methyl violet as indicator. Each ml of 0.01N HC104 is equivalent to 9.52 mg of nystatin. In applying this procedure to the molecular weight determination of nystatin, the authors252 obtained an equivalent weight of 952 for a purified sample of nystatin (see Section 6.2). It is also stressed that the results of potentiometric titrations of nystatin will not provide any measure for the biological activity of a given sample. Attempts at utilizing the addition of bromine or iodine monochloride as the basis for a direct titrimetric determination of nystatin in glacial acetic acid have been reported by Udvardy et al.lg2; however, in either case it was found that halogen addition to the olefinic linkages of nystatin fell short of the theoretically calculated values for six double bonds over a wide range of experimental conditions. Nevertheless, at 105OC and a reaction period of 2 min., iodine monochloride uptake was shown to be equivalent to the saturation of four double bonds. A quantitative version of the latter reaction - involving the dissolution of nystatin in a glacial acetic acid/sulfuric acid mixture, reaction with an excess quantity of a 0.1N iodine monochloride solution, addition of excess potassium iodide solution after the reaction and, finally, back-titration with 0.1N sodium thiosulfate solution - was adopted by the investigators as a means to estimate the tetraene content of a large number of nystatin batches in an effort to correlate the results of chemical assays with biological activity determinations. 6.11 Microbiological Methods Agar diffusion microbiological assays are in general

NYSTATIN

407

use by regulatory agencies42 253 254 for the determination of nystatin in pharmaceutical products. Turbidimetric, tube dilution and respiration inhibition procedures, as well as automated methods , are discussed in respective reviews5116911 7 0 1 255,256. In addition to these conventional antibiotic assay procedures, nystatin activity assays based on its mode of action (membrane disruption, followed by cytoplasmic leakage) have been proposed. They include the measurement of specific conductance changes resulting from the efflux of ionic intracellular constitwnts259, the analysis of released potassium ions260 and of yeast cell constitutents, specifically ninhydrin-positive m i n e products261; the latter method is an automated procedure. 1

I

Nystatin in animal feeds is measured by an a ar diffusion method following extraction with m e t h a n ~ l ~ ~ ~ , ~ ' De~. termination of nystatin in blood, other body fluids, animal tissues and pharmaceutical dosage forms has been described and reviewed169, 256. Sensitivity of the agar diffusion method is approx. 3 units per ml of blood serum, and that of the microscale turbidimetric method is approx. 1 unit per ml.

408

7.

GERD W. MICHEL

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31. 32. 33. 34. 35. 36.

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37. 38. 39. 40.

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NY STAT I N

41 1

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412

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70. 71. 72. 73. 74. 75.

76. 77. 78.

79. 80.

81. 82. 83.

84,

85. 86.

I

Vanek, L. Dolezilova, and 2. Rehacek, J. Gen. Microbiol. 18,649 (1958). I.M. Tereshin, "Polyene Antibiotics - Present and Future", E.R. Squibb Lectures on Chemistry of Microbial Products, 1974. National Center for Antibiotic Analysis, FDA, Department of Health, Education and Welfare, Washington, D.C., 1973. J.W. Lightbown, M. Kogut, and K. Uemura, Bull. Wld. Hlth. Org. 29, 87 (1963). B. Arret, D.P. Johnson, and A. Kirschbaum, J. Pharm. Sci. 60, 1689 (1971). a. "Code of Federal Regulations", Title 21, 141.101141.111, January 1, 1971, U.S. Government Printing Office, Washington, D.C. b. "Code of Federal Regulations", Title 21, Parts 130-146c, and Parts 147 to End, January 1, 1971, U.S. Government Printing Office, Washington, D.C. c. Federal Register, Volume 36, p.7233-7238 (Title 21, Part 148k), U.S. Government Printing Office, Washington, D.C., 1971. Federal Register, Volume 39, p.43833 (Part 449), U.S. Government Printing Office, Washington, D.C., 1974. I. Sunshine, ed., Handbook of Analytical Toxicology, The Chemical Rubber Co., Cleveland, 1969, p.314. Official Methods of Analysis of the Association of Official Analytical Chemists, llth Edition (W. Horwitz, ed.), Association of Official Analytical Chemists, Washington, D.C., 1970, p.954/960. N. Coy, U.F. Nager, and O.T. Ratych, The Squibb Institute for Medical Research, Personal Communication. H. Jacobson and Q. Ochs, ':he Squibb Institute for Medical Research, Personal Communication. B. Toeplitz, The Squibb Institute for Medical Research, Personal Communication. E. Fralick and G.W. Michel, The Squibb Institute for Medical Research, Unpublished Data. J.D. Dutcher, D.R. Walters, and O.P. Wintersteiner, in T.H. Sternberg and V.D. Newcomer, eds., Therapy of Fungus Diseases, An International Symposium, Little, Brown and Co., Boston/London, 1955, p.168. R.M. Evans, The Chemistry of Antibiotics Used in Medicine, Pergamon Press, London, 1965, p.170. F.S. Parker, Applications of Infrared Spectroscopy in Biochemistry, Biology, and Medicine, Plenum Press, New York, 1971, p.400. L.J. Bellamy, The Infra-red Spectra of Complex Molecules, 2. Edition, John Wiley & Sons, Inc., N . Y . , 1958. 2.

-

NYSTATIN

87. 88.

89. 90.

91. 92. 93. 94.

95. 96. 97. 98. 99.

100.

101. 102. 103.

104. 105. 106. 107. 108.

109.

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414

110.

111. 112. 113.

114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124.

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-

-

-

125. 126. 127. 128. 129. 130.

-

NYSTATIN

131. 132.

133. 134. 135. 136. 137.

138. 139. 140.

141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152.

41 5

L. F e u e r , I . I n c z e f y , a n d S. I s t v a n , G e r . O f f e n . 152685~. 2 , 0 6 3 , 0 3 0 ( 1 9 7 1 ) ; C.A. A. Benda, M. Bucko, J . Dasek, J. P a l k o s k a , R. Roubicek, and J. Z a j i c e k , Czech. P a t e n t 124,390 ( 1 9 6 7 ) ; C.A. 69: 1798c. T.S. Bobkova and I . N . Koysharova, A n t i b i o t i k i 2, 40 (1957). L.A. Popova, A n t i b i o t i k i 2, 1 4 ( 1 9 6 0 ) . L.A. Popova, G.F. Z a v i l e i s k a y a , N.T. Dygern, and G . D . P e s t e r e v a , A n t i b i o t i k i 6, 34 ( 1 9 6 1 ) . L.A. Popova and N.E. S t e p a n o v a , A n t i b i o t i k i 868 (1962). E.P. Yakovleva, E.S. L a n t a s , a n d E . I . I o f i n a , 4 t h S c i . Conf. L e n i n g r a d . R e s . I n s t . A n t i b i o t . , L e n i n g r a d , 194 ( 1 9 6 5 ) ; B.A. 48: 92431 (1967). M. Musilkova, F o l i a M i c r o b i o l . ( P r a g u e ) 6, 175 ( 1 9 6 1 ) . B.S. Nugumanov, E.G. Toropova, and N . S . Egorov, A n t i 489 ( 1 9 7 3 ) . biotiki I . Aburatami, K. K a s h i i , K. Minoura, K. T e r a i , Y. I m a n s h i , and T. S u g a i , J . Osaka C i t y Med. C e n t r e 91 (1959). J. V a n d e p u t t e and W. Gold, U.S. P a t e n t 2 , 7 8 6 , 7 8 1 ( 1 9 5 7 ) ; C . A . 51: 8388e. J. V a n d e p u t t e , U.S. P a t e n t 2 , 8 3 2 , 7 1 9 ( 1 9 5 8 ) ; C.A. 52: 14095i. J . D . D u t c h e r and J. V a n d e p u t t e , U.S. P a t e n t 2 , 8 6 5 , 8 0 7 ( 1 9 5 8 ) ; C.A. 53: 655033. J . G . R e n e l l a , U.S. P a t e n t 3 , 5 1 7 , 1 0 0 ( 1 9 7 0 ) ; C . A . 73: 86548b. R.C. E s s e , U.S. P a t e n t 3 , 5 1 7 , 1 0 1 ( 1 9 7 0 ) ; C . A . 73: 69838d. H. Mendelsohn, U.S. P a t e n t 3 , 5 0 9 , 2 5 5 ( 1 9 7 0 ) ; C.A. 28897d. Y . Okami, R. U t a h a r a , S. Nakamura, and H. Umezawa, J. A n t i b i o t i c s ( J a p a n ) , S e r . A , 98 (1954). M. Urks, C . D . C e r k e s , and 2 . S e v e r a , Med. Prom. S.S.S.R. 1 4 , 2 1 ( 1 9 6 0 ) ; C . A . 55: 87549. J. Gyimesi, K. Magyar, B. Kasszan, and V. S e n k a r i u k , Hung. P a t e n t 149,132 ( 1 9 6 2 ) ; C . A . 58: 5458g. E . Nevole, Czech. P a t e n t 110,992 ( 1 9 6 4 ) ; C.A. 6 1 : 15940~. French P a t e n t 1 , 4 8 8 , 5 3 5 ( E . R . S q u i b b & S o n s ) , 1967; C . A . 68: 9 8 6 4 3 ~ . J . V a n d e p u t t e and U.F. Nager, U.S. P a t e n t 3 , 3 3 2 , 8 4 4 ( 1 9 6 7 ) ; C.A. 67: 811299.

2:

1,

=,

8,

-

2:

z,

-

-

-

GERD W. MICHEL

41 6

153.

154. 155. 156.

D.M. T r a k h t e n b e r g , E . I . Rodionovskaya, Z.V. G o r d i n a , L . I . R o s t o v t s e v a , G . I . Kleiner, A.M. Nagle, a n d V. L a z d i n a , Med. Prom. S.S.S.R. 18 ( 1 9 6 0 ) ; C.A. 55: 2019d. G . I . K l e i n e r , V.Y. L a s d i n y a , a n d D.M. T r a k h t e n b e r g , U.S.S.R. P a t e n t 123,287 ( 1 9 5 9 ) . M. Matsuoka and K. Kitamura, Jap. P a t e n t 1 3 , 7 4 8 ( 1 9 6 1 ) ; C.A. 56: 7445e. G . I . K l e i n e r , N.V. I o n o v a , D.M. T r a k h t e n b e r g , a n d L . I . Rostovtseva, A n t i b i o t i k i 200 ( 1 9 6 1 ) . V. Panes and V. Pecak, Czech. P a t e n t 1 0 6 , 4 2 8 ( 1 9 6 3 ) ; C.A. 60: 1 0 7 9 f . V.S. z p e j e v , Czech. P a t e n t 1 1 6 , 9 5 8 ( 1 9 6 5 ) ; C.A. 167972. S. I s t v a n , Hung. P a t e n t 152,192 ( 1 9 6 5 ) ; C.A. 17094e. I . K . K a r p i t s k i i , M.P. E l i z a r o v s k i i , G . I . K l e i n e r , E . I . Dangovet, D.M. T r a k h t e n b e r g , and P.A. K a l i n i n , U.S.S.R. P a t e n t 167,609 ( 1 9 7 0 ) ; C.A. 73: 7231s. A.P. Bashkovich, Y.D. S h e n i n , T.V. KoGnko, V.Y. R a i g o r o d s k a y a , M.P. Karpenko, N.A. f i a s n o b a e v a , N.G. V a s i l ' e v a , N.P. Bogdanova, and O.N. Ekzemplyarov, 31 ( 1 9 6 7 ) . a i m . - F a r m . Zh. S. Boteanu, S. Balliu-Mutu, E. A i t e a n u , and 0. Ludu, 681 (1969). Farmacia ( B u c h a r e s t ) G . I . K l e i n e r a n d N.V. I o n o v a , A n t i b i o t i k i 5, 712 (1963). A.D. Kuzvkov, G.B. L o k s h i n , Y.V. Zhdanovich, and K.M. Khryascheva, A n t i b i o t i k i 422 ( 1 9 6 6 ) . G.B. L o k s h i n , Y.V. Zhdanovich, A.D. Kuzovkov, T . I . Volkova, V.Y. Shtamer, a n d G . I . Kleiner, A n t i b i o t i k i 1 2 , 196 ( 1 9 6 7 ) . G.B. L o k s h i n , Y.V. Zhdanovich, A.D. Kuzovkov, G . I . K l e i n e r , S.M. Chaikovskaya, V.Y. Shtamer, and T . I . Volkova, U.S.S.R. P a t e n t 1 7 9 , 4 2 9 ( 1 9 6 6 ) ; C.A. 2076a. A.E. Tebyakina, S.M. Chaikovskaya, and T.G. Venkina, 547 ( 1 9 6 1 ) . Antibiotiki A.E. E l k o u l y , N.A. E l s a y e d , and A.M. M o t a w i , Mfg. Chem. Aerosol N e w s , August 1 9 7 3 , p.37. D.C. Grove and W.A. R a n d a l l , Assay Methods o f A n t i b i o t i c s - A L a b o r a t o r y Manual ( A n t i b i o t i c s Monographs N o . 2 ) , Medical E n c y c l o p e d i a , I n c . , N e w York, 1 9 5 5 , p.116. J . R . Gerke a n d M.E. Madigan, A n t i b i o t i c s Chemother. 11, 227 (1961), I. Horvath a n d I. Koczka, N a t u r e 1305 ( 1 9 6 4 ) .

14,

-

5,

157. 158. 159. 160.

161.

65:

63:

L,

162. 163. 164. 165.

17,

11,

-

166.

65:

167. 168. 169.

170. 171.

6,

-

203,

NYSTATI N

172. 173. 174. 175. 176. 177. 178. 179. 180. 181.

182. 183.

184. 185. 186.

187. 188. 189. 190. 191. 192. 193.

417

B.M. Trivedi and N.B. Shah, Indian J. Pharm. 32, 156 (1970). G.B. Lokshin, Y.D. Zhdanovich, and A.D. Kuzovkov, Antibiotiki 11,590 (1966). Y.D. Zhdanovich, G.B. Lokshin, and A.D. Kuzovkov, Antibiotiki 12 , 122 (1967). I. Boudru and C. Bouillet, J. Pharm. Belg. 27, 305 (1972). V.A. Tsyganov and N.G. Vasilieva, Antibiotiki 16,47 (1971). A.Capek and A. Simek, Folia Microbiol. (Prague) 16, 364 (1971). T.Y. Skvirskaya, G.N. Naumchik, A.I. Pavlova, and M.F. Ugleva, Farmatsiya (Moscow) 18,13 (1969). M. Hermansky and M. Vondracek, Czech. Patent 139,880 (1971); C.A. 76: 90049~. J.S. Hydro, T G Squibb Institute for Medical Research, Personal Communication. J.F. Alicino, The Squibb Institute for Medical Research, Personal Communication. Federal Register, Volume 37, p.865-6 (Title 21, Part 148k) U.S. Government Printing Office, Washington, D.C.r 1972. J. TrAfouel, J. Cheymol, R. Paul, H. Phnau, G . Hagemann, J. Comar, R. Romain, M. Philippe, J. Vignalon, and A. Quevauviller, Therapie 14,573 (i959). L. Dryon, J. Pharm. Belg. 21, 433 (1966). V.B. Korchagin and L.N. Savushkina, Antibiotiki 8,634 (1963). C. Sherman, T. Platt, M. Massey, and E. Ivashkiv, The Squibb Institute for Medical Research, Personal Communication. C. Sherman, The Squibb Institute for Medical Research, Personal Communication. E. Aiteanu and M. Medianu, Rev. Chim. (Bucharest) 20, 28 (1969). Federal Register, Volume 2, p.18922-19193 (Title 21, Part 449), U.S. Government Printing Office, Washington, D.C., 1974. L.O. Bolshakova and B.G. Belenky, Antibiotiki 10,707 (1965) V.B. Korchagin and A.A. Korobitskaya, Antibiotiki 8, 1049 (1963). A. Udvardy, A. David, G. Horvath, and A. Toth, Proc. Conf. Appl. Phys. Chem. 2nd 1971, Part 1, p.255. L.G. Wayland and P.J. Weiss, J. Pharm. Sci. 57, 806 (1968).

.

-

418

GERD W . MICHEL

194. K.V. Unterman, Antibiotiki 10,867 (1965). 195. V. Valenti, The Squibb Institute for Medical Research, Personal Communication. 196. E.D. Etingov, G.V. Kholodova, V.O. Kul'bakh, and A.I. Karnatushkina, Antibiotiki 17,301 (1972). 197. H. Laubie, Bull. SOC. Pharm. Bordeaux 99, 3 (1960); C.A. 54: 17793d. 198. 0. Szucs, Gy&gyszer&szet 2, 469 (1961); C.A. 57: 4764i. 199. B. Ozsoz, Turk Ijiyen Tecrubi Biyol. Dergisi 22, 59 (1962); C.A. 57: 9957d. 200. W.H. Unterman, Rev. Chim. (Bucharest) 12, 415 (1961). 201. W.H. Unterman, Rev. Chim. (Bucharest) 12, 504 (1961). 202. S. Ochab, Diss. Pharm. 17,323 (1965). 203. W.H. Unterman, Rev. Chim. (Bucharest) 13, 618 (1962). 204. L. Mazoi and K. Papay, Acta Pharm. Hung. 32, 59 (1962). 205. J.C. Chang, A.B. Honig, A.T. Warren, and S. Levine, J. Pharm. Sci. 52, 536 (1963). 206. M.M. Amer and A.A. Habib, Talanta 22, 605 (1975); see also GyAgyszer6szet 18,472 (1974). 207. B.Z. Chowdhry, Talanta 23, 79 (1976). 208. V. Betina, in M. Lederer, ed., Chromatographic Reviews, Volume 7, Elsevier Publishing Company, Amsterdam/London/New York, 1965, p.119-178. 209. K. Macek, I.M. Hais, J. Kopeck$, and J. Gasparic, Bibliography of Paper and Thin-Layer Chromatography 1961-1965, Elsevier Publishing Company, Amsterdam/ London/New York, 1968, p.578. 210. Z. RehAcek, Folia Microbiol. (Prague) 8, 228 (1963). 211. A.P. Struyk, I. Hoette, G. Drost, J.M. Waisvisz, T. van Elk, and J.C. Hoogerheide, in H. Welch.and F. Marti-Ibanez, eds. , Antibiotics Annual 1957-1958, Medical Encyclopedia, Inc., New York, 1958, p.878. 212. R. Fiigner and G. Bradler, Z. Allgem. Mikrobiol. 3, 173 (1963). 213. V. Sevcik, M. Podojil, and A. Vrtiskovh, Cesk. Mikrobiol. 2, 175 (1957). 214. V. Betina, J. Chromatogr. 78, 41 (1973). 215. A. Ammann and D. Gottlieb, Appl. Microbiol. 3, 181 (1955). 216. D.H. Peterson and L.M. Reineke, J. Amer. Chem. SOC. 72, 3598 (1950). 217. W.A. Ritschel and H. Lercher, Pharm. Ztg., Ver. Apotheker-Ztg. 106,120 (1961); C.A. 62: 397i. 218. R. Paris and J.-P. Theallet, Ann. Pharm. Franc. 20, 436 (1962). 219. K.V. Rao and W.P. Cullen, J. h e r . Chem. SOC. 82, 1127 (1960)

-

-

-

.

NYSTAT I N

220.

221.

222. 223.

C . P . S c h a f f n e r , I . D . Steinman, R . S . S a f f e r m a n , and H. L e c h e v a l i e r , i n H. Welch and F. M a r t i - I b a n e z , e d s .

226. 227. 228. 229.

230.

231. 232. 233. 234. 235. 236. 237. 238. 239. 240.

241. 242.

,

A n t i b i o t i c s Annual 1957-1958, Medical E n c y c l o p e d i a , I n c . , New York, 1958, p.869. G . H . Wagman and M . J . W e i n s t e i n , Chromatography of A n t i b i o t i c s , J o u r n a l of chromatography L i b r a r y Volume 1, E l s e v i e r S c i e n t i f i c P u b l i s h i n g Company, Amsterdam/London/New York, 1973, p.55/130. J . Burns and D . F . Holtman, A n t i b i o t i c s and Chemot h e r a p y 9, 398 ( 1 9 5 9 ) . Y.M. Khokhlova, A.V. Puchnina, E.F. Oparysheva, L.M. Golovkina, and N.O. B l i n o v , Izv. Akad. Nauk SSSR, S e r . B i o l . 2, 433 ( 1 9 6 6 ) ; C.A. 65: 3668c. V . B e t i n a and P. Nemec, Nature 1111 ( 1 9 6 0 ) . V . B e t i n a and P . Nemec, Chem. Z v e s t i 853 ( 1 9 6 1 ) ; C . A . 58: 5453d. Y . V . Zhdanovich, G . B . Lokshin, and A.D. Kuzovkov, 42 ( 1 9 6 7 ) ; C.A. 68: 33147k. Khim.-Farm. Zh. S.N. L i t v i n e n k o , Lab. Delo 8,39 ( 1 9 6 2 ) ; C.A. 58: 2322d. A . A s z a l o s , S. Davis, and D. F r o s t , J. Chromatoqr. 37, 487 ( 1 9 6 8 ) . T. Ikekawa, F. I w a n i , E . A k i t a , and H. Umezawa, J . A n t i b i o t . (Tokyo), S e r . A , 56 ( 1 9 6 3 ) . G. Zweig and J. Sherma, e d s . , Handbook o f Chromatog r a p h y , Volume 1, CRC Pre ss, The Chemical Rubber Co., C l e v e l a n d , 1972, pp.458, 459, 751. E. A k i t a and T. Ikekawa, J . Chromatogr. 250 ( 1 9 6 3 ) . S . Ochab, D i s s . Pharm. Pharmacol. 22, 351 ( 1 9 7 0 ) ; C.A. 74: 797322. F. Tarqos and J. Metzqer, The Squibb I n s t i t u t e f o r Medical R e s e a r c h , P e r s o n a l Communication. F. T a r q o s , The Squibb I n s t i t u t e f o r Medical R e s e a r c h , P e r s o n a l Communication. C. M a t h i s , B u l l . SOC. Chim. F r . (l), 93. P.-A. Nussbaumer, Pharm. A c t a Helv. 43, 462 ( 1 9 6 8 ) . 0. Kocy and N . C o l e , The Squibb I n s t i t u t e f o r Medical R e s e a r c h , P e r s o n a l Communication. J . K e i n e r , R. Hiittenrauch, and W. P o e t h k e , Pharm. Z e n t r a l h a l l e 108,525 ( 1 9 6 9 ) . M.H.J. Zuidweg, J . G . Oostendorp, and C . J . K . BOS, J. Chromatogr. 42, 552 ( 1 9 6 9 ) . E . Meyers and D.A. Smith, J. Chromatogr. 129 (1964). V . B . Korchagin, G.B. Lokshin, and V . I . N i r e n b e r c h i k , Antibiotiki 1047 ( 1 9 6 6 ) ; C.A. 66: 40745r. N.O. B l i n o v and A.S. Khokhlov, A n t i b i o t i k i S , 751 (1963).

-

224. 225.

419

187,

15,

I,

16,

12,

-

1973

14,

11,

GERD W. MICHEL

420

243. 244. 245. 246. 247. 248. 249. 250. 251. 252. 253. 254. 255. 256. 257.

258. 259. 260. 261.

E. Stahl, ed., Thin-Layer Chromatography, SpringerVerlag, Berlin/Heidelberg/New York, 1965, p.488. B.S. Finkle, E.J. Cherry, and D.M. Taylor, J. Chromatogr. Sci. 9, 393 (1971). H.J. Burrows and D.H. C a l m , J. Chromatogr. 53, 566 (1970). S. Omura, Y. Suzuki, A. Nakagawa, and T. Hata, J. Antibiot. 26, 794 (1973). W. Mechlinski and C.P. Schaffner, Abstr. 13th Interscience Conf. on Antimicrobial Agents and Chemotherapy, Washington, D.C., 1973, Paper 143. W. Mechlinski and C.P. Schaffner, J. Chromatogr. 2, 619 (1974). S. Ochab, Diss. Pharm. Pharmacol. 24, 205 (1972); C.A. 77: 44438t. H.W. Unterman and S. Weissbuch, Pharmazie 29, 752 (1974). F. Icha and J. Strosova, Czech. Patent 114,468 (1965); C.A. 64: 6418f. L. M&& and K.M. PApay, Z. Anal. Chem. 184,272 (1961). "Code of Federal Regulations", 21 CFR 436.105, 1975, U.S. Government Printing Office, Washington, D.C. Minimum Requirements of Antibiotic Products, Ministry of Health and Welfare, Jap. Government, Tokyo, 1961. J.R. Gerke, J.D. Levin, and J.F. Pagano, in F. Kavanagh, ed., Analytical Microbiology, Volume I, Academic Press, Inc., New York/London, 1963, p.387. T.B. Platt, J.D. Levin, J. Gentile, and M.A. Leitz, in F. Kavanagh, ed., Analytical Microbiology, Volume 11, Academic Press, Inc., New York/London, 1972, p.147. Official Methods of Analysis of the Association of Official Analytical Chemists, 12th Edition (W. Horwite, ed.), Association of Official Analytical Chemists, Washington, D.C., 1975, p.811. T.B. Platt and A.G. Itkin, J. Assoc. Offic. Anal. Chem. 57, 536 (1974). D.M. Isaacson and T.B. Platt, Bacteriol. Proc. 1968, 1. S. Clements-Jewery, Antimicrob. Agents Chemother. 9, 585 (1976). W.G. Evans and J.E. Bodnar, Adv. Autom. Anal. Technicon Int. Congr. 1972 (Pub. 1973) ?, 45.

-

NYSTATIN

421

The above references attempt to cover the literature through 1972 (Chemical Abstracts, Volume 76). In addition to several more recent publications also included, the following additional papers related to analytical aspects of nystatin have come to the author's attention during the preparation of this profile: 262. M.M. Amer et al. Application of orthogonal functions to determination of nystatin in the presence of its degradation products J. Pharm. Pharmacol. 27, 377 (1975). 263. M.V. Bibikova et al. On the possibility of early identification of organisms producing polyenic antibiotics Antibiotiki 20, 675 (1975). 264. E.D. Etingov et al. Ionization of acid-base groups of polyenic antibiotics in aqueous solutions Antibiotiki 20, 678 (1975). 265. V.A. Weinstein et al. Studies on association of nystatin and amphotericin B in non-aqueous solvent systems Antibiotiki 20, 688 (1975) 266. E. Jereczek et al. Use of tris (dipivaloylmethane) europium in NMR studies of some structural elements of antibiotics of the polyene macrolide group Inst. Nucl. Phys., Cracow, Rep. 1973,No. 819/(PL) (Pt. 2), 232. 8.

ACKNOWLEDGMENT

The author expresses his appreciation to Dr. T.B. Platt for his contribution of the section on microbiological assay methods; to Dr. N.S. Semenuk and his associates of the Science Information Department of the Squibb Institute for Medical Research for their assistance in the literature search; to Ms. E. Fralick for a thorough review of the manuscript; and to Ms. F. Kaiser for her expert secretarial support and for her patience in the preparation and correction of this monograph.

PROPARACAINE HYDROCHLORIDE

Daisy B. Whigan

424

1.

2.

3. 4. 5.

6.

DAISY B. WHIGAN

TABLE OF CONTENTS Description 1.1 Name,Formula, Molecular Weight 1.2 Appearance, Color, Odor Physical Properties 2 . 1 Spectra 2 . 1 1 Infrared Spectra 2.12 Nuclear Magnetic Resonance Spectra 2.13 Ultraviolet Spectra 2.14 Mass Spectra 2.15 Fluorescence Spectra 2.2 Crystal Properties 2 . 2 1 Crystallinity 2.22 Polymorphism 2.23 Differential Thermal Analysis 2.24 Thermal Gravimetric Analysis 2.25 Differential Scanning Calorimetry 2.26 X-Ray Powder Diffraction 2.27 Melting Range 2.3 Solution Data 2 . 3 1 Solubility 2.32 pKa 2.33 Phase Solubility Analysis Synthesis Stability-Degradation Analysis of Intermediate Compound and Hydrolysis Products Methods of Analysis 6.1 Identification Tests 6.2 Elemental Analysis 6.3 Spectrophotometric Analysis 6 . 3 1 Ultraviolet Spectrophotometric Ana lysis 6.32 Fluorescence Spectrophotometric Analysis 6.4 Titrimetric Procedures 6 . 4 1 Nonaqueous Titration 6.42 Titration with Sodium Nitrite 6.43 Spectrophotometric Titration with Nitrous Acid 6.5 Colorimetric Methods 6 . 5 1 With Bratton-Marshall Reagent

PROPARACAINE HYDROCHLORIDE

With Sodium 1,2-Naphthoquinone-4sul f onate 6.6 Chromatographic Procedures 6.61 Paper Chromatography 6.62 Thin-layer chromatography Analysis of Hydrolysis Products in Body Fluids and Tissues Serum Protein Binding Drug Metabolism References 6.52

7. 8. 9. 10.

425

426

DAISY B. WHIGAN

Description 1.1 Name, Formula, Molecular Weight Proparacaine h y d r o c h l o r i d e i s 2 - ( d i e t h y l a m i n o ) e t h y l 3-amino-4-propoxybenzoate monohydrochloride. Chemical A b s t r a c t s l i s t i n g s a r e under t h e heading benzoic acid,3-amino-4-propoxy-2( d i e t h y 1 a m i n o ) e t h y l e s t e r , monohydrochloride. The Chemical A b s t r a c t s R e g i s t r y Number i s 5875-06-9. I t i s a l s o known a s proxymetacaine hydrochloride. Common t r a d e names a r e Ophthaine, Alcaine, and Ophthetic, 1.

-0-CH2 CH2N (C2H5) 2

CH3CH2CH27

.H C 1

Appearance, Color, Odor Proparacaine h y d r o c h l o r i d e i s a white o r f a i n t buff c r y s t a l l i n e , o d o r l e s s powder. 1.2

Physical P r o p e r t i e s 2 . 1 Spectra 2 . 1 1 Infrared Spectra The i n f r a r e d spectrum o f proparac a i n e h y d r o c h l o r i d e compressed i n a potassium bromide p e l l e t i s shown i n Figure 1. The spectrum was o b t a i n e d on a Perkin-Elmer Model 6 2 1 g r a t i n g i n f r a r e d spectrophotometer. The following assignments have been made f o r s t r u c t u r a l l y s i g n i f i c a n t bands' : Waveleng t h ycm'l Assiqnment 3420,3280 NH2 s t r e t c h 2700,2640 HC1 1700 Ester G O 1610,1585,1510 Aromatic C=C =C-0 ( e s t e r and 1295,1200 aromatic e t h e r ) 2.

PROPARACAIN E HYDROCHLORIDE

427

obtained the infrared spectrum of proparacaine hydrochloride from a mineral oil dispersion on a Perkin-Elmer spectrophotometer Model 157. The following spectral assignments were made: Wavelength,cm-l 3470 3300 2620 2500 171 5 1630 1600 870

Assignment -NH2 group - N H ~group H'N of trisubstituted amine H'N of trisubstituted amine C=O Ester Phenyl ring Phenyl ring CH aromatic

The discrepancies in the spectral wavelengthsof the two interpretations could be attributed to calibration differences of the di fferent instruments used2.

WAVELENGTH (MICRONSI

FREQUENCY (CM’)

F i g u r e 1.

I n f r a r e d Spectrum of P r o p a r a c a i n e Hydrochloride i n a Kl3r P e l l e t .

PROPARACAINE HYDROCHLORIDE

429

2.12

N u c l e a r Maqnetic Resonance S p e c t r a F i g u r e 2 shows t h e n u c l e a r m a g n e t i c resonance spectrum of proparacaine h y d r o c h l o r i d e i n d e u t e r a t e d d i m e t h y l s u l f o x i d e . The s p e c t r u m was o b t a i n e d on a Perkin-Elmer R12B NMR s p e c t r o m e t e r u s i n g t e t r a m e t h y l s i l a n e a s an i n t e r n a l r e f e r e n c e . S p e c t r a l a s s i g n m e n t s 3 a r e r e c o r d e d i n Table 1. Table 1

CH2 H 3C - CH2 HC 1

Proton Position

1 2 3 4 5 6

7

a 9 10 11

N+H

*d

( N o . of

* Peaks)

'CH2- CH3

Coupling c o n s t a n t J (HZ)

1.27 ( t ) 3.17 ( 9 ) 3.44 ( t ) 4.61 ( t ) 7.32 ( 9 ) 7.40 (d) 6.90 (d)

7.0 7.0 6.0 6.0 9.0 9.0 1.0

5.00 ( b )

---

4.00 ( t )

6.0

1.75 ( m ) 1.00(t) 11.35 ( b )

= doublet, t = t r i p l e t , q = q u a r t e t , m = m u l t i p l e t , b = broad

--6.5

---

w%3

8-25-76

DM%

P

0 w

-

5 -5

Figure 2.

I

. .

--7.

7--7---*---

9-72 GMJ

1.

I

4

5

6

7

8

NMR Spectrum of P r o p a r a c a i n e H y d r o c h l o r i d e i n D e u t e r a t e d

D i m e t h y l s u l f oxide.

*

I *t#l

PROPARACAINE HYDROCHLOR IDE

431

2.13

U l t r a v i o l e t Spectra The u l t r a v i o l e t s p e c t r u m o f p r o p a r a c a i n e h y d r o c h l o r i d e i n m e t h a n o l , ca. 1 2 pg/ml, i s shown i n F i u r e 3 ( 1 n s t r u m e n t : C a r y 1 5 ) . H e f f e r e n and co-workers' a t t r i b u t e d t h e f o l l o w i n g chemical s t r u c t u r e s r e s p o n s i b l e f o r the u l t r a v i o l e t a b s o r p t i o n of s u b s t i t u t e d benzoic a c i d esters: Chemical S t r u c t u r e Approximate Wavelength, nm Carbonyl d i r e c t l y 225 attached t o aromatic ring Amino c o n j u g a t e d w it h ca rbonyl Ethers conjugated with carbonyl

300

270

The u l t r a v i o l e t maxima o b s e r v e d f o r proparacaine hydrochloride agree very w e l l with t h e above assignments. A l l t h r e e peaks a r e a l s o o b s e r v e d when e t h y l a l c o h o l 6 , w a t e r 5 , 7, a n d a q u e o u s base4 a r e used a s s o l v e n t i n s t e a d of methanol. F i g u r e 4 shows t h a t t h e u l t r a v i o l e t a b s o r p t i o n o f p r o p a r a c a i n e i s d e p e n d e n t on pH. The e f f e c t of t h e pH o f t h e s o l u t i o n on t h e u l t r a v i o l e t absorption of proparacaine hyd ro ch lo rid e w a s A t a c i d i c pH, e x t e n s i v e l y s t u d i e d by H e f f e r e n 8 . t h e a r o m a t i c amine forms a p o s i t i v e l y c h a r g e d ammonium i o n t h u s n u l l i f y i n g t h e p a r t i c i p a t i o n o f t h e amino g r o u p i n r e s o n a n c e w i t h t h e a r o m a t i c r i n g . The pH p r o f i l e o f t h e s p e c t r a p r e s e n t e d b y H k f f e r e n showed an i s o b e s t i c p o i n t a t 243 nm.

432

k

E0

h

'D

Ultraviolet Spectrum of Proparacaine Hydrochloride So1vent:Methanol - 1nstrument:Cary 15

433

a,

C

.rl

k 04

0

WI

k

m JJ

d

A k

I

Ii

2.14

Mass S p e c t r a The l o w r e s o l u t i o n mass s p e c t r u m o f p r o p a r a c a i n e h y d r o c h l o r i d e , T h i s w a s o b t a i n e d on a n S q u i b b S t a n d a r d L o t 41519-003, i s shown i n F i g u r e 5. A s s o c i a t e d E l e c t r i c a l I n d u s t r i e s Model MS-902 M a s s S p e c t r o m e t e r e q u i p p e d w i t h a frequency-modulated a n a l o g t a p e r e c o r d e r . The major The p a r e n t i o n , M+, o f t h e compound a t m / e 294 i s weak. i o n a t m / e 8 6 i s d u e t o t h e c l e a v a g e o f t h e bond b e t a t o t h e t e r t i a r y a m i n e nitrogen. T h i s c l e a v a g e i s a n t i c i p a t e d i n t h e f r a g m e n t a t i o n of amines. Mass s p e c t r a l a s s i g n m e n t s of p r o m i n e n t i o n s a r e g i v e n b y t h e f r a g m e n t a t i o n p a t t e r n below48.

-1 f.i m/e 99-H

P

m/e

I -

w P

H3C-

CH2-CH2-

CH2

0

195 +H

m/e

m/e

178

m/e

295 (M+

178

_ I

+

H)

m/e

m/e

136

+

CH3CH = CH2

222

N

~ ' 2 ~ 5

\C2H5

$00

hlISN31NI 3 A I l t 1 1 3 t l

435

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5 0

k

.d d

k

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5

Q)

C

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rd

.d

k

rd

0 k

rd a

PI

0

W

k

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U

4J

a v)

111 111

c

2 0

4J

-d

7 0

ffl

d Q)

I

p: d

Q)

In

k

7 tn

-4 L4

436

DAISY 6. WHIGAN

Fluorescence S p e c t r a Proparacaine h y d r o c h l o r i d e e x h i b i t s I t s e x c i t a t i o n and f l u o r e s n a t i v e fluorescence4. cence s p e c t r a i n methanol a s recorded on a PerkinElmer Fluorescence Spectrophotometer Model 204 a r e reproduced i n Figure 6. The f l u o r e s c e n c e of prop a r a c a i n e h y d r o c h l o r i d e v a r i e s w i t h pH. I t i s most i n t e n s e i n 0 . 1 N sodium hydroxide where a concentrat i o n of 0 . 0 5 pg per m l had an i n t e n s i t y t h a t was f i v e times t h a t of t h e blank. I n 0.1N s u l f u r i c a c i d , t h e f l u o r e s c e n c e i s quenched. Fluorescence c h a r a c t e r i s t i c s of p r o p a r a c a i n e h y d r o c h l o r i d e i n a l i m i t e d l i s t of s o l v e n t s a r e p r e s e n t e d i n Table 2 . 2.15

Table 2 Fluorescence C h a r a c t e r i s t i c s of Proparacaine Hydrochloride Solvent Water Sodium Hydroxide, 0.1N Phosphate B u f f e r , pH 7.0 Me thano 1

Excitation Maximum ,nm 3 16

Fluorescence Maximum, nm 460

300

3 96

316 3 18

454 44 0

I n 0.1g sodium hydroxide, t h e r e i s a l i n e a r r e l a t i o n s h i p between t h e f l u o r e s c e n c e i n t e n s i t y and t h e c o n c e n t r a t i o n of p r o p a r a c a i n e hydrochloride up t o 5 pg p e r m l .

437

PROPARACAINE HYDROCHLORIDE

Figure 6. Excitation and Fluorescence Spectra of Proparacaine Hydrochloride 1nstrument:Perkin-Elmer Fluorescence Spectrophotometer Model 204 Solvent: Methanol I

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i

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m m

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,

438

DAISY B.WHIGAN

crystal Properties 2.21 Crystallinity P r o p a r a c a i n e h y d r o c h l o r i d e forms small r o s e t t e s o r bunches of n e e d l e s w i t h p l a t i n i c It a l s o forms r o s e t t e s of l o n g t h i c k bromide'. needles with 5 - n i t r o b a r b i t u r i c acid44. Photomicrog r a p - , s of c r y s t a l s formed w i t h c h l o r o p l a t i n i c a c i d , p i c r o l o n i c a c i d , and p o t a s s i u m permanganate w e r e t a k e n b y Rich a n d C h a t t e n l o . 2.2

2.22

Polymorphism N o polymorphism h a s b e e n r e p o r t e d

f o r proparacaine hydrochloride. However, K o e h l e r and Feldmann'l s u g g e s t e d t h e p o s s i b i l i t y o f polymorphism i n t h e s o l i d t e t r a p h e n y l b o r a t e derivative

.

2.23

D i f f e r e n t i a l Thermal Analysis(DTA) Jacobson12 c o n d u c t e d t h e d i f f e r e n t i a l t h e r m a l a n a l y s i s of p r o p a r a c a i n e h y d r o c h l o r i d e on a DuPOnt 900 Thermo-analyzer w i t h a The thermot e m p e r a t u r e r i s e of 15O p e r m i n u t e . gram o f p r o p a r a c a i n e h y d r o c h l o r i d e (Squibb House S t a n d a r d L o t 41519-003) showed a s h a r p endotherm a t 1 8 1 O C which c o r r e s p o n d s t o t h e m e l t of t h e d r u g (See S e c t i o n 2.27 f o r M e l t i n g R a n g e ) . 2.24

Thermal G r a v i m e t r i c A n a l y s i s (TGA) Thermal g r a v i m e t r i c a n a l y s i s o f p r o p a r a c a i n e h y d r o c h l o r i d e was c o n d u c t e d on a DuPont T h e r m o g r a v i m e t r i c A n a l y z e r Model 900.Working w i t h p r o p a r a c a i n e h y d r o c h l o r i d e , Squibb S t a n d a r d L o t 41519-003, Jacobson12 found no w e i g h t l o s s b e f o r e 150OC. The compound w a s h e a t e d a t a r a t e o f 15O p e r m i n u t e u n d e r a n i t r o g e n sweep. 2.25

D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) Va1entil3 determined t h e p u r i t y of p r o p a r a c a i n e h y d r o c h l o r i d e by DSC. A s c a n n i n g r a t e o f 0.625 deg/min and a s e n s i t i v i t y o f 2 m i l l i c a l / s e c were used. Using a Perkin-Elmer DSC Model lB, t h e p u r i t y of p r o p a r a c a i n e h y d r o c h l o r i d e l o t 46016-064

PROPARACAINE HYDROCHLORIDE

439

was c a l c u l a t e d t o b e 99.94 mol p e r c e n t . 2.26 X-Ray Powder D i f f r a c t i o n The x - r a y powder d i f f r a c t i o n p a t t e r n of p r o p a r a c a i n e h y d r o c h l o r i d e was o b t a i n e d by Ochsl4 on a P h i l l i p s X-Ray Powder D i f f r a c t o m e t e r , Type 120-101-11, a t a v o l t a g e of 35 kv a n d a c u r r e n t of 1 0 mA. Theosample w a s i r r a d i a t e d b y a copper s o u r c e a t 1 . 5 4 A . Data d e r i v e d from the spectrum ( F i g u r e 7 ) of p r o p a r a c a i n e h y d r o c h l o r i d e , Squibb S t a n d a r d Lot 41519-003, a r e l i s t e d i n T a b l e 3. Table 3 X - R a y Powder D i f f r a c t i o n P a t t e r n of Proparacaine Hydrochloride 1 n s t r u m e n t : P h i l l i p s X-Ray Powder D i f f r a c t o m e t e r

1(2q

0

*

d' ( A )

**

I/IO***

0.663 12.56 7.04 0.460 10.02 8.83 0.176 7.84 11.29 0.197 6.95 12.74 1.000 6.52 13.59 15.63 5.67 0.212 5.46 16.22 0.140 0.178 5.12 17.33 4.97 17.84 0.357 4.52 19.62 0.518 4.17 21.32 0.483 3.81 0.179 23.36 0.299 3.64 24.47 0.122 25.15 3.54 0.360 3.37 26.42 0.300 27.02 3.30 0.497 3.26 27.36 0.261 3.05 29.31 2.69 0.153 33.31 2.65 0.226 33.82 2.51 0.115 35.77 *Twice t h e a n -q l e of i n c i d e n c e o r r e f l e c t i o n ** d ( i n t e r p l a n a r d i s t a n c e ) = 2 sin 8 0

*** R e l a t i v e

o f 1.000.

=

1.539 A

i n t e n s i t y b a s e d on h i g h e s t i n t e n s i t y

OPP

PROPARACAl NE HY DROCH LOR I DE

44 1

2.27

Melting Range The m e l t i n g r a n g e f o r U.S.P. prop a r a c a i n e h y d r o c h l o r i d e i s s p e c i f i e d a s 178O t o 185OC. C l i n t o n , et. al. reported a melting range of 182-183.3OC. Squibb S t a n d a r d p r o p a r a c a i n e h y d r o c h l o r i d e Lot 41519-003 gave a m e l t i n g range o f 1820 t o 184OC. Monguzzi and c o - w ~ r k e r sr~e p~o r t e d a m e l t i n g range of 180' t o 182OC. 2.3 S o l u t i o n Data 2.31 S o l u b i l i t y Approximate S o l u b i l i t y of P r o p a r a c a i n e Hydrochloride a t Room Temperature 1 7 Solvent

S o l u b i l i t y (mq/ml)

> 50

Water Dimethylsulfoxide Chloroform Ethanol Benzene Hexane Ethyl Acetate Ether 2.32

50 30 7 < 0.1 (0.1 (0.1 (0.1

pKa

Hef f e r e n 8 determined t h e a p p a r e n t d i s s o c i a t i o n c o n s t a n t of t h e a r o m a t i c amino group: R

-

+

NH3

R

-

NH2

+

H+

Using a s p e c t r o p h o t o m e t r i c method d e s c r i b e d by F l e x s e r , Hammett, and Din the a p p a r e n t pK' a i s 3.22 (Kh = 6.03 x lo-') 2.33

Phase S o l u b i l i t y A n a l y s i s The p u r i t y of p r o p a r a c a i n e hydroc h l o r i d e h a s been determined by phase s o l u b i l i t y a n a l y s i s 6 . The a n a l y s i s i s c a r r i e d o u t by e q u i l i b r a t i o n i n a b s o l u t e e t h a n o l a t 23OC f o r 24 hours, Proparacaine h y d r o c h l o r i d e Lot N o . B R - 1 assayed 99.8% pure by phase s o l u b i l i t y a n a l y s i s .

442

DAISY 6 . WHIGAN

3.

xnthesis Proparacaine h y d r o c h l o r i d e h a s been s y n t h e s i z e d 1 9 by t h e s e q u e n c e o f r e a c t i o n s shown i n F i g u r e 8. The f o u r s t e p s y n t h e s i s s t a r t s w i t h p-hydroxybenzoic a c i d . This is e t h e r i f i e d with n-propylbromide i n t h e p r e s e n c e of p o t a s s i u m hydroxide. The r e s u l t i n g compound i s n i t r a t e d t o g i v e 3-nitro-4-propoxy-benzoic a c i d (11). The a c i d c h l o r i d e i s formed w i t h t h i o n y l c h l o r i d e and r e a c t e d w i t h B-diethylaminoethanol t o y i e l d 2( d i e t h y 1 a m i n o ) e t h y l 3-nitro-4-propoxybenzoate (111). T h i s i n t e r m e d i a t e i s reduced w i t h hydrogen c a t a l y t i c a l l y , t o produce p r o p a r a c a i n e hydrochloride (IV)

.

C l i n t o n and co-workers15 s y n t h e s i z e d 3 - n i t r o - 4 propoxybenzoic a c i d (11) b y a l k y l a t i o n o f 4hydroxy-3-nitrobenzoic acid w i t h p r o p y l p-toluenes u l f o n a t e i n x y l e n e s o l u t i o n . The f r e e a c i d i s produced by s u b s e q u e n t a l k a l i n e s a p o n i f i c a t i o n o f t h e ester. Following t h e Williamson r e a c t i o n , Monguzzi and c o - ~ o r k e r s o~b~t a i n e d I1 d i r e c t l y from 4 - c h l o r o 3 - n i t r o b e n z o i c a c i d b y r e a c t i n g i t w i t h sodium n-propoxide i n d i m e t h y l s u l f o x i d e s o l u t i o n . The n i t r o g r o u p i n t h e i n t e r m e d i a t e I11 h a s a l s o been s u c c e s s f u l l y c o n v e r t e d t o t h e correspondi n g amino g r o u p b y 15,20 a ) iron-hydrochloric acid reduction b) c a t a l y t i c hydrogenation with palladium/ charcoa 1 c a t a l y s t22,23 c ) c a t a l y t i c r e d u c t i o n w i t h Raney n i c k e l 24 15 d ) c a t a l y t i c r e d u c t i o n w i t h pla tinum o x i d e Proparacaine h y d r o c h l o r i d e h a s been r e c r y s t a l l i z e d from a b s o l u t e ethanol2', from methanol19, a n d from a b s o l u t e a l c o h o l - e t h y l a c e t a t e 1 5 .

0

F i g u r e 8.

S y n t h e t i c R e a c t i o n S c h e m e for P r o p a r a c a i n e H y d r o c h l o r i d e

STEP I '

O

H

S T E P 11:

+

D COOH

CH3CH2CH2Br ,

CH3CH2CH20

CH3CH2CH20

a

CH3CH2CH20

CO :H

I1

02N

S T E P 111: CH3 CH2 CH2

SOCl2 D

e w

O

CH3

H (C2H5 ) 2 * H C 1

CH3CH2Cz::@-OCH2CH2N 02N

I S T E P IV:

H?

CH3 CH2

C H 3 CH2 H2N

-

IV

COOH

4.

Stability-Deqradation P r o p a r a c a i n e h y d r o c h l o r i d e i s c h e m i c a l l y s t a b l e a s a s o l i d a t room temperat u r e f o r a t l e a s t two y e a r s 2 5 . The w h i t e c r y s t a l l i n e powder d i s c o l o r s o n h e a t i n g Liquid formulations of proparacaine hydrochloride a r e and exposure to a i r g . However, s o l u t i o n s s t a b l e up t o a t l e a s t two y e a r s 1 6 i n t h e a b s e n c e of a i r . w i l l d i s c o l o r i n t h e p r e s e n c e of air2’. P r o p a r a c a i n e h y d r o c h l o r i d e undergoes h y d r o l y s i s when b o i l e d i n c h l o r i c a c i d f o r 60 m i n u t e s 2 6 ( F i g u r e 9 ) .

HC 1

Heat

P

P P

OCH2 CH2 CH3 Proparacaine

F i g u r e 9.

.

2N hydro-

COOH

+ QNH2 OCH2 CH2 CH3

3-amino-4-propoxybenzoic a c i d H y d r o l y s i s of P r o p a r a c a i n e

(CH3CH2) 2NCH2 CH20H B-diethylaminoethano 1

PROPARACAINE HYDROCHLOR ID€

445

Analysis of I n t e r m e d i a t e Compound and Hydrolys i s Produ c t s Traces of t h e n i t r o i n t e r m e d i a t e (I11 i n Figure 8 ) i n proparacaine hydrochloride have been determined p o l a r o g r a p h i c a l l y by Kocy45. A Leeds and Northrup Electrochemograph type E equipped w i t h a s a t u r a t e d calomel e l e c t r o d e and a dropping mercury e l e c t r o d e was used. The e l e c t r o l y t e b u f f e r used i s pH 4.0 a c e t a t e b u f f e r c o n t a i n i n g 0.001; dodecyltrimethylammonium c h l o r i d e (DTAC) a s a maxima suppressor. The " s t a n d a r d a d d i t i o n technique" allows a q u a n t i t a t i v e method f o r determining a s l i t t l e a s 0.1% of I11 i n proparac a i n e hydrochloride. The n i t r o i n t e r m e d i a t e h a s an average r e d u c t i o n p o t e n t i a l of -0.37 v o l t s (vs. S.C.E.) 5.

3-Amino-4-propoxy-benzoic a c i d , a h y d r o l y s i s product of proparacaine, has been s e p a r a t e d from proparacaine by l i q u i d - l i q u i d e x t r a c t i o n . The f r e e a c i d remains i n pH 6.8 b u f f e r while proparacaine i s e x t r a c t e d i n t o chloroform 27 The aqueous l a y e r i s assayed s p e c t r o p h o t o m e t r i c a l l y f o r 3-amino-4-propoxy-benzoic a c i d . When t h e pH of t h e aqueous l a y e r i s lowered t o 4 , t h e f r e e a c i d i s e x t r a c t e d i n t o chloroform26. S o l u t i o n s of t h e f r e e a c i d were s p o t t e d on s i l i c a g e l t h i n - l a y e r p l a t e s and developed i n two s e p a r a t e s o l v e n t systems: Sys tem I , acetone: benzene :chloroform ( 20 :40 :40) :and System 11, benzene: chloroform: a c e t i c a c i d (20:80: 1 0 ) . The p o s i t i o n of t h e f r e e a c i d , r e l a t i v e t o c a f f e i n e , was 0.7 i n System I and 1.6 i n System 11.

.

Diethylaminoethanol, a l s o a h y d r o l y s i s product of proparacaine, has been determined i n plasma by a c o l o r i m e t r i c method with methyl orange 28

.

446

DAISY 6.WHIGAN

6.

Methods o f A n a l y s i s 6.1 Identification Tests U.S.P. m e t h o d s 1 i n c l u d e t h e c h a r a c t e r i s t i c u l t r a v i o l e t s p e c t r u m of p r o p a r a c a i n e h y d r o c h l o r i d e ( S e c t i o n 2.13) f o r i t s i d e n t i f i c a t i o n . Infrared s p e c t r o s c o p y ( S e c t i o n 2 . 1 1 ) may be u s e d t o i d e n t i f y t h e drug. The p r i m a r y a r o m a t i c amino g r o u p i s i d e n t i f i e d b y r e a c t i n g w i t h a q u e o u s sodium n i t r i t e , c o o l i n g t h e m i x t u r e , and t h e n a d d i n g a s o l u t i o n o f (3-naphthol i n sodium h y d r o x i d e . The s c a r l e t - r e d p r e c i p i t a t e formed d o e s n o t d i s s o l v e upon a d d i t i o n o f a c e t o n e1 T h i n - l a y e r chromatography ( S e c t i o n 6 . 6 2 ) and p a p e r c h r o m a t o g r a p h y ( S e c t i o n 6 . 6 1 ) h a v e been u t i l i z e d f o r i d e n t i t y purposes. Photomicrog r a p h s of p r o p a r a c a i n e c r y s t a l l i n e d e r i v a t i v e s ( S e c t i o n 2 . 2 1 ) have been used as an a d j u n c t t o o t h e r p h y s i c a l methods f o r c h a r a c t e r i z a t i o n . Formation of s o l i d d e r i v a t i v e s ( T a b l e 4) and t h e determination of t h e melting ranges and t h e i n f r a r e d s p e c t r a of t h e s e d e r i v a t i v e s p ro v id e f u r t h e r parameters for i d e n t i f i c a t i o n .

.

Table 4 Propa r a c a i n e D e r i v a t i v e s Derivative M e l t i n q Ranqe ( O C ) Chloroplatinate 195.5-198.5 Flavianate 162.0-163.0 ( d e c ) Methiodide 145.0-147.5 122-124 Picrate 138.0-140.0 Reineckate 151-158 St y p h n a t e Tetraphenyl143-147 boratea

131.5-132.0 a

Polymorphism h a s been s u g g e s t e d

Reference 10

15 10

11 10 10

11 10

11

PROPARACAINE HYDROCHLORIDE

6.2

447

E l e m e n t a l A n a l y s i s (as C16H26N203-HC1) Element

Carbon Hydrogen Nitrogen Chlorine

% Theory

58.08 8.23 8.47 10.71

% Reported Ref.6 Ref. 15

8. 56 10.88

58.04 8.11 8.54 10.85

C h l o r i d e s may be d e t e r m i n e d 6 b y r e a c t i n g t h e sample w i t h e x c e s s s i l v e r n i t r a t e i n t h e p r e s e n c e of n i t r o b e n z e n e and n i t r i c a c i d . The excess s i l v e r n i t r a t e i s t i t r a t e d w i t h potassium o r ammonium t h i o c y a n a t e u s i n g f e r r i c ammonium sulfate a s the indicator. 6.3

Spectrophotometric Analysis 6.31 u l t r a v i o l e t Spectrophotometric Analysis Since proparacaine displays a high d e g r e e o f a b s 3 r p t i o n i n t h e 220 t o 320 nm r a n g e , u l t r a v i o l e t spectroscopy ( Sectio n 2.13) provides In local a c o n v e n i e n t means €or i t s a s s a y . a n e s t h e t i c f o r m u l a t i o n s , t h e p r e s e n c e o f some vasoconstrictor agents, preservatives,and s a l t s w i l l not interfere i f these materials e i t h e r a)do n o t d i s p l a y absorption i n t h i s a r e a or b) a r e d i l u t e d t o t h e p o i n t where t h e i r a b s o r p t i o n i s negligible. I n more c o m p l i c a t e d f o r m u l a t i o n s p n p a r a c a i n e h a s been e f f e c t i v e l y s e p a r a t e d p r i o r t o u l t r a v i o l e t a n a l y s i s b y e x t r a c t i o n from a n 11 a l k a l i n e medium i n t o e i t h e r e t h e r ' o r c h l o r o f o r m

.

I n t h e presence of s t r o n g a c i d s , t h e a r o m a t i c amine forms a p o s i t i v e l y c h a r g e d ammonium i o n and t h e peak due t o t h e p a r t i c i p a t i o n o f t h e amino g r o u p i n r e s o n a n c e i s n u l l i f i e d This o b s e r v a t i o n h a s been u t i l i z e d i n (Figure 4). determining propoxycaine i n t h e presence of p r o c a i n e 3 0 . The same phenomenon c o u l d b e a p p l i e d t o t h e determination of proparacaine i n t h e presence of procaine.

448

DAISY B. WHIGAN

6.32

Fluorescence Spectrophotometric Analysis Although f l u o r o m e t r i c procedures f o r t h e a s s a y of pr opar acaine h y d ro ch lo rid e have n o t b e e n reported, t h e y s h o u l d be f e a s i b l e b e c a u s e t h e n a t i v e f l u o r e s c e n c e o f p r o p a r a c a i n e hydroc h l o r i d e i n 0.1g sodium h y d r o x i d e i s s u f f i c i e n t l y s t r o n g (Section 2.15). 6.4

T i t r i m e t r i c Procedures 6 . 4 1 Nonaqueous T i t r a t i o n P r o p a r a c a i n e h y d r o c h l o r i d e c a n be t i t r a t e d w i t h good p r e c i s i o n u s i n g a c e t o u s p e r c h l o r i c a c i d29

.

6.42

T i t r a t i o n w i t h Sodium N i t r i t e T h i s a s s a y has b e e n d e s c r i b e d f o r

is a n isomer o f p r o p a r a c a i n e . p r o p ~ x y c a i n ewhich ~~ I n t h i s a s s a y , t h e p r i m a r y a r o m a t i c amine u n d e r g o e s d i a z o t i z a t i o n and t h e e n d - p o i n t i s d e t e r m i n e d b y starch-iodide paper e x t e r n a l i n d i c a t o r . F e r r o c y p h e n s o l u t i o n , which h a s b e e n u s e d a s a n i n t e r n a l i n d i c a t o r f o r sodium n i t r i t e t i t r a t i o n s 46 , may be u s e d i n s t e a d of t h e cumbersome e x t e r n a l i n d i c a t o r . Although t h i s t i t r a t i o n h a s n o t been reported f o r proparacaine, the presence of a p r i m a r y a r o m a t i c amino g r o u p i n p r o p a r a c a i n e suggests a p p l i c a b i l i t y of this t i t r a t i o n .

6.43

Spectrophotometric T i t r a t i o n with N i t r o u s A c i d31

I n t h i s t i t r a t i o n , absorbance measurements a r e made d u r i n g t h e t i t r a t i o n o f t h e p r i m a r y a r o m a t i c amine w i t h n i t r o u s a c i d . The a b s o r b a n c e r e a d i n g s a r e d e p e n d e n t on t h e s p e c t r a of n i t r o u s a c i d and t h e d i a z o d e r i v a t i v e formed. In p l o t t i n g t h e a b s o r b a n c e s a g a i n s t t h e volume o f t i t r a n t added, t h e i n t e r s e c t i o n o f s t r a i g h t l i n e s o f d i f f e r e n t slopes ( p r i o r t o a n d a f t e r r e a c t i o n T h i s t i t r a t i o n has completion) i s t h e end-point. been a p p l i e d t o the d e t e r m i n a t i o n of propoxycaine. S i n c e i t depends on t h e d i a z o t i z a t i o n o f the p r i m a r y a r o m a t i c amine, t h i s t i t r a t i o n s h o u l d be

PROPARACAINE HYDROCHLORIDE

449

applicable t o the determination of proparacaine.

C o l o r i m e t r i c Methods 6 . 5 1 With B r a t t o n - M a r s h a l l R e a g e n t The u t i l i t y o f t h e B r a t t o n M a r s h a l l r e a g e n t i n t h e a n a l y s i s of p r i m a r y A p p l i c a t i o n of t h i s a r o m a t i c amines i s w e l l known. r e a g e n t t o t h e a n a l y s i s o f p r o p a r a c a i n e hydroc h l o r i d e h a s been d e s c r i b e d b y P o e t 3 3 . 6.5

Add 5 m l o f 0.15 h y d r o c h l o r i c a c i d and 35 m l o f d i s t i l l e d w a t e r t o a 100 m l v o l u m e t r i c f l a s k c o n t a i n i n g a b o u t 4 mg o f p r o p a r a c a i n e h y d r o c h l o r i d e . Add 2 m l o f 1%sodium n i t r i t e , w a i t 2 m i n u t e s t h e n add 10 m l o f 0.5% ammonium s u l f a m a t e . A f t e r 3 m i n u t e s add 1 0 m l o f 0.1% B r a t t o n - M a r s h a l l r e a g e n t (N-l-naphthylethylenediamine h y d r o c h l o r i d e ) i n 70% p r o p y l e n e g l y c o l . D i l u t e t o t h e mark w i t h d i s t i l l e d w a t e r and measure t h e a b s o r b a n c e a t 550 nm a g a i n s t a Reagent Blank. 6.52

With Sodium 1,2-Naphthoquinone-4sulfonat e I n t h i s assay procedure, t h e y e l l o w sodium 1,2-naphthoquinone-4-sulfonate, i n t h e presence of a l k a l i , r e a c t s with t h e primary amine t o y i e l d a h i g h l y c o l o r e d o r a n g e - r e d p r o d u c t . The e x c e s s y e l l o w r e a g e n t i s t h e n b l e a c h e d w i t h sodium t h i o s u l f a t e a f t e r making t h e s o l u t i o n s l i g h t l y a c i d i c with a c e t a t e buffer. This p r o c e d u r e h a s been a p p l i e d t o t h e a s s a y o f l o c a l a n e s t h e t i c s i n c l u d i n g p r o p ~ x y c a i n ea n ~d~ c o u l d b e extended t o t h e d e t e r m i n a t i o n of proparacaine. 6.6

Chromatographic Procedures 6 . 6 1 P a p e r Chromatography K o e h l e r and Feldmann'l d e s c r i b e d t w o paper chromatographic systems used i n s e p a r a t i n g and i d e n t i f y i n g l o c a l a n e s t h e t i c s including proparacaine. The d r u g s a r e e x t r a c t e d from t h e i r d o s a g e forms and t h e n s u b j e c t e d t o p a p e r c h r o m a t o g r a p h i c a n a l y s i s u s i n g Whatman N o . 1 p a p e r . I n t h e s o l v e n t system b u t y l a l c o h o 1 : h y d r o c h l o r i c

4 50

DAISY

B. WHIGAN

a c i d : w a t e r (30:5:35.5) t h e Rf f o r p r o p a r a c a i n e i s 0.45 and i n t h e s y s t e m b u t y l a l c o h o 1 : a c e t i c a c i d : water (40:10:50) t h e Rf i s 0.79. To l o c a t e t h e s p o t s , t h e d r i e d s t r i p s a r e e i t h e r viewed u n d e r a n u l t r a v i o l e t lamp i n a d a r k room o r s p r a y e d w i t h a modified Dragendorff r e a g e n t ( a c i d i f i e d mixture o f p o t a s s i u m i o d i d e , b i s m u t h s u b n i t r a t e , and i o d i n e i n w a t e r ) . An a l t e r n a t i v e s p r a y s o l u t i o n i s an a c i d i c s o l u t i o n o f potassium permanganate i n water. I n t h e g e n e r a l s c r e e n i n g of n i t r o g e n e o u s bases, C l a r k e 9 u s e s a s o l u t i o n o f c i t r i c a c i d i n a m i x t u r e o f 130 m l o f w a t e r and 870 m l of n-butanol a s t h e s o l v e n t system. The Whatman p a p e r N o . 1 i s p r e - t r e a t e d b y d i p p i n g i n a 5% s o l u t i o n o f sodium d i h y d r o g e n c i t r a t e a n d d r y i n g I n t h i s system,proparacaine a t 2 5 O C f o r one h o u r . h a s a n Rf o f 0.52. 6.62

Thin-Layer Chroma t o q r a p h y T h i n - l a y e r chromatography u s i n g s i l i c a g e l p l a t e s h a s been r e p o r t e d f o r proparac a i n e . Using a s o l v e n t s y s t e m o f s t r o n g ammonium h y d r o x i d e solution:methanol(3:200), t h e Rf o f p r o p a r a c a i n e i s 0.5g9 a n d i t s p o s i t i o n r e l a t i v e t o c o d e i n e i s 1 . 8 2 6 . The s p o t s may b e l o c a t e d b y a c i d i f i e d i o d o p l a t i n a t e s p r a y o r b y p-dimethylaminobenzaldehyde s p r a y . A l t e r n a t i v e l y , t h e spots may be l o c a t e d b y v i e w i n g u n d e r u l t r a v i o l e t l i g h t . L o c a l a n e s t h e t i c s h a v e been a n a l y z e d u s i n g t h i n - l a y e r c h r o m a t o g r a p h y b y Fuwa a n d c o - ~ o r k e r s ~ ~S e. p a r a t i o n o f t h e d r u g s w a s e f f e c t e d on s i l i c a g e l p l a t e s u s i n g t h e s o l v e n t s y s t e m , benzene:acetone:ammonium h y d r o x i d e ( 8 0 : 2 0 : 1 ) . The spots w e r e i d e n t i f i e d b y t h e E h r l i c h

(p-dimethylaminobenzaldehyde) r e a g e n t . A n a l y s i s of H y d r o l y s i s P r o d u c t s i n Body F l u i d s and T i s s u e s Reed a n d Cravey26 r e p o r t e d t h e d e t e r m i n a t i o n of 3-amino-4-propoxybenzoic a c i d ( A ) i n body f l u i d s

7.

PROPARACAINE HYDROCHLORIDE

451

and t i s s u e s . They p r e p a r e d t u n g s t i c a c i d p r o t e i n f r e e f i l t r a t e s from b l o o d , l i v e r , k i d n e y , a n d b r a i n . U r i n e and g a s t r i c specimens d i d n o t undergo f i l t r a t e p r e p a r a t i o n . With e a c h specimen, t h e pH was a d j u s t e d t o 4 and e x t r a c t e d f i v e times w i t h chloroform. The o r g a n i c phase was t h e n e x t r a c t e d w i t h 0.064N sodium h y d r o x i d e and t h e aqueous l a y e r , c o n t a i n i n g A , was measured s p e c t r o p h o t o m e t r i c a l l y . T a b l e 5 shows t i s s u e c o n c e n t r a t i o n s found i n a s i n g l e case. Table 5 Tissue Concentrations of Hydrolysis Product a s E q u i v a l e n t Proparacaine Specimen Blood Brain Lung Liver Kidney Urine Stomach

mq/100 m l o r 100 g 1.5 0.4 1.2 1.7 1.6

None d e t e c t e d None d e t e c t e d

For f u r t h e r i d e n t i f i c a t i o n , t h e aqueous l a y e r i s a c i d i f i e d and back e x t r a c t e d i n t o c h l o r o f o r m . The chloroform e x t r a c t i s e v a p o r a t e d and t h e r e s i d u e i s s u b j e c t e d t o t h i n - l a y e r chromatography (See Section 5 ) . I t is s p e c u l a t e d t h a t t h e s t r o n g n a t i v e fluorescence of proparacaine hydrochloride ( S e c t i o n 2.15) could provide a s e n s i t i v e t e c h n i q u e € o r i t s a s s a y i n body f l u i d s and t i s s u e s .

Serum P r o t e i n B i n d i n Dastugue a n d c o - ~ o r k z r s s~t u~d i e d t h e b i n d i n g of some d r u g s i n c l u d i n g p r o p a r a c a i n e h y d r o c h l o r i d e w i t h b o v i n e se ru m p r o t e i n s . P r o p a r a c a i n e hydroc h l o r i d e w a s d i s s o l v e d i n 5 m l of serum and d i a l y z e d a t 4OC a g a i n s t a p h o s p h a t e - c h l o r i d e b u f f e r of p H 7.4 f o r 48 h o u r s . The c o n c e n t r a t i o n of p r o p a r a c a i n e i n t h e d i a l y z a t e was d e t e r m i n e d by 8.

452

DAISY 6 . WHIGAN

measurin t h e u l t r a v i o l e t absorbance a t 268 nm. 3% t a b u l a t e s t h e amount of protein-bound Table 6 drug depending on t h e c o n c e n t r a t i o n of drug i n t h e serum. Table 6 Serum P r o t e i n Binding o f Proparacaine Hydrochloride D r u g Concentration ug/ml s e r u m 25 50 100 2 00 300 400 9.

% Bound D r u g 46.4 33.6 26.4 21.9 19.4 19.6

D r u q Metabolism

The pharmacology of p r o p a r a c a i n e h y d r o c h l o r i d e h a s been i n v e s t i g a t e d by d i f f e r e n t workers37. 3 8 9 39, There i s no evidence of blood l e v e l s t u d i e s 47. f o r proparacaine h y d r o c h l o r i d e i n t h e l i t e r a t u r e . Proparacaine h y d r o c h l o r i d e hydrolyzed by guinea p i g l i v e r 3 7 O C i n t h e presence of 0.067M (pH 7 . 2 ) , 456 bmole o f drug i s gram of f r e s h t i s s u e per hour.

is rapidly At homogena tes4'. phosphate b u f f e r hydrolyzed p e r

I n a s i n g l e c a s e where a person p u r p o r t e d l y i n h a l e d about 500 mg of a white c r y s t a l l i n e m a t e r i a l purchased a s "super-cocaine" , Reed and Cravey26 i d e n t i f i e d t h e m a t e r i a l t o be proparacaine hydrochloride. Working w i t h t h i s case, t h e y r e p o r t e d t h e h y d r o l y s i s of p r o p a r a c a i n e i n body f l u i d s . T h i s o b s e r v a t i o n , s i m i l a r t o t h e h y d r o l y s i s of proparacaine when h e a t e d i n 2 g hydrochloric a c i d (Figure 9), i s t y p i c a l of t h e metabolic pathway found with o t h e r amino a l c o h o l e s t e r t y p e a n e s t h e t i c s 4 l 9 42. Hydrolysis i s a c c e l e r a t e d by enzymes i n t h e l i v e r , o t h e r t i s s u e s ,

PROPARACAINE HYDROCH LOR ID€

453

and plasma43 9 49. About 2 hours a f t e r t h e a d m i n i s t r a t i o n of t h e d r u g , Reed and Cravey found no p r o p a r a c a i n e i n s a m p l e s o f t h e b l o o d , b r a i n , l u n g , and u r i n e . Some amounts o f t h e h y d r o l y s i s p r o d u c t , 3-amino-4p r o p o x y - b e n z o i c a c i d , were found i n t h e b l o o d , b r a i n , l u n g , l i v e r , and k i d n e y .

I n s t u d y i n g t h e f a t e o f p r o c a i n e i n man, B r o d i e , L i e f , and P o e t 2 8 found t h a t some diethylaminoethanol is excreted i n t h e u r i n e while some o f i t i s f u r t h e r m e t a b o l i z e d i n t h e body. I t i s s p e c u l a t e d t h a t t h e d i e t h y l a m i n o e t h a n o l formed from t h e h y d r o l y s i s o f p r o p a r a c a i n e f o l l o w s t h e same f a t e i n man.

454

10.

1. 2.

3. 4. 5. 6. 7.

0.

9. 10.

11. 12.

13. 14.

15. 16.

17. 18. 19.

20. 21.

DAISY E. WHIGAN

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and Lee, R.,

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45.

46.

47. 48. 49.

Acknow l e d q m e n t The a u t h o r g r a t e f u l l y a c k n o w l e d g e s t h e u n s e l f i s h g u i d a n c e o f D r . J. M. Dunham i n t h e p r e p a r a t i o n of t h i s p r o f i l e .

PROPYLTHIOURACIL

Hassari Y. A hod-Etiein

458

HASSAN Y . ABOUL-ENEIN

CONTENTS Analytical Profile - Propylthiouracil 1.

2.

3. 4. 5.

6.

Description 1.1 Nomenclature 1.11 Chemical Names 1.12 Generic Name 1.13 Trade Name 1.2 Formulae 1.21 Emprical 1.22 Structural 1.23 Wiswesser Line Notation 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Odor Physical Properties 2.1 Crystal Properties 2.11 Crystallinity 2.12 X-ray diffraction 2.13 Melting Range 2.2 Solubility 2.3 Identification 2.4 Spectral Properties 2.41 Ultraviolet Spectrum 2.42 Infrared Spectrum 2.43 Nuclear Magnetic Resonance Spectrum 2.44 Mass Spectrum and Fragmentometry Synthesis Stability, Decomposition Production and Metal Comp1exes Metabolism Method of Analysis 6.1 Titrimetric Methods 6.11 Aqueous 6.12 Non-Aqueous 6.2 Colorimetric 6.3 Ultraviolet Spectrophotometric 6.4 Chromatographic Analysis 6.41 Paper Chromatography 6.42 Column Chromatography 6.43 Thin Layer Chromatography 6.44 Gas Chromatographic Analysis References Acknowledgement

PROPY LTHlOURAClL

1.

459

Description 1.1

Nomenclature 1.11

4-one pyrimidin-4-one midine hydropyr imid ine pyr imidinone

Chemical Names 2-Thio-4-oxo-6-propyl-lI3-pyrimidine 2-Thio-6-propyl-lI3-pyrimidine1,2-Dihydro-6-propyl-2-thioxo4-Hydroxy-2-mercapto-6-propylpyri4-0xo-6-propyl-2-thio-lI2,3,4-tetra-

2,3-Dihydro-6-propyl-2-thioxo-4 (1H)-.

6-Propyl-2-thiouracil.

1.12

Generic Name: Propylithouracil.

1.13 Trade Name: Propacil, Propycil, Prothyran, Procasil, Propyl-thyracil, Thyroestat 11. 1.2

Formulae C7 H10 N2 0s

1.21

Emprical:

1.22

Structural:

H

Keto tautcawr 1.23

en01 tautmer

Wiswesser Line Notation: T6MYMVJ BUS F3

1.3

Molecular Weight:

170.23

1.4

Elemental Composition C, 49.39%; H I 5.92; N, 16.46%; 0, 9.40%; S, 18.84%.

460

HASSAN Y . ABOUL-ENEIN

1.5

Appearance, Color, Odor:

White to pale cream-colored crystals or microcrystalline powder of starch-like appearance to the eye and to the touch: odorless: taste, bitter. 2.

Physical Properties 2.1

Crystal Properties 2.11

Crystallinity Propylthiouracil is a microcrystalline solid. Ashley (1) described a procedure for the preparation of distinctive crystals of propylthiouracil for the purpose of identification. The crystals are

follows:

prepared

as

Dissolve a few crystals of the sample in a drop of 0.1N NaOH on a slide, acidify by allowing a drop of 10% H SO to coalesce gradually with the solutio;. 4Gently rock the slide to mix and examine microscopically. A typical photomicrograph of these crystals is shown in Fig.1.A. Furthermore, crystals are obtained by quickly smearing a drop of saturated solution of the sample in 75% alcohol at 70° over the whole surface of the slide with a small glass rod, and allowing the solvent to evaporate at room temperature. Photomicrograph of these crystals is shown in Fig.1.B. Kassau (2) described the crystallinity of some pyrimidine derivatives including propylthiouracil by microsublimation. 2.12

X-ray Diffraction Although the X-ray diffraction of propylthiouracil is not described in the literature. et a1 (3) described the elemental crystal strNisi ucture €or the reaction product between propylthiouracil and formaldehyde under acidic condition, 8propyl 6H-pyrimido [2,1-d] [1,3,5] oxathiazin-6-one. 2.13

Melting Range

USP XIV(4)specifie.s a melting range €or propylthiouracil between 219 - 221O as a criteria of acceptability.

Fig. 1-A by acid precipitation Fig. 1 : Photomicrograph of

Fig.1-B from alcohol propylthiouracil crystals.

HASSAN Y . ABOUL-ENEIN

462

Table I shows the melting range of propylthiouracil reported in the literature Table I m.p. ,C

0

Reference

218-221 219-221 220 219 215-216 2.2

Solubility Propylth&ouracil is sparingly soluble in water (1:900 at 20 ) ; soluble in 100 parts boiling water, in 60 parts of ethanol; in 60 parts of acetone. Practically insoluble in ether, chloroform, benzene. Freely soluble in aqueous solutions of ammonia and alkali hydroxides. A saturated aqueous solution is neutral or slightly acidic to litmus. 2.3

Identification The following identification tests are published in B.P. 1973(7) as a part of the identification of propylthiouracil. These tests are identical to the identification tests of methylthiouracil with the exception of the melting point. To a boiling saturated solution, add an (a) equal volume of a freshly prepared solution containing 0 . 4 % w/v of sodium nitroprusside, 0 . 4 % w/v of hydroxylammonium chloride, and 0.8% w/v of sodium carbonate; a greenish blue color is produced. (b) To 25 mg of propylthiouracil, add bromine solution drop by drop with completely dissolved, cool, and add 10 ml of barium hydroxide solution; a white precipitate is produced. Bucher (10) introduced a modification to the above mentioned test in which excess bromine water was added then the excess bromine was removed by treating the solution until the solution was colorless and then the test was done as described before. Metto and deFigueiredo (11) described a color test for thiouracil and its homologes as follows :

PROPY LTH IOU RACl L

463

To a sample of the material add 0.5 ml 0.1 N NaOH and 10 ml of water; then introduce 10% CuS04 dropwise until an excess is present. Propylthiouracil gives a dark gray precipitate becoming bluish and then purplish gray. Another color reagent was described by Nilsson (12) which can detect 1.3 mcg/ml of propylthiouracil in solution. Solutions required for the test were : 0.2 g 0-toulidine in 5% acetic acid, 1% CuC12 in water and 5% sodium acetate in water. A drop of each was mixed on a spot plate and a drop of propylthiouracil solution was added. An intensive blue color developed. Propylthiouracil gives an orange-red color with 2,6-dichloroquinone chloroimide reagent which is sensitive enough to render the color test an excellent colorimetric analytical method of the drug in tablets which will be discussed later in the chapter (13). The complex of propylthiouracil-chloroimide could be seperated from chloroform solution as an orange-red needles (m.p. 172O with decomposition). Bucher (10) reported a procedure for identification of thiouracil and its homologs through the preparation of their benzylthio ether derivatives (propyJthiouraci1 benzylthio ether derivative m.p. 131-932 ) . p-Nitrobenzyl khio ether derivative (m.p. 193 ) has been reported for propylthiouracil as a mean of identification of the drug (5). 2.4

Spectral Properties 2.41

Ultraviolet Spectrum Propvlthiouracil in neutral methanol absorbs ultraviolet*Gadiation at 275nm (a 15800) and at 214 nm (am 15600) as shown in Fig YA. In alkaline medium it shows 3 maxima at 315.5 nm (am10900), 260 nm (am 10700) and at 207.5 (am 15400) as shown in Fig. 2B.

-a1 (14) published a Galimberti et detailed study on the ultraviolet spectrophotometry of several derivatives. He reported that the replacement of an oxygen atom at C 2 by sulfer caused

464

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PROPYLTH IOURACI L

467

a big shift to longer wavelength with increased absorption. Furthermore, the authors attributed the appearance of 3 maxima in case of the thiouracil and derivatives as compared to two maxima for the uracil homologs in alkaline medium (pHll-12) to the double enolization. Informations with regard to the ultraviolet behavior of the drugs containing a thioamideCONHCS - over a pH range from 1 to 13 were discussed by Stanovnik and Tisler (15). Their data indicated that the dipolar structure I was common with these compounds (Structure I €or propylthiouracil at pH 7-8).

oQ

2.42

Infrared Spectrum The infrared spectrum of propylthiouracil is shown in Fig 3. T h e spectrum was obtained on a Beckman IR4 spectrophotometer from KBr pellet. The structural assignments have been correlated with the following band frequencies: Frequency (cm -I) 3120 3020-2910

Assignment NH Stretching imide CHI CH2, CH3 stretching SH stretching

2580 (weak since the Keto form predominates) C=O imide carbonyls 1650 Other fingerprint bands characteristic to propylthiouracil a e 1550, 1440, 1240, 1190, 1160, 880 and 810 cm-f Further information with regard to the infrared spectra of propylthiouracil is given in several references (8, 16).

468

HASSAN Y . ABOUL-ENEIN

2.43

Nuclear Magnetic Resonance Spe ctrum

A typical NMR spectrum of propylthiouracil is shown in Fig. 4. The sample was dissolved in deutrated dimethyl sulfoxide (DMSO-d6). The spectrum was determined on a Varian T-60 N M R spectrometer with TMS as the internal standard.

The following structural assignments have been made for Fig. 4 . Chemical Shift

(b)

Assignment

Triplet at 0.93

-CH2CH2CH3

Multiplet centered at 1.60 Multiplet centered at 2.50 (Solvent protons at 2.63 for DMSO-d5). Singlet at 5.66

-CH2CH2CH3

Broad singlet at 12.6

-

-CH2CH2CH3 -

Olefinic proton at C5 2-NH imide groups exchangeable with D20.

Further information concerning the interpretation of the NMR spectrum of propylthiouracil can be obtained from Sadtler NMR catalog(l7) and also from CRC Atlas of spectral data (8). 2.44

Mass Spectrum and Fragmentometry

The mass spectrum of propylthiouracil obtained by conventional eleltion impact ionization shows a molecular ion M at m/e 170. The M ion peak has about 85% relative intensity (Fig. 5 ) . The base peak is at m/e 68. The mass fragmentation mechanism of propylthiouracil is shown in Scheme I. It follows the same fragmentation pattern of uracil and derivative which has been established by several authors (18, 19, 20). It involves the loss of HCNX (X=O or S ) , and later verified by Hecht et a1 (21).

4 69

I

9 0

H

170 I

142

1 P

51

Fig. 5

:

Mass Spectrum of propylthiouracil (EI).

=35

m

0' I

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a m

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472

HASSAN Y. ABOUL-ENEIN

The first step in the fragmentation is a retro-Diel-Alder decomposition with a loss of HCNS and the production of ion radical which is only of minor importance since it immediately undergoes the following paths : (a) (b)

(c) (d) 3.

A loss of CO to give an abundant ion at m/e 8 3 , A loss of propyl radical to give an abundant ion at m/e 68, which subsequently base CO to give an ion at m/e 40. A loss of HN=C-CHion to give ketene 37 m/e 41. A loss of CH = C = 0 to give an ion m/e 7 0 .

Svnthesis CH3 CH2 CH2 CO CH2 COOC2 H5 + H2N-CS-NH2 1)NaOEt EtOH

Propylthiouracil is prepared by the condensation of ethyl 3-oxocaproate with dry thiourea in the presence of a base (22). Several authors had modified the above synthetic procedure for patent purposes yet the principle still the same (9, 23, 24, 25). 4.

Stability, Decomposition Product and Metal Complexes:

Propylthiouracil is a relatively stable compound at room temperature. It is recommended to that it should be kept in a well-closed containers protected from light.

PROPYLTH IOURACIL

473

Propylthiouracil forms metal complexes with divalent metals e C U + ~ ,Pb+2, Cd+2, Ni+2 and Zn+2 but not with Fe 'Fe+2 , Co+2 , Ca+2 or Mn+j. Garret and Weber(26, 27) published detailed studies on these metal complexes of thiouracil and analogs regarding their structures, stability constants, so 1ub i1ity ana1yses and spectrophotometric properties.

$3;

5.

Metabolism

Interest in the metabolism of antithyroid drugs has recently been focused on 6-propyl-2-thiouracilI one of the current drug of choice in the treatment of hyperthyroidism. Propylthiouracil is readily metabolized after administration to humans and rats and the major metabolite in urine, plasma and bile has been identified as propylthiouracil glucuronide (28, 2 9 , 30, 31, 32, 33). Other metabolites identified in rat bile and urine are shown in Fig. 6. These include :

S-methyl-6-propylthiouracil (minor metabolite) 6-Propyluracil (minor metabolite) 6-propylthiouracil sulfenic acid] Identified 6-propylthiouracil sulfonic acid] in rat thyroid 6-propylthiouracil sulfate I extracts &sbarats-Schhbaum et a1 ( 3 4 ) reported that in highly alkalinized guinea pig urine, propylthiouracil disulfide was isolated. The sulfer group of propylthiouracil appears to be the major site of alteration, biotransformation at this site results in a total or major loss of antiperoxidase activity (35). None of the metabolites isolated and identified was as active as the parent compound ( 3 5 ) . Several metabolites of propylthiouracil remain unidentified. It has been reported that the plasma half-life in hours of methimazole (another drug of choice in treatment of hyperthyr0idism)was 2 - 5 times that of propylthiouracil (36)

.

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474

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PROPYLTHIOURACIL

6.

475

Method of Analysis 6.1.

Titrimetric Methods 6.11.

Aqueous Several titrimetric methods were developed €or analysis of propylthiouracil.

1. Simple titration with standard NaOH, in neutral alcohol solutions, the N.N.R method. This method is simple. Both phenolphthalein or thymolphthalein being used as indicator. Yet the presence of stearic acid interfers with the assay and it should be removed by extraction with petroleum ether before titration (37). 2. Silver nitrate method: Berggren and Kirsten (38) introduced a modification to the above mentioned method. Acetone was used to extract propylthiouracil from most of the tablet exciepients. Acetone extract was neutralized by adding HN03 or 0.1 N NaOH using 1, 2, 5-dinitriphenol as indicator. To the neutralized solution, water was added and a certain volume of 0.1 N AgNO 3 was added and the solution was titrated with 0.1 N NaOH to a persisting blue color (bromothymol blue was used as indicator). This method was found satisfactory yet if stearic acid was present, it should be removed before addition of AgNO It was adopted by USP 3' XVIII.

3. Mercuric acetate titrimetic method: Abbot (39) described a method for determination of thiouracil and analogs by titrating the solution with 0.05M Hg (OAc12 using 0.5% diphenylcarbazone in ethanol as indicator. The method was adopted by B.P. 1973 and USP X I V issue because excipients of starch, sucrose, acacia, rodin, calcium carbonate, stearic acid or magnesium stearate did not interfere. 4. Potassium Bromate titrimetric method: The bromometric method was developed by Wojahn and Wempe (40, 41, 42) and was reported to be more satisfactory and accurate method than USP XIV procedure using mercuric acetate method, since the presence of lactose in propylthiouracil tablets interfered with the mercuric salt method. To an

4 76

HASSAN Y . ABOUL-ENEIN

alkaline solution of propylthiouracil bromination was effected by 0.1 N KBr03 and KBr in presence of 25% HC1. After one hour, an excess of 0.1 N NaAs02 was added and back-titrated with 0.1N KBr03 with p-ethoxychrysoidine as the indicator. 6.12

Non Aqueous Backe-Hansen (43) reported a nonaqueous titration method for propylthiouracil using sodium methoxide in benzene and methanol in a solution of dimethylforamide or pyridine (against thymol blue or azoviolet as indicator). Lithium methoxide 0.1 N in benzene and methanol had also been used instead of sodium methoxide (5). 6.2.

Colorimetric

A number had been developed thiouracil in pure tablets and animal

of colorimetric analyticalmthcds for the determination of propylform, pharmaceutical formulations, feeds.

(a)

The use of Grote reagent: Doden et a1 (44) had applied the use of Grote's reagents to determine different thiouracils quantitatively. The absorption maxima at 660 nm was measured. The reaction obeyed eer's Law ove the concentration range 0 . 5 x lo-' to 3 x M. Brueggeman and Schole (45) applied the color reaction of Grote reagent with thiouracils for the quantitative determination of thiouracils in feeds. Bucci and Cusmano (46) reported a similar colorimetric method using Grote's reagent as modifiedvchristeinsen (47). The authors claimed that the method was suitable for the analysis of thiouracild in very small amounts ( 2 0 0 p.p.m) in the presence of other biological substances. (b)

2,6-Dichloroquinone Chloroimide reagent: McAllister and Howells (13) reported a method for analysis of propylthiouracil intablets using 0.4% solution of 2,6-dichloroquinone chloroimide in aldehyde-free absolute ethanol. The yellow color obtained from such reaction was extracted in chloroform and optical density of the solution was compared with a standard graph.

PROPY LTHlOURAClL

477

Ruthenium c h l o r i d e c o l o r r e a c t i o n : R e i n h a r d t (48) d e s c r i b e d a c o l o r i m e t r i c method f o r d e t e r m i n a t i o n o f p r o p y l t h i o u r a c i l and s i m i l a r compounds. I t w a s b a s e d on t h e react i o n between t h e t h i o c a r b a m i d e l i n k a g e CONHCS and RuCl i n s t r o n g a c i d medium. The c o l o r devcl o p e d w a s measured a t 520 nm and compared w i t h a standard carve. (C)

-

-

Isopropylamine - c o b a l t acetate reagent: H o l t and M a t t s o n ( 4 9 ) d e v e l o p e d a c o l o r i m e t r i c a s s a y f o r compounds c o n t a i n i n g t h e g r o u p s - CONHCO - and - CONHCS - , i n c l u d i n g p r o p y l thiouracil. A c o l o r d e v e l o p e d w i t h t h e r e a c t i o n of t h e s e compounds w i t h i s o p r o p y l a m i n e r e a g e n t ( 5 0 m l of t h e amine made t o 200 m l w i t h d r y c h l o r o f o r m ) and c o b a l t a c e t a t e (made o f 0.259 i n 200 m l m e t h a n o l ) . The c o l o r w a s measured a t 530 nm and compared w i t h s t a n d a r d s . The method was s e n s t i v e t o a c o n c e n t r a t i o n of 1 m c g / m l . (d)

(el

-

Hydroxylamine h y d r o c h l o r i d e Sodium n i t r o p r u s s i d e r e a g e n t : Doden and Kopf ( 5 0 ) p u b l i s h e d a c o l o r i m e t r i c method f o r t h e d e t e r m i n a t i o n o f t h i o u r a c i l and a n a l o g s u s i n g h y d r o x y l a m i n e h y d r o c h l o r i d e and sodium n i t r o p r u s s i d e i n t h e p r e s e n c e o f sodium b i c a r b o n a t e , bromine and p h e n o l . The g r e e n i s h b l u e c o l o r d e v e l o p e d w a s compared w i t h s t a n d a r d c u r v e . Potassium i o d a t e - a c e t i c a c i d color reaction: A method b a s e d on t h e c o l o r d e v e l o p e d by t h e r e a c t i o n o f p r o p y l t h i o u r a c i l and i t s m e t h y l a n a l o g , w i t h K I O and a c e t i c a c i d w a s u s e d t o determine these drug2 i n t a b l e t s ( 5 1 ) . (f)

Powdered t a b l e t s c o n t a i n i n g a p p r o x i m a t e l y 50 mgs, i n 1 0 m l e t h a n o l and 30 m l o f w a t e r , w e r e a l l o w e d t o s t a n d € o r 30 m i n u t e s . The f i l t e r e d s o l u t i o n ( 0 . 5 m l ) , 5 m l o f K I 0 3 ( 0 . 5 w/v% s o l u t i o n ) , and 2 m l of a c e t i c a c i d were d i l u t e d with water. The c o l o r a t 4 6 5 nm w a s measured a f t e r 80 minutes. The s o l u t i o n s were s t a b l e f o r a f u r t h e r 30 m i n u t e s . A c a l i b r a t i o n c u r v e w a s made f o r comparison.

478

HASSAN Y . ABOUL-ENEIN

6.3.

Ultraviolet Spectrophotometric:

The alkaline solution of propylthiouracil shows two peaks, one at 2 3 4 nm and the other at 2 6 0 nm, the first maximum has a higher molar absorptivity. The ultraviolet absorption of propylthiouracil in ammoniacal solution at 2 3 4 nm is used as a sentive criteria for its analysis in pure and tablet forms ( 5 2 , 5 3 , 5 4 ) . This method is sensitive to a concentration of 7 . 5 mcg/ml and satisfactory results are obtained. Yet, Bruggeman and Schole (45) used the absorption maximum at 2 6 0 nm for the analysis of propylthiouracil in feeds. 6.4.

Chromatographic Analysis:

Paper: The chromatographic behavior of propylthiouracil and related analogs were discussed by several authors (55, 5 6 , 5 7 ) , for the purpose of separation and identification in biological fluids and pharmaceutical preparations. Table I1 summariz e s the solvent systems and visualizing agents used 6.41.

Table I1 Solvent System

Visualising agent

C 6 H 6 :EtOH 16 : 6

RuC 1

AmOH : H 2 0

Reference 55

I vapor or 56 dicklorobemzapinone chloroimide and alkali

Column Chromatography: et a1 ( 3 2 , 3 3 ) separated Lindsay and purified propylthiouracil from its S-methyl derivative and other metabolites using column chromatography on Bio-Gel P-2 columns ( 2 0 0 - 4 0 0 mesh) with water and on DEAE-Sephadex A-25 columns eluting with freshly prepared 0.1 M ammonium acetate. 6.42.

Thin Layer Chromatography: _ a1 _ (58) described a Begliomini et procedure for the seperation and identification of several antithyroid drugs includ ing propylthiouracil 6.43.

PROPYLTHIOURACIL

479

i n a n i m a l f e e d s and b i o l o g i c a l s a m p l e s by means of t l c on s i l i c a g e l G . The s o l v e n t s y s t e m was a mixt u r e of 5 0 m l c h l o r o f o r m , 6 m l i s o p r o p o n o l , and 0.1 m l glacial acetic acid. Amounts up t o 1 mcg were d e t e c t e d by t h i s method. Propylthiouracil showed R f v a l u e of 0 . 8 1 w h i l e i t s m e t h y l homologs moved s l o w e r R f 0 . 6 5 . O t h e r s o l v e n t s y s t e m s used t o i d e n t i f y p r o p y l t h i o u r a c i l and i t s m e t a b o l i t e s and d e r i v a t i v e s were p u b l i s h e d by L i n d s a y et a1 ( 3 2 , 3 5 ) and summer i z e d i n T a b l e 111. T a b l e I11 Solvent System

Developer

0 . 0 5 M Ammonium a c e t a t e 1 M Ammonium a c e t a t e e t h a n o l 15:75 C6H6: i s o p r o p a n o l 6 : l

uv uv uv

Hexane : a c e t o n e : e t h a n o l 60:20:2 Hexane : a c e t o n e 3:l

uv uv

Gas C h r o m a t o g r a p h i c A n a l y s i s : Although a number of methods a r e available f o r t h e determination of propylthiouracil and i t s a n a l o g s , y e t a l l t h e s e methods are l a r g e l y b a s e d on t h e p r o p e r t i e s o f t h e s u l f h y d r y l g r o u p s (-SH). However, t h e same p r o p e r t i e s a r e a l s o common t o t h e C=S and -S-S- g r o u p s . 6.44.

F r a v o l i n i and B e g l i o m i n i ( 5 9 ) d e v e l o p e d a s i m p l e , r a p i d g a s c h r o m a t o g r a p h i c method f o r d e t e r m i n a t i o n of t h i o u r a c i l s i n a n i m a l f e e d s . The method w a s s e l e c t i v e and s e n s i t i v e ( a b l e t o d e t e c t 0 . 1 mcg of t h i o u r a c i l s ) . The c h r o m a t o g r a p h i c s e p a r a t i o n was c a r r i e d o u t on d i a l k y l a t e d t h i o u r a c i l s p r e p a r e d a c c o r d i n g t o Wheeler ( 6 0 ) . The b e s t r e s u l t s were o b t a i n e d w i t h a g l a s s column c o n t a i n i n g Chromosorb was a s o l i d s u p p o r t and 3 % SE-30 polymer The p r o c e d u r e methyl s i l i c o n e as t h e l i q u i d phase. p e r m i t e d s i m u l t a n e o u s i d e n t i f i c a t i o n and d e t e r m i n a t i o n of a v a r i e t y o f t h y r o s t a t i c p r o d u c t s . A typic a l chromatogram i s shown i n F i g . ?

480

HASSAN Y. ABOUL-ENEIN

Retention times were similar to those reported by other authors (61) except that the 5-methyl and 6-methyl thiouracils eluted in the order shown in Table IV. Table IV &tention Tines of Thioracils Compounds 2-Thiouracil 5-Methyl-2-thiouracil 6-Methyl-2-thiouracil 6-Propyl-2-thiouracil 6-Phenyl-2-thiouracil

Retention times, Sec. 160 178 190 322 774

0

A

Gas A = C = D =

a

U D-

E

Fig. 7:

s

t

u)

I

I

I

U 1 2 1 0 8 6 4 TIME, MINUTES

2

0

Chranatograph of thiouracils. 2-thouracil B = 5-methyl-2-thiouracil 6-methyl-2-thiouracil D = 6-propyl-2-thiouracil 6-pheny1-2-thiouracil.

482

HASSAN Y. ABOUL-ENEIN

REFERENCES 1.

J. -Pharm. Pharmacol., &lo1 M.G. Ashley, -

2.

E. Kassau, Deut. Apoth. Ztg.,

3.

C. Nisi, M. Calligaris, S . Fabrissin and M. DeNardo, J. Org. 36, 602 (1971). - Chem., -

4.

The United States Pharmacopeia XIV, Mack Printing Co., Easton, Pa. 1975.

5.

"Specifications for the Quality Control of Pharmaceutical Preparations" 2nd Ed., World Health Organization, Geneva, 1967, p. 502.

6.

Merck Index, 8th edition, Merck Rahaway, N.J., p.878.

7.

British Pharmacopeia, London Her Majesty's Stationary Office, 1973, p. 401.

8.

"CRC Atlas of Spectral data and Physical constants of Organic Compounds" edited by J.G. Grasselli, CRC Press, Cleveland, Ohio, 1973, p. B 980.

9.

M.M. Mosnier, French patent, 1, 012, 739 (1952); through C.A. 52, 4701 (1958).

108, 424

b

(1953). (1968)

Co. Inc.,

10. K. Bucher, Pharm. Acta Helv., 26, 145 (1951). 11. J . M . Mettello Metto and A.P. de Fisueiredo. Rev. brasil farm., 31, 17 (1949); th;ough C.A. 43, 8 3 1 1 ( 1 9 = ) .

12. G. Nilsson, Sci. Rev. (Holland), 89, 86 (1957); (1957); through C.A. 51, 8577 (1957). 13. R.A. McAllister and K.W. Howells, J. -Pharm. Pharmacol., 4, 259 (1952). 14. P. Galimberti, V. Gerosa and M. Melandri, Ann. Chim. (Rome), 48, 457 (1958) 15. B. Stanovnik and M. Tisler, Farm.Vestnik., 14, 61, 6894 (1964). 129 (1963); through C.A. -

483

PROPY LTH IOU RAClL

16.

"The A l d r i c h L i b r a r y o f I n f r a r e d S p e c t r a " by C . J . P o u c h e r t , A l d r i c h C h e m i c a l C o . , Milwank e e l W i s c o n s i n , 1970, p. 994G.

17.

S a d t l e r NMR C a t a l o g , S a d t l e r R e s e a r c h Laborat o r i e s , I n c . , P h i l a d e l p h i a , Pa. 1 9 7 0 , s p e c t rum No. 8367M.

18.

J.M.

19.

J. N i s h i w a k i ,

T e t r a h e d r o n , 2_2,

20.

J . U l r i c h , R.

T e o u l e , R. Massot, A . ,2 , 1 1 8 3 ( 1 9 6 9 )

R i c e , G.O.

Dudek, M.

S O ~,. 87, 4569 ( 1 9 6 5 ) .

Barker, J. Amer. - Chem. 3117 ( 1 9 6 6 ) .

.

Mass. Spectrm.

Cornu, Org.

21.

S.M. H e c h t , A.S. G u p t a , N.J. L e o n a r d , Biophys. A cta,182, 444 (1969). -

22.

G.W. A n d e r s o n , I . F . H a l v e r s t a d t . W . H . M i l l e r , R.O. R o b l i n Jr. , J . Amer. Chem. SOC. , 67,. ~-

Biochem.

2197 ( 1 9 4 5 ) .

and L. Pod, Acad. Rep. P o p u l a r e Romine, Baza C e r c e t a r i S t i i n t . Ti miso ara, S t u d i i Ce r ce t a ri .

23.

J. D i c k , J. R i s t i c i ,

24.

S t i i n t e Chim., 8 , 233 ( 1 9 6 1 ) ; t h r o u g h C . A . 3429h ( 1 9 6 3 )T

25.

U. L i p p o l d , German p a t e n t 8 5 9 , 8 9 3 ( 1 9 5 2 ) ; t h r o u g h C.A. 5 0 , 7851 ( 1 9 5 6 ) .

26.

E.R.

58,

-

G a r r e t t and D . J . 1383 (1970).

Weber, J. Pharm. Sci.,

G a r r e t t and D . J . 6_0, 8 4 5 ( 1 9 7 1 ) .

Weber, -J. Pharm. Sci.,

59,

27.

E.R.

28.

B. M a r c h a n t . W . D . A l e x a n d e r . J . W . K . Robertson and J . H . ' L a z a r u s , Metabolism, 2 0 , 289 (1972).

29.

P.D. P a p a p e t r a u , B. M a r c h a n t , H . Gauvas, and W.D. A l e x a n d e r , Biochem. P h a r m a c o l . , 21, 363 ( 1 9 7 2 ) .

484

HASSAN Y . ABOUL-ENEIN

30.

D.S. Sitar and D.P. Thornhill, J. Pharmacol. Exp. Ther.183, 440 (1972)

31.

R.H. Lindsay, J.B. Hill, K. Kelly and A. Vaughn, Endocrinology, 94, 1689 (1974)

32.

R.H. Lindsay, B.S. Hulsey and H.Y. Aboul-Enein, Biochem. Pharmacol., 24, 463 (1975) and references were citedtherein.

33.

R.H. Lindsay, A. Vaughn, K. Kelly and P.V. P b o u l Enein, Biochem. Pharmacol., in press.

34.

M.L. Desbarats - Schonbaum, L. Endrenyi, E. Koves, E. Schonbaum and E.A. Seller, Europ. J. Pharmacol., 19, 104 (1972)

35.

R.H. Lindsay, H.Y. Aboul-Enein, D. Morel, and S . Bowen, J. Pharm. -Sci 63, 1383 (1974) * I -

36.

D.W. Alexander, V. Evans, A. MacAulay, T.F. Brit.Med.J.,2, -Gallagher Jr., J. Londono, 290 (1969).

37.

G. Smith, J. Assoc. Office.=. 196 (1955)

38.

A. Berggren and W. Kirsten, Farm. Revy, 50, 45, 6348 (1951). 245 (1951); through C.A. -

39.

C.F. Abbott, J. -Pharm. Pharmacol., 5, 53 (1953).

40.

H. Wojahn and E. Wempe, Pharm. Zentralhalle, 92, 124 (1953); through C.A. 48, 3635 (1954)

41.

H. Wojahn and E. Wempe, Arch. Pharm., 286, 344 (1953); through C.A. c11-1954).

42.

Pharm.ActaHelv., 28, 336 (1953). H. Wojahn, -

43.

17, K. Backe-Hansen, Medd. Norsk. Farm. Selskap, 63 (1955); through C.A.0,113(1956).

44.

W. Doden, R. Kopf and H. Specker, Arch. exptl. Path. Pharmakol., 213, 467 (1951)hrough C.A. 46, 8520 (1952).

.

Chemists, 33,

-

PROPY LTH IOURACIL

485

45.

J. Bruggeman and J. Schole, Landwirt. Forsch., 21, 1 3 4 ( 1 9 6 7 ) ; through C.A. 68, 2 0 9 9 (1968).

46.

F. Bucci and A.M. Cusmano, Boll.Lab.Chim. Provinciali., 13., 2 0 6 ( 1 9 6 2 ) ; through C.A. 57., 1 5 2 4 1 h ( 1 9 6 2 ) .

47.

H. Christeinsen, J. -Biol. - Chem.,

48.

F. Reinhardt,

49.

W.L.

50.

W. Doden and R. Kopt, Arch. exptl. Path. makol. 213, 5 1 ( 1 9 5 1 ) ; through C.A. 46, 383 d (1952).

51.

S.

Bruno, Boll. Chim. Farm., 1 0 2 . , 4 7 8 ( 1 9 6 3 ) ; through-. F 1 5 m a (1963).

52.

G.

Smith, J. Assoc. 34, - Offic. - Agr. Chemists., -

-

Z.

160, 4 2 5

(1945).

Physiol. Chem., 293, 268 ( 1 9 5 3 )

Holt and L.N. 1389 (1949).

Mattson, Anal. 21, -Chem., -

u-

576 ( 1 9 5 1 ) .

53.

G.

Smith, J. Assoc. Offic. Agr. Chemists., 35, -~ 572 (19F2).

54.

"Official Method of Analysis of AOAC", W. Horwitz editor, 11th ed., 1970., Association of Official Analytical Chemist, Washington D.C. 1970, p . 691.

55.

F. Reinhardt, Mikrochim. Acta, 2 1 9 ( 1 9 5 4 ) ; through C.A. 48, 6 3 2 5 h n 5 4 ) .

56.

M. Lederer and H. Silberman, Anal. Chim. Acta -

57. 58.

6,

133 (1952).

A.C. Shabica and E. Solook, Federation Proc.,

9,

314 ( 1 9 5 0 ) .

u.,

A. Begliomini, A. Fravolini, Arch. v e t . 21, 63 ( 1 9 7 0 ) ; through C.A. 73, 8 6 5 9 5 g (1970).

59.

A. Fravolini and A. Begliomini, -J. Assoc. Offic. Agr. Chemists, 48, 908 ( 1 9 6 5 ) .

486

HASSAN Y. ABOUL-ENEIN

60.

H.L. Wheeler and D.F. McFarland, Amer. - Chem. J., g., 101 ( 1 9 0 9 ) .

61.

A. Zamorani and P.G. Pifferi., Chemie Industria, 45, 966 (1963).

ACKNOWLEDGEMENT The author expresses appreciation to Mr. Altaf Hussain Naqvi for typing the manuscript.

SODIUM NITROPRUSSIDE

Richard Rucki

488

RICHARD RUCK1

INDEX

1.

Descr i p t i on

1.1 1.2 2.

Name, Formula, M o l e c u l a r Weight Appearance, Color, Odor

Physical Properties

2.1 2.2 2.3 2.4 2.5

2.6 2.7

2.8

2.9

I n f r a r e d Spectrum Raman Spectroscopy U l t r a v i o l e t / V i s i b l e Spectrum Fluorescence Spectrum Opt ica 1 R o t a t i o n D i f f e r e n t i a l Scanning C a l o r i m e t r y Thermogravimetric A n a l y s i s Solubility Crystal Properties

2.9.1 2.9.2

Crystal Structure X-Ray D i f f r a c t i o n

3.

Syn thes is

4.

S t a b i li t y and Degradation

4.1

Sol i d S t a b i l i t y

4.2

S t a b i l i t y i n Solution

5.

Drug M e t a b o l i c Products

6.

Toxicity

7.

Methods o f A n a l y s i s

7.1 7.2

7.3 7.4 7.5 7.6 7.7 7.8 7.9

Elemental A n a l y s i s I dent i f i c a t i o n Tests Thi n-Layer Chroma tograph i c A n a l y s i s Spectrophotometric A n a l y s i s Colorimetric Analysis Polarographic Analysis Cou lometr i c A n a l y s i s T i t r i m e t r i c Analysis Miscellaneous Methods of A n a l y s i s

8.

Acknowledgements

9.

References

SODIUM NITROPRUSSIDE

1.

489

Description

1.1

Name, Formula, M o l e c u l a r Weight Sodium n i t r o p r u s s i d e i s d i s o d i u m p e n t a c y a n o n i t r o s y l I t i s a l s o known as sodium f e r r a t e (2-) dihydrate. n i t r o f e r r i c y a n i d e and sodium n i t r o p r u s s i a t e . The d i h y d r a t e i s t h e common f o r m o f t h e compound and i s assumed i n t h i s r e p o r t e x c e p t where s p e c i f i e d as an h y d ro u s .

-2

NC

ON

.2H20

d

Sodium N i t r o p r u s s i d e

C N OFeNa2.2H20

5 6

Mol ecu 1 a r Weight :

297.95 1.2

Appearance, C o l o r , Odor Red-brown, p r a c t i c a l l y o d o r l e s s , c r y s t a l s o r powder.

2.

Physical Properties

2.1

I n f r a r e d Spectrum The i n f r a r e d s p e c tru m o f sodium n i t r o p r u s s i d e i s p r e sented i n Figure 1 ( I ) . The spectrum was r e c o r d e d on a P e r k i n - E l m e r Model 621 G r a t i n g I n f r a r e d S p e c t r o ph o to m e te r (Su rv e y C p d i t i o n s ) . The sample was d i s p e r s e d i n F l u o r o l u b e t o r e c o r d t h e spectrum i n t h e r e g i o n o f 4000-1340 cm-l and i n m i n e r a l o i l f o r t h e Assignments f o r t h e bands r e g i o n of 1340-370 c m -l . i n F i g u r e 1 a r e l i s t e d i n T a b l e I ( 1 ) . These assiclnments a r e i n agreement w i t h thase r e p o r t e d i n t h e 1 iterature (2-4).

E

3

.U 0

v)

0

al

.-

-u

L

m

I C

-

%

33NVlllWSNVLll %

490

SODI UM N IT ROP R USS I D E

TABLE I

Infrared Assignments for Sodium Nitroprusside

Band (cm-I)

Assignment

3628

Asymmetric OH stretch

3547

Symmetric OH stretch

2174

-C:N

2144, 2157, 2162

-CzN radial stretch

1942

N 4 stretch

1614, 1618.5, 1624

OH bend i ng

662

Fe-N-tD linear bending

65 1 49 1 46 1 418.5

Fe-N stretch Fe-C:N

axial stretch

bending

Fe-C- axial stretch Fe-CEN bending

49 1

RICHARD RUCK1

492

2.2

Raman Spectroscopy Raman spectra o f single-crystal sodium nitroprusside have been utilized for structure elucidation by a number of investigators (2, 5-8). The use of a He-Ne laser to excite the oriented crystal has been reported (2,8). Polycrystal sodium ni troprusside rapidly oxidized when subjected to laser excitation ( 9 ) .

2.3

Ultraviolet/Visible Spectrum The ultraviolet/visible spectrum of sodium nitroprusside (750 mg of sodium nitroprusside/lOO ml o f water v s . water in the reference cell) in the region o f 240 to 700 nm exhibits two maxima at 390-395 nm (molar absorptivity, E = 20.4) and at about 500 nm (appears as a shoulder). The instrument used was a Cary 14 Recording Spectrophotometer. The visible portion of the spectrum is shown in Figure 2 (10). These results are in agreement with UV/visible data reported previously in the 1 i terature ( 1 1-13). The existence of the maximum at 500 nm as a distinct absorption band (a distinct elec ronic transition) has been confirmed by the determ nation of the polarized crystal spectrum of a sing e crystal of sodium ni troprusside dihydrate (12).

2.4

Fluorescence Spectrum Sodium nitroprusside exhibits no fluorescence in acidic, basic o r neutral media ( 1 4 ) .

2.5

Optical Rotation A 0.6% (w/v) solution of sodium nitroprusside in water exhibited no optical rotation between 650 and 220 nm (15).

2.6

Differential Scanning Calorimetry DSC scans for typical lots of sodium nitroprusside at a scan rate of 20"C/minute exhibit two very broad endotherms, the first between about 125 and 180°C and the second between about 320 and 360"C, followed immediately by an exotherm. The endotherms do not correspond to sample melt and have the typical appearance of volatile material leaving the system. Anhydrous sodium nitroprusside did not exhibit the

SODIUM NITROPRUSSIDE

FIGURE 2 V i s i b l e A b s o r g t i o n S p e c t r u m o f Sodiuni Nitroprusside

493

RICHARD RUCK1

494

f i r s t endotherm ( 1 6 ) . The temperature of each endotherm corresponds t o a weight loss i n t h e TGA (Section 2.7). Thermal a n a l y s i s o f sodium n i t r o p r u s s i d e has been r e p o r t e d i n the l i t e r a t u r e (17-19). 2.7

Thermogravimetr ic A n a l y s i s TGA scans f o r t y p i c a l l o t s o f sodium n i t r o p r u s s i d e e x h i b i t two d i s c r e t e w e i g h t losses. The f i r s t occurs between 100 and 190°C and accounts f o r 12 t o 13% o f sample weight ( t h e o r e t i c a l w e i g h t loss f o r d i h y d r a t e i s 12.09%)). The second occurs between about 280 and 390°C and accounts f o r 17.6 t o 19.9% o f sample w e i g h t ( t h e o r e t i c a l w e i g h t l o s s f o r cyanogen, (CN) is 19.85% o f anhydrous sample w e i g h t ) (16). f i e ident i f i c a t i o n of the second w e i g h t l o s s as cyanogen i s speculative. Chamberlain and Greene (17,18), u s i n g dynamic gas e v o l u t i o n a n a l y s i s , have r e p o r t e d t h a t t h e thermal decomposition of c y a n o n i t r o s y l f e r r a t e s i n v o l v e t h e e v o l u t i o n of w a t e r , (CN) and NO. G e r i t i l e t a l . (19) and Mohai (20,21f have r e p o r t e d TGA d a t a for sodium n i t r o p r u s s i d e .

2.8

S o l u b i 1 it y Approximate s o l u b i l i t y d a t a o b t a i n e d for a sample o f sodium n i t r o p r u s s i d e a t 25°C a r e l i s t e d i n Table I I ( 2 2 ) . E q u i l i b r a t i o n t i m e was 20 hours f o r each system.

SOD IUM

N IT ROPR USSl DE

49 5

TABLE I I

S o l u b i l i t y o f Sodium N i t r o p r u s s i d e Solvent

S o l u b i 1 i t y (mg/ml)

Water

>200

95% E t h a n o l

1.1

Absolute Ethanol

5.0

Methanol

100- 200

Acetone

lnsolub

Diethyl Ether

I n s o l ub

Chloroform

Insolub

Benzene

0.2

lsopropyl Alcohol

0.1

Hexane

0.1

Ethyl Acetate

0.3

Normal S a l i n e

>200

2.9

Crystal Properties

2.9.1

Crys t a 1 S t r u c t u r e Sodium n i t r o p r u s s i d e o c c u r s as r e d d i s h brown ( o r r u b y - r e d ) c r y s t a l s ; anhydrous ( l y o p h i 1 i z e d ) sodium n i t r o p r u s s i d e e x i s t s as a l i g h t o ra n g e , u n i f o r m powder ( 2 3 ) . The c r y s t a l s t r u c t u r e o f sodium n i t r o p r u s s i d e has been s t u d i e d v i a X -ray d i f f r a c t i o n , i n f r a r e d and Raman a n a l y s i s (2-4, 24-27). The c r y t a l i s o r t h o r h o m b i c w i t h space g ro u p G"-Pnnm. The n i t r o p r u s s i d e i o n 1 i e s 2b or1 t h e m i r r o r p l a n e and has a p p r o x i m a t e l y C1,, symmetry. The u n i t c e l l c o n t a i n s f o u r f o r m u l a u n i t s o f t h e t y p e Na Fe ( C N ) N O ' 2 2H20.+ Thz c r y s t a l s t r u c t u r e i s compAsed o f Na , F S ~ C N ) ~ N O ~ and -, H 0 uni t s . The 2 Fe(CN) NO i o n s occupy 5 i t e s o f C 5ym5 m ~ t r y , ~ a ntdh e H 0 m o l e c u l e s occupy C 1 2 sites. The I i g a n d s a r e c o l i n e a r w i t h t h e

496

RICHARD RUCK1

metal atom, which i s d i s p l a c e d s l i g h t l y i n t h e d i r e c t i o n o f t h e NO group from t h e p l a n e o f the f o u r pseudo-equivalent CN groups. Each sodium i o n 1 i e s a t the c e n t e r o f a d i s t o r t e d octahedron composed of f o u r CN groups and two water molecules. These octahedra share edges i n such a way t h $ t each CN group i s c o o r d i n a t e d t o two Na i o n s , as i s each water molecule. The n i t r o s o group i s c o o r d i n a t e d o n l y t o Fe+2 (24). The w a t e r molecules a r e n o t i n v o l v e d i n any s i g n i f i c a n t hydrogen bonding w i t h t h e Fe(CN) NO2- ion; they merely s e r v e t o f i l l t h e ehpty space i n t h e l a t t i c e (2, 24, 26). S t u d i e s o f t h e i n f r a r e d (28, 29) and Mbssbauer (30) s p e c t r a i n d i c a t e a l a r g e amount o f back bonding between t h e metal and t h e n i t r o s y l l i g a n d . Although t h e f o r mal charge o f i r o n and n i t r o s y l i n t h e comp l e x has been a m a t t e r of c o n t r o v e r s y , t h e general consensus appears t o be t h a t Fe and NO have formal chaKges o f +2 and + I , r e s p e c t i v e l y (12, 24, 31-35). 2.9.2

X- Ray D i f f r a c t i on The X-ray powder d i f f r a c t i o n p a t t e r n o f a proposed house standard of sodium n i t r o p r u s s i d e i s presented i n Table I l l (36). The i n t e r p l a n a r spacings agree w i t h those r e p o r t e d i n t h e l i t e r a t u r e u s i n g a molybdenum t a r g e t ( 3 7 ) .

Ins t rumen t a 1 Cond i t i ons Instrument Generator Tube T a r g e t Optics

Goniometer Detector

GE Model XRD-6 Spectrogon i ometer 50 KV, 12.5 mA 0 Copper (Cu Km = 1.5418A) 0.1" D e t e c t o r s l i t M.R. S o l l e r s l i t 3" Beam s 1 i t Ni Filter 4" T a k e - o f f angle Scan a t 0.4" 20/minute Sealed p r o p o r t i o n a l counter 1 . 7 5 KV ( f r o n t ) , 0 . 9 5 KV ( r e a r ) . Pulse h e i g h t selector E l 5

SODIUM NITROPRUSSIDE

Recorder Sample

v o l t s , window o u t . Time c o n s t a n t = 2.5 seconds Range = 1000 c/sec f u l l scale Synchronized w i t h goniometer a t l"/2.5 minutes Ground a t room temperature

TABLE I I I Sodium N i t r o p r u s s i d e

11.60 15.61 19.00

21.65 23.08 27. I4 31.25 33.30 35.65

A

*;':

7.63 5.68 4.67 4.10

3.85 3.29 2.86 2.69 2.52

21

55 58 98 29 34 100

16 25

d (interplanar distance) = n h / 2 s i n 0 1/10

497

= r e l a t i v e i n t e n s i t y (highest

i n t e n s i t y = 100)

498

3.

RICHARD RUCK1

Synthesis Sodium nitroprusside is commonly prepared by the oxidation of potassium ferrocyanide with dilute nitric acid and subsequent neutralization o f the liquid with sodium carbonate (38). The reaction scheme i s shown below (39)

+ 6 HNO + H2[(NO)Fe(CN) 5] + 4 KNO 3 + 3 + NH NO + C02 4 3

K4[Fe(CN)6].3H20

H2[(NO)Fe(CN)5]

+ Na2C03 H20

2 Na2[(NO)Fe(CN) 5].2H20

+

+ CO 2

4. Stabi 1 i ty and Degradation

4.1

Solid Stability Sodium nitroprusside crystals have been reported to be stable in air (40). Even in the dry, solid state, however, the compound is somewhat light sensitive (Section 4.2) and should be protected from light (41, 4 2 ) . Small amounts of moisture could facilitate the photodegradation o f dry sodium ni troprusside (41). In closed, amber vials at 25"C, sodium nitroprusside in the sol id state remains suitably stable for at least 24 months (measuring absorbance maximum at 394 nm; Section 4.2) ( 4 3 ) .

4.2 Stability in Solution Sodium nitroprusside in solution is extremely photosensitive, undergoing rapid and numerous reactions, many o f which are undefined. Literature descriptions o f the photodecomposition products of nitroprusside are, in some cases, contradictory. 2In direct sunlight [Fe(CN) NO] ultimately yields Prussian blue, HCN and NO 144). Kapatos et al. (45) have reported that photoirradiation o f solutions o f nitroprusside yield Prussian blue and NO, while Wolfe and Swinehart (46) report a similar reaction in unbuffered solutions o f nitroprusside: Na2[Fe(CN) 5NO] + h v (X>300 nm) + Na[Fe"'Feli (CN) 61 (sodium salt o f Prussian b l u e ) + NO + (CN) 2 + H C N

499

SOD I UM N IT R OP R USS I D E

I n s o l u t i o n s b u f f e r e d a t pH 6, W o l f e and S w i n e h a r t ( 4 6 ) have r e p o r t e d f o r m a t i o n of t h e pentacyanoaquof e r r a t e ( I I I ) , a g r e e i n g w i t h s e v e r a l o t h e r papers

(47-50) [Fe(CN)

:

5

+

hy(X>jOOnm)

-f

[FelII(CN)

H

5 2

O]*-

+ NO

The pentacyanoaquof i - r a t e ( ',I$ undergoes r a p i d e q u i l i b r i u m w i t h [F e 2 'lP(CN)lO] (SO, 51). II P h o t o a q u a t i o n t o y i e l d [Fe (CN) H O I 3 - and NO has been d e s c r i b e d most f r e q u e n t l y a2 $he p r i m a r y phot o c h e m i c a l r e a c t i o n o f n i t r o p r u s s i d e ( 1 1,34,52-54). M i t r a and coworkers (34) f o u n d a pH decrease upon p h o t o l y s i s and a t t r i b u t e d t h i s t o h y d r o l y s i s o f t h e rii t r o s y l c a t i o n :

NO

+

+ H20

++

[F e ' I (CN) 5H20]3-

+ 2H+ +

NO;

The p e n t a c y a n o a q u o f e r r a t e ( 1 I A-undergoes e q u i l i b r i u m w i t h [Fe2 (CN) ] (55). 10

rap i d

P h o t o r e d u c t i o n o f [Fe(CN) NO]2- t o [Fe(CN) in 5 ?he orangeaqueous s o l u t i o n has been r e p o r t e d (56). t o - b l u e c o l o r change o f sodium n i t r o p r u s s i d e solut i o n upon s t a n d i n g and e x p o s u re t o l i g h t has been a t t r i b u t e d t o t h e change o f f e r r i c t o f e r r o u s i o n

(57, 58). When p r o t e c t e d f r o m l i g h t , aqueous s o l u t i o n s o f sodium n i t r o p r u s s i d e have been r e p o r t e d t o be s t a b l e f o r as l o n g as s i x months (11,59,60). I n aqueous s o l u t i o n t h e n i t r o p r u s s i d e i o n r e a c t s w i t h a w i d e v a r i e L y o f i n o r g a n i c and o r g a n i c subs t anc e s t o f o r m u s u a l l y h i g h l y c o l o r e d r e a c t i o n 52, 61-71). pi-oducts (50, S p e c t r o p h o t o m e t r i c measurements have most o f bzen used t o d e t e r m i n e s t a b i l i t y o f sodium n p f u s s i d e , w i t h most emphasis on t h e i n c r e a s e absorbance a t 350-395 nm w i t h d e g r a d a t i o n ( I 5 3 , 5 5 , 6 0 ) . Curce has d e v e l o p ed a s t a b i 1 i t y

en t roin ,119 S O ,

500

RICHARD RUCK1

i n d i c a t i n g method by complexing i r o n i n any form o t h e r than n i t r o p r u s s i d e w i t h a z i d e and measuring t h e r e s u l t i n g absorbance a t 560 nm ( 7 2 ) . P o l a r o g r a p h i c s t a b i l i t y s t u d i e s (11, 73) have i n d i c a t e d t h a t the f i r s t two p o l a r o g r a p h i c waves ( S e c t i o n 7.6) decrease i n l i m i t i n g c u r r e n t w i t h d e g r a d a t i o n , b u t spectrophotometry i s a much more s e n s i t i v e method f o r d e t e c t i n g photodegradation ( 1 1 ) .

5.

Drug M e t a b o l i c Products When g i v e n i n t r a v e n o u s l y , sodium n i t r o p r u s s i d e r a p i d l y lowers b l o o d pressure by p e r i p h e r a l v a s o d i l a t a t i o n and r e d u c t i o n i n p e r i p h e r a l r e s i s t a n c e as a r e s u l t of a d i r e c t a c t i o n on t h e b l o o d vessel w a l l s , independent o f autonomic i n n e r v a t i o n (74-78). Blood p r e s s u r e can be maint a i n e d a t any l e v e l depending on t h e r a t e o f i n f u s i o n ( ~ 7 ~ 5 8 ) The . hypotensive a c t i o n i s a t t r i b u t e d t o the 79, 80) o f the n i t r o p r u s n i t r o s o (NO) group (57, 75-77, s i d e r a d i c a l and i s augmented i n b o t h doqs and humans by autonomic g a n g l i o n b l o c k i n g agents (57,76). The drug has an immediate e f f e c t , w i t h d e s i r e d b l o o d pressure l e v e l s u s u a l l y a t t a i n e d w i t h i n 0.5 t o 2 minutes. Upon d i s c o n t i n u a t i o n o f t h e i n f u s i o n , b l o o d p r e s s u r e r a p i d l y r i s e s to previous levels, usually w i t h i n 1 t o T h i s evanescence o f t h e d r u g ' s 10 minutes (81-84). e f f e c t i s due t o r a p i d d e s t r u c t i o n o f t h e a c t i v e n i t r o p r u s s i d e r a d i c a l which i s s l o w l y converted i n t h e body t o cyanide. T h i s conversion i s a t t r i b u t e d t o t h e i n t e r a c t i o n of t h e f e r r o u s i o n i n n i t r o p r u s s i d e w i t h f r e e s u l f h y d r y l groups i n e r y t h r o c y t e s ( r e d b l o o d c e l l s ) and o t h e r t i s s u e s (57,76,79,85,86). I n v i v o and i n v i t r o s t u d i e s have shown t h a t n i t r o p r u s s i d e l i b e r a t e s cyanide when c o n t a c t e d w i t h l i v e r (85). whole blood, washed e r y t h r o c y t e s , plasma, and u r i n e (76,87-89). The r e l e a s e o f cyanide i s non-enzymatic (76,79,85,87), and i t s slow t i m e course p r e c l u d e s t h e r e a c t i o n from b e i n g t h e mechanism o f a c t i o n o f n i t r o p r u s s i d e (76,79). The cyanide i s then conv e r t e d by t h e h e p a t i c enzyme rhodanase ( t r a n s s u l f u r a s e ) t o t h i o c y a n a t e (79,90). A small amount o f t h e t h i o c y a n a t e i s o x i d i z e d back t o cyanide by a t h i o c y a n a t e oxidase present i n e r y t h r o c y t e s (91,92) and perhaps a l s o by a r e v e r s a l of the rhodanase system (93). Boxer and R i c k a r d s . ( g $ ) found these two compounds t o be i n dynamic e q u t l t b r r u m b u t t h a t the equilibrium i n v i v o i s f a r i n the d i r e c t i o n of t h i o cyanate. The h a l f - l i f e f o r e x c r e t i o n o f t h i c y a n a t e i s a p p r o x i m a t e l y seven days w i t h normal r e n a l f u n c t i o n ( 9 5 ) . A m e t a b o l i c scheme (57) i s presented i n F i g u r e 3.

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O r a l a d m i n i s t r a t i o n o f sodium n i t r o p r u s s i d e f o r l o n g p e r i o d s does n o t s i g n i f i c a n t l y l ow er b l o o d p r e s s u r e ; the e f f e c t s are s i m i l a r t o tho5e o f thiocyanatc given o r a l l y ( 7 6 ) . S i n c e t h i o c y a n a t c accumul ates i n b l o o d w i t h p r o l o n g e d n f u s i o n o f sod um n i t r o p r u s s i d e , t h i o c y a n a t e o r c y a n i d e may be respons b l e f o r some l a t e r e f f e c t s o f t h e d r u g ( 5 7 76,77)

.

6.

Toxicity Sodium n i t r o p r u s s i d e has few s de e f f e c t s , none o f w h i c h u s u a l l y r e q u i r e s discontinuance o f therapy, provided t h a t dosage i s r e a s o n a b l e (57,58,79,81 ,82,96). Acute t o x i c i t y was i n i t i a l l y t h o u g h t due p r i m a r i l y t o f o r m a t i o n o f c y a n i d e , b u t subsequent s t u d i e s (74) have i n d i c a t e d t h a t t h e im m edia te t o x i c i t y o f t h e d r u g i s p r o b a b l y due t o s e v e r e h y p o t e n s i o n , caused by e x c e s s i v e l y h i g h r a t e s of i n f u s i o n (57,58,79). Johnson (74) e s t i m a t e d t h e r a t i o between d e p r e s s o r and t o x i c dosages a s 1 : l O . C a u t i o n s h o u l d b e e x e r c i s e d i n t r e a t m e n t w i t h sodi um n i t r o p r u s s i d e s i n c e i t s i mme d i ate m e t a b o l i c p r o d u c t s a r e Prolonged treatment t h i o c y a n a t e and c y a n i d e ( S e c t i o n 5 ) . may r e s u l t i n e l e v a t e d serum t h i o c y a n a t e l e v e l s , e s p e c i a l l y i f r e n a l f u n c t i o n i s i m p a i r e d (57,76,97). Toxic symptoms o f e x c e s s i v e e l e v a t i o n o f t h i o c y a n a t e i n t h e b l o o d i n c l u d e f a t i g u e , nausea, weakness and loss o f appet i t e (58,76). I n a p a t i e n t w i t h severe renal i n s u f f i c i enc y , l o n g - t e r m sodium n i t r o p r u s s i d e a d m i n i s t r a t i o n r e s u l t e d i n h y p o t h y r o i d i s m , caused b y t h i o c y a n a t e i n h i b i t i o n o f t h e u p t a k e and b i n d i n g o f i o d i n e by t h e t h y r o i d (97). A l t h o u g h s i g n i f i c a n t l e v e l s of t h i o c y a n a t e have appeared i n b l o o d d u r i n g c h r o n i c o r a l a d m i n i s t r a t i o n o f n i t r o p r u s s i d e (76), e l e v a t e d l e v e l s have n o t been o b s e r v e d w i t h i t s s h o r t - t e r m use (81) or d u r i n g p r o l o n g e d , i n t r a venous use (98) i n p a t i e n t s w i t h normal k i d n e y f u n c t i o n .

A s m a l l amount o f t h i o c y a n a t e i s o x i d i z e d back t o c y a n i d e i n t h e body ( S e c t i o n 5 ) . Elevated blood cyanide l e v e l s i n v i v o . have been r e p o r t e d f o l l o w i n g sodium n i t r o p r u s s i d e a d m i n i s t r a t i o n (87,88,92,94), but i n the vast majori t y o f cases t h e amounts have been s m a l l . Even w i t h d i r e c t a d m i n i s t r a t i o n o f t h e r a p e u t i c doses o f t h i o c y a n a t e , b l o o d c y a n i d e amounts were smal 1 and idel 1 below l e t h a l concent r a t i o n s (91,32). Vesey e t a ] . (88) found a s i g n i f i c a n t r i s e i n plasma c y a n i d e l e v e l s a f t e r sodium r i i t r o p r u s s i d e i n f u s i o n and a s i m u l t a n e o u s d e c r e a s e i n plasma v i t a i i l i n B I2 ( 9 9 ) , a l t h o u g h t h e r e were no a d v e r s e e f f e c t s on t h e

SO DIU M N ITROPR USS IDE

503

p a t i e n t s . S i n c e t h e l i v e r s e r v e s a s t h e main r e g u l a t o r y s y s t em o f c y a n i d e d e t o x i f i c a t i o n ( S e c t i o n 5 ) , sodium n i t r o p r u s s i d e s h o u l d be used w i t h c a u t i o n i n p a t i e n t s w i t h i m p a i r e d 1 i v e r f u n c t i o n (57,88,97,100). Sodium n i t r o p r u s s i d e i n f u s i o n t o baboons was s t u d i e d and, on a w e i g h t c o r r e c t i o n b a s i s , i t has been r e p o r t e d t h a t t h e s m a l l e s t t o x i c dose i n t h e baboon g i v e n o v e r 2 h o u r s i s e q u i v a l e n t t o 320 mg/hour i n man, and 1-1/2 t h e mean t o x i c dose e q u i v a l e n t t o 518 mg/hour ( 1 0 1 ) . I n t r a v e n o u s LD has been d e t e r m i n e d t o be 8.4 2 0.3 1 . 1 mg/kg mg/kg i n m i c e , 5 ? l . 2 2 1 . 1 mg/kg i n r a t s , 2.8 i n r a b b i t s , and a p p r o x i m a t e l y 5 mg/kg i n dogs (102). LD i n m i c e has been d e t e r m i n e d t o be 48 2 2.9 mg/kg o r z ? l y and g r e a t e r t h a n 2000 mg/kg t o p i c a l l y (103).

-

7.

Methods of A n a l y s i s

7.1

E lem e n ta l A n a l y s i s An e l e m e n t a l a n a l y s i s o f a s t a n d a r d sample o f sodium n i t r o p r u s s i d e (as t h e d i h y d r a t e ) i s p r e s e n t e d i n T a b l e I V . Water was d e t e r m i n e d by K a r l F i s h e r t i t r a t i o n ( 04). TABLE I V E 1emen t a 1 A n a l y s i s o f Sodium N i t r o p r u s s i d e

E 1 emen t

% Found

C

20.14

20.12

H

1.34

1.40

N

28.21

29.68

"3

15.44

14.98

Fe

18.74

18.72

12.09

12.03

H2° 7.2

% Theory

I d e n k i f i c a t i o n Tests The v i s i b l e a b s o r p t i o n s p e ctrum ( S e c t i o n 2.3) i s s p e c i f i e d b y U S P X I X as t h e i d e a t i f i c a t i o n t e s t f o r sodium n i t r o p r u s s i d e ( 1 0 5 ) . The i n f r a r e d spectrum ( S e c t i o n 2.1) may also be used f o r i d e n t i f i c a t i o n of

RICHARD RUCK1

504

the drug. For t h e dosage fo rm , USP X I X s p e c i f i e s m i x i n g sodium n i t r o p r u s s i d e w i t h a s c o r b i c a c i d and d i u t e H C I , f o l l o w e d by d r o p w i s e a d d i t i o n o f sodium h y d r o x i d e T.S. t o p ro d u c e a t r a n s i e n t b l u e c o l o r 105). A number o f o t h e r q u a l i t a t i v e c o l o r r e a c t ons have been r e p o r t e d ( 106- 1 10)

.

7.3

T h i n - L a y e r C h ro m a to g ra p h i c A n a l y s i s

A number o f TLC systems f o r t h e s e p a r a t i o n o f sodium n i t r o p r u s s i d e from i t s - m e t a b o l i t e s , thiocyanate (SCN ) and c y a n i d e (CN ) , a r e l i s t e d i n T a b l e V (111). S i l i c a g e l s t a t T o n a r y phases were used i n each, and n i t r o p r u s s i d e was d e t e c t e d w i t h 1% Na S i n O.%N NaOH, SCN- w i t h 0.1% FeCl i n 0.5N H C l , an8 C N - w i t h t h e method o f 0 . Wasc3wik e t ( 1 12). A good s e p a r a t i o n o f t h e t h r e e substances i s p o s s i b l e u s i n g t h e f i r s t s y s te m l i s t e d ( s o l v e n t f r o n t 10 cm) f o l l o w e d by t h e second s y s tem ( s o l v e n t f r o n t 14 cm), r e s u l t i n g i n d i s t a n c e ? f r o m s t a r t i n g p o i n t f o r CN , r , i t r o p r u s s i d e and SCN of 0, 45 and 99 mm, respectively (111).

al.

TABLE V T h i n - L a y e r C h ro m a to g ra p h i c Systems f o r Sodium N i t r o p r u s s i d e

-!

f

Va 1 ues

Solvent

CN-

n- pr opan o l :H20 (10:2)

--

--

--

n - b u t a n o l :2N NH ( I : 1) (organ i c LhasJ)

0

0.20

0.71

0

0.44

0.77

0

0.95

0.85

n - p r o p a n o l :H 0 (1 0 : 1 ) 2 n-butano1:n-propanol: d i b u t y l a m i n e ( 4 5 : 4 5 : 10)

7.4

N i troprusside

SCN-

Spectrophotometric Analysis Sodium n i t r o p r u s s i d e may be a n a l y z e d s p e c t r o p h o t o m e t r i c a l l y by u t i l i z i n g t h e m o l a r a b s o r p t i v i t y v a l u e ( E = 20.4) a t t h e m a x i m u m i n t h e v i s i b l e spectrum a t 394 nm ( 1 1 ) .

SODIUM NITROPRUSSIDE

7.5

505

Colorimetric Analysis Small amounts o f n i t r o p r u s s i d e have been d e t e r m i n e d c o l o r i m e t r i c a l l y as t h e i s o p horone complex by measuri n g absorbance a t 495 nm i n pH 10.2 b u f f e r ( 1 1 3 ) . An i n d i r e c t c o l o r i m e t r i c method f o r sodium n i t r o p r u s s i d e d e t e r m i n a t i o n , c o n s i s t i n g of p r e c i p i t a t i o n w i t h 1 , l O - p h e n a n t h r o l i n , s e p a r a t i o n and measurement o f t h e e x t i n c t i o n c o e f f i c i e n t o f t h e f i l t r a t e , has been r e p o r t e d ( 1 14- 1 15)

.

7.6

P o l a r o g r a p h i c Ana l y s i s Sodium n i t r o p r u s s i d e has been d e t e r m i n e d p o l a r o g r a p h i c a l l y by a number o f w o r k e r s . A t t h e d r o p p i n g merc u r y e l e c t r o d e , t h r e e r e d u c t i o n waves were observed a t -0.4, -0.6 and -1.2 v o l t s vs. S C E . The f i r s t t w o waves were r e p o r t e d t o i n v o l v e one e l e c t r o n each as c a l c u l a t e d from t h e n i n the l l k o v i c equation, a r e independent o f t h e hydrogen i o n c o n c e n t r a t i o n i n t h e pH r a n g e 6 t o 10, and a r e r e v e r s i b l e , w h i l e t h e t h i r d wave i s i r r e v e r s i b l e and t h e v a l u e of n i s 2 (31,ll6,117). Zuman and Kabat (118,119) c o n f i r m e d t h a t t h e f i r s t two waves were o n e - e l e c t r o n reduct i o n s , and deduced t h a t t h e t h i r d wave was a twoe l e c t r o n r e d u c t i o n , b u t c o n s i d e r e d a1 1 t h r e e waves t o be i r r e v e r s i b l e . More r e c e n t s t u d i e s (11,73,120) have r e p o r t e d t h e f i r s t two waves o n l y . A typical p o l a r o g r a m o f sodium n i t r o p r u s s i d e , showing t h e f i r s t t w o waves, i s shown i n F i g u r e 4 (120). The c u r r e n t o f t h e f i r s t p o l a r o g r a p h i c r e d u c t i o n wave a t a b o u t -0.33 v o l t s v s . Ag/AgCI r e f e r e n c e e l e c t r o d e i n aqueous pH 7.2 b u f f e r i s used t o assay t h e dosage f o r m (50 mg d r y - f i 1 l e d v i a l ) (105,120). P hotodegrad a t i o n o f sodium n i t r o p r u s s i d e has a l s o been d e t e r mined b y f o l l o w i n g t h e d e c re ase i n 1 i m i t i n g c u r r e n t o f t h e f i r s t two p o l a r o g r a p h i c waves (11,731.

7.7

Coulornetric Analysis C o u l o m e t r i c s t u d i e s o f n i t r o p r u s s i d e , u s i n g a rnerc u r y c a th o d e and a s i l v e r anode, have i n d i c a t e d t h a t t h e second and t h i r d r e d u c t i o n waves i n v o l v e two and f o u r fa ra d a y s p e r mole o f e l e c t r o d e r e a c t i o n , respect i v e l y , w h i l e the products o f reduction i n t e r f e r e d w i t h t h e d e t e r m i n a t i o n o f n for t h e f i r s t wave

506

RICHARD RUCK1

FIGURE

4

Polarogram o f Sodium N i troprusside

SODIUM N ITROP RUSSl D E

507

(121,122). I t has a 1 5 0 been r e p o r t e d t h a t c o n t r o l I c d p o t e n t i 2 1 c o u l o 8 i i e t r i c t i L r s t i o n was n o t s t o i c h i o nie t r i c , p ro b s b 1 y due t o Loinpe t i ng background r c a c tions (120).

7.8

T i t r i m e t r i c Analysis Sodium n i t r o p r u s s i d e i s assayed by d i s s o l v i n g t h e sample i n w a t e r and t i t r a t i n g w i t h 0. IN s i l v e r n i t r a t e . The e n d p o i n t i s d e t e r m i n e d p o t e n t i o m e t r i c a l l y , using a s i l v e r - s i l v e r chloride electrode system. Each rnl o f 0.1N s i l v e r n i t r a t e i s e q u i v a l e n t t o 14.90 mg o f Na2TFe(CN) N0].2H20 ( 1 0 5 ) . A l t e r n a t i v e l y , m e r c u r i c n i t r a t z has been used as t i t r a n t , and p o l a r i z e d p l a t i n u m e l e c t r o d e s and s i l i c o n - r u b b e r based h a l i d e - s e l e c t i v e membrane e l e c t r o d e s have been used as i n d i c a t o r e l e c t r o d e s ( 1 2 3 ) . Titrim e t r i c d e t e r m i n a t i o n o f n i t r o p r u s s i d e w i t h mercurous i o n has been d e s c r i b e d by Tomicek and Kubi k (124). An i n d i r e c t t i t r i m e t r i c method f o r n i t r o p r u s s i d e , u s i n g a f l u o r e s c e n t e n d p o i n t , has been r e p o r t e d (125). A f t e r d e c o m p o s i t i o n o f n i t r o p r u s s i d e w i t h NaOH and Na2Ni(CNI4 and f i l t r a t i o n , t h e n i c k e l i s t i t r a t e d w i t h Na EDTA w i t h bisglycinemethylenedichlorofluorescein as mezal l o f l u o r o c h r o m i c i n d i c a t o r .

7.9

M i s c e l l a n e o u s Methods o f A n a l y s i s

N i t r o p r u s s i d e has been d e t e r m i n e d g r a v i m e t r i c a l l y u s i n g d i a n t i p y r y l p h e n y l m e t h a n e ( 1 2 6 ) , and by p r e c i p i t a t i o n o f n i c k e l hydroxide i n the r e a c l i o n o f n i c k e l c y a n i d e w i t h a l k a l i n e n i t r o p r u s s i d e (127). The l a t t e r method i s more s e l e c t i v e t h a n t h e f o r m e r , b u t c y a n i d e , f e r r i c y a n i d e , and l a r g e amounts o f ferrocyanide w i 1 1 i n t e r f e r e ( 1 13). A m i c r o c r y s t a l t e s t , one i n w h i c h t h e p r e c i p i t a t e formed by t h e c h e m i c a l r e a c t i o n between a substance and a r e a g e n t i s examined w i t h a mi croscope, has been r e p o r t e d f o r the detet-rni n a t i o n o f sodi urn n i t r o p r u s s i d e (128). The v a r i a t i o n o f e q u i v a l e n t c o n d u c t i v i t i e s o f aqueous s o l u t i o n s o f sodium n i t r o p r u s s i d e has been s t u d i e d as a f u n c t i o n o f th e i o n i c c o n c e n t r a t i o n (129).

508

8.

RICHARD RUCK1

Acknow I edgiilen t-5 The a u t h o r w i s h e s t o a c k n o w l e d g e t h e a s s i s t a n c e o f M i s s E. R o l l e r i , t h e S c i e n t i f i c L i t e r a t u r e D e p a r t m e n t , and t h e Research Records O f f i c e o f Hoffrnann-La Roche I n c . i n the preparation of t h i s analytical p r o f i l e .

SOD I UM N ITROPR USSl DE

9.

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e,

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lo,

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E,

28,

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14,

3,

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54.

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16,

E,

69. 70.

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73. 74. 75. 76. 77. 78. 79. 80.

81.

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lo.

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2,

and West, D.X., J. I n o r g . Nucl. Chem. 3837 (1970). 434 (1915). Cambi, L., A t t i . Accad. Nazl. L i n c e i

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CA E : 1 3 5 1 , 1916. Rybkina, A.A., Ref. Zh. Khim. A b s t r . No. 16V155. CA E : 6 8 7 9 t , 1971. Stiono, T., N i i , H. and Shinra, K., Kogyo Kagaku Zzsshi 72, 1669 (1965). CA 72:21437z, 1970.

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Burce, G. and B o e h l e r c J . , Hoffmann-La Roche Inc., I n t e r n a l Report, November 29, 1976. Hamilton, C.M. and S t e w a r t , D., Hoffmann-La Roche Inc., I n t e r n a l Report, June 10, 1974. Johnson, C.C., Arch. I n t e r n a t . Pharm. 480 (1929). Page, I . H . , J. Amer. Med. Assn. j>z, 1311 (1951). Page, I.H., Corcoran, A.C., Dustan, H.P. and Koppanyi, T., C i r c u l a t i o n 11, 188 (1955). S r h l a n t , R.C., Tsagaris. T x’ and Robertson, R.J., Amer. J. C a r d i o l . 2, 51 ( I ). G’iatia, S.K. and F r o h l i c h , . U . , Amer. Heart J. 367 ( I J73). 576 (1974). Veriier, I . R . , Postgrad. Med. J. Glusa, E.. Markwardt, F . and Stuerzebecher, J., Haemos tas i s 2, 249 ( 19714) . Jonc;, G.0.N. and Colc, P . , B r i t . J . Anaesthesia 8011 (1968).

S,

5,

0,

2,

512

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hloraca, P. e t a l . , Anesthesiology 23, 193 (1962). Schiffmann, H. and Fuchs, P., A c t a G a e s t h . Scand. (Suppl.) 3, 704 (1966). Katz, R.L. and Wolf, C . E . , H i g h l i g h t s o f C l i n i c a l Anesthesiology, Ed. by Nark, L.C. and Ngai, S.H., Harper and Row, New York, N.Y., 1971, p. 48. 89 (1942). H i l l , H.E., Aust. Chem. I n s t . J . Proc. M i c h a e l i s , L., J. B i o l . Chem. 777 (1929). Smith, R.P. and Kruszyna, H., J. Pharmacol. Exp. Ther.

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99.

100. 101 *

102. 103. 104.

.

.

g,

5,

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513

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673 (1954). CA 3 : 6 7 4 9 e , 1955. 119. Zuman, P. and Kabat, M., Chem. L i s t y

5,368

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143,

L i t e r a t u r e surveyed through October,

1976.

SULPHAMERAZINE

Richard D.C. Woolfenden

516

RICHARD D. G . WOOLFENDEN

CONTENTS 1.

2.

3. 4.

5.

6.

Description 1.1. N a m e , F o r m u l a , M o l e c u l a r W e i g h t 1.2. A p p e a r a n c e , Colour, Odour, T a s t e Physical Properties 2.1. I n f r a - r e d Spectrum 2.2. Ultraviolet Spectrum 2.3. Fluorescence and Phosphorescence Spectra 2.4. Mass S p e c t r u m N.M.R. Spectrum 2.5. 2.6. M e l t i n g Range 2.7. D i f f e r e n t i a l Thermal A n a l y s i s 2.8. Thermal G r a v i m e t r i c A n a l y s i s 2.9. X-ray D i f f r a c t i o n 2 . 1 0 . Polymorphism 2.11. S o l u b i l i t y 2 . 1 1 . 1 . I n Aqueous B u f f e r s a n d Urine 2.11.2. In Solvents 2.12. Dissociation Constant 2.13. P a r t i t i o n C o e f f i c i e n t s S y n t h e s i s and P u r i f i c a t i o n 3.1. Chemical S y n t h e s i s 3.2. Purification Salts 4.1. Organic S a l t s 4.2. Metal Complex S a l t s Chemical S t a b i l i t y Hydrolysis 5.1. Pyrolysis 5.2. Photolysis 5.3. Methods o f A n a l y s i s I d e n t i f ic a ti o n 6.1. Elemental Analysis 6.2. T i t r i m e t r i c Assay P r o c e d u r e s 6.3. 6.3.1. Diazometric T i t r i m e t r y 6 . 3 . 2 . Non-Aqueous T i t r i m e t r y 6.3.3. B r o m o m e t r i c T i t r i m e t r y 6.3.4. Argentometric T i t r i m e t r y 6.3.5. Complexometric T i t r i m e t r y 6.3.6. Thermometric T i t r i m e t r y 6.4. S p e c t r o p h o t o m e t r i c Assay Procedures 6.4.1. Infra-red Spectroscopic Methods

SULPHAM E R A2 IN E

Ultraviolet Spectroscopic M e t h o ds 6 . 4 . 3 . C o l o r i m e t r i c Methods 6.5. Chromatographic Procedures 6 . 5 . 1 . High P e r f o r m a n c e L i q u i d Chromatography 6 . 5 . 2 . Gas Chromatography 6.5.3. T h i n L a y e r Chromatography 6 . 5 . 4 . P a p e r Chromatography 6 . 5 . 5 . I o n Exchange a n d P a r t i t i o n Chromatography 6.5.6 Electrophoresis 6.6. E l e c t r o c h e m i c a l Methods 6.6.1. Polarography 6 . 6 . 2 . Ion S e l e c t i v e E l e c t r o d e s 6.7. Bioassay Estimation i n Biological Fluids Pharmacology 8.1. Metabolism 8.2. Absorption,Distribution,Excretion 8 . 2 . 1 . I n Humans 8 . 2 . 2 . I n Animals 8.3. Toxicity 8 . 3 . 1 . Acute T o x i c i t y 8.3.2. Chronic Toxicity 8.3.3. C l i n i c a l Toxicity P r o t e i n Binding Pharmacodynamics Acknowledgements R e f e r e n ce s 6.4.2.

7. 8.

9. 10. 11.

51 7

518

1.

RICHARD D. G.WOOLFENDEN

Desc-ription 1.1. N a m e , F o r m u l a , M o l e c u l a r Weiqht

1 G e n e r i c names - S u l p h a m e r a z i n e ; Methylpyrimal; Sulphamethyldiazine. N o m e n c l a t u r e - The f o l l o w i n g nomenclat r e i s u s e d i n Chemical Abstracts: N - (4-methyl-2-pyrimidinyl) s u l p h a n i l amide ; 4-amino-N- ( 4 - m e t h y l - 2 - p y r i m i d i n y 1) b e n zene s ul ph on a m i de

P

.

Structure Chemical Abstracts R e g i s t r y N o . (127-79-7)

C11H12N402S 1.2.

M o l . w t 264.30.

A p p e a r a n c e , C o l o u r , Odour, Taste

2

White o r f a i n t l y y e l l o w i s h w h i t e a l l i n e powder which i s o d o u r l e s s has a s l i g h t l y b i t t e r t a s t e . It s t a b l e i n a i r b u t slowly darkens exposure t o l i g h t . 2.

crystbut

is

on

Phys i ca 1 Proper t i e s 2.1.

I n f r a - r e d Spectrum The i n f r a - r e d s p e c t r u m o f s u l p h a m e r a z i g e ( S q u i b b sample P083425) w a s recorde d i n K B r a n d i s shown i n F i g u r e 1. A s s i g n m e n t s f o r t h e more i m p o r t a n t a b s o r p t i g n 5 b a n d s are l i s t e d i n T a b l e 1. '

--

15' 4000

j

I

3500

3000

Fig. 1

-

I

1

2500

2000

,

,

I ,lj,I I 1800

! - I ?

1600

*

I

I

,

1400 FREQUENCY (CM')

,

v 1200

1000

-

e

800

Infra spectrum of sulphamerazine (KBr pellet)

600

400

200

520

RICHARD D. G.WOOLFENDEN

TABLE 1

I n f r a r e d assignments f o r Sulphamerazine Frequency ( c m - l )

NH asymmetric s t r e t c h i n g . NH symmetric s t r e t c h i n g . CH a s y m m e t r i c s t r e t c h i n g . 3 CH3 symmetric s t r e t c h i n g . NH2 s c i s s o r i n g . C = C stretching, skeletal

3490 3380 2960 2870 1 6 30 1600) 15 7 0 ) 1500) 1325

v i b r a t i o n s o f aromatic ring. SO asymmetric s t r e t c h o v s r l a p p i n g C-N s t r e t c h ing vibration. SO symmetric s t r e t c h i n g A r 8 m a t i c CH i n p l a n e benuing S-N s t r e t c h i n g . C-H o u t of p l a n e deformation.

.

1160 109 2

.

890 8 35

2.2.

A s s i gnmen t

U l t r a v i o l e t Spectrum The u l t r a v i o l e t s p e c t r u m of s u l p h a m e r a -

zine i n 0 1 M hydrochloric acid solution

3

e x h i b i t e d a b s o r p t i o n maxima a t 243 nm and I n 0 . 1 M sodium a t 307 nm ( F i g u r e 2 ) . hydroxide s o l u t i o n sulphamerazine behaves a s t h e sodium s a l t e x h i b i t i n g one main peak w i t h t w o maxima a p p e a r i n g a t 243 nm and 257 nm as shown i n F i g u r e 3. The hypsochromic s h i f t of t h e 307 nm maximum t o 257 nm i n a l k a l i n e s o l u t i o n i s due t o i o n i z a t i o n of t h e s u l p h o n a m i d e f r a c t i o n of the molecule. The u l t r a v i o l e t s p e c t r u m o s u l p h a m e r a z i n e h a s been r e c o r d e d i n water (mexima a t 243 and 257 nm) and125% e t h a n 01 (maximum a t 2 7 1 nm) The E values e v a l u a t e d f o r t h e aforemention&8msystems a r e given i n Table 2 .

6

.

522

I

.

SULPHAMERAZI NE

523

TABLE 2

values € o r sulphamerazine i n E l c m var ious s o l v e n t systems Solvent

1%

Band (nm)

0 . 1 M H C 1 aqueous

0.1M NaOH aqueous Water 95% Ethanol

243 3 07 243 257 2 43 257 271

Elc m

R e f e r e n ce

5 79 625 2 00 896 883 875 822 835

7 3 3 3 3 6

6 6

2 . 3 . F l u o r e s c e n c e and P h o s p h o r e s c e n c e

’-

N Subs t i t U t e d s u 1phon a m i de s c o n t a i n i n g a n-electron d e f i c i e n t heterocyclic r i n g s y s t e m are g e n e r a l l y weakly o r non-f l u o r e s c e n t . S u l p h a m e r a z i n e i s s u c h a s u l p h o n a m i d e and i t s l a c k of f l u o r e s c e n c e h s been d e m o n s t r a t e d by The p r e s e n c e o f t h e G i f f o r d and co-workers 8 h e t e r o c y c l i c r i n g a t t h e N - p o s i t i o n produced a marked q u e n c h i n g of f l u o r e s c e n c e o v e r t h e pH r a n g e s t u d i e d . T P i s o b s e r v a t i o n w a s a g e n e r a l f e a t u r e of N - s u b s t i t u t e d h e t e r o c y c l i c s u l p h a n i l a m i d e s and i t was c o n s i d e r e d t h a t t h e s e compounds p r e f e r e n t i a l l y a b s o r b e d l i g h t v i a an n+n* t r a n s i t i o g which i s known t o d e t r a c t from f l u o r e s c e n c e

.

.

S u l p h a m e r a z i n e h a s been shown t o e x h i b i t a phosphorescence spectrum o r i g i n a t i n g from a t r a n s i t i o n i n t h e lowest e x c i t e d t r i p l e t G i f f o r d and l e v e l i n t e aromatic nucleus. co-workers’ p r o d u c e d t h e p h o s p h o r e s c e n c e p d e m i s s i o n s p e c t r u m of s u l p h a m e r a z i n e a t 7 7 K u s i n g a Qaird-Atomic SF 1 0 0 - E s p e c t r o f l u o r i meter f i t t e d w i t h a p h o s p h o r o s c o p e a t t a c h m e n t , t h e e x c i t a t i o n s p e c t r u m showing a maximum a t 310nm (A,) and t h e emission s p e c t r u m a maximum a t 412nm(Ap). The d e l a y e d l u m i n e s c e n c e l i f e t i m e ( T ) was 0 . 8 s e c o n d s .

524

2.4.

RICHARD D. G.WOOLFENDEN

Mass S p e c t r u m

The mass s p e c t r u m of s u l p h a m e r a z i n e shown i n F i g u r e 4 was o b t a i n e d on an AEI-MS 9 0 2 mass s p e c t r o m e t e r by d i r e c t Temple i n t r o d u c The fragment i o n i n t o t h e s o u r c e a t 90°C t a t i o n p a t t e r n s which can b e a s s i g n e d t o more i m p o r t a n t i o n s a r e shown i n scheme I 11

.

s b ~ ~ ,

Cambon and co-workers’’ have s t u d i e d t h e mass s p e c t r a of s e v e r a l s u l p h a p y r i m i d i n e s a n d showed t h a t p r e f e r e n t i a l f r a g m e n t a t i o n occur r e d t o e l i m i n a t e SO The f r a g m e n t a t i o n patterns w e r e attrib6ted to localization of t h e c h a r g e s on t h e h e t e r o a t o m s . The w o r k e r s c o n s i d e r e d t h e p e a k s a t m / e = 2 0 0 and m / e = 1 9 9 a s extremely important corresponding t o t h e removal o f S O and S02H t o g i v e t h e f o l l 2 owing i o n s :

.

m/e = 200

m/e

= 199

2 . 5 . N.M.R.Spectrum P u a r and Funkel’ h a v e r e c o r d e d t h e 6 0 MHz spectrum of sulphamerazine i n dimethyl s u l p h o x i d e - d c o n t a i n i n g T.M.S. a s i n t e r n a l s t a n d a r d ( F i g u r 8 5 ) . The s t r u c t u r a l d a t a i s presented i n Table 3.

N . M. R.

The n a t u r a l abundance 1 3 C m a g n e t i c r e s o n a n c e s p e c t r u m o f s u l p h a m e r a z i n e h a s b e e n comp a r e d w i t h a s e f j e s of o t h e r s u l p h o n a m i d e s by The s p e c t r a w e r e d e t e r Chang and F l o s s mined a t 25.15 MHz u s i n g t h e p u l s e F o u r i e r t r a n s f o r m t e c h n i q u e . Chemical s h i f t s w e r e a s s i g n e d w i t h t h e a i d of o f f - r e s o n a n c e and s e l e c t i v e p r o t o n d e c o y g l i n g t e c h n i q u e s as w e l l as by l o n g - r a n g e C p r o t o n c o u p l i n g patterns.

.

525

526

RICHARD D. G. WOOLFENDEN

528

TABLE 3 NMR S p e c t r a l A s s i g n m e n t s o f S u l p h a m e r a z i n e

Proton A s s i gnmen t

f

2H p-substituted 2 H benzene r i n g

Chemical s h i f t , 6 (ppm)

J

10,ll

(Hz)

6.57d 7.70d

9 .o 9.0

6.86d 8.30d

5 .O 5.0

protons

2.29s 5.95b,s 11.12b,s

3H CH3 2H NH2 1 H NH

s = singlet; 2.6.

d = doublet;

b = broad.

M e l t i n g Range The m e l t i n g r a n g e q u o t e d i n t h e U.S.P. A m e l t i n g p o i n t o f 234OC X 1 X i s 234-239OC. was o b t a i n e d f o r a U.S.P. g r a d e s a m p l e o f s u l p h a m e r a z i n e u s i n g D . T . A . 3.

2.7.

D i f f e r e n t i a l Thermal A n a l y s i s Using a S t a n t o n R e d c r o f t Thermal Analy-

ser Model 671 a t a h e a t i n g r a t e o f 2OoC m i n - l , i t w a s found t h a t U.S.P. g r a d e s u l p h a m e r a z i n e gave a s h a r p m e l t i n g e n d o t h e r m a t 234OC 3 ( F i g u r e 6 ) . This w a s r a p i d l y followe d by d e c o m p o s i t i o n . The h e a t o f f u f i o n ( A H f ) e v a l u a t e d by Yang and G u i l l o r y was 8 . 6 8 k . c a l . m o l - l , a t a f u s i o n f s m p e r a t u r e of quote a 236OC w h e r e a s Sunwoo ancllEisen v a l u e o f 7.54 & . c a l . m o l , a t a f u s i o n tempe r a t u r e of 2 4 2 C. Yang and G u i l l o r y a l s o q u o t e d a n e n t r o p y of f u s i o n o f 1 7 . 1 e . u . f o r sulphamerazine. 2.8.

Thermal G r a v i m e t r i c A n a l y s i s The t h e r m o g r a v i m e t r y of s u l p h a m e r a h a s been s t u d i e d by Cook and H i l d e b r a n d S u l p h a m e r a z i n e e x h i b i t e d n o w e i g h t loss up t o a t e m p e r a t u r e of 26OoC, b u t between 26OoC a n d 396OC a r a p i d w e i g h t loss o c c u r r e d f o l l o w e d

Hine .

529

530

RICHARD D. G . WOOLFENDEN

b y a l e s s r a p i d l o s s b e t w e e n 526OC a n d 690OC. The TGA c u r v e , t h e r e f o r e , e x h i b i t e d p l a t e a u s a t temperature ranges 396-526OC. N o r e s i d u e remained a t t h e e n d of t h e h e a t ing period. A l t h o u g h n o a t t e m p t w a s made t o i d e n t i f y t h e g a s e o u s p y r o l y s i s products Cook and H i l d e b r a n d h y p o t h e s i s e d t h a t s u l p h u r d i o x i d e would p r o b a b l y s p l i t o u t f r o m t h e sulphamerazine molecule i n a similar manner t o s u l p h o n e s a n d a l k y l s u l p h o n y l chlorides. 2.9.

X-ray

Diffraction

O c h s 1 7 h a s recorded t h e X-ray p o w d e r d i f f r a c t i o n p a t t e r n f o r a sample of s u l p h a m e r a z i n e (see F i g u r e 7 and T a b l e 4 ) . Yang a n d G u i l l o r y 1 4 a n d L e n n o x l 8 h a v e a l s o rep o r t e d X-ray powder d i f f r a c t i o n d a t a f o r sulphamerazine. TABLE 4 X-Ray Powder D i f f r a c t i o n Data of S u l p h a m e r a z in e ( P 0 8 3 4 2 5 ) Interplanar D i s t a n c e s R e l a t ive I n t e n s i t i e s

,.

Q*

1/10

10.72 0.117 7.65 0.130 7.03 0.949 0.315 6 . 76 6 . 35 0.199 0.885 6.02 5.46 0.636 5.14 0. 760 4.72 0.207 0.971 4.37 4.11 0.545 0.432 3.95 0.322 3.89 0.257 3.30 0.307 3.74 1.000 3.67 0.324 3.53 0.207 3.27 3.22 0.286 0.142 3.05 2.94 0.133 2.90 0.397 2.76 0.456 0.278 2,38 *Interplanar distance d = n X 2 sin 0

u

8

8

8

a

8

8

6

?

RICHARD D. G.WOOLFENDEN

532

2.10.

Polymorphism During e x t e n s i v e s t u d i e s on polymorphism i n s u l p h o n a m i d e s u s i n g X-ray d i f f r a c t i o n , i n f r a r e d and D. T . A . t e c h n i q u e s Yang a n d G u i l l o r y l 4 found t h a t sulphamerazine w a s among t h o s e s u l p h o n a m i d e s i n which polymorphism c o u l d n o t be d e t e c t e d .

2.11.

Solubility

2.11.1.

I n Aqueous B u f f e r s a n d U r i n e The s o l u b i 1i t y of sulphame r a z i n e i n a q u e o u s media i s i m p o r t a n t i n c l i n i c a l p r a c t i s e and t h e r e f o r e , it h a s m a i n l y been d e t e r m i n e d i n a q u e o u s b u f f e r s a n d u r i n e i n t h e a p p r o x i m a t e pH r a n g e of 6-8 a t 37OC. T y p i c a l v a l u e s are g i v e i n T a b l e 5 a l o n g w i t h t h o s e o f t h e N'-acetyl derivative. TABLE 5

The S l u b i l i t y o f S u l p h a m e r a z i n e and -I-acetyl d e r i v a t i v e i n aqueous p h o s p h a t e b u f f e r and u r i n e a t 3 7 O F M e d i um

M/30 P h o s p h a t e buffer,pH 6 . 1 Urine,pH 5.9 Urine,pH 6 . 9 Urine,pH 7.9 2.11.2.

R e f e r e n ce S o l u b i 1i t y mg./ml., Sulphag-Acetylm e r a z i n e s u l phame r a zi n e 19 40 53

37 66 310

76 175 650

19,20 1 9 ,2 0 19,20

In Solvents The a p p r o x i m a t e s o l u b i l i t i e s o f s u l p h a m e r a z i n e i n some s o l v e n t s are given i n Table 6 .

SU LPHAME RAZ I NE

533

TABLE 6

Sulphamerazine s o l u b i l i t i e s i n some s o l v e n t s

S o l u b i 1i t y

Solvent

Reference

mg. / m l . w a t e r ,2 0 : ~ Water,37 Water,100 C 1 . 5 N Aqgeous NaOH,22 C E t h a n o l ,2 2 O C I s o p r o p a n o l ,2 2 C

g

16 30 3 30 290

6 6 6 21

3 30 174

6 22

2.12.Dissociation Constant The d i s s o c i a t i o n of t h e p r i m a r y a r o m a t i c amine f u n c t i o n o f some s u l p h o n a m i d e s has2kjeen s t u d i e d by S a l v e s e n and S c h r o d e r - N i e l s o n u s i n g s p e c t r opho tome t r i c and p o t e n t iome t r i c methods. I n 065M aqueous sodium c h l o r i d e s o l u t i o n a t 2 4 C t h e pKal v a l u e r e p r e s e n t i n g t h e p r i m a r y amine d i s s o c i a t i o n of s u l p h a m e r iven a s 2.29. K o i z u m i a n d coazine w workers" Zuoted a pKal v a l u e of 2 . 2 6 . Krebs and SpeakmanZ5 d e t e r m i n e d t h e pK of a number o f s u l p h o n a m i d e s f r o m s o l u b i l i t y data using the following relationship.

s

=

s0

(1

+

10PH-PKa)

where S i s t h e s o l u b i A i t y of t h e compound a t a p a r t i c u l a r pH and S i s t h e s o l u b i l i t y of t h e u n i o n i s e d compound. ghese workers o b t a i n = 4 1 mg./lOOml.) e d a pK v a l u e of 6 . 9 5 ( S f o r t h e a& s s o c i a t i o n o f t h e s ulphonamide group of sulphamerazine i n a s o l u t i o n o f ioni c s t r e n g t h 0 . 1 a t 38OC. Using&he same p r i n c i p l e S j o g r e n and O r t e n b l a d obtained a pKa v a l u e of 7 . 0 5 . Both t h e s e reports assume8 t h a t t h e s u l p h o n a m i d e s b e h a v e d as monob a s i c a c i d s . The a u t h e n t i c i t y of t h e s e pKa2 v a l u e 2 6 h a s been c o n f i r m e d by W i l l i and Meier , who u s i n g a p o t e n t i o m g t r i c method, o b t a i n e d a v a l u e o f 6.84 a t 2 0 C a t an i o n i c s t r e n g t h o f 0.1.

534

RICHARD D. G.WOOLFENDEN

2.13 P a r t i t i o n C o e f f i c i e n t s D u r i n g t h e i r s t u d i e s on some pharmacokinet i c a s p e c t s of e r t a i n s u l p h o n a m i d e s Koizumi and co-workers2' g e n e r a t e d p a r t i t i o n c o e f f i c i e n t d a t a a t 37OC between an a q u e o u s p h a s e c o n t a i n i n g u n i o n i s e d d r u g and t h e s o l v e n t s c a r b o n t e t r a c h l o r i d e I b e n z e n e , c h l o r o f o r m and Suzuki and c o - ~ o r k e r s ~ ~ isoamyl acetate. also gener ated s i m i l a r d a t a u s i n g isoamyl The r e s u l t s f o r a l c o h o l a s $he o r g a n i c p h a s e . s u l p h a m e r a z i n e are g i v e n i n T a b l e 7 . TABLE 7 P a r t i t i o n C o e f f i c i e n t s f o r sulphamerazine o 24,27 a t 37 C Organic Phase Partition Coefficient CC14 0.022 0.202 'gH6 CHCl 2.4 Isoamyl acetate 2.1 Isoamyl a l c o h o l 2.1

3. S y n t h e s i s and P u r i f i c a t i o n 3.1.Chemical

Synthesis

Two p r i m a r y s y n t h e t i c r o u t e s have b e e n used t o p r e p a r e sulphamerazine , t h e s e b e i n g v i a t h e r e a c t i o n b e t w e e n 2-amino-4-methylpyrimidine w i t h c e r t a i n d e r i v a t i v e s o f benze n e s u l p h o n y l c h l o r i d e and a l s o by a c o n d e n s a t i o n p r o c e s s b e t w e e n s u l p h a g u a n i d i n e and c e r t a i n r i n g f o r m i n g compounds. R o b l i n and c o - w o r k e r s 2 8 f i r s t s y n t h e s i z e d s u l p h a m e r a z i n e by t h e a c t i o n of p - a c e t a m i d o b e n z e n e - s u l p h o n y l c h l o r i d e on 2-amino4 - m e t h y l p y r i m i d i n e i n a weakly b a s i s o l v e n t s u c h a s p y r i d i n e t o g i v e t h e N'-acetyl d e r i v a t i v e o f s u l p h a m e r a z i n e H y d r o l y t i c dea c e t y l a t i o n of t h i s i n t e r m e d i a t e was a c h i e v e d under e i t h e r a c i d i c o r b a s i c c o n d i t i o n s . Using a c e t a n i l i d e as t h e s t a r t i n g m a t e r i a l the various steps involved i n t h e s y n t h e s i s The p - n i t r o d e r i v a a r e shown i n Scheme 2 . t i v e of b e n z e n e s u l p h o n y l c h l o r i d e c o u l d a l s o

.

p?

I

t

'

o u

u

G

d

ktd 0

5

o u

QG

c o

N k

a, 4

c 5

ilal

I

*n I

Y

z 0, 0

v)

I Z

4I

+

I 0

n I

"n i 2,

0

0

I

I

(Y

z

I

@ +T

535

a,

c

N

-4

a k

5

al

c a LII

7

rl

N

a

al

Ll

5

a

G

m

7

rl

h

rl

a,

I I

7z w

N

X

kw8 u

v)

n

+ I " X

\ &

z z I

0

I

z

rl

u

.c

=,

0

&

0, N

I

Q

0

z

536

I

m

X

!2w u

v)

SULPHAMERAZIN E

537

b e u s e d l t h e f i n a l s t a g e o f t h e s y n t h e s i s req u i r i n g a c a t a l y t i c r eductio n of t h e n i t r o group t o g i v e t h e f i n a l p r o d u c t . I n t h e s e c o n d major method a number o f r i n g f o r m i n g compounds were c o n d e n s e d w i t h sulphaguanidine t o produce sulphamerazine. T y p i c a l l y s u l p h a g u a n i d i n e h a s been c o n d e n s e d with ch orovinylmethyl ketone i n a l k a l i n e medium2$ as i l l u s t r a t e d i n Scheme 3. I n t h i s case t h e c o n d e n s a t i o n mechanism i n v o l v e d t h e removal o f a m o l e c u l e o f w a t e r and a m o l e c u l e of h y d r o c h l o r i c a c i d t o g i v e t h e f i n a l p r o d u c t . O t h e r r i n g f o r m i n g compounds which h a v e been u s e d i n c l u d e a c e t o a c e t a l d e h y d e acet a l s 3 0 , a c e t a l d e h y d e m t h y l a c e t a l s 3 l 1 and d i a l k y l a m i n o b u t e n y n e s35

.

3.2.

Purification Crude s u l p h a m e r a z i n e i s u s u a l l y u r i f i e d v i a i t s sodium s a l t . I n one method31; t h e pH of t h e medium w a s a d j u s t e d t o 1 0 . 5 by t h e add i t i o n of c a l c i u m h y d r o x i d e . The s o l u t i o n was b o i l e d and sodium d i t h i o n i t e a d d e d . D e c o l o r i z a t i o n was t h e n a c h i e v e d u s i n g a c t i v a t e d c h a r c o a l . On c o o l i n g t o room t e m p e r a t u r e t h e s o l u t i o n was a c i d i f i e d w i t h a c e t i c a c i d and t h e p r e c i p i t a t e d s u l p h a m e r a z i n e i s o l a t e d by f i l t r a t i o n . I f required t h i s product c o u l d be r e c r y s t a l l i s e d from a q u e o u s a l c o h o l o r b e n z e n e . A number o f v theme have been d e s c r i b e d

4. S a l t s 4 . 1 . Organic S a l t s

B a r r y and P ~ e t z e rp r~e p~a r e d t h e c e t y l -

m e t h y l ammonium s u l p h a m e r a z i n e d i h y d r a t e s a 1t which w a s found t o 3 9 a v e a m e l t i n g p o i n t o f 126OC. Schonhafer prepared the diethylamin o e t h a n o l s a l t of s u l p h a m e r a z i n e which w a s found t o g i v e a 30% aqueous s o l u t i o n of pH9.2 -9.5. Winnek38 p r e p a r e d a q u e o u s s o l u t i o n s of s t r e p t o m y c i n s u l p h a t e and b a r i u m o r c a l c i u m s a l t s of c e r t a i n sulphonamides i n v a r y i n g p r o p o r t i o n s t o g i v e s a l t s of s t r e p t o m y c i n c o n t a i n i n g 1 , 2 or 3 moles of s u l p h o n a m i d e .

538

RICHARD D. G.WOOLFENDEN

The s t r e p t o m y c i n d i s u l p h a m e r a z i n e s a l t was found t o h a v e a water s o l u b i l i t y o f a b o u t 10%.

4.2.

Metal Complex S a l t s V a r i o u s complex s a l t s o f s u l p h o n a m i d e s w i t h m u l t i v a l e n t metals h a v e b e e n p r e p a r e d . Complex s a l t s o f c o b a l t , s u l p h a m e r a z i n 3 g a n d e t h y l e n e d i a m i n e were p r e p a t j e d b y E r d o s at a t e m p e r a t u r e o f less t h a n 5 C f o r 2 4 h o u r s f o l l o y g d by p r e c i p i t a t i o n w i t h 100 m l . e t h a n o l . Shakh prepared the c o b a l t , n i c k e l , copper and z i n c complexes o f s u l p h a m e r a z i n e a n d f o u nd them t o b e i n s o l u b l e i n w a t e r , a l c o h o l , e t h e r , c h l o r o f o r m , a c e t o n e and b e n z e n e . T h e s e complexes w e r e f o u n d t o b e s o l u b l e i n a c i d s o l u t i o n b u t were decomposed by 10%sodium The molar r a t i o o f h y d r o x i d e o r ammonia. s u l p h a m e r a z i n e t o metal w a s 2:l. Lee has s t u d i e d i n d e p t h t h e f o r m a t i o n o f c o p p e r comp l e x e s o f tile s u l p h o n a m i d e s , d e a l i n g 1 1 t h t h e i r p r e p a r a t i o n from c o p p e r grjetate , t h e i r , the4geters e n s i t i v i t y t o micro-organisms m i n a t i o n of t h e i r s t a b i l i t y 4 $ o n s t a n t s , a n d t h e i r s t r u c t u r e assignments The c o p p e r complex o f s u l p h a m e r a z i n e w a s p r e p a r e d by t r e a t i n g an a l c o h o l i c s o l u t i o n o f t h e s u l p h o n amide w i t h an aqueous s o l u t i o n o f c u p r i c acet a t e a t pH 7-9. The complex was i s o l a t e d a s g r e y n e e d l e s , w a s less s e n s i t i v e t o m i c r o organisms t h a n sulphamerazine and had a s t a 6 b i l i t y c o n s t a n t o f 9 . 6 8 a t 2 5 C. TQe s t r u c t u r e o f t h e qomplex w a s d e t e r m i n e d by i n f r a r e d s p e c t r o s c o p y which e x h i b i t e d a s h i f t i n t h e S = 0 a b s o r p t i o n band f r o m 7 . 6 2 ~ f o r s u l p h a m e r a z i n e t o 7 . 7 9 ~i n t h e c o p p e r complex. From t h e i n f r a r e d d a t a i t w a s d e d u c e d t h a t t h e c o p p e r c h e l a t e d between t h e S-0 group of t h e s u l p h o n a m i d e s a n d a h e t e r o c y c l i c n i t r o g e n atom a s f o l l o w s

.

539

SULPHAMERAZINE

5 . Chemical S t a b i l i t y 5.1.Hydrolysis The k i n e t i c s of t h e a c i d c a t a l y s e d hydrol y s i s of some s u l p h a n i a m i d o p y r i m i d i n e s h a s been s t u d i e d by Z a j a c 48 The h y d r o l y s i s r a t e was f o u n d t o f o l l o w f i r s t o r d e r k i n e t i c s i n e a c h c a s e , t h e r a t e b e i n g d e p e n d e n t on t h e hydrogen i o n c o n c e n t r a t i o n . The r e s u l t s of t h e s t u d y a l s o showed t h a t t h e s u b s t i t u t i o n o f m e t h y l o r methoxy g r o u p s w i t h i n t h e p y r i midine n u c l e u s i n c r e a s e d t h e h y d r o l y s i s rate. Thus t h e h a l f l i f e o f ghe s u l p h a m e r a z i n e hyd r o l y t i c p r o c e s s a t 6 0 C (333OK) w a s f o u n d t o be 6 7 . 9 h o u r s compared t o 9 4 . 7 h o u r s f o r s u l phadiazine the p a r e n t sulphanilamidopyrimidine.

.

A u t e r h o f f and S c h m i d t 4 6 a l s o s t u d i e d t h e h y d r o l y s i s of c e r t a i n sulphanilamidopyrimidines. Using TLC combined w i t h e l e m e n t a l , a n a l y t i c a l and s p e c t r o s c o p i c t e c h n i q u e s t h e s e investigations identified sulphanilic acid, s u l p h a n i l a m i d e , 2-amino-4-methylpyrimidine and 2-hydroxy-4-methylpyrimidine as t h e dec o m p o s i t i o n p r o d u c t s of s u l p h a m e r a z i n e

.

5.2.Pyrolysis The p y r o l y t i c de c o m p o s i t i o n o f s u l p h a n i l a m i d o p y r i m i d i n e s w a s a l s o s t u d i e d by A u t e r h o f f and S c h m i d t 4 6 . The compounds w e r e p l a c e d i n t o t e s t t u b e s and h e a t e d i n an o i l b a t h t o between 230 and 28OoC. Y e l l o w i s h w h i t e sublimates appeared i n t h e upper p a r t of t h e t e s t t u b e s which were s u b s e q u e n t l y examined by TLC on Merck K i e s e l g e l F254 u s i n g n - b u t a n o l , a c e t i c a c i d , w a t e r ( 8 0 , 2 0 , 2 0 ) as s o l v e n t s y s tem. S u l p h a m e r a z i n e ( R f 0 . 5 9 ) w a s f o u n d to decompose t o 2-amino-4-methylpyrimidine (Rf 0.48) i n 92% y i e l d . 5.3. P h o t o l y s i s N a i t o and M i ~ o g u c h is ~ t u~d i e d t h e p h o t o l y t i c decomposition of c e r t a i n s u l p h a drugs and t h e i r b e n z o y l d e r i v a t i v e s i n a q u e o u s a l k a l i n e s o l u t i o n u s i n g a s t e r i l i z a t i o n lamp. A n u l t r a v i o l e t s p e c t r o p h o t o m e t r i c a s s a y method

RICHARD D. G.WOOLFENDEN

540

showed t h a t a b o u t 5 0 % o f s u l p h a m e r a z i n e w a s decomposed o v e r a p e r i o d o f 8 h o u r s w h e r e a s t h e benzoyl d e r i v a t i v e w a s completely stable. The same s a m p l e s s t o r e d i n t h e dark e x h i b i t e d no decomposition. 6 . Methods of A n a l y s i s 6.1. Identification Two i d e n t i t y t e s t s a r e g i v e n i n t h e U.S.P.XlX, one b e i n g an i n f r a r e d a b s o r p t i o n t e s t and t h e o t h e r a m i c r o c h e m i c a l test. I n t h e l a t t e r method a s a m p l e of sulphamerazine i s suspended i n w a t e r and t h e s u s p e n s i o n made a l k a l i n e w i t h sodium h y d r o x i d e . On t h e a d d i t i o n of c u p r i c s u l p h a t e s o l u t i o n an o l i v e g r e e n p r e c i p i t a t e i s formed which t u r n s d a r k g r e y on s t a n d ing. T h i s t e s t h a s been s u c c e s s f u l l y used48t49to d e t e c t sulphamerazine i n t h e p r e s e n c e of t h e r s u l p h o n a m i d e s . T u r c z a n and Medwickl’ h a v e i n c l u d e d s u l p h a m e r a z i n e i n a c l a s s i f i c a t i o n scheme f o r t h e i d e n t i f i c a t i o n o f s u l p h o n a m i d e s by N.M.R.spectroscopy

.

6.2.

Elemental Analysis The e l e m e n t a l c o m p o s i t i o n o f s u l p h a m e r a z i n e ( S q u i b b b a t c h PO 83425) w a s obt a i n e d by Young50 w i t h t h e f o l l o w i n g res u l t s :Element Carbon Hydrogen Nitrogen

% Theory

Sulphur

12.13 12.11

oxygen 6.3.

% Found

49.98 4.58

50.08 4.55

21.20

21.31 12.12

-

T i t r i m e t r i c Assay P r o c e d u r e s

6.3.1.Diazometric

Titrimetry

S u l p h a m e r a z i n e may b e t i t r a t e d i n strongly acid solution with a standard s o l u t i o n o f sodium n i t r i t e , t h e e n d - p o i n t b e i n g d e t e c t e d w i t h an e x t e r n a l o r i n t e r n a l i n d i c a t o r , o r by an e l e c t r o m e t r i c procedure. The d i a z o m e t r i c t e c h n i q u e i s

SU LPHAMERA2 IN E

t h e o f f i c i a l method of t h e U.S.P. sulphamerazine .

541

X1X f o r

E l - S e b a i a n d c o - ~ o r k e r sh~a v~e e v a l u a t e d t h e sodium s a l t of 4- ( b e n z y 1 a m i n o ) a z o benzene-4 ' - s u l p h o n a t e a s an i n t e r n a l i n d i c a t o r f o r sulphonamide t i t r a t i o n s a n d c l a i m t h a t i t p r o v i d e s a r a p i d , s h a r p and e a s i l y d e t e c t e d c o l o u r change which i s s t a b l e f o r 30 m i n u t e s . More a c c u r a t e r e s u l t s w e r e obt a i n e d t h a n w i t h an e x t e r n a l i n d i c a t o r s u c h as s t a r c h i o d i d e p a p e r . O t h e r i n t e r n a l i n d i c a t o r s which have been s u c c e s s f u l l y u s e d are c y a n o b i s (1,1 0 - p h e n a n t h r o l i n e ) i r o n (II)f3 and t r o p a e o l i n 00 w i t h m e t h y l e n e b l u e as c o n t r a s t medium53. The d i a z o m e t r i c method h a s been u s e d f o r t h e determination of sulphamerazine i n tab l e t d o s a g e forms w i t h o u t i n t e r f e r e n c e from e x c i p i e n t s s u c h a s s t a r c h , l a c t o s e , calcium c a r b o n a t e , sodium b i c a r b o n a t e , magnesium s t e a r a t e s t e a r i c a c i d , g e l a t i n , gum a c a c i a , and t a l c 5 4

.

6.3.2.

Non-Aqueous T i t r i m e t r y The p o w e r f u l e l e c t r o n w i t h d r a w i n g s u l phony1 g r o u p i n s u l p h o n a m i d e s r e n d e r s t h e amide hydrogen atom a c i d i c s o t h a t t h e s e drugs can be c o n v e n i e n t l y t i t r a t e d w i t h a s u i t a g h e b a s e i n a non-aqueous m e d i u m . Faber t i t r a t e d sulphamerazine i n p y r i d i n e s o l u t i o n u s i n g sodium m e t h o x i d e d i s s o l v e d i n a m i x t u r e o f benzene and m e t h a n o l ( 3 : 1)a s titrant and thymol b l u e i n m e t h a n o l as i n d i c a t o r . A c i d i c t a b l e t e x c i p i e n t s were n a t u r a l l y found t o i n t e r f e r e . Sulphamerazine h a s a l s o been determined56 i n t e t r a m e t h y 1u re a w i t h t e t r a b u ty 1ammonium h y d r o x i de ( 0 . 1 M ) as titrant, t h e e n d - p o i n t b e i n g determined using e i t h e r potentiometry or a thymol b l u e i n d i c a t o r . 57

More r e c e n t l y Davis and co-workers as a s u i t e v a l u a t e d 3-methyl-2-oxazolidone a b l e s o l v e n t f o r t h e non-aqueous t i t r a t i o n o f s u l p h o n a m i d e s on t h e b a s i s t h a t i t s h i g h d i e l e c t r i c c o n s t a n t and wide l i q u i d r a n g e contributed t o its outstanding solvent

542

RICHARD D. G.WOOLFENDEN

T e trabutylammonium h y d r o x i d e properties. was u s e d as t i t r a n t and p o t e n t i o m e t r y as end- p o i n t de t e ct i o n

.

6.3.3

Bromometric Titrimetry 58 The b r o m o m e t r i c methods o f Wojahn and Conway59 a r e w e l l e s t a b l i s h e d a n d have been a p p l & d , w i t h e x c e l l e n t r e s u l t s by D e Reeder t o t h e assay of sulphamerazine i n mixtures with o t h e r sulphonamides. R e c e n t l y , however, some a t t e n t i o n h a s been p a i d t o t h e improvement o f t h e d e t e c t i o n o f e n d - p o i n t i n h e b r o m o m e t r i c method. E j i m a and co-workers6' t i t r a t e d a number o f sulphonamides , i n c l u d i n g sulphamerazine , by a c o u l o m e t r i c method i n v o l v i n g brominat i o n with e l e c t r o l y t i c a l l y g en erated bromine i n an a q u e o u s s o l u t i o n of h y d r o c h l o r i c a c i d and p o t a s s i u m b r o m i d e . The e n d - p o i n t w a s d e t e c t e d p o t e n t i o m e t r i c a l l y . A coulom e t r i c met&d w a s a l s o a d o p t e d by E b e l and co-workers i n which e x c e s s o f e l e c t r o l y t i c a l l y g e n e r a t e d bromine was t i t r a t e d w i t h cuprous i o n s t o a p o t e n t i o m e t r i c end-point. A spectrophotometric t i t r a t i o n with bromide-bromate s o l u t i o n h a s b e e n d e v e l o p ed63, t h e drug being d i s s o l v e d i n a mixture of concentrated hydrochloric acid- a c e t i c a c i d ( 2 :8 ) Q u a n t i t a t i v e recoveries f o q s u l p h a m e r a z i n e were r e p o r t e d as 9 8 . 4 3 0.58% w i t h bromination t i m e of 5 minutes.

.

6.3.4.

Argentometric T i t r i m e t r y The p r i n c i p l e o f t h e a r g e n t o m e t r i c method i s t h a t some s u l p h o n a m i d e s f o r m i n s o l u b l e s i l v e r s a l t s . The s u l p h o n a m i d e s a r e p r e c i p i t a t e d by t h e a d d i t i o n o f excess standard s i l v e r n i t r a t e s o l u t i o n , t h e prec i p i t a t e removed by f i l t r a t i o n , and t h e excess s i l v e r n i t r a t e t i t r a t e d with standa r d ammonium t h i o c y a n a t e u s i n f e r r i c alum as t h e i n d i c a t o r . D e R e e d e r 6 I s u c c e s s f u l l y a p p l i e d t h e above method t o t h e d e t e r m i n a t i o n of a m i x t u r e o f s u l p h a m e r a z i n e , s u l p h a d i a z i n e and s u l p h a m e t h a z i n e .

SULPHAME RAZlNE

6.3.5.Complexometric

543

Titrimetry

Abdine and S a ~ e d e v ~ e l~o p e d a complexo m e t r i c a s s a y f o r s u l p h a m e r a z i n e . The sample was d i s s o l v e d i n a l k a l i n e s o l u t i o n and p r e c i p i t a t e d w i t h e x c e s s c o p p e r s u l The p h a t e s o l u t i o n i n pH 6 b o r a t e b u f f e r . e x c e s s c o p p e r w a s t h e n d e t e r m i n e d by t i t r a t i o n w i t h t h e d i s o d i u m s a l t o f E.D.T.A.using 1- ( 2 - p y r i d y l a z o ) - 2 - n a p h t h o l as i n d i c a t o r . S u l p h a m e r a z i n e h a s a l s o been e s t i m a t e d 6 6 i n combined s u l p h a d r u g s by p r e c i p i t a t i o n w i t h e x c e s s c o p p e r a c e t a t e f o l l o w e d by t h e d e t e r m i n a t i o n o f t h e r e s i d u a l c o p p e r by The s e l e c t i v e complexing w i t h E . D . T . A . p r e c i p i t a t i o n and c o m p l e x o m e t r i c a s s a y o f m i x t u r e s of s u l p h a m e r a z i n e , s u l p h a t h i a z o l e , and s u l p h a d i a z i n e were a l s o d i s c u s s e d . 6.3.6.Thermometric T i t r i m e t r v Bark and G r i m e 6 7 d e v e l o p e d a thermometric a s s a y f o r s e v e r a l s u l p h o n a m i d e s i n c l u d i n g s u l p h a m e r a z i n e . The s u l p h a m e r a z i n e was d i s s o l v e d i n t h e minimum volume o f 0.1M aqueous sodium h y d r o x i d e s o l u t i o n and t h e pH a d j u s t e d t o between 8 . 0 and 9.18 w i t h 0.1M n i t r i c a c i d s o l u t i o n . The s o l u t i o n w a s t i t r a t e d with standard s i l v e r n i t r a t e solu t i o n and t h e d a t a c a l c u l a t e d from t h e res u l t i n g e n t h a l p o g r a m . E x c i p i e n t s s u c h as l a c t o s e , s t a r c h , and magnesium s t e a r a t e d i d n o t i n t e r f e r e . D e t a i l s o f t h e a p p a r a t u s req u i r e d f o r t h i s a s s a have been d e s c r i b e d by Bark and B a t e 6 8 , 6 J . Sulphonamides have a 1s o been d e t e r m i n e d by a c a t a l y t i c t h e r m o m e t r i c t i t r a t i o n t e c h n i q u e . The p r i n c i p l e o f t h e method i s t h a t weak a c i d s a r e t i t r a t e d w i t h a b a s e i n non-aqueous media u s i n g a c r y l o n i t r i l e as a thermometric i n d i c a t o r . Thus , a t t h e endp o i n t t h e a c r y l o n i t r i l e undergoes a l k a l i catalysed anionic polymerization with a c o r r e s p o n d i n g e v o l u t i o n o f h e a t which i s Greenhow and S p e n c e r 7 0 de t e r m i n measured. e d s u l p h a m e r a z i n e by t h i s t e c h n i q u e u s i n g d i m e t h y 1f o r m a m i de a s t h e n on - aq ue ou s so 1ve n t and 0.1M o r 0.01M tetra-n-butylammonium

544

RICHARD D. G . WOOLFENDEN

hydroxide i n methanol-toluene or isopropan0 1 - t o l u e n e a s titrant. The lower p r a c t i c a b l e l i m i t o f d e t e r m i n a t i o n w a s shown t o b e 0.0001 m.equiv. of d r u g . I n t e r f e r e n c e s were e v i d e n t i n t h e p r e s e n c e o f a c i d i c e x cipients

.

6.4.

S p e c t r o p h o t o m e t r i c Assay P r o c e d u r e s

6 . 4 . 1 . I n f r a r e d S p e c t r o s c o p i c Methods

The a p p l i c a t i o n of i n f r a r e d s p e c t r o scopy t o t h e q u a n t i t a t i v e a s s a y o f s u l phonamides h a s b e e n o f l i m i t e d i n t e r e s t as r e f l e c t e d by a d i s t i n c t l a c k o f publ i c a t i o n s i n t h i s f i e l d . However, D o l i n s k y 7 l d e t e r m i n e d s u l p h a m e r a z i n e and s u l p h a d i a z i n e i n m i x t u r e by t h i s t e c h n i q u e u s i n g c a r b o n d i s u l p h i d e as s o l v e n t . O i and M i y a ~ a k i ’ a ~l s o d e t e r m i n e d s u l phamerazine i n m i x t u r e w i t h s u l p h a t h i a z o l e u s i n g d i m e t h y l f o r m a m i d e as s o l v e n t . 6.4.2.Ultraviolet

S p e c t r o s c o p i c Methods

U l t r a v i o l e t spectrophotometry has f o u n d some u s e i n t h e d e t e r m i n a t i o n o f sulphamerazine. Since t h i s drug i s normally incorporated i n t o a double or t r i p l e s u l p h o n a m i d e f o r m u l a t i o n t h e methods most commonly a v a i l a b l e i n v o l v e i t s determination i n the presence o e or t w o o t h e r s u l p h o n a m i d e s . Marzys 5,9s de s c r i b e d a method f o r t h e a s s a y o f s u l p h a m e r a z i n e i n t h e p r e s e n c e of s u l p h a d i a z i n e and s u l p h a t h i a z o l e w i t h o u t p r i o r s e p a r a tion. Following t h e d etermin atio n of s u l p h a d i a z i n e by t h e 2 - t h i o b a r b i t u r i c a c i d c o l o r i m e t r i c method d i r e c t u l t r a v i o l e t spectrophotometry w a s used t o measure t h e q u a n t i t i e s o f s u l p h a m e r a z i n e a n d s u l p h a t h i a z o l e . The r e s u l t s were t h e n c a l c u l a t e d by s o l v i n g t w o s i m u l t a n e o u s e q u a t i o n s . Using a s i m i l a r p r i q t i p l e Zajac74 and R a p a p o r t a n d Shakh determined s u l p h a m e r a z i n e i n f o r m u l a t i o n s w i t h o t h e r s u l p h o n a m i d e s . The u s e of a comp u t e r programming t e c h n i q u e f o r r e s o l v i n g t h e u l t r a v i o l e t s p e c t r a of t r i p l e s u l phonamide t a b l e t s c o n t a i n i n g s u l p h a m e r a -

-

SULPHAMERAZINE

545

z i n e h a s been d e s c r i b e d by Madsen and R~bertson~~. 6.4.3.

C o l o r i m e t r i c Methods A number o f c o l o r i m e t r i c methods have been d e s c r i b e d f o r t h e d e t e r m i n a t i o n o f s u l p h o n a m i d e s which are a p p l i c a b l e t o s u l p h a m e r a z i n e . P r o b a b l y t h e most w e l l known i s t h e B r a t t o n and M a r s h a l l method77 which i n v o l v e s d i a z o t i z a t i o n o f t h e p r i m a r y amine f u n c t i o n w i t h a c i d i c sodium n i t r i t e s o l u t i o n , decomposing t h e excess n i t r i t e w i t h s u l p h a m i c a c i d f o l l o w e d by c o u p l i n g t h e d i a z o compound w i t h N- (1naphthy1)-ethylenediamine. In general t h i s method h a s found i t s g r e a t e s t a p p l i c a t i o n i n t h e assay of s m a l l a m 1 w g a paper s ulphon a m i t h i n l a y e r ~&,iP,iS,QS c h r o m a t o g r a p h i c p r o cedure

78ybso;f

.

O t h e r c o l o r i m e t r i c methods h a v e been d e v e l o p e d , b u t have n o t b e e n as w i d e l y used a s t h e B r a t t o n ang4Marshall procedeveloped a d u r e . T u l u s and Guran method f o r s u l p h a m e r a z i n e and o t h e r s u l phonamides u s i n g t h e p o t a s s i u m s a l t o f 1,2-naphthoquinone -4-sulphonic a c i d as t h e c o u p l i n g a g e n t . The u s e of d i m e t h y l aminobenzaldehyde f o r t h e q u a n t i t a t i v e assay of sulphamerazine f o l l o w i n g paper c h r o m a t o g r a p h i c s e p a r a t i o n h a s been s t u d . c o l o r i m e t r i c determinai e d by L ~ i s e * ~ A t i o n f o r sulphamerazine i n a t a b l e t dosa g e form u s i n g 9 - c h l o r o - a c r i d i n e h a s been d e v e l o p e d by S t e w a r t and co-workers86 who f o u n d t h a t t h e r e s u l t s compared e x c e l l e n t l y w i t h t h o s e o b t a i n e d by t h e B r a t t o n and M a r s h a l l method. 6.5.

Chromatographic Procedures

6.5.1.High

Performance L i q u i d Chromatography

Kram87 q u a l i t a t i v e l y studied the b e h a v i o u r of some 2 1 s u l p h o n a m i d e s by H.P.L.C. Using a s t a i n l e s s s t e e l column packed w i t h s p h e r i c a l s i l i c e o u s p a r t i c l e s coated with a s t r o n g anion exchanger t h e

546

RICHARD D. G.WOOLFENDEN

r e t e n t i o n t i m e s o f t h e d r u g s were establ i s h e d using a mobile phase of 0 . 0 1 M sodium b o r a t e c o n t a i n i n g v a r i o u s l e v e l s of sodium n i t r a t e . From t h e s e s t u d i e s t h e optimum sodium n i t r a t e l e v e l s were p r e d i c t e d f o r t h e s e p a r a t i o n of s u l p h a m e r a z i n e , s u l p h a d i a z i n e and s u l p h a m e t h a z i n e , t h e o f f i c i a l trisulphapyrimidines. A q u a n t i t a t i v e H.P.L.C. assay f o r t h e t r i s u l p h a p y r i m i d i n e s h a s b e e n r e p o r t e d by P o e t and u s i n g a " Z i p a x " SCX(DuPont) c a t i o n e x c h a n g e column w i t h 0 . 2 M d i s o d i u m p h o s p h a t e b u f f e r s o l u t i o n (pH 6 .O) as t h e mobile phase. Sulphadimethoxine w a s chose n a s t h e i n t e r n a l s t a n d a r d . The recomme n d e d p r e s s u r e of 1000 p s i g p r o d u c e d a s c l v e n t f l o w r a t e o f 0 . 7 - 0 . 8 ml./min. res u l t i n g i n a 15-20 m i n u t e s e p a r a t i o n t i m e . A n a l y t i c a l d a t a w a s o b t a i n e d f o r f o u r representative lots of t a b l e t formulations and t w o s u s p e n s i o n f o r m u l a t i o n s , t h e c a l c u l a t e d c o e f f i c i e n t s of v a r i a n c e f o r rep l i c a t e i n j e c t i o n s r a n g i n g from 0.9 t o 4.0%.

Westlie and c o - w ~ r k e r shave ~ ~ develope d a l i q u i d - s o l i d chromatographic assay p r o c e d u r e which i s a p p l i c a b l e t o t h e t r i s u l p h a p y r i m i d i n e s . A MicroPak S i - 1 0 column w a s u s e d i n c o n j u n c t i o n w i t h a m o b i l e phase c o n s i s t i n g o f chloroform, methanol, ammonia 2 5 % ( 3 6 5 , 7 5 , 10) f l o w i n g a t a r a t e of 0.73ml. /min. Sulphathiazole w a s i n c l u d e d as a n i n t e r n a l s t a n d a r d . A H.P.L.C. p r o c e d u r e f o r t h e s e p a r a t i o n of a r a n g e o f s u l p h o n a m i d e s u t i l i s i n g s i l i c a g e l a s t h e column p i n g h as been d e s c r i b e d by Cobb a n d H i l l The s e p a r a t i o n was a c h i e v e d on a 25cm. s t a i n l e s s s t e e l column o f i n t e r n a l d i a m e t e r 4 m.m. p a c k e d w i t h S p e r i s o r b S5W 5 u m d i a m e t e r s p h e r i c a l s i l i c a g e l p a r t i c l e s . The m o b i l e p h a s e c o n s i s t e d of a m i x t u r e o f c y c l o hexane, anhydrous e t h a n o l , g l a c i a l a c e t i c a c i d ( 8 5 . 7 , 1 1 . 4 , 2 . 9 ) and t h e e l u t i o n was m o n i t o r e d a t 260nm u s i n g a

8% .

SULPHAMERAZ INE

547

C e c i l CE 2 1 2 v a r i a b l e w a v e l e n g t h d e t e c t o r . I n i t i a l separations w e r e obtained using c y c 1ohe xane -e t h a n o 1 m i x t u r e s of v a r i a b l e c o m p o s i t i o n and i t w a s found t h a t i n c r e a sing the ethanol content decreased t h e The a d d i t i o n o b s e r v e d r e t e n t i o n times. of s m a l l amounts o f a c e t i c a c i d s i g n i f i c a n t l y i n c r e a s e d column e f f i c i e n c y w i t h o u t a l t e r i n g r e s o l u t i o n . A t a flow r a t e of 2 m l . /min. t h e d e s c r i b e d m o b i l e p h a s e r e s u l t e d i n a 1 3 minute r e t e n t i o n t i m e f o r sulphamerazine.

The u s e o f h i g h p e r f o r m a n c e i o n p a i r p a r t i t i o n chromatography f o r t h e s e p a r a t i o n of s u l p h o n a m i d e s h a s b e e n g a n v e s t i Their g a t e d by K a r g e r and co-workers e f f o r t s represented a f e a s i b i l i t y study on t h e s e p a r a t i o n of 1 2 s u l p h a d r u g s usi n g a s i l i c a gel/CT (Reeve A n g e l ) s u p p o r t , a s t a t i o n a r y phase c o n s i s t i n g of a c a t i o n i c c o u n t e r i o n ( t e t r a b u t y l ammonium i o n ) b u f f e r e d t o a pH of 9 . 2 and a m o b i l e p h a s e of n - b u t a n o l , hexane ( 2 5 , 7 5 ) Under t h e s e c o n d i t i o n s s u l p h a m e r a z i n e w a s shown t o have a r e t e n t i o n t i m e of 13-14 m i n u t e s .

.

.

6.5.2.

Gas Chromatography The main g a s c h r o m a t o g r a p h i c method reported i n the l i t e r a t u r e for the determ i n a t i o n o f s u l p h a p y r i m i d i n e s i n v o l v e d an i n i t i a l hydrolytic step, the resulting v o l a t i l e 2 - a m i n o p y r i m i d i n e s b e i n g measure d . Turczangl d e v e l o p e d s u c h a method f o r quantitatively assaying the individ u a l sulphonamides, i n c l u d i n g sulphamerazine, i n the o f f i c i a l trisulphapyrimidines. Concentrated s u l p h u r i c a c i d w a s added t o t h e sample r d t h e m i x t u r e h e a t e d i n an oven a t 1 3 0 C f o r 1 h o u r . The s o l u t i o n w a s made a l k a l i n e and 2-amino-4 , 6 - d i m e t h y l p y r i d i n e added a s i n t e r n a l s t a n d a r d . Theocomponents were t h e n s e p a r a t e d a t 1 5 0 C on a column p a c k e d w i t h 5% SE-30 + 5 % Carbowax 2 0 M on Chromosorb W u s i n g flame i o n i z a t i o n d e t e c t i o n . E x c e l l e n t r e c o v e r i e s were a c h i e v e d f o r t h e t r i s u l p h a p y r i m i d i n e s i n b o t h s y n t h e t i c mixt u r e s and s e v e r a l commercial t a b l e t p r e parations.

548

RICHARD D. G.WOOLFENDEN

Daung2 f o u n d t h a t t h e above method was uns u i t a b l e f o r t h e d e t e r m i n a t i o n of s u l p h a merazine i n p o u l t r y f e e d s a t l e v e l s rangi n g between 0.002 and 0 . 0 5 % . The method adopted required the preparation of a relati v e l y c l e a n e x t r a c t o f t h e f e e d f o l l o w e d by an e x t r a c t i o n s t e p u s i n g e t h y l a c e t a t e . T h e r e s i d u e r e m a i n i n g a f t e r e v a p o r a t i o n of t h e e t h y l acetate w a s m e t h y l a t e d w i t h d i a z o methane and t h e n a c y l a t 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 n h y d r i d e . The a c y l d e r i v a t i v e s w e r e f o u n d t o b e e a s & l y s e p a r a t e d on a 10% DC-200 column a t 230 C. D e t e c t i o n w a s achi e v e d by e l e c t r o n c a p t u r e . N o i n t e r n a l s t a n d a r d was u s e d , t h e r e s u l t s b e i n g e v a l u a t e d by comparing s t a n d a r d a n d s a m p l e peak heights. Roeder and S t u t h e g 3 d e v e l o p e d a g a s c h r o m a t o g r a p h i c method f o r t h e s u l p h o n a mides a n d t h e i r N 4 - a c e t y l m e t a b o l i t e s i n b l o o d and u r i n e . The method w a s a p p l i c a b l e t o s u l p h a m e r a z i n e . The s u l p h o n a m i d e s were e x t r a c t e d f r o m t h e b l o o d and u r i n e s a m p l e s and t h e n m e t h y l a t e d w i t h d i a z o m e t h a n e . The m e t h y l d e r i v a t i v e s were d e t e r m i n e d u s i n g a column o f 3% OV 101 on Gaschrom Q w i t h a 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 of 5 % f o r t h e f r e e s u l p h o n a m i d e s and 7 % f o r t h e a c e t y l conjugates

.

The s i m u l t a n e o u s q u a l i t a t i v e a n a l y s i s o f 1 4 s u l p h a d r u g s and t h e i r i n d i v i d u a l 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 by g a s l i q u i d c h r o m a t o g r a hy w e r e p e r f o r m e d by Nose a n d co-workers 92A on s o l u t i o n s o f d i m e t h y l f o r mamide d i a l k y l a c e t a l d e r i v a t i v e s o f t h e drugs i n acetone. The d e r i v a t i v e s c o u l d be d e t e c t e d w i t h an e l e c t r o n c a p t u r e d e t e c t o r with a highly s e n s i t i v e response following s e p a r a t i o n u s i n g 10%O V - 1 0 1 on Chromosorb G HP (80-100 m e s h ) , 5 % X E - 6 0 on Gas-Chrom Q (80-100 mesh) o r 5 % OV-225 on G a s Chrom Q ( 80-1000mesh) a t t e m p e r a t u r e s between 2 2 0 a n d 2 4 0 C. However, t h e r e t e n t i o n t i m e s f o r s u l p h a m e r a z i n e v a r i e d between a b o u t 4 0 t o 80 m i n u t e s .

SU LPHAME RAZ I NE

6.5.3.

549

T h i n L a y e r Chromatography A number o f t h i n l a y e r c h r o m a t o g r a p h i c methods have been d e v e l o p e d f o r t h e 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 a n a l y s i s o f s u l phamerazine and r e l a t e d s u l p h a d r u g s . C e r t a i n d e t a i l s of t h e s e methods are summarise d i n T a b l e 9 a n d some s p o t l o c a t i n g r e a g e n t s a r e g i v e n i n T a b l e 11.

B i c a n - F i s t e r and Kajganovic8O reCOgniSe d t h e p o t e n t i a l of t h i n l a y e r chromatography a s a more r a p i d t e c h n i q u e t h a n p a p e r chromatography f o r t h e q u a n t i t a t i v e a s s a y of t r i p l e s u l p h a c o n t a i n i n g p r e p a r a t i o n s such a s t a b l e t s , s u p p o s i t o r i e s , and suspens i o n s . Using a K i e s e l g e l G l a y e r combined w i t h t h e s o l v e n t s y s t e m c h l o r o f o r m , metha n o l ( 9 0 ,10) a q u a n t i t a t i v e s e p a r a t i o n o f s u l p h a m e r a z i n e , s u l p h a t h i a z o l e and s u l p h a d i a z i n e was a c h i e v e d . F o r t h e s e p a r a t i o n of c e r t a i n m i x t u r e s of s u l p h a m e r a z i n e, sulphacetamide , sulphamethazine and sulphad i a z i n e t h e s o l v e n t s y s t e m c h l o r o f o r m , metha n o l , 258 ammonia s o l u t i o n ( 9 0 , 1 5 , 2 . 4 ) w a s found t o be b e t t e r . F o l l o w i n g e l u t i o n f r o m t h e a d s o r b e n t t h e s e p a r a t e d sulphonamides were a t f i r s t a s s a y e d by a U.V. method b u t B i c a n - F i s t e r and K a j g a n o v i c f o u n d t h a t t h e K i e s e l g e l G gave a h i g h c o n t r i b u t i o n t o t h e b l a n k a b s o r b a n c e . They , t h e r e f o r e ,a p p l i e d t h e B r a t t o n and M a r s h a l l c o l o r i m e t r i c method, e x c e l l e n t r e c o v e r i e s b e i n g o b t a i n e d f o r a l l t h e sulphonamides p r e v i o u s l y mentioned. L i m i t s of e r r o r f o q sulphamerazine ranged between - 3 . 2 % t o 4 . 1 % i n s y n t h e t i c mixt u r e s with t h e o t h e r sulphonamides.

-

B r u n n e r 8 1 d e v e l o p e d a t h i n l a y e r method f o r t h e a n a l y s i s of t r i s u l p h a p y r i m i d i n e preparations containing sulphamerazine,sulp h a d i a z i n e and s u l p h a m e t h a z i n e u s i n g s i l i c a g e l GF p l a t e s a n d a s o l v e n t s y s t e m comprisi n g chloroform, methanol, ammonia(30,12,1). Again t h e B r a t t o n and M a r s h a l l colorimetric method was u s e d r e s u l t i n g i n e x c e l l e n t r e c o v e r i e s . A c o l l a b o r a t i v e s t u d y 8 2 on t h e u s e of t h i s method found t h a t the c o e f f i c i e n t s of v a r i a n c e f o r t h e i n d i v i d u a l compounds r a n g e d from 0 . 7 6 t o 1 . 6 6 . The t r i -

TABLE 9

Thin l a y e r chromatography of sulphamerazine Adsorbent K i e s e l g e l G.

S i l i c a g e l G: impregnated with fluorescein. Polyamide CM1011. ln

8

S o l v e n t System a)Chloroform,methanol (90,lO). b ) Chlorof orm,methanol, 25 % ammonia ( 9 0 , 1 5 ,2 . 5 ) Chloroform,ethanol, h e p t a n e (1,1,1) containi n g 1 . 2 % water. a ) C h l o r o f o r m , 95% e t h a n o l (90,lO). b)Ethyl acetate,95% ethanol (80,20). c )Water, 95% e t h a n o l

-

Ref.

Use -

-Rf

Q u a n t i t a t i v e assay f o r trisulphapyrimidine preparations.

80

0.57 I d e n t i t y test.

94

I d e n t i t y test.

95

0.79 0.83

It

11

0.59

11

II

(60,40).

P l a s t e r of P a r i s imp re gn a t e d w i t h z,inc f e r r o c y a nide. Silica gel G impregn a t e d w i t h sodium h y d r o x i d e .

a ) 0.03M a q u e o u s a c e t i c acid. b ) 1.74M a q u e o u s a c e t i c acid. c ) 3.33M a q u e o u s a c e t i c acid. a)Chloroform,methanol (4,1). b )A c e ton e, methanol ( 4 1).

0.01 I d e n t i t y t e s t .

0.17

11

11

0.38

11

II

0.56 I d e n t i t y test. 0.61

11 II

96

97

TABLE 9 ( c o n t ' d )

T h i n l a y e r c h r o m a t o g r a p h y of s u l p h a m e r a z i n e Adsorbent Silica gel G impregnated with potassium hydrogen s u l p h a t e . S i l i c a g e l G.

Chloroform,carbon tetrachloride,methanol ( 7 , 2 I 1).

0.34

Identi t y test.

97

E t h y l acetate ,methanol

0.59

I d e n t i t y test.

97

0.47

I d e n t i t y test.

98

(981).

S i l i c a g e l G. m

?

S i l i c a gel.

S i l i c a g e l GF. S i l i c a g e l GF.

S i l i c a gel H imp re gn a t e d w i t h sodium h y d r o x i d e . Silica gel G precoated p l a t e s (Analt e c h ) .

Ref. -

Use -

S o l v e n t System

a)Ethyl acetate,methanol, 25% a m m o n i a ( 1 7 , 6 , 5 ) . b)Petroleum e t h e r I chloroform n - b u t a n o l ( l , l , l ) . ChloroformI methanol (951 5 ) E t h y l acetate ,methanol (9I l l . Chlorof o mI met h a n o l ammonia ( 3 0 , 1 2 , l ) . Chloroform,methanol

0.6 7 0.29

I d e n ti t y t e s t .

98

0.6 3

I d e n t it y t e s t

.

99

-

(9I1).

Acetone,n-heptane,metha n o 1 , 2 8 - 3 0 % ammonia,nbutanol(72,21,9,10,10).

II

II

0.31

Quantitative assay for trisulphapyrimidine preparations. Quantitative assay f o r f e e d c o n c e n t r a t e s or p r e m i xe s. I d e n t i t y t e s t and q u a n t i t a t i v e assay i n animal tissues

.

81

100 a3

TABLE 9 (con t ' d )

Thin l a y e r chromatography of s u l p h a m e r a z i n e Adsorbent

S o l v e n t System

Use -

Ef

pH 7 . 4 aqueous v e r o n a l acetate. Polyamide 11. a)pH 7 . 4 aqueous v e r o n a l acetate. b ) pH 7 . 4 aqueous v e r o n a l a c e t a t e c o n t a i n i n g 10% acetone. S i l i c a gel. Chloroform,methanol, ammonium hydroxide (30,12,1). S i l i c a gel 60 a ) Chlorof o m , e t h a n o l (Merck p r e c o a t e d ) . (9,l). b)Chloroform,ethanol, ammonium h y d r o x i d e , (8,2,0.1). C Chloroform, e t h a n o l , dioxane, a c e t i c acid, (8,lr1,0-1). d Ethyl a c e t a t e ,dioxane, a c e t i c acid (8,2,0.1) S i l i c a g e l G.

.

-

-

0.33 0.20 0.49 0.46

RM-structure a c t i v i t y correlation. R -structure activity M correlation.

Ref. 101

101

II

Q u a n t i t a t i v e a s s a y for 102 t r i s u l phapyrimi d i n e t a b l e t s and o r a l s u s p e n s i o n s Q u a n t i t a t i v e assay i n 1 28 human u r i n e .

553

SU LPHAME RAZINE

s u l p h a p y r i m i d i n e s have a l s o been a s s a y e d by a comb&d T.L.C. i n s i t u densitometric method

.

-

One o f t h e more common s u l p h o n a m i d e m i x t u r e s used i n animal t h e r a p y c o n t a i n s s u l p h a m e r a z i n e w i t h s u l p h a q u i n o x a l i n e ,s p h a t h i a z o l e , and s u l p h a m e t h a z i n e . C i e r i showed t h a t t h e s u l p h a m e r a z i n e , s u l p h a m e t h a z i n e and s u l p h a t h i a z o l e c o n t e n t s o f t h e s e m i x t u r e s were b e s t d e t e r m i n e d by a t h i n l a y e r method r a t h e r t h a n by t h e ga chromatog r a p h i c method p r o p o s e d by Dam” ( r e v i e w e d Using s i l i c a g e l H i n s e c t i o n 6.5.2 . ) i m p r e g n a t e d w i t h sodium h y d r o x i d e and c h l o r o f o r m , m e t h a n o l ( 9 0 , l O ) as t h e s o l v e n t s y s t e m C i e r i a s s a y e d t h e i s o l a t e d components by a n u l t r a v i o l e t a b s o r p t i o n method which a l l o w e d t h e components t o b e d e t e r m i n e d w i t h i n 2-3% of t h e a c t u a l amounts p r e s e n t .

Yto

.

T h i n l b 2 y e r chromatography i s now t h e o f f i c i a l method f o r t h e d e t e r m i n a t i o n of s u l p h a m e r a z i n e , s u l p h a d i a z i n e and s u l p h a m e t h a z i n e i n t r i s u l p h a p y r i m i d i n e t a b l e t s and o r a l s u s p e n s i o n s h a v i n g r e p l a c e d t h e p a p e r c h r o m a t o g r a p h i c method o f t h e U.S.P.XVII1. The method i n v o l v e s t h e u s e of s i l i c a g e l as a d s o r b e n t combined w i t h c h l o r o f o r m , m e t h a n o l , ammonium h y d r o x i d e ( 3 0 , 1 2 , 1 ) a s s o l v e n t s y s t e m . The s e p a r a t e d sulphapyrimidines a r e q u a n t i t a t i v e l y determined u s i n g t h e B r a t t o n and M a r s h a l l colorimetric procedure.

u. s . P . x1x

A t h i n l a y e r chromatographic screeni n g method f o r t h e e s t i m a t i o n o f s u l p h a m e r a z i n e and o t h e r s u l p h o n a m i d e r e s i d u e s i n p o u l t r y t i s s u e s h a s b e e n r e p o r t e d by P h i l i p s and T r a f t ~ n * ~ The . minimum d e t e c t a b l e amount o f sulphonamide was f o u n d t o b e a b o u t 2 pg o r 0 . 0 4 p.p.m. u s i n g a 50g. s a m p l e . To d e t e r m i n e t h e r e p r o d u c i b i l i t y of t h e method 0.1 p.p.m. o f a s e r i e s o f s u l phonamides was added t o 50g. p o r t i o n s of l i v e r t i s s u e , t h e n r e - i s o l a t e d and a s s a y e d by d i r e c t c o l o r i m e t r y and by t h e p r o p o s e d t h i n l a y e r method. The mean recoveries were 88 and 81% r e s p e c t i v e l y . The recover-

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RICHARD D. G. WOOLFENDEN

i e s of s u l p h a m e r a z i n e were r e s p e c t i v e l y 9 1 a n d 80%. T.L.C. h a s a l s o been u s e d f o r t h e e s t i mation of sulphamerazine i n b i o l o g i c a l f l u i d s ( s e e s e c t i o n 7 ) and f o r t h e examinat i o n of s u l p h a m e r a z i n e d e c o m p o s i t i o n p r o d u c t s ( s e e s e c t i o n s 5 . 1 and 5 . 2 ) .

6.5.4.

P a p e r Chromatography P a p e r c h r o m a t o g r a p h y w a s o r i g i n a l l y used extensively f o r the separation, identif i c a t i o n and q u a n t i t a t i v e a n a l y s i s of s u l phonamide m i x t u r e s . A number o f a p p l i c a t i o n s a r e summarized i n T a b l e 10 and some s p o t l o c a t i o n a g e n t s a r e g i v e n i n T a b l e 11. Sulphamerazine h a s been q u a n t i t a t i v e l y determined i n mixtures with o f $ ~ r g ~ y l # y ~ i amides by a number o f w o r k e r s Most methods u s e d Whatman N o . 1 p a p e r , t h e main v a r i a t i o n b e i n g i n t h e c o m p o s i t i o n of t h e m o b i l e s o l v e n t s y s t e m . The B r a t t o n a n d M a r s h a l l c o l o r i m e t r i c method h a s been exa t't t'o f the t e n s i v e l y used f o r t h e i s o l a t e d components 7 9 , 1 8 ! i , P O 2 - ? 0 S , P l P

6.5.5.

Ion-Exchange and P a r t i t i o n Chromatography Hutchins and C h r i s t i a n 1 1 3 a s s a y e d s u l phamer a z i n e by a n i s o t o p e d i l u t i o n t e c h n i q u e a f t e r + p r i o r s e p a r a t i o n on an Amberli f541R1 2 0 ( H ) column. G i l m e r and P i e t r z y k r e p o r t e d t h e d i s t r i b u t i o n voef f i c i e n t s of several s u l p h o n a m i d e s on H -form,macroporous a n d g e l - t y p e r e s i n s f o r a number o f water-organic solvent mixtures. A mixture of sulphabenzamide, sulphacetamide, sulphad i a z i n e , s u l p h a m e r a z i n e and s u l p h a p y r i d i n e w a s s u c c e s s f u l l y s e p a r a t e d by u s i n g 4 0 , 5 2 , 6 4 , 7 7 and 9 0 % d i m e t h y l s u l p h o x i d e s o l u t i o n s as e l u t r i a n t s . S e l z e r and Banes '15 r e p o r t e d a column c h r o m a t o g r a p h i c method for t h e s e p a r a t i o n , d e t e c t i o n and e s t i m a t i o n of s u l p h o n a m i d e r e s i d u e s i n milk. The r e c o v e r y o f s u l p h a -

TABLE 10

P a p e r Chromatography o f S u l p h a m e r a z i n e Paper Whatman N o . 1

Whatman No.1

m

%

Ascending o r D e scendinL a Ascending

-

Whatman N o . 1 imp re gn a t e d w i t h 4 % aqueous potassium dihydrogen phosphate Whatman N o . 1

Descending

Whatman N o . 1 impregnated w i t h ace t o n e , f o r m a m i de (70,30)

A s ce n d i n g

Descending

S o l v e n t System

-Rf

Use -

Ref.

S e p a r a t i o n of 103 metabolic prod u c t s from b i o l o g i ca 1 m a t e r i a Is. 104 a ) B u t a n o l , g l a c i a l a c e t i c 0.50 I den t i t y t e s t . a c i d , w a t e r (50,15,60). b ) B u t a n o l , a m m o n i a , w a t e r 0.34 (40,10,30). Butanol s a t u r a t e d I d e n t i t y test. 105 with water. B u t a n o l ,ammonia water (40,10,50).

0.3

-

B u t a n o l , 3% a q u e o u s ammonia ( u s e t h e organic layer). Chloroform,methyl chloroform(55,S).

0.29

-

I d e n t i t y test 79 and q u a n t i t a t i v e as s a y . Quantitative 106 assay f o r trisulphapyrimidines i n t a b l e t s and ora l suspensions.

TABLE 10 (cont ' d )

P a p e r Chromatoqraphy of Sulphamerazine Paper

Ascending o r De s c e n d i n u

Whatman N o . 1 impre gna t e d with acetone, f ormamide (70,301. Whatman N o . 1

Ascending

Methylene c h l o r i d e .

Descending

Whatman No.1

Ascending

Whatman No. 2

Circular

Butano1,absolute e t h a n o l , 2 N ammonia (10,2,4). 0.2N aqueous EDTA containing 20% ammonia. Butano1,acetic a c i d , w a t e r (5,1,4).

Ln

m

Ln

Use

S o l v e n t Sy s tern

-

Ref.

Q u a n t i t a t i v e assay 107, f o r trisulphapyri111 midines i n t a b l e t s and o r a l suspensions.

0.24 S t a b i l i t y assay.

109

0.87 I d e n t i t y t e s t .

109

-

I d e n t i t y t e s t and q u a n t i t a t i v e assay.

110

TABLE 11

V -i s u a l i z a t i o n Methods f o r t h i n l a y e r a n d p a p e r c h r o m a t o g r a p h y of s u l p h a m e r a z i n e Spot Colour

Reagent

T.L.C. U.V. (254n.m. ) - f l u o r e s c e n c e

2.

E h r l i c h s r e a g e n t - 1 % dimethylaminob e n z a l d e h y d e + 1-108 conc.HC1 i n 95% ethanol. B r a t t o n and M a r sha l l r e a g e n t a )I N HC1, b ) 5 % N a N O Z , C ) O . 1%N - 1 - n a p h t h y l ) ethylenedlamine dihydrochloride.

3.

quenching

.

1.

Dark b l u e black. Yellow.

Reddishpurple.

94

95,97,98

Copper s u l p h a t e -1-5% CuS04. 5H20 i n w a t e r . Brown.

5.

Fluorescein-1% i n acetone, w a t e r ( 3 , 1 ) , f l u o r e s c e n c e - q u e n c h i n g a t 254 nm.

Dark b l u e black.

97

6.

Copper a c e t a t e - s a t u r a t e d s o l u t i o n i n methanol. C e r i c sulphate-2% i n water containing

Brown.

99

Y e 1l o w i s h purple.

99

5 % conc.H2S0 4 '

106,107, 108,111 79,105 109,110

94,96, 97,98

4.

7.

R e f e r e n ce P.C. -

97,99

112

558

RICHARD D. G. WOOLFENDEN

m e r a z i n e from m i l k w a s f o u n d l & be 8 3 % a t t h e 0 . 5 p.p.m. l e v e l . M i l l e r developed a p a r t i t i o n column c h r o m a t o g r a p h i c method f o r t h e s e p a r a t i o n and q u a n t i t a t i v e a s s a y o f t r i s u l p h a p y r i m i d i n e s . The s u l p h a p y r i m i d i n e s were q u a n t i t a t i v e 1y t r a n s f erre d i n acetone t o t h e t o p of a potassium bicarbona t e i m p r e g n a t e d C e l i t e 545 column. S u l p h a m e t h a z i n e was e l u t e d f i r s t u s i n g 10% n-butanol i n e t h e r s a t u r a t e d with 0 . 1 N aqueous potassium b i c a r b o n a t e s o l u t i o n . S u l p h a m e r a z i n e w a s t h e n e l u t e d w i t h 2 0 % nb u t a n o l i n e t h e r s a t u r a t e d w i t h 0 . 1 N aqueous p o t a s s i u m b i c a r b o n a t e a n d f i n a l l y sulphadiazine w a s e l u t e d with 40% n-butanol i n e t h y l a c e t a t e s a t u r a t e d w i t h w a t e r . The s e p a r a t e d compounds were t h e n a s s a y e d by u l t r a v i o l e t spectrophotometry. When t h e v a l i d i t y o f t h e method w a s s t u d i e d c o l l a b o r a t i v e l y s e v e r a l d i f f i c u l t i e s were e n c o u n t e r e d w i t h h i g h column b l a n k s which w e r e a t t r i b u t e d t o t h e q u a l i t y of t h e nHowever, t h e o v e r b u t a n o l and C e l i t e u s e d . a l l r e s u l t s were s a t i s f a c t o r y w i t h a n o v e r a l l s t a n d a r d d e v i a t i o n of 2.58%. Rader 1 1 7 a p p l i e d t h e c o n c e p t of i o n p a i r i n g t o t h e s e p a r a t i o n o f some s e l e c t e d s u l p h o n a m i d e s by p a r t i t i o n c h r o m a t o g r a p h y . One p r o c e d u r e h a s b e e n a p p l i e d t o t h e s e p a r a t i o n of s u l p h a m e r a z i n e , s u l p h a m e t h a z i n e and s u l p h a d i a z i n e by i o n - p a i r f o r m a t i o n w i t h t h e t e t r a b u t y l a m m o n i u m i o n f o l l o w e d by s e p a r a t i o n on a C e l i t e 545 column.The i s o l a t e d sulphapyrimidines w e r e then q u a n t i t a t i v e l y measured by u l t r a v i o l e t s p e c t r o p h o t o m e t r y . 6.5.6.

Electrophoresis The e l e c t r o p h o r e s i s ( 4 0 0 V , 1mA p e r cm. , 15OC, 6 0 m i n . , d e v e l o p e r p-dimethylaminob e n z a l d e h y d e ) of s e v e r a l s u l p h o n a m i f f s w a s s t u d i e d by K i n o s h i t a and c o - w o r k e r s at v a r i o u s pH v a l u e s a d j u s t e d by C l a r k - L u b s ' , Sorensen I s o r Kolthof f ' s b u f f e r s o l u t i o n s . Sulphamerazine w a s found t o m i g r a t e towards t h e anode. The procedure w a s u n s u i t a b l e f o r t h e i d e n t i f i c a t i o n of s u l p h a m e r a z i n e , s u l p h a g u a n i d i n e and s u l p p f g i a e i n e i n a t e r n a r y mixture. Garber has generated

559

SULPHAMERAZINE

paper e l e c t r o p h o r e t i c mobility d a t a f o r s e v e r a l sulphonamides, i n c l u d i n g sulpham e r a z i n e , u s i n g 1%, 5 % and 10%a c e t i c a c i d as s o l v e n t . 6.6. 6.6.1.

E l e c t r o c h e m i c a l Methods Polarography Using p o l a r o g r y g b y c o u p l e d w i t h microc o u l o m e t r y Okazaki studied the electrode r e a c t i o n s of s e v e r a l s u l p h a p y r i m i d i n e s The optimum c o n d i t i o n s f o r t h e p o l a r o g r a p h i c r e d u c t i o n o f s u l p hame r a z i n e w e r e d e t e r m i n e d , a l i n e a r p l o t b e i n g o b t a i n e d of d i f f u s i o n c u r r e n t a g a i n s t c o n c e n t r a t i o n f o r 0.1-l.0m.M s o l u t i o n s o f t h e d r u g i n pH 3 . 0 and 9 . 0 aqueous b u f f e r s . The r e d u c t i o n w a s shown t o t a k e p l a c e w i t h i n t h e p y r i m i d i n e n u c l e u s by comparison w i t h t h e p o l a r o g r a p q i t b e h a v i o u r of 2 - a m i n o p y r i m i d i n e . Okazaki applied t h e method t o t h e d e t e r m i n a t i o n of s u l p h a merazine i n t a b l e t s , i n j e c t a b l e s , s y r u p s and o i n t m e n t s .

.

Woodson a p p l i e d t h e p r i n c i p l e s of d . c . a n d a . c . p o l a r o g r a p h y ko t h e r e d u c t i o n of a number of p h a r m a c e u t i c a l s i n an a p r o t i c Using a d r o p p i n g organic solvent system. mercury e l e c t r o d e a g a i n s t a s i l v e r w i r e ref e r e n c e t h e d . ~ .h a l f - w a v e p o t e n t i a l of s u l p h a m e r a z i n e i n a c e t o n i t r i l e - 0.1M t e t rabutylammonium p e r c h l o r a t e a s s o l v e n t s y s t e m w a s found t o be - 1 . 9 5 ~ . The co -5 responding d e t e c t i o n l i m i t w a s 1 x 10 moles/ litre. The p o l a r o g r a p h i c b e h a v i o u r of t h e S c h i f f b a s e of s y j g h a m e r a z i n e h a s been A l i n e a r response t o s t u d i e d by Donev c o n c e n t r a t i o n was f o u n d and t h e method was subsequently applied t o the determination of s u l p h a m e r a z i n e i n t h e b l o o d plasma and u r i n e of animals dosed o r a l l y .

.

6 . 6 . 2 . Ion S e l e c t i v e E l e c t r o d e s

Hazemoto and co-workers 124 c o n s t r u c t e d an e l e c t r o d e s e n s i t i v e t o s u l p h a d r u g s u s i n g s u l p h a m e r a z i n e and s u l p h i s o m i d i n e a s

RICHARD D. G. WOOLFENDEN

560

e x a m p l e s . The e l e c t r o d e s e n s i n g s y s t e m cons i s t e d o f a l i q u i d membrane c o n t a i n i n g a n i r o n (11)- b a t h o p h e n a n t h r o l i n e c h e l a t e . Rapid and N e r n s t i a n r e s p o n s e s w e r e e x h i b i t e d agai n s t s o l u t i o n s o f sulpham r a z i n e g a n g i n g i n c o n c e n t r a t i o n between 10-5 and 10 M. High s e l e c t i v i t y w a s obtained i n the presence of u r e a , g l y c i n e , a m i n o p y r i n e and p-aminobenzoic a c i d which are s u b s t a n c e s known t o interfere i n the usual colorimetric analysis of s u l p h a drugs. I n c o n t r a s t s m a l l amounts o f sodium t r i c h l o r o a c e t a t e and a s p i r i n p r o duced an a p p r e c i a b l e e f f e c t i n t h e m e a s u r e d potential. 6.7.

Bioassay A method f o r t h e m i c r o b i o l o g i c a l a s s a y of s u l p h o n a m i d e s , i n v o l v i n g m e a s u r i n g t h e zone o f i n h i b i t i o n o f E s c h e r i c h i a c o l i s t r a i n 9 on a g a r p l a t e s , h a s bf5f: d e v e l o p e d A linear by C a n t e l l i F o r t i a n d F r a c a s s o p l o t w a s o b t a i n e d f o r l o g c o n c e n t r a t i o n aga i n s t i n h i b i t i o n zone d i a m e t e r a l o n g w i t h a s e n s i t i v i t y of 6-50 u g . / m l . o f 1 8 s u l p h o n a mides t e s t e d s u l p h a m e r a z i n e was t h e f i f t h most a c t i v e .

.

S h i b a t a and c o - w o r k e r s 126 d e v e l o p e d a b i o a s s a y method f o r t h e d e t e r m i n a t i o n o f s u l p h o n a m i d e s , i n c l u d i n g s u l p h a m e r a z i n e ,usi n g B a c i l l u s m e g a t e r i u m as t h e c h a l l e n g e organi s m .

7. Estimation i n Biological Fluids Longene c k e r lo3d e v e l o p e d a p a p e r chromat o g r a p h i c method f o r t h e d e t e r m i n a t i o n of s u l p h a m e r a z i n e i n t h e p l a s m a of c h i c k e n s f e d w i t h a mixture of s u l p h a d i a z i n e , sulpham e r a z i n e and s u l p h a t h i a z o l e . The b l o o d sample was drawn f r o m t h e h e a r t and t r a n s f e r r e d t o a t e s t tube containing potassium Following c e n t r i f u g a t i o n t h e p l a oxalate sma w a s s p o t t e d o n t o Whatman N o . 1 p a p e r which w a s t h e n d e v e l o p e d by t h e a s c e n d i n g t e c h n i q u e u s i n g a n - b u t a n o l , ammonia,water ( 4 0 , 1 0 , 5 0 ) e m u l s i o n as t h e s o l v e n t s y s t e m . The s e p a r a t e d s u l p h o n a m i d e s were t h e n l o c a t e d u s i n g p-dime t h y 1aminoben z a l d e h y d e re-

.

SULPHAM E RAZ IN E

561

a g e n t and u l t i m a t e l y d e t e r m i n e d u s i n g t h e B r a t t o n and M a r s h a l l c o u p l i n g t e c h n i q u e . I n t h e case of blood a n a l y s i s b e t t e r separa t i o n s were a c h i e v e d when 0.1% of nonae t h y l e n e g l y c o l m o n o s t e a r a t e (a n o n i o n i c s u r f a c t a n t ) was added t o t h e d e v e l o p i n g s o l vent. For t h e a n a l y s i s o f u r i n e t h e a d d i t i o n of t h i s m a t e r i a l w a s unnecessary. A h o r i z o n t a l c i r c u l a r p a p e r chromatog r a p h i c method € o r t h e q u a n t i t a t i v e e s t i mation o f sulphamerazine i n ood and u r i n e h a s been d e v e l o p e d by Sinha’”. The chromatograms were r u n i n a c i r c u l a r chromatog r a p h i c chamber by b o t h t h e c e n t r a l and l a t e r a l f l o w p r o c e s s e s . The d r u g w a s l o c a t e d and e s t i m a t e d u s i n g p-dime t h y l a m i n o b e n z a l dehyde a s t h e c o l o r i m e t r i c r e a g e n t . The method gave r e p r o d u c i b l e r e s u l t s .

O r t e n g r e n and T r e i b e r 1 2 * h a v e r e v i e w e d t h e v a r i o u s c h r o m a t o g r a p h i c methods a v a i l a b l e f o r t h e e s t i m a t i o n of s u l p h o n a m i d e s i n b i o l o g i c a l materials. An e x t e n s i o n o f t h e i r report described the q u a n t i t a t i v e analysis of s u l p h o n a q i d e s ( i n c l u d i n g s u l p h a m e r a z i n e ) and t h e i r N - a c e t y l m e t a b o l i t e s i n human u r i n e u s i n g t h i n l a y e r chromatography. F o r a minimum d r u g c o n c e n t r a t i o n o f 10 pg./ml. t h e s a m p l e was s p o t t e d d i r e c t l y on t o t h e B e l o w t h i s minimum c o n c e n t r a t i o n i t plate. w a s n e c e s s a r y t o s a t u r a t e t h e u r i n e sample w i t h ammonium s u l p h a t e f o l l o w e d by e x t r a c t i o n with e t h y l acetate. The r e s i d u e from t h e e t h y l a c e t a t e l a y e r was t h e n d i s s o l v e d i n a s m a l l amount o f a c e t o n e and s p o t t e d on The s e p a r a t e d s u l p h o n a m i d e t o thg p l a t e . and N - a c e t y l m e t a b o l i t e were u l t i m a t e l y estimated using densitometry. An e x t e n s i v e review of a q u a n t i t a t i v e method f o r t h e d e t e r m i n a t i o n o f t h e b a c t e r i o s t a t i c a l l y a c t i v e f r a c t i o n of s u l p h o n a mides and t h e sum o f t h e i r i n a c t i v e m e l$38” i n body f l u i d s i s g i v e n by R i e d e r The B r a t t o n and M a r s h a l l c o l o r i m e t r i c a s s a y was u s e d f o r a l l q u a n t i t a t i v e measurem e n t s . The p r o c e d u r e w a s a p p l i c a b l e t o t h e a n a l y s i s o f b l o o d p l a s m a , serum, i n t e r s t i t i a l f l u i d and u r i n e .

.

is$:

562

R I C H A R D D. G. W O O L F E N D E N

Methods f o r t h e d i r e c t measurement o f sulphonamides i n b i o l o g i c a l f l u i d a v e been d e s c r i b e d by Hawking and Lawrence ? 39

.

8. Pharmacology 8.1.Metabolism Sulphameraz i n e u n d e r g o e s t h r e e main t y p e s of m e t a b o l i c t r a n s f o r m a t i o n t h e s e bei n g a c e t y l a t i o n , g l u c u r o n a t i o n a n d hydroxylation. Acetylation i s the m o s t important of t ese t r a n s f o r m a t i o n s t h e p r o d u c t b e i n g t h e N -acetyl derivative. The p r o c e s s t a k e s place i n the liver t o varying degrees i n man, monkeys, m i c e , r a t s , r a b b i t s a n d most o t h e r a n i m a l s e x c e p t dogs. Various a s p e c t s of t h e metabolism o f sulphamerazine have been d i s c u s s e d . 132-134-

k4

8.2.Absorption,DistributionIExcretion 8.2.1.In

Humans

Sulphamerazine i s absorbed c h i e f l y from the g a s t r o i n t e s t i n a l t r a c t following oral a d m i n i s t r a t i o n a n d h a s a t e n d e n c y t y e more r a p i d l y a b s o r b e d t h a n 1 2 p p h a d i a z i n e 31:

.

Murphy and co-worke rs reported certain o b s e r v a t i o n s on t h e a b s o r p t i o n , d i s t r i b u t i o n and e x c r e t i o n o f s u l p h a m e r a z i n e f o l l o w i n g o r a l , subcutaneous , i n t r a v e n o u s and r e c t a l a d m i n i s t r a t i o n t o humans. S t u d i e s on t h e d i s t r i b u t i o n o f 1 5 y l p h a merazine have been d e s c r i b e d . Iiri de monstrated the e x c r e t i o n of sulphamerazine i n t o t h e h y q p p a r o t i d s a l i v a , R u m l e r and co-workers demonstrated t h e r a p i d t r a n s p o r t of s u l p h a m e f g 6 i n e a c r o s s t h e human p l a c e n t a , and Boger studied the extent t o which d i f f u s i o n of s u l p h a m e r a z i n e and o t h e r sulphonamides i n t o t h e c e r e b r o s p i n a l f l u i d depended on t h e i r c o n c e n t r a t i o n s i n t h e An e x t e n s i v e stu d y of t h e c i r c u l a blood. t i o n of s u l p h o n a m i d e s , i n c l u d i n g s u l p h a m e r a z i n e , i n t h e human o r g a n i s m h a s been r e p o r t -

SU LPHAME R A 2 IN E

e d by A l l i n e

140

563

.

A comparison o f t h e r e n a l e x c r e t i o n r a t e s o f s u l p h a m e r a z i n e and s u l p h a d i a z i n e i n human a d u l t s w i t h n o r m a l l t f n a l f u n c t i o n h a s been c o n d u c t e d by E a r l e Sulphameraz i n e e x h i b i t e d a lower o v e r a l l c l e a r a n c e r a t e indicating extensive reabsorption v i a t h e r e n a l t u b u l e s and B i n d i n g t o plasma p r o t e i n s whereas t h e N - a c e t y l d e r i v a t i v e was e x c r e t e d r a t h e r t h a bsorbed. f45e acompared t h e reR e i n h o l d and co-workers n a l c l e a r a n c e s o f s u l p h a m e r a z i n e and s e v e r a l o t h e r s u l p h o n a m i d e s i n man w i t h t h a t o f i n u l i n ( n o n - r e a b s o r b e d by t h e r e n a l t u b u l e s )

.

.

8 . 2 . 2 . In Animals The a b s o r p t i o n and e x c r e t i o n o f s u l p h a as been m e r a z i n e i n m i c e , r a t s and monkey s t u d i e d by Schmidt and c o - ~ o r k e r s * ~ ’ , t h e r e s u l t s b e i n g i n good a g r e e m e n t Y3k h f ot hl looswe o b t a i n e d by Welch and co-workers i n g e x p e r i m e n t s i n a n i m a l and human s u b j e cts. F l o r e s t a n o and co-workers 144 compared t h e b l o o d c o n c e n t r a t i o n s p r o d u c e d i n dogs , swine and c a t t l e f o l l o w i n g t h e p a r e n t e r a l a d m i n i s t r a t i o n o f s ulphame r a z i n e and s e v e ra 1 o t h e r s u l p h o n a m i d e s . The t i s s u e r e s i d u e d e p l e t i o n of s u l p h a m e r a z i n e i n s h e e p h a s been i n y s S t i g a t e d by R i g h t e r and coworkers P r i o r t o t h i s work L e h r 146 demonstrated t h e d i s t r i b u t i o n of sulphameraz i n e ( i n a t r i p l e sulphonamide m i x t u r e ) i n t h e b l o o d , l u n g and b r a i n of r a t s and r a b b its.

.

The mechanism o f t h e r e n a l t u b u l a r exc r e t o r y t r a n s p o r t o f s e l e c t e d sulphonamides h a s b e e n l q j s c u s s e d by Despopoulos and Callahan

.

564

RICHARD D. G.WOOLFENDEN

8.3. Toxicity 8.3.1.Acute

Toxicity

When g i v e n o r a l l y t o w h i t e m i c e as t h e of sulphamerazine w a s sodium s a l t t h e LD a b o u t 2 . 3 3 5 / k g . , a?? d e a t h s o c c u r r i n ithin 2 4 hours S c h m i d t and c o - w ~ r k e r s ~ ~ ~ h a v e d i s c u s s e d t h e r e l a t i v e t o x i c i t i e s of s u l p h a m e r a z i n e , s u l p h a m e t h a z i n e and s u l p h a d i a z i n e . The o r a l a c u t e t o x i c i t y of s u l p h a m e r a z i n e i n mice w a s f o u n d t o be 3 . 3 g . / k g . a t a corresp o n d i n g b l o o d 4 c o n c e n t r a t i o n of 148mgm. %.The LD50 of t h e N - a c e t y l d e r i v a t i v e was 0 . 7 g . i kg. a t a c o r r e s p o n d i n g b l o o d l e v e l of 6 6 mgm.%.

.

8.3.2.Chronic

Toxicity

Welch and co-workers 135 h a v e s t u d i e d t h e chr onic t o x i c i t y of sulphamerazine i n r a t s , dogs and monkeys. The c o m p a r a t i v e chr onic t o x i c i t i e s of sulphamerazine, sulphad i a z i n e and sulphamethazine p p j been r e p o r t e d by Schmidt and c o - w o r k e r s

.

8.3.3.Clinical

Toxicity

The v a r i o u s t o x i c m a n i f e s t a t i o n s which have been o b s e r v e d d u r i n g t h e c l i n i c a l u s e of s u l p h a m e r a z i n e i n c l u d e r e n a l damage, a c u t e l o i n p a i n , n a u s e a and v o m i t t i n g , s k i n r a s h , f e v e r , leY%pf$,a, t h r o m b o c y t o p e n i a , a n d psychosis Of a l l t h e s e m a n i f e s t a t i o n s t h e p r o b l e m o f r e n a l damage h a s r e c e i v ed the greatest attention. The more common t y p e s of r e n a l damage r e s u l t e d f o l l o w i n g t h e deposition of drug and/or drug metabolite c r y s t a l s i n t h e k i d n e y and u r i n e ( c r y s t a l l u r i a ) . S u l p h a m e r a z i n e i t s e l f h a s b e e n sj$yn f t 3 p r o d u c e r e n f48dfqnage i n b o t h a n i m a l s , b u t as w i t h o t h e r s u l and humans phonamides t h e i n c i d e n c e o f r e n a l damage h a s b e e n r e l a t e d t o t h e pg d e p e n d e n t s o l u b i l i t y o f t h e d r u g and i t s N - a c e t y l d e r i v a t i v e ( s e e section 2 . 1 1 . 1 ) . The a d m i n i s t r a t i o n of an a l k a l i w i t h t h e d r u g h e l p e d t o oy5fcome t h e p r o b l e m of c r y s t a l l u r i a b u t L e h r pointed o u t t h a t adequate a l k a l i z a t i o n cannot always be accomplished i n e v e r y p a t i e n t s i n c e i n

.

SULPHAMERAZ INE

565

c e r t a i n c a s e s s u c h a s c a r d i a c and r e n a l i n s u f f i c i e n c y a l k a l i z a t i o n was c o n t r a i n d i c a t ed. The i n c i d e n c e o f r e n a l damage w a s e v e n t u a l l y ove rcome w i t h t h e adv t r i p l e s u l p h o n a m i d e f o r m u l a t i o n ss 8 f l ? m s . 9.

P r o t e i n Bindinu The r e l a t i o n s h i p between t h e b l o o d l e v e l s a t t a i n e d by s u l p h a m e r a z i n e and i t s deg r e e of b i n d i n y 5 f ~p l a s m a h a s b e e n d i s c u s s In v i t r o experiments e d by G i l l i g a n c o n d u c t e d w i t h pH 7 . 4 b l o o d p l a s m a c o n t a i n i n g 10mgm.% of s u l p h a m e r a z i n e and 7 % of p r o t e i n r e v e a l e d t h a t o n l y 1 6 % of t h e d r u g wasl,€geely d i f f u s i b l e . Beyer and co-workers d u r i n g s t u d i e s on t h e r e n a l e l i m i n a t i o n o f s u l p h a m e r a z i n e by t h e dog showed t h a t a t plasma l e v e l s o f 6 mgm.% t h e p r o p o r t i o n bound t o plasma p r o t e i n w a s 3 6 . 5 % . D i a l y s i s and e l e c t r g k j o r e s i s were u s e d by Dessi and B a r a t t i n i t o determine t h e int e r a c t i o n o f s u l p h a m e r a z i n e w i t h t h e serum p r o t e i n of t h e r a b b i t . The f a c t o r s i n f l u e n c i n g t h e d e g r e e of b i n d i n g were t h e d e g r e e of i o n i z a t i o n of t h e d r u g and t h e pH of t h e medium. I n v i v o , s u l p h a m e r a z i n e was f o u n d t o b e bound t o t h e p r o t e i n t o t h e e x t e n t of 3%.

.

-

S cho 1t a n 15*showed t h a t t h e p r o t e i n sulphonamide r a t i o i n human and a n i m a l serums f o l l o w e d t h e F r e u n d l i c h a d s o r p t i o n i s o t h e r m . A r e l a t i o n between b i n d i n g capa c i t y , t i s s u e d i s t r i b u t i o n and c u r a t i v e act i o n w a s demonstrated.

The i n t e r d e p e n d e n c e between t h e e l i m i n a t i o n by g l o m e r u l a r f i l t r a t i o n a n d p l a s ma p r o t e i n b i n d i n g of some s u l p h o n a m i d e was examined by P o r t w i c h and co-workers 9 5 9 P r o t e i n b i n d i n g w a s measured w i t h a n u l t r a c e n t r i f u g e a n d t h e e l i m i n a t i o n r a t i o by i n u l i n c l e a r a n c e under t u b u l a r blockade. With s u l p h a m e r a z i n e , which i s r e s o r b e d b u t n o t s e c r e t e d , t h e k i d n e y e l i m i n a t i o n was f o u n d t o b e d e p e n d e n t on t h e d e g r e e of p r o t e i n binding.

.

566

RICHARD

D. G. WOOLFENDEN

M o r i g u c h i and c o - w o r k e r s studied t h e b i n d i n g of sulphonamides, i n c l u d i n g s u l p h a m e r a z i n e , t o b o v i n e serum a l b u m i n dem o n s t r a t i n g a c o r r e l a t i o n between b i n d i n g constant, decreased i n v i t r o b a c t e r i o s t a t i c a c t i v i t y and pKa. In aq6fxtension of t h i s work Wada and M o r i g u c h i s p e c t r o Q h ot o m e tr i c a l l y e v a l u a t e d t h e b in d in g of N - a c e t y l s u l p h o n a m i d e s f g 2 b o v i n e s e r u m a l b u m i n .Agren a l s o showed a c o r r e l a t i o n and c o - w o r k e r s between pK,, pH and b i n d i n g t o human a l b u m i n in vitro. The d e g r e e of b i n d i n g o f s u l p h a m e r a z i n e p r e s e n t e d as a f u n c t i o n o f p H i n c r e a s e d from t h e a c i d i c t o t h e b a s i c s i d e of t h e pK v a l u e i n d i c a t i n g t h a t t h e a n i o n i c form i g more bound t h a n t h e u n c h a r g e d species. The r e l a t i o n s h i p between s t r u c t u r e a n d b i n d i n g of s u l p h o n a m i d e s t o b o v i n e serum albumin w a s s t u d i e d by Hsu and co-worker$63. u s i n g a f l u o r e s c e n c e p r o b e t e c h n i q u e . The wyrk e s t a b l i s h e d t h a t t h e s u b s t i t u e n t a t t h e N - p o s i t i o n p l a y e d an i m p o r t a n t r o l e i n t h e b i n d i n g t o h y d r o p h o b i c p r o t e i n s i t e s . The methyl group a t t h e 4 - p o s i t i o n w i t h i n t h e pyrimidine r i n g of sulphamerazine a p p a r e n t l y s i g n i f i c a n t l y increases t h e binding of the drug t o albumin. O t h e r s t u d i e s on t h e b i n d i n g o f s u l p h a have b e e n r e p o r t -

mepg&gg8to p r o t e i n s ed 10. P ha rmacodyn ami c s

The k i n e t i c mechanisms o f t h e a b s o r p t i on o f t h e s u l p h o n a m i d e s t h r o u g h t h e l i p o i d a1 b a r r i e r and t h e r e l a t i o n s h i p o f a b s o r p t i o n r a t e s and oil-water p a r t i t i o n c o e f f i c i e n t h a s 9 g e n i n v e s t i g a t e d by Koizumi a n d co-workers An a b s o r p t i o n r a t e vs. pH p r o f i l e w a s o b t a i n e d from e x p e r i m e n t s i n which m a l e r a t s were o r a l l y d o s e d w i t h s o l u t i o n s of t h e d r u g a t v a r i o u s pH v a l u e s . S u l p h a m e r a z i n e e x h i b i t e d a v a r i a b l e r a t e of a b s o r p t i o n , t h e r a t e r e a c h i n g a maximum a t a r o u n d pH 6-7 a n d t h e n f a l l i n g o f f u n d e r m o r e a l k a l i n e c o n d i t i o n s showing t h a t t h e u n i o n i z e d form w a s a b s o r b e d predominantly.

.

SULPHAMERAZINE

567

However, a c c o r d i n g t o t h e o r y t h e pH a t which s u l p h a m e r a z i n e was c o m p l e t e l y unionized w a s c a l c u l a t e d t o be 4 . 7 . T h i s d i s c r e p a n c y w a s a t t r i b u t e d t o cert a i n c h a r a c t e r i s t i c s of g a s t r i c j u i c e and t h e s i t e o f a b s o r p t i o n i n t h e stomach. The a b s o r p t i o n r a t e of t h e u n i o n i z e d form o f l s u l p h a m e r a z i n e was f o r d t o be 0 . 0 7 h r . compared t o 0 . 0 9 h r . f o r sulphamerazine. T h i s and o t h e r k i n e t i c d a t a gave a l i n e a r c o r r e l a t i o n w i t h t h e r e c i p r o c a l of t h e p a r t i t i o n c o e f f i c i e n t d e t e r m i n e d between i s o amyl a c e t a te and water s u g g e s t i n g t h a t t h e elementary pr ocesses of ab so rp tio n followed t h e model shown below.

drug i n stomach

+

k2 d r u g a t __+ interface

drug i n plasma

-1

T h a t t h e h y d r o p h o b i c i n t e r a c t i o n between s u l p h o n a m i d e s and t h e i n t e s t i n a l memb r a n e formed an i m p o r t a n t f a c t o r i n t h e i r a b s o r p t f g g was shown by Nogami and coworkers A physico-chemical approach b a s e d on t h e a d s o r p t i o n of s u l p h o n a m i d e s from pH 7 . 4 a q u e o u s s o l u t i o n by c a r b o n b l a c k was u s e d a s a model. The e x p e r i m e n t s showed t h a t t h e i n t r o d u c t i o n o f a m e t h y l group i n t o t h e pyrimidine r i n g , a s i n t h e case of sulphamerazine, n o t only increased t h e a d s o r p t i o n on t o c a r b o n b l a c k b u t a l s o i n c r e a s e d t h e b i n d i n g t o b o v i n e serum a l bumin and i n c r e a s e d t h e r a t e of a b s o r p t i o r ) from t h e r a t s m a l l i n t e s t i n e . A good c o r n r e l a t i o n was a l s o o b t a l n e d between t h e deg r e e o f a b s o r p t i o n and t h e p a r t i t i o n coe f f i c i e n t i n n-butanol w a t e r .

.

A u g u s t i n e and S w a r b r i c k l 7 O u s e d a t h r e e - p h a s e model c e l l e m p l o y i n g an i s o pentyl acetate liquid l i p i d b a r r i e r t o test t h e i n v i t r o t r a n s p o r t rates of a series

568

RICHARD D. G . WOOLFENDEN

of N1-substituted h e t e r o c y c l i c salphonamides, i n c l u d i n g s u l p h a m e r a z i n e . C o r r e l a t i o n s were found between t h e i n v i t r o t r a n s p o r t r a t e s ( d e t e r m i n e d as a f u n c t i o n o f p H ) , p a r t i t i o n c o e f f i c i e n t s i n isopentyl acetate-aqueous b u f f e r , and i n v i v o g a s t r i c , i n t e s t i n a l and r e c t a l a b s o r p t i o n d a t a . The s t u d i e s i n d i cated t h a t t h e maximum r a t e o f t r a n s p o r t o c c u r r e d a t a pH i n t e r m e d i a t e between t h e two pK v a l u e s o f e a c h d r u g and t h a t it w a s relate8 t o t h e f r a c t i o n of unionized drug. The u s e o f h i g h p e r f o r m a n c e l i q u i d chromatography f o r q u a n t i t a t i v e s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s of sulphonamides h a s been i n v e s t i g a t e d by Henry and co-workers171. The r e t e n t i o n volumes f o r a g r o u p o f s u l phonamides which i n c l u d e d s u l p h a d i a z i n e , s u l p h a m e r a z i n e and s u l p h a m e t h a z i n e w e r e obt a i n e d i n t h r e e d i f f e r e n t H.P.L.C. columns and s u b s e q u e n t l y c o r r e l a t e d w i t h l o g p a r t i t i o n c o e f f i c i e n t ( n - o c t a n o l - w a t e r ) ,pKa, and biological activity. T a r a s z k a and F o r i s t l ’ * d i s c u s s e d s u c h k i n e t i c a s p e c t s as h a l f l i v e s f o r a b s o r p t i o n and e l i m i n a t i o n as w e l l a s l i m i t i n g s o l u b i l i t i e s i n connection with the administrat i o n of t h e t r i p l e s u l p h a s s u l p h a d i a z i n e , s u l p h a m e r a z i n e and s u l p h a m e t h a z i n e Two s i m p l e h y p o t h e t i c a l cases were p r e s e n t e d : a ) t h e s e l e c t i o n of t h e r a t i o of t w o d ru g s with d i f f e r e n t rate c o n s t a n t s f o r absorpt i o n and e l i m i n a t i o n t o o b t a i n a v e r a g e a s y m p t o t i c serum l e v e l s o f e a c h d r u g on mult i p l e d o s e a d m i n i s t r a t i o n and b ) t h e select i o n of t h e r a t i o of t w o d ru g s w i t h d i f f e r e n t r a t e c o n s t a n t s f o r a b s o r p t i o n and e l i m i n a t i o n , and d i f f e r e n t s o l u b i l i t i e s t o minim i s e t h e r i s k o f c r y s t a l l u r i a . The l a t t e r was e x t e n d e d t o t h e t r i p l e s u l p h a s on t h e b a s i s o f s o l u b i l i t y and human blood d a t a g i v i n g an optimum r a t i o o f 1:3:4 f o r s u l p h a d i a z i n e , s u l p h a m e r a z i n e and s u l p h a methazine r e s p e c t i v e l y .

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11. Acknowledgements The a u t h o r w i s h e s t o t h a n k M r . J . E . F a i r b r o t h e r of E.R. S q u i b b and Sons L t d . , Moreton, England f o r h i s e d i t o r i a l assi s t a n c e i n t h e p r e p a r a t i o n o f t h i s prof i l e and M r s . M. Watson f o r h e r i n v a l u a b l e h e l p and p a t i e n c e i n t h e t y p i n g o f t h e manuscript.

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RICHARD D. G.VdaOLFENDEN

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71. 72. 73. 74. 75.

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, .

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76. 77. 78. 79.

80. 81. 82. 82A. 83. 84. 85. 86. 87. 88. 89. 89A. 90. 91. 92. 93. 94. 94A. 95. 96. 97.

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111. 112. 113. 114. 115. 116.

117. 118.

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.

.

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575

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119. -120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143.

2, 16-21,(1969). C.Garber, Proanalysis, Through C.A. 74,9124b. 11, 1142-46, Y.Okazaki, Bunseki Kagaku, (1962). 11, 1239-42, Y.Okazaki, Bunseki Kagaku, (1962). A.L.Woodson, Anal.Chem. ,42, 242-8, (1970). B. Donev, Nauchni Tr. ,V i s G h Veterinarnomed Inst. Sofia, 23, 431-8, (1973). N.Hazemoto, N z a m o , and Y.Kobatake,J.Pharm. Sci. 65, 435-6, (1976). G.CanGlli Forti and M.E.Fracasso, Riv. Farmacol. Ter. 2, 301-7, (1971). M.Shibata, K.Shigemori hnd Y.Imamura,Chemotherapy (Tokyo), 22, 1424-9, (1974). A.Sinha, J.Proc.Inst.Chem.(India), 3,2824, (1959). B Ortengren and L. R. Treiber, Res Commun . Chem. Pathol. Pharmacol., 2, 339-57,(1974). 17, 1-21, (1972) J.Rieder, Chemotherapy, J.Rieder, Chemotherapy, 22, 84-87, (1976). F.Hawking and J.S.LawrenZ,The Sulphonamides. H.K.Lewis and Co.,Ltd.,London, (1950). J.V.Scudi and Y.C.Jelinek,J.Pharmacol1811 218-23, (1944). J.N.Smith and R.T.Williams, Biochem J. ,42, 351-56, (1948). H.G.Bray, H.J.Lake, and W.V.Thorpe, Biochem. J. 48, 400-6, (1951). A.DZelch, P.A.Mattis, A.R.Latven,W.M.Benson and E.H.Shiels, J.Pharmaco1.77,357-911 (1943) . F.D.Murphy, J.K.Clark and H.F.Flippin, Am. J.Med. Sci - 1 - 205, 717-26, (1943). K.Iiri, Shika Igaku, 31, 96-125, (1968). W. Rumler , H .J .Woraschk, I.Ri chter,C A. Gruendig, and W.Weigel,Gesundheitsw, 25,19003, (1970). W.P.Boger, Antibiotic Med. and Clin.Therapy, 6, 32-40, (1959) M.Allinne, A n n . p h a r m . f r a n c . 4 , 5 6 - 7 2 , ( 1 9 4 6 ) . D.P.Earle, J.Clin.Investiga~ion,23,914-2Ol (1944). - 279-87, J.G.Reinhold, J.Pharmacol.83, (1945). L.H.Schmidt, H.B.Hughes,E.A.Badger, and 1,17-42,(1944). I.G.Schmidt, J . P h a r m a c o 1 . 8-

.

.

.

.

-

576

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144.

H.J.Florestano, M.E.Bahler, H.E. Blair, and G.R.Burch, N.Amer.Vet. ,34, 17-20, (1953). 145. H.F.Righter, J.M.WorthiGton,and H.D.Mercer, J.Agr.Food Chem.,20, 876-78, (1972). 601-04,(1950). 146. D.Lehr,Brit.Med.J,2, 147. A.Despopoulos and PTX.Callahan, Am.J. 203, 19-26, (1962). Physiol. 148. J.K.Clark, H.F.Flippin, and F.D.Murphy, Am.J.Med.Sci. ,205,846-51(1943). 149. W.H.Hal1 and W.W.Spink, J.Am.Med.Assoc., 123, 125-31, (1943). 150. H.F.Dowling, E.Dunnoff-Stanley, M.H.Lepper, - 103-5, and L.K.Sweet, J.Am.Med.Assoc. ,125, (1944). 151. D.Lehr, J.Uro1. 55, 548, (1946). 152. R.E.Irvine, Clin.J.,E, 319, (1950). 153. A.L.Sahs, Neurology, 1, 394, (1951). 154. G.Hagerman, Nord. MedT,g, 1223,(1944), 241 1944, (1944). 64 ,393-401, 155. D.Lehr, Proc.Soc .Exptl.Biol.Med. ,(1947). 155A. D.R.Gilligan, J.Pharmacol.79, 320-8, (1943). 156. K.H.Beyer, L.Peters, E.A.P=ch and H.F. RUSSO, J.Pharmacol182, 239-46,(1944). 157. P.Dessi and M.A.Bar=tini, Boll.Soc.Ita1. Biol.Sper. 36, 595-98, (1960).Through C.A. 57 ,13126e. 15 8. W. Scholtan, Arzne imitte 1-Forsch.11, 707-20, (1961).Through C.A. 56,5336e. 159. F-Portwich, H.Buettner, and K.Engelhardt, 41, 447-51(1963).Through Klin.Wochschr, C.A.59,12043~. 160. I.Moriguchi, S.Wada and T.Nishizawia,Chem. Pharm.Bul1. (Tokyo), 16, 601-5 (1968). 161. S.Wada and I.Moriguchi, Chem.Pharm.Bul1. (Tokyo), 16, 1440-4, (1968). 162. A.Agren, R.Elofsson, and S.O.Nilsson, Acta - 48-56, Pharmacol. Toxicol, Supp1.,29, (1971). 163. P.Ilsu, J.K.H.Ma, H.W.Jun, and L.A.Luzzi, J.Pharm.Sci. ,63, 27-31, (1974). 164. J.Rieder, ArzGimittel-Forsch.,13r 81-8, (1963). 165. K.Marcinkowski, Wiss,Z.Karl-Marx-Univ.Leipzig , Math-Naturwiss. Reihe , 17, 85-6 , (1968). 166. R.Elofsson, S.O.Nilsson, and B.Kluczykowska, Acta Pharm. Suecica,8,465-74, (1971). 8I 2 1- 5 , ( 197 2 ) 16 7. F P Meyer ,Ac ta B io1.fied Ger .2 -

..

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168. 169. 170. 171. 172.

,c,

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.

TRIAMCINOLONE HEXACETONIDE

VIadirnir Zbinovsky and George P. Chrekian

VLADlMlR ZBINOVSKY AND GEORGE P. CHREKIAN

580

CONTENTS

1.

Description

1.1 Name, Formula, Molecular Weight 1.2 Appearance, C o l o r , Odor 2.

Physical Properties 2.1

Infrared Analysis Nuclear Magnetic Resonance Spectrum U1 traviolet Spectrum Mass Spectrum Optical Rotation Melting Point Thermogravimetric Analysis Differential Thermal Analysis Solubility 2.10 Crystral Properties

2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

3.

Synthesis

4.

Stability, Degradation

5.

Pharmacodynamic Studies

6.

Methods of Analysis 6.1 6.2 6.3 6.4 6.5

Elemental Analysis Direct Spectrophotometric Analysis Colorimetric Analysis Polarographic Analysis Chromatographic Analysis 6.51 6.52

Thin Layer Column

TR IAMCINOLONE HEXACETONIDE

58 1

Trlamclnolone Hexacetonlde 1.

Description 1.1

Name, Formula, Molecular Weight

Triamcinolone hexacetonlde is 9-Fluor0-118,16a,l7, 21-tetrahydoxypregna-l,4-dlene-3,2O-dlone cyclic 16,17-acetal It I s also known as with acetone 21-(3,3-dimethyl-butyrate). Pregna-l-4-diene-3,2O-dione, 21-(3,3-dlmethyl-1-oxobutoxy)-9f luoro-ll-hydroxy-16,17-[ (1-methylethy1idene)bls (oxy)I-, (118, 16a)-.

*'CH*OCOCHzC(CH,)3 19

c3 0 H4 1 Fo7

1.2

1

MOL. Wt.:

532.65

Appearance, Color, Odor White to cream colored, odorless crystalline powder.

2.

Physical Properties 2.1

Infrared Analysis]

The infrared spectrum of trlamclnolone hexacetonlde (Lederle House Standard No. 48550-115) I s presented In Figure

582

VLADlMlR ZBINOVSKY AND GEORGE P. CHREKIAN

1. The spectrum was taken in a KBr pellet. The following bands (CM-1) were assigned to triamcinolone hexacetonide: a.

b. c. d.

Characteristic 20-one: 1745 Characteristic OAc: 1715 Characteristic Characteristic

for 21-OAc=0 in the presence of for 20-one in the presence b f 21 for c1, B unsaturated 3-One: 1664 for double bond system, A-1; 4 :

1 6 1 8 , 1605

e. f.

Characteristic for C-0 stretching bands of 1 6 ; 17 acetonide: 1078, 1063 Characteristic for Cis CH of the A - 1 , 4 system: 890

2.2

Nuclear Magnetic Resonance Spectrum’

The NMR spectrum Figure 2 was obtained by dissolving 40 mg of Lederle House Standard No. 48550-115 in 0 . 5 ml of deuterochloroform plus one drop of hexadeutero dimethyl sulfoxide. Tetramethyl silane was added to the solution as internal standard. The spectrum is a single scan on an HAl O O D Varian Spectrometer. The spectral assignments of triamcinolone hexacetonide are shown in Table I. 2.3

U1 traviolet Spectrum

The X max. of the triamcinolone hexacetonide 15,500. (Lederle House Standard No. 48550-115) is 238 nm, E

FIGURE 1 Infrared Spectrum of Triamcinolone Hexacetonide in KBr Pellet; Instrument: Perkln - Elmer 21

FIGURE 2

NMR Spectrum of Triamcinolone Hexacetonide Containing Tetramethylsilane as Internal Standard. Instrument: HA-100D

Ln m P

TR I AMCINOLONE HEX ACETON I DE

585

TABLE I

1

NMR Spectral Assignments of Triamcinolone Hexacetonide

Protons at

Chemical Shift ( 5 )

C1

7.29

d; J1,2

c2

6.34

dd; J1,2 J2,4

c4

6.14

m

c11

4.41

m

c16

5.03

m

C18

0.97

S

c19

1.58

8

c2 1

4.86

d

c2 1

5.07

d

Acetonide Methyl

1.24

s

Acetonide Methyl

1.44

S

2.34

S

1.08

S

p

10.0

m

10.0, 2.0

Jg em = 1 9 ABq

Side Chain at C ~ L 0 C CH2

a

s = singlet; d = doublet; m = multiplet; ABq = AB quartet; dd = doublet of doublets; J = coupling constant in Hz

2.4

Mass Sprectruml

The mass spectrum of triamcinolone hexacetonide was run on an AEI MS-9 instrument and is shown in the Figure 3 . The molecular i o n at m/e 532 is of low intensity. The major fragment ions in the high mass region are observed at m/e 517 (loss of CH3), 512 (loss of HF) 474 (loss of C3H6O), 459 (108s of CqHg0). The base peak in the spectrum appears at m/e 375

FIGURE 3 Low Resolution Mass Spectrum of Triamcinolone Hexacetonide. Instrument: AEI MS-9

3 75

*a 30

en 10

. , . . 50 SPECC

. . . . . . . . . . . . . . . . . . . . . . . too

150

37811 L R T R I R N C I N O L O N E H E X R C E T O H I D E

900

250

(

.

~

S T E P nRSS;l.

.

. 350

300 I*8,S

.

1%

,

.

.

. Lloo

.

,

.

~, 950

..

. . . , 500

,

.

I

550

TnrAMClNOLONE HEXACETONIDE

587

and results from cleavage of the bond between C-17 and C-20 with loss of CeH1303. Intense ions at m/e 122 and 121 are indicative of a CL-GYS conjugated dimone in the A ring. 2.5

Optical Rotation

The optical rotation was determinedl for triamcinolone hexacetonide in chloroform solution at conc. 1.13%. [ a ] 25

D

2.6 271

-

272' 2.7

+

goo + 2

Melting Point The melting point of triamcinolone hexacetonide is (decomposition). Thermogravimetric Analysis2

A thermal gravimetric analysis was performed on triamcinolone hexacetonide on a House Standard (No. 48550-115) using a DuPont Model 950 instrument revealed < 0.2% weight loss up to 18OoC indicating no significant amount of volatile matter such as water and low boiling organic solvents. The analysis was performed using a nitrogen sweep and a programmed heating rate of 5'C/min. 2.8

Differential Thermal Analysis2

Differential thermal analysis on triamcinolone hexacetonide (House Standard) using a DuPont Model 990 instrument gave a thermogram displaying aosingle sharp meltingdecomposition endotherm centered at 300 with no indication of any other phase change. The heating rate was programmed at a rate of 1O0C/min. 2.9

Solubility

Solubility determinations at 25OC were carried out on Lederle House Standard No. 48550-115 and are presented in Table 11.

588

VLADlMlR ZBINOVSKY AND GEORGE P. CHREKIAN

TABLE I1 SOLUBILITY OF TRIAMCINOLONE HEXACETONIDE AT 25OC. %

mdml

WIV

H20

0.5

0.050

Hexane

1.3

0.130

Benzene

4.2

0.420

MeOH

6.5

0.650

1-Oc t a n o l

7.3

0.730

E t h y l A c e tate

7.9

0.790

1-Bu t ano 1

11.3

1.130

Abs. Ethanol

11.4

1.140

1-Propanol

11.5

1.150

Dioxane

21.5

2.150

Methyl-Ethyl Ketone

35.4

3.540

Acetone

36.6

3.660

172.6

17.260

Solvent

Chloroform

2.10 C r y s t a l P r o p e r t i e s Triamcinolone hexacetonide does n o t form polymorphic forms when r e c r y s t a l l i z e d from s o l v e n t s used f o r demonstration of polymorphism i n tria mc in o lo n e 3 and triamcinolone d i a c e t a t e 4 , Mesley5 who in s p e c te d tr ia mc in o lo n e a c e t o n i d e by i n f r a r e d spectroscopy was n o t a b l e t o demons t r a t e polymorphic forms i n t h i s compound. The x-ray powder d i f f r a c t i o n p a t t e r n of triamcinol o n e hexacetonide6 (Led erle House Standard No. 48550-115) is pr e s e nt e d i n Table 111.

TRIAMCINOLONE HEXACETONIDE

589

TABLE I11 POWDER X-RAY DIFFRACTION PATTERN OF TRIAMCINOLONE HEXACETONIDE Relative Intensity** d (Ao)* 15.70

0.06

13.10

0.13

10.80

0.10

8.80

0.03

7.30

0.07

6.65

0.04

5.90

1.00

5.50

0.01

5.15

0.17

4.75

0.15

4.60

0.01

4.34

0.05

4.13

0.05

3.63

0.07

3.44

0.03

3.32

0.02

3.10

0.12

2.63

0.05

2.58

0.01

2.47

0.03

2.37

0.05

2.14

0.01

2.08

0.02

*d = (interplanar distance)

nX

2 sin 8 , X = 1.539A0

**Based on highest intensity of 1.00 Radiation: Kal, and Ka2 Copper 3.

Synthesis Trlamclnolone acetonide, whose synthesis was described

590

V L A D l M l R ZBINOVSKY AND GFORGF P. CHREKIAN

p r e ~ i o u s l y ~is , ~used as starting material for synthesis of trlamclnolone hnxacetmide. The synthesis consists of reacting triamcinolcneoacetonide with tert, butylacetyl chloride in pyridine at +4 C and is shown in Figure 4 . 4.

Stability, Degradation

Triamcinolone hexacetonide seems to be quite stable vivo, no enzymatic deacetonization or deesterification was observed and 902 of the compound was excreted unchanged in dogs. Triamcinolone hexacetonide is very stable as a solid. It does not lose its physical appearance and chemical potency when stored at room temperature for more than ten years in an absence of ltght.

It has been reported1' that hydrocortisone and prednisolone when exposed to ultraviolet light or ordinary fluorescent laboratory light in alcoholic solution undergo photolytic degradation of the A-ring, Since triamcinolone hexacetonide has the same A-ring as prednisolone it probably also is labile under these conditions.

L. L. Smith et a 1 reported15 that the 21-acetate group in triamcinolone diacetate is easily split off with subsequent oxidation rearrangement and degradation of one side chain in mildly alkaline solution. Since triamcinolone hexacetonide also has an ester group on 21-carbon, it is probable, that this side chain can be easily hydrolysed by the similar mechanism. 5.

Pharmacodynamic Studies

In a single intravenous dose of the radioactive triamcinolone hexacetonide administered to the dog, the plasma concentrations of total and ether extractable radioactivity exhibited a biphasic disappearance curve with half lives of about 0.6 to 6 hours for the initial and final phases respectively.1° Throughout the 7 hour period in which measurable concentration of radioactivity were present, the ratio of plasma to whole blood concentrations was 1.98, indicating little or no penetration to erythrocytes. In expertments with dogs and cats, 3.ess than 10% of the radioactivity of the oral dose was absorbed and 90% was excreted in feces. No deacetonization or deesterification of triamcinolone hexacetonide was observed and the compound was excreted unchanged. Only small amounts were metabolized into C14

FIGURE 4.

CH20-

I C=O

m

i

H CH, I

1

CHI -0-C-C-C-CH3

I

0 " ' H ICH, - - -

0, /CH3

o/c\

+ HCI

592

VLADlMlR ZBINOVSKY AND GEORGE P. CHREKIAN

three more polar, unidentified products. Intra-articular dose of triamcinolone hexacetonide was released from the site of injection at much slower but steady rate than was the case for triamcinolone acetonide and other related compounds. The half life of radioactivity in this case was about 60 days.

6.

Methods of Analysis

6.1 Elemental Analysis for C30H41F07, Lederle House Standard No. 48550-1152 Element

% Theory

C

67.65 7.76 3.57

H F

Found -

67.79 7.60 3.62

6.2 Direct Spectrophotometric Analysis The UV absorption maximum at 238 nm has been extensively utilized for assay purposes especially when methanol was used for elution of triamcinolone hexacetonide from thin layer chromatographic plates. Triamcinolone hexacetonide has a distinct infrared spectrum, which can be used in qualitative and quantitative analysis.

6.3

Colorimetric Analysis

Blue tetrazolium, the most common reagent used for colorimetric determination of adrenocortical steroids, cannot be applied to triamcinolone hexacetonide, since a-ketol group is not available. Isonicotinic acid hydrazide (INAH) is used instead to produce yellow derivative of the triamcinolone hexacetonide which has absorption maximum at 380 nm.12 The color is due to hydrazone formation from A 1,4 -3 keto group.

6.4 Polarographic Analysis The polarogram of triamcinolone hexacetonide was obtained by scanning the sample from (-) 0.85 Volts vs. SCE to (-)1.38 Volts vs. SCE using differential pulse mode of operation with full scale range of 3.0V. A single reduction

TRIAMCINOLONE HEXACETONIDE

593

wave appeared at Ep (-1 1.12 V. vs. SCE, when 0.1M tetrabutylammonium chloride, adjusted to pH 3.5 with phosphoric acid as supporting electrolyte was used. The concentration of triamcinolone hexacetonide was 30 ppm and a well defined peak could be obtained down to 3 ppm. Other parameters for the polargram, shown in Figure 5 were: modulation amplitude of 50 mV, scan rate 2 mV sec-l, drop rate 1 sec. -l, and a current sensitivity of 2pA full scale. 6.5

Chromatographic Analysis

6.51

Thin Layer

Separation of triamcinolone hexacetonide from 1,2-dihydro triamcinolone acetonide, 1,2-dihydro triamcinolone hexacetonide and triamcinolone acetonide present as minor components has been accomplished by this method. Silica Gel GF precoated plates (Analtech Inc.) were used with benzene, Skellysolve C, methanol and p-dioxane-water mixture as developing solvent. Development time was approximately 45 minutes. The approximate Rf values (after rechromatography) were 0.50 for triamcinolone hexacetonide, 0.21 for 1,2-dihydro triamcinolone acetonide, 0.60 for 1,2-dihydro triamcinolone hexacetonide and 0.16 for triamcinolone acetonide. Compounds were eluted with methanol and quantitatively determined spectrophotometrically at 238 w. 6.52

Column

The Chromatronix Model 3100 instrument was used for High pressure Liquid Chromatography in quantitative determination of triamcinolone hexacetonide in presence of triamcinolone acetonide. Spherical siliceous packing, was used, employing dichloromethane and isopropanol for the mobile phase. Steroids were eluted and determined at 254 m. Retention time for triamcinolone hexacetonide was 3.5 min.; triamcinolone acetonide can be eluted in 18 min. When the measured peak areas and/or peak heights of standards were plotted, a linear relationship resulted between areas or heights and concentration.

594

VLADlMlR ZBINOVSKY AND GEORGE P. CHREKIAN

Fig. 5. Differential Pulse Polarogram of Triarncinolone Hexacetonide in 0.1M Tetrabutylammonium Chloride buffer, pH 3.5

I

-1.12

10.0cm

I -0.80

1

I

I

I

I

I

-1.00

I

1

I I

-1.20

POTENTIAL (VOLTS vs S.C.E.)

I

I

-1.40

595

TR IAMCINOLONE HEXACETONIDE

REFERENCES 1.

W. Fulmor, L e d e r l e L a b o r a t o r i e s , p e r s o n a l communication.

2.

L. M. Rrancone, L e d e r l e L a b o r a t o r i e s , p e r s o n a l communicat ion.

3.

G . Michel, K. F l o r e y , A n a l y t i c a l P r o f i l e s of Drug Sub-

stance,

A, 380

(1972)

4.

L. L. Smith and M. Halwer, J. Am. Pharm. ASSOC., Ed., 48 348 (1959).

5.

R. J . Mesley, Spectrochimica Acta,

6.

P . Monnikendam, L e d e r l e L a b o r a t o r i e s , p e r s o n a l communica-

22

Sci.

889 (1966).

tion.

1,

7.

K. F l o r e y , A n a l y t i c a l P r o f i l e s of Drug S u b s t a n c e s , 397 (1972).

8.

S. B e r n s t e i n , R. H. Lenhard, W. S . A l l e n , M. Heller, R. L i t t e l l , S. M. S t o l a r , L. Feldman and R.H. Blank, J . Am. Chem. S O C . , 81, 1689 (1959).

9. 10.

M. Heller, S. S t o l a r and J . B e r n s t e i n , J. Org, Chm., 5044 (1961).

26,

J . A. Morrison, L e d e r l e L a b o r a t o r i e s , p r i v a t e communicat ion.

11. A. Michaleides, L e d e r l e L a b o r a t o r i e s , p e r s o n a l communication.

27,

12.

E. J. Umberger, Anal. Chem.,

13.

P. P. Ascione, L e d e r l e L a b o r a t o r i e s , p e r s o n a l communica-

768 (1955).

tion.

14.

15.

s,

W. E. Hamlin, T. Chuleki, R. H. Johnson and J . G . Wagn e r , J. Am. Pharm. ASBOC., 253 (1963) and D. R. Burton and W. C. T a y l o r , J . Am. Chem. S O C . , 244 (1958); J. Chem. SOC., 2500 (1958).

80,

L. L. Smith, M. Marx, J . J . J. Gabardini, T. F o e l l , V. E. O r i g o n i and J. J. Goodman, J. Am. Chem. SOC., 4616 (1960).

82,

ADDENDA AND ERRATA

598

ADDENDA AND ERRATA

Affiliations of Editors and Contributors Volume 5, p. vii Correct affiliation: Z.L. Chang, Abbott Laboratories, North Chicago, Illinois Bendroflumethiazide Volume 5, p. 13 Fig. 6, Correct formula for bendroflumethiazide (1) 0 0 \\ //

Volume 5, p. 16 Add Section 6.53: Column Chromatographic Aialysis. A column chromatographic method, using a sodium carbonate column and chloroformacetic acid ( 9 8 + 2 ) and U.V. readout has been described by F. R. Fazzari, Journal of the A.O.A.C., 59, p. 96 (1976). Propoxyphene Hydrochloride Volume 1, p. 316 Add Section 4.7:

HPLC Analysis

An HPLC method for tablets and capsules has been described by R. K. Gilpin, J. A. Korpi and C. A. Janicki, J. Chromat., 107, p. 115 (1975).

CUMULATIVE INDEX Italic numerals refer to Volume numbers. Acetaminophen, 3, 1 Acetohexamide, 1, 1;2,573 Alpha-Tocopheryl Acetate, 3, 11 1 Amitriptyline Hydrochloride, 3, 127 Amphotericin B, 6, 1 Ampicillin,2, 1;4,517 Bendroflumethiazide, 5, 1; 6,597 Betamethasome Dipropionate, 6 , 4 3 Cefazoli, 4 , 1 Cephalexin, 4 , 2 1 Cephalothin Sodhm, 1, 319 Cephradine, 5 , 2 1 Chloral Hydrate, 2, 85 Chloramplienicol, 4,47, 5 17 Chlordiazepoxide, 1 , 15 Chlordiazepoxide Hydrochloride, 1, 39; 4,517 Chloroquine Phosphate, 5,61 Chlorprothixene, 2 , 6 3 Clidinium Bromide, 2, 145 Clnnazepam, 6 , 6 1 Clorazepate Dipotassium, 4 , 9 1 Cloxacillin Sodium, 4 , 113 Cyclizine, 6 , 8 3 Cycloserine, I , 53 Cyclothiazide, 1,66 Dapsone, 5 , 87 Dexamethazone, 2, 163; 4 , 5 18 DiatrboicAcid,4, 137;5, 556 Diazepam,1,79;4,517 Digitoxin, 3, 149 Dioctyl Sodium Sulfowccinate, 2, 199 Diperodon, 6,99 Diphenhydramine Hydrochloride, 3, 173 Disulfiram, 4,168 Echothiophate Iodide, 3, 233 Ergotamine Tatrate, 6, 113 Erthromycin Estolate, 1, 101;2,573

Estradiol Valerate, 4 , 192 Ethynodiol Diacetate, 3, 253 Fenoprofen Calcium, 6, 161 Flucytosine, 5 , 115 Fludrocottisone Acetate, 3, 281 Fluorourbcil, 2, 221 Fluphenazine Enanthate, 2,245; 4 , 5 2 3 Fluphenazine Hydrochloride, 2,263; 4,518 Gluthethimide, 5 , 139 Halothane, 1 , 119;2,573 Hydroxyprogesterone Caproate, 4, 209 lodipamide, 3, 333 Isocarboxazid, 2,295 Isoniazide, 6, l b 3 Isoproparnide, 2 , 315 Isosorbide Dinitrate, 4 , 225; 5,556 Kandmycin Sulfate, 6,259 Ketamine, 6,297 Levarterenol Bitartrate, 1 , 4 9 ; 2,573 Levallorphan Tartrate, 2, 339 Levodopa, 5 , 189 Levothyroxine Sodium, 5 , 2 2 5 Meperidine Hydrochloride, 1, 175 Meprobamate, 1,209; 4 , 5 19 Methadone Hydrochloride, 3, 365;4,519 Methaqualone, 4 , 2 4 5 , s 19 Methotrexate, 5 , 283 Methyclothiazide, 5 , 307 Methyprylon, 2, 363 Metronidazole, 5,327 Minocycline, 6,323 Nitrofurantoin, 5,345 Norethindrone, 4,268 Norgestrel, 4 , 294 Nortriptyline Hydrochloride, 1,233; 2,573 Nystatin, 6, 341 Oxazepam, 3,441

599

CUMULATIVE INDEX

Phenazopyridine Hydrochloride, 3,465 Phenelzine Sulfate, 2,383 Phenformin Hydrochloride, 4,319;5,429 Phenoxymethyl Penicillin Potassium, I , 249 Phenylephrine Hydrochloride, 3,483 Piperazine Estrone Sulfate, 5,375 Primidone, 2,409 Procainamide Hydrochloride, 4, 333 Procarbazine Hydrochloride, 5,403 Promethazine Hydrochloride, 5,429 Proparacaine Hydrochloride, 6,423 Propiomazine Hydrochloride, 2,439 Propoxyphene Hydrochloride, I , 301; 4,5 19; 6,598 Propylthiouracil, 6,457 Reserpine, 4,384;5,557 Rifampin, 5,467 Secobarbital sodium, 1,343 Spironolactone, 4,431 Sodium Nitroprusside, 6,487 Sulphamerazine, 6,5 15

Sulfamethoxazole, 2,467; 4,520 Sulfasalazine, 5,515 Sulfisoxazole, 2,487 Testolactone, 5,533 Testosterone Enanthate, 4,452 Theophylline, 4,466 Tolbutamide, 3,5 13;5,557 Triamcinolone, I , 367;2,571;4,520,523 Triamcinolone Acetonide, 1,397 ;2,57 1; 4,520 Triamcinolone Diacetate, 1,423 Triamcinolone Hexacetonide, 6,579 Triclobisonium Chloride, 2,507 Triflupromazine Hydrochloride, 2,523; 4,520;5,557 Trimethaphan Camsylate, 3,545 Trimethobenzamide Hydrochloride, 2,55 1 Tropicamide, 3,565 Tybamate, 4,494 Vinblastine Sulfate, I , 443 Vincristine Sulfate, I, 463

A 8 7 C 8 0 9

€ 0 F 1 G Z H 3 1 4 J 5

600