Profiles of Drug Substances Vol 06

Analytical n Profiles Of

Drug Substances Volume 6

Edited by

Klaus Florey The Squibb Institute for Medical Research

New Brunswick, New Jersey

Contributing Editors

Norman W. Atwater Glenn A. Brewer, Jr. Jack P. Comer

Salvatore A. Fusari Bruce C. Rudy Bernard Z. Senkowski

Compiled under the auspices of the Pharmaceutical Analysis and Control Section

Academy of Pharmaceutical Sciences

Academic Press New York San Francisco London 1977 A Subsidiary of Harcourt Brace Jovanovich. Publishers

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.

ACADEMIC PRESS, INC. 11 1 Fifth Avenue, New York, New York 10003

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

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 70-1 87259

ISBN 0-1 2-260806-2 PRINTED IN THE UNITED STATES OF 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 AND 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 Phar- maceutical 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) M. Bunow (NIH) I. Asher (0s) S. Delgado(BD) B. Baer (NIH) V. Folen (BD) B. Baron (BD) C. Graichen (BF) W. Benson (BD) R. Gryder ( 0 s ) W. Brannon (BD) I. Levin (NIH) J. Blakelp (BD) M. Maienthal (BD) R. Bradky (NM) G. Mazzola (BF)

G. Schwartzman (BD) 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 eta / .

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

4.1 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

4. Spectral Properties (Other)

1.1 Drug Properties Amphotericin B is a macrocyclic, polyene anti-

biotic 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 channe l s a c r o s s ce l l membranes i s b e i n g widely i n v e s t i g a t e d (4-6). The absence 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 of Ampho- t 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.

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 hypocho les t e ro l emic agen t . Amphotericin B has a l s o been used (8) t o t rea t c a n i n e p r o s t a t i c h y p e r p l a s i a (d 30% r e d u c t i o n i n gland s i z e ) . 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 , lO) may d i scourage such 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 is 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 ) , 88 mg/kg f o r Fungizone and 1320 mglkg f o r t h e methyl ester. The corresponding in t r avenous dosages a r e ove r an o r d e r of magnitude lower ( 3 ) .

I n can ine experiments ( 7 ) , o r a l l y a d m i n i s t e r e d

1 .2 Chemical P r o p e r t i e s Amphotericin B i s an amphoter ic , mac rocyc l i c

hep taene w i t h a mycosamine suga r head group. It 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 can 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 based on i ts 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 ) .

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 s o l v e n t s (Sec t ion 2 . 3 ) . V i b r a t o r g r i n d i n g d r a m a t i c a l l y 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 i n f r a r e d a b s o r p t i o n s p e c t r a ( S e c t i o n 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 (Sec t ion 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 con t in - genc ie 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 . Th i s r e p o r t t r ies t o ana lyze 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 .

Amphotericin B is 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 -

1 .3 The U . S. Standard 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 markets t h e d rug under t h e name Fungizone. The f i n a l s t a g e s of manufacture i n c l u d e p r e c i p i t a t i o n from aqueous methanol (pH c o n t r o l l e d by H C 1 t hen 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 moi s tu re . Samples were d r i e d f o r 3 hours a t 6OoC (4 5 mm p r e s s u r e ) b e f o r e measuring potency, 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 . There 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 anda rd e x i s t s ; i t is n o t f u r t h e r

4 IRVlN M. ASHER etel.

considered here . There is a l s o an i n t e r n a t i o n a l s t anda rd (WHO) f o r Amphotericin B (12).

1 .4 Chemical Composition 1.41 Empir ical Formula and Molecular Weight

(1% = 12.000)

(a) c47 H73 N017 MW = 923.62

i n agreement wi th r ecen t x-ray (13) and mass spec t romet r i c (14) measurements; accepted by USP-XIX (15) , supersedes:

(b) c46 H73 NO20 MW = 959.62

repor ted i n Reference (11,16).

1.42 Elemental Composition

(a ) C47 H73 NO17 r equ i r e s :

C 61.12% H 7.96% N 1.52% 0 29.45%

Reference 1 found:

C 60.40% H 8.38% N 1.62% --

with negat ive r e s u l t s f o r halogens, s u l f u r , and a c e t y l and methoxyl groups, f o r samples prepared by t h e methods of Reference 1.

(b) C46 H73 NO20 requ i r e s :

C 57.58% H 7.67% N 1.46% 0 33.34%

and Reference 1 7 found:

C 57.17% H 7.80% N 1.20% 0 29.98%

f o r un t rea ted U.S. s tandard Amphotericin B , c o n s i s t e n t wi th t h e CHN r e s u l t s of References 18,19. ( I n t h e l a t te r Ampho- t e r i c i n B w a s d r i e d 3 hours 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 tandard (3 hours , 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 those of Reference 1.

Notice t h a t t h e oxygen con ten t of Reference 17 is c o n s i s t e n t w i th 1.41(a) r a t h e r than 1.41(b) .

AMPHOTERICIN 6 5

The f u l l CHNO ana lys i s of Reference 1 7 is cons i s t en t wi th t h e hydrochloride sal t of 1 .41(a) p lus 1 . 5 waters of hydrat ion. (Variat ion i n water content a lone can only p a r t i a l l y r e so lve the d iscrepancies noted above.) However, tests (20) f o r C1 i n the U.S. s tandard were negat ive (6 0.11%).

(c ) The Karl F isher tes t gave 6 .36% water content f o r t he unt rea ted U.S. s tandard (21). The s tandard e x h i b i t s a 4-5% loss on dry ing a t 6OoC under a vacuum. A t atmospheric pressure , thermal grav imet r ic a n a l y s i s (Sect ion 2 . 1 2 ) i nd ica t e s an 3.5% weight l o s s between 60- 100°C. appears t o be incorporated i n t o the l a t t i c e ; t h e Amphotericin B de r iva t ive inves t iga t ed i n Reference 1 3 incorporated t h r e e te t rahydrofuran molecules and one water molecule per u n i t ce l l .

Although some of t h i s water may be adsorbed, some

1 .5 S t ruc tu re The following s t r u c t u r e is based on x-ray

c rys t a l log raph ic s t u d i e s of N-iodoacetyl Amphotericin B , tri- te t rahydrofuran monohydrate c r y s t a l (13). It corresponds t o formula 1.41(a) .

COOH

AMPHOTERICIN B

The r i g i d heptaene chain e longates t h e macrocycle, such t h a t one s i d e (polyene) is hydrophobic, whi le t he o t h e r s i d e ( a l i p h a t i c ) is hydroph i l l i c due t o t h e presence of seven hydroxyl groups and an ester carbonyl group. This may account f o r i ts 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 idue is a t tached t o one end, providing a f r e e amino group. There is an i n t e r n a l hemi-ketal r ing . It has been suggested (14) t h a t t he ketal-form may be i n equi l ibr ium with an open keto-form i n so lu t ion . However, recent 13C-NMR r e s u l t s (22) confirm t h e presence o f the ketal-form i n DMSO s o l u t i o n (Sect ion 4.2), and provide no evidence f o r a keto-form i n t h a t environment.

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 s e v e r a l d e t a i l s .

This s t r u c t u r e supersedes an earlier, partial

6 IRVIN M. ASHEA eta / .

1.6 Phys ica l Desc r ip t ion Bright yellow powder. Microscopic examination

r e v e a l s prisms o r needles 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 (11); b u t t h i n , i r r e g u l a r fragments (roughly 5-15 long, less than 0 . 3 p t h i c k ) i n the U.S. s tandard (25). The fragments tend t o clump i n t o l a r g e ( + 8 0 ~ diameter) c l u s t e r s . drug manufacture may a l s o convert some c r y s t a l s t o an amorphous form (24; Sec t ion 2.2) . A t y p i c a l photomicrograph of t he s tandard is shown i n Figure 1.

The g r ind ing process used i n

2 . PHYSICAL PROPERTIES 2 . 1 Thermal P r o p e r t i e s

2 . 1 1 D i f f e r e n t i a l Thermal Analysis (DTA) DTA scans (25) show a g radua l , approxi-

mately l i n e a r decrease from 35 t o 135OC wi th peaks near 157 and 209°C (Figure 2 ) . The sample begins t o decompose above 2 O O 0 C , wi thout mel t ing . The 157°C t r a n s i t i o n i s accompanied by a change i n co lor from b r i g h t yellow t o brown-orange which. begins around 130°C, and inc reases p rogres s ive ly . This presumably r e f l e c t s an endothermic chemical change invo lv ing t h e chromophore.

2.12 Thermal Gravimetr ic Analysis (TGA) TGA scans (25) show an N 3.5% weight l o s s

s t a r t i n g below 65°C which reaches completion nea r 90°C (Figure 2 ) . A f u r t h e r r educ t ion i n weight begins near 18OoC and l e v e l s of f near 220"C, wi th maximum s l o p e nea 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 spec t ive ly .

2.13 Melt ing Po in t We f ind no evidence of t he mel t ing i n

Amphotericin B up t o 250°C, a t which temperature the a n t i - b i o t i c has a l ready decomposed. This i s c o n s i s t e n t wi th Reference (l), but perhaps n o t Reference (16,18). Vaporizat ion is de tec t ed (26) above 25OoC i n a mass spec t ro - meter (vacuum 4 t o r r ) . Tr ime thy l s i ly l - e the r d e r i v a t i v e s of Amphotericin B may vapor ize 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 "unt rea ted"

(unground, unheated) U.S. s t anda rd Amphotericin B demon- s 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 d- spacings are given i n Table 1 and Figure 3 ( s o l i d curve) . Unground samples hea ted 15 minutes a t 158OC produce a p a t t e r n wi th less i n t e n s e peaks, s l i g h t l y s h i f t e d d-spacings and increased background (Figure 3, do t t ed curve) . 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.

m

U

\ J e -

E

100%

90 %

80%

/

70 DTA

I I 210

157

AMPHOTERICIN B

I I I I I I 1

120 160 200 240 280 40 80

TEMPERATURE ( " C )

Figure 2. Differential thermal analysis (DTA) and thermal gravimetric analysis (TGA) scans of Amphotericin B.

AMPHOTERICIN B 9

d (A)

18.0 9.30 7.73 7.42

* 6.30 5.82 5.14 4.82 4.65

TABLE 1 X-Ray Powder D i f f r a c t i o n Data

f o r Amphotericin B (Untreated Sample)

111, d (A) I/Io

23 3.87 17 6 3.79 16

12 3.49 1 2 10 3.33 16 9 1 3.22 13 21 2.925 B 11 33 2.775 9 1 7 2.460 B 4

7 2.370 4 5 2.315 B 4

46 2.240 B 11 90 2.040 B 7

100

T = t r i p l e t 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 e thy lene g l y c o l dimethyl formamide (1) a c e t i c a c i d (1) propylene g lyco l (1) pyr id ine methanol * isoamyl a l c o h o l water benzyl a l coho l 1.4-dioxane e thano l e t h y l e s t e r ace tone e t h y l a c e t a t e e thylene-C 1 isoamyl a c e t a t e

methyl e t h y l ketone i s o p r . a l c o h o l CHC1-j benzene c-hexane p e t . e t h e r CCl4 t o l uene iso-octane

cs2

30. - 40. 6.40 2.60 2 . - 4. 1. - 2. 1. - 2 . 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

X0.2 - 0.4 mg/ml f o r anhydrous methanol i n Reference 1.

I 1

24

AMPHOTERICIN B

632 i

I 1 I 20 16 12

28( D EG R E ES 1

Figure 3 . X-ray powder diffraction patterns of "untreated" (unheated, unground) Amphotericin B 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 (%30%). Ordinate for the magnified (x2.5) insert i s 4 x lo2 cps.

) and an aliquot heated to 158' C for 15 minutes (----).

AMPHOTERICIN B 11

changes indicate the introduction of additional strain in the crystal lattice and an increase in the amorphous (non- crystalline) fraction of the sample ( 2 4 ) . Otherwise, the two patterns are highly similar.

ground 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). 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 ) .

N-iodoacetyl derivative (tri-tetrahydrofuran monohydrate crystal) is given in Reference 13 (see Section 1.5).

In contrast, the diffraction pattern of vibrator-

Such a pattern is

A complete structural determination of the

2 . 3 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. Table 2 , unless otherwise noted, are part of a previous FDA study ( 2 7 ) .

aids solvation (1,ll):

The solubility data of

Ionization of the acidic and amino groups often

CH30H dime thylf ormamide

neutral insoluble 0 . 2 - 0 . 4 mg/ml 2-4 mg/ml acidic 0.1 mg/ml 3-5 mg/ml 60-80 mg/ml basic 0.1 mg/ml 2-3 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 sterol- containing natural membranes ( 4 - 6 ) .

2 .4 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. Amphotericin 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

Comparison with N-acetyl-

AMPHOTERICIN B (VIBRATOR GROUND) i s

- -~ - __ -~ - - . -

I I I

24 20 16 12 8 SCATTERING ANGLE, 28(DEGREES)

Figure 4. X-ray powder diffraction of Amphotericin B ground in a vibrator (3 min., 2 mg. at a time). a phase transition to an amorphous form; little crystalline Amphotericin B remains.

The dramatic decrease in peak heights and increase in background demonstrate

AMPHOTERICIN B 13

constant Kt = 1000 with r e spec t t o t h e n e u t r a l molecule) .

2.5 Aggregation Measurements (29) of t he u l t r a v i o l e t absorp t ion of

aqueous so lu t ions of Amphotericin B as a func t ion of concen- t r a t i o n do not obey the 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 aggregates 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 he presence of Na+-desoxycholate and phosphate). The aggregate mass is approximately unaffected by the add i t ion of up t o 35% C2H50H, but drops p rec ip i tous ly t h e r e a f t e r . S imi la r e f f e c t s a r e observed i n the i n t e n s i t y of t he 349, 367, 386, 409 nm u l t r a v i o l e t absorp t ion bands; however, t h e 328 nni band is a f f ec t ed by even 10% C2H50H. of exc i ton ic i n t e r a c t i o n s between t h e heptaene chromophores of t he aggregate . The aggregate mass was ca l cu la t ed using a (measured) va lue of 290. ml/mg f o r dn/dc, t he change i n the index of r e f r a c t i o n with concent ra t ion of Amphotericin B.

3. SPECTRAL PROPERTIES (OPTICAL)

The d a t a a r e explained i n terms

3.1 U l t r a v i o l e t Amphotericin B has a h ighly 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 absorp t ion spectrum i n DMSO, CH30H s o l u t i o n s (Figure 5). The sharp , i n t ense bands a r i s e from - n* t r a n s i t i o n s of the heptaene chromophore. The same spectrum occurs i n heated samples (15 minutes, 158"C), bu t with 25% less abso rb t iv i ty . The in t ense 406, 382, 363, 345 nm. qua r tup le 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 Federal Regis te 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.

( so lub i l i zed by DMSO o r Na+-desoxycholate) a r e considerably d i f f e r e n t (Figure 6 ) , and change f u r t h e r upon t h e add i t ion of l e c i t h i n and/or cho le s t e ro l (31,32). These changes appar- e n t l y r e f l e c t t he presence of l a r g e , l a b i l e aggregates i n such aqueous so lu t ions ( see Sect ion 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 absorpt ion s p e c t r a i n var ious H20: 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 water y i e l d t h r e e concent ra t ion-sens i t ive bands (33). The t r a n s i t i o n moment (or ien ted along the heptaene chain) l i e s wi th in 6" of t he water i n t e r f a c e ; t he add i t ion of cho le s t e ro l t i l t s t h i s upward t o approximately 35".

Spec t ra of Amphotericin B i n aqueous s o l u t i o n

C2H50H systems may be found i n Reference (29).

3.2 In f r a red L i t e r a t u r e s p e c t r a of Amphotericin B are contra-

We f ind t h a t both types can be obtained a t d i c to ry (1,18,34). Two b a s i c types of s p e c t r a a r e seen (Figures 7a,b) .

14 IRVlN M. ASHER era / .

I 1 ' 1 ' 1 ' I '

6

AMPHOTERICIN B (DMSO/CH30H)

303

n AMPHOTERICIN ''

363

WAVELENGTH (nm)

Figure 5. Ultraviolet absorption spectra of Amphotericins B and A in DMSO/CH30H solution (concentrations respectively 5.45, 8.32 pg/ml).

AMPHOTERICIN B 15

1 .4

1.2

0.8

0.4

300

a. H ~ O

b. H;O + CHOLESTEROL 3 C. H 2 0 + CH30H I I C .

I

a.

I I I I 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 1 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 d I I I I

350 400

WAVELENGTH (nm)

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 I I

I I I I I I I I

450

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 era / .

I I I I

A M P H O T E R I C I N 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). stretch regions resulting from differences in sample preparation.

Note the changes in the C=C and C=O

AMPHOTERICIN B 17

Type I

(625) 664 69 7 (732) 762 79 5 812 818 (837)sh 8 5 1 (878) 889 ( 8 9 8 ) s h 9 16 (9 31) s h (953) s h ( 9 7 2 ) s h ( 9 8 1 ) s h 1009 1041 10 70 1109 1132 1164sh 1186 1210sh 1233sh 1272sh ( s h ) 1324 (1338) s h (1371)sh 1381 1401 1448 1556* (1628)B 1692" (1710)sh+

( s h ) 2918d

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

NOTES:

TABLE 3 I n f r a r e d S p e c t r a

Type I1 T e n a t i v e Assignment

OH Out -of -p lane Bend ( ? )

( s h ) P y r a n o s e Ring B r e a t h i n g ( C ) (792) ( 8 0 4 ) s h

CH Bend (GI

888 CH Bend, CH3 Rock

' P y r a n o s e Ring V i b r a t i o n ( C )

CH Out-of -p lane 1010 Bend (trans p o l y e n e ) 1040

:?:6" } CO Asym. S t r e t c h (COC, COH) 1130 (1173)B (1188)B COC Asym. S t r e t c h (COC=O)

(1230) s h 1269 } CH2 Wag, Bend ( s k e l e t a l ) (1291) 1322

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

1712B* 2859*

2925* 1 (29 79 ) s h 3015

1 3 39 OB

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)

B = b r o a d , s h = s h o u l d e r , s l = s l a n t , S = s o l v e n t p e a k s , ( ) = weak, 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 = a s y m m e t r i c , * = 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 or 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.

18 IRVlN M. ASHER e t a / .

room temperature, i n the same medium ( i . e . , KBr p e l l e t o r Nujol mull) depending on t h e method of sample p repa ra t ion (24). (Figure 5a; Reference 1 ,18 ) , whi le v i b r a t o r ("wigglebug") ground powders y i e l d type I1 s p e c t r a (Figure 5b; Reference 34) o r a more even mixture of the two types (Figure 7c).

Type I s p e c t r a are cha rac t e r i zed by a sharp 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 cons iderable subs t ruc tu re (e .g . , 800-950 c m - l r eg ion) . I1 s p e c t r a a r e charac te r ized by a broad C=O s t r e t c h band nea r 1 7 1 2 cm-1, a 1566 c m - l C=C s t r e t c h band and less - reso lved subs t ruc tu re . In "mixed" spectra (Figure 5 c ) , super- p o s i t i o n g ives a C=O 1692, 1710 c m - 1 double t . Spec t ra of DMSO s o l u t i o n s conta in 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 (Sec t ion 2.2) show t h a t type I1 s p e c t r a r ep resen t an amorphous phase induced by v i b r a t o r gr inding (24); similar polymorphism has been observed i n t h e Cinchona a l k a l o i d s (35). The broad shoulder observed near 1710 cm-I i n Figure 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 tandard ( c f . 1 .3 ) . Hand- gr inding 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 .

Handground powders t y p i c a l l y y i e l d type I s p e c t r a

Type

Heating the sample t o 120°C has l i t t l e e f f e c t on the spectrum. In c o n t r a s t , t h e s p e c t r a of samples heated above t h e chemical t r a n s i t i o n near 157°C (Sec t ion 2.1) resemble Type T I , even when handground. This i s c o n s i s t e n t wi th t h e -30% inc rease i n the amorphous f r a c t i o n observed us ing x-ray powder d i f f r a c t i o n (24) .

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 given i n Table 3 . Four ie r t ransform i n f r a r e d s p e c t r a confirm the ex i s t ence of many of t he weaker peaks. The 1692 cm-1 peak is a c t u a l l y a very c lose doublet .

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 the 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 idechains from 41°C t o r ~ 4 5 " C (as monitored by frequency s h i f t s i n t h e CH s t r e t c h region; Reference 36) . Because of t he high i n f r a r e d a b s o r p t i v i t y of water , such measurements r equ i r e the use of narrow, IRTRAN sample cells .

The i n f r a r e d absorp t ion f requencies of Amphotericin

The add i t ion of Amphotericin B t o aqueous

3.3 RAMAN Laser Raman s p e c t r a of Amphotericin B (37) a r e

presented i n Figure 8 and Table 4. v i s i b l e absorp t ion resonant ly enhances modes coupled t o the chromaphore.

The presence of a s t r o n g

The in t ense peak near 1562 cm-l corresponds t o

AMPHOTERICIN B 19

CH 3 OH Solution

1 1 1 1

1800 ls00 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.


Powder

922

100 7 (1014)sh 1142

1159

1202 1298 1562 1608

16 35

(1645) sh

TABLE 4 Resonance Raman Spectra (cm-l)

CH30H Ref. (36) KBr P e l l e t Assignment

C=CC, HCC in- plane Bend

(9 80 1

995 1011 100 7

1136sh

1156

119 8 (1298) 1559 1602

16 39

(1666)

1140sh 1131sh

1161 1152

1201 (1195) 1 2 8 7

1562 1554 1607 (1597)

1624 1640 16 36

(1661)

1136sh CC S t r e t c h ,

1156 } In-plane HCC Bend (mixed with'

(1198) C=C S t r e t c h )

C=C S t r e t c h ( in t ense )

C=O S t r e t c h (mixed wi th C=C S t r e t c h )

AMPHOTERICIN B 21

almost pure C-C s t r e t c h , whereas t h e weak 1635-1645 c m - 1 modes a l s o conta in cons iderable 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 no t be observed, even us ing a dye laser. Notice t h a t s e v e r a l of t h e Raman modes are not i n f r a r e d a c t i v e (compare Sec t ion 3.2) .

So l id - s t a t e s p e c t r a d i f f e r only s l i g h t l y from those i n CH30H o r DMSO s o l u t i o n (37). However, our r e s u l t s d i f f e r markedly from previous observa t ions of w e t Amphoter- i c i n B powder smeared on f i l t e r paper (38); i n p a r t i c u l a r , w e observe a peak near 1010 cm-1. 1010 cm-1 Amphotericin peak i n previous s p e c t r a w a s used t o i n t e r p r e t caro tenoid s p e c t r a (38) .

Spec t ra of heated Amphotericin B powder (15 minutes a t 158°C) d isso lved i n CH30H (pH 5.) appear normal, d e s p i t e t h e change i n sample co lo r (Sec t ion 2 .1) . However, lowering the pH t o ( 1 causes immediate decomposition i n t o a product i n which the i n t e n s i t y of t he prominent 1156, 1559 cm-1 peaks is markedly reduced.

The supposed absence of a

3.4 ORD, CD, S p e c i f i c Rota t ion The s p e c i f i c r o t a t i o n , [& ] ~ 2 4 c of Amphotericin B

has been given as -33.6' and +333' i n 0.1N methanol ic H C 1 and "acidic" DMF respec t ive ly ( 1 , l l ) . However, c l o s e r i n v e s t i - ga t ion (39) shows t h a t t he s p e c i f i c r o t a t i o n i s h igh ly pH dependent. It is approximately +285 and pH 1 .0 , and +413 a t pH 2 . 1 , i n DMF (2.5 mg/ml) . (The "pH" was measured wi th a Beckman pH-meter wi th one g l a s s and one K C 1 e l e c t r o d e ) .

i n H 2 0 , CH30H/H20, and H20/cholesterol (32) are given i n Figure 9. The corresponding 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 given i n F igure 10.

A l l CD peaks i n CH30H/H20 c l o s e l y match Amphoter- i c i n B u l t r a v i o l e t absorp t ion f requencies ; 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 he s t rong 340-420 nm. quadruple t , and negat ive f o r t he weak 260-290 nm. t r i p l e t (Figure 9 c ) . The CD s p e c t r a of DMSO-solubilized Amphotericin B i n H20 and H20/cholesterol are less complex, oppos i t e i n s i g n and an order of magnitude more in t ense . P repa ra t ions of Squibb Fungizone (Amphotericin B s o l u b i l i z e d i n H 2 0 by Na+-desoxycho- l a t e ) are s imilar but even more o p t i c a l l y a c t i v e (Figure 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 sp l ays apprec iab le changes only i n the 260-300 nm region , whereas i n a c i d i c DMF both reg ions show cons iderable changes. I n a c i d i c DMF, t he r o t a t i o n near 2 7 1 , 392, 413 nm. is p o s i t i v e and the maximum near 290 nm. becomes a minimum (Figure l o ) . ORD measurements (41) i n n e u t r a l CH30H somewhat resemble those i n a c i d i c DMF; however, t he 286 nm. band is ass igned t o an

C i rcu la r dichroism (CD) s p e c t r a of Amphotericin B

+ 2000

t 1500

5 + 1000

B

4

u D

OI + 500

-

0.

a . n 2 0

b. n 2 0 + CHOLESTEROL

C . H 2 O t C H 3 0 H

2 5 0 300 350 400 450

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). Ampho- tericin 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 Refer- ences 1,11 should be viewed with caution.


impuri ty . Reduction wi th Na-borohydride has l i t t l e e f f e c t on the ORD s p e c t r a , sugges t ing t h e absence of t h e ketone (and presence of t he hemi-ketal) form i n n e u t r a l methanol.

3.5 Fluorescence The f luorescence 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 inco rpora t ion i n t o l e c i t h i n v e s i c l e s (31). This 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 presence of e p i c h o l e s t e r o l bu t no 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 luorescence emission f o r 340 nm e x c i t a t i o n is cons iderable between 410-500 run, wi th broad maxima near 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 300- 345 nm, wi th broad maxima nea 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 the 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 de tec t ion ; Reference 42).

4. SPECTRAL PROPERTIES (OTHER) 4.1 Proton NMR

A t y p i c a l 60 MHz proton NMR spectrum of Ampho- t e r i c i n B i n DMSO-db s o l u t i o n (43) is presented i n Figure l l a . The broad s i g n a l s can only b e l o o s e l y i d e n t i f i e d wi th s p e c i f i c chemical groups. Subs t ruc ture is p resen t ( c . f . t he 1.19 pprn broad m u l t i p l e t ) bu 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 has 13 exchangeable pro tons (10 hydroxyl, 2 amino, 1 a c i d ) . Rapid exchange between H20 and Amphotericin protons g ives rise t o a combined OH s i n g l e t . Its p o s i t i o n is h igh ly v a r i a b l e and depends upon the e x t e n t of Amphotericin-H20 hydrogen bonding, and thus H20 concen- t r a t i o n . Pos i t i ons between 3.8 and 4.7 ppm are t y p i c a l (19, 43).

The 220 MHz spectrum (Figure l l b ) reso lved cons iderable d e t a i l (e. g . , more than 10 resonant s i g n a l s between 0.7 - 1.7 ppm), a l though 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 demonstrate t h e presence of a hemi-ketal r i n g i n DMSO-d6 s o l u t i o n (22) consis- t e n t w i th the s o l i d - s t a t e conformation of Reference 13 . There is no evidence of an equ i l ib r ium wi th a keto-form. un-derivat ized Amphotericin B , t h e hemi-ketal and hemi- a c e t a l (mycosamine C-1) carbons appear a t 9 7 . 1 and 95.9 ppm respec t ive ly ; they are r e s p e c t i v e l y a s i n g l e t and a double t i n of f-resonance measurements. The l a c t o n e and COO- carbonyl

I n

N u1

60 MHz i\ 0

AMPHOTERICIN B

D M S

1 CH

OH

I I I 1 7.50 6.75 5.00 3.75 2.50 1.25

Figure 11. 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.


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 the U.S. s tandard (45) appears i n F igure 1 2 .

4 .3 Mass Spectrometry Early mass spec t romet r i c a t tempts a t s t r u c t u r a l

e l u c i d a t i o n were not completely success fu 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 per-

dg-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 th s t r u c t u r e 1.5 (TMS = t r ime thy l - sa l ine ) . The fragmentat ion p a t t e r n of Amphotericin B is f a r more complex than 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 lose chemical resemblance. Addi t iona 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 he TMS-ether d e r i v a t i v e are presented i n Table 5. Despi te genera l agreement s e v e r a l c h a r a c t e r i s t i c ions d i f f e r by 1-2 amu, o r are no t observed (Table 6 ) .

The M-150 fragment (m/e 1637) r ep resen t s t h e l o s s of C02 CH3, and TMS:OH from the molecular ion ; fragments f , g, h , i rep resen t t he l o s s of a d d i t i o n a l TMS:OH. Fragment 1 (m/e 1346) r ep resen t s M-150 minus a doubly s u b s t i t u t e d mycosamine fragment (m/e 201). Fu r the r l o s s e s of TMS:OH from fragment 1 y i e l d fragments m, n , 0, q , r.

t o f ragmentat ion (46). The t r i p l y TMS-substituted mycosamine-ester fragment g ives rise t o an intense m / e 362 (80.5%) peak; charge r e t e n t i o n on the oppos i t e s i d e of t h e l inkage w a s less common (m/e 378, 4.05%). No suga r fragments were found wi th a l l fou r l a b i l e hydrogens rep laced (m/e 434, 450).

The g lycos ide l i nkage i s p a r t i c u l a r l y vu lne rab le

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 paper p r e t r e a t e d wi th 0.3M K3PO4 b u f f e r (pH 3 .0) . developed 6-7 hours wi th 80% propanol . The mob 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 Ampho- t e r i c i n A. However, t h e low pH damaged the a n t i b i o t i c s , p revent ing longer development. High-pressure l i q u i d techniques (Sect ion 5.3) are p r e f e r a b l e f o r automation, quan t i t a t i o n , and co 1 l e c t ion .

Alternate methods (51) u t i l i z e Whatman No. 1 paper p r e t r e a t e d wi th McIlvaine 's b u f f e r , equ ib ra t ed over so lven t f o r 1 hour , and developed f o r 5 hours. The r e s u l t s are :

Spot

Solvents Rf(A) Rf(B) pH T(OC)

Sec-butanol: H20: C a C 1 2 0.82 0.64 3.2 37 (20 m l : 80 m l : 200 mg)

"C-NMR Amphotencin B (DMSO)

100 200 ppm 0

Figure 12 . 13C-NMR spectrum of Amphotericin B in DMSO-d6 solution (saturated).


I/BASE

0 . 7 4 % 0.14% 0 . 5 1 % 1 . 4 4 % 1 . 4 0 % 1 . 7 5 % 0 . 9 6 % 0 . 7 6 % 3.72% 0 . 8 9 % 3.16% 0 . 0 9 % 1 . 0 4 % 1 . 5 6 % 2.70% 3.14% 0 . 6 3 % 3.11% 1 . 1 5 % 0 . 6 1 % 1.08% 0 . 3 1 % 0 . 5 0 % 1 . 8 8 % 0 . 1 6 % 1 . 0 4 % 2.88% 0 . 0 9 % 1 . 6 1 % 0.80% 0.36% 2.28% 1.27% 1.28% 0.98% 0.95% 2.29% 1.61% 1.90% 1 . 2 6 % 0.74% 1 . 0 5 % 0 . 6 1 %

TABLE 5 High Mass Portion of the Spectrum of

Amphotericin B-TMSI

MAS s - 706.5 7 0 7 . 3 708.4 711.3 715.6 7 1 6 . 4 720.5 7 2 2 . 3 723.5 724.2 7 2 6 . 1 729.8 731.5 734.5 735.4 737.6 738.5 741.4 745.3 7 4 6 . 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 7 6 9 . 3 7 7 0 . 8 7 7 1 . 3 773.0 777.5 778.4 781.6 782.5 785.5

790.2 7 8 8 . 8

I/BASE

0 . 1 1 % 0 . 7 1 % 1 . 3 0 % 1 . 3 3 % 0 . 2 4 % 0 . 8 7 % 0 . 4 1 % 3.63% 2.83% 2.64% 0.29% 1 . 0 5 % 0 . 5 8 % 0 . 8 7 % 3.23% 2.71% 0 . 2 1 % 1 . 0 5 % 2.08% 3.24% 2.52% 2 . 0 5 % 1 . 9 6 % 1 . 2 3 % 0 . 1 8 % 0 . 5 3 % 0.40% 0 . 7 1 % 1 . 3 6 % 1 . 7 6 % 0.71% 0 . 9 2 % 0 . 5 6 % 2.53% 0 . 5 6 % 1 . 5 0 % 0 . 2 8 % 0 . 7 6 % 0 . 1 6 % 0 . 5 1 % 1 . 9 6 % 2.79% 1.52%

MASS

791.5 793.5 7 9 4 . 3 7 9 6 . 3 798.7 804.3 805.5 806.6 807.3 810.5 8 1 1 . 3 813.7 815.5 8 1 7 . 1 818.2 819.6 820.3 8 2 3 . 3 826.5 8 3 5 . 2 8 3 6 . 1 837.2 838.4 839.3 840.4 8 4 1 . 1 844.0 8 4 6 . 8 848.5 851.0 852.0 853.2 857.0

865.4 866.3 867.5 868.3 868.9 869.6 877.0 881.3 882.4

8 6 1 . 5

AMPHOTERICIN B 29

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 9 3 3 . 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 . 1 1 % 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 l l i O . l 1122 .1 1123 .1 1134.0 1148.5 1151.3 1 1 5 3 . 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

30 IRV lN M. ASHER era/ .

0 .13% 0 .94% 0 . 3 5 % 1.00% 0 .37% 1.01% 0.72% 0 . 8 4 % 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

Mt M-TMSi

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

1594.5 1607.9 1641 .2 1650 .5 1652 .5

1572 .8

TABLE 6 Comparison of Character i s t i c Ions of

Ampho t e r i c i n B-INSi

Reference 14 Reference 46

mf e m/e

1787 1714

M- 1 50 ( e ) 1b37 1 6 2 4

1534 1532.4* (8) 1457 1455.5*

1444 1445 .O* fh ) 1367 1366.4 (1) 1346 1345.6 ( 1 ) 1277 1278.0* (m) 1 2 5 h 1255.8 (n) 1166 ( 0 ) 1076 1076.5

986 985.6 (P) 89 h ( r ) aot 806.6 (k) 71 b

( f ) 1567 1549.3*

( j ) 988 987.7

(4 ) 897.9*

* Measurements d i f f e r by 1 mu.

X R . I n t e n s i t y

Not Observed N o t Observed 2 . 0 0 . 3 0.9 0 . 0 5 1.8 0.6 0.5 1 .5 Not Observed 1.2 0.6 0 . I 1 . 3 3 . 6 Not Observed

AMPHOTERICIN 6 31

Same (paper n o t 0.86 0 .41 3.2 37 e q u i l i b r a t e d )

Acetone: H 2 0 (8:Z) 0.77 0.59 4 . 6 25

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 de te rmined 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 Thin Layer (TLC) Most u s a b l e s o l v e n t systems f o r t h i n - l a y e r

chromatography (TLC) of Amphoter ic in B c o n t a i n a l c o h o l (Table 6 ) . So lven t sys t em G shou ld s e p a r a t e Amphoter ic in B (Rf 0.32) from Amphotericin A. S o l v e n t systems G,J s h o u l d s e p a r a t e Amphotericin B (Rf = 0 .32 , 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 .65, 0.54 r e s p e c t i v e l y ) . O the r r e f e r e n c e s are found i n Refe rence 3.

5 . 3 High-pressure L i q u i d (HPLC) Using a Waters A s s o c i a t e s (Mi l fo rd , Mass,) ,p c18

column, h igh -p res su re l i q u i d chromatography (HPLC) cou ld s e p a r a t e s o l u t i o n s of Amphoter ic in B from small amounts of an accompanying d e g r a d a t i o n p roduc t i n a v a r i e t y o f a c i d i c methanol systems. The contaminant ranged 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 sys t ems of Re fe rence 53.

more d i f f i c u l t , b u t can b e ach ieved u s i n g t h e f o l l o w i n g p rocedure (53): 20% CH30H/80% DMF t o 100% CH30H ove r 5 minu tes , s t r a i g h t o r concave g r a d i e n t , 1 . 5 ml/min f low, a b s o r p t i o n monitored a t 280 nm. S e p a r a t i o n r e q u i r e s less than 20 minutes . Maximum r e s o l u t i o n (na r rowes t peaks ) 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 ach ieved i n CH30H, d e s p i t e ea r l ie 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 (54 ) . The B/A u l t r a v i o l e t absorbance r a t i o is 0.6 n e a r 280 nm.

u s i n g VYDAC-RP (30-44 pm) columns w i t h H20:CH30H:tetrahydro- f u r a n (420:90:45) f o r Amphoter ic in B (3.4 minu tes ) and n y s t a t i n (3 .0 , 3.4 minu tes ) are t o o s imilar t o d i f f e r e n t i a t e between them. The method o f Refe rence 5 3 is 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 .

The u s e f u l s e p a r a t i o n of Amphoter ic in A and B i s

The r e t e n t i o n times found by o t h e r worke r s (55)

5 .4 Gas C o n t r o l l e d p y r o l y s i s fo l lowed by g a s chromato-

graphy o f t h e r e s u l t i n g f r agmen t s ( > 30) gave 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 Amphoter ic in B (56 ) .

5.5 E l e c t r o p h o r e s i s 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 Amphoter ic in B ,

W N

HPLC (,+tC18) 280 nm

AMPHOTERICIN A

AMPHOTERICIN B

AMPHOTERICIN

B7 rA 11 10.60

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 of 1. mg/ml. Waters p c18 column was used with a methanol/dimethylforamide solvent system as described in the text.

A

The absorption of effluent was monitored at 280 nm.

AMPHOTERICIN B 33

TABLE 7 Solvent Systems for Thin Layer Chromatography

Solvent !!€ Sys tem

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

B

C

D

E

F

G

H

I

J

K

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

N-butanol:CH$OOH:H20 (3:l: 1)

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

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

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

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

N-butanol (H20 saturated)

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

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

Butan-l-ol:ammonia:methanol:H~0 (20: 1: 2: 4)

0.6

0.5

0.45

0.4

0.4

0.32

0.2

0.19

0.18

0.07

Reference

47

50

50

48

50

50

49

50

49

48

47


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 rop 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 Fusa 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 muci lagenosa Saccharomyces c e r e v i s i a e Sporotr ichum s c h e n c k i i

Trichophyton megnini Trichophyton mentagrophytes Trichophyton g a l l i n a e Trichophyton rubrum Trichophyton t o n s u r a n s Monosporium apiospermum

( y e a s t phase)

M I C w a s > 40 Pgfml f o r :

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

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 wernecki Fonsecaea p e d r o s o i Fonsecaea compactum Geotrichum s p .

Microsporum gypseum Nocardia a s t e r o i d e s Nocardia a s t e r o i d e s mexicana Nocardia b r a s i l i e n s i s Nocardia madurae Ph i l aophora v e r r u c o s a Sporotr ichum s c h e n c k i i

( m y c e l i a l phase )

Note: From Refe rence 1; M I C ()lg/ml) measured on second 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 var ious e l e c t r o l y t e systems have been repor ted (57).

6. ISOLATION In the o r i g i n a l method of Vandeputte, e t a l . , ( l ) ,

Streptomyces nodosus (M 4575) whole b ro th i s mixed wi th isopropanol (1 : l ) and ad jus t ed 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 he a lcohol evaporated, and the r e s u l t i n g powder (40-70% pure) washed wi th water and acetone, and vacuum dr i ed . S lur ry ing wi th 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 the s l u r r i e d wi th 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 wi th water while maintaining pH 5. The p r e c i p i t a t e (75-80% pure) i s aga in d isso lved i n a c i d i c DMF, d i l u t e d wi th pure methanol, and p r e c i p i t a t e d with water. Amphotericin A (65-70%) r e s u l t s from adding water t o the A f i l t r a t e , and dry ing the p r e c i p i t a t e . p r e c i p i t a t i o n can be repeated t o remove the remaining Amphotericin B . )

(Methanolic C a C 1 2 s o l u b i l i z a t i o n and water

7. STABILITY

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 ap id ly a t pH 1 2 (1). 70°C (pH 7) i s ha l f t h a t a t 3OoC (1 ) . Solu t ions i n phosphate- c i t r a t e bu f fe r ( 5 < p H < 7 ) are apparent ly s t a b l e (58) . In dext rose in fus ions a t room temperature , Amphotericin B aggregates i n the presence of N a C l (25% reduct ion of a c t i v i t y wi th in 4 hours ) .

s o l u t i o n s (pH>4) d id not decrease apprec iab ly dur ing an 8- hour exposure t o 100-foot candles of ambient f l uo rescen t l i g h t (59). Af t e r 3 days exposure t o l i g h t i n o t h e r exper i - ments, b i o l o g i c a l (but no t co lo r ime t r i c ) assays showed a 26% los s i n a c t i v i t y (60) .

only ~ 1 7 % l o s s of potency. In c o n t r a s t , 15 minutes a t 158OC (above the chemical t r a n s i t i o n of Sec t ion 2 . 1 1 ) i s s u f f i c i e n t t3 cause an ~ 2 1 % l o s s of potency ( 2 1 ) . Vibra tor gr inding of t he sample a t room temperature causes an 2, 30% loss of potency (average a c t i v i t y 688 mcg/min, r a t h e r than 986 mcg/min; Reference 61) as measured by t h e Saccharomyces Cervisiae assay of Reference 30.

Dry Amphotericin B powder appears s t a b l e f o r long pe r iods

The s t a b i l i t y a t

The a c t i v i t y of aqueous, c l i n i c a l l y prepared dext rose

Heating dry samples f o r 16 hours a t 105°C r e s u l t s i n

8. ANTIMICROBIAL PROPERTIES AND ASSAYS

B are given i n Table 8 f o r s e v e r a l organisms (1). Stock Minimal i n h i b i t o r y concent ra t ions (MIC) of Amphotericin

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 he fungi were p l a t e d on agar (broth d i l u t i o n assays g ive 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 observa t ion .

Candida a lb i cans , o r Candida t r o p i c a l i s are descr ibed i n References (1,3,16) . The Code of Federa l Regulat ions (30) p r e s c r i b e s a microbio logica l aga r d i f f u s i o n assay s u i t a b l e f o r pharmaceut ical formulat ions us ing Saccharomyces c e r e v i s i a e (ATCC 9763). Addi t iona l b i o l o g i c a l assays can be found i n Reference 3 and are summarized i n Table 9 .

The b inding of Amphotericin B t o 5. c e r e v i s i a e has been inves t iga t ed using f luorescence (62) . Weak, r e v e r s i b l e binding occurs even a t O°C and i n the presence of metabol ic i n h i b i t o r s ; i t appears t o a f f e c t on ly t h e o u t s i d e of t he membrane. I n c o n t r a s t , an t imic rob ia l 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 rong , i r r e v e r s i b l e b inding t o the membrane. This s t r o n g b inding , which can be blocked by cool ing t o O°C o r by metabol ic i n h i b i t o r s , apparent ly d i s r u p t s t he deeper hydrophobic po r t ions of t h e membrane. Enhanced f luorescence assays are repor ted t o be l i n e a r i n t h e range 0 . 1 - l 0 . p (62).

Amphotericin B a c t i v i t y wi th a s e n s i t i v i t y of about 0 .01 mcg/ml (63). An equa l ly s e n s i t i v e t u r b i d i m e t r i c microbio- l o g i c a l assay (64) has been developed f o r use wi th small samples (e .g . , 2 5 ~ ~ 1 of serum 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 spectrophotometry of s imple DMSO e x t r a c t s , making use of a c o r r e c t i o n f o r t h e h igh b a s e l i n e abso rp t ion (64).

Assay procedures u t i l i z i n g Saccharomyces c e r e v i s i a e ,

Serum and u r ine can be assayed by agar d i f f u s i o n f o r

9 . AMPHOTERICIN A Amphotericin A (C46C73N019, Reference 13) is i s o l a t e d

from Streptomyces Nodosus, along wi th Amphotericin B which i t c lose ly resembles (1) . It 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 thus r e a d i l y d i s t ingu i shed from Amphotericin B by i ts u l t r a v i o l e t abso rp t ion spectrum: 2 2 8 , 280, 291, 304, 318 nm (1,18) . I ts 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 "ac id ic" DMF) is a l s o d i s t i n c t i v e (1 ,3 ; bu t see Sec t ion 3.4). In c o n t r a s t , i n f r a r e d s p e c t r a (1 ,18,34) are h igh ly similar, bu t no t i d e n t i c a l t o Amphotericin B.

s a t u r a t e d propanol o r bu tano l , and CH3COOH than 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 he l a t t e r proper ty w a s used i n i t s o r i g i n a l

Amphotericin A i s f a r more s o l u b l e i n CH30H, DMF, water-

AMPHOTERICIN B 37

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

Amphoter ic in B

Type of Sample Method T e s t C u l t u r e Refe rence

Formulated and D i f f u s i o n S a cc h a romy ces 65 unformulated c e r e v i s i a e p r o d u c t s N.C .Y.C . 87

D i f f u s i o n Saccharomyces 66 c e r e v i s i a e ATCC 9763

T u r b i d i m e t r i c Candida 64 t r o p i c a l i s ATCC 13803

Body F l u i d s D i f f u s i o n Paeci lomyces 6 3 v a r i o t i MSSC 5605 N I A I D

T u r b i d i m e t r i c Candida 64 (Micro s c a l e ) t r o p i c a l i s

ATCC 13803

Animal Feeds D i f f u s i o n Sac c h a r omy c es 6 7 c e r e v i s i a e ATCC 9763


i s o l a t i o n (1). Amphotericin A can (presumably) be sepa ra t ed from Amphotericin B and n y s t a t i n by the th in - l aye r chromatographic 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 separa ted from Amphotericin B by high-pressure l i q u i d chromatography (Sect ion 5 .3) .

t e r i c i n B (59) and i s usua l ly encountered a s a contaminant of t h e la t ter . Amphotericin A i s cons iderably more s e n s i t i v e t o c a t a l y t i c hydro lys i s , and is thus less s t a b l e i n aqueous isopropanol (1).

Amphotericin A is s e v e r a l times less a c t i v e than Ampho-

AMPHOTERICIN B

REFERENCES

39

1. J . Vandeput te , J. L. Wachtel , and E. T. S t i l l e r , Ant 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 Refe rence , Med. Econ. I n c . , L i t t o n Pub. ( O r a d e l l , N J , 1970) .

3. A. H. Thomas, The A n a l y s t , 101, 321, 1976.

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 . , (Sp r inge r Ver l ag ; B e r l i n , 1967) , 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, Proc. 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 , Proc. Nat. Acad. S c i . (USA), 60, 1201, 1968.

9 . J . M. T . Hamil ton-Mil ler , Bact. Review, 37, 166 , 1973.

10. F. R. K e i m , J. W. P o u t s i a k a , J . Kirpan and C . H. Keysse r , Sc i ence , 179, 584, 1973.

11. The Merck Index , Merck & Co., (Rahway, N J , 1968) .

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 Hea l th Org., 47, 343, 1972.

13. W. Mech l insk i , C . P. S h a f f n e r , P. Ganis , and G. A v i t a b i l e , Te t r ahedron L e t t . , H : 3 8 7 3 (1970); P. Gan i s , G. A v i t a b i l e , W . Mech l insk 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 Spec. , - 1:20 (1974).

15. The U. S. Pharmacopeia, 1 9 t h Ed. , USP Convent ion, I n c . , ( R o c k v i l l e , MD, 1975) .


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. Graichen, BF, FDA, unpubl ished d a t a (1976).

18. Index 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 Col lege , PA, 1967).

19 . E. R . Squibb & Sons, I n c . , unpubl ished d a t a (1972).

20. A. Wong and B. Baer, N I H , unpubl ished d a t a .

21 . S. Delgado and L. Wayland, BD, FDA, unpubl ished d a t a .

22 . 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 , manuscr ipt submi t ted .

23. A. C . Cope, J. Am. Chem. SOC. , 3 : 4 2 2 8 (1966)

2 4 . G. Schwartzman, I . M. Asher, V. Fo len , W. Brannon, and J . Taylor , FDA, manuscript submi t ted .

25. M. Maientha l , BD, FDA, unpubl ished d a t a (1976).

26. W. Barron, BD, FDA, unpubl ished 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 Chemo- therapy , 9:277 (1959).

28 . E. D . Et ingov, G. V . Kholodova, V. 0. Kul 'bakh, and A. I. Karnatushkina, 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 Regula t ions , ' ' 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. Bi t tman, W. C . Chen, and 0 . R. Anderson, Biochemis t ry , 13: 1364 (1974).

32 . 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 41

Compounds of Pha rmaceu t i ca l I n t e r e s t , A.O.A.C. (Washington, D. C . , 1972).

35. A. L. Hayden and 0. R . Sammul, J. Am. Pharm. Assoc. , 49: 497, 1960.

36. I . M. Asher, FDA, I. Lev in , N I H , manusc r ip t i n p r e p a r a t i o n .

37. M. Bunow, I . Asher , and I. Lev in , unpub l i shed d a t a (1976) .

38. L . R i m a i , M. E . Heyde and D . G i l l , J . Am. Chem. SOC., - 95:4493 (1973).

39. S . Delgado, BD, FDA, manusc r ip t i n p r e p a r a t i o n .

40. K. W. Henry, EDRO, FDA, unpubl ished d a t a (1976) .

41. C . N . Chong and R. W . R icha rds , Te t r ahed . L e t t . , 5053, 19 72.

42. F. Schroede r , J . F. Holland and L . L. B i e b e r , Biochem- i s t r y , 11, 3105 (1972).

43. E. S h e i n i n , BD, FDA, unpub l i shed d a t a (1976) .

44. R. B rad ley , N I H , unpubl ished d a t a (1976).

45. G . Mazzola, BD, FDA, unpubl ished d a t a (1976) .

46. R. Barron, BD, FDA, unpubl ished d a t a (1976).

47. M. Kalasz, V. S z e l l , J . Gyimesi, K. Magyar, I. Horva th , and I. Szabo, Acta Mic rob io l . Acad. S c i . Hung., 19, 111, 1972.

48. L . Dryon, J. Pharm. Be lg . , 2, 433, 1966.

49. S . Ochab, D i s snes Pharm. Pharmac., 22, 351, 1970.

50. J . B lake ly , BD, FDA, unpubl ished d a t a (1976).

51. J . Semar, The Squibb I n s t i t u t e f o r Medical Resea rch , unpubl ished d a t a (1964).

52. E. Meyers and D . Smith, 3. Chromatog., 14, 129 (1964) .

53. B. Smith, BD, FDA, manusc r ip t i n p r e p a r a t i o n .


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., 25, 401, 1973.

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, 102 , 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 S. I. Schwartz, Applied Microbiol., 17, 497, 1969.

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, J- Antibiot., I&, 56 , 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

I N D E X

A n a l y t i c a l P r o f i l e - Betamethasone D i p r o p r i o n a t e

1. D e s c r i p t i o n 1.1 Name, Formula, Molecular Weight 1 .2 Appearance

2 . P h y s i c a l P r o p e r t i e s 2.1 I n f r a r e d Spectrum 2 . 2 Nuc lea 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 Mel t ing 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 Thermogravimetr ic 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 . S y n t h e s i s

4 . S t a b i l i t y

5. Method of A n a l y s i s 5 .1 Elemental A n a l y s i s 5.2 Thin Layer Chromatographic A n a l y s i s 5 . 3 L iqu id 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. Re fe rences

BETAMETHASONE DIPROPIONATE 45

1. D e s c r i p t i o n

1 . 1 Name, Formula , M o l e c u l a r Weight The chemica l name f o r be t ame thasone d i p r o p i o n a t e i s

9a - f luo ro -11B-hydroxy- l6~-me thy l - l7~2 l -d ip rop iony loxy-p regna - 1 ,4-d iene-3 ,20-d ione .

2 8H3 7 Fo 7 M o l e c u l a r Weight 504.6

1 .2 Appearance Betamethasone 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.

2. P h y s i c a l P r o p e r t i e s

2 . 1 I n f r a r e d Spectrum (IR)

is 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 as a m i n e r a l o i l m u l l on a Perk in-Elmer Model 180 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 spec t rum are l i s t e d i n T a b l e I . 1

The i n f r a r e d spec t rum of be t ame thasone d i p r o p i o n a t e

P m

Figure 1

INFRARED SPECTRUM OF BETAMETHASONE DIPROPIONATE

WAVELENGTH, MICRONS

I I I 1 I I I I I 1 I I 1 1 1 1 I I

2.5 3 4 5 6 7 8 9 10 12 14 18 22 35 50

1001

I I 1 I I I I 1700 1400 1 1 0 0 800 500 200

0 4Ooo 3500 3000 2500 2000

FREOUENCY (CM-’1


Table I

I R Assignments f o r Betamethasone Dip rop iona te

* Frequency (cm-l) I n t e n s i t y C h a r a c t e r i s t i c of

3300 m 0-H s t r e t c h 3025, 3000 W C-H s t r e t ch ,A1y4 1755, 1728 s , d C=O s t r e t c h , 17,21-dipro-

1660 s C=o s t r e t c h , 3-ketone 1620, 1608 s , d C=C s t r e t c h , A1,4-diene 1189 S C-0 s t r e t c h , p r o p i o n a t e

1068 m C-0 s t r e t c h , 11-hydroxyl

p i o n a t e , 20-ketone

ester

* s = s t r o n g , m=medium, w=weak, d=doub le t

2.2 Nuclear Magnetic Resonance Spectrum (NMR)

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 Varian XL-100-15 s p e c t r o m e t e r a t ambient t empera tu re i n CDC13 s o l v e n t w i th a c o n c e n t r a t i o n of 20 mg/ml. s h i f t s a r e r e p o r t e d i n ppm ( 6 ) downfield 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 Table II.2

The 100 MHz F o u r i e r t r ans fo rm p ro ton NMR spectra of

Chemical

Tab le I1

NMR Assignments f o r Betamethasone Dip rop iona te

8 21CH20CCH2CH3

I

// 0

P cn

Figwe 2 NMR SPECTRUM OF BETAMETHASONE DIPROPIONATE

, ..... ., . , , , . . . . ... ' . ' . :". " ~ ' ' ' ' ~ ' ' ' ' ~ : .'. , , I. . , .;. .


P r o t o n

C 1 3-CH 3 C16-CH3 C10-CH3 1 la-H 21-H 21'-H

* 1 18-0-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 1.09

C17 and C21 P r o p i o n a t e 2.42 me thy l s

methylenes

Mu1 t i p l i c i t y

S i n g l e t Doublet Sing l e t Mu1 t i p l e t Doublet Doublet Doublet Broad s i n g l e t Doublet of

d o u b l e t s J1, =10 Hz; J 2 , -1.5 Hz

Doublet T r i p l e t

Q u a r t e t

*Chemical s h i f t and coup l ing c o n s t a n t v a r y w i t h concen- t r a t i o n and t empera tu re , b u t d i s a p p e a r s when D20 is added.

2 .3 Mass Spectrum The mass spectrum of betamethasone 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 Var i an MAT CH5 medium r e s o l u t i o n s i n g l e focus ing (magnet ic 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 Varian SS-1OOC d a t a system, a t a probe t e m p e r a t u r e of 170°C and a s o u r c e t empera tu re o f 25OoC. 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 raph i n

A l i s t i n g of t h e prominent f r agmen t s and t h e i r

The d a t a

F i g u r e 3 . 3

r e s u l t i n g masses a r e g i v e n i n Tab le 111.

-~

o

50


Table 111

Mass Spectrum Assignments for Betamethasone Dipropionate

Mass

505

484

417

-

410

343

336

333

315

295

277

267

223

147

- Ion Fragments Lost

M+ 1

M-20 HF

M-87 C H ~ O ~ C H ~ C H ~ P

M-94 HF+CH3CH2COOH

M-161 R

CH20CCH CH +CH3CH2COOH 2 3

M-168 HF+2CH3CH2COOH

9 M-171 C O C H ~ O ~ : C H ~ C H ~ + C 2 ~ 4 ~ ~

M-189 C O C H ~ O ~ C H ~ C H ~ + C H ~ C H ~ C O O H 0

fl COCH20CCH2CH3+CH CH COOH+HF

3 2 M-209

C O C H ~ O ~ C H ~ C H ~ + C H 9 CH C O O H + H ~ O 3 2

M-227

91 M-237 COCH20CCH2CH3+CH3CH2COOH+C0


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

Loss - - I o n - Mass

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 spectrum o f be t ame thasone d ip ro -

p i o n a t e was scanned from 350 t o 210 nm, a s i n g l e maxima was observed a t 238 nm @ = 1 . 5 7 ~ 1 0 4 ) . 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 me thano l .

The spectrum i n

2.5 Op t i ca l R o t a t i o n

s p e c i f i c r o t a t i o n s : 4 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

26'

2 7'

BE

TA

ME

TH

AS

ON

E D

IPR

OP

ION

AT

E

53

Figum 4

ULTR

AVIO

LET SPECTR

UM

OF B

ETAM

ETHA

SON

E DIPR

OPIO

NA

TE

NA

N0 M

ETE

RS


2.6 Mel t ing Range Betamethasone d i p r o p i o n a t e me l t s i n a 3' r ange

between 1700 and 179OC w i t h decompos i t ion , when t h e USP XvIT.1 c l a s s Ia p rocedure i s used.5

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 (DSC) The DSC c u r v e f o r betamethasone d i p r o p i o n a t e ob-

t a i n e d a t a scan ra te of 10°C/min. i s shown i n F i g u r e 5 . The c u r v e was r eco rded w i t h a DuPont 900 D i f f e r e n t i a l Thermal Analyzer under an atmosphere of n i t r o g e n f lowing a t 200 cc/min. A s i n g l e endotherm w a s obse rved , t h e e x t r a p o l a t e d o n s e t of m e l t i n g occur red a t 175OC.6

2 .8 Thermogravimetr ic A n a l y s i s (TGA)

p i o n a t e e x h i b i t e d no weight l o s s on a scan from 27O t o 175OC a t 10°C/min.7

The TGA c u r v e f o r s t a n d a r d betamethasone d ip ro -

2.9 S o l u b i l i t y

i s l i s t e d i n Tab le I V . The s o l u b i l i t y d a a f o r betamethasone d i p r o p i o n a t e s

Tab le 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

So lven t

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 thano l (USP) Ethano l (USP) 85% - Water 15% (v /v ) E the r E t h y l Acetate Methanol Mineral O i l Petroleum E the r P o l y e t h y l e n e Glyco l 400 Propylene Glyco l Water

S o l u b i l i t y mglml, 25OC

> l o o 30

>loo >loo >loo

45 30

5 70 55 <O .05 <0.03 26

7 <0.04

BETAMETHASONE D IPROPIONATE

Figure 5 DSC OF BETAMETHASONE OIPROPIONATE

55


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 spectrum of betamethasone

d i p r o p i o n a t e is p r e s e n t e d i n Tab le 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).

Tab le V

Xray Data f o r Betamethasone D i p r o p i o n a t e

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

I/I'

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

100 71

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 3.030 3.020

I/I'

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

3 . S y n t h e s i s Betamethasone d i p r o p i o n a t e i s p repa red 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 or tho- p r o p i o n a t e and toluene-p-sulphonic a c i d t o y i e l d betamethasone 17,21-ethylorthopropionate.~O r e a c t e d w i t h ace t ic a c i d t o y i e l d betamethasone 17- p r o p i o n a t e . l l T h i s i n t e r m e d i a t e p roduc t is t h e n t r e a t e d w i t h 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 w i t h d i l u t e h y d r o c h l o r i c a c i d . T h i s y i e l d s t h e c r u d e 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

T h i s compound i s t h e n

BETAMETHASONE D IPROPIONATE 57

4. S t a b i l i t y 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 aque-

ous suspens ions as 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 co r re spond ing- 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 betamethasone a l c o h o l . 13 amounts of more p o l a r p r o d u c t s were observed which a l t h o u g h n o t i d e n t i f i e d , can be assumed t o be f u r t h e r breakdown pro- d u c t s of t h e dihydroxy a c e t o n e s i d e cha in .14

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 oxida- t i o n i n t h e s o l i d s ta te . Hea t ing of t h e compound a t 75 C f o r 6 months i n t h e p re sence 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

i s minor d e g r a d a t i o n of t h e drug.16 pec ted t h a t s o l u t i o n s of betamethasone d i p r o p i o n a t e are 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 degrada- t 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

A t t h e ex t r emes of pH, l a r g e

Over long p e r i o d s of exposure t o f l o u r e s c e n t l i g h t , t h e r e It shou ld a l s o b e ex-

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 betamethasone d i p r o p i o n a t e are p r e s e n t e d below. 18

Element

C H F

Theory

66.65 7.40 3.77

Found

66.54 7.18 3.65

5 . 2 Thin Layer Chromatographic A n a l y s i s (TLC) A TLC system which i s used i n t h e a n a l y s i s of be 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 scend ing chromatography us ing ch1oroform:acetone (7: 1) as t h e deve lop ing sol- v e n t .


A f t e r t h e s o l v e n t i s al lowed t o ascend 1 5 c m , 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 unde r a sho r twave 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 betamethasone d i p r o p i o n a t e band. The approx ima te Rf v a l u e is 0.5. '9

5.3 L iqu id Chromatographic A n a l y s i s

t h e s e p a r a t i o n and d e t e c t i o n of be t ame thasone d i p r o p i o n a t e was developed 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 .

A h i g h p r e s s u r e l i q u i d chromatography system f o r

20

Tab le V I

Column: Permaphase ODs* (DuPont) packed i n a I m x 2mm ( i . d . ) s t a i n l e s s s t ee 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 Phase: A c e t o n i t r i 1 e : w a t e r ( 1 : 3 )

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 monopropionates 5

Betamethasone d i p r o p i o n a t e 7

(degassed f o r 5 m i n u t e s u s i n g vacuum)

*ODs - O c t a d e c y l s i l a n e

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 Direct UV a b s o r b a n c e s may b e c a r r i e d ou t on be ta -

methasone d i p r o p i o n a t e . d i p r o p i o n a t e i s p repa red c o n t a i n i n g approx ima te ly 0.02 mg/ml i n methanol . The a b s o r p t i o n spectrum of t h i s s o l u t i o n i s t h e n r eco rded between 350 and 220 nm and compared t o a s i m i -

2 1 l a r s o l u t i o n of t h e s t a n d a r d .

A s o l u t i o n of be t ame thasone

5.5 C o l o r i m e t r i c A n a l y s i s The c o l o r i m e t r i c a n a l y s i s f o r betamethasone d ip ro -

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 betamethasone 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 approx ima te ly 0.016 mg/ml i n e t h a n o l (USP). To 20.0 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 hydrox ide s o l u t i o n (10 m l of tetramethylammonium hydrox ide , 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 ) . water b a t h f o r 45 minu tes . 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 lowed t o c o o l . The a b s o r p t i o n spectrum 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 450 nm (hmax = 525 m).23

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


6. R e f e r e n c e s

1.

2 .

3.

4 .

5.

6 .

7.

8.

9 .

10.

11 .

12 .

13 .

14 .

15.

16 .

E c k h a r t , C . and McGlo t t en , J . , Sche r ing -P lough Corp . , P e r s o n a l Communicat ion.

B r a m b i l l a , R. and McGlo t t en , J . , Sche r ing -P lough Corp . , P e r s o n a l Communicat ion.

B a r t n e r , P . and McGlo t t en , J . , Sche r ing -P lough Corp . , P e r s o n a l Communicat ion.

E c k h a r t , C . and McGlo t t en , J . , Sche r ing -P lough Corp . , P e r s o n a l Communicat ion.

R o s e n k r a n t z , B . , Sche r ing -P lough Corp . , P e r s o n a l Commun i c a t i o n .

G l i s s o n , R. and R o s e n k r a n t z , B . , Sche r ing -P lough Corp . , P e r s o n a l Communicat ion.

I b i d .

R o s e n k r a n t z , B . , Sche r ing -P lough Corp . , P e r s o n a l Communicat ion.

S a n c i l i o , F. D . , Sche r ing -P lough Corp . , P e r s o n a l Communicat ion.

E l k s , J . , May, P. J . , and Weir, N . G . , US P a t e n t 3 ,312 ,591 (1967) .

E l k s , J . , May, P. J . , and Weir, N . G . , US P a t e n t 3 ,312 ,590 (1967) .

I b i d .


Guttman, D . , J . An. Pharm. ASSOC. , 47, 773 (1958) .


I b i d .


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 Commun ic at ion.

22. Mader, W. J. and Buck, R. R., Anal. Chem. 24, 666-667, (1952).

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

CLON AzEPAM

Walter C. Window

62 WALTER C . WINSLOW

Contents

1.

2.

3 .

4.

5.

6.

7.

8.

9.

Analytical Profile - Clonazepam

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

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

Synthesis

Stability Degradation

Drug Metabolism and Pharmacokinetics

Toxicology

Methods of Analysis 7.1 Elemental Analysis 7.2 Phase Solubility Analysis 7.3 Chromatographic Analysis 7.4 7.5 Spectrophotometric Analysis 7.6 Polarographic Analysis 7.7 Titrimetric Analysis

Electron Capture Gas Liquid Chromatography

Acknowledgements

References

CLONAZEPAM

1. Description

63

1.1 Name, Formula, Molecular Weight

Clonazepam is (5-[2-chlorophenyl]-l,3-dihydro-7- nitro-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] listed in Table 1.

The spectral assignments are

1.

2.

3.

4 .

5.

6.

7.

Table 1

NH stretching: 3250-3100 CM-I

Aromatic CH stretching: 3076, 3056 CM-’

Carbonyl stretching: 1696 CM-’

Aromatic Ring: 1615, 1582 CM-’

Asymmetric NO2 stretching: 1540 CM-l

Symmetric NO2 stretching: 1339 CM-’

Aromatic CH out-of-plane bendin : 4 adjacent free H’s: 750 CM- ? 2 adjacent free H ’ s : 844 CM-I

I\

I

I I 8

0

0

0

0

(0

* 3

3N

VllI W

SN

Wl%

64

CLONAZEPAM 65

2.2 Nuclear Magnetic Resonsance Spectrum (NMR)

The NMR spectrum of 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

Chemical Proton Shift 6 (ppm) Multiplicity Coupling Const. J (Hz)

4.48 Singlet --- a

7.55-7.85 Mu1 t iple t --- b

C 7.93 Doublet 2.5

d 8.50 Doublet (2 Sets) 2.5 (meta coupling)

e 11.30 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 ]

L

a ;d a

PI N

(D

c 0

u

a, a

m

4

4

f

66

CLONAZEPAM 67

FIGURE 3

UV Scan of Clonazepam

0.1

0.i

0.E

0.5

w 0 2 a E 0.4

m a E:

0.3

0.2

0. I

0

1 1 1 1 250 300 350 400

NANOMETERS

68 WALTER C . WINSLOW

2.4 Mass Spectrum

The low resolution mass spectrum of clonazepam is shown in Figure 4. [4] 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:

The spectrum was run on a

Mass (m/e) Species

315 M+ 314 M+ - H 298 M+ - OH 286 M+ - CHO 2 80 M+ - C1 268 314 - NO2 252 280 - CO, 280 - CHzN 2 40 268-CO 2 34 280-NO 205 240-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 Mass Calculated C H N 0 151.0541 151.0548 1 2 7 0 0 0 177.0596 177.0578 1 3 7 1 0 0 205.0763 205.0767 1 4 9 2 0 0 213.0354 213.0327 1 4 3 3 0 0 2 34.0 79 7 234.0794 15 10 2 1 0 240.0437 240.0455 1 4 9 2 0 1 252.0531 252.0536 1 4 8 2 3 0 252.0760 252.0773 14 10 3 2 0 268.0433 268.0404 1 5 9 2 1 1 2a0.0701 280.0723 15 10 3 3 0 286.0370 286.0383 1 4 9 3 2 1 287.0308 287.0349 15 10 1 3 1 298.0362 298.0381 1 5 9 3 2 1 314.0294 314.0333 1 5 9 3 3 1 315.0364 315.0411 15 10 3 3 1

_ _ _

5 2 a

at N

0

m m

2

69

70 WALTER C. WINSLOW

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 heating rate of 10°C/minute is shown in Figure 5. endothermic transition, corresponding to the melting of the compound, i s observed from 238.6"C to 240.2"C. [ 6 ]

A single

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 equili- brated for 3 hours at 25"C, are given in Table 4 .

Table 4

Solvent

Water 95% Ethanol Absolute E t hano 1 Methanol Isopropanol Chloroform Ethyl Ether Benzene Ace tone Ethyl Acetate Propylene Glycol

Solubility mg/ml

<o. 1 5.6 4.7 8.6 2 . 3

0.7 0.5

15

31 10

5.2

U

vl n

71


2.10 Crys ta 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 presented below. [ 7 l Instrument Condi t ions

Instrument GE Model XRD-6 Generator

Camera Guider-DeWolff 11, w i t h Pt-Rh Sample Screen

X-Ray Target Chromium (CrK, = 2.2909A)

Focus Line

Voltage 50 KV

Current 12.5 mA

Atmosphere Helium

Exposure T i m e 2 H r s .

Film

0

I l f o r d X-Ray Film I n d u s t r i a l G 0

28 d (A) * I / Io**

17.89 7.37 0.54 22.30 5.92 1.00 22.58 5.85 0.57 23.33 5.67 0.32 26.30 5.03 0.11 25.02 5.29 0.10 27.49 4.82 0.29 27.93 4.75 0.29 30.21 4.40 0.47 30.84 4.31 0.49 33.59 3.96 0.19 34.39 3.88 0.54 34.72 3.84 0.60 36.16 3.69 0.38 36.78 3.63 0.76 38.91 3.44 0.15 39.47 3.39 0.56 41.50 3.23 0.27 42.04 3.19 0.16

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

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 ] p-nitroaniline in a modified Friedel-Crafts reaction to yield 2-amino-5-nitro-2’-chlorobenzophenone. The amino- ketone is then condensed with bromoacetyl bromide to form 2-bromoacetamido-5-nitro-2’-chlorobenzophenone. This compound is isolated and converted to the corresponding acet- amido 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]

o-Chlorobenzoyl chloride is reacted with

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 po- tentiating inhibitory mechanisms in the subcortical brain structure responsible for the propagation of seizure activity ,

Clonazepam, even in pg doses, protected mice from pen- tetrazole 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

+

E O

w

f 8

z

8

z

74

U v1

FIGURE 7 Decomposition of Clonazepam via H y d r o l y s i s

7 %N TI _Ic 02Na&+i"2 COOH

C I NH2

\ \ CLONAZEPAM I


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 CS 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 % Found

C 57.37 57.07

H 3.17 3.19

c1 N

0

1 1 . 2 8 1 1 . 2 3

13 .43 13.31

14.75 15 .20 (by difference)

-

0 F

\

/

I’

-

0

I’ yp \/

(u

w a

I 2-X

77


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[ 21] is ethyl acetate:carbon tetrachloride 50:50. In both systems, 20 p1 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 System I1

Species Rf Rf

Clonazepam .46 .43

Bromacetamido Impurity .56

Aminoacetamido Impurity .64

Carbostyril .60

Benzophenone .90

7.4 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 these methods is reported to have a sensitivity of approximately 1 ng/ml. [ 2 2 ]

Each of

FIGURE 9 Phase S o l u b i l i t y Analysis of Clonazepam

n e "

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

I 1 1 I 1 1 I I

SYSTEM COMPOSITION: rng SOLUTE/g SOLVENT

0 10 20 30 40 50 60 70 80 90 I

EQUILIBRATION 20 HOURS AT 25°C EXTRAPOLATED SOLUBILITY 10.72 mg/g SOLVENT

I I 1 1 I 1 1 I I

SYSTEM COMPOSITION: rng SOLUTE/g SOLVENT

0 10 20 30 40 50 60 70 80 90 I


7.5 Spectrophotometr ic Analysis

Spectrophotometr ic 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 sopropanol , [ 12] however, as hydro lys i s products of 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 ( see s t a b i l i t y s e c t i o n ) , t he absence of t hese s p e c i e s a t apprec i ab le l e v e l s should be confirmed by TLC.

7.6 Polarographic Assay

Clonazepam e x h i b i t s a d u a l r educ t ion wave which may be a t t r i b u t e d t o t h e r educ t ion of t h e 4,5-azometh- i 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 polarographic r educ t ion of t hese groups f o r va r ious 1,4-benzodiazepines i n 0.1N H C 1 i n 20% methanol are s u f f i c i e n t l y sepa ra t ed f o r q u a n t i t a - t i v e work based on t h e r educ t ion of t h e azomethine group a t about - 0.6V vs . SCE. L i n e a r i t y w a s ob- t a ined between sample concen t r a t ion and t h e d i f f u - s i o n c u r r e n t . The polarographic a s say of clonazepam has been performed i n aqueous systems by D e S i l v a e t a 1 . , [ 1 6 ] w i th a s e n s i t i v i t y of 0.5 - 0.75 pg/ml.

7.7 T i t r i m e t r i c Analys is

Clonazepam i s assayed by d i s s o l v i n g t h e sample i n a c e t i c anhydride and t i t r a t i n g wi th 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 ac id . The endpoint may be determined p o t e n t i o m e t r i c a l l y us ing a g l a s s calomel e l e c t r o d e system o r , al ter- n a t i v e l y , by adding 5 drops of N i l e Blue hydro- c h l o r i d e i n d i c a t o r (1% i n g l a c i a l a c e t i c a c i d ) t o t h e sample and t i t r a t i n g t o a yellow-green endpoint . Each m l of 0.1N p e r c h l o r i c a c i d is equ iva len t t o 31.57 mg of clonazepam.

8. Acknowledgements

The au tho r wishes t o acknowledge the a s s i s t a n c e of D r . K. Blessel, D r . R . I . F ryer and t h e photo- graphic and graphic s e r v i c e s departments of Hoffmann-LaRoche i n t h e p repa ra t ion of t h i s p r o f i l e .

CLONAZEPAM 81

9. References

1.

2.

3. 4. 5. 6.

7.

8.

9.

10. 11.

12. 13.

14. 15. 16.

17.

18.

19.

20.

21. 22.

23.

Waysek, E. and Go, M.V., Hoffmann-La Roche Inc., Personal Communication Johnson, J., Hoffmann-La Roche Inc., Personal Communication Data on File, Hoffmann-La Roche Inc. Benz, W., Hoffmann-La Roche Inc., Personal Communication Data on File, Hoffmann-La Roche Inc. Ramsland, A., Hoffmann-La Roche Inc., Personal Communication Chiu, A.M., Hoffmann-La Roche Inc., Personal Communication 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) Propper, R. and Niemczyk, H., Hoffmann-La Roche Inc., Internal Report Chase, G., Hoffmann-La Roche Inc., Internal Report Mayer, W., Erbe, S., Wolf, G., and Voigt, R., Phmmazie,

Johnson, J.B. , Hoffmann-Lzoche Inc. , Internal Report Blum, J.E. , Haefely, W. , Jalfre, M. , Polc, P . , and Schaerer, K. , Arzneim. -Forsch., 23, 377-389 (1973) , CA, 79: 190 t (1973) Eschzoff , E. , Arzneim. -Forsch., 23, 390 (1973) Data on File, Hoffmann-La Roche Inc. DeSilva, J.A.F., Puglisi, C.V., Munno, N., Journal of Pharmaceutical Sciences, 63, 520 (1974); CA, %:99138h (1974) Naestoft, J. , Larsen, N.E. ,

DeSilva, J.A.F. , Bekersky, I., Journal of Chromatography, 3, 447-460 (1974) Scheidl, F., Hoffmann-La Roche Inc., Personal Communication Guastella, J. and Laureano, C., Hoffmann-La Roche Inc., Internal Report Gomez, R., Hoffmann-La Roche Inc., Internal Report Brooks, M.A. , DeSilva, J.A.F. , Talanta, 22, 849-860 (1975) Senkowski, B . Z . , Levin, M.S., Urbigkit, J.R., Wollish, E.G. , Analytical Chemistry, - 36, 1991 (1964)

- 29, 700-707, (1974); CA, 82:1601636 (1975)

Journal o f Chromatography, - 93, 113-122 (1974)

CYC LIZIN E

Steven A . Benezra

04 STEVEN A. BENEZRA

INDEX

Analytical Profile - Cyclizine

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 Melting Range 2 . 6 Differential Scanning Calorimetry 2.7 Solubility

3 . SYNTHESIS

4 . STABILITY

5. DRUG METABOLISM AND PHARMACOKINETICS

6 . METHODS OF ANALYSIS

6 . 1 Elemental Analysis 6 . 2 Nonaqueous Titration 6 . 3 Thin Layer Chromatography 6 . 4 Gas Chromatography 6.5 High Pressure Liquid Chromatography 6 . 6 Fluorimetry 6.7 Colorimetry

CYCLlZlNE

1. DESCRIPTION

85


Cyclizine is 1- (diphenylmethyl) -4-methylpiperazine .

H

Mol. Wt. 266.40 C18H22N2


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;; aromatic C-H stretch 1448 cm-l C-C skeletal vibration 1372 cm C-N stretch (tertiary amine) 745,698 cm-I mono-substituted benzene

Numerous other bands are in agreement wi h the f 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. following assignments can be made for the observed signals.

The

1.

86

Figure 1. Infrared Spectrum of Cyclizine

10

-r- I I 1 I I I I I

9 8 7 6 5 4 3 2

-r- I I 1 I I I I I I I

10 9 8 7 6 5 4 3 2 I 0

PPm Figure 2. 100 MHz NMR Spectrum of Cyclizine


Proton No. of Chemical Po sition Protons Shift (ppm) Multiplicity

a b

d C

3 2.27 singlet 8 2.43 singlet 1 4.21 singlet 10 7.19-7.44 mu 1 tip le t

(C 1 H (b)

@lo(!- N N-CH3 I \ /

n (a)

(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 Molar Wavelength Maximum (nm) Absorptivity of Minimum (nm)

269 540 263 742 258 694 253 (sh) 548 225 1.13 lo4

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


Figure 4 . 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.

The molecular Ion of m/e 266 is present but

2.5 Melting Range

3 The melting range reported in the N.F. XIV for

cyclizine is 106°C to 109°C using the class I procedure.

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

Solvent Solubility gm/ml

Water <0.1 mg/ml Ethanol 0.17 Chloroform 1.1 Ether 0.17

3. 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. pH 11.5 decompose to N-methylpiperazine, benzhydrol and benzophenone.

At 105°C cyclizine suspensions at

7

= 7.1 caI/mole

I I I I I I I I

TEMPERATURE OC Figure 5. DSC Thermogram of Cyclizine

(0 w

OH CI I I

Figure 6 . Synthesis of Cyclizine


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 50 mg t.i.d. for 6 days. 5

6 . METHODS OF ANALYSIS


10 Theoretical (%) Found ( X )

C 81.33 80.93 H 8.32 8.33 N 10.52 10.50

6.2 Nonaqueous Titration

Dissolve 0.3 g in 75 ml glacial acetic acid.

3

Titrate with 0.1 N perchloric acid using crystal violet indicator. Each ml of 0.1 N perchloric acid is equivalent to 0.01332 g of cyclizine.

6.3 Thin Layer Chromatography

A variety of thin layer chromatographic systems have been used for cyclizine. All visualization was done with short wave W.

They are given in Table 11.

CYCLlZlNE 95

TABLE I1

Thin Layer Chromatograph Systems for Cyclizine

Ad so r ben t

silica gel

silica gel

silica gel

silica gel

silica gel

0.1 M NaOH coated Si02 plates

Si02 plates

Si02 plates

SiO plates

SiO plates

0.1 M NaOH coated

0.1 M NaOH coated

0.1 M KHSO coated 4 2

0.1 M KHS04 coated

2

Mobile Phase R -f-

cyc1ohexane:diethylamine: 0.55

benzene:ethanol:NH OH 0.61

benzene (95:15:5)

(95: 15 : 5) 4

methano1:chloroform (1:2) 0.60

ethy1acetate:methanol:NH OH 0.67

ch1oroform:isopropyl alcohol: 0.45

cyc1ohexane:benzene: 0.55

methanol 0.46

4 (17 : 2 : 1)

5% aq. NH40H (74:25:0.6)

diethylamine (75:15:10)

acetone 0.27

methanol 0.41

95% ethanol 0.16

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.

Ref

11

-

11

11

11

11

12

12

12

12

12


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 ternpera- ture 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 . 1 1 Infrared Spectrum 2 . 1 2 Nuclear Magnetic Resonance Spectrum 2 . 1 3 Mass Spectrum 2 . 1 4 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 . 2 1 Elemental Analysis 6 . 2 2 Ultraviolet Spectrophotometry 6 . 2 3 Titrimetry 6.24 Chromatography Analysis in Biological Fluids and Pharmacokinetics References

DIPERODON 101

1. D e s c r i p t i o n

1.1 Name: Diperodon Diperodon ~ s L ~ J i s d 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. It is a l s o known as 1 ,2 -p ropaned io l , 3 - ( l - p i p e r d i n y l ) - , b i s (phenycarbamate) monohydrate.

1 . 2 Formula and Molecular Weight

415.49

1 . 3 Hydra t e s

monohydrate and anhydrous p h y s i c a l l y s t a b l e compound .

Diperodon h a s been r e p o r t e d t o ex is t i n b o t h t h e orms w i t h t h e former b e i n g t h e i

1 . 4 S a l t s The h y d r o c h l o r i d e s a l t is t h e o n l y r e p o r t e d s a l t

of pha rmaceu t i ca l i n t e r e s t 5 .

1 .5 Appearance, Color , Odor and Taste Diperodon occur 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. P h y s i c a l P r o p e r t i e s

2 . 1 S p e c t r a

2 .11 I n f r a r e d Spectrum

reco rded 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 ass ignmen t s from t h i s spectrum are p r e s e n t e d i n Tab le I.

The I R spectrum od d ipe rodon h y d r o c h l o r i d e S t r u c t u r a l

Figure 1. Infrared Spectrum of Diperodon Hydrochloride

DIPERODON 103

Table I

Infrared Spectrum of Diperodon HC1

-1 IR Absorption Band (cm ) Assignment

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

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

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 )

-CH2-(aliphatic ring)

Impurity

-H- CH

-1-0-CH -

-C-0-CH-C

0 2

9 2

-CH- ( aroma tic) 0 11

-0C-NH-C H 6 5 +

- C-N- C H

No.

6

-

-

4

2

1

5

2

1

Proton Assignment

8.2

7.5

6.7

5.7

4.5

2.7

0.1,0.2

-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 spectrum of

diperodon from a s o l i d probe i n s e r t i o n is d e p i c t e d i n F i g u r e 3. The ex t r eme ly weak i n t e n s i t y of t h e p a r e n t i o n peak a t 397 m / e i s t y p i c a l of carbamates which undergo the rma 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 . O the r s t r u c - t u r a l a s s ignmen 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 Tab le 111.

Table I11

Mass S p e c t r a l F ragmen ta t ion of Diperodon

MassICharge (m/e) Assignment A

98 L b - C H 2 -

119

12 4

1 4 1

158

0 0 -NH-t-

-CH2CHCN

N -CH CH- CHOH C C N-CH2-C=CHOH

+ 260 l o s s of 119 and H

No compara t ive l i t e r a t u r e spec t rum is a v a i l a b l e .

2.14 U l t r a v i o l e t Absorp t ion Spectrum The u l t r a v i o l e t a b s o r p t i o n spec t rum

of a ~ x ~ O - ~ M s o l u t i o n of diperodon i n H C 1 is shown i n F i g u r e 4. Maximal a b s o r t i o n occ r ed a t 233 nm w i t h a mola r abso r -

-'cm-l. The a b s o r p t i o n spectrum w a s p t i v i t y of 2 . 6 ~ 1 0 1 mole a l s o r eco rded i n h e p t a t e with-? A-Tax of 234 nm and a molar 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 comparat ive 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 p rocedure of t h e N a t i o n a l Formulary!

8

2.2 O p t i c a l R o t a t i o n The o p t i c a l r o t a t i o n o f d ipe rodon h a s a n

assymetric c e n t e r and t h e me thy l e t h y l ke tone s o l v a t e s of t h e

Ln K1

0

n!

0

0

d!

.

30h

38'2

39

5

3h

S

32s

30s

38

2

09

2

Oh

2

022

00

2

08

I

09 1

Oh

I

0.2

00

08

09

Oh

02

0

106

-. 0

0

0

s Q

0

2 0

0

33

NV

WO

S9

V

C

0 a 0

w 0

107

108 JORDAN L. COHEN

d- and 1- forms were repor ted8to be [aID i- 14.5 and [aID-14.3" r e spec t ive ly . Water w a s t h e so lven 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 epor t ed

a mel t ing poin t of 106.5"C f o r diperodon and a range of 197- 198°C f o r i ts hydrochlor ide sal t . Current compendia3,101ist t he mel t ing range f o r t h e hydrochlor ide between 195 and 200°C with decomposition.

2 . 4 S o l u b i l i t y Diperodon is p r a c t i c a l l y in so lub le i n water

but i s moderately s o l u b l e i n a l coho l and very s o l u b l e i n most non-polar so lven t s . The hydrochlor ide sa l t i s s o l u b l e i n a lcohol , s l i g h t l y so lub le i n e t h y l a c e t a t e , ace tone and water ( l e s s than 1%) and i n s o l u b l e i n most organic 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 repor t ed ly increased by t h e add i t ion 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 is repor t ed t o form 1:l so lub le complexes wi th 1 ,3 ,5 - t r in i t robenzane .I2 These i n t e r - a c t i o n s are pos tu l a t ed t o involve the t e r t i a r y amino group and a r e probably charge- t ransfer and hydrophobic i n na tu re . A s i g n i f i c a n t s p e c t r a l change is observed a t 475 nm.

2.5 Di s soc ia t ion Constant Diperodon is a t e r t i a r y amine and i s weakly

bas i c . Aqueous s o l u t i o n s of 1% diperodon hydrochlor ide have a pH of 5 .1 . l ' Although the d i s s o c i a t i o n cons t an t i s not spe- c i f i c a l l y repor ted i n the l i t e r a t u r e a pKa of 8.44 can be es t imated from t h i s in format ion .

2.6 Dipole Moment The d ipo le morlrent of diperodon is n o t a v a i l -

ab 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

hydrochlor ide has been determined and is summarized i n Table IV . I 3

DIPERODON 109

Tab le 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 1/10 28 111, 2.26 -13 4.29 -50 2.95 -16 4.58 -23 3.13 - 34 5.10 -50 3.22 -18 5.89 -60 3.49 -27 7.06 -23 3.64 -28 9.39 -22 3.94 -25 11.42 -100

3. S y n t h e s i s Diperodon is one of s e v e r a l pheny lu re thane 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 have demonostrated 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 has been P a t e n t e d , 16involves t h e consenda t ion 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 re sence of a l k a l a i and then condensa t ion of t h e r e s u l t i n g 1-piperidinopropane-2,3- d i o l (11) w i t h pheny l i socyana te (111) . The s y n t h e s i s is 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,-> R2NCH2 CHOHCH 20H

(1) (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 is 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 be 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 acetate . The f r e e b a s e can t h e n be o b t a i n e d by add- i 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 hydro- c 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 be d r i e d ove r anhydrous sodium s u l f a t e , f i l t e r e d and evapora t ed .

110 JORDAN L. COHEN

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

5. S t a b i l i t y and Compat ib i l i ty Diperodon hydrochlor ide is 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 should be s t o r e d i n non- a l k a l i n e g l a s s con ta ine 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 base and g e n e r a l l y a trace of ac id 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 paper can a l s o l ead t o p r e c i p i t a t i o n of t he f r e e base and less of potency of diperodon hydrochlor ide s o l u t i o n s . So lu t ions of t he hydroch lo r ide a l s o appear t o decompose over t i m e t o produce t r a c e amounts of a n i l i n e . This is a c c e l e r a t e d by h e a t i n dur ing 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 of a l k a l a i . g 8 A maximal pH of 4 . 8 is recommended f o r diperodon hydrochlor ide s o l u t i o n s and s o l u t i o n s wi th t r a c e s of c loud iness o r c o l o r should not be used. Diperodon monohydrate, which i s not in - compatible wi th t r a c e s of a l k a l a i has been u t i l i z e d more re- c e n t l y i n non-solut ion dosage forms inc lud ing l o t i o n s and ointments.5

6. Methods o f Analysis 6 . 1 I d e n t i f i c a t i o n Tests

Diperodon has 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 in - f r a r e d spectrophotometry * ' Condit ions f o r paper e lec t rophor- es is19and paper and th in- layer chromatography20have a l s o been e s t ab l i shed . The x-ray d i f f r a c t i o n p a t t e r n has a l s o been re- ported13(see s e c t i o n 2.7) . have been repor ted t o d i s t i n g u i s h diperodon hydrochlor ide from o t h e r a n e s t h e t i ~ s . ~ A white 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 so lubized by excess ammonia . Addit ion of H C 1 , sodium n i t r a t e and be tanaphthol produces a whi te p r e c i p i t a t e which darkens t o yel low and then orange upon s tanding . Diperodon hydrochlor ide r e a c t s wi th ch lo r ide t o g ive an organge-yellow p e r c i p i t a t e .

Several s p e c i f i c chemical tes ts

6.2 Q u a n t i t a t i v e Ana ly t i ca l Methods

5 6 . 2 1 Elemental Analysis Chlor ide is determined by g rav ime t r i c ana l -

y s i s fol lowing the a d d i t i o n of s i l v e r n i t r a t e t o an ammonia s o l u t i o n . Nitrogen is analyzed us ing a modified Kje ldahl determinat ion. Selenium oxychlor ide is used i n p l ace of copper s u l f a t e a s a c a t a l y s t and a four hour , r a t h e r than two hour , d i g e s t i o n i s used.

DIPERODON 111

6.22

o in tmen t i n v o l v e s

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 rocedure f o r d ipe rodon a chromatographic 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 rug u s i n g an alumina 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 as 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 measuring 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 molar a b s o r p t i v i t y of d ipe rodon 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 r ange t o b e ach ieved .

6 . 2 3 Ti t ra t i o n The compendia1 a s s a y f o r d ipe rodon i n v o l v e s

t i t r a t i o n i n ace t ic 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 t o 39.75 mg of diperodon. 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 mercu r i c 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 .

4 Diperodon h y d r o c h l o r i d e can be

4

6.24 Chromatography A q u a n t i t a t i v e t h i n - l a y e r chromatographic

method u s i n g pho todens i tome t ry h a s been r e p o r t e d . 20

7 . Ana lys i s i n B i o l o g i c a l F l u i d s 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 conce rn ing 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 , metabol ism o r pha rmacok ine 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. Davies 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 JORDAN L. COHEN

1. 2. 3.

4. 5 .

6.

7 .

8.

9. 10. 11. 12 . 13.

14. 1 5 .

16 . 1 7 .

18.

19 .

20.

21.

Re fe rences The N a t i o n a l Formulary, XlV, p.232 (1975) . Merck, Index, 8 t h Ed., p.385 (1968) . Remington's P r a c t i c e of Pharmacy, 1 4 t h Ed., p.1076 (1970) . J .S . Scan lon , General P r a c t i c e , 27, 1 3 (1964) . Diperodon Hydroch lo r ide Brochure , S.B. P e n i c k and Co., New York, N . Y . ( 1962) . These s p e c t r a were k i n d l y p rov ided by D r s . K . F l o r e y , B. T o e p f i t z and A. Cohen, Squibb Med ica l Resea rch , 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 and W.R. Brode, J. Am. Chem. SOC., 64, 112 (1942) . T.H. R ide r , J . Am. Chem. SOC. , 52, 2115 (1930) Un i t ed 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 (1973) . 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 . Pharmacol . Exper. The rap . , 5, 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 ) , 31 (1968). T.H. R i d e r , J . Am. Chem. SOC., 52, 2583 (1930) T.H. R ide r and A . J . H i l l , J . Am. Chem. SOC., 52 , 1528 (1930) . U.S. P a t e n t 2 E.S. Cook, K . 24, 269 (1935 E.S. Cook and (1937) . 0. S c h e t t i n o , 89353 (1965) .

004,132 (1935) Bambach and T.H. R i d e r , J . Am. Pharm. Assoc.

T.H. R ide r . J. Am. Pharm. ASSOC., 26, 222

Farmac Ed. P r a t . 20, 40(1965); C.A. 62:

V . J o k l and A. Muchora, Acta. Fac. Pharm. Behemoslov., - 11 :23 (1965); C.A. 64: 14024g (1966) . J . W . Becher , The Assay and 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, School of Pharmacy, 1962. 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 1930 t o J u l y , 1976 i n c l u s i v e .

,

ERGOTAMINE TARTRATE

Bo Kreilgard

114 BO K R E I L G ~ R D

CONTENTS

1. D e s c r i p t i o n 1.1 N a m e 1 . 2 Formula and Molecular Weight 1 . 3 Appearance, C o l o r , Odor and Taste

2 . P h y s i c a l P r o p e r t i e s 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 S p e c t r a Nuclear Magnetic Resonance Spectrum U 1 t r a v i o l e t Spectrum F 1 uo re s c e n ce and P ho s pho re s ce nce Mass Spectrum O p t i c a l Ro ta t ion Mel t ing 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 D i s s o c i a t i o n C o n s t a n t s

3. P roduc t ion and S y n t h e s i s

4 . Degradat ion o f Ergotamine T a r t r a t e 4 . 1 Chemistry o f Ergotamine Degrada t ion 4 . 2 S t a b i l i t y i n Pha rmaceu t i ca l Dosage Forms

5. Drug Metabolism

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 T e s t s 6 . 2 Element A n a l y s i s 6 .3 Spec t ropho tomet r i c A n a l y s i s

6 .3 .1 U l t r a v i o l e t 6 .3 .2 C o l o r i m e t r i c 6 .3 .3 F luo rescence

6 . 4 Non-Aqueous T i t r a t i o n 6.5 Chromatographic A n a l y s i s

6 .5 .1 Paper Chromatography 6.5.2 Thin Layer Chromatography 6 .5 .3 Column Chromatography 6.5.4 High P r e s s u r e L i q u i d Chromatography

7. Determina t ion i n B i o l o g i c a l Systems

8. Determina t ion i n Pha rmaceu t i ca l P r e p a r a t i o n s

9. References

ERGOTAMINE TARTRATE 115

1. Description

1.1 Name

Ergotamine Tartrate (1-3) is the (+)-tartrate salt of (6aRI9R)-N-((2R,5S,1OaS,l0bS)-5-phenyl- methyl-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-hexa- hydro-indolo[4,3 -fglquinoline-9-carboxamide.

1.2 Formula and Molecular Weight

r

COOH

CHOH

CHOH

COOH

I

I

I

2

(C33H3s05N5) 2"24H606 Molecular Weight: 1313,43

1.3 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-


tamine 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 Spec t ropho tomete r , Model 457. The I R spectrum o f e rgotamine base u s i n g t h e K B r as w e l l as t h e n u j o l t echn ique h a s been re- p o r t e d by Cromp and Turney ( 4 ) and Hofmann ( 5 ) .

2 .2 Nuclear Magnet ic Resonance Spectrum 1 The H -NMR spec t rum 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 rgo tamine 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 hour s ) 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 c o n t a i - n ing 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 spectrum w a s r e c o r d e d on a Jeol JNM- C-60HL i n s t r u m e n t . The s p e c t r a l a s s ignmen t s of some of t h e p r o t o n s are p r e s e n t e d i n Table 1. A d e t a i l e d spec t ra l a n a l y s i s o f s e t o c l a v i n e , which has a s t r u c t u r e s imi 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 been r e p o r t e d ( 6 ) . The 13C-NMR spectrum of e rgotamine and e rgo tamin ine have been pub l i shed 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 spec t rum of e rgo tamine 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 spec t rum of e r g o t a - mine s a l t s and e rgo tamine 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 abou t 317 nm and a minimum a t abou t 270 nm. Maximum wavelengths and molar a b s o r p t i v i t i e s are p r e s e n t e d i n Tab- l e 2 .

2 . 4 F luo rescence and Phosphorescence

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 and i s o l y s e r g i c a c i d t y p e are 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 of t h e 1 0 , l O a doublebond conjuga- t e d w i t h t h e i n d o l e group c a u s e s loss of t h e f l u o r e s c e n c e ( 1 1 , 1 4 1 . F luo rescence s p e c t r a o f e rgotamine i n aque0u.s s o l u t i o n (pH 2 . 1 and 10.8) and i n e t h a n o l are shown i n F i g u r e 4 . There i s a hypsochromic 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 and from aqueous 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 ) de- s 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 l u o r e s c e n c e i n t e n s i t y of e rgotamine . The f l u o r e s c e n c e i n t e n s i t y of e rgo tamine i n


Table 1

Pro ton

NMR S p e c t r a l Assignments of Ergotamine T a r t r a t e

Number of Chemical Mu1 t i p 1 i c i t y p r o t o n s S h i f t (ppm)

NH ( i n d o l e

NH (amide)

Aromatic p r o t o n s

C-lO'b,-OH 1 + .

3 , NE-CH

J Tartrate,-OH

H2° C-10 ' a , -H

C-5' ,-H

C - 2 ' ,-CH 3

* *

1 10.9 Broad s i n g l e t

1 9 .5 Broad s i n g l e t

10 6.9-7.4 Mu1 t i p l e t

* 5 6.3-6.7

1 6 . 3 T r i p l e t

1 4 .5 T r i p l e t

3 1 . 5 Broad s i n g l e t

~ ~~ ~~ ~ ~~

* Exchangeable wi th 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 Solut ion ( 8 ) .

Table 2

Compound

Ergotamine t a r t r a t e

Ergotamine t a r t r a t e

Ergot a m i ne t a r t r a te

Ergo tamine t a r t r a t e

Ergotamine

A Ergo t a m i ne p!

Ergo tamine

Ul t r av io l e t Spectral Charac t e r i s t i c s

Reference max, nm Solvent

1% t a r t a r i c ac id 240, 318 7.72(at 318 nm) 8

1% t a r t a r i c ac id 317 7.34 9

0.01 N H C 1 317 -7.50 2

0.01 M t a r t a r i c acid 317.5 8.00 10

Ethanol 318 7 .24 11

Methylene chlor ide 308-310 8.59 1 2

A

Ethanol 311 8.60 13

122 BO K R E I L G ~ D

7.

c

2 W t 2

- @ 5 '

- W 0 2 W 0 v) W

a 2 0 3

Y

0

W A V E L E N G T H (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 rgo tamine i n : (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). Emission s p e c t r a ( r i g h t ) of e rgo tamine i n (1) water a t pH 2 . 1 (Aex 325 nm) : ( 2 ) water a t 10.8 ( A e 318 nm); (3 ) e t h a n o l (Aex 318 my (17).


aqueous s o l u t i o n i s h i g h l y dependen t on t h e p H o f t h e s o l u t i o n showing a lmost e q u a l i n t e n s i t y i n t h e pH-range 1 - 9 and maximum i n t e n s i t y a t pH -11 ( 1 7 , 1 8 1 . Data on f l u o r e s c e n c e o f e rgo- t amine a r e summarized i n Table 3 .

The phosphorescence spec t rum 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 and 6 1 3 nm ( 1 3 ) .

2 . 5 Mass SDectrum

S e v e r a l a u t h o r s have r e p o r t e d on t h e mass spec t rum o f e rgo tamine ( 2 0 - 2 3 ) . The low r e s o - l u t i o n mass spec t rum 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 peak) i s a b s e n t i n t h e spectrum o b t a i n e d by 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 " r e a s o n a b l e - s i z e d " p a r e n t i o n i s o b s e r v e d u s i n g 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 between 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 ' ) , fo l lowed 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 p a r t . I o n b ( m / e = 2 6 7 ) i s i d e n t i c a l w i t h 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 mole- c 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 p e r c e n t o f t h e t o t a l i o n c u r r e n t ( 2 3 ) . O t h e r i m p o r t a n t f r agmen t s a r e shown i n Scheme I ( 2 1 , 2 3 ) .

C h a r a c t e r i z a t i o n o f e r g o t a m i n e 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 b a s e d on t h e i o n s c , 1, & and 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 f r a g m e n t s i n c l u d e t h e me thy l g roup a t C - 2 ' and t h e b e n z y l g r o u p a t C - 5 ' .

t amine h a s a l s o been r e p o r t e d ( 2 0 , 2 1 1 . 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 rgo-

2 . 6 O p t i c a l r o t a t i o n

Carbons 6a , 9 , 2 ' , 5 ' , 1 0 ' a and 1 0 ' b o f e rgo tamine a re asymmetr ic , r e s u l t i n g i n 6 4 pos- s i b l e c o n f i g u r a t i o n a l i somers . However, o n l y i somers w i t h changed c h i r a l i t y a t C-9 and 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 ro- t a t i o n o f e rgo tamine and some o f i t s i somers

Table 3

D a t a on Fluorescence of Ergotamine

Solvent

Ethanol

Water

Ethanol

Water (pH 2.1)

- Water (pH 10.5) 3

Ethanol

Water (pH 2-6)

Water ( p H 8-14)

Acetone

Temper a t u r e

25OC

2 5OC

77OK

ambient

ambient

ambient

ambient

ambient

ambient

Exc i t a t ion wavelength (nm)

3 20

3 20

3 20

325

318

318

335

325

350

Maximal Emission wavelength (nm)

404

432

383

43 5

422

402

43 5

4 2 5

400

Reference

1 3

1 3

13

17

1 7

17

18

l a 19

loor 80

'0 (i)

1 2 5 ( h )

50

1 2 0 ( k )

w 100

2 4 4 ( d )

[L [L 3 V

- 6 E I

2

I- 0

a

- 4 +

U 0

bp

150 200 250 300 M'E

Figure 5. Mass Spectrum of Ergotarnine ( 2 2 ) .

q2

0 I

qj- 0

I 0 a

-

0

+- -

I 1

+*

.- -

+

4J k

w

fm

0

+I 0

ro .lJ

c,

H

H

w X

U

m

126


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 s p e c t r u m of e r g o t a r n i n e i n m e t h a n o l h a s b e e n r e p o r t e d ( 2 5 ) a n d i s r e p r o d u c e d i n F i - g u r e 6 .

2 . 7 M e l t i n g r a n g e

The 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 re- p o r t e d :

18OoC (decomp. (1)

-190Oc ( 2 ) 203OC (decomp. ) ( 2 9 )

2 . 8 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 a n d M o - l 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 f o l l o w s :

S o l v e n t A p p r o x i m a t e Tempera- R e f e - s o l u b i l i t y t u r e r e n c e

rng/rnl

Water 2 . 5 30 ( 3 3 ) I1

0 , l N H C 1

-2 Ambient ( 1 1 2 ) 3 . 5 30 ( 3 3 )

0 , l M p h o s p h a t e b u f f e r ( p H 6 . 6 5 ) 0 . 0 1 30 ( 3 3 ) E t h a n o l 2- 3 Ambient ( 2 , 3 4 1 C h l o r o f o r m -1 Ambient ( 2 )

The base, 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 :300 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 lmost i n s o l u b l e i n wa te r a t room tempera- t u r e ( 2 9 ) .

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 seve- r a l a u t h o r s ( 1 7 , 3 5 - 3 7 ) . V i r t u a l l y q u a n t i t a t i v e e x t r a c t i o n o f e r g o t a r n i n e f rom a q u e o u s 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 . B e r a n and 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 K R E I L G X R D

Table 4

O p t i c a l R o t a t i o n f o r Ergotamine

and some of i t s isomers

Compound

Ergotamine

Ergo tamine

Ergotamine

Ergo tamine

Ergo tamine

Ergo tamine

Ergotamine

Ergo tami ne

Ergo t a m i ne

Ergotamine

Ergo t a m i ne

Ergotamine

Ergo t a m i ne

Ergotamine

Ergotamine

[al

-181O

-166'

-150°

-160°

-192O

-12.7O

-8.6O

-155O

+40°

-466O

-375O

-309O

-174O

-152O

-145O

Ergotaminine +4 50° 0 Ergotaminine +369 0 Ergotaminine +462 0 Ergotaminine +397

Ergotaminine +49 7O

Ergotaminine +381°

Ac i -e rgo tamine -32O

Aci-ergo tamine +258O

A , r-lm

589

546.1

589

589

546.1

589

546.1

589

5 89

365

405

436

546

578

589

Condi t ions CHC13(c=l) ,25 0 C

CHC13 ( c= l ) ,20 0 C

C H C 1 3 ( c = 1 ) ,20°C

CHC13 (c=l ) ,2O0C

CHC13 (c=l ) ,2O0C p y r i d i n e (c=l. 0) 20 0 C

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

CHC13, 20°C

e t h a n o l ,2OoC

CHC13 (c=O .6) , 20°C

CHC13 (c=0.6) ,2O0C

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

CHC13 (c=0.6) ,20°C

CHC13 (c=O. 6 ) ,20°C

3

CHClj(c=0.6) ,20 0 C

546.1 CHC13, 20°C

C H C l (c=O. 5) ,2O0C 3 589

546.1 C H C l (c=0.5) ,2O0C 3 589 0 p y r i d i n e ( c = O .5 ) , 2 O C 546.1 p y r i d i n e ( c = 0 . 5 ) ,20 0 C

589 CHC13, 2O0C

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

p y r i d i n e ( c = 1 . 2 ) ,20 0 C 589

Reference

26

27

28

29

29

29

29

30

30

31

31

31

31

31

31

27

29

29

29

29

30

32

32

0

0

a-

0

Lo

m

0

c*l m

0

00

(v

0

=r

(v

0

0

(v

E

C

I

I- CY

r

W

W

w e 3

1 29


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.

has been shown t h a t e rgotamine t a r t r a t e forms molecu la r complexes wi th 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 pe rmi 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 .

Using t h e phase s o l u b i l i t y t e c h n i q u e i t

2 . 9 D i s s o c i a t i o n C o n s t a n t s

Due t o t h e low s o l u b i l i t y o f e rgotamine 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, cou ld n o t b e de te rmined by c o n v e n t i o n a l t i t r a - t i o n methods. An a p p a r e n t pKa v a l u e of 6 . 4 a t 2 4 O w a s o b t a i n e d 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 of e rgotamine i n 2% c a f f e i n e (39 ) i n accordance 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 s o l u b i l i t y method. A pKa v a l u e of 5 . 6 i n 80 p e r c e n t aqueous m e t h y l c e l l o s o l v e h a s been re- p o r t e d ( 5 ) .

3 . Produc t ion and S y n t h e s i s

Ergotamine w a s o r i g i n a l l y produced by i s o l a - t i o n of t h e a l k a l o i d from t h e fungus C l a v i c e p s Pur- purea ( 3 0 , 4 0 ) . Methods o f i s o l a t i o n o f e rgo tamine and p r e p a r a t i o n of t h e t a r t r a t e s a l t have been de- s c r i b e d ( i . e . : 5 ,29 ,42 ) . The complete s y n t h e s i s of e rgotamine was n o t r e p o r t e d u n t i l 1 9 6 1 (43) (Scheme 11) . Methylbenzyloxymalonic ac id -hemi -es 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-proline- l ac tam (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 ve ry l a b i l e and i s t h u s immediately t r e a t e d w i t h Pd/H2 t o c l e a v e t h e benzy l g roup (IV) . (IV) c y c l i z e s spon taneous ly 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 stereo- isomer wi th 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 so la - t e d . The carbe thoxy group a t C - 2 ' i s t r ans fo rmed i n t o an amino group(V1) through 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 reac- t 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 ch loroform and t r i b u t y l a m i n e t o form ergotamine . 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 a1 ( 4 4 ) . An i m - proved s y n t h e s i s o f e rgo tamine u s i n g ( S ) - ( - 1 -methyl-benzyloxy-malonic-hemi-acid c h l o r i d e h a s been r e p o r t e d ( 4 5 ) .


*

1. - COOH

2 . - C o c l HCI ' H2N V

3. - CON3 CH2C6H5

s c - C I

N-CH3 8 HCI $i V I

H H

ERGOTAM I NE

SCHEME I1 Synthesis of Ergotarnine ( 4 3 ) .

132 BO K R E I L G ~ D

4 . Degradat ion of e rgotamine t a r t r a t e

4 . 1 Chemistry of e rgotamine d e g r a d a t i o n

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

1 Ergot;;:e $ Ergot;y;ine 1 Aci-ergotamine z Aci-ergotaminine

i c t c

Lysergic acid amide Lysergic acid

I lsolysergic acid amide I 1 lsolysergic acid

Lumi compounds

I

i e

Oxidation products

Scheme 111. Degradat ion scheme f o r e rgotamine . 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 . b: 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-2 ' ( t h e a c i - i n v e r - s i o n ) . c: h y d r o l y s i s . d: fo rma t ion o f lumi compounds. e: o x i d a t i o n .

Ep imer i za t ion a t C-9 w i t h fo rma t ion 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 , e rgo tamin ine , i s t h e m o s t impor t an t r o u t e of d e g r a d a t i o n ( 4 6 - 4 8 ) . I n a c i d i c s o l u t i o n s e r g o t a l k a l o i d s e p i - m e r i z e a t c -2 ' , t h e s o - c a l l e d a c i - i n v e r s i o n ( 3 2 , 4 7 , 4 9 ) . Hydro lys i s of t h e f o u r e rgo tamine 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 corresponding is0 compounds (27 ,47 ,48 ,50 -53) . Upon exposure t o l i g h t , p a r t i c u l a r l y UV-l ight , e r g o t a l k a l o i d s add a molecu le w a t e r t o t h e 1 0 , l0a-doublebond (15,551 as shown:

133 ERGOTAMINE TARTRATE

CO- R

r? CO-R

J 7 N- CH,

H

N-CH,

N - C H , H O

A l l compounds mentioned above are a b l e t o undergo o x i d a t i o n ( 5 6 ) . One of t h e expec ted de- g r a d a t i o n p roduc t s i s t h e 2-0x0-3-hydroxy-2,3- d ihydro d e r i v a t i v e s ( R = p e p t i d e 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 deg rades when exposed t o l i g h t , humid c o n d i t i o n s and h igh t empera tu re (34).

4 . 2 S t a b i l i t y i n Pharmaceut ica l Dosage Forms

S e v e r a l s t u d i e s on t h e s t a b i l i t y of ergotamine t a r t r a t e i n aqueous s o l u t i o n have been done (47 ,48 ,55 ,57 ,58 ,59 ,60 ,66) . However, i n some of t h e s t u d i e s non- spec i f i c methods o f a n a l y s i s were used.

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 proceeds r a t h e r f a s t a t t h e pH of op t ima 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 drug


are fo rmula t ed t o c o n t a i n an e q u i l i b r i u m mix- t u r e of t a r t ra tes o f ergotamine and ergotami- n i n e ( 1 , 2 , 6 3 ) . I n accordance w i t h t h i s , inve- s 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 ergotamine showed a c o n t e n t of o n l y 50-60 p e r c e n t of ergotamine ( 6 1 , 6 2 ) . A t p H = 3.6 such a mix- t u r e appea r s t o be 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 whi 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 is 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 - mat ion of lumi compounds h a s a l so been i n v e s t i - g a t e d ( 5 5 ) . I f s o l u t i o n s c o n t a i n i n g e rgo tamine 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 gas fo rma t ion of l u m i compounds and ox i - d a t i o n are ve ry s l o w p r o c e s s e s ( 4 7 , 4 8 1 .

longed p e r i o d s i n t a b l e t s . I n t a b l e t s c o n t a i - n ing e rgotamine t a r t r a t e , p h e n o b a r b i t a l and a mix tu re of 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 of ergotamine was observed t o d e c r e a s e g r a d u a l l y du- r i n g t i m e of s t o r a g e ( 6 4 ) . I n accordance w i t h t h i s t h e c o n t e n t of e rgotamine 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 (65 ) 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 about 2 y e a r s when 4 mg t a r t a r i c a c i d b lended w i t h 4 0 mg lactose were used as 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, used a method which on ly d e t e c t e d l o s s of a c t i v e drug through o x i d a t i o n .

Ergotamine t a r t r a t e i s n o t s t a b l e f o r pro-

5. Drug Metabolism

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 ab- s o r p t i o n , metabolism and e x c r e t i o n of e rgo tamine ( 6 6 ) .

E rgo 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 ge- n e r a l p o o r l y and i r r e g u l a r l y absorbed from t h e ga- s t r o i n t e s t i n a l t r ac t and a l a t e n t p e r i o d of -30 m i - n u t e s w a s observed ( 3 2 , 6 6 ) . C a f f e i n e 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 rgotamine t a r t r a t e and reduces 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 ve ry r a p i d l y from t h e b lood a f t e r i n t r a - venous i n j e c t i o n (67 -69) . Only a minor amount of t h e drug 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 de- t 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

mation on metabolism of ergotarnine seems t o b e a v a i l a b l e i n t h e l i t e r a t u r e .

135

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 t es t s

Ergotamine t a r t r a t e can 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 and 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 s e c t i o n 2 ) . Various chromatographic methods such 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 rov ide 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 produced when 0 .3 mg a lka - l o i d i s d i s s o l v e d i n 1 . 0 m l g l a c i a l ace t ic 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 ) as FeCl and 0 . 1 % g l y o x y l i c a c i d ) and 1 . 0 m l of 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 ) . Th 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, modi f ied s l i g h t l y , i s used i n USP X I X (1) is based on 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 a lmost a lways p r e s e n t as a n impur i ty i n g l a c i a l a c e t i c a c i d . The van Urk r e a c t i o n i s based on condensa t ion 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 one molecule p-dimethylaminobenzah dehyde fo l lowed by a F e ( I I 1 ) - c a t a l y z e d oxida- t i o n of t h e condensa te ( 5 , 7 1 , 7 2 ) . O the r c o l o r t e s t s have been r e p o r t e d by C l a r k e ( 7 3 ) . E r g o - tamine t a r t r a t e h a s been i d e n t i f i e d by means of 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 roduc t s ( 7 4 ) and by o s c i l l o p o l a r o g r a p h y ( 7 5 ) .

3

6 . 2 Elemental a n a l y s i s

The e l emen ta l composi t ion of e rgotamine 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 hour s a t 60° and a t a p r e s s u r e below 1 mm Hg t o remove water i s :

Carbon 6 4 . 0 1 % Hydrogen 5.83% Ni t rogen 1 0 . 6 6 % Oxygen 1 9 . 4 9 %

136 80 KREILGARD

6.3 Spec t ropho tomet r i c A n a l y s i s

6 . 3 . 1 U l t r a v i o l e t

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 rgotamine t a r t r a t e can 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 be 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 absorbance of t h e f i n a l samp- l 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 depending on t h e s o l v e n t (see s e c t i o n 2 . 3 ) .

Ergotamine t a r t r a t e i n a t a r t a r i c a c i d so- l u t i o n obeys B e e r ' s l a w a t 271 and 318 run i n t h e c o n c e n t r a t i o n r ange (1-10) x (8,761. 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 as impu- r i t i es i n hydrogenated a l k a l o i d s can be d e t e r - mined by measurement of t h e u l t r a v i o l e t absorbance ( 1 0 ) . By r e a d i n g t h e absorbance a t 2 7 1 , 283 and 318 nm o f a degraded ergotamine 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 of f o r m a t i o n of l u m i compounds and o f o x i d a t i o n p r o d u c t s cou ld b e e s t i m a t e d ( 8 ) .

6.3.2 C o l o r i m e t r i c

The m o s t w ide ly used 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 rgo tamine i s t h e r e a c t i o n w i t h p-dimethylaminobenzaldehyde (8 t71 r72 t79-97) . 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 used i s named as e i t h e r t h e van Urk r e a g e n t ( 7 1 ) I t h e Maurice Smith r e a g e n t (89 ) 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 . Se- v e r a l a g e n t s have been sugges t ed t o b r i n g abou t t h e o x i d a t i o n of t h e condensa te (see sec- t i o n 6 . 1 ) such a s l i g h t ( 8 9 , 9 3 , 9 5 ) , FeC13 ( 8 0 , 9 3 , 9 5 ) , hydrogen pe rox ide (80 ,96 ,97 ) and s o d i - um n i t r i t e ( 9 5 ) . The absorbance of t h e f i n a l sample i s g e n e r a l l y measured a t 550 nm. The method i s n o t s p e c i f i c f o r e rgo tamine . 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 as 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 . Measuring absorbance a t 546 and 586 nm f o l l o w i n g t h e van Urk r eac t ion and c a l c u l a t i o n s assuming a two-component system w i l l l e a d t o an estimate of t h e amount of l u m i d e r i v a t i v e s i n a g i v e n ergotamine t a r t r a t e sample ( 8 ) . I t h a s been sugges t ed 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-


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 ) . Ergotamine ta r - t r a t e has a l s o been ana lysed 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 wi th m i d o p y r i m i d i n e ( 1 0 0 ) . Ion- p a i r format ion between ergotamine and t ropae - o l i n 0 0 0 ( t h e sodium s a l t of 4-[(2-hydroxy- napthy1)azolbenzol-sulphonic a c i d ) h a s been used 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 ergotamine (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 e rgotamine t a r t r a t e i n t a b l e t s h a s been d e s c r i b e d by Hoo- p e r e t a1 ( 1 7 ) . The t a b l e t s were e x t r a c t e d wi th an a c i d i c aqueous s o l u t i o n , which a f t e r be ing 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 benzene. A f t e r e v a p o r a t i o n of benzene 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 and 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 ead wi th an e x c i t a t i o n wave- l e n g t h of 318 nm and an emis s ion wavelength 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 be 0 . 0 0 2 ug p e r m l and t h e s t a n d a r d cu rve w a s l i n e a r up t o 5 pg p e r m l ( 1 7 ) . I t i s r e p o r t e d t h a t t h e s t a n d a r d c u r v e 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 so lu - t i o n i s non- l inea r i n t h e range 10-60 ug p e r m l ( 1 0 2 ) . I n o r d e r t o i n c r e a s e s e n s i t i v i t y f l u o r i m e t r i c d e t e c t o r s have been used i n ana- l y s i s of ergotamine by h igh performance l i q u i d chromatography ( 1 9 , 1 0 3 ) . For d e t e r m i n a t i o n of e rgotamine t a r t r a t e i n pha rmaceu t i ca l dosage forms by q u a n t i t a t i v e t h i n l a y e r chromatography, e l u t i o n fo l lowed 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 used (61). The f l u o r e - scence i n t e n s i t y o f e rgo tamin ine i s 2.5 f o l d g r e a t e r t han t h a t of e rgotamine ( 1 9 ) . F l u o r i - m e t r y h a s been used t o de te rmine 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 a lka - l o i d s e .g . d ihydroergotamine ( 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 mixture of a c e t i c anhydr ide and g l a c i a l ace t ic a c i d (1) o r a mix tu re o f d ioxane and g l a c i a l a c e t i c a c i d ( 2 ) c a n be t i t r a t e d w i t h pe rch lo - r i c a c i d i n g l a c i a l ace t ic a c i d . The endpo in t can be observed p o t e n t i o m e t r i c a l l y o r by us ing


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 .05 - 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 e rgotamine 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 ch lo ro - form e x t r a c t i o n o f an a l k a l i n e aqueous so lu - t i o n and subsequent t i t r a t i o n wi th 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 ( 8 4 ) .

6 .5 Chromatography

6 .5 .1 Paper chromatography

S e v e r a l paper chromatographic sys tems f o r e rgotamine are summarized i n Tab le 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 are o u t l i n e d i n Table 6 . O the r r e p o r t s on PC of e rgotamine a r e found i n r e f e r e n c e s (92 ,106 ,107 ,159) .

Q u a n t i t a t i o n of e rgotamine fo l lowing pa- p 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 Thin Layer Chromatography (TLC)

A v a r i e t y o f TLC systems have been developed f o r e rgotamine and most of t h e s e are summarized i n Tab le 7 . Methods used f o r de tec- t i o n and i d e n t i f i c a t i o n of e rgotamine on t h e p l a t e are summarized i n Tab le 8.

Q u a n t i t a t i o n of e rgotamine fo l lowing TLC i s done us ing e i t h e r e l u t i o n t echn ique ( 8 , 111-114,120) or i n s i t u scanning (62,115-119) . 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 water-methanol 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 (112 ,120) . Measurement o f t h e e l u a t e i s done us ing e i t h e r W-spec t rophotometry o r c o l o r i m e t r y . The in s i t u measurement i s done u s i n g UV-ref lectance ( 1 1 6 1 , f l u o r i m e t r y (62 ,115 ,117) o r t r ansmis - s i o n of p l a t e s sp rayed wi th t h e van Urk t y p e r e a g e n t ( 1 1 9 ) . TLC sys tems have a l s o been 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 rgotamine and t h e most impor- t a n t d e g r a d a t i o n p roduc t e rgo tamin ine can b e de te rmined i n pha rmaceu t i ca l s by us ing t w o se-

N o . Paper -- 1. S c h l e i c h e r and

S c h h l l no. 2043b

2 . Whatman no.1

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

2

w W

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

5. Whatman no.1

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

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

Table 5

Paper Chromatography Systems f o r Ergotamine

Impregnat ion

Ace tone- f o r m a m i de (6 :4)

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

C i t r i c acid-phos- p h a t e b u f f e r (pH=5.6)

Ethanol-formamide (1:l)

E t h a no 1 - f o r m a m i de (1:l)

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

Dimethylpthala t e

S o l v e n t system

Benzene

Formami de-water (4:6) (pH=5.2 wi th Formic a c i d )

Benzene-ethanol (95%) (9:l)

Benzene

Chloroform

Formamide-O.066M Na2HP04 s o l u t i o n

(4:6)

Formamide-ci t ra te b u f f e r (pH 4.4)

( 2 : 8 )

Rf

0.17

-

0 . 3 7

0.35

0 .05

0.86

0 . 0 5

0.38

Appl i ca t i o n Reference

S e p a r a t i o n of 12 ,15 e r g o t a l k a l o i d s

S e p a r a t i o n of 16 e r g o t a l k a l o i d s

4 1

54

70

S e p a r a t i o n from 47 d e g r a d a t i o n pro- d u c t s

S e p a r a t i o n from 47 d e g r a d a t i o n pro- d u c t s

No.

8. -

9.

10.

11.

4

P 0

1 2 .

Paper

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

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

Whatnian no. 1

Whatman no.1

Whatman no. 1

13. Whatman no. 1

14. Whatman no .1

Impregnat ion

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

Formamide- benzoi c a c i d ( 2 5 : l )

5% sodium dihydrogen c i t r a t e

None

None

None

None


Formami de- 0 .1 N KOH ( 2 : 8 )

E t h e r

2.4 g of c i t r ic acid i n water-butanol (65:435)

Methylethylketone- acetone-formic a c i d - w a t e r (40:2:1:6)

Methylethylketone- d ie thylamine-water (921:2:77)

10 p a r t s of methyl i s o b u t y l k e t o n e s a t u - r a t e d w i t h 1 par t of 4% formic acid

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 methanol and 1 p a r t of 4% formic acid

Rf

0.00 -

0.24

0.65

0.80

0.91

0.16

0.47

A p p l i c a t i o n Reference

S e p a r a t i o n from degrada- t i o n p r o d u c t s

S e p a r a t i o n f r o m de grada- t i o n p r o d u c t s

I d e n t i f ica t i o n

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

47

47

78

104

104

104

104


Tab le 6

V i s u a l i z a t i o n o f Ergotamine o n Pape r Chromatograms

N o . Treatment - 1. U l t r a v i o l e t l i g h t

( A = 254 or 366 nm)

R e s u l t Re fe rence

B lue 15,78,104

2 . p-dimethylaminobenzaldehyde B l u e - v i o l e t 16,54,78, r e a g e n t ( v a r i o u s modif ica- 104 t i o n s )

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

Table 7

Thin Layer Chromatography Systems f o r Ergotamine

Solvent system

1. Heptane-tetrahydrofuran- toluene-chloroform (5:4:1:5)

2. Heptane-tetrahydrofuran- toluene- e thy 1 a c e t a t e (10:8: 3:9)

'p 3. Heptane- te t rahydrofuan- to luene (2:4:5) N

4. Heptane-tetrahydrofuran- to luene ( 2 : 4 : 1 )

5. Heptane-tetrahydrofuran- to luene (1 :4 :1 )

6. Tetrahydrofuran-toluene ( 4 : l )

7. Benzene-cyclohexane- d i e thy lamine ( 5 : 2 : 0.0 1 )

Sorbent

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

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

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





Rf Appl ica t ion and comment Reference

0.04 Separa t ion o f e r g o t 128

-

a l k a l o i d s

0.14 Separa t ion of e r g o t a l k a l o i d s

0.05 Separa t ion o f e r g o t a l k a l o i d s

0.08 Separa t ion of e r g o t a 1 ka l o i d s



0.05 Sepa ra t ion of e r g o t a l k a l o i d s

128

1 2 8

1 2 8

128

1 2 8

128

Solvent system

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

9 . Dibuthylether-dichloro- me thane-diethylamine (60:40:0.2) s a t u r a t e d with f ormamide

10. Chloroform-methanol (9:l)

P 11. Chloroform-methanol-

concent ra ted ammonia (18:l:O.Ol)

1 2 . Benzene-chloroform e thano l ( 2 : 4 : 1 )

13. Methanol-chloroform ( 2 : 8)

14. Diethylamine-chloroform (1:9)

Sorbent - Rf Appl ica t ion and comment Reference

S i l i c a g e l , formamide 0.17 Sepa ra t ion of e r g o t 129 impregnated a 1 ka lo i d s

S i l i c a g e l , formamide 0.29 Separa t ion of e r g o t 129 imp reg na t e d a l k a l o i d s

S i l i c a g e l

S i l i c a gel

S i l i c a g e l G

S i l i c a g e l G

S i l i c a g e l G

0.39 Separa t ion from o t h e r l y s e r g i c acid type compounds

0.25 Separa t ion from o t h e r l y s e r g i c a c i d type compounds

0 .62 Quan t i t a t ive a n a l y s i s

0.65 I d e n t i f i c a t i o n of e r g o t a l k a l o i d s

0.09 I d e n t i f i c a t i o n of e r g o t a l k a l o i d s

130

130

131

132

132

Solvent system Sorbent

15. Methanol-chloroform- concentrated ammonia (20: 80 : 0.2 )

Silica gel G

16. Chloroform-ethanol (96:4) Aluminiumoxide G

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

18. Benzene-N ,N-dimethyl- Silica gel G formamide (13 : 2 )

P P

- Rf Application and comment Reference

0.75 Identification of ergot 13 2 alkaloids

0.58 Identification of ergot 132 alkaloids

0.31 Quantitative'analysis 114

0.31 Quantitative analysis 114

19. Chloroform- die thy lethe r- Aluminium oxide G 0.01 Quantitative analysis 114 water (7:1:2)

20. Benzene-n-propanol-NH (1 M) Silica gel 3

(100:10:2)

21. Chloroform-ethanol-acetone Silica gel G (6: 4: 4)



22. ChLoroform-ethanol (9: 1) Silica gel G 0.27 Quantitative analysis 120

Silica g e l GF (Merck) 0.56 Usefulness of azeotropic 133 0.1 N Na CO

irnpregna ted

mixtures in TLC 254

23. Dichloromethane-methanol

2 3 (92.7:7.3)


24. Chloroform-ethanol (92: 8)

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

26. Acetone-cyclohexane (67.5:32.5)

d

P u1

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

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

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

3 0. Hep tane-carbonte tra- c h l o r i d e - p y r i d i n e (1:3: 2 )

31. Chlorofonn-acetone- d ie thylamine (5:4:1)

Sorbent - Rf A p p l i c a t i o n and comment Reference

S i l ica g e l GF254(Merck) 0.44 Usefu lness o f a z e o t r o p i c 133 0.1 N Na2C03

impregnated

mixtures i n TLC

S i l i c a g e l GF254(Merck) 0.16 Usefu lness of a z e o t r o p i c 133 0.1 N Na2COj

impregnated

mixtures i n TLC

Si l ica g e l GF254(Merck) 0 .31 Usefu lness of a z e o t r o p i c 133 0.1 N Na2C03

impregnated

m i x t u r e s i n TLC

S i l i c a g e l G 0.29 S e p a r a t i o n f r o m degrada- 61 t i o n p r o d u c t s

S i l ica g e l G , 1% KOH 0.16 S e p a r a t i o n from degrada- 134 impregnated t i o n p r o d u c t s

S i l i c a g e l G

S i l i ca g e l G

0.43 T e s t i n g of p u r i t y o f 135 e r g o t a l k a l o i d s

0.16 T e s t i n g of p u r i t y of 135 e r g o t a l k a l o i d s

S i l i c a g e l 0.24 Q u a n t i t a t i v e a n a l y s i s 163

Solvent system

32. Chlorof om-me thano l ( 9 : 1)

33. Chloroform-methanol (7:l)

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

35. Chloroform-methanol (17: 3 )

36. Chloroform-methanol (4 : 1)

37. Morpholine-toluene (1:9)

38. Chloroform-methanol (9: 1)

39. Acetone

40. Acetone-chloroform (4:l)

41. Acetone-methanol (4 : 1)

Sorbent

S i l i c a g e l

S i l i c a g e l GF254

S i l i c a g e l GF 254

S i l i c a g e l G

S i l i c a g e l G

S i l i c a g e l

S i l i c a g e l

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

S i l i c a g e l F254 (Merck, p recoa ted )

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

R f

0.50

0.52

0.36

-

0.64

0.75

0.22

0.58

0.32

0.39

0.63

Appl ica t ion and comment

Q u a n t i t a t i v e a n a l y s i s





Separa t ion from o t h e r e r g o t a l k a l o i d type compounds

Sepa ra t ion from o t h e r e r g o t a l k a l o i d type compounds

I d e n t i f i c a t i o n , sepa- r a t i o n from LSD



Reference

136,137

8

8

1 1 2

112

138

138

139

139

139


42. Chloroform

43. Chloroform-acetone ( 6 : l )

44. Chloroform-methanol (4: 1)

45. Chloroform-me t h a n o l (9 : 1)

47. Methanol-acetate b u f f e r ( p H 4 .5) ( 9 : l )

48. Chloroform-cyclohexane- i sopropylamine (5 :5 :1)

49. Chloroform-cyclohexane- d ie thylamine (5: 5: 1)

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

So rbe n t

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

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

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

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

S i l i c a g e l F?,, (Merck, p r e c o a t e d ) -2-

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

S i l i c a g e l F (Merck, precoa ted) 254

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

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

- R f A p p l i c a t i o n and comment Reference

0.00 I d e n t i f i c a t i o n , sepa- r a t i o n from LSD


0 .62 I d e n t i f i c a t i o n , sepa- r a t i o n from LSD


0.69 I d e n t i f i c a t i o n , sepa- r a t i o n f r o m LSD


0.11 I d e n t i f i c a t i o n , sepa- r a t i o n f r o m LSD

0 .02 I d e n t i f i c a t i o n , sepa- r a t i o n f r o m LSD

0 .20 I d e n t i f i c a t i o n , sepa- r a t i o n from LSD

139

139

139

139

78,139

139

139

139

139

Solvent system

51. Acetone

52. 1,1, l - t r ichloroethane- methanol (9: 1)

53. l , l , l - t r i ch lo roe thane - methanol (96:4)

54. 1,1, l- tr ichloroethane- methanol (98:2)

A

P m

55. l,l,l-trichloroethananol- methanol (99: 1)

56. n-Butanol-citric acid- water (870 n1:b.B g:130 ml)

57. Benzene-acetone-diethyl- ether-ammonium hydroxide (25%) (4:6:1:0.3)

Sorbent

Aluminium oxide F

(Merck , precoated) 254

254 Aluminium oxide F

(Merck, precoated)

A l u m i n i u m oxide F254

(Merck, precoated)

A l u m i n i u m oxide F254

(Merck , precoated)

254 Aluminium oxide F

(Merck, precoated)

Cellulose (Merck) sprayed with 5% sodium dihydrogen c i t r a t e and d r i ed

S i l i c a g e l G

- Rf Application and comment Refereme

0.48 I d e n t i f i c a t i o n , sepa- 139 r a t i o n from LSD

0 .52 I d e n t i f i c a t i o n , sepa- 139 r a t i o n from LSD

0.20, I d e n t i f i c a t i o n , sepa- 139 r a t i o n from LSD




0.65 I d e n t i f i c a t i o n 140

Solvent system Sorbent

58. Benzene-chloroform (4 : 5 ) S i l i c a g e l G s a t u r a t e d with formamide and mixed wi th 10% me- t h a n o l

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

S i l i c a g e l G

60. Chloroform-ethanol (9:l) Aluminium oxide

6 1. D i - i sop ropy 1 - e t h e r - t e tra- S i l i c a g e l , formamide d

ID P hydrofuran-toluene-di- impregnated ethylamine (70: 15 : 15: 0 . 1 )

62. Dioxane-cyclohexane- Polyamide d ie thylamine (10: 20 : 0 . 5 )

63. Chloroform-cyclohexane- Po 1 yamide d ie thylamine (10:20:0.5)

64. 2-Butanon-cyclohexane- Polyamide d ie thylamine ( 2 0 : 3 0 : 0 . 5 )

6 5. Ethanol- c h l o r o form- Pol yam ide acet ic a c i d (20:200:0.5)

- Rf A p p l i c a t i o n and comment Reference

0.41 I d e n t i f i c a t i o n 140

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

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

0.11 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

0.05 S e p a r a t i o n of a lka- l o i d s




140

141

142

143

143

143

143

Solvent system

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

67 . Cyclohexane-ethylacetate n-propanol-dimethylamine ( 30: 2.5: 0.9: 0.1)

68. Water-ethanol-dimethyl- mine (88:12:0.1)

69. Chloroform-ethanol (10 : 1)

70. Chloroform-methanol (9 : l ) A

m 0

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

Sorb en t

Po lyamide

Rf Application and comment Reference

0.10 Separation of alkaloids 143

-

Po lyami de 0.01 Separation of alkaloids 143

Polyamide 0.03 Separation of alkaloids 143

Silica gel 0.51 Quantitative analysis 113

Silica gel G, 0.1 N 0.51 Identification NaOH impregnated

Cellulose, formamide 0.06 Identification impregnated

144

145

ERGOTAMINE TARTRATE

Table 8

151

Visual iza t ion of Ergotamine on Thin Layer P l a t e s

No. Treatment - Resul t Reference

1. p-dimethylaminobenzal- B h e - v i o l e t 58 ,78 ,113 , dehyde Reagent (var ious 120,132, modi f ica t ions) 135 , 1 3 7 , 138

139 , 144

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

3. I n s i t u scanning

4. Iodine vapor

5. Dragendorff Reagent

6. Ammoniated copper su lpha te

7. Xanthydrol-hydrogen peroxide

8. Ninhydrin-cadmium ace ta t e

Blue

Violet-brown spo t s on l i g h t green background

Blue-violet

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

62 , 115 , 116 , 117 , 119

143

5 8

58 ,140 ,141

140

Red-violet spo t s 140 on pink background

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 78

11. F e r r i c ch lor ide- g lyoxyl ic ac id

Blue 1 2 4

152 BO KREILGKRD

p a r a t e C e l i t e 545 columns (147 ,148) . The f i r s t column i s impregnated w i t h sodium b i c a r - bona te and t h e a l k o l o i d bases are e l u t e d w i t h ch loroform. The second column is impregnated wi th a 20% c i t r i c a c i d s o l u t i o n and ergotami- n i n e i s e l u t e d w i t h a p o r t i o n o f ch loroform. Ergotamine i s e x t r a c t e d w i t h ch lo ro fo rm from t h e ex t ruded s u p p o r t which i s suspended i n an 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 c o n t e n t i n each f r a c t i o n i s de termined u s i n g t h e van Urk r e a c t i o n . T h i s method h a s been adapted 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) and i n a n a l y s i s o f t a b l e t s c o n t a i n i n g e rgotamine t a r t r a t e , t ropa - ne 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 me- t hod , however, does 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 , such 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 accoun t .

been de termined u s i n g C e l i t e 545 impregnated wi th formamide as t h e s t a t i o n a r y phase and benzene-pe t ro leumether ( 9 : 1) as t h e mobi le phase ( 1 4 9 ) .

Ergotamine can be q u a n t i t a t i v e l y sepa ra - t e d from o t h e r e r g o t a l k a l o i d s us ing an aluminium o x i d e column and methylene c h l o r i d e ad- d ing i n c r e a s i n g amounts of methanol as e l u t i n g s o l v e n t (12,151. Carless (150) used columns of c e l l u l o s e impregnated w i t h a pH 3 .0 McIlva- ne 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 mobile phase 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 abou t 80% e rgo tamine i s recovered (150) .

Ergotamine and e rgo tamin ine i n d rugs have

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

Within 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 chromatographic methods f o r t h e ana ly- s is o f e rgotamine have been developed. Ad- s o r p t i o n chromatography is c a r r i e d o u t on si l i- ca g e l w i th 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 as mobile phases (19 ,103 ,153) . A reverse phase Bondapak pheny l /Coras i l o r pBondapak c18 column wi th a c e t o n i t r i l e - a q u e o u s ammonium car- bona te b u f f e r as mobi le phase , p e r m i t s s epa ra - t i o n of e rgotamine from i t s d e g r a d a t i o n pro- d u c t s (154 ,155) . I n c r e a s e d s e n s i t i v i t y i n t h e


a n a l y s i s of ergotamine cou ld be ach ieved by us ing p i c r i c a c i d as c o u n t e r i o n i n forming an i o n - p a i r which on t h e s i l i c a g e l column i s di- 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 mobile o r g a n i c phase ( 1 5 2 ) . An i n c r e a - s ed s e n s i t i v i t y r e l a t i v e t o common f luor ime- t r i c d e t e c t o r s (19,151) cou ld b e ach ieved by us ing a h igh -p res su re Xenon arc lamp w i t h an i n t e g r a l c o l l i m a t i n g mirror as e x c i t a t i o n source ( 1 0 3 ) . A l s o UV-detectors a t 254 nm have been used (153-155) t o d e t e c t e rgotamine . Recent ly Bethke e t a1 (156) d e s c r i b e d a r eve r - se phase HPLC method wi th s o l v e n t g r a d i e n t which i n less t h a n 20 minutes enab led them t o de te rmine t h e c o n t e n t of e rgotamine a s w e l l as 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 pharmaceut ica l p r e p a r a t i o n s .

7 . Determinat ion i n B i o l o g i c a l Systems

Ergotamine have been ana lyzed i n plasma by TLC fo l lowed by i n s i t u f l u o r i m e t r y ( 1 1 5 ) . Kopet & D i l l e (69) used t h e van Urk react ion t o de termine e rgotamine i n b lood and t i s s u e s , w h i l e a n a l y s i s of e rgo tamin ine i n plasma i s done u s i n g HPLC equipped wi th 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 rgotamine t a r t r a t e have been done moni tor ing 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 3H- labe l led e rgotamine t a r t r a t e ( 3 2 ) .

8. Determina t ion i n Pharmaceut ica l P r e p a r a t i o n s

The fo l lowing methods have been a p p l i e d t o a n a l y s i s of ergotamine t a r t r a t e i n pharmaceut i - cals:

Color imet ry References 81,91,92,99,157

F luor ime t ry 1 7 Paper chromatography 47 Column chromatography 64,148,149 Thin l a y e r chromatography 8 ,6 l ,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 f e r ence s

1.

2.

3 .

4.

5.

6.

7.

8.

9. 10.

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).

--

11. A.Stoll. and W-Schlientz, Helv.Chim.Acta - 38, 585 (1955).

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

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). W.Soffe1 and H.Rochelmeyer, Pharm.Ztg. - 101, 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).


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.

T.Inoue, Y.Nakahara and T.Niwaguchi, Chem. Pharm.Bul1. 20, 409 (1972). L.C .Craig I 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. S toil I A.Stol1 I

J. Sage1 I R. Schmid

M .A. Zosl col. 7, - -

- Helv.Chim.Acta 28, 1283 (1945). Schweiz.Apoth.Ztg. 60, 341 (1922). Pharm.Weekb1. 107, 119 (1972). t and A.Fanchamp, Europ.J.Clin.Pharma- 213 (1974). io, H.V.Mauldinq and J.J.Windheuser,

J.Pharm.Sci. - 58, 222 (1969). 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.Zog- lio, Third Migraine Symposium 24.-25.April (1969). Ed. by A.L.Cochrane. Heinemann, Lon- don 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. z, 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 . Kornf eld I E . J . Fornef eld , G . B. Kline I M.J.Mann, R.G.Jones and R.B.Woodward, J.Amer. Chem.Soc. - 76, 5256 (1954). 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).

--

- -

156

50.

BO KREILGKRD

W.A.Jacobs and L.C.Craig, J.Biol.Chem. 104, 547 (1934).

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.

W.A.Jacobs and L.C.Craig, J.Biol.Chem. - 106, 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). M.Sahli and M.Oesch, Pharm.Acta Helv. - 40, 25 (1965). J.M.G.J.Frijns, Pharm.Weekb1. - 106, 865 (1971). 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.Kas- sem, J.Drug Res. 2, 159 (1973). L.S.Goodman and A.Gilman, The Pharmacological Basis of Therapeutics, Fourth Ed. The MacMil- lan 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). H.W. van Urk, Pharm.Weekb1. 66, 473 (1929) M.P6hm, =h.Pharm. 286, 509719531. E.G.C.Clarke, Isolation and Identification Drugs , The Pharmaceutical Press , London 19 D.L.Andersen, J.Chromatogr. 41, 491 (1969) G.Dusinsky and L.Faith, (1967).

- - narm.Weekb1. 66, 473 (1929)

E.G.C.Clarke, Isolation and Identification Druss , The Pharmaceutical Press. London 19

- 286 , 50971953) . -- D.L.Andersen, J.Chromatogr. 41, 491 (1969)

ith, Pharzzie -- 22, 475

of 69. -

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, Farm.Po1. &, 513 (1961). N.L.Allport and T.T.Cocking, Quart.J.Pharm. Pharmacol. - 5, 341 (1932).

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).

81. 82. 83. 84.

85. 86.

87. 88.

89. 90.

91.

92. 93. 94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

104. 105.

106.

107.

108.

P.Horak, Cesk.Farm. 17, 37 (1968). Y.Kazutaka. T.KawataS. T.Tabata, S.Fukushima and M.Ito,.J.Pharm.Soc: (Japan) - 73, 268 (1953). V.Pedersen, Arch.Pharm.Chemi 62, 675 (1955). F.Schlemmer, P.H.A.Wirth and TPeters, Arch. Pharm. 274, 16 (1936). M.I.Smith, Pub.Health Rep. 45, 1466 (1930). J.W.Strong and F.A.Maurina, J.Amer.Pharm. Ass.Sci.Ed. 42, 414 (1953). F.D.Snel1 anTC.T.Snel1, Colorimetric Methods of Analysis. Including Photometric Methods. Volume IV AA, Van Nostrand Reinhold Comp. 1970. R.Voigt and F.Weiss, Pharmazie 13, 319 (1958). R.Voigt, Mikrochlm.Acta, 619 (1959). F.Wokes and H.Crocker, Quart.J.Pharm.Pharma- - - col. 4 , 420 (1931). L.E.Michelon and W.J.Kelleher, Lloydia 26, 192 (1963). E.Schulek and G.Vastagh, Dan.Tidsskr.Farm. 13, 101 (1939). L.Vida and G.Vastagh, Acta Pharm.Hung. - 37, 6 7 (1967). E.Graf and E.Neuhoff, Arzneimittel-Forsch. 4, 397 (1954). H.J. van der Pol, Pharm.Weekb1. - 106, 515 (1971). H-Wachsmuth and L. van Koeckhoven, J.Pharm. Belg. 2, 378 (1963). E.M.Karacsony and B.Szarvady, Planta Medica - 11, 169 (1963). 1.Gyenes and K.Szasz, Magy.Kem.Foli. 61, 393 (1955). R.J.Perchalski, J.D.Winefordne der, Anal.Chem. 47, 1993 (1975 L.Reio, J.ChromaGgr. 68, 183 L.Wichlinski and J.Trzebinski, Pharm. 20, 32 (1963). P.Heinanen, L.Tuderman and E.N Apt.Lethi 46, 133 (1957). K.Macek, A.Cerny and M.Semonsk 388 (1954). J.Kolsek, Mikrochim.Acta, 1500

..-

!r and B.J.Wi 1 . (1972). Acta Pol.

'issilz , Suom y , Pharmazie

(1956).

1-

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.Spei- chert, 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 J.Pharm.Sci. 53, 306 (1964). M.Amin and W.Spp, J.Chromatogr. 118 , (1976).

i

225

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-Chroma- tog:. 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). A.Peuch, C.Duru and M.Jaaob, J.Pharm.Belg. - 29, 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, Farma- - cia (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 (1966 1 .

-

M.Zinser and C.Baumggrte1, Arch.Pharm. 297, 158 (1964).


136.

137.

138.

139.

140.

141.

142.

143.

144.

145.

146.

147.

148. 149.

150. 151.

152.

153.

154.

155.

156.

157.

158.

159.

Y.Petrova. T.Tomova and L.Fili~ova. Farmat- siya (Sofia) 22, 9 (1972). E.Stah1, Di innzh ich t -Chromatograph ie , Ein Laboratoriumhandbuch, 2.Ed. Springer, Ber- lin 1967. G.V.Alliston and M. J. de Faubert Maunder, J.Pharm.Pharmaco1. 23, 555 (1971). R.Fowler, P.J.Gomm and D.A.Patterson, J.Chromatogr. 72, 351 (1972). K.C.Guven and TGuneri, Eczacilik Bul. -- 17, 46 (1975). K.C.Guven and L.Eroglu, Eczacilik Bul. 10, 53 (1968). J.Reichelt and S.Kudrnac, J.Chromatogr. 87, 433 (1973). H.-C.Hsiu, J.-T.Huang, T.-B.Shih, K.-L.Yang, K.T.Wang and A.L.Lin, J.Chin.Chem.Soc. - 14, 161 (1967).

--

W.N.French and A.Wehrli, J.Pharm.Sci. - 54, 1515 (1965). K.Teichert, E-Mutschler and H.Rochelmeyer, Deut.Apoth.Ztg. 100, 283 (1960). F.Sita, V.Chmelova and K.Chme1, Cesk.Farm. - 22, 234 (1973). T.G.Alexander, J.Ass.Offic.Agr.Chem. - 43, 224 (1960). T.G.Alexander, J.Pharm.Sci. 52, 910 (1963). J.J.A.M. van de Langerijt, Pharm.Weekb1. - 95, 133 (1960). J.E.Carless, J.Pharm.Pharmaco1. 5, 883 (1953). I.Jane and B.B.Wheals, J.ChromatEgr. - 84, 181 (1973). W.Santi, J.M.Huen and R.W.Frei, - 115, 423 (1975). J.D.Witter,Jr. and J.H.Kluckhoh togr.Sci. 11, 1 (1973). ApplicationSheet No DS 042, Wa

- J.Chromatogr.

.n , J .Chroma-

ters Associ- ates,Mass., U.S.A. Application Sheet No.AN 118, Waters Associ- ates,Mass., U.S.A. H-Bethke, B.Delz and K.Stich, J.Chromatogr. 123. 193 (1976). - S.Czyszewska, F.Kaczmarek, L.Lutomski and H.Speichert, Herba Pol. 12, 87 (1966). V.Prochazka, F.Kavda, M.Eucha and J.Pitra, Cesk.Farm. 2, 493 (1964). 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.

FENOPROFEN CALCIUM

Christine K , Ward and Roger E. Schimer

162 CHRISTINE K. WARD AND ROGER E. SCHIRMER

1. 2.

3. 4. 5.

6 . 7.

8 .

9. 10.

CONTENTS

D e s c r i p t i o n Phys ica 1 P r o p e r t ies 2.1 C r y s t a l Charac te r i s t ics

2.1.1 C r y s t a l . Forms and Hydra t e s 2.1.2 M e l t i n g Range and 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 Spectrum 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 Nuclea r Magnet ic Resonance Spec t rum 2.7 Mass Spectrum S y n t h e s i s D e g r a d a t i o n of Fenoprof e n C a lc ium Me t a bol i s m of Fenopr of e n 5.1 M e t a b o l i t e s of Fenoprofen 5.2 Pharmacokine t ics E lementa 1 A n a l y s i s Chromatographic Methods of A n a l y s i s 7 .1 Th in Layer Chromatography 7.2 Gas Chromatography 7.3 High Pressure 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 lc ium) Spec t ropho tomet r ic A n a l y s i s A n a l y s i s of Fenopro fen i n B i o l o g i c a l Samples

FENOPROFEN CALCIUM 163

1. Descr i p t i o n

p h e n y l l p r o p i o n a t e d i h y d r a t e ( I ) .

- Fenoprofen Calcium is c a l c i u m 2- (3-phenoxy-

ii [ coo- ‘I 2 1 -2H$ I. 2

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

2 . P h y s i c a l P r o p e r t i e s - 2 . 1 . 1 C r y s t a l Forms and Hydra t e s -- F e n o p r o f e n c i u m occurs a s a

c r y s t a l l i n e d i h y d r a t e which is s table from 94% t o less t h a n 1% r e l a t i v e humid i ty a t room tempera tu re . Only one c r y s t a l form has been obse rved f o r t h e d i h y d r a t e .

2 . 1 . 2 Mel t ing Range and 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 open pan the

thermcgram of Fenoprofen Calc ium (!ee f i g u r e 1) e x h i b i t s a large endotherm n e a r 94 C c o r r e s p o n d i n g t o a loss of water accompanied by col lapse 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 , as i n a m e l t i n g p o i n t t u b e , t h $ endotherm a p p e a r s a t h i g h e r temperature (118-123 C ) and 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 sample . 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

-80

-70

TGA

-60

DTA -50

-40

-30 94

Figure 1


d

13.40 9.70 7.31 6.70 6.06 5.79 4.83 4.47 4.27 4.07 3.89 3.75 3.53

2.1.3 X-ray Powder Pa t t e rn of Fenoprofen f!a lc i u m

1/11

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

3.40 05 3.27 30

d

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

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

1.76 02

2.2 S o l u b i l i t y

S o l u b i l i t y Solvent <mg/m 1 1 Temperature

Methanol 8 1- Hexano 1 11 Chloroform 0.01 Cyc lohexane -0.01 Water 2.5 Buffer pH 1.2 0.12

4.0 0.28 6 . 0 3.30

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

25 O C

25OC

2.3 pKa Water 4.5 66% D i m e t hylf ormamide/34$ water 7.6

2.4 Electronic Spectra - 2.4.1 Ul t rav io le t Absorption Spectrum

The u l t r a v i o l e t spectrum of Fenoprofen Calc ium i n methanol is shown i n Figure 2 . The spec t r e x h i b i t s maxima a t 266, 272, and 278 nm w i t h E @ , _I values of 61.3, 70.0, and

A bill

63.2, respec t ive ly .


0.5

0 I I I i

250 300 350 380 F i g u r e 2 . U l t r a v i o l e t Spectrum of Fenoprofen

Calcium


2.4 .2 O p t i c a l Rotat ion Althousch Fenoprofen 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 co r re spond ing e n a n t i o - mer ic ac ids . 1

ta12i C = 1 - i n CHC1, d - (+I-Fenoprofen Acid +46.0’ 1- (- -Fenopr of e n A c i d -45.7O

2 . 5 I n f r a r e d Spectrum The i n f r a r e d sDectrum of Fenomofen

Calcium spec t rum w a s ob ta ined u s i n g a Beckman IR12 I n f r a r e d Spec t rophotometer . Major band a s s i g n - ments a re as f o l l o w s :

i n a KBr d i s k is g i v e n i n F i g i r e 3. The

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

1560 (very s t r o n g , broad) CO; a s y m m e t r i c and and 1420 symmetric s t r e t c h i n g

-

h y d r a t e

1490, 1440 and 1450 aromat ic r i n g s t r et c hing

1260 t o 1210 (several C-0-C asymmetric e t h e r bands) s t r e t c h i n g

930 t o 695 ( s e v e r a l p r i m a r i l y aromat ic out bands) of p l a n e bend i n g .

2.6 Nuclear Magnetic - Resonance Spectrum T h e y nmr spectrum of

Fenoprofen Calc ium 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 are as fo l lows:

-

Assignment -- Band P o s i t i o n , ppm

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

aroma t i c p r o t o n s

-CH-

1.35 (double t , J = 7Hz) -CH3 - 3 . 7 0 ( q u a r t e t , J = 7Hz) -

E

a,

E nl 0 E

a, E

a a,

k

rd k w

c H

m

a, k

168


2.7 Mass Spectrum

p resen ted i n F i g u r e 5 . The mass spec t rum of Fenoprofen is

3. S y n t h e s i s

The s y n t h e s i s of Fenoprofen Calcium is presen ted i n F i g u r e 6 .

4 . S t a b i l i t y

and heat. For example, s t o r a g e a t 135 C f o r s i x days r e s u l t s on ly i n t h e loss of t h e waters of hydratio!. Samples stored for over 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 .

induced by expos ing aqueous s o l u t i o n s of t h e drug 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 pho to - f r ies rea r r angemen t s occur l ead ing t o a mixture of t h e f o l l o w i n g isomeric b ipheny l s2 :

Fenoprofen is q u i t e s t a b l e t o a c i d % base ,

However, d e g r a d a t i o n of Fenoprofen c a n be

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 observed i n any f o r m u l a t i o n .

I

I

,yY' . ' I 1 I t . I I I, / 1 1 1 1 1 ~ 1 ' 1 1 1 1 1 1 1 ' 1 ' 1 1 1 ' 1 ' 1 ' 1

200 220 240 260 280 300 320 340 F i g u r e 5. Mass Spectrum of Fenoprofen Calcium

w 0

k a

0

c (u

b4 c h

rn

a

-4

r.4

172

F ENOP RO F EN CALCIUM 173

Table 1

Urinary Metabolites of Fenoprofen3'4

H3c\c00H

(unchanged Fenopr of en )

I1

0 d COOH

3%

455

2%

42%

F i r s t un ident i f i ed a c i d l a b i l e conjugate 35 Second un ident i f i ed a c i d l a b i l e conjugate 5%

I74 CHRISTINE K. WARD AND ROGER E. SCHIRMER

5. Metabolism

5.1 M e t a b o l i t e s The p r i n c i p l e rou tes of metabolism of

Fenoprofen invo lve hydroxy la t i o n of t h e t e r m i n a l phenyl group and c o n j u g a t i on 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 metabolites i n human u r i n e a r e p r e s e n t e d i n Tab le 1.

5 .2 Pharmacokine t ics -*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 Fenoprofen 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 ra l doses . 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 pa rame te 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 pa rame te 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

range from 38 .6 t o 47 .8 ml/min and suggest that t u b u l a r r e s o r p t i o n of Fenoprofen o c c u r s .

Rena 1 c l e a r a n c e v a l u e s f o r Fenoprof e n

6 . E lement a 1 Ana l y s is

E l e m e n t

Ca C H 0

Calcium Fenoprof e n D i hydra t e -- Anhydrous

7 .67 68.94

5 . 0 1 18.37

7.17 64 .50

5 . 4 1 2 2 . 9 1

7. Chromatographic Methods of A n a l y s i s

7 . 1 Thin Layer Chromatography S e v e r a l t h i n l a y e r sys t ems have been

r e p o r t e d f o r s e p a r a t i o n of Fenoprofen from its s y n t h e t i c p r e c u r s o r s and metabolites. These sys tems are summarized i n T a b l e 2 . S i l i c a ge l p l a t e s were used in a l l cases. The Roman numerals r e f e r t o t h e structures given in Figure 6 .

f D

kab=0.15 min-1 -

kab= absorption rate constant kd= elimination rate constant

f D = fraction of dose absorbed x dose

V = volume of the plasma compartment

PLASMA COMPARTMENT I rn -

f /V = 0.14 Q-1 kd=0.005 min-1

1

Figure 7. Pharmacokinetics of Fenoprofen Calcium

O

3

:I O

*I O

4 O

rl

Ic

ID

m

00 In

10

(0

cv

cv 0

Q

E

Q

N

: In

176

Q,

(D

21 8

In

In

H

In

Q,

El z

(0

(0

3

W

0

El :

m

m

3

u(

0

(D

Q,

>I w

In

W

W

bm

"I

Iy" m

m

v)

5

dl

0

dn

.r

O

al4

v d

rl

+o

a

lc

bb

CU

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D

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I V V I VII VIII IX R e f . - - - - - - - Solvent System

17. E t h y l e t h e r 63 49 60 54 66 62 7

18. E t h y l e t h e r - 86 82 89 56 96 91 7 acetic a c i d (98-2)

19. E t h y l e t h e r - 87 82 89 87 98 91 7 acetic a c i d (95- 5 1

20. E t h y l e t h e r - 92 85 92 91 98 92 7 acetic a c i d

A

CO -I (90- 10)


7.2 G a s Chromatography Calc ium Fenoprofen r a w materials and

f o r m u l a t i o n h a v e been a n a l y z e d by gas chromato- g raphy . The sample 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 aqueous 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 chloro- f o r m , d r y i n g t h e chloroform o v e r anhydrous sodium s u l f a t e and e v a p o r a t i n g t h e chloroform as necessary t o c o n c e n t r a t e t h e sample . The Fenoprofen acid is s i l y l a t e d by warming at 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 ) t r i f l u o r o a c e t a m i d e and t h e n 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-diphenylbenzene 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 iqu id Chromato r a p h y Fenoprof e n Calc ium c a d b y

h i g h p r e s s u r e l i q u i d chromatography u s i n g t h e f o l l o w i n g c o n d i t i o n s :

Column :

Tempera ture :

S o l v e n t Flow Rate :

Detector:

Sample S i z e :

E l u t i n g S o l v e n t :

I n t e r n a l S t a n d a r d :

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

Ambient ( approx ima te ly 25OC )

100 ml/hr (-1100 p s i )

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

Approximate ly 22 mcg on c o lumn

600 m l d e i o n i z e d water 400 m l a c e t o n i t r i l e and 20 ml glacial acetic a c i d

p -ch lo robenzo ic acid

Table 3

Condit ions for G a s Chromatography of S i l y l a t e d Fenoprofen

w p ~ ~ ~ ~ i on R e f A rox. Liquid Phase Solid Support Length ID Been Temp. Tfme - - L -

3.8$, W98 D iat opor t S 3 f t . 3 mm 175OC 4 min 8

1.0$, W98 Gas Chrom Q 2 f t . 3 mm 15OoC 4 min

0.5%,OV17 Chromosorb G- 4 f t . 3 mm 175OC 2 min HP

1.%, oV17 Chromosorb G- 3 f t . 3 mm 14OoC 6 . 6 min. AW DMCS


8 . T i t r i m e t r i c D e t e r m i n a t i o n

The c a r b o x y l a t e f u n c t i o n may be de te rmined

Calcium c a n be de te rmined by t i t r a t i o n w i t h

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 perchloric ac id u s i n g g lac ia l acet ic ac id a s t h e s o l v e n t .

0.05 M EDTA u s i n g Calcon i n d i c a t o r . About 1.5 g of Fenoprofen 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 are t h e n t i t ra ted t o t h e blue end- p 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 70 m l of water, 2 m l of 10% sodium hydroxide , 1 d r o p of 1% g e l a t i n , 3 d r o p s of 10% KCN and 2 drops of Ca lcon i n d i c a t o r s o l u t i on .

9. - S p e c t r o p h o t o m e t r i c D e t e r m i n a t i o n s

Fenoprofen acid has been de te rmined by measur ing t h e a b s o r b a n c e a t 272 nm i n methanol s o l u t i o n s ac i d i f i e d w i t h acet ic acid.

measuring t h e a b s o r b a n c e a t 270 nm i n a pH 7.5 phosphate b u f f e r s o l u t i o n .

Fenoprofen Calcium has been de te rmined by

10, A n a l y s i s of Fenoprofen i n Biological Samples

Fenoprofen has been de te rmined i n blood plasma samples8 by gas chromatography f o l l o w i n g e x t r a c t i o n . Fenoprofen was first extracted i n t o hexane from t h e a c i d i f i e d plasma sample , 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 sodium hydroxide s o l u t i o n , and f i n a l l y extracted 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 aqueous s o l u t i o n t o a b o u t 3. The hexane w a s e v a p o r a t e d and t h e f e n o p r o f e n 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 . The ca rbon 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 175 O C .


1.

2. 3.

4.

5.

6.

7.

8 .

References --- W.S. Marshall , U.S. P a t e n t 3,600,437 (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. Chernish A.S. Ridolfo, C.M. Gruber, Jr., - - J. Pharmacol, E X ~ . Ther. 183, 449(1972) A . Rubin, S.M. Chernish, R. Crabt ree , C.M. Gruber, Jr., L. Helleberg,B.E. Rodda, P. Warrick, R.L. Wolen, and A.S. Ridolfo, Curr . Med. R e s . Opinion 2, 529(1974). A . Rubin, B.E. Rodda, P. Warrick, A.S. Ridolfo, and C.M. Gruber, Jr., J. Pharm. Sc. 60, 1797 (1971)

A . Rubin, B.E. Rodda, P. Warrick, A.S. Ridol fo , and C.M. Gruber, Jr., J . Pharm S c . 61, 739 (1972 ) R.H. Bishara , J. Ass. Off. Anal Chemists 56, 657 (1973)- - - - J .F. Nash, R. J. Bopp and A . Rubin, J . Pharm Sc. 60, 1062(1971).

- - -

- - - - - A -

- -- -

- - - -

ISONl AZID

Glenn A. Brewer

184 GLENN A. BREWER

CONTENTS

1. D e s c r i p t i o n 1.1 N a m e , Formula , Molecular W e i g h t 1 . 2 A p p e a r a n c e , Color, Odor, T a s t e

2 . 1 Spectra 2. P h y s i c a l a n d C h e m i c a l P r o p e r t i e s

2 . 1 1 I n f r a r e d S p e c t r u m 2.12 U l t r a v i o l e t S p e c t r u m 2 . 1 3 C h e m i l u m i n e s c e n c e 2 .14 F l u o r e s c e n c e S p e c t r u m 2 . 1 5 N. M. R. Spectrum 2 . 1 6 E. S. R. S p e c t r u m 2 .17 Mass S p e c t r o m e t r y

2 . 2 1 M e l t i n g 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. 2 .24 E lec t r i ca l Moment 2 . 2 5 E l e c t r i c a l C o n d u c t i v i t y 2 .26 C r y s t a l c h a r a c t e r i s t i c s 2 .27 X-Ray D i f f r a c t i o n

2 . 3 1 Water S o l u b i l i t y 2 .32 S o l u b i l i t y i n S o l v e n t s

2 .4 P h y s i c a l P r o p e r t i e s o f S o l u t i o n 2 . 4 1 PH 2 . 4 2 D i s s o c i a t i o n C o n s t a n t 2 .43 P h o t o l y s i s C o n s t a n t 2 . 4 4 O x i d a t i o n P o t e n t i a l

2 . 2 P h y s i c a l P r o p e r t i e s of t he S o l i d

2 . 3 S o l u b i l i t y

3. Metal Complexes 4. His tory , S y n t h e s i s a n d M a n u f a c t u r i n g 5. S t a b i l i t y 6. A n a l y t i c a l C h e m i s t r y

6 . 1 I d e n t i t y T e s t s 6 . 2 Methods o f A n a l y s i s

6 . 2 1 Genera l R e v i e w s 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 185

6.27 Grav ime t r i c Methods 6 .28 Mic rob io log ica l and Enzymatic Methods 6.29 Misce l laneous Methods

6 . 3 1 Paper Chromatography 6.32 Thin-Layer Chromatography 6.33 Ion Exchange chromatography 6 .34 Other Chromatographic Methods

6 .4 Determina t ion 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 Genera l Reviews 6.42 Colorimetric Methods 6 .43 T u r b i d i m e t r i c Method 6 .44 F luo r i m e t r i c Methods 6 .45 E lec t rochemica l Methods 6 .46 Gasometr ic Methods 6.47 Misce l laneous Chemical Assays 6 .48 Mic rob io log ica l Assays 6 .49 Chromatographic Assays

6 .3 Chromatographic Methods

7 . Drug Metabolism 8 . Biopharmaceut ics 9. References

186 GLENN A. BREWER

1. Description 1.1 Name, Formula, Molecular Weiqht

Acid Hydrazide, INH, Isonicotinoylhydrazine, Isonicotinyl hydrazide, Isonicotinylhydrazine, Tubaz i d, I Sonia z i dum .

Chemical names - 4-Pyridinecarboxylic acid hydrazide, pyridine-4-carboxyhydrazide, pyridine- y-carboxylic acid hydrazide.

Generic names - Isoniazidl, Isonicotinic

2 Chemical Abstracts Registry No. 54-85-3

(++)-c0mNH2

C6H7N30 Mol. Wt. 137.14

Colorless or white crystalline powder 1.2 Appearance, Color, Odor, Taste

which is odorless and has at first a slightly sweet and then bitter taste3.

2. 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 made , 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.

4

Frequency (cm-l) Ass iqnmen t 3300-3000 Bonded NH and C-H 1670 c=o 1560 Amide I1 1640 NH2 deformation 1610t 1500/

ring C=C and C=N

WAVELENGTH (MICRONS)

FRMUENCY (W')

F i g u r e 1:Infrared spectrum of isoniazid as a KBr pe l l e t .


A

8

FREQUENCY (W')

F i g u r e 2 : I n f r a r e d 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 189

2.12 U l t r a v i o l e t Spectrum Numerous a u t h o r s have r eco rded t h e

u l t r a v i o l e t spectrum o f i s o n i a z i d i n a number of 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 the 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 spec t rum h a s been noted. Zommer13 h a s r eco rded the s p e c t r a of t h e hydrazones of i s o n i a z i d and a c e t o n e o r p-hydroxy- benza ldehyde.

The u l t r a v i o l e t spectrum 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 approximate ly equa l maximima a t 213 nm ( E Y i m 437) and 265 nm ( E l % 417) . The minimum occur s a t 233 nm.

The spec t rum i n d i s t i l l e d w a t e r shows a b road peak a t 261 nm (EFgm 306) w i t h o u t a d e f i n e d minimum. There i s a shou lde r a t 208 nm. I n d i l u t e a l k a l i (0.01N aqueous a l k a l i ) t h e spectrum taken immediately shows a s h o u l d e r a t 266 nm ( E F i m 293 295 nm (Elcm !% 284) . On s t a n d i n g t h e s e peaks s h i f t so t h a t a t 2 hour s t h e r e a r e peaks a t 256 nm ( E l %

A t 24 hour 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 excep t t h a t i t occurs more r a p i d l y .

me thano 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 t h o s e i n w a t e r excep t t h a t t h e a b s o r p t i o n maximima g e n e r a l l y occur a t s l i g h t l y lower wave- l e n g t h s .

1 c m

and peaks a t 272 nm (EFgm 298) and

1 7 3 ) , 262 nm ( E E m 170) and 325 nm (Ergm 7 6 ) . 1 c m

The u l t r a v i o l e t spectrum t aken i n

2.13 Chemiluminescence Caenl5 h a s observed a weak

chemiluminescence of i s o n i a z i d when s o l u t i o n s a re o x i d i z e d wi th sodium h y p o c h l o r i t e . The lumj nes- cence i n c r e a s e s w i t h pH from 10 .2 t o 13. The maximum of t h e emiss ion cu rve i s a t 0.552 p corresponding t o an energy o f 5 1 K c a l , Two t h e o r i e s f o r t h e observed luminescence a r e o f f e r e d , b o t h of which depend on t h e p re sence o f f r e e OH and H02 r a d i ca 1 s .

190 GLENN A. BREWER

2.14 F luo rescence S p e c t r u m I s o n i a z i d shows an i n t e n s e

f l u o r e s c e n c e spectrum 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 c l eavage of the p y r i d i n e r i n g w i t h cyanogen bromide. T h i s f l u o r e s c e n c e is t h e basis o f s e v e r a l s e n s i t i v e methods t o de te rmine 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 (See 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 minutes 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

14 emiss ion peak a t 415 nm . A f t e r i s o n i a z i d i s re- a c t e d w i t h cyanogen bromide 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 found a n a c t i v a t i o n maximum a t 312 nm and a f l u o r e s c e n c e maximum a t 392 nmI4.

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 ca rbony l compounds ( S e c t i o n 6 .24) .

I s o n i a z i d a l s o f l u o r e s c e s when

2.15 N. M. R. Spectrum S e v e r a l a u t h o r s have s t u d i e d t h e

n u c l e a r magnet ic resonance spectrum o f t h e h d ra - z 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 ~ g s t u 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 of i s o n i a z i d and *zO exchanged i s o n i a z i d a re shown i n F i g u r e s 3 and

420. The 60 MHz NMR spec t rum of i s o n i a z i d , i n d ime thy l su l fox ide -d6 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 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 hydraz ino p r o t o n s r e sonances a t (ppm) 4.60 (broad , 2H, exchanged) and 10.15 (broad , l H , exchanged) . The a r o m a t i c p r o t o n s r e sonances appear a s m u l t i p l e t s a t 7 . 7 3 ( 2 H ) and 8 .70 ( 2 H ) . ( F i g u r e s 3 and 4 ) . The complex p a t t e r n of t h e r e sonances , o t h e r t h a n t h e expec ted d o u b l e t s , s u g g e s t s 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 r i n g . However, t h e h i n d e r e d r o t a t i o n a round t h e N-C=O as w e l l as C-aryl bonds can n o t be r u l e d o u t . The NMR spec t rum i n methanol -d4 w a s s imi l a r t o F igure 4 , t h e hydraz ino p r o t o n s hav ing been 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 the spectrum excep t a downf ie ld s h i f t o f t h e aromatic

a, a

.d

3 W

d

5

m d

h

a, E

.d

a

0

k

a, U

5 5

al a c 0 m .d

w 0

191

a

a, m

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al

a, a

-rl X

0

192

ISON I AZ I D 193

pro tons resonance by 0 . 1 ppm. 2.16 E. S. R. Spectrum

H i l l e r b r a n d and co-workers19 used E l e c t r o n Spin Resonance t o s t u d y charge 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 copper ions .

2.17

t a t i o n p a t t e r n F i g u r e 5 shows o b t a i n e d on w i t h a d a t a m / e 137 and d i r e c t bond e l i m i n a t i o n

an

Mass Spec t romet ry G i l l i s 2 1 has d i s c u s s e d t h e fragmen- f o r i s o n i a z i d and s i m i l a r compounds. t h e e l ec t ron - impac t mass spectrum A E I MS902 mass s p e c t r o m e t e r equipped

a c q u i s i t i o n system. The M+ o c c u r s a t t h e f ragment i o n s r e s u l t from e i t h e r c l eavage ( m / e 106, 78) o r th rough t h e of HCN from t h e p y r i d y l r ing(m/e 5 1 ) .

m / e 106 ]

m / e 51 m / e 78

2 . 2 P h y s i c a l Propert ies of t h e S o l i d 2 . 2 1 Mel t ing 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 of i s o n i a z i d i s used a s s p e c i f i c a t i o n i n t h e Uni ted S t a t e s Pharma- ~ o p o e i a ~ ~ and European Pharmacopoeia3. The m e l t i n g p o i n t o c c u r s between 170 and 174OC.

2 .22 D.T.A. and D.S.C. D i f f e r e n t i a l t he rma l 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 ga ined i t s c u r r e n t popular i ty243 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 re sence o f z i n c , copper and ' iron s a l t s and mercu r i c o x i d e g i v e s an abnormal D.T.A. p a t t e r n .

D r . Jacobson27 h a s shown t h a t t h e

194

1 O O t

GLENN A. BREWER

1

3793 ISONIHZID LOT 866434

90-

80-

70.-

60-

58--

48-

3 0 -

20-

_I

t-

t-

t- Z W 0

a

-- 10 o

- - 5 w

-Fl+T-r 3 6

I

I WR

I - t- a 1 :

l -

J 0 3 ( 2 ,

. - - i 4 d +

MFISS/CHRRGE INTENSITY SUM = 4 4 6 7 2 BRSE PERK 2 =24.36

Figure 5:Low-resolution mass spectrum of isoniazid.

ISON IAZlD 195

Squibb House S tanda rd 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 thermal a n a l y s i s equipment.

de te rmined t o be 99.95 mole p e r c e n t u s i n g a Pe rk in E l m e r DSC-1B d i f f e r e n t i a l s cann ing color- imeter27.

The p u r i t y of t h i s s t a n d a r d w a s

2.23 T.G.A. Thermogravimetry can be used t o

de termine 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 soniaz id . When t h e Squibb House S tanda rd w a s t e s t e d no loss on d r y i n g was recorded27.

2.24 Electrical Moment Lumbroso and Barassin’* de te rmined

t h a t t h e e lec t r ica l moment of i s o n i a z i d w a s 2.92 I.L.

2.25 E lec t r i ca l Conduc t iv i ty

compressed t a b l e t of i s o n i a z i d w a s de te rmined a t t empera tu res between 50 and 150 0 C 29.

The e lec t r ica l c o n d u c t i v i t y of a

2.26 C r y s t a l Characteristics Bhat and co-workers30 have 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 are or thorhombic, space g roup P 212181, w i t h a , 14.915 (15) b, 11.400 (10) c, 3.835 ( 5 ) ~ , d (measured) = 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

f o r i s o n i a z i d i s shown i n F i g u r e 6 . 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:

The powder x-ray d i f r a c t i o n c u r v e 3 f

I n t e r p l a n a r D i s t a n c e s R e l a t i v e I n t e n s i t i e s d (ANGSTROMS )

8.84 7.30 6.10 5.64 5.25 4 .49 3.69 3 .51 3.42 3.36 3.27 3.10 3.04 3 .01 2.80 2.63 2.47 2.42 2.33

0.098 0.408 0.398 0 .451 1.000 0.502 0.296 0.398 0.102 0.068 0.197 0.235 0.060 0.058 0.170 0.076 0.168 0.115 0.187

2 . 3 S o l u b i l i t y 32 2 .31 Water S o l u b i l i t y

F o u r t e e n grams of i s n i a z i d -re so lub le i n 100 m l o f water a t 25OC. grams are s o l u b l e i n 100 m l of water a t 4OoC.

Twenty-six

2.32 S o l u b i l i t y i n S o l v e n t s 3 2 ~ 3 3

S o l v e n t S o l u b i l i t e thanol (25OC) 2 g/100 m f e t h a n o l ( b o i l i n g ) 10 g/100 m l ch loro form 0 . 1 g/100 m l e t h y l e t h e r v e r y s l i g h t l y

s o l u b l e benzene i n s o l u b l e

1 0 4

M

co

7 0

60

M

re

M

ie

ut

- 0

, 1 0 0 I.,

I h h

30

20 4 c

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

198 G L E N N A. BREWER

2.4 Physical P rope r t i e s of Solu t ion 2.41 pH

The pH of a s o l u t i o n ( 1 i n 10) should be between 6.0 and 7.523.

2.42 Dissoc ia t ion Constant There i s a discrepancy i n t h e

l i t e r a t u r e on the d i s s o c i a t i o n cons t an t s of i soniaz id . This 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.

Fal lab34 determined t h e basic d i s - soc i a t ion cons tan t a s 3 x 10-11 measured conduct- omet r ica l ly . t h a t t h e 1st b a s i c cons tan t should be a sc r ibed t o t h e pyr id ine n i t rogen and t h e 2nd t o t h e hydrazine group. This i s cont ra ry t o previous work by Cingolani and G a ~ d i a n o ~ ~ .

t h e d i s s o c i a t i o n cons tan ts po ten t iome t r i ca l ly as

Cani6 and Djordj evi635 e s t a b l i s h e d

Nagano and c o - ~ o r k e r s ~ ~ determined

PK1 = 2.13, PK2 = 3.81, PK3 = 11.03. Salvesen and G l e n d r a n c ~ e ~ ~ de te r -

mined t h e d i s s o c i a t i o n cons tan ts i n 1 . O M sodium ch lo r ide s o l u t i o n a s K1 = 9.80 x and K2 = 1.42 10-4.

Zommer and Szuszkiewicz'l have e s t ab l i shed pK1 = 10.75 and pK2 = 11.15 and protona t ion cons tan ts cf 3.57 f o r t h e py r id ine N and 1.75 f o r t h e hydrazide N.

Rekker and N a ~ t a ~ ~ found t h a t sol- u t ions of i s o n i a z i d became yellow a t p H 10 and 2.7. The co lo r i s r e v e r s i b l e on changing t h e pH. They explained t h i s behavior on the b a s i s of t h e e x i s t - ance of two p o s i t i v e ions , 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 values a r e pK' = 2.00, pK" = 3.6 and pK"' = 10.8.

2.43 Photolysis Cons tan t Salvesen and Eiki113' e s t a b l i s h e d

t h e photo lys i s cons tan ts f o r i s o n i a z i d a t 20°C and 370 nm i n M NaCl s o l u t i o n as kl = 1.00 x lo'* and k2 = 1.45 x 10-4.

ISON I AZ I D 199

2.44 Ox ida t ion 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 by vu 1 t e r in40 .

S o l u t i o n i n Ef 1 N H C 1 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 . These complexes have been used i n t h e d e t e r m i n a t i o n of i s o n i a z i d (see S e c t i o n s 6.22, 6 .25 and 6 . 2 9 ) .

Tamura and Nagano4I have de te rmined t h e c o n s e c u t i v e fo rma t ion c o n s t a n t s f o r t h e complex formed between 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 de t e rmined were k l = 35, k2 = 0.57, k3=52.5. A t h i g h c o n c e n t r a t i o n s o f i s o n i a z i d y e l l o w 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 i n d i c a t i n g t h a t c o n t r a r y t o t h e p o l a r o g r a p h i c d a t a t h a t t h e 2 : l complex is more s tab le 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 stepwise f o r m a t i o n c o n s t a n t s w e r e k l = 12.2 , k2 = 12.6 , and k3 = 3.4.

The Same a u t h o r s 4 2 s t u d i e d the 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 and C u ( I I ) , Zn, N i ( I I ) , C o ( I 1 ) and Mn (11).

The complexes o f copper and 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. H i s t o r y , S y n t h e s i s and Manufac tu r inq 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 by Meyer and

M a l 1 Y s o 0 i n 1912 by h e a t i n g a m i x t u r e of iso- n i c o t i n i c ac id and h y d r a z i n e above 30OoC. 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 by Chor ine501 a n d b y Huant502 i n 1945. The d rug w a s r e p o r t e d as 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 by Farbenfabr iken-Bayer , A. G. , Hoffmann-LaRoche, Inc . and E. R. Squibb & Sons , Inc .

The

200 GLENN A. BREWER

i n 1952503.

t h e condensa t ion of h y d r a z i n e w i t h a y - s u b s t i t u t e d py r i d i n e .

Hydrazine can be d i r e c t l y condensed 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 re- a c t i o n i s u s u a l l y removed by a z e o t r o p i c d i s t i l l a -

Es te rs o f i s o n i c o t i n y l a c i d can be hydro lyzed and t h e r e s u l t i n g a c i d condensed w i t h hydraz ine . 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 .

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 a c i d . The co r re spond ing a c i d c h l o r i d e i s made w i t h t h i o n y l c h l o r i d e . 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 i s o n i a z i d 4 9 . I n a m o d i f i c a t i o n o f t h i s procedure t h e a c i d c h l o r i d e i s reacted 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 then 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 one can o x i d i z e 2 , 4 d i - me thy lpyr id ine w i t h se len ium and s u l f u r i c a c i d .

T h e mix tu re i s n e u t r a l i z e d w i t h ammonia. A mix tu re o f i s o n i c o t i n i c ac id , 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.

The b a s i c method of manufac ture o f i s o n i a z i d i s

tion44945946947,

Y-Picol ine can be o x i d i z e d i n 70% s u l f u r i c

5. S t a b i l i t y The s t a b i l i t y of i s o n i a z i d h a s been 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 and i n v a r i o u s pharmaceu- t 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 hydraz ine group w i t h n a t u r a l l y occur ing a ldehydes 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 complexat ion o f i s o n i a z i d w i t h metal ions .

Lewin and H i r ~ c h ~ ~ have shown t h a t non- ionic c h e l a t i n g mater ia l can l a r g e l y p r e v e n t t h e degra- d a t i o n of i s o n i a z i d when n e u t r a l and a l k a l i n e s o l u t i o n s are au toc laved . 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 re sence o f hydrogen peroxide .

u n s t a b l e i n human o r rabbi t plasma w h i l e it i s Poole and M e ~ e r ~ ~ reported t h a t i s o n i a z i d 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 buf fered aqueous solu- t i o n s a t pH values 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 temp- e ra tu re s .

Kakemi and c o - ~ o r k e r s ~ ~ have s tud ied t h e degradation of i son iaz id i n aqueous s o l u t i o n under anaerobic condi t ions. Alka l ine hydro lys i s under aerobic condi t ions y i e l d s a mixture of i s o n i c o t i n i c ac id , isonicot inamide and 1 , 2 d i i s o n i c o t i n o y l hydrazine p lus small amounts of u n i d e n t i f i e d products. Under anaerobic condi t ions i s o n i c o t i n i c ac id and 1 , 2 d i i s o n i c o t i n o y l hydrazine were t h e p r i n c i p a l products. When EDTA was added t o the r eac t ion mixture only i s o n i c o t i n i c ac id was formed. F i r s t order k i n e t i c s were followed.

Inoue55 found t h a t a t pH 3 . 1 unde r anaerobic condi t ions i s o n i a z i d hydrolyzes t o form i s o n i c o t i n - i c ac id . Pseudo f i r s t o rde r k i n e t i c s a r e followed. A t lower pH values t h e e f f e c t of bu f fe r type can be seen . Act iva t ion energ ies were c a l c u l a t e d for the hydro lys is by d i f f e r e n t i o n i c spec ies .

Horioka and c o - ~ o r k e r s ~ ~ found t h a t losses of i son iaz id were encountered when t h e drug was blended with var ious a n t i a c i d prepara t ions . The e f f e c t of temperature, 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 oxida t ion i n aqueous so lu t ion , b u t i n t h e presence of sucrose the i son iaz id reac ted with t h e aldo- hexoses formed on invers ion . The r eac t ion with sucrose could be i n h i b i t e d by t h e add i t ion of 0.3% sodium c i t r a t e .

a s condi t ions were kept anaerobic t h a t t h e decompo- s i t i o n of i s o n i a z i d i n t h e pH range 3 t o 7 followed f i r s t o rder k i n e t i c s . They repor ted t h a t a 1% so lu t ion of t h e drug was 37 times more s t a b l e a t PH 6 than 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 spec ies on t h e r a t e of t h e r eac t ion was noted.

r eac t ion between l a c t o s e and i s o n i a z i d i n t h e

Pawelczyk and c o - ~ o r k e r s ~ ~ found t h a t a s long

Wu and co-workers59 i n v e s t i g a t e d t h e browning

202 G L E N N A. B R E W E R

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. Thin-layer chromatography was used t o demonstrate t h e presence of i s o n i c o t i n o y l hydrazones of l a c t o s e and hydroxymethylfurfural .

InoueG0 has e s t a b l i s h e d t h e e f f e c t of t h e presence of copper (11) ions on t h e r a t e of oxida- t 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 products were i s o n i c o t i n i c a c i d , isonicot inamide, 1,2-diisonicotinoylhydrazine, i son ico t ine - carboxaldehyde and i son ico t inoy l hydrazone. The copper che la 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 rde r r eac t ion and t h e r a t e i s determined by t h e r a t i o of the concent ra t ion of che la t ed spec ie s p re s en t .

Inoue and Ono61 have e s t a b l i s h e d the k i n e t i c s of t h e degradat ion of i s o n i a z i d i n t h e presence of Managenese (11). Shchukin62 has s tud ied t h e r eac t ion of copper (11) with i son iaz id .

of the sodium methanesulfonate 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 s ~ ~ have demonstrated t h a t i s o n i a z i d i n syrup formulat ions undergoes hydrazone formation with t h e f ree glucose t h a t i s present . Absorption of t h i s hydrazone i s r epor t ed t o be impaired. The au thors suggest t h e u s e of s o r b i t o l a s a replacement f o r sucrose.

Kakemi and co-workers63 s t u d i e d t h e s t a b i l i t y

6. Ana ly t i ca l Chemistry 6 . 1 I d e n t i t y T e s t s

A l a r g e number of i d e n t i t y t e s t s have - -

been e s t ab l i shed f o r i s o n i a z i d . Most of t h e s e are colorimetric and a re r epor t ed below i n t a b u l a r form.

Reaqen t p- Dimethylaminobenza ldehyde A l k a l i n e Na2Fe (CN) 5NO Q C F e (CN) 6J + l i g h t Dini t r o c h lorobenzene o -n in i t robenzene Reduction w i t h Zn/HCl and

ph e n y l h ydra z i n e 1 , 2 Naphthoquinone- 4 -Su l fon ic acid + 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

E t h y 1 en i c d i ca rbox y 1 i c a c i d s ( fumar ic , m a l e i c 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 acid N i nh y d r i n BrCN and NaOH Benzyl c h l o r i d e NaOH D r a g e n d o r f f ' s Reagent

8 w Dimethylglyoxime

rnlnr i n t e n s e ye l low i n t e n s e o range p ink p u r p l e v i o l e t

ye 1 low

b r i g h t r e d orange t o r e d

r e d r e d ye 1 low

--

-- brown r e d r e d orange green-b lue f l u o r . b l u e f l u o r e s c e n c e r e d

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

73

74,75,76 77 78 7 9 80 81

82 8 3 84,85 85 85 86,94

I n a d d i t i o n to t h e s e c o l o r r e a c t i o n s a number of c o l o r e d p r e c i p i t a t e s can be formed on t h e a d d i t i o n of me ta 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 A m 0 3

HgC12 cuso4 Hg2C12

S e02

K I A u I Lead a c e t a t e + K I KBr

~ 2 0 5 . 1 2 ~ 0 3 0 e Picro lonic a c i d

Tannic a c i d V i t a l i l s reagent Mecke' s reagent Frghde' s reagent Mandelin' s reagent Alloxan D i s u I f imides M e thy1 iodide K2 C r 2 0 7 Pho s phomo 1 yb d i c a c i d P i c r i c Acid Reineckel s s a l t Styphnic ac id

N

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 Effervescence followed by black and c o l o r l e s s c r y s t a l s P r e c i p i t a t e Green-ye1 low PPt Yellow mass Rose-sienna Blue Red White ppt

-- Yellow needles

-- --

Reference

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

74 ,94

86 8 6 , 9 3 86 8 6 86 86 86 8 9 90 91 92 92 93 ,94 94 94

Kay 's r e a g e n t V a i l l e f s r e a g e n t Na 2 P t B r 6

94 94 94

F e i g l and co-workersg6 have r e p o r t e d a spot t e s t i n which i s o n i a z i d i s q u a t e r n i z e d and py ro lyzed w i t h Na2S203 a t 180OC. i s used f o r d e t e c t i o n .

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 d e t e c t i o n o f i s o n i a z i d .

A c i d i f i e d Fe B e (CN) 6-7

6.2 Methods of A n a l y s i s 6 . 2 1 Genera l Reviews

Deltombe99, S l o u f l O O , Robles and Unzueta'Ol, Ya lc indag lo2 , Brandys l03 and Garcia and co-workerslo4 have a l l p u b l i s h e d rev iews 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 .

6.22 Color imet r ic methods A number of a u t h o r s have formed hydrazones of i s o n i a z i d w i t h

v a r i o u s a ldehydes 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 de te rmine t h e d rug . O f t h e v a r i o u s a ldehydcs used , p-dimei?hylaminobenza ldehyde

?R and vanill in113' 197yPgghave a l so been used . ears t o be t h e m o s t 0 ular105,106, lo7; lo8, log, Ilo, lll. Benza1dehydes7, I 6 O ,

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 benza ldehyde i n sodium b i c a r b o n a t e s o l u t i o n . The abso rbance o f t h e hydrazone is measured a t 302 nm. The absorbance a t 375 nm (background) is s u b s t r a c t e d as a correct ion169.

Sodium 1,2-naphthoquinone-4-sulfonate reacts 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 produce an orange-red color w i t h a maximum a t 480 nm1149115. 2-3-Dichloro-1,4-naphthoquinone reacts

206 GLENN A. BREWER

with i s o n i a z i d t o g ive a b l u e c o l o r i n a l k a l i n e ~ o l u t i o n ~ l ~ , ~ ~ ~ . pharmaceutical p repara t ions which a l s o contain sodium aminosa l icy la te l l8 . 4-naphthoquinone has a l s o been regorted1l9.

The r eac t ion i s use fu l wi th

An as say u t i l i z i n g 1,

I son iaz id reduces phosphomol bda te

a similar r eac t ion molybdophosphotungstate g ives a b l u e color122. An assay u t i l i z i n g molybdic a c i d i n a l k a l i n e acetone s o l u t i o n has also been repor ted l2?

I son iaz id r e a c t s with cvano en

i n a l k a l i n e so lu t ion to molybdenum b l u e 120,lYl. In

chlor ide124~125~126, chlororhodanaminel27~1 2 8 o r cyanogen bromide129 t o form g lu t acon ic dialdehyde which can then be condensed with b a r b i t u r i c o r 2- t 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 colored polymethine dyes.

I son iaz id reduces f e r r i cyan ide t o ferrocyanide. The amount of ferrocyanide can be determined by the add i t ion of f e r r i c ion t o y i e l d a b l u e colorl30,131.

r e a c t s with i son iaz id t o g i v e a yellow chromogen 132

Sodium pentacyanoaminoferroate

I son iaz id r e a c t s with 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 ive a purp le color133,74,134. l-Fluoro-2,4 dini t robenzene

135 a l s o r e a c t s i n a s i m i l a r manner . I son iaz id forms colored complexes

with many metals which can be used i n a n a l y t i c a l methods. can be formed wi th ammonium vanadate 13g~y9’~358, f e r r i c c h l o r i d e and 2,21 b i - ~ y r i d i n e l ~ ~ , copper139 and Nickel (11) and f e r r i c i0nl40.

Reineckels s a l t forms a water i n so lub le p r e c i p i t a t e with i son iaz id . This p r e c i p i t a t e d i s so lves i n acetone and t h e concentra- t i o n of i s o n i a z i d can be determined co lo r ime t r i c -

The fol lowing compounds have a l s o aiiy141, 142.

been used i n co lo r ime t r i c assays for i aon iaz id .

Reagent Reference ch loropi crin 504 epichlorohydrin 14 3 n inh ydri n 144 t r iphen y 1 t e t razol i um ch lor ide 14 5 9-chloroacridine 146 dinitrobenzoic acid 14 7 p-aminosalicylate-HVO~ 148

p-ni trophenyldiazonium f luoroborate 14 9 7-chloro-4 nitrobenzo-2-oxa-1,3-diazole 150 acid chrome dark blue 151

N- (4-pyridyl) pyridinium chloride 152 picryl chloride 174

1,2,4-aminonaphtholsulfonic acid 77

2 -bromo-1-acetonaphthone 112

6.23 Spectrophotometric Methods

absorbance of isoniazid in the ultraviolet as a means of determining the concentration of the drug. In many methods the a in alkaline and acid solution as an identity test 1659237.

an ultraviolet assay162,163,164.

A number of authors have utilized the strong

Y @:f!74 y3, iQ%, ar39:'iB; I P S Fh Isoniazid can be determined in the presence of p-aminosalicylate by

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 yellow- green fluorescence. The compound has an excitation maximum at 495 m and an

208 GLENN A. BREWER

emiss ion 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 bromide t o form g l u t a c o n d i a l d e h y d e . A S c h i f f ' s base i s t h e n formed w i t h 4-aminobenzoic a c i d which has a n e x c i t a t i o n maximum a t 336

6 .25 T i t r i m e t r i c Methods A l a r g e v a r i e t y o f t i t r i m e t r i c

methods have been 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 f o r m u l a t e d p r o d u c t s .

A ser ies o f r e v i e w s h a v e 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 methods of a n a l y s i s i n t h e U . S. P. 2 3 , B. P. 174 and European Pharmacopoeia3 a r e t i t r i m e t r i c methods.

t i t r a t i o n i s u t i l i z e d . 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 b y t h e a d d i t i o n o f po ta s s ium i o d i d e . 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 e t h o x y c h y s o i d i n e a s an i n d i c a t o r .

a r e summarized i n t h e Table.

I n t h e U .S .P ,23 a n i t r i t e I n t h e B.P. 174 t h e

I n t h e European Pharmacopoeia3 a d i r e c t

The v a r i o u s t i t r i m e t r i c methods

Reaq e n t mr , m r 0 3 , KI m r Br2 -

~ 1 0 3 , KI H I , K2Cr207, K I ~ ~ 1 0 4 , I C 1, K I

N

8 non-aqueous

non-aqueous non-aqueous

non-aqueous Cd++ Cd++ c U + + , NH4SCN CU++, N H ~ S C N

T i t r a n t t h i o s u 1 f a t e K B r 0 3 a l k a l i KBrO3 m r 0 3 t h i o s u l f a t e K I O ~ t h i o s u l f a t e t h i o s u 1 f a t e t h i o s u l f a t e t h i o s u l f a t e

HC lo4 I 2

NaN02 HC 104 HClOq N a C l O 4 NaOMe Complexon 111 C a C 1 2 A m 0 3 EDTA

I n d i c a t o r

e t h o x y c h r y s o i d i n e pheno lph tha l e i n m e t h y l o r a n g e po t e n t iome t r i c s t a r c h e t h o x y c h r y s o i d i n e s t a r c h s t a rch s t a r c h s tarch thermome t r i c c r y s t a l v i o l e t o r m e t h y l v i o l e t

s tarch

- Sb e l e c t r o d e g l a s s electrode p o t e n t i o m e t r i c thymol b l u e eriochrome Black T

methylthymol B l u e

R e f e r e n c e 1 7 5 , 1 7 7 , 1 7 9 , 1 8 2 1 7 6 , 1 0 6 , 1 8 0 , 1 8 4 1 7 8 181,186 1 8 1 , 1 8 3 , 185 , 187 7 4 , 1 8 8 , 1 9 2 , 1 9 6 18 9 1 9 0 , 1 9 3 , 1 9 5 1 9 1 1 0 6 , 1 9 4 198 200 2 1 7 , 2 1 1 , 2 1 0 , 2 0 9 , 2 0 8 , 2 0 7 , 2 0 6 , 2 0 4 , 2 0 5 , 2 1 6 , 2 1 3 , 2 0 1 , 7 4 , 5 7 , 2 0 3 , 2 14 233 2 1 2 , 2 7 6 215 2 18 2 02 2 1 9 , 2 2 1 220 222

mu r e x i de 223 , 224

E

% m ~m d

ma

,

k

a, a

m a a, a

.ti a

0

.ti

E k

4J m

m

a, c -ti

5 rl h

C

a,

a, a 0

k

c, u

a,

aE

E 000

E

7

5 k

4J m

m

d

a,

c, nl

d

m a,

Ik .d a

U

Ill - 4J m

C a, rn

a, cr W

m

H

H

C 0

3 dd d

0 a

0

mE

cn

C

OD

N

Uf

:

m

m

9

d

h

h

Hd

'

o"+ 0-

cn+ z

33 tn

uu 4

I I

I I

I

210

Nesslerl s reagent sodium d i e t h y l - cuso4 d i thiocarbama t e

2 56

I 2 hydraz i n e s t a r c h 2 57 ammonium hexani t r o - a-naphtho- 2 58 c e r a t e (IV) f lavone

C e (So4) 2 Mohrrs s a l t P t e l e c t r o d e 259 C e (NO3 14 P t e l e c t r o d e 2 60

K2Cr207 Mohrfs s a l t diphenylamine 261 isopropenyl t r i - ch loroace ta t e NaOH bromphenol b lue 262 KOH K3Fe (CN) 6 P t e l e c t r o d e 263,264 K 3Fe (CN) 6, KOH, H2S04, K I t h iosu I fa t e s t a r c h 265

-

-

Y 6.26 Electrochemical Methods A number of au tho r s have d e t a i l e d polarographic

methods f o r i son iaz id . The reduct ion appa ren t ly occurs i n two s t e p s ( t o t a l of 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 we l l s epa ra t ed t o be u t i l i z e d a n a l y t i c a l l y , so t h a t t h e s i n g l e wave i s used. comes more nega t ive a t h igher pH values over t h e 279,280,2Kl

Vallon and c o - ~ o r k e r s ~ ~ ~ i n t h e a s say of i son iaz id . Okuda and co-workers284 re- a c t e d i s o n i a z i d with 1,2-naphthoquinone-4-sulfonic a c i d and have then used polarography t o measure t h e r e a c t i o n product.

The h a l f wave p o t e n t i a l be- b u t t h e h e i g h t d i d not change r e a t l y

H range s t u d i e d 266,153,267,266,269,270,271,272,273,274,275,27 4 ,278,

A.C.Polarography has been used by Sato282 and

se erpl au ho r s h ve r e r t e d coulo e t ' c gethods for t h e a n a l y s i s of i s o n i a z i x with ekectroccemica??y genera te8 c6 io r ine 285,286 o r bromine286,287,288,289.290.

212 GLENN A. BREWER

6.27 Gravimetric Methods Rela t ive ly few gravimet r ic assays

have been reported f o r i soniaz id . This i s probably because of the l a r g e number of co lor imet r ic , t i t r i m e t r i c and electrochemical 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 gravimetr ic methods.

assay using p i c r i c ac id t o form a water i n so lub le s a l t .

c i p i t a t e i soniaz ide a s t he C u ( I 1 ) o r Hg(I1) s a l t s . The s a l t s a r e redissolved i n hydrochlor ic ac id and the metal i s then r ep rec ip i t a t ed a s t h e s u l f i d e which i s determined gravimet r ica l ly .

can be measured by d i r e c t gravimetry. The benzyl- idene de r iva t ive can be determined e i t h e r grav imet r ica l ly o r v o l ~ m e t r i c a l l y ~ ~ ~ . I soniaz id can be quaternized and t h e s a l t can be then measured volumetr ical ly o r grav imet r ica l ly2 95. The phosphotungstate of i son iaz id can be determined gravime t r i c a 11~164 .

Leal and Alves 234 have reported an

Akiyama and co-workers291 pre-

The zinc292 and cadmium293 s a l t s

6.28 Microbioloqical and Enzymatic Methods Several agar d i f fus ion microbio-

l o g i c a l assays u t i l i z i n g s t r a i n s of a c t e r ium have been reported f o r i s o n i a ~ i d ~ ~ ~ ~ ~ ~ ~ .

systems and a number of these might be se l ec t ed a s t h e bas i s of enzymatic assays. Examples of enzyme systems which a r e i n h i b i t e d a r e pea cotyledon amine oxidase, c a r r o t r o o t L-glutamic decarboxylase and wheat seedl ing t r a n s a m i n a ~ e ~ ~ ~ . The i n h i b i t i o n i s reversed by the presence of ke to ac ids .

I soniaz id i n h i b i t s many enzyme

6 . 2 9 Miscellaneous Methods Osci l lometr ic t i t r a t i o n s have been

Isoniaz id can used t o determine i s o n i a ~ i d ~ ~ ~ j 301. be assayed gasometr ical ly a f t e r oxidat ion with iodate3O2 o r ferr icyanide303 J 304.

Conductometric t i t r a t i o n s with sodium hydroxide o r hydrochlor ic a c i d have been

used t o measure i s o n i a z i d conten t305 ,306.

ke tone . The copper c o n t e n t of t h e c h e l a t e i s de termined i n t h e o r g a n i c phase by a tomic a b s o r p t i o n spec t romet ry307 .

3 08

The copper 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

I s o n i a z i d i n pu re s o l u t i o n s can be de te rmined by r e f r a c t o m e t r y

6 .3 Chromatographic Methods 6 . 3 1 Paper Chromatoqraphy

Numerous paper chromatographic systems have been used 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 used 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 roduc t s . S i n c e i s o n i a z i d a b s o r b s s t r o n g l y 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 or q u a n t i t a t i n g t h e drug 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 ab le of some pape r chromatographic sys tems i s g iven below: S o l v e n t S y s t e m D e t e c t i o n U s e 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 Ur ine m e t a b o l i t e s 3 10 Isoamyl a lcohol -water - CNBr , Microb i o 1. Ur ine m e t a b o l i t e s 3 1 1 a c e t i c a c i d (50 : 50 : 1 . 5 ) I sopropanol -water (85 : 15) -- U r i n e m e t a b o l i t e s 312,313 Butano 1-ammonia -- D e r i v a t i v e s 3 14 1st Dimension sec. b u t a n o l - w a t e r ( s a t u r a t e d )

5 w

2nd Dimension isoamyl CNBr- o-phenyl- Ur ine a l c o h o l - a c e t o n e - a c e t i c enediamine d imethyl - ac id -wa te r (56:24:6: 14 ) benza ldehyde

Butanol-10% NH4OH(lO:2) Butanol sat.ammoniaca1 Impurities 3 16 circular with silver nitrate Butanol-water(4:l) dimethylaminobenzaldehyde Dosage forms 3 17 ascending 2,4-lutidine-isoamyl methanolic dinitro- Impu ri ties 3 18 alcohol-water (5: 100: 9) chlorobenzene Butanol-HC1-pet. ether or iodine-platinic other basic 319 Butanol-HCl-H20(paper sat. iodide substances with KC1 solution)

(d) Phenol-isopropanol-water

(a)Butanol-ethanol-water

(b)Butanol-pyridine-water (16:4: 3 ) (c) Ethanol-l.5N NHqOH-water ultraviolet (17: 1:2)

(16 : 1:5) 0.5 ammonium chloride (a)Butanol saturated with water (b) Propanol-water ( 8 0 : 2 0 )

u 1 travio le t

--

I (2:2:1)

1

metabolites 320

metabolites 32 1 metabo 1 i tes 322,323 in urine

CI4 and Me tab0 1 i tes 324 spray reagents

(a) 1sopropanol-25% NH20H(85: 15) (b) Isopropanol-water (85: 15)

Metabolites 325

(c) Isopropanol-formic acid-

Pyridine-Water (65:35)

Isopropanol-NH40H-water (7: 1:2) 1 FeC13 and Metabolites 326 Butanol-acetic acid-water ( 5 : 1:4) ( a ) E thyl me thyl ketone-acetone-

(b )E thy 1 me thy 1 ketone -di e thy lam ine - (c) Methyl isobutylketone-formic

-- 1 water (80: 1O:lO)

K 3 F e (CN) 6

1 formic acid water(40:2:1:6)

water (921: 2 : 77)

acid-water(ketone sat.with 4%

(d)Chloroform-methanol-formic acid- 2 Ln formic acid)

water(CHC13 sat.with 1 part H20 and 1 part 4% formic acid)

(e)Benzene-ethylmethyl ketone- formic acid-water(9 parts benzene plus 1 part ketone sat.with 2% formic acid)

(benzene sat. with 2% formic acid)

(f)Benzene-formic acid-water

327

2,4,6 trinitrobenzene-sulfonic

chlorani 1 ic acid (c) 1.4M potassium phosphate buffer pH 7.0 acid

la) ISO-propanol-water (17 : 3) (b)Butanol-acetic acid-water(4:1:5)

Butanol-acetone-water(45:5:50) Butanol-phosphoric acid-water(3:1:3) (a)Butanol S a t . with water in atmosphere copper sulfate

in ethanol then 0.1% benzidine in 50% aqueous ethanol

(b)95% ethanol-M ammonium acetate(7:3)

-- I 1 of NH3

adjusted to pH 5

6.32 Thin-layer Chromatrogaphy In recent years several authors have developed thin-layer

chromatographic system for isoniazid. These are presented in tabular form. N

3 28

329 330 33 1

- m

System Detect ion Chloroform-methanol (8: 2) Folin-Ciocalteu Chloroform-acetone-diethylamine or Phospho- (5:4:1) Cyclohexane-chloroform-diethylamine (4:5:1)

Acetone-methanol-NJQOH (50: 50: 1) dimethylamino-

mo 1 ybda t e

Butanol-phosphoric acid-water(3:1:3) --

benzaldehyde 5:l mixture 10% cuso4 and 10% NH~OH

\ (a) Pkthanol (b) Chloroform (c) Ethanol

Use Ref separation 332 from other drugs

derivatives 330 identification 333

identity 3 34 test

IS opropanol-acetone (6 : 4) --

chloroform-methanol (6:4) --

Chloroform-methanol (125:60) UV iodine

separation of hydrazine separation of isonicotinic acid hydrazone with lactose

335

335

59

Ninhydrin or separation from 336 I drugs of abuse

(b)same solvent but

(c)Ethyl acetate-cyclohexane- 0.5% H2S04

(a!Ethyl acetate-cyclohexane- dioxane-methanol-water- NH40H(50:50:10:10:1.5:0.5)

(50:50: 10: 1O:O. 5: 1.5)

NHqOH-methanol-water (70:15:2:8:0.5)

NHqOH-methanol(56 : 40 : 0.4 : 0.8) \ (d)E thyl acetate-cyclohexane-

(e ) same but (7 0 : 15 : 5 : 10) (f )E thyl acetate-cyclohexane-

Methanol-NH4OH-H20(100:1:4) KMnO4 bromothymol other drugs NH40H ( 5 0 : 40 : 0.1 )

blue 337

338 254 nm U.V. iron chloride- hexacyanoferrate,molybdo- phosphoric acid. Folin- Ciocalteu, potassium permanganate,ammoniacal

13N ammonia(90:10:0.2) silver nitrate,amminepenta-

(a) Chloroform-methanol- 13N ammonia (90: 10: 1)

(b ) Benz ene-;oe thano 1 - diethylamine (90 : 10 : 1)

(c) Chloroform-hexanol-

(d)Chloroform-ethyl acetate cyanoferrate,iodoplatinate, 13N ammonia ( 5 0 : 5 0 : 1)

(e ) ch loro f orm-a ce tone- acetic acid (90: 10: 1)

(f) Benzene-acetone- diethylamine (50 : 50 : 1)

(g ) Chloroform-acetone-

iodine,Dragendorff, cinna- maldehyde triphenyltetra- zolium, di thioca rbama te or ammonium molybdate 1 3 rn acetic acid(50:50:1)

Nishimoto and T~yoshima~~' 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.

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. with 1% mercurous nitrate to reveal isoniazid as black spots.

Wijnne and c o - w o r k e r ~ ~ ~ ~ found that isoniazid could be

Kawale and c o - w o r k e r ~ ~ ~ ~ sprayed thin-layer plates

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 + + + .

ISONIAZID 219

No a d s o r p t i o n occur s on r e s i n i n t h e Ba++, Mg++, Ca++ o r Na+ forms.

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 of Dowex 1-X8 i n t h e py ruva te form.

chromatographic method f o r t h e s e p a r a t i o n of i s o n i a z i d from some d e g r a d a t i o n p roduc t s . I s o n i a z i d i s adsorbed on a weak c a t i o n exchanger such a s Amber l i te 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 adsorbed and i s de termined c o l o r - i m e t r i c a l l y u s i n g cyanogen bromide. To de t e rmine 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 through a column o f s t r o n g an ion exchanger such a s Dowex 1-X8 i n t h e c h l o r i d e form. The amide is unchanged and i s n o t adsorbed on t h e r e s i n . Another degrada- t i o n product,1,2-diisonicotinoyl hydraz ide i s determined by a d j u s t i n g t h e sample t o pH 8 . 9 w i t h b o r a t e b u f f e r and de te rmin ing t h e absorbance a t 329 nm.

P e t e r s and 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 of m e t a b o l i t e s of i s o n i a z i d us ing 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 color r e a c t i o n s were used t o d i f f e r e n t i - a t e t h e groups of m e t a b o l i t e s .

p -aminosa l i cy l i c a c i d from i s o n i a z i d us ing a Dowex 2 - X 8 column. Inoue and c o - w ~ r k e r s ~ ~ ~ used a system s i m i l a r t o t h a t of Kakemi e t a1343 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 roduc t s .

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 by paper 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 paper was connected w i t h an i o n exchange paper 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 dioxane. The s h a r p zones on the i o n exchange paper 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

graphed s e v e r a l d rugs on CM-82 carboxymethyl c e l l u l o s e c a t i o n exchange paper u s i n g a water -

H e l l e r and c o - ~ o r k e r s ~ ~ ~ s e p a r a t e d

Kakemi - e t -9 a~~~~ developed a

Fan and Wald348 s e p a r a t e d

Lewandowski and S y b i r ~ k a ~ ~ O

Darawy and Mobarak3" chromato-

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.

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 chromato- graphy356,357, 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.

Smolarek and Dlugosch353 separated

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 adi~tou~~’ studied a

number of color reactions to determine their application to blood level assays. Ginoulhiac also made a literature review of blood level methods, A critical review of methods for isoniazid determination has been written .

360

364

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

Dimethylaminobenzaldehyde Dimethylaminobenza ldehyde

Dimethylaminobenzaldehyde

Dimethylaminobenzaldehyde

N 5 Vanillin

Vanillin

Vani 11 in

Van i 11 in

Cinnama ldehyde

Cinnama ldehyde

Pretreatment of sample Type of specimen Ref. acid hydrolysis serum & urine 361,370,

none urine extraction into plasma and isoamyl alcohol- urine ether from alkaline solution deproteinization serum with ~ ~ 1 0 ~ deproteinization serum and with trichloroacetic tissues acid none serum

deproteinization serum with trichloroacetic acid extraction with serum organic solvent extraction with milk propanol deproteinization serum with trichloroacetic acid extract ion with serum butanol-chlorof orm

375 362,363 365,366, 367,3 68, 369.

37 1

372

373,376, 377 374,432,

375 433,434

3 78

379,380, 429,430

38 1

o- Ni t robenz a ldehyde

S a 1 i cy la 1 dehyde - Fe C 1

S a li cy la ldehyde

Salicylaldehyde

Glutaconic aldehyde

@-diketone

N

E Catecho1

Cat echo 1 H2 02 - CNB r CNBr

A Ika line hydrolysis- CNBr NHqV03-H2S04

KCN, Chloramine T- barbituric acid

deproteinization with trichloroacetic acid extraction into isoamyl alcohol-ether from alkaline solution none

extraction with acetone deproteinization with trichloroacetic acid none

deproteinization with trichlor oace t ic acid automated method deproteinized serum deproteinized trichloroacetic acid deproteinized trichloroacetic acid acid hydrolysis

serum

serum

bio log ica 1 fluids cadavers

plasma

biological ma teria Is ci tra t ed blood serum serum &

urine biological fluids urine

urine

382

383

3 84

385

386

387

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-4-

Naphthoquinone-4- sulfonic acid Naphthoquinone-4- sulfonic acid 2,4,6-trinitrobenzene- sulfonic acid Dinitrochlorobenzene Dinitrochlorobenzene

sulfonic acid

K3Fe (CN) 6 Sodium pentacyanoaminoferroate K3Fe (CN) 6

r.J

Ni tropen tacyano- f erroate Na ph thoqu ino ne

Na ph thoq i non e H202,CNBr, aniline

Picryl chloride

KMn04,BrCN,NH3

deproteinization

extraction methyl isobutyl ketone

Zn (OH) 2

deproteinized serum

deproteinized tissue deproteinized with sodium tungstate deproteinized with phosphoric acid

--

tr i ch loracetic acid tungstic acid extraction BuOH, Et20

Py tein-free fiPtra te

urine biol. fluids urine

urine

whole blood

urine serum

serum tissue, urine spina 1 fluid serum

spinal fluid urine blood blood urine plasma urine spinal fluid plasma

402

403,404,405, 406,407 408

409,410

411 412,413

4 14 415,416,417

4 18

419

420

42 1 422

423,424

42 5

4-pyr idy lpyr id in ium t r i c h l o r a c e t i c a c i d plasma 426 dichloride,NaOH,HCl f i l t r a t e KBrO3+ methyl o range a c i d t u n g s t a t e b l o o d 427 Zn powder + heat -- u r i n e 428

6.43 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 is 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 can be measuEed t o d e t e r m i n e the amount of i s o n i a z i d 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 hydrox ide and z i n c s u l f a t e .

Wagner and co-worker-431 have 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

6.44 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 methods €or i s o n i a z i d have been

Hedr ick and c o - ~ o r k e r s ~ ~ ~ abso rbed 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 r e p o r t e d . on p H 6 .5 A m b e r l i t e XE-64 i o n exchange r e s i n . The i s o n i a z i d was e l u t e d w i t h

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 320 nm. A s l i t t l e a s 0.05 y/m1 o f s e r u m can be de termined .

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 and Putsch438 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 and e l u t i o n from i o n exchange 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 cyanogen bromide. Wi lson , 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 o f i s o n i a z i d w i t h pentane-2 ,4-d ione i n an a s s a y i n se rum.

metr ic 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 and i s o n i c o t i n y l g l y c i n e i n serum and u r i n e . Boxenbaum and Riegelman441 have a l s o deve loped 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 b lood .

3 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 p H 8 .7 b u f f e r . The P

E l l a r d , Gammon and Wallace440 have developed s p e c i f i c f l u o r i -

M i c e l i , Olson and Weber442 have 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 se rum. A s l i t t l e a s 2 5 p1 of serum can be used i n t h e assay .

0' B a r r , Kei th and B la i r444 have compared 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 lec t rochemica l Methods

Peters, Morse and Schmidt 443 and f 1 U O r i -

Lauermann and Otto445 hydro lyzed i s o n i c o t i n i c a c i d hydraz ide 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 wi th 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 by t h i s method i n t h e a n a l y s i s of 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 ie l sch and Giefer401 was used. method was less t i m e consuming.

b i o l o g i c a l f l u i d s w i t h o u t p r i o r s e p a r a t i o n .

The p o l a r o g r a p h i c

Kane 446 de termined i s o n i a z i d i n

6.46 Gasometr ic Methods The hydraz ine group i n i s o n i a z i d can

be r e a d i l y decomposed i n t o n i t r o g e n gas . S e v e r a l 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 lood and u r i n e l e v e l a s s a s .

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 by t h e p re sence of p- a 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 iven i n con junc t ion w i t h i s o n i a z i d . 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 n i t r o g e n gas .

t o s e l e c t i v e l y use copper , i r o n and chromium azometry t o de te rmine i s o n i a z i d and i t s v a r i o u s m e t a b o l i t e s i n u r i n e .

S t r i c k l a n d and Hen te l 1;47 r e a c t e d

I t o and c o - w ~ r k e r s ~ ~ ~ 7 450 were able

6.47 Misce l laneous Chemical Assays

i s o n i z i d i n c e r e b r o s p i n a l f l u i d by iodomet r i c ti t r a t i o n .

a radioimmunoassay f o r t h e 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 i o l o g i c a l f l u i d s .

V e r r o t t i and B a r d e l l i 4 5 1 de termined

Schwenk and c o - ~ o r k e r s ~ ~ * employed

6.48 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 i t i wi ty Ref. Mycobacterium phlei agar diffusion 2.5-30 y/ml 453 Koch bacilli turbidimetric -- 454 tubercle bacteria cord formation -- 455 bacilli vertical diffusion -- 456 Mycobacterium

tuberculosis HV37 vertical diffusion -- 457 Mycobacterium agar diffusion -- 458

tubercu 10s is

tuberculosis vertical diffusion > 0.49 y/m1 459 N

N Mycobacterium m

H37Rv and H37Ra assay of isoniazid 378 BGG in milk-agar

diffusion

Mycobacterium tuberculosis

--

vertical diffusion vertical diffusion

vert i ca 1 di f €us ion tube dilution vertical diffusion for urine

460 46 1

462

463,464

ISON IAZlD 22 7

Bartmann and F re i se465 s t u d i e d t h e t i s s u e b ind ing of i s o n i a z i d w i t h t h e mic rob io log 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 wh i l e mice and guinea p i g t i s s u e gave 80% b ind ing . Incuba t ing the t i s s u e a t an e l e v a t e d t empera tu re d i d n o t r a i s e t h e recovery .

N i ~ h i ~ ~ ~ , Poole 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 co-workers 433 and O’Barr & a4!* a l l compared v a r i o u s chemica 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 workers conclude t h a t t h e two methods gave comparable r e s u l t s .

6.49 Chromatographic Assays

complex and many workers have s e l e c t e d chromato- g r a p h i c a s s a y s t o measure t h e drug i n t i s s u e and b i o l o g i c a l f l u i d s . These methods p rov ide 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 iven by many chemica l methods.

systems a r e g iven i n s e c t i o n 6.3. Many of t h e s e methods could probably be used 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 iven i n t h i s s e c t i o n have been developed j u s t f o r t h i s purpose.

Makino and c o - ~ o r k e r s ~ ~ ~ fo l lowed t h e metabolism 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 paper chromatography (water 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 wi th 0.02M phosphate 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 ac id-water ( 4 : 1 : 5 ) ) .

s a t u r a t e d w i t h wa te r and isoamyl a l c o h o l as developing s o l v e n t s .

hydrazones of i s o n i a z i d and py ruv ic and a-ke to- 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 paper chromatog-

The metabolism o f i s o n i a z i d i s

A l a r g e number of chromatographic

L e ~ s c h n e r ~ ~ g used sec -bu tano l

I ~ a i n s k y ~ ~ ~ s e p a r a t e d t h e

raphy. Sezaki 470 s e p a r a t e d i s o n i a z i d from

pyrazinamide i n u r i n e by means of Amber l i t e IRA-400. B e l l e s and Li t t leman471 used 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 s ~ ~ ~ use Dowex 1-XlO to separate these as well as the hydrazone of glucuronic acid.

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.

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

Peters, Miller and Brown346

Okudaira and co-~orkers~’~ used

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.

TLC to determine isoniazid and other drugs in cadavers.

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.

from isoniazid by counter-current distribution (butanol-ethylene dichloride- 9: 1 - 2M phosphate buffer pH 5.1).

three conjugated metabolites of isoniazid by paper chromatography. Cuthbertson et used paper chromatography to determine isonicotinoylglycine. They used the following systems:

Fartushnyi and S ~ k h i n * ~ ~ have used

Cattaneo, Fantoli and Ferrari478

Hughes479 separated acetylisoniazid

Ozawa and Kiyomoto 480 isolated

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 i ~ ~ ~ used a isopropanol:water(85:15) system to separate the hydrazones of pyruvic and a -ketoglutaric acid from

i son i az id. 7. Druq Metabolism

very reactive molecule and can undergo non-enzymatic reactions in the body. general metabolic pattern is indicated in diagram.

Non-enzymatic Enzymatic

The drug metabolism of isoniazid is unusually complicated in that it is a A

acetylisoniazid

0 II

isonicotinoylhydrazones of glucose, a-ketoglutaric acid, aldehyde

pyruvic acid etc. or N ketone N ID

0

isonicotinamide

N , N * diisonicotinoylhydrazide

230 GLENN A. BREWER

The major metabolite of isoniazid is N-acetylisoniazid. The rate of acetylation is genetically ~ o n t r o l l e d ~ ~ ~ , ~ ~ ~ 485. It has been established that the slow acetylation is a auto- soma1 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.

absorption of derivatives of isoniazid in the stomach and intestine. The authors report a rough correlation between degree of absorption and lipid- water partition coefficient.

Bi opha rmaceut i cs Kakemi and co-workers 498 determined the rate of

ISONIAZID 23 1

9.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12 .

13.

14.

15.

16.

17 .

References

G r i f f i t h s , M. C. ; Dickerman, M. J. and Mil ler , L.C. ; USAN and t h e USP D i c t i o n a r y of Druq Names page 152 (1975) . 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 (1974) . Anon; European Pharmacopoeia Volume 1 ,page 310 (1969) . Prevorxek, D. ; B u l l . SOC. chim. France, 795-801 (1958) ;C.A. 53 21160b(1959). Nagano, K. ; Kinosh i t a , H. and Hirakawa,A. ; Chem. Pharm.Bul1. l2,1198-1206 (1964) ;C.A. - 62 2759a (1965) . T o e p l i t z , B . ; E. R. Squibb and Sons: P e r s o n a l Communication. Lapp, C. ; B u l l , SOC. pharm. Nancy 3 4 , 14-17 (1957) ; C.A. 53 21145d (1959) . Grebennik, L. I. ;Farmakol. i Toks ikol . 24, 233-7 (1961) ;C .A. 55 26263a (1961) . Pinyazhko, R. M. ; Farmatsevt . Zh. 2 0 , 18-22 (1965) ; C.A. 64 7969e (1966) . Kracmar, J . ; Kracmarova, J. and Zyka, J . ; Pharmazie 23, 567-73 (1968) ; C . A . a 31719r (1969) . Zommer, S. and Szuszkiewicz , J. ;Chem.Anal. - 14, 1075-83 (1969) : C.A. 2 89616n (1970) . Celechovsky, J . ; Greksakova, 0. and Polasek ,E . ; Acta Fac. Pharm. l7 ,51-5 (1969) ; C . A . B 112880a (1970) . Zommer, S. ; A c t a Pol. Pharm. 29,533-5 (1972) ; C.A. 78 158502k (1973) . Dunham, J. ; E. R. Squibb and Sons ;Pe r sona l Communication. Caen, G. ; J. Chim.phys. - 51,60-3(1954);C.A. - 48 10438e (1954) . T i t o v , E. V. ;Kapkan, L. M. ;Rybachenko,V. I. and Korzhenevskaya, N . G . ; Reakts Sposobnost O r q . Soedin 2, 673-81 (1968) ; C.A. 70 67448r(1969) . T i t o v , E.V. and Kapkan, L.M.; Dokl.akad.Nauk -- SSSR 184,1342-5 (1969) ; C.A. - 70 114411k (1969) .

232 GLENN A. BREWER

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

Titov, E. V. :Kapkan, L. M. and Chervinskii,A. Yu. : Teor. Eksp. Khim. &3, 202-9 (1972) : C.A. 77 74367m (1973). Hillerbrand M. :Lohmann, W. : Penka,V. and Ehling, U.: Z.Naturforsch, Tell C =,33-6 (1975) :C.A. - 82 169663~ (1975). Puar, M . S . : E. R. Squibb and Sons: Personal commun i ca t ion, Gillis, R. G. : Orq. Mass Spectrom. 2,1107-11 (1971) : C.A. 75 150910j (1971). Funke, P.T. : E. R. Squibb and Sons:Personal Communications. Anon: United States Pharmacopoeia XIX page 272 (1975). Mattu, F. and Pirisi, R . : Chimica g, 188-9 (1953) : C.A. 48 1897b (1954). Mattu, F. and Pirisi, R.: Rend.seminar. fac.sci. univ. Caqliari 25,96-117 (1955) : C. A. 50 767211 (1956). Pirisi, R. : Rend. seminar. fac. sci. univ. Caqliari

Jacobson, H.and Valenti, V. : E. R. Squibb and Sons:Personal Communication. Lumbroso, H. and Barassin, J. : Bull. SOC. Chim. France, 3190-7(1964):C.A. - 62 10322e(1965). Ziolkowski, J. and Guminiski, K. : Roczniki -- Chem. 38, 1807-13 (1965) ;C.A. - 63 9168b (1965). Bhat, T.N.: Singh, T.P. and Vijayan, M. : Acta Crystallogr. ,Sect.B B30, 2921-2 (1974) : C.A. 82 50126j (1975). Jacobson, H. and Ochs, Q.: E. I?. Squibb and Sons : Personal Communication. Del Rio-Estrada, C. and Dougherty, H.W. ; Kirk- Othmer Encyclopedia of Chemical Technoloqy 2nd Vol 21 Page 528-533 John Wiley & Sons,Inc. (1970). Clarke, E.G. C. : Isolation and Identification of Druqs page 383, The Pharmaceuti ca 1 Press London (1969) . Fallab, S.: Helv.Chim.Acta 36, 3-S(1953): C.A. 3 7296c (1953). Cani6,V.D. and Djordjevie,R.Dj, -geoqrad 22,

- 25,88-95(1955) :C.A. - 50 7668~ (1956).

193-9(1956) :C.A.= 16026f (1958) .

ISON I AZlD 233

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

C i n g o l a n i and Gaudiano; Rend. 1st. s u p e r . s a n i t > l7, 601 (1954) . Nagano, K. ;Tsukahara , H. ; K i n o s h i t a , H. and Tamura, Z . ; Chem. Pharm.Bul1. 11,797-805 (1963) ; C . A . 5 9 486711 (1963) . S a l v e s e n , B. and Glendrange , J .H . ; Medd.Norsk. Farm.Selsk 28 272-8(1966);C.A. 66 68866e(1967) . S a l v e s e n , B. and E i k i l l , S .A. ; E d d . Norsk. Farm.Selskap 27,135-44 (1965) ;C .A.& 9507f (1966) . V u l t e r i n , J. : T a l d n t a 10 891-8 (1963) : C.A. - 59 121423. (1963) . Tamura, Z . and Nagano, K. ;Chem. Pharm.Bul1. 11,

Tamura, Z . and Nagano, K. ; i b i d 999-1013 (1963) ; C.A. 59 14868a (1963) . I s h i d a t e , M. : Anal. Chem. Proc. In te rn .Symp. , Birminqham Univ. , Birminqham, Enql. 178-82 (1962) :C.A. 59 10967b(1963) .

Thomassen, 1I .B. :Dan P a t . 8 7 , 2 2 8 ( J u l y 7 ,1959) ; C.A. - 54 7742 (1960) . Pavlov, L . N . ; G a n g r s k i i , P . A . and Polyachenko, V.M. ; U . S . S . R . P a t 106 ,220 ( J u l y 25, 1957);C.A. 51 16563 (1957) . Pasedach, H. and S e e f z d e r , M . : G e r . P a t 1 .116 ,667 (Nov. 19 , 1 9 6 1 ) ; C . A . z 14247(1962) . Frehden, 0 . and Laza rescu , G. ;Rev.Chim. 13 91-5(196i ) ;C.A.57 11158i (1962) . Takizawa, S. : ,Tap. Pa t . 7472 (Nov. 15 ,1954) ; C . A . - 50 9447 ( 1 9 5 6 ) . Magrane, D . ; Span .Pa t . 202,45O(May 28 ,1952) : C .A. - 50 i7323 ( 1 9 5 6 ) . Lock, G. : Pharm. Ind . 14 ,366-8 (1952) : C. A. - 47 10531 ( 1 9 5 3 ) . I sh ikawa, M. ; J apan . Pa t . 2 6 2 9 ( A p r i l 21 ,1955) ; C . A . - 5 1 14832 (1957) . Lewin, E. and H i r s c h , J. G. ;Am. Rev. T u b e r c . 7 1 732-42(1955);C.A.= 2918e(1956) . Poole,N. F. and Meyer,A. E. , ; Proc. SOC. E x p t l . B i o l . - - Med. 104,560-2 (1960) ; C . A . = 661b(1961) . Kakemi, K . : S e z a k i , H. and Inoue, S. ;Yakuqaku Z a s s h i 86 163-8(1966);C.A.= 17362c(1966) .

793-6(1963);C.A.% 14867f (1963) .

234

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

GLENN A. BREWER

Inoue,S.; Yakuqaku Zasshi 87,883(1967); C.A. 68 21441~ (1968). Horioka, M.;Tsuruoka, Michio and Shinmura,Y.; Kyushu Yakuqakkai Kaibo 23 31-6 (1969) ;C.A. - 72 93307e (1970). Hald, J. G. ; Dan. Tidsskr. Farm. 43,156-9 (1969) ; C.A. 72 157196 (1970). Pawelczyk, E; Herman, T. and Sukowski, R.; Diss Pharm. Pharmacol 2 481-9(1969);C.A.72 - 3572911 (1970). Wu, Wen-Hung;Chin,Ting-Fong and Lach, J.Pharm. - - Sci. 59 1234-42 (1970) ;C.A. 73 101974h(1970). Inoue,S.;Yakugaku Zasshi 91 - 81-7(1971);C.A.74 - 111385b(1971). Inoue,S. and Ono,Y. : ibid 91 88-94 (1971) ; C. A . 7 4 111386~ (1971). Shchukin, V. A. ; Sb.Aspir. Rab. , Kaza v. Univ. , Estestr.Nauki.Gio1. ,Khim. ;171-80(1972) : C.A. 81 6776b(1974). Kakemi, K.; Sezaki, H., Nudai, T.;

Inoue, S. and Ono,Y. ; - 91 95-100 (1971);C.A. - 74

Nishikiori, K. : Yakugaku Zasshi 111387d (1971). Rao, K. V. N. ; Kai Radhakrishna, S C.A. - 76 117460k

asam, S. ; Menon, N.K. and

(1972). ; Bull WHO 45,625-32 (1971) :

Rekker, R.F. and Nauta, W. Th;Pharm Weekblad

Feigl, F. and Mannheimer, W.A. : Microchemie ver.Mikrochim Acta - 40,355-8(1953);C.A.47 - 7376b (1953).

- 99,1157-65 (1964), C.A. 63 4 3 5 ~ (1965).

Lucas,V. ;Rev.brasil. farm. - 33,471-3 (19521 : C.A. 47 9216e(1953). Cooper, P. ; Pharm. J. 177, 495-6 (1956) ; C.A. 51 6945g (1957). Laubie,H. : Bull. soc. pharm.Bordeaux 90,106-8 (1952) : C.A. 46 9019g (1952). Murti, P.S.; Rao, G. ;Bala, B. and Ra0,P.V. Krishna; Anal.Chim.Acta 71,202-4 (1974) ; C.A. 81 85757~ (1974). Garattini,S. : Atti SOC. lombarda sci-rnpd. biol. g, 107-9 (1953) ;C.A. 48 1895b (1954).

ISONIAZID 235

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

8 2 .

83.

84.

85.

86.

87.

88.

89.

90.

Yavorsl k i i , N . P. :Farmalsevi Zh. l7 ,9-12 (1962) : C.A. 58 13722d (1963) . Sgnchez, J.A. : Rev.Asoc.bioquim.Arqentina 17, 321-3 (1952) :C.A. 47 6822a (1953) . S c o t t , P .G .W. ; J.Pharm. and Pharmacol. 4,

Yarvarsl k i i , N . P. : Farmatsevt . Zh. 20,29-33 (1965) : C.A. 64 7971a (1966) . Cat taneo , C.: F a n t o l i , V. and B e l a s i o , L.; Ann. 1st. "Car lo F o r l a n i n i " 20,59-67 (1960) : . C.A. 55 12520d (1961) . Gomez, S.A. : Arch.bioquim.quim. y farm. , Tucumgn. 11, 69-95 (1957) :C.A. 55 908c (1961) . Poggi, A.R. : Mattu, F. : P i r i s i , R . and M i l l e t t i , M. : Chimica 8,238-41(1953) :C.A.48 - 1880b (1954) . Waksmundzki, A. and Romanowski, H. : Ann. Univ. Mariae Curie-Sklodowska,Lublin-Polonia,Sect.AA,

Ohkuma, S. and Yasumasa, K. : J. Pharm. Soc. Japan 76,894-6 (1956) ; C.A. 51 953b (1957) . Neuze l , E. and LeDuc, Y. ;Bul l .Soc. Pharm. Bordeaux 100 159-79(1961): C.A. 56 8682a(1962) . DIAmico,E. and RUSSO,R. : A g g i o r n T e d i a t . - 17 227-32 (1966) ; C.A. 9 9334e (1968) . Dospeux, P. F. : Chim.Analyt. 53 251-3 (1971) : A.A. 22 1129 (1972) . Yavorski i ,N. P. : Aptechn. Delo &48-50 (1965) ; C.A. 63 5452h (1965) . Leuschner, F. ; Natu rwis senscha f t en 40,554 (1954) ; C.A. 48 8698b (1954) . Calabrug, J . A . G . and Fresneda, M. F. :Galen ica Acta 6 221-37 (1953) C.A. 48 7499b (1954) . C a s t e l , P . : O r z a l e s i , H. and Dubois , A . : Trav. SOC. pharm. M o n t p e l l i e r l 2 ,73 -4 (1952) : C.A. - 48 10297e (1954) . Watanabe, T . ; E i s e i Sh iken jo d k o k u 74 111-12 (1956):C.A. - 5 1 15339f (1957) . Neuzi1,E. : B u l l . soc.pharm.Bordeaux 91 122-30 (1953) ; C.A. 48 329a (1954) . Runge, F. ;Enge lb rech t , H. J. and Franke, H. : Pharmazie 12 8-13 (1957) ; C.A. 52 15830a (1958) .

681-6 (1952) ;C.A. 47 1334d(1953) .

- 8,71-6(1953) ;C.A. 51 8374b(1957) .

236 GLENN A. BREWER

91.

92.

93.

94.

95.

96.

97.

98.

99.

Mladenovie, M. ;Blagoj evi6,Z. and Zivanov, C. : J. pharm.Belq. 13 559-63 (1958) :C.A. - 53 13504c (1959). Shilov,Yu. M and Chichiro,V. E. :Aptechn. Delo

11' yasov,Ya. Z. : Sudebno-Med.Ekspertiza,Min. Zdravookhr. SSSR 2,43-5 (1965) :C.A. 67 29824r (1967). Stebletsova,Zh.D. ;Farm.Zh. (Kiev)= 31-4 (1969) : C.A. 70 99672a (1969). Gull k0,R.N. : Yavorskii,N. P. :Garasevich,S. N. and Pershina,G. I. : Farmatsiya (Moscow) - 22,29-32 (1973): C.A. 80 19649~(1974). Feigl,F., Anger, V. and Goldstein, D.: Helv. Chim.Acta 43,2139-42(1960): C.A. - 55 131783. (1961). Popkov,V.A. : Farmatsiya (Moscow) l6,77 (1967) : C.A. 66 98547u (1967). Amelink,F. ;Pharm. Weekblad 87,821-36(1952) : C.A. 3 5773d (1955). Deltombe, J. : J. pharm. Belq. S 59-75 (1953) :

- 12,65-8(1963);C.A. 61 10534b (1964).

C.A. 47 9559f (1953). 100. Slouf,A. : ??eskoslov. farm.2 168-71 (1953) :

C.A. 49 2032e (1955). 101. Robles G. G. and Unzueta,L.: Actas y tabaios

conqr.peruano quim., 403-6(1953) :C.A.a 4653a (1957).

C.A. 66 111301f (1967).

12934v (1968).

.-

102. Yalcindag 0. N. : Kim. Sanayi &47-55 (1966) :

103. BrandF, J. ;Farm. Pol. - 23 430-4 (1967) :C.A. - 69

104. Garcia,M.T. :Castello,J.M. and Colome, J. : -. 5,311-15 (1973) ;C.A. 82 160277s (1975).

Arzneimittel-Forsch. - 3,45-7 (1953) :C.A. 5 5479d(1953).

105. Gemeinhardt, K. and Rangnick, G. F. :

106. Battistessa,M. J. and Pross, H. :Rev. farm 95,

107. Kidani, I. : NakashSa,T. ;Kochi,Y and Kasahara, 113-16(1953) ;C.A.49 92275. (1955).

S. : Buil.Nat1. Hyg.Lab. - 72, 95-7(1954) : C.A. 49 6 0 3 5 ~ (1955). -

ISONlAZlD 237

108.

109.

110.

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

Machek,G. ~Scientia Pharm. 24, 11-17 (1956) : C.A. 9690h (1956). Inandar, M.C. : Padlia, S. and Shah, V.N. ; Indian J. Pharm. 32,62-3 (1970) :C.A.B 38599x (1970). Spinkova, V. ; Pharm.Acta Helv 46,643-8 (1971) ; C.A. 76 37469e( 1972). Deodliar, R , D . ; Shastri, M.R. and Ganatra, J. P. : Indian. J.Pharm. 32,99-101 (1970) : C.A. 3 91302~ (1970). Hirose, S. and Uno, T.; Yakuqaku Zasshi 8 l ,

Deeb,E. N. :Drua Standards 22, 194-9 (1954) ; C.A. 49 2027d (1955). Pratt, E.L. : Anal .Chem.g ,814-16(1953) :C.A.47 - 9391f(1953). Nielsch, W. and Gief er,L. ;Mikrochim,Acta, 17-26 (1960) ;C.A.E 1097b 1973). Akatsuka,M.:Yakuqaku Zasshi 83,227-33(1963): C . A . 59 7475f (1963). Ahou-Ouf ,A.A. ; Taha, A.M. and Saidhom,M.B. :

1623-5 (1961) ;C.A. 56 10286h(1962)

J. Pharrr.Sci. - 62 1700-2 (1973) :C.A.E 7009v (1974). Elliston, S.C. and Hammond, M.D. : Analyst 90,

Pavlyuchenkova, L. P.and Veksler, M.A. : Farmatsiya (Moscow) 23,29-33 (1974) : C.A. - 81 54487e (1974). Ozawa, H. and Kiyomoto,A. : J. Pharm. SOC. Japan - 72,1059-60 (1952) : C.A.46 11047a (1952). Akiyama,T. :Yabuuchi,T. and Shiono,K. : Kyoto Yakka Daigaku Gakuho 1, 48-56(1959); C.A. - 54 6031e(1960). Bose,B.C. and Vijayvargiya,R.:Indian J.Pharm.

Colarusso,R. J. :Schmall,M. ; Wollish,E. G. and Shafer, E.G. E. :Anal. Chem. 30,62-5 (1958) : C.A. 52 4931b(1958).

298-300(1965); C.A. 63 5454~(1965).

- 28,328-30 (1966) :C.A. - 66 108307~~ (1967).

Nielsch,W. and Giefer, L.: Z. anal.Chem. 171, 401-lO(1960);C.A. - 54 8428f(1960). Fried, R. ;Mitt.Deut. Pharm.Ges. 32,157-9 (1962) : C.A. 58 2323b(1963).

238 GLENN A. BREWER

126.

127.

128.

12 9.

130.

131.

132.

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

143.

Mesnard, P. ;Devaux,G. and Fauquet, J. : Sci.Pharm.Proc 25th 2,77-86 (1965) :C.A.e 1098701 (1968). Aliev,A. M. and Beisenbekov,A. S. : Azerb.Med. a. 48,85-8 (1971) :C.A.n 39337e (1972). Aliev,A. M. and Beisenbekov, A. S. : Farmatsiya (Moscow) 22,35-8 (1973) :C.A. 2 1291202 (1973). Mesnard, P. ; Arch. Pharm. 22,105-21 (1966) ; C.A. 66 11887711 (1967). Jacobs, M.B. : J.Am. Pharm.Assoc. 42 346-8 (1953) ;C.A. 47 956%~ (1953). Blanc, P. ;Bertrand, Po and Liandier,L. : Lyon pharm,Spec.m-4 (1956) :C.A.= 22185c (1959). Naito,T. : Shirai, H. and Oda N. :Bull. Naqoya City Univ. Pharm.Schoo1, 34-7 (1955) : C.A. 50 17331f (1956). Ballard, C.W. and Scott, P.G.W. ;Chemistry and Industry, u(l952) : C.A. 47 823b(1953). Schoog, M.; Mhch. med.Wochschr.94,2135-7 (1952) ;C.A. 47 2645i (1953). S i l , J. ;Agrawal, K. C. and Dutta, B.N. :=. Cult. 28 472-4 (1962) ;C.A. 61 6868b (1964). Alessandro, A. :G_iorn. med. militare 104,195-7 (1954) :C.A. 48 13790d (1954). Ra0,P.V. : Ra0,G.B.B. and Murti, P.S. : Microchim Acta , 979-84 (1974) : C.A. 82 64615e (1975). Avanza, H. L. : Jornada Med. 53, 673-4 (1955) : C.A. 50 7003f (1956). Zommer, S. and Lipiec, T. : Acta Polon.Pharm.

Munson, J.W. and Connors, K.A. ;J. Pharm.Sci. - 61 211-13 (1972) : C.A.76 103829a(1972). Danek, A.: Dissertationes Pharm.u,237-42 (1959) :C.A. 54 13555h(1960). Teodorescu,Gr. and Dinescu, A.:Bul.Inst. Politeh. Bucuresti 2349-52 (1964) :C.A.G 14045e (1965). Roth, H. J. and Schrimpf; Arch. Pharm. 293,

- 20,229-32 (1963) ;C.A. 62 1517g (1965).

22-8 (1960) ;C.A. - 54 16286d (1960).

ISON I AZ ID 239

144.

145.

146.

147.

148.

149.

150.

151.

152.

153.

154.

155. 156.

157.

158.

159.

160.

161.

Tsuji, A. and Hojo, M.:Bunseki Kaqaku 11,

Vasileva-Aleksandrova, P. ; Aleksandrov, A. and Kovacheva, E.: Nauch.Tr. Vissh.Pedagoq. Inst. Plovdiv, Mat. , Fiz. ,Khim. ,Biol. 6,

Stewart, J. T. and Settle, D.A.; J.Pharm.Sci.

Akiyama, T. and Fujimoto, K. ; Kyoto Yakka Daiqaku Kakuho &32-6 (1966) ; C.A. - 68 34582w (1968). Deltombe, J. and Stainier, R. : J. Pharm. Belq. - 17,239-40 (1962) : C.A. 61 5461a(1964). Chekryshkina, L.A.: Farmatsiya(Moscow) 23 44-6 (1974); C.A. 81 126821j (1974). Tan, H. S. I. : J. Pharm.Sci. 62 993-7 (1973) : C.A. 79 45897v (1973). Kydymor, G. I. and Sidorova, E. F. : Nauch.Tr., Perm. Farm. Inst. 4 64-7 (1971) : C.A. 79 149340 Z( 1973). Teodorescu N. and Bebesel, E. ;Farmacia 14

Neuss, J.D. : Seagers, W.J., and Mader W. J. : J.Am. Pharm.Assoc. 41, 670-73 (1952) : C.A. 47 1545b (1953). Carol, J. : J.Assoc.Offic Agr.Chemists 33, 722-5(1953): C.A. 48 10300i (1954). Goldman, D.S.; Science 120, 315-16(1954). Pinyazhko, I.R.M. : Farmatsevt.Zh. 20, 17-21 (1965) : C.A. 64 7969h (1966). Kracmar, J.; Kracmarova, J. and Zyka, J.; Cesk. Farm. l7, 68-69 (1968) :C.A. 69 5266a (1968). Ghe A.M. and Peretti, A.; Ric.Sci. 38,937-41 (1968) : C.A. - 71 395111 (1969). Tieas, D. : Wiss. Beitr. ,Martin-Luther-Univ. Halle-Wittenberg 12, 152-4 (1968) :C.A. - 74 74412k (1971). Welsh, L.H. : J.Assoc. 0ffic.Agr. Chemists 40, 807-14 (1957) : C.A. 51 170973. (1957). Welsh, L.H. : ibid 41, 496-8 (1958) : C.A. 52 20894i (1958).

1255-62 (1962) ;C.A. - 58 8852h (1963).

111-16(1968) : C.A. 71 74096~(1969).

64,1403-5 (1975) : C.A. 83 168545~ (1975).

743-750 (1966) ; C.A. 67 102843~ (1967).

240 GLENN A. BREWER

162.

163.

164.

165.

166.

167.

168.

169.

170.

171 .

172.

173.

174. 175.

176.

177.

178.

179.

180.

Du t t , M.C., and Chua, T .H . ; J. Pharm. Pharmacol. - 16,696-9 (1964);C.A. - 6 1 15936a (1964) . S a t t l e r , H. ; Pharm.Ztq. 111,1395-1405 (1966) ; C.A. 66 40761t (1967) . S a r k a r , B. N . ; I n d i a n J. Pharm. 34, 58-61 (1972) ; C.A. 77 1 3 0 6 5 8 ~ (1972) . S t e i n k e , G. and Schmidt , F. ;Krankenhaus-Apoth.

Momose, T . ; Ueda, Y . ; Mukai,Y. and Watanabe, K . ; Yakuqaku Z a s s h i 8 0 , 225-8 (1960) ; C.A. 54 11861d(1960) . Uno, T. and Taniguchi , H . ; J apan A n a l y s t 20, 997-1002(1971); A . A . - 25 447(1973) . B a r t o s , J . ; Annls . pharm. f r . 29, 71-74(1971); A . A . - 2 1 3502 (1971) . Anon.: O f f i c i a l Methods of A n a l y s i s of t h e A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r a l Chemists 9 t h Ed. page 526 32.289 (1960) . KGhni, E . ; J acob , M. and G r o s s g l a u s e r , H . ; Pharm. Acta Helv. 29,233-50 (1954) ;C.A. - 49 3469b (1955) . Berka, A. and Z$ka,J . ;Pharmazie l3 ,81-92 (1958) ; C.A. 53 1634f (1959) . Duda, H. and c s t e , U. ; Deut.Apoth.Ztq. - 110,593-8 (1970) ; C.A. 73 91265m (1970) . Blake, M . I . ; Bode, D. and Rhodes, H . J . ; J. Pharm. S c i . 63,1303-6 (1974) ;C.A. - 8 2 35088e (1975) . A n o n . ; B r i t i s h Pharmacopoeia page 256 (1973) . Haugas, E.A. and Mitchell , B.W. ;J. Pharm. and Pharmacol. 4 687-92 (1952) : C.A. - 47 lOlOa (1953) . Wojahn, H. iArzne imi t te1-Forsch . 2, 324-6 (1952) ;C.A. 46 10052a (1952) . Coppini,D. ;Cameroni,R. and Monzani,A. ; R i c e r c a sc i . 22,1783-4(1952) ; C . A . z 5310b (1953) . Rosentha ler ,L . ; Pharm.Acta. Helv. 30,69-72 (1955) ; C.A. 49 10123a (1955) . Kochi, Z . ; J. Pharm. SOC. Japan 75,748-50 (1955) ; C.A. 49 135969 (1955) . Horn, D. ;Pharmazie - 8, 646-7 (1953) ; C.A. - 5 1 464933 (1957) .

- 24,20-2(1974);C.A. 81 96483f (1974) .

IS0 N I A2 I D 24 1

181.

182.

183.

184.

185.

186.

187.

188.

189.

190.

191.

192.

193.

194.

195.

196.

197.

198.

199.

200.

Modrzejewski, B. and Zommer, S. :Chem.Anal.

Miszczuk, B. and Taborska, H.:Przemyst Chem. 11,706-10 (1955) : C.A. 52 208953. (1958). Zn$ik,F. : Einkova, 07and mrbl, J. : Collection Czechoslov. Chem. Commns. 24,

Laszlovszky, J. :Acta Pharm.Hung. 30,101-9 (1960) : C.A. - 54 19268~ (1960). Vulterin, J. : Collection Czech. Chem. Commun. 28, 1393-1400 (1963) : C.A. 59 10767h Vulterin, J.:Cesk.Farm. l2,391-3(1963): C.A. 62 15994c (1965). Wihchurch, R. : J. Assoc. Off. Anal. Chem. 56,

Canb'ick, T. : J. Pharm. Pharmacol. 4 407 (1952) : C.A. - 46 83253. (1952). Domleo, A.P. : J.Pharm.and Pharmacol. 2,

Struszygski, M. and Bellen, Z. Przemyst. Chem.

Montequi, F. : Farm.nueva - 18,lO-14(1953) : C.A. 47 6603d(1953). Noronha da Costa, A. : Rev.brasi.1. farm.33,

Kattionis,A.: Chim.Chronika 2,78-80(1952): C.A. 47 11665i (1953). Berka, A. and Zijka, J. ;Chem.Listy 50,314-16 (1956) ;C.A. 50 7651a (1956). Spacu, P. and Teodorescu, G. :Bul. inst.

- 7,659-66 (1962) : C. A. 57 13889e (1957) .

2695-8 (1959) ; C.A. 54 8429g (1960).

(1963).

1464-6(1973):C.A. 80 87544d(1974).

117-18 (1953) ;C.A. 47 4251d(1953).

- 32,40-l(1953) ;C.A.G 85869. (1953).

343-50(1952); C.A. 47 7736d(1953).

politeh Bucuresti l8,47-50(1956):C.A.51 - 176071.1 (1957). Tsupikov, M. T. : Aptechn. Delo. u,55-7 (1964) : C.A. 61 - 9361c (1964). Laipanov, A. Kh. and Lobanov,V. I. : Farm. Zh,

Nambisan, P.N.K. and Nair, C.G.R. Indian J. Chem. 10,665-6 (1972) : C.A. 78 37750f (1973). Laipanov,A. Kh. and Lobanov,V. I. : Farmatsiya 22.

Greenhow,E. J. :Chem. Ind. 14,697-8 (1973) :C.A.79 121642a (1973).

2344-8 (1973) ;C.A. 2.B 140435f (1973).

48-9(1972) ;C.A.m 23619d(1973).

242 GLENN A. BREWER

201.

202.

203.

204.

205.

206.

207.

208.

209.

210.

211.

212.

213.

214.

215.

216.

217.

218.

219.

220.

Alicino, J. F. : J.Am. Pharm.Assoc.~,401-2 (1972) : C.A. 46 97863. (1946). Anastasi,A.; Mecarelli,E. and Novacic,L.; Mikrochemie Ver, Mikrochim Acta 40,53-9 (1952);C.A.g 8317h(1953). Pilar de 10s Rios de Bascones; Rev.sanidad y asistencia social. &221-30(1956) ;C.A. 2 4652e (1957). Kahane,E. and Sackur,O. ;Ann. pharm. frans. 11

Kashima , T. : Bu 11, Na t 1. Hyq. Lab. 2 145- 50 (1954) :C.A.% 7186h(1955). Sell&, E. and Flores, E.S.;Galenica Acta f3, 291-349 (1955) ;C.A.z 2234i (1957). Mizukami,S. and Hirai,E.;Yakugaku Zasshi 79,

Keller, W. and Weiss, F.;Pharmazie 12,462-71 (1957) :C.A.% 3260b(1958). KO. I.S. : Kim,J.B. and Choi,B.K. Repts.Nat1. Chem.Lab. 2,50(1959) ;C.A.% 11385a (1960). Tao,T. and Yu,H.Y.;Yao Hsueh Hsueh Pao g,

Solomon-Ionescu,I.;Popescu,D. and Enache,St.; Rev.Chim.l4,532 (1963) ,C.A.g 3950c(1964). Vajgand,V. and Pastor, T.;Glasnik,Hem.Drustva, Beoqrad 28,1-7(1963);C.A.= 15118g(1964). Boichinov, K. and Karatodorov, K. Farmatsiya l4,

Nicke1,P. Pharm.Ztq. 113,1609-12 (1968) ;C.A. - 70 31693~ (1969). Devani,M. B. and Shishoo, C. J. : J. Pharm. Sci.

Shishov, C. J. and Devani,M. B. : J. Pharm. Sci.59,

Sel1,E. : Farm.Po1.~,761-6(1970) ;C.A. - 74 130450k( 1971). Va jgand,V. J. : Pastor,T. J. and Bjelica,L. J. : Glasn. hem. Drust. ,Beogr.=, 345-352 (1970) : A.A. 21 4158(1971). Budgx%sk$,B. : Pharmazie 10,567-9 (1955) ;C.A. - 50 5985f (1956) . Korbl, J. ;Collection Czech. Chem. Communs. 25

175-82(1953);C.A.g 7375d(1953).

454-7(1959) ;C.A. 53 16471b(1959).

206-16 (1960) : C.A.56 7427f (1962).

12-17 (1964) ;C.A.G 6868~ (1964).

- 59 90-2 (1970).

92-3 (1970) yC.A.72 47410k(1970).

76-84 (1960) : C.A. - 54 13988d (1960).

ISON IAZlD 243

221.

222.

223.

224.

225.

226.

227.

228.

229.

230.

231.

232.

233.

234.

235.

236.

237.

238.

239.

240.

Adamova/,E. and Zqka, J. ;Ceskoslov. farm. 4, 9- lO(1955) :C.A. - 54 3858a (1960). Grecu, I. and Curea, E. : Farmacia €3,503-6 ( 1960) : C.A.55 19136d(1961). Tatsuzawa,M.:Bunseki Kagaku - 11,1055-9(1962); C.A. 57 16748f (1962). Ciogolea,G. :Mora it,G. :Teodorescu,N. and Petroniu, L. : Farmacia 2 401-8 (1964) : C. A. - 61 13131e (1964). van Pinxteren, J.A. C. and Verloop,M. E. : Pharm. Weekblad 99,1125-33 (1964) ;c.A. 63 1659f (1965). Blagojevic, Z. and RadosavljevicJ. ;ACta Pharm.

Blagojevic,Z. and Radosavl jevz,J.#lCta Pharm. Jugoslav. 17,131-6 (1969) :C.A. 70 60882u (1969). Blagojevic,Z. and Radosavljevic, J.; Gyoqyszereszet U, 121-4 (1969) :C.A. TL 33486h (1969).

175-82 (1970) : C.A. 73 10502m (1970). Barrosa,M. T. ;Rev. Port. Farm. 19,206-9 (1969) : C.A. 74 15757d(1971).

JUqOSlav. l6,27-30(1963) ;C.A.65 571b(1966).

Pszonicka,M. and Skwara,W. ;Chem.Anal fi,

es 2 Kasse6A.A. and El-Marakby,M.M. :J.Drucr R - 79-87 (1969) ;C.A.a 67753~ (1971) Soliman,R. and Bela1,S.A. ;Pharmazie 29,204 (1974):C.A.g 111504j(1974). Armestar,A.M. ;conar.farm.bioauim.P eruano v convenci6n farm. Norte,Actas y traba jos 3 1 5 - 1 6 ( 1 9 5 3 ) ; C . A . 4 ~ 7 1 9 3 h ( 1 9 5 5 ) . Lea1,A. M. and Alves, M.A. :Rev. port. farm.2,

Merz,K. W. and Schirm,M. ;Naturwissenschaften

Ra0,R.S. ;Rao,G.B.B. and Rao, P.V.K. : Chem.Ana1. 19,927-30 (1974) :C.A.= 51131~ (1975). Businelli,M. and Rocchi,B. :Farm. sci. 1 tec. - 7,153-60(1952);C.A.* 9019i(1952). Deys,H. P. :Pharm.Tijdschi.Belqie 36,117-20 (1959);C.A.!3J 7067g(1960). Spacu, P. and Teodorescu,Gr. ;Rev. chim.8, 42-3 (1957) : C.A.2 1552af1958) Danek,A. ;Acta Polon. Pharm. 18,229-34 (1961) : C.A. 56 8842d(1962).

175-9(1952);C.A.= 5635f(1953).

- 39,570-1 (1952) ;C.A. 48 1629d(1954)

244 GLENN A. BREWER

241.

242.

243.

244.

245.

246.

247.

248.

249.

250.

251.

252.

253.

254.

255.

256.

257.

258.

259.

Franchi,G. ;Farm. sci. e tec.Z,640-4 (1952) : C.A.47 4044b (1953). Akiyama,T. :Fujiwara,M. and Ichida H. : Bull. Kyoto Coll. Pharm. fl, 18-20 (1956) : C.A. 51 4650a (1957). Sbnchez, J. A. ; Rev.Asoc.bioquim.Argentina

Mitchel1,B. W. ; Haugas, E.A. and McRoe, C. S. ; J. Pharm.and Pharmaco1.?,42-5 (1957) :C.A. - 5 1 608713 (1957). Kum-Tatt, L. and Yan-Hon,H. : J. Pharm. Pharmacol.

Spacu, P. ;Teodorescu,Gr. and Gavanescu,D. :

- 17,324-6(1952) ;C.A.g 6822b(1953).

14,123-4(1962) ;C.A.X 2337b(1962). J Y

-

Bul. inst. politch. Bucuresti - 18,51-4 (1956) : C.A.51 176073. (1957). Ibadov,A. Yu. and Drebentsova,N. F. ;Dokl. Akad.Nauk Uz.SSR 21 39-41(1964) :C.A. - 61 10538a (1964). Nair,V. R. and Nair, C.G. R. ;Analytica chim. Acta 57,429-34(1971);A.A.23 799(1972). Pinzauti,S. ;Dal Piaz,v. and LaPorta, E. : J. Pharm. Sci. 63,1446-8 (1974) : C. A. 82 7701f (1975) . Gowda, H. S. and Rao, G. G. ;Z.Ana 1. Chem. - 9 1 6 5

Budggi'nsk$,B. :Collection Czechoslov. Chem. Communs.26,781-7 (1961) :C.A.z 14174b(1961).

-

36-8 (1959) : C. A. 53 11765h (1959) .

Ra0,P.V.K. and Rao, G.B.B. ;Analyst 96, 712-15 (1971) ;C.A.E 6762r (1972). Rao, P. V. K. and Rao,G. B. B. ;Freseniug Z.Ana1. - Chem. =,360-1(1971) :C.A.E 37475d(1974). Eremina,Z. I. and Antonchik, I.A. :Farm.Zh. (Kiev)

Rao, P, V. K. :Rao,G.B.B. and Ra0,R.S. :Anal. Chim. Acta 65,227-30 (1973) :C.A.E 57733b(1973). Tatsuzawa,M. :Bunseki Kagaku 10,129-33 (1961) : C.A.55 23931e(1961). Panwar,K. S. :Rao,S. P. and Gaur, J. N. ;Anal. Chim. Acta 25,218-21 (1961) :C.A. 55 26865d (1961). Ra0,G.G. and Rao, P.V.K. ;Tzanta ll, 1489-96 (1964) :C.A.62 1239h(1965) Gein,L.G. and Sumbaikina,Z.A. ;Farmatsiya l6, 41-3 (1967) : C.A. 67 84919a (1967).

- 27,79-81(1972) :C.A.z 92916~(1972).

ISON I AZlD 245

260.

261.

262.

263.

264.

265.

266.

267.

268.

269.

270.

271.

272.

273.

274.

275.

276.

277.

278.

Pinzauti,S. ;Dal Piaz V. and La Porta, E. q Farmaco Ed. Fra t . 29,136-42 (1974) :C.A. 81 29576a (1974) .' Rao P . V. K. and Rao , G. B . B . : Res . Ind. =,14 3-4

- -

(1972) : C.A. 80 19640g (1974). Volodina,M.A. and Karandi, I. V. :Vestn. Mosk. Univ. ,aim l3,357-8(1972) :C.A.E 147343n(1972). Vulterin,J. and Z?ka,J.:Chem.Listy - 48 839-42 (1954) :C.A.% 13521a(1954). Sakurai,H. ;Kimura,T. and Senoo,S. : Yakuzaigaku 16 39-41(1956);C.A.a 9089a(1957). Akiyama,T. ;Fujiwara,M. and Ichida,H. : Kyoto Yakka Daiqaku Gakuh6 5,42-4(1957);C.A.= 3262i (1958). Tachi,I. and Nagata,V. :Kaqaku no Ry6iki 6,

Anastasi,A.;Mecarelli,E. and Novacic,L.: Mikrochemie ver Mikrochim.Acta 40,113-20 (1952) :C.A.G 3185a(1953). Maruyama,M. : J. Pharm. SOC. ,Japan 72,1213-4 (1952) : C.A. 47 417g (1953). Varela,G. :Anales. real SOC. espaz. fls.y.qulm

Liberti,A. ;Cervone,E. and Cattaneo,C. : Giorn.biochim. 1,440-6 (1952) :C.A.49 - 98883. (1955). Tsa0,King-Hung:Loo,Yun-Chieng and Tang, Teng-Han. ;Yao Hsu'eh Hsueh Pao 4,121-6(1956) : C.A.52 6716a (1958). Maruyama,M. : Ann. Rept. Takamine Lab.g,l22-7 (1956) : C.A. 54 19310a (1960). Sato,H. :Eisei Shikenjo Hskoku 77, 39-44 (1959) : C.A. 55 14281i (1961). Lund,H. :ActaChprn. S u . U , 972-8 and 1077-86 (1963) ;C.A.S 13598g (1963). Vajgand,V. and Pastor,T. :Glasnik Hem.Drustva, Beoqrad 27,263-70 (1962):C.A.s 13326c(1963). Va jgand,V. and Pastor T. : J. Electroanal. Chem.

Lund,H. :Abhandl.Deut.Akad.Wiss. Berlin K1. Chem. ,Geol.Biol. 434-42 (1964) ;C.A. - 62 8674d (1965). Brandys, J. ; D i s s . Pharm. Pharmacol. 18,319 (1966) : C.A. 66 22276j (1967).

490-l(1952) ; C.A.46 11045f (1952).

f f

- 48B 713-15 (1952) : C.A. 47 8 3 2 3 ~ (1953).

S 40-8 (1964) 7C.A. 62 4619f (1965).

246 GLENN A. BREWER

279.

280.

281.

282.

283.

284.

285.

286.

287 .

288.

289.

290.

291.

292.

293.

294.

295.

S c h l i t t , L . :Rink,M. and V. S t a c k e l b e r g , M. : J. E l e c t r o a n a l . Chem. 13, 10-20 (1967) , C.A. 66 5798p(1967) . Turczan, J. W. : J . A s s . O f f ic . Anal. Chem. 50,

Kitaev,Yu. P. and BudnikoV,G.X. ; 1zv.Akad. Nauk SSSR, Ser.Khim. 554-61 (1967) C . A . 6 7 90282d (1967) . S a t o , H. : E i s e i S h i k e n j o Idkoku 7 8 , 2 1 - 4 (1960) :C.A. 55 24395a (1961) .

Va l lon , J . J . ;Bad inand ,A . and Bichon,C.: Anal. Chim.Acta =,93-8 (1975) : C . A . 8 3 1 5 2 4 3 5 ~ (1975) . Okuda,Y. :Ohara,M. :Horino,S. ;Masak,H.; Furukawa, Y. and Yamasaki, M. ; Rev. Polaroq . - 9,112-15 (1961) ; C . A . g 7307e (1964) . Kalinowski,K. :Acta Polon. Pha rm. l l , 113-16 (1954) : C . A . = 14123b(1954) . Ka l i nowsk i , K. and Zwierzchowski, Z . ; Acta Polon.Pharm. 20,309-13(1963) ; C . A . G 1517h (1965) . S to i cescu ,V . ; I v a n , C. and Bera1,H. ;Rev. Chim. - 19,484(1968) : C . A . z 6568h(1969) . Brandys,J.;Diss.Pharm.Pharmacol.z,87-92 (1969) ; C.A. 72 3 8 2 5 7 ~ (1970) . Curran, D. J T a n d Cur l ey , J . E . ;Analy t . Chem.

Jenn ings , V . J . ; Dodson, A. and Harr i son ,A. : Ana lys t - 99 145-8 (1974) ; C . A . G 57876s (1974) . Akiyama, T. :Fuj i ta ,M. ; Fujiwara,M. and Kawashima, K. ;Bunseki Kagaku 2, 701-3 (1956) : C.A. 52 155f (1958) . Grecu , I . and C u r e a , E. rRev.Chim.2, 162-3 (1962) :C .A .x 11856 i (1962) . Grecu, I. : Curea, E. and P i t i s , M.: Acad.Rep. Populare Romine, F i l i a l a C l u j , S t u d i i C e r c e t a r i Chem. 13 213-23 (1962) : C.A. - 6 1 100279 (1964) . van P i n x t e r e n J. A . C . and Verloop, M.E. : Pharm. Weekblad 100, 189-95 (1965) : C . A . 6 3 5454a (1965) . Zivanov, D . ; B l a g o j e v i c , 2 . and Mladenovic,M. : Arhiv . Farm. 14,273-8 (1964) :

652-4 (1967) :C .A.e 6 7 6 5 8 ~ ( 1 9 6 7 ) .

- 42,373-7(1970) :A.A. 20 2497(1971) .

ISONIAZID 24 7

296.

297.

298.

299.

300.

301.

302.

303.

304.

305.

306.

307.

308.

309.

310.

311.

312.

313.

C . A . 62 2671h(1965) , Tabenkin,B. : Dolan,B. and Johnson , M.G. : Proc. SOC. E x p t l . B i o l . Med. 80,613-15 (1952) : C . A . 46 10254b(1952) . C e r o t t i , G . : A t t i SOC lombarda s c i med biol. - 7 , 7 6 ( 1 9 5 2 ) : C . A . Q 2248d(1953) . I f r im ,A. and Con ive r , L. : L u c r % r i l e p r e z e n t a t e conf . n a t l . farm. , B u c h a r e s t

Suzuki,Y. ; P h y s i o l . P l a n t a r u m l 9 , 2 5 7 - 6 3 ( 1 9 6 6 ) : C . A . 6 4 20211c(1966) . Va jgand,V. J. and P a s t o r , T. J. : G l a s . H e m . Drus . , Beoqrad. 3 l , 9-17 (1966) : C.A. 67 39955y( 1967) .

674-8(1958) ~ c . A . 5 3 9354h(1959) .

Dusinsky,G. and F a i t h , L . :Pharmazie 22, 475-82 (1967) : C.A. 68 53280b (1968) . McKennis J . H . and Yard, A.S.: U . S . Dept.Com., O f f i c e Tech. Se rv . PB Rept . 143 ,914 (1957) ; C.A. 55 17375i (1961) . H a r t i n g , H. J . A m . Pharm.Assoc. 42,323-4( 1953) ; C.A. 47 7163f (1953) . Kazuo,T. :Yakuzaiguku 32,150-4 (1972) :C.A. 79 149305s (1973) .

- -

- -

Artamonov, B. P. and Kupina, N . A . :Med. Prom.

N i k o l i c , K. and Cupic,Z. :Ark. Farm. I B e l q r a d e )

Kidani ,Y. : Inagak i ,K . :Saotome,T. and Koike,H. : Buneski Kayaku 22 896-9(1973):C.A.79 1 3 9 6 7 2 ~ (1973) .

B e l i l o v s k i i , Ya . E. ; Farmats iya 2 1 ,62 -4 (1 972) : C.A. 77 130636n ( 1 9 7 2 ) .

SSSR 19,61-3 (1965) ;C.A.64 4871f (1966) .

- 17,201-4(1967) ; C . A . e 5 4 2 9 8 ~ ( 1 9 6 8 ) .

- --

- -

- -

Mattu,F. ;Rend. s emina r . f a c . s c i . u n i v . C a g l i a r i

Roth, L. J. and Manthei,R.W. ; Proc. SOC. Exp. Biol.Med. 8 1 , 5 6 6 ( 1 9 6 2 ) : C.A.48 9438f ( 1 9 5 4 ) .

- 22,92-106(1952);C.A.48 - 4 1 7 9 f ( 1 9 5 4 ) .

- - C e r i o t t i , G . :Def rancesch i ,A . ;DeCarne r i , I. and Zamboni,V. ; B r i t . J. Pha rmaco l .8 356 (1953) : C . A . 48 885g(1954) . Defra&eschi ,A. and Zamboni, V. : B i o c h i m . Biophys.Acta 1 3 304 (1954) ; C . A . B 90822.1 (1955) . Iwainsky,H.:Arzneimitt,Forsch.7,745(1957): - C.A. 52 5665c(1958) .

248 GLENN A. BREWER

314.

315.

316.

317.

318.

319.

320.

321.

322.

323.

324.

325.

326.

327.

328.

329.

330.

331.

332.

Savoia,F. ; E o l l . soc. ital.bio1. sper. ,30,29-30 (1954) ;C.A.E 9438f (1954). Leuschner,F. :Naunyn-Schmiedebergs Arch.expt1. Pathol. Pharmakol. 221,323-7 (1954) ;C.A.48 - 88539 (1954). Itai,T.:Oba,T. and Kamiya,S. :Bull.Natl.Hyg. - - Lab. 72,87-90(1954) ;C.A.e 6350h(1955). Unverricht,W. ;Schattmann,K. and Senft,G. ; Arztl.Wochschr.2 838(1954) ;C.A.9 1280h(1955). Ishikawa,M. and Kikkawa, I. ;Ann. Repts. Shionoqi Research Lab. 4, 39-42 (1954) : C. A. 50 156299 (1956). Fischer, P. and Burgen, A. ; Pharm.Acta. Helv.

Abe,M. ;Sci. Rept. Res. Inst. Tohoku. Univ. ,Ser. C. 8,1(1958):C.A.= 17529i(1958). Albert,A. and Rees,C. W. ; Biochem. J. 6 l , 128 (1955) :C.A.e 16197c(1955). Boone, I. U. ;Magee,M. and Turney,D. F. :J. Biol. -- Chem.221,781(1956) iC.A.50 15784h(1956). Boone, I . U . ;Strang,V.G. and Rogers,B. S. ; Am, Rev.Tuberc.76 568 (1957) iC.A.53 3361c (1959). Diller,W. ;KrUger-Thiemer,E. and Wempe,E. : Arzneimittel-Forsch.2 423-9(1959):C.A.= 21363b (1959). Barreto, R. C. R. : J. Chromatoq.Q,344 (1963) : C.A. 57 6247a(1962). Greulach,V.A. and Haesloop, J. G. :Anal. Chem.

Reio, L; J. Chromatoq. 47,60-8 5 (1970) : C. A. 73 21216~ (1970). LaRue,T.A. :J. Chromatogr.=,784-5 (1968) : C.A. 68 74892t(1968) Barreto, R. C. R. ; J. Chromatoq.g,416-19 (1961) : C.A. 56 12295f (1962). Gonnard,P. ;Camier,M. and Bcigne,N. : B o l l . Soc.Chem.Biol.46 407-ll(1964) ;C.A. - 61 6867d (1963). Pallini,V. :Vasconetto,C. and Ricci, C. : E o l l . Soc.Ital.Biol.Sper. 41 673-5 (1965) :C.A.64 - 2401f (1966). Schmid, E. : Hoppe, E. : Meythaler , Chr. and Zicha,L. :Arzneimittel-Forsch. 13,969-72 (1963) i

..

* a

- 31,518-42(1956) ;C.A.s 6945b(1957).

- 33 1446-7 (1961) ;C.A.% 25577f (1961).

ISON I AZ ID 249

333.

334.

335.

336.

337.

338.

339.

340.

341.

342.

343.

344.

345.

346.

347,

348.

349.

350.

C.A.60 7351c (1962). Thomas, J. J. and Dryon, L. ;J. Pharm.Belq. -7 19 481-504 (1965) : C.A.63 1660e (1965). Guven, K. C. ;Eczacilik Bul. 9, 186-9 (1967) ; C.A. 69 5256x(1968). Spinkova, V. : Cesk. Farm. 16,138-42 (1967) ; C.A. 67 47125t (1967). Kaistha,K.K. and Jaffe,J.H.:J.Pharm.Sci. 6 l , 679-89 (1972) : C.A. 77 57236a (1972) . Garcia Carro,A. J. ; An-Real Acad-Farm. 2,

DeSagher, R.M. :De Leenheer, A. P. and Claeys, A. E. ; J. Chromatoqr. 106 357-62 (1975) :C.A. - 83 157v (1975). Nishimoto,Y. and Toyoshima, S.: Yakuqaku Zasshi 87,27-32 (1967) : C.A. - 66 98544r(1967). Wijnne,H. J.A. : Bletz,E. and Frijns, J. M. : Pharm. Weekbl. 102,959-70 (1967) :C.A. - 67 102844d (1967). Schmidt, F. ; Krankenhaus-Apoth. 23,lO-12 (1973) : C.A.79 458661 (1973). Kawale,G. B. : Joglekar,V. D. :Barve,V. P. and Mahal, H. S. :Sci. Cult.38,373-376 (1972) : A.A. - 25 3438(1973). Tsuji,A. and Sekiguchi, K.;Nippon Kagaku Zasshi 81,847-52 (1960) :C.A.S lOOlSd(1961). Heller,A. :Kasik, J.E. :Clark, L. and Roth,L. J. : Anal. Chem. 33,1755 (1961). Kakemi,K., Sezaki,H. and Inoue,S. ; Yakugaku Zasshi 85,674-9(1965);C.A.s 12974a(1965). Peters, J.H., Miller, K.S. and Brown, P.: Anal. Biochem 12,379 (1965) : C.A. 63 8896a (1965). Peters, J.H., Miller, K . S . and Brown, P.: J. Pharm. Exp. Ther. 150 298 (1965) : C.A. 63 8896a (1965). Fan, M.C. and Wald, W.G. :J.Assoc.Offic.Agr. Chemists 48,1148-50 (1965) : C.A. 64 6407d (1966). Inoue, S.;Ogino,A. and Ono,Y.:Yakuraigaku

Gwandowski,A. and Sybirska,H. :Chem.Anal.u,

211-36(1973) ;C.A.m 74377b (1974).

26,302-7(1966) ;C.A.e 99472g(1968),

319-24(1968) :C.A.E 73784t (1968).

250

351.

352.

353.

354.

355.

356.

357.

358.

359.

360.

361.

362.

363.

364.

365.

366.

367.

368.

369.

370.

GLENN A. BREWER

Darawy, Z. I. and Mobarak , Z. M. : Pharmazie 28,

Barreto,R. C. R. and Sabino,S. 0. : J. Chromatoq. - 13,435(1964) :C.A.S 13542e(1964). Smolarek, W. and Dlugosch, G. : Naturwissenschaften 4 l , 18 (1954) :C.A. 48 - 10996i (1954). Barret0,R.C.R. ;J. Chromatog.?, 180(1962) ;C.A. - 57 6247a (1962). Russell, D. W. : J. chromatoq. 19,199-201 (1965) : C.A. 63 1065211 (1965). CaloT. :Cardini, C. and Quercia,V. :Boll.Chim. -- Farm. 107,296-9 (1968) : C. A. 69 80244v (1968) . CAlo,A. : Cardini,C. and Quercia,V. ;J. Chroma-

Gelbicova-Ruzickova, J. :Novak, J. and Chundela,B. :Proc. Conf .Appl. Phys. Chem. 295-300 (1971) ; C.A. 76 90104h (1972). Terze, B. and Dadiotou ,M. T. : Chim. Chronika l7, 45-8(1952) :C.A.u 9862f (1953). Ginoulhiac, E. : Rass. med. =,86-9 (1952) : C.A. - 48 13791b (1954). zuschwabedissen, 0. M. ;Deut. med. Wochschr. 78,

Smolarek,W. and zah1,R. ;Deut.med.Wochschr.

Schattmann,K. :Deut. med. Wochschr. 79,758-9 (1954) :C.A.S 8851b(1954). zuSchwabedissen, 0. M. :Deut. med. Wochschr.2, 759-60(1954) ;C.A.s 8851~(1954). Kelly,J.M. and Poet,R.B. :Am. Rev.Tuberc.65, 484-5 (1952) : C.A. - 47 124785 (1953). Wojahn, H. and Wempe, E. :Arzneimittel-Forsch. - 3 191-2(1953);C.A.47 5983d(1953). Marenzi,A.D. and Gomez, C. J. ;Rev. asoc.mdd. arqentina 66,379-80(1952):C.A.C 7013b(1953). Wojahn,H. and Wempe,E. ;Arzneimittel-Forsch.&, 294-5(1954) :C.A.% 8856e(1954). Ma losetti, H. : Balea, E. and Addiego,A. :Ana les. fac.med.Montevideo - 40,13-17 (1955) :C.A.x 16924g (1956). Peukert,D. ;Arzneimittel Forsch.Z,304-6 (1957) : C.A. 51 1304733 (1957).

37-39 (1973) ;A.A. 25 1155 (1973).

toqr. 3 7 , 194-6 (1968) : C.A. 70 6564d (1969).

104-5 (1954) ;C,A.47 4406d(1953).

- 78,273-4(1953) :C.A.G 5978f (1953).

ISON IAZlD 251

371.

372.

373.

374.

375.

376.

377.

378.

379.

380.

381.

382.

383.

384.

385.

386.

387.

388.

389.

Waldron-Edward, D. M. : Irish J. Med. Sci. 363, 130-6 (1956) : C.A. 54 13228e (1960). Litman, I. I. :Tr.Leninqrad. 1nst.Usoversh. Vrachoi 49 165 (1966) : C.A. 2 34297s (1968) . Deeb,E. N. and Vitagliano,G. R. : J.Am. Pharm.

Yantchitch,M. :Ann. biol. clin. 12,328-9 (1954) : C.A. 49 1126f (1955). LeifhXt,H. C. and Smith,E. R. B. ;Am. J. Clin. Pathol. - 31,142-7 (1959) :C.A.= 9349d(1959)'. Tansini,G. :Perna,G. and Trotta,E. :Atti. soc. lombarda sci. med. biol. 13,258-62 (1958) : C. A. 53 13251e (1959). Pedenko,E. P. and Kozlovskii,I. Z. ;Lab.Delo 249 (1974) : C.A. 81 114327g (1974) . Ruffo,G. :Arch.Vet. Ita1.&,245-55(1965) :C.A.e 11143b (1966). Jessamine,A.G. :Hamilton,E. J. and Eidus,L. : Can.Med.Assoc. J. 89,1214-17 (1963) :C.A. - 60 853633 (1964). Eidus,L. and Hamilton, E. J. :Clin. Chem. l0,

Assoc. 44,182-5 (1955) : C.A. 49 7633b (1955).

581-8 (1964) :C.A.s 123079 (1964). Eidus,L. and Harnanansingh,A. M.T. :Clin. Chem. - 17,492-4 (1971) ;C.A. - 75 74372t (1971). Rinaldi,M. R. and Cragnolin0,N.A. :Rev.asoc. bioquim. - arqentina 18,162-3 (1953) :C.A. - 47 12479f (1953). Aoki,S. :Terai, I. and Mori,K. : Iryo 6,33-5 (1952) : C.A.3 4941h (1953). Guillaume, J. :Tacquet,A. and Berthelot, J . Y . : Rev. franc. gtudes clin. et biol. 2,729-33 (1960) :C:A. - 55 7531i (1961). Fartushnyi,A. F. ; Sud. -Med. Ekspertiza ll, 26-31 (1968) :C.A. - 70 18570r (1969). Prescott,B. ;Kauffmann,G. and James,W.D. ; Proc.Soc.Exptl.Bio1.Med. - 84,704-6 (1963) : C.A. - 48 4620g (1954). Lever,M. ;Biochem. Med.5,65-71(1972) : C. A. - 76 123789r (1972). Hashmi,M.H. :Adil,A.S. :Malik,F.R.and Ajma1,A. I. Mikrochim.Acta 772-7 (1969) : C.A. 71 59223u (1969) Bartels, H. and Spring, P. :Chemotherapy -9 21

/

1-lO(1974) ;C.A. - 82 92852~(1975),

252 GLENN A. BREWER

390.

391.

392.

393.

394.

395.

396.

397.

398.

399.

400.

401.

402.

403.

404.

405.

406.

407.

408.

409.

G inou lh iac , E. ; R u s s , med. 29,86-9 ( 1952) : C . A . 4 6 9646f (1952) . Defranceschi ,A. and Zamboni,V. ;Giorn.biochim.

%lscher,F.M. ;Minerva p e d i a t . - 7,160-1 (1955) C.A. 49 14867f (1955) . Wol lZberg ,O. ; K l i n . W o c h s c h r . z , 906-7 (1952) ; C.A. 47 2087i (1953) . Kel lner ,H. and SEdhof , H . ;K l in . Wochschr.31,

Rober t s , R. W. andDeuschle,K.W. : Am. Rev. R e s p i r a t . D i seases 80,904-8 (1959) : C. A. - 55 10551d (1961) . Paunescu,E. ;Acad. Rep. P o l u l a r e R o m i n e , I n s t . B ioch im. ;S tud i i C e r c e t a r i Biochim. 4 117-22 ( 1 9 6 1 ) ; C . A . s 16081a(1962) . Paunescu,E. and Buzescu,M.;Rumanian Med.Rev.

Smirnov,G.A. and Kozul i t syna ,T . I. :Lab. Delo

Maier, N. - 128 ,213-17(1964) ;C .A.g 8773a(1964) . Nielsch,W. : Chemiker-Ztq. - 8 2 494-9 (1958) : C.A. 53 6913d(1959) . N i e l s c h , W. and Gief er , L. ;Arzne imi t t e l -For sch .

Rapi , G . ; Sper imenta l e , Sez . chim. bio 1.4,l l- 2 2 (1953) ; C . A . 4 7 10604a (1953) . Rubino,G. F. and B r a c c o , M . ;Minerva med.

Nardini,F.B. and P e r i t i , P. ; B o l l . SOC. i t a l . b i o l . s p e r . 29,356-7(1953) 7 C . A . G 12317h(1954) . Short ,E.A. :Lancet266,656-7 - (1954) ;C.A.49 - 13598d (1955) . Bre t ton i ,B . ;Arch. i t a l . sc i . farmacol .2 ,227-8 (1955) ; C . A . E 1971b(1956) . Sh i r a i ,Y . ;Ohishi,K. ;Suchi ro ,K. ;Motoike,H. and Adachi,R. ;Seikagaku 29,557-62 (1957) ; C . A . B 10551a (1961) . Shor t ,E . I . : T u b e r c l e - 42 218-26(1961) :C.A. - 62 llOOla (1965) . Dymond, L. C. and R u s s e l 1 , D . W. ; C l i n . Chim.Acta

1,405- 16 (1952) 7 C . A . e 9082h(1955) .

287-8(1953);C.A.47 7014g(1953) .

2,38-40 (1961) ; C.A. 57 10502g (1962) . 48-52 (1964) ; C . A . S 11039f (1964) .

and Moisescu,V. ; B e i t r . K l in . Tuberk.

- 9 636-41,700-07 (1959) ; C.A. - 54 5 8 0 3 ~ (1960) .

1136-7(1952) ; C . A . B 12212g(1954) .

- 27,513-20 (1970) ; C.A. 72 1 1 9 6 6 5 ~ (1970) .

ISON I A 2 ID 253

410.

411.

412.

413.

414.

415.

416.

417.

418.

419.

420.

421.

422.

423.

424.

425.

426.

427.

Russel1,D.W. ;Clin. Chirn.Acta 2,367-73 (1971) : C.A. 74 97428j (1971). Morvillo,V. and Garattini,S. ;Boll. SOC. it&. biol. sper.28,388-90(1952) :C.A.% 217c(1954). Porcellati,G. and Preziosi, P. ;Boll. SOC. ital. biol.sper.29,269-71(1953) ;C.A.48 1577d(1954). Ioffe,R.A. ;Lab.Delo 47(1967) ;CZ.& 84348t (1967). Jacobs,M.B. : Science -,142-3(1953) ;C.A.47 - 11302d (1953). Scardi,V. and Bonavita,V. ;Arch. ital. sci. farmacol. 1,206-11 (1957) ;C.A.a 16647d(1957). Scardi,V. and Bonavita,V. Clin. Chem.3,

Holecek,V. and Herlik, J. ;Rozhledy Tuberk.

Caste1,P. ;Carli,G. ;Gras,G. and Cambou,P. : Trav. soc. pharm.Montpellier l5,27-36 (1955) : C.A.49 14080e(1958). Bjornesj0,K.B. and Jarnulf ,B. : Scand. J. Clin. Lab. Invest. 20 39-4C (1967) ; C.A. 67 107109~ (1967). Brettoni,B. :Boll. SOC. ital. biol. sper. 28

728-31(1957);C.A.= 4738~(1958).

- 24,3-6(1964);C.A.60 16400h(1964).

939-42(1952);C.A.= 8809b(1953). Oba,Y. :Igaku Kenkyu - 31,124-42 (1961)C.A.z 27505i(1961). Ginoulhiac,E. and Tenconi,L. ;Giorn.mal. infettive e parassitar - 4 63-7(1952);C.A.e 9400a (19.52). Cuthbertson,W. F. J. : Ireland,D. M. ;Wolf f ,W. and Kuper,S.W.A. :Brit.Med. J. - I 609-11 (1954) ; C.A. - 48 13777d(1954). Hunter,G. ;Brit.Med. J. I 585 (1955) ;C.A.s 10407e (1955). Rubin,S.H. ;Drekter,L. ;Scheiner, J. and

-

DeRitter,E. ;Diseases of the Chest - 21 439-49 (1952):C.A.46 - 6686i(1952). Prescott,B.:Katz,S. and Kauffrnan,G.; J.Lab.Clin.Med. 44, 600-3 (1954) :C.A.s 1131c(1955). Kinoshita,Y. and Moriyama,S.;Bull.Naqoya City Univ.Pharm.Schoo1 1,37-9(1953):C.A.50 - 9489h (1956).

254 GLENN A. B R E W E R

428.

429.

430.

431.

432.

433.

434.

435.

436.

437.

438.

439.

440.

441.

442.

443.

Movrin, M. :Bull.Sci. Cons.Acad. Sci.Arts RSF Youqos1avie.Sect.A. 14,141-2 (1969) :C.A.B 690292 (1969). Hodgkin,M. M. ;Hsu ,A. H. E. :Varughese, P. and Eidus, L. : Int. J. Clin. Pharmacol. ,Ther. Toxicol.

Varughese,P. : Hamilton,E. J. and Eidus, L. : Clin. Chem. 20 639-41 (1974) : C. A. 81 99149n (1974). Wagner, J. :Kraus, P. and Ve$e?!ek,B. ;Rozhledy Tuberk 16,211-13 (1956) :C.A.z 9498a(1956). Maher, J. R. :Whitney, J. M. :Chambers, J. S. and Stanonis, D. J. :Am. Rev. Tuberc. Pulmonary Diseasg - 76 852-61(1957):C.A.= 3345a(1959). Goedde,H. W. : Schloot,W. and Benkmann,H. G. : Chemotherapia 3 6 1 - 7 (1967) :C.A.a 61487a (1967). Bracco,M. and Savio,E. : Ann.Med. Sondalo

Wareska,W.:Polski Tvqodnik Lekarski 8,1736 (1953):C.A. 48 14082c(1955). Hedrick,M. T. :Rippon, J. W. :Decker, L. E. and Bernsohn, J. : Anal.Biochem. 5 85-98 (1962) : C.A. 58 743e(1963). Scott,E.M. and Wright,R. C. ; J.Lab. Clin.Med.

Reiss,O.K. :Morse,W. C. and Putsch,R. W. : Amer. Rev. Resp.Dis.96 111-14 (1967) :C.A. - 67 80847c (1967). Wilson,D.M. :Lever,M. and Smal1,C.W. : Amer. J. Med. Technol.2 451-3 (1973) :c.A.g 34m(1974). Ellard,G.A. :Gammon, P.T. and Wallace,S.M. : Biochem. J. 126 449-58 (1972) :C.A. - 76 13548711 (1972). Boxenbaum, H. G. and Riegelman, S. : J. Pharm. Sci.

Miceli, J.N. : Olson, W.A. and Weber,W.W. :

- 7,355-62(1973) yC.A.79 112860t(1973).

- 9 201-5 (1961) ;C.A.!5& 14560f (1962).

- 70 355-60 (1967) : C.A.67 72217g (1967).

- 63 1191-7 (1974) ; C.A.82 38374f (1975).

Biochem.Med. 12 348-55 (1975) :C.A.= 157655d (1975). Peters, J.H. :Morse,C.W. and Schmidt,L.H. : Am. Rev. Respirat.Diseases 91,225-31 (1965) : C.A. 63 2245b(1965).

ISON IAZlD 255

444.

445.

446.

447.

448.

449.

450.

451.

452.

453.

454.

455.

456.

457.

458.

459.

O'Barr, T. P. ;Keith, D. J. and Blair, E.B. ; Amer.Rev.Resp.Dis. 107 472-4(1973) iC.A.79 270202 (1973). Lauermann, I. and Otto, L. ; Wiss. Z. Martin- Luther Univ. ,Halle-Wittenberq, Math. - Naturwiss. Reihe 17,561-7 (196 8) : C.A. 71

Kane, P. 0. ; Nature 183,1674 (1959) ; C.A. - 55 7528i (1961). Strickland,R.D. and Hentel, W. ; Am. J. Clin. Pathol. 24,988-92 (1954) and Tech.Bul1 Reqistry Med. Technoloqists 24,168-72 (1954) i C.A. 48 12376e (1954). Harting, H. and v, Gerzanits, P. ; Acta Med. et Biol. 2,643-48(1954) iC.A.50 9484a(1956). Ito,F. :Nasu,Y. :Mizobata,H. :Yuasu, M. and Nishi, K. : Kekkaku 33 345-7 (1958) ;C.A. - 52 20351h (1958). Ito, F. ; Nasu,Y. and Nishi, K. ; Med.J.Osaka - Univ. 2 567-71 (1958) :C.A.% 13253h( 1959). Verrotti,M. and Bardelli,N. ; Rass. studi sichiat.4l,472-5(1952)C.A. 47 4410h(1953).

ichwenk,R. .Kell K. -Tse K S.ad Sehon A.H. :

BBnicke, R. :Naunyn-Schmiedeberg' s Arch. exptl. Patho1.u. Pharmakol. 216,490-3 (1952) ;C.A.47 - 4937i(1953). Cascio,G. and Purpura,R. ;Bull. SOC. ital.bio1. sper. 28,1947-8 (1952) :C.A.% 6361c (1955). Grosset, J. ; Grumbach,F. and Canetti,G. i Ann. inst. Pasteur 92,752-9 (1957) ;C.A. - 51 156783. (1957). Strupczewska-Januszowa, H.; Gruzlica 2, 481-4(1961) ;C.A.z 15768i(1962). Pregowski,W.; Zukowska, H. and Szymska,E.; Przeglad Lekar. 16,310-13 (1960) ;C.A.Z 4974a (1962). Ginsburg, T. S . and Poddubny , A, F. :Lab Delo 9,

Finn,E. R. and Vil' derman, A.M. Zdravookhranenie 39-43 (1964) ;C.A. - 63 1077f (1965).

28801b (1969). -

hem.& 108b61(19?51 ;C.A.a3 125474r w+-

37-40 (1963) rC.A.62 67959 (1965).

256 GLENN A. BREWER

460.

461.

462.

463.

464.

465.

466,

467.

468.

469.

470.

471.

472.

473.

474.

475.

476.

477.

Tagawa,C.;Nippon Kagaku Rhohogakukai Zasshi

Vil derman,A. M. : Finn, E. R. and Evgrafova, Z.A. ;Vopr.Borl by s Tuberkulezom,Minsk,Sb

Sen,P. K. ;Saha, J. R. and Chatterjee,R. ; Indian J. Med.Res. @,19-27(1972) ;C.A.c 42992v (1972). Kharizanova,T. and Simova,V.;s. Nauchnoizsled. Khim. -Farm. Inst. S 353-8 (1972) ;C.A.B 38384x(1973). Kharizanova,T. and Simova,V. ;Advan. Antimicrob.Antineoplastic Chemother.; Proc. Int. Conqr. Chemother., 7th; 1971 Part 2 697-9;C.A.z 73417g(1973). Bartmann,K. and Freise,G. ;Beitr. Klin. Tuberk 127 546-60(1963);C.A.& 5880g(1964). Nishi,K.;Kekkaku 33 272-3(1958);C.A.52 - 16463i(1958). Poole,N. F. and Meyer, A. E. : Proc. SOC. Exptl. Biol.Med. - 98 375-7 (1958) ;C.A.= 20340i(1958). Makino,K. :Kinoshita,T. and Itoh,T. :Nature 173, 36(1954);C.A.% 5367f(1954). Leuschner,F.:Arzneimittel-Forsch.4,686 (1954) :C.A.B 2555g(1955). Sezaki,H. ;Yakuqaku Zasshi 78,1211-15 (1958) ;C.A.= 8259e(1959). Belles,Q. C. and Littleman,M. L. : Am. Rev. Respirat. Diseases 8 l , 364-72 (1960) : C.A.55 11519~ (1961). Abiko,Y. :Onoue,K. ;Yamamura,Y. : Nakazono, I. and Yoshida,T. : J. Biochem. (Tokyo)48,

Okudaira,M. ;Kuchii,M. and Hasaki,K. ;Irt’o 23, 1283-90(1969); C.A.73 - 23657r(1970). Siege1,D. ;Z. g e s . inn.Med.u. ihre Grenzqebiete - 13 66-68 (1958) ; C.A. 54 23032e (1960). Barreto,R. C. R. : J. Chromatoq.1 82-5 (1962) ; C.A. 57 6246i(1962). Barreto, R. C. R. and Sabino,S. 0. ; J. Chromatoq.

Fartushnyi,A. F. and Sukhin,A. P. ;Sud. -Med. Ekspert. - 16,39-43 (1973) ;C.A.E 78879q(1974).

- 13,156-64 (1965) : C.A. 64 147769 (1966) .

119-22 (1964) C.A. 64 4104f (1966).

838-45(1960) ;C.A.x 14691d(1961).

- 9 180-6(1962) ;C.A.x 9395f (1963).

ISON IAZ I D 257

478.

479.

480.

481.

482.

483.

484.

485.

486.

487.

488.

489.

490.

491.

492.

493.

Cattaneo,C. ;Fantoli,U. and Ferrari, S. : Ann. 1st. “Carlo Forlanini” 27, 78-93 (1967) ;C.A.B 11373d(1968). Hughes, H. B. ; J. Pharm. Exp. Ther. 109, 444 (1453) ; C.A. 48 2241i (1954). Ozawa,H. and Kujomoto,A.;Iqaku To Seibutsauqaku 27,110 (1953) ;C.A.S 1580f (1954). Cuthbertson,W. F. J. ; Ireland,D. M. and Wolff ,W. : Bi0chem.J. 55,669(1953);C.A.48 1462i(1954). Zamboni,V. and Defranceschi; Biochim. Biophys.Acta. 14,430 (1954) ; C.A. 3 12218d (1954). Mandel, W. ; Heaton,A. D. ; Russele, W. F. and Middlebrook, G. ; J. Clin. Invest. 38,1356-1365 (1959) ; C.A. 54 2590e (1960). Evans,D.A. P. ;Manley,K.A. and McKusick,V.A. ; Br. med. J. 485-491 (1960) ; C.A. 55 11636g (1961). Sunahara,S.;Urano,M. and Ogawa,M.;Science - 134, 1530 (1961). Goodman,L.S. and Gilman, A.; The Pharmacological Basis of Therapeutics 5th Ed page 1205 MacMillan Publishing Co.Inc. New York. Goldstein,A. ;Aronow, L.and Kalman, S.M. ; Principles of Druq Action: The Basis of Pharmacoloqy 2nd Ed. page 452, John Wiley and Sons, New York. Venkataraman, P. : Eidus, L. ; Ramachandian, K. and Tripathy, S. P. ; Tubercle (G.-B. ) 46 , 262 (1965). Hirtz,J.L.;Analytical Metabolic Chemistry of Druss pages 191-198 (1971) ,Marcel Dekker Inc. ,New York. Grebennik,L. I. ;Sov.Med. 3l,47-51(1968) ; C.A.69 3436117. (1968). Russel1,D.W. ; Clin. Chem.Acta. 4 l , 163-8 (1972);C.A.z 52461f(1973). Ritter, W. ; Prax. Pneumol. - 27 139-45 (1973) ; C.A. 79 61303d (1973). El1arxG.A. and Gammon, P. T. ;Advan.Antimicrob. Antineoplastic Chemother. , Proc. Int. Conqr. Chemother. 7th 45-6 (1971) ;C.A.E 100376b (1973).

258 GLENN A. BREWER

494.

495.

496.

497.

498.

499.

500.

501.

502. 503.

504.

Mitchell, J. R. and Jollow,D. J. ;Druq Interact.

Iwamoto, T. :J. Pharm. SOC. Japan 74 36-9 (1954) i C.A. & 5362b(1954). Nakazono, I. and Hirata,H. ;Kekkaku 3 4 101-6(1959) ;C.A.S 25288e(1960). Toth,B. and Shimizu,H. ;Eur. J. Cancer 9, 285-9(1973) :C.A.B 101439m(1973). Kakemi,K. ;Arita,T. ;Sezaki,H. and Takasugi, N. :Chem. Pharm.Bul1. JJ 551-7 (1965) ;C.A.Q 7511c(1965). 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 . 9 393(1912) ;C.A.6_ 2073(1912). Chorine,V. : Compt. rend, acad. sci. 220,150 (1945). Huant,E.:Gazette des Hopitaux Aug.15(1945).

Tuberculosis.3rd ed.,The Williams & Wilkins C Co. ,Baltimore, 1958. Kirschbaum,A. ; Pharm.Acta Helv. 27 229-33 (1952) : C.A.

65-79(1974) :C.A.U 92730g(1975).

Long,E.S.:The Chemistry and Chemotheraw o f

47 2429b (1953).

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


2.1 . 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

8 .1 . Identification

8 . 2 . Determination of Sulfate

8 .3 . Loss on Drying

8.4. Microbiological Assay

8 .5 . Assay of Kanamycin B

KANAMYCIN SULFATE 26 1

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

262 PAUL J. CLAES et a/ .

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)-( 1 --4)-0-[3-amino-3- - deoxy-CY -D-glucopyranosyl-( 1 - 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-6-

deoxy-CY-D-glucopyranosyl-( 1 - 411 -2-deoxy-. The numbering is given in the formula below. The carbon atoms of the 2-deoxy-

6'

1' 1' I

,2 0

OH \I 0

Kariadlycin A free base : C,8H36N4011

Kanatnycin A monosulfate monohydrate :

M.W. 484.50

C18H36N401 1 0H2S04'H20 M.W. 600.59

KANAMYCIN SULFATE 263

streptamine ring are numbered as 1,2,3, ..., those of the amino sugar moieties linked at C-4 and C-6 of 2-deoxystrepta-

mine 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 con-

tent. The latter is more readily soluble in water and is

designated in the Brit. Ph. Add. 1975

fate. The sulfate (SO ) content calculated for the monosul-

fate monohydrate is 15.99 2. In most commercial samples the

sulfate content varies from 16 to 16.4 %3'4. The monosulfate

monohydrate is reported in the U.S. Ph. XIX and in the Brit.

Ph. 1973 under the heading kanamycin sulfate. To avoid con-

fusion the designation kanamycin monosulfate should be pre-

ferred. The limits of the pH ( 1 2 aqueous solution) given in

the Code of Federal Regulations7 and in the Eur. Ph.

from 6.5 to 8.5.

2 as kanamycin acid sul-

4

5

6

8 are

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.

1.8 H SO

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.

18 36 4 1 1 ' are respectively 23.95 and 26.14 X . It is obvious

2 4

Kanamycin B (11) and kanamycin C (111) are two minor

components of the antibiotic complex. They differ from kana-

mycin A in the nature of the amino sugar linked to the

4-position of the 2-deoxystreptamine moiety (2,6-diamino-

2,6-dideoxy-D-glucose for I1 and 2-amino-2-deoxy-D-glucose

264 PAUL J. CLAES et a / .

for 111). Kanamycin B, also referred to as bekanamycin, is

available as its sulfate salt under the name Kanendomycin

(Meij i) . R

6 " CH20H

I

HO

I

' 1"

2 I1 kanamycin B : R1 = R = NH2, R3 = H, R4 = OH

I11 kanamycin C : R - NH2, R2 = OH, R3 = H, R4 = OH

I V

1

1 2 amikacin : R = H, R - NH,, R3 = L(-)-CO-CH-(CH,),-NH,,

R4 = OH - ..- - 1

OH

2 3 V tobramycin : R1 = R - NH2, R - H , R4 = H


The two antibiotics amikacin (or BB-8) (IV) and tobramy-

cin (V) are structurally related to the kanamycins. The for-

mer is obtained by selective !-acylation of kanamycin A at the I-amino group with L(-)y-amino-CY-hydroxybutyric acid . The latter is a 3’-deoxykanamycin B produced by Streptomyces

tenebrarius .

9

10

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 1 1.3. Definition of International Unit

The International Reference Preparation is a sample

of kanamycin monosulfate (17 .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 8 12 U/mg .


2.1. Spectra

2 . 1 1 . Infrared Spectra

Infrared spectra of kanamycin monosulfate monohy-

. 12 drate and of the free base have been published by Maeda

These spectra are typical for polyhydroxy polyamino com-

pounds. However, no characteristic bands, which would permit

differentiation from related aminoglycosidic antibiotics,

are present.

266 PAUL J. CLAESetal.

2.12. Ultraviolet Spectrum

12 Kanamycin free base and its sulfate salts show end

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 pre-

sented in the figure I . The spectrum was obtained by dissol-

ving 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 anome-

ric 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 Chemical Shift* Coupling Constant

2-Ha

2-He

3"-H

1.22 (m)

1.96 (m)

2.90 (m)

J = 13 Hz, Jaa= 12 Hz gem

J~ = 13 Hz, J = 4 HZ gem ea

2"-H 3.48 (m) J = 3.8 Hz, J = 10 Hz

2'-H 3.55 (m) J = 3.3 Hz, J = 9.5 Hz

3'-H 3.77 (m)

I "-H 5.03 (d) J = 3.8 Hz

1 '-H 5.79 (d) J = 3.3 Hz

d = doublet; m = multiplet; gem = geminal; a = axial; e = equatorial.

3-(trimethylsilyl)propane-l-sulfonate as internal standard. They can be converted into b-values, referring to TMS, by adding 0.48 ppm.

I The 6-values, given in this table, refer to sodium

3'" 4

h)

U m

Fig. 1. PUR spectrum (lo0 Mc) of kanamycin A free base, taken in D 0 with sodium 3-(trimethylsilyl)propane-l-sulfonate as internal standard.

2

268 PAUL J. C U E S et a/ .

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

al. . L

13 - Spectral data, observed for a solution of the monosul-

fate of kanamycin A in D 0 solution and for its tetrahydro-

chloride, are given in Table 11. It can be seen that protona-

tion of the amino groups causes a downfield shift of some of

the protons. This effect is less pronounced for the monosul-

fate.

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.

2

* Table 11. Chemical Shift Values observed for Kanamycin A

14 Salts L_ *** Protons Kanamycin mono sul f a te Kanamycin 4 DC1

2-Ha 1.48 (m) 1.98 (m)

2-He 2.16 (m) 2.6 (m)

CHO, - Cfi20

CHN, - CE2N

anomer i c protons

2.95 - 4.2 (m) 2.95 - 4 . 2 (m)

5.09 (d) and 5.52 (d) 5.18 (d) and 5.58 (d)

* ** Saturated solution in D20 ***

6-Values relative to sodium 3-(trimethylsily1)propane- I-sulfonate as internal standard.

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

derivatives of kanamycin A (N-acetyl-N,O-methyl- - - - and - N-acetyl- - 0-trimethylsilylkanamycin) have been determined and interpre- ted by De Jongh et a1.I8. The electron impact spectra of both


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-trimethyl- - silyl derivative. Other diagnostic peaks, observed in the

spectra of both derivatives, result from a cleavage of glyco-

sidic bonds or C-0 bonds connecting a hexose to the deoxy-

streptamine unit. The m/e values of these peaks reveal the

sequential arrangement and the gross structure of the saccha-

ride- 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

are also described in the paper of De Jongh et al. 18 .

Mass spectra of the underivatized free bases of kanamy-

cin 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 f-aminog moiety lucose 1

m/e 306(a),A k m / e 162(a), 530 (b) ,720 (c) 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 ) ,

(c) - N-Acetyl-0-trimethylsilylkanamycin A : m/e 1156 (M) ,

+

+ m/e 806 (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

_-_----------- 12

Maeda

[a]:+ 146' (c 1 , 0.1N H 2 S 0 4 ) , % + 70.737 Cron et al. 21 -- - 22 [

for the monosulfate

150.5' (c 1 , 0.2E H 2 S 0 4 ) , % + 72.917 Claes et al. -- ---------------

12 Maeda

mmoles HCI a d d e d - Fig. 2. Electrometric titration curve of kanamycin A free

base.


%) For each of these[(XID values the mole@tilar rotations (

were calculated. The %, 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 kana-

mycin 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.

commerc

taining

Crystal Properties

The X-ray powder diffraction pattern obtained24 for a

a1 sample of kanamycin monosulfate monohydrate con-

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 10 cps; time constant 1 s

Scanning speed : 0.5" 20 per minute

3


Chart speed : 10 mm/min.

Table 111. X-Ray Powder Diffraction Data

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

111: - 10 21 12 24 28 24 22 16

100 41 9 7

10 47 58 6 6 2 7

41 16 26 20

d* (A) I/Io ** d* (A) 1/17 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

1 1 12 31 4 3 7 2 2 7 2

1 1 2 2 5 8 6 3 6 5 7

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 1 1 2 3 2 5 3 4 3 2 2 2 2

* n h 2 sine

d = - = interplanar distance

** 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 monohy-

drate has been determined by X-ray analysis 25 .

2.5. Melting Range

The following melting (decomposition) temperatures

have been reported :


for the free base of kanamycin A ______--______----__------ -__-- 12

Claes et al.

250' Maeda

255" (decomp.)

for the monosulfate

22 -- __-----------------

12 268-276" (decomp .) Maeda

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 transi-

tion was noted27 below 250' in the differential thermal ana-

lysis (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.

Kanamy c in Solvent Solubility References

monosulfate water 350 m g / m l 12

5 0 Z aq.MeOH 5 mg/ml 12 1 part in 8 parts 6,8,23

acid sulfate water 1 part in 1 part 6,8

The solubility of kanamycin sulfate in water at various

pH values is given in a paper by Granatek -- et a1.28. Solubi-

lities in various solvents are also given in this paper.


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

carbon and adjusted to pH 8.0-8.2 with NH OH. Addition of

methanol affords a precipitate of the crude crystalline mono-

sulfate monohydrate of kanamycin A, which is recrystallized

from water-methanol or water-methylcellosolve. The biosynthe-

sis of kanamycins has been studied by Kojima et al.

review of the biosynthesis of aminocyclitol antibiotics is 31 given by Rinehart et al. .

4

29,30. A

-0 -

3.2. Chemical Synthesis

Total synthesis of the kanamycins A, B and C was

achieved in 1968 by s. Umezawa and coworkers 3 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 experi-

ments. The following results, taken from their paper, illus-

trate that both forms are extreqely stable as powders. After

storage for 4 months at 56' on qverage no loss of activity


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 po-

tency. The crystalline monosulfate can be heated as a powder

for 6 hr at 150" without loss of activity .

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 loo", 97 Z of the biologi-

cal 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-amino-

3-deoxy-D-glucose.

4

During determination of the structure of kanamycin A

Kanamycin, like the related antibiotics neomycin, para-

neomycin 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 NaOH 36 .

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 Davies . A recent article by Haas and D ~ w i n g ~ ~ describes the isolation

and assay of these enzymes. The kanamycin A-modifying enzymes,

37

Enzyme

Kanamycin acetyltransf erase (KAT)

Gentamicin acetyl- transferase I11

2 (GATIII)

Gentamicin adenyl- transferase (GAdT)

Neomycin phospho- transferase I WTI)

(NPTII)

Neomycin phospho- transferase I1

Table IV. Kanamycin A Modifying Enzymes

Cof actor Modification induced in kanamycin A

Acetyl coenzyme A Acetylation of the 6'-amino group

Acetyl coenzyme A Acetylation of the +amino group (of deoxystreptamine)

ATP

ATP

ATP

Adenylylation of the 3'-hydroxyl group

Phosphorylation of the 3 '-hydroxyl group

Phosphorylation of the 3 '-hydroxyl group

Other Substrates

Neomycins, kanamycin B, gentamicin Cia, gentamicin C2, tobramycin, butirosins, ribostamycin, sisomicin, BB-K8 (amikacin)

Kanamycins B & C, gentamicins, sisomicin, ribostamycin, tobramycin, lividomycins

Kanamycins B & C, gentamicins, t obramyc in

Kanamycins B & C, neomycins, lividomycins, ribostamycin, gentamicins A & B

Kanamycins B & C, neomycins, butirosins, ribostamycins, gentamicins A & B


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 dis-

cussed 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 LL et al.40. These

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 n ~ ~ in 1966. A com-

parative pharmacokinetic study of kanamycin and amikacin (a

semisynthetic aminoglycoside antibiotic derived from kanamy-

cin 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 pri-

marily by glomular filtration, and urinary excretion of the

278 PAUL J. CLAESeral.

unchanged antibiotic accounted for 83 % of the dose. No

protein binding of kanamycin by human serum was observed

Kanamycin sulfate is poorly absorbed from the gastrointesti-

nal tract and large amounts of kanamycin are recovered in the

stools of patients given the drug by mouth

45-47

42 . The distribution of kanamycin in tissues, after paren-

teral administration, has been studied by several

authors 48-50


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 charac-

teristic melting point (about 235’ with decomp.) of the crys-

talline picrate salt of kanamycin is also useful as identi-

fication test. The procedure is described in the Brit. Pharm.

Codex5’ and in the Eur. Ph. . 8

Thin layer chromatography (TLC) on silica gel H with a

6 solvent system consisting of 3.85 % aqueous amonium acetate

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 commer-

cial samples of kanamycin (section 8.62, solvent system VI)

is also a specific method for the identification of kanamy-

cin A23. The chromatography is carried out on Merck pre-


coated silica gel plates with a 15 2 aqueous solution of

KH PO as a solvent system. Spots are visualized by the color

reaction with ninhydrin or by a spray consisting of a 0.2 2

alcoholic solution of 1,2-dihydronaphthalene and sulfuric

acid 9N - in a ratio of 1:1, followed by heating f o r 5 to 10

min at 150". Differentiation of kanamycin from related amino-

glycoside antibiotics is based on Rf values and the color

observed after visualization with the 1,2-dihydroxynaphtha-

lene 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

2 4

a 7 2 aqueous solution of KH PO

system 8'23 (section 8.62, system VII).

is used as a solvent 2 4

8.2. Determination of Sulfate

The limits for the sulfate content (SO ) are for 6 4

kanamycin monosulfate, from 15.7 to 17.3 2 (Brit. Ph. 1973 )

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 kana-

mycin and in related aminoglycoside antibiotics has been

reported by Roets and Vanderhaeghe53. In this method the sul-

fate ion is titrated with BaCl 0.1M, using thorin as indica-

tor, after fixation of the kanamycin free base by ion exchan-

ge on a column filled with a suitable strongly acidic resin

in the H form (e.g. Dowex 50W-X8, 200-400 mesh).

8

2 -

+ 8

and consists in the precipitation of the sulfate with a known

amount of BaCl

The most convenient method is described in the Eur. Ph.

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 adapted

from a complexometric titration described by Anderegg et - al.54. 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.

3Y4

8.3. Loss on Drying

The water present in kanamycin monosulfate monohy-

drate 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 ano-

ther 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 phos-

phorus pentoxide. This treatment does not alter the X-ray

powder The limits for this loss on drying is 3 % 6 8

(Brit. Ph. ) and 1.5 Z (Eur. Ph. ). The values actually ob-

served under these conditions vary from 0.2 to 0.7 2.

For kanamycin acid sulfate the same procedure (3 hr at

2 60' in vacuo over P 0 ) is recommended in the Brit. Ph. (Add.

1975)

copoeias is 5 2 .

2 5 and in the Eur. Ph.7. The limit given in both Pharma-


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 sul-

fates 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 mini-

mum 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

ATCC 6538P or NCTC 6571. The FDA prescribes Staphylococcus

aureus ATCC 6538P. Details of the FDA procedure can also be

found in refs. 55 and 56. No detailed description of the

-- 7 --

--L_

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

B-component given in the Code of Federal Regulations is 5 2 .

A method using column chromatography on Dowex 1-X2 ion-

-7

exchange 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

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.

2 4

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 identi-

fied22 by degradation and mass spectral studies as paromamine

and as 6-0-(3-amino-3-deoxy-CY-D-glucopyranosyl)deoxystrep- - tamine.


8.6. Chromatographic Analysis

8.61. Paper Chromatography

The solvent system of Peterson and Reinecke5', which

consists of water-saturated butanol containing 2 Z p-toluene

sulfonic acid, has been used by a number of authors

for differentiation of kanamycin A from the B- and C-compo-

nents. 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 bioautogra-

phyl y60, ninhydrin reaction 62'63 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').

1,21 ,60-63

Differentiation of kanamycin from related antibiotics by

paper chromatography using a combination of several solvent

systems has been described in a number of papers

review article on paper chromatography of antibiotics has

been published recently .

64-67. A

68

8.62. Thin Layer

Various TLC systems for separation of kanamycin A

from its congeners (kanamycin A and B) and from other water-

soluble basic antibiotics have been reported. Details are

given in Table V. Separation of kanamycin A from tuberculo-

static antibiotics such as rifamycin SV, capreomycin, viomy-

cin, cycloserine, and streptomycin (or dihydrostreptomycin)

has been reported by Voigt and Maa Bared 69 .

V8Z

Po

N

uw

uu

~~

uu

mw

u~

l~

u

Um

We

WW

WW

- -

-N

UN

N

W

WN

R-

N

U

Ll

WL

l

00

w

m

c"

P

W

N

W

WW

P

Ln

CW

Ll

0

NO

,

w-

(0 -

W

N

WL

l

Ll

w

v1

P

WW

W

R

>u

C.

WN

VI

WL

-L

nQ

I

VI

WN

~O

WN

UN

CO

O u

oo

c.

f-

W

WL

n

R

h-

W

W 00

W

uc:

v1

N

NW

Ln

-w

Ln

w-

-

wm

o

m

0

0

oc

l

- -

uu

u

0-0

WV

I~

V

tW

Ln

h I::

Ll

0.. -

W

m

U -

*P

o.

o

am

VI

N

I> m 0

System

Plate

Revelation

Reference

Kanamycin A

Kanamycin B

Kanamycin C

Neamine

Neoaycin,,

Paromamine

Paromomycin,,

Cent

arni

cin C,

Spec

tino

rnyc

in

Streptomycin

Dihy

dros

trep

t.

Viomycin

Capreomycin

Poly

niyx

in B

Eacitracin


v i s u a l i n a r i o s - o l - t p e , a s t i b i o t i c s _ . s e d _ i s ~ - ~ ~ ~ ~ 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-dihydroxynaph-

talene) 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 naphthoresor-

cinol

OR = as NR, but with orcinol instead of naphthoresorcinol

DN = p-dimethylaminobenzaldehyde-ninhydrin reagent

NP = sodiumnitroprusside-permanganate reagent

ON = oxidized nitroprusside reagent

CT = chlorine-tolidine reagent 81 MS = Mathis-Schmitt reagent

Solvent systems for the TLC procedures given in Table V

I

69

69

79

80

--------- ------------------ ---------- --------------_ : Upper layer of CHCl -MeOH-17 % ammonium hydroxide

3 70 ( 2 : 1 : 1) 70 I1 : n-Propanol-pyridine-HOAc-water ( I5 : 10 : 3 : 10)

I11 : CHCl -MeOH-28 2 ammonium hydroxide-water (1:4:2:1) 72 3 tank saturated overnight

IV : 10 % aqueous solution of NaH PO 2H20-MeOH-EtOAc 2 4' 52 (8: 7 :3)

V : NH40Ac (3.85 g) in water (100 ml) (tank saturated 78 overnight)

VI : Aqueous solution of KH PO 15 2 (tank saturated over- 2 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 separa-

ting power is mainly due to the presence of a poly-

carboxylic 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 polycarboxy- 8,23 late resin (carbomer) is added to the silica gel .

VII : Aqueous solution of KH PO 7 % (tank saturated over- 2 4 8,23 night)

VIII : n-Propanol-EtOAc-water-25 % ammonium hydroxide- 73 pyridine-3.85 % in water (100:20:60:20:10:200)

IX : EtOH-EtOAc-water-25 % ammonium hydroxide-pyridine- 73 3.85 % NH40Ac in water (100:20:60:10:200)

X : MeOH-EtOAc-water-25 % ammonium hydroxide-pyridine-

3.85 % NH40Ac in water (100:20:60:20:10:200) 73

73 XI

XI1 : Water-sodium citrate-citric acid (100:20:5)

XI11 : n-Propanol-pyridine-HOAc-water (15:10:3:12)

XIV

: 25 % Ammonium hydroxide-water-Me2C0 (16:144:40) 74

75

: MeCOEt-MeOH-isopropanol-7.9N - ammonium hydroxide (10:8:5:3:7) (tank and plate saturated - double deve 1 opment )

: 1.5M NaOAc (adjusted to pH 8.5) containing 1.OM - NaCl and 10 % s-butanol

76

XV 77

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'


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 - kieselguhr G (1:2) activated for 1 hr at

1 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 61 exclusion chromatography, was introduced by Rothrock et al.

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 proce-

dure have been reported82. Other applications in the field of

aminoglycoside antibiotics have been reviewed recently by

Umezawa and Kondo . 83

Experimental details of ion exclusion chromatography can

be found in several papers 61 y72y82-84. Most of the separa-

tions 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

288 PAUL J. CLAESet a / .

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

with CO -free water. Detection systems based on a continuous

measurement of electric conductivity, optical rotation and

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).

2

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 aminogly-

coside antibiotics from culture f iltrates12. The antibiotic

is adsorbed on the carboxylic resin in the Na

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 83 - 4

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

+ + or NH4 and

.

exchanger such as Zipax SCX (Dupont) has been reported

recently . 87


8.64. Gas Liquid

Tsuj i 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 antibio- 88

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 amino-

glycoside 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


been described by Maeda et a1 .92. The spots of the kanamycins

were visualized with ninhydrin. Mobilities relative to ala-

nine (Rm values) are 1.82 for kanamycin A , 1.92 for kanamy-

cin 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. Resolu-

tion of antibiotic mixtures in serum samples by high-voltage 95 electrophoresis on agarose is described by Reeves and Holt

L-

.

9. Determination in Body Fluids

Since kanamycin acid, like other aminoglycoside antibio-

tics, 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 pro-

cedures have been worked out for this purpose. Sabath % al. 96' 97 described a microbiological assay method. Interfe-

rence by penicillins and cephalosporins can be eliminated by

a treatment of the serum with a "broad-spectrum" /j-lacta-

mase 96'98. 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 compari-

son of fluorescent intensity with that of reference samples

on TLC plates after reaction with 7-chloro-4-nitrobenzo-2-

oxa-l,3-di'azole, has been developed by Benjamin -L et al. . Enzymatic assays employing aminoglycoside-modifying enzymes

(section 5) have been introduced recently 101y102 . In these

procedures the antibiotic is enzymatically modified in the

L

100


presence of a radiolabeled cofactor. Kanamycin acetyltrans-

ferase (KAT) l o ' and gentamicin acetyltransf eraselo2 have been

used in the assay of kanamycin.

10.

1 .

2.

3.

4.

5.

6.

7 .

8.

9.

10.

1 1 .

12.

13.

14.

15.

16.

17.

Reference Cited

H. Umezawa, M. Ueda, K. Maeda, K. Yagishita, S. Kondo, Y. Okami, R. Utahara, Y. Osato, K. Nitta and T. Takeuchi, - J . Antibiotics, Ser.A, 2, 181 (1957).

British Pharmacopoeia, Addendum (1975).

J. Hoebus and H. Vanderhaeghe, unpublished results.

M. Dubost, unpublished results.

United States Pharmacopoeia, XIX,

British Pharmacopoeia (1973).

Code of Federal Regulations, 3, § 148h (1972).

European Pharmacopoeia IV, t o be published.

H. Kawaguchi, T. Naito, S. Nakagawa and K. Fujisawa, - J. Antibiotics, 25, 695 (1972).

--

--

K.F. Koch and J.A. Rhoades, Antimicrob. &. Chemother.- 1970, 309 (1971). - J.W. Lightbown, P. de Rossi and P. Isaacson, K. World Hlth. Organ. , 47, 343 (1972).

K. Maeda, "Streptomyces Products Inhibiting Myco- bacteria", J. Wiley, New York, 1965, p. 61.

H. Naganawa, S. Kondo, K. Maeda and H. Umezawa, - J. Antibiotics, 24, 823 (1971).

P.J. Claes, S . Toppet and H. Vanderhaeghe, unpublished results.

K.F. Koch, F.A. Davis and J.A. Rhoades, - J. Antibiotics, 26, 745 (1973).

N. Yamaoka, T. Usui, H. Sugiyama and S. Seto, - Chem. Pharm. Bull., 22, 2196 (1974).

F.K. Koch, J.A. Rhoades, E. Hagaman and E. Wenkert, 2. Am. Chem. %. , 96, 3300 (1974).

-- e

-

--

-7

292 PAUL J. CLAESet a/ .

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

D.C. De Jongh, E.B. Hills, J.D. Hribar, S. Hanessian and T. Chang, Tetrahedron, L 29, 2707 (1973).

P.J. Daniels, M. Kugelman, A.K. Mallams, R.W. Tkach, H.F. Vernay, J. Weinstein and A. Yehaskel, Chem. Commun., 1629 (1971).

P.J.L. Daniels, A.K. Mallams, J. Weinstein, J.J. Wright and G.W.A. Milne, J. Chem. SOC., Perkin I, 1078 (1976).

M.J. Cron, D.L. Johnson, F.M. Palermiti, Y. Perron, H.D. Taylor, D.H. Whitehead and I.R. Hooper, J. Am. Chem. S O ~ . , 80, 752 (1958).

P.J. Claes, H. Vanderhaeghe and F. Compernolle, Antimicrob. %. Chemother., - 4, 560 (1973).

H. Vanderhaeghe and P. Claes, unpublished results.

C. De Ranter, unpublished results.

- - -

- - --

G. Koyama, Y. Itaka, K. Maeda and H. Umezawa, Tetrahe- dron Letters, 1875 (1968). - M. Draguet and R. Bouch6, unpublished results.

Bristol Laboratories, report.

A.P. Granatek, S. Duda and F.H. Buckwalter, Antibiot. Chemother., 10, 148 (1960).

M. Kojima, Y. Yamada and H. Umezawa, &. Biol. Chem., 32, 467 (1968).

-- c

M. Kojima, Y. Yamada and H. Umezawa, s. Biol. Chem., - 32, 1181 (1969).

-- K.L. Rinehart and R.M. Stroshane, J. Antibiotics, 29, 319 (1976).

- - S. Umezawa, K. Tatsuta and S. Koto, Bull. Chem. SOC. Japan, 42, 533 (1969).

---

S. Umezawa, S. Koto, K. Tatsuta, H. Hineno, Y. Nishimura and T. Tsumura, Bull. Chem. z. Japan, 42, 537 (1969).

S. Umezawa, S. Koto, K. Tatsuta and T. Tsumura, K. Chem. SOC. Japan, 42, 529 (1969).

M. Nakajima, A. Hasegawa, N. Kurihara, H. Shibata, T. Ueno and D. Nishimura, Tetrahedron Letters, 623 ( 1968).

G.H. Wagman and M.J. Weinstein, J. Med. Chem., I, 800 ( 1964).

-- -

--

- - -


37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

R. Benveniste and J. Davies, Annu. Rev. Biochem., 42, 471 (1973).

M.J. Haas and J.E. Dowding, Methods 611 (1975).

Enzymoloa, 43,

B. Weisblum and J. Davies, Bacteriol. e., 493 (1968).

E.F. Gale, E. Cundliffe, P.E. Reynolds, M.H. Richmond and M.J. Waring, "The Molecular Basis of Antibiotic Action", J. Wiley, London, 1972, p . 304.

R. Benveniste and J. Davies, Antimicrob. &. Chemother., - 4 , 402 (1973) .

C.M. Kunin, Ann. N.Y. Acad. Sci 132, 811 (1966). - -- - -.' - B.E. Cabana and J.G. Taggart, Antimicrob. 9. Chemother., - 3, 478 (1973).

B.M. Orme and R.E. Cutler, =. Pharmacol. Therap., - 10, 543 (1969).

R.C. Gordon, C. Regamey and W.M.M. Kirby, Antimicrob. &. Chemother., - 2 , 214 (1972). W. Scholtan and J. Schmid, Arzneimittelforsch., 13, 288 ( 1 963) . H. Welch, W.W. Wright, H.I. Weinstein and A.W. Staffa, Ann. N.Y. Acad. Sci., 76, 66 (1958) . H. Okubo, Asian Med. J., M, 309 (1968).

-

- -- - - -- -

K. Fukaya and 0. Kitamoto, Progr. Antimicrob. Anti- cancer Chemother., 1 , 503 (1970), Proc. 6th Intern. Congr. Chemother. (T969) . - -- K. Matsumota, S. Arai and K. Yokoyama, Progr. Antimi- crob. Anticancer Chemother., 1 , 500 (1970), Proc. 6th Intern. Congr . Chemother. ( 19z9). - c_- -- British Pharmaceutical Codex, 1968. M. Dubost, C. Pascal, B. Terlain and J.P. Thomas, - J. Chromatogr., 86, 274 (1973).

E . Roets and H. Vanderhaeghe, 2. Pharm. Pharmacol., 24, 795 (1972).

G. Anderegg, H. Flaschka, R. Sallmann and G. Schwarzen- bach, Helv. Chim. Acta. , 37, 113 (1953) . B. Arret, D.P. Johnson and A. Kirshbaum, J. Pharm. s., - 60, 1689 (1971).

-

- -

294 PAUL J. CLAESetal .

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

F. Kavanagh, "Analytical Microbiology", Vol. 11, p . 123, Academic Press, New York (1972).

F. Kavanagh, "Analytical Microbiology", Vol . 11, p . 44, Academic Press, New York (1972).

T. Wakazawa, M. Abe, Y. Sugano and S. Kawaji, 2. Antibiotics, Ser.A, 14, 187 (1961). D. Peterson and L. Reinecke, J. &. +. +., 72, 3598 (1950).

K. Maeda, M. Ueda, K. Yagishita, S. Kawaji, S. Kondo, M. Murase, T. Takeuchi, Y. Okami and H. Umezawa, - 3. Antibiotics, Ser.A, 10, 228 (1957). J.W. Rothrock, R.T. Goegelman and F.J. Wolf, Antibiotics Annu., 796 (1958-1959).

T. Wakazawa, Y. Sugano, M. Abe, S. Fukatsu and S. Kawaji, J. Antibiotics, Ser.A, 14, 180 (1961). D.A. Johnson and G.H. Hardcastle Jr., U.S. Patent, 2,967,177, January 3, 1961. Chem. Abstr., 55, P6792e (1961).

T. Miyaki, H. Tsukiura, M. Wakae and H. Kawaguchi, - J. Antibiotics, Ser.A, 2, 15 (1962). V. Betina, J. Chromatogr., 15, 379 (1964). M . J . Weinstein, G.M. Luedemann, E.M. Oden and G.H. Wagman, Antimicrob. &. Chernother.-1963., 1 (1964).

S. Kondo, M. Sezaki and M. Shimura, J. Antibiotics, Ser.A, 17, 1 (1964). Chem. Abstr., c, 4681 (1964). V. Betina, Methods in Enzymology, - 43, 100 (1975).

R. Voigt and A.G. Maa Bared, J. Chromatogr., 36, 120 ( 1 968).

T. Ikekawa, F. Iwami, E. Akita and H. Umezawa, - J. Antibiotics, Ser.A, 16, 56 (1963).

-

- - -

- -

- -

E. Roets and H. Vanderhaeghe, Pharm. Tijdschr. Belgi;, 44, 57 (1967). - H. Maehr and C.?. Schaffner, - J. Chromatogr., 2, 572 (1967).

B. Borowiecka, Diss. Pharm. Pharmacol., 22, 346 (1970). J.P. Schmitt and G. Mathis, &. Pharm. Franq., 28, 205 (1970).

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.


Y. Ito, M. Namba, N. Nagahama, T. Yamaguchi and T. Okuda, J. Antibiotics, Ser.A, 17, 218 (1964).

J.K. Paunz, - J. Antibiotics, 2, 677 (1972).

N.R. Chatterjee, Indian J. s., 2, 1282 (1975).

C. Vickers, 2. Pharm. Pharmacol., 2, Suppl., 17s (1966).

H. Fishback and J. Levine, Antibiot. Chemother., - 3 , 1159 (1953).

E. Stahl, "Dunnschichtchromatography", Springer Verlag, Berlin, p. 822 (1967).

J.P. Schmitt and G. Mathis, Ann. Pharm. Frang., 26, 727 (1968).

S . Inouye and H. Ogawa, - J. Chromatogr., 13, 536 (1964).

- -

--

H. Umezawa and S. Kondo, Methods 263 (1975).

H. Vanderhaeghe, J. Tott6 and P. Chim. Belges , 77, 597 (1968).

T. Ottaka and M. Yaguchi, Liquid Work, 5 , Varian Associates, Palo

-

- -

- in Enzymology, 43,

Claes, E. e.

Chromatography at Alto, California (1973).

K.S. Andrews and M.W. Coleman, Communication presented at the Annual General Meeting of Electrophoresis and Chromatography Group of the Chemical Society of London, November 1975.

D.L. Mays, R.J. Van Apeldoorn and R.G. Laubach, - J. Chromatogr., 120, 93 ( 1 976).

K. Tsuji and J.H. Robertson, Anal. Chem., 42, 1661 (1970).

K. Tsuji and J.H. Robertson, Methods - in Enzymology, 43, 213 (1975).

S. Omoto, S. Inouye and T. Nida, - J. 430 (1971).

M. Margosis and K. Tsuji, J. Pharm. - - ( 1 973).

Antibiotics, 2,

Sci. , 62, 2946 - K. Maeda, A. Yagi, H. Naganawa, S. Kondo and H. Umezawa, - J. Antibiotics, 22, 635 (1969).

H. Umezawa and S. Kondo, Methods in Enzymology, 43, 279

S. Ochab, Pol. J. Pharmacol. Pharm 25, 105 (1973).

- - ( 1 975) .

- - -., -


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).

98. S.A. Stroy and D.A. Preston, a. Microbiol., - 21, 1002

99. P. Noone, J.R. Patton and D. Samson, Lancet, 2, 16

100. D.M. Benjamin, J.J. McCormack and D.W. Gump, Anal.

101. M.J. Haas and J. Davies, Antimicrob. &. Chemother., - 4 ,

(1971).

(1971).

Chem., 45, 1531 (1973). - - 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



2 . 1 Spectral

2 . 1 1 Infrared Spectrum 2 . 1 2 Nuclear Magnetic Resonance

Spectrum 2 . 1 3 Ultraviolet Absorption Spectrum

2 . 2 Mass Spectrum 2 . 3 Differential Thermal Analysis and

2 . 4 Solubility 2 . 5 Optical Rotation 2 .6 Ionization Constant 2 . 7 Crystal Properties

Melting Point

2 . 7 1 Derivative Crystallinity 2 . 7 2 X-Ray Diffraction

3 . Synthesis

4 . Decomposition

4.1 Metabolic Decomposition 4 . 2 Chemical Decomposition


5 . 1 Elemental Analysis of the Hydrochloride 5 . 2 Ion-Pairing Colorimetric and Fluorescence 5 . 3 Ultraviolet 5 . 4 Differential Thermal Analysis 5 . 5 Non Aqueous Titration 5 . 6 Tritium Labeling 5 . 7 Chromatography

5 . 7 1 Paper Chromatography 5 . 7 2 Thin Layer Chromatography 5 . 7 3 Gas Chromatography

KETAMINE 299

5 . 7 4 Liquid Chromatography

6. Determination in Body Fluids

7. References

300 WILLIAM C. SASS AND SALVATORE A. FUSARl

1. Description

1.1 Name, Formula, Molecular Weight'

Ketamine is 2-(2-~hlorophenyl)-2-(methyl- amino)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


Ketamine and the hydrochlor'de are both odorless, white crystalline powders. i 2. Physical Properties

2 . 1 Spectral

2 . 1 1 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 phenyl- hydrogen bending respectively. (1200 cm-1 is CHC13)

2.12 Nuclear Magnetic Resonance Spectrum

Figure 3 shows the proton magnetic

WAVELENGTH MICRONS 2.5 3 4 5 6 7 8 9 10 12 14 18 22 3550

t-- - -

-- - - -

4- I

4000 3500 300C 2500 2 W C 17CO -i40C l!i)O 800 500 200 FREQIJENCY 'C M')

Figure 1. (1601.0 cm-

infrared Spectrum of Ketamine Base in Chloroform. is polystyrene reference peak)

k 0

k a

a c

H

N

302

SOLVENT TEMPERATURE FILTER BANDWIDTH R . F . FIELD SWEEP TIME SWEEP WIDTH SWEEP OFFSET SPECTRUM AMP. INTEGRAL AMP.

D20

4 cps 2 5 OC

0 . 2 mG 2 5 0 sec 500 cps 0 c p s 16 2.5

,/-- w 0 w

1 I I 1 I I I *

8 .0 7 . 0 6 .0 5.0 PPM(6) 4 . 0 3.0 2 . 0 1.0 0

Figure 3. 60 MC NMR Spectrum of Ketamine Hydrochloride in D20.


J’ lox

1% a 1 cm W a v o Ionurh (nm)

2 76

269

W a v o Iongth (nm)

20 4

23.2

Figure 4 . Hydrochloride in 0.1 N Hydrochloric Acid.

Ultraviolet Spectrum of Ketamine

KETAMINE 305

resonance spectrum of ketamine hydrochloride in D20 at 60 y g . Hz. been made:

The following assignments have

Structural Assignments

L P F ! # of Protons and Description

2.0 5 - protons of cyclohexanone ring. Shape of absorption peak is typical of cyclo- hexyl ring protons

2.6

3.5

4 .8

5 - Sharp peak is N-CH3, rounded - peak at 2.7 ppm represents 2 protons of cyclohexanone ring

hexanone 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

1 - One of protons on cyclo-

2 - Two protons. Total inte- gration is 19 spaces; sub- tract 5 spaces for D20 blank to give 14 spaces or two protons. These are exchangeable protons so that they are -NH - and H-C1 - protons

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 of 20.4 and 23.2 respectively.

264 nm. (a 1%, 1 cm. = 16.6), 269 nm. (a 1%, In 0.1N sulfuric acid, maxima at


W o v e length (nm)

301

2 74

268

261

2 5 X \

1% al cm

5.0

7.0

9 8 10.5

\ m O R S 8 N 0 t i N N 4 8 5 3 3 0

W o v e length (nm)

Figure 5 . Ultraviolet Spectrum of Ketamine Base in 95X methanol - 0.01N sodium hydroxide.

K ETAM IN E 307

1 cm. = 23.2), and 2 6 nm. (a 1%, 1 cm. = 20.3)

Figure 5 represents the alkaline

have been reported. 2i:

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 base6~30 is 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

Ketamine reportedly melts at 92-930'. A 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 ml. of methanol 14 ml. of 95% ethanol (USP) 60 ml. of chloroform 60 ml. of absolute ethanol

One gram of the hydrochloride is incom- pletely dissolved in 60 ml. of acetone, ether, benzene, DMF, or dioxane.

RRW D R T R DR. SRSS. PARKE D F I V I S : B R S E FORM 16-JUN-76 FI 52 TIC = 8118 3.96 M I H RGNGE so THRIJ 250 THRESHOLU = 0 . 0 0

fl/E I? I f l / E R I M/E R I

se) .~5m 0.16 7 6 . 3 ~ 0 . 3 1 183.201 0.52 53.258 0 . ? G 7i.117 0.35: 110.484 0.50

32 0 . 1 8 112:?02 0.28 l? 0.16 214.900 8 . R 6

5 9 . 177 0. 16 69 0.21: 116.19? 0.64 62.89R 0. 19 89 0.25 117.302 n.csr 64.E18 0.19 .375 0.22 123.972 0.16 67.314 0.14 91.548 i3.18 125.630 1.06 78.302 17.18 9H.173 0.34 127.4'34 0.36 72.537 3 . 5 0 101.466 0 .42 128.392 R.28 i > . 9 9 5 n.52 li32.232 1.28 123.44% 0.20

M/E R I

130.949 B . 3 6 171.5rlc 8.38 132.378 8.52 137.833 R.74

138.982 8.70 i:?.-e2 1.74

148.183 1.82 141.351 0 . 2 4 143.93Z 0.92 144.OIJtl 1 . 12 14F.355 1.66

M Y E

146.875

1 ~ 0 . ~ 3 9 148.333

151.681 352.718 154.3 12 356.858 164.637 165.8 14 i 66. 820 168.130

R I

0.26 0. 18

3.62

1.94 6.24

1.30 1.72

8.82

0.80

8 . 2 4

I .sa

f l /E P I

169.ias 0.80 171.283 8.16 174.244 1.94 175.468 0.36 178.322 @.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

Figure 6. Mass Spectrum of Ketamine Base

KETAM IN€ 309

Figure 7. Differential Thermal Analysis Melting Curve.



Isolation of the d(+) isomer of the hydrochloride from a-racemzc mixture using d- camphorsulfonic 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. and 100 mg./ml. solutions of the hydrochloride are 4.63, 4.16, and 3.92 respectively.

The pH of 10, 50,

2.7 Crystal Properties

2.71 Derivative Crystallinity 24

In latinic iodide solution, rhom- boidal 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 Diffracto- meter6929 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 5.8 5.30 1.4 7.43 100.0 4.87 42.1 6.92 3.0 4.63 5.9 6.44 15.6 4.55 12.1 6.14 11.6 4.32 1.5 5.90 1.8 4.14 7.9

KETAM INE 31 1

4.13 3.72 3.57 3.45 3.35 3.25 3.22 3.17 3.15 3.05 2.92 2.90

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

2.70 35.2 2.63 2.7 2.44 3.4 2.11 1.8 2.02 4.0 1.83 1 . 8 1.79 2.6 1.75 2.6 1.64 2.6 1.44 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 levels (half-life 10 min. 11 min. 17 rnin.l2, and 25 min.13) due to distribution of drug to the tissues is followed by a first order decrease in plasma lev 2.5 hours. 2

ith a half-life of about fP2X

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.

intact drug excreted, the decomposition scheme shown in Figure 9 has been suggested.llp15,25 indication of Another report33 suggests that if present, protein binding does not exceed 12%.

In addition to small amounts of the

No otein binding was observed. 11


1. A1220 P A r - C N 2 . N a O H

I11

1. aq. C H 3 0 H , NaOH

2 . H C 1 * Ar*-CH=NOH

i1

0 1. BrMgcl 11

C U C l A r / ' > C 7 2 . H 2 0 , H C 1 H

IV

0ch3 F1 C H 3 0 N a 1

*=2 C H 3 0 H , A

B r - A,/'= -f cc14

v

VIII

NCH 3

A r /cm * H C 1 * HO /'

I X

= a c1

VI

VII

pc. /

X

k e t a m i n e h y d r o c h l o r i d e

F igure 8. Syn the t i c Procedure

H

H

a

aJ U

rd M

3

'r)

U C

P

0

H

H

8 4 !-l H

H

H

a

PI

+J rd bo

H

W

*

a,

I 3

m

9 U

0

0

*

4

6-

U

U

313

314 W I L L I A M C. SASS A N D S A L V A T O R E A. FUSARI

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 hydro- lyze to (2-chlorophenyl)(l-hydroxycyclopentyl) methanone (111), the primary product of this reaction. 2-(2-chlorophenyl)-2-hydroxycyclo- hexanone (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

E 1 emen t Found3 Theory

% C 57.05-57.29 56.94 % H 6.49-6.61 6.25 % N 4.95 5.11 % C1 (total) 25.88-26.02 25.86 % C1 (ionic) 13.06 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. Ti 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.

KETAM INE 315

A r * A r

Ketamine Hydrochloride

c1 9 0: - I V

Ketamine Base

A r

I11

1

I

Figure 10. Chemical Decomposition


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 recrystallized sample which contains less than 1 x 10-3 mole % impurity.

Figure 7 is a thermogram of a

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 9 15

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 31 7

5.72 Thin Layer Chromatography

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 used25~27 to separate ketamine (Rf = 0.58) and the N-dealkylated metabolite (Rf = 0.41) 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-hydroxycyclo- penty1)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.

Two of the four tritium labeled

5.73 Gas Chromatography

Since gas chromatography allows

The use of all glass systems22

rapid, quantitative analysis of ketamine and its degradation roducts, numerous systems have been utilized.20,51 and the avoidance of evaporation to dryness13 have been suggested to avoid degradation. Chroma- tographic 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

11 1% ECNSS-M

12 3% OV-17 3% (100/120 Gas Chrom Q)

13 1% OV-101 and 3% succinamine polymer on (100/120 Gas Chrom Q)

Gas Chrom P) 15 1% ECNSS-M (80/100

20 2.5% SE-30 (80/100 Chromasorb G)

21 1% DDTS Gas Chrom Q

Column Temp. Detector Internal Standard

155O FID, EC o-trifluoromethyl

195O E.C. of hepta- o-Bromo analog"

and o-Bromo analogs*

fluorobutyryl derivative

158O FID CL-392

170°

200°

180'

FID

FID

o-trifluoromethyl analog*

Pent ob arb i t a 1

TABLE I1 (Continued)

Ref.

22

2 3

26 w, W

28

25 9

27

Column

0.5% polyethylene- glycol (20,000 M) (80/100 Chromasorb G) silinized

2YL SE-30 (80/100 Chromasorb G)

0.5% PEG 20000 M (80/100 Chromasorb G - DMC S )

10% UCW-982 (80/100 CWAW-DMCS)

1% Carbowax 20-M (60/80 Gas Chrom G AW - DMC S )

Column Temp.

@ 3O/min. 98-180O

zooo

90-2oooc.

27OoC.

21oOc.

Detector Internal Standard

FID methyldiphenylamine

FID

FID

FID

FID

Pen tobarb i t a1

Carbo thes in

- (all separated as p-nitrobenzamides)

*analogs of ketamine


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 J s tritium labeling18 5J11J19,25,27J gas ?f'€ES~fT~!!B, 22 25-28,31 and liquid thin layer chr

chromatography

the determination of ketamine and its metabolites from body fluids.

techniques have been applied to

KETAMINE 32 1

7 .

1. 2 . 3 .

4 .

5 .

6 .

7 .

8 .

9 .

1 0 . 11.

1 2 .

13.

1 4 .

1 5 .

1 6

1 7 .

18

1 9 .

20 .

21

22 9

References (Current to June, 1 9 7 6 )

The Merck Index, Eighth Edition, 599 ( 1 9 6 8 ) .

Wheeler, L.M., Parke, Davis & Co., Personal Communication. Fusari, S.A., Parke, Davis & Co., Personal Communication. Chang, J.H., Parke, Davis & Co., Personal Communication. Sass, W.C., Parke, Davis & C o . , Personal Communication. Nordin, I.C., Parke, Davis & Co., Personal Communication. O’Connor, R.E., Parke, Davis & Co., Personal Communication. McCarthy, D.A., Parke, Davis & Co., Personal Communication, Chem. Abs. 6 5 , 5414h ( 1 9 6 6 ) . Chang, T., uazko, A.J., Int. Anesthesiol. Clin. 1 2 , 157-77 ( 1 9 7 4 ) . Chang,T., Glazko, A.J., Anesthesiology - 3 6 ,

Hodshon, B.J., Ferrer-Allado, T., Brechner, V.L., et. al., Anesthesiology - 3 6 , 506-8 ( 1 9 7 2 ) . Philip, J., Parke, Davis & C o . , Personal Communication. Chang, T., Dill, W.A., Glazko, A.J., Fed. Proc. - 2 4 , 268 ( 1 9 6 5 ) . Dill, W.A., Chucot, L., Chang, T., Glazko, A.J., Anesthesiology - 3 4 , 73-6 ( 1 9 7 1 ) . Nishijima, M., Fujii, A., Kojima, T., et. al., Jap. J. Anesthesiol. 2 1 , 881-5 ( 1 9 7 2 ) . Blackburn, C.E., Ober,R.E., J. Labelled Compounds 2, 38 ( 1 9 6 7 ) . Glazko, A.J., Parke, Davis & C o . , Personal Communication. Finkle, B.S., Cherry, E.J., Taylor, D.M., J. Chromatogr. Sci. 9 , 393-419 ( 1 9 7 1 ) . Jenden, D.J., Roch, R . , Booth, R., J. Chromatogr. Sci. 10, 1 5 1 - 3 ( 1 9 7 2 ) . Wieber, J., HengstmaE, J., In: Ketamin, Neue 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

RX Bull, 3 , 5-10 ( 1 9 7 2 ) .

401-4 ( 1 9 7 2 ) .

- 6 9 , 146-50 ( 1 9 7 3 ) .


23. 24.

25 .

26.

27 .

28 .

29 .

30 .

31.

Moffat, A.C., J. Chromatogr. - 113, 69-95 (1975) . Clarke, E.G.C., Isolation and Identification of Drugs, 1969 , The Pharmaceutical Press, 1 7 Bloomsbury Square WC1, London, England. Kochhar, M.M., et. al., Res. Commun. Chem. Pathol. Pharmacol. - 1 4 , 367-76 , June 76 . Wieber, J., et. al., Anaesthesist 2 4 , 260-3, June 75.

- Kochhar, M.M., et. al., Clin. Toxicol. - 9 ( 1 ) , 2 0 - 1 . 1976 . Needham, L.L., et. al., J. Chromatogr. - 1 1 4 ,

Krc, J., Parke, Davis & Co., Personal Communication. Leavett, R., Michigan State University, Personal Communication. Cohen, M.L., Trevor, A.J., J. Pharmacol. Exp. Ther., - 189, 351-8 , May 1 9 7 4 .

220-2, 1 2 NOV. 75.

MINOCYCLINE

VMimir Zbinovsky and George P. Chrekian

324 VLADlMlR ZEINOVSKY AND GEORGE P. CHREKIAN

CONTENTS

1. Description



2 . 1 Infrared Analysis 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectra 2 . 4 Mass Spectra 2.5 Optical Rotation 2 . 6 Thermogravimetric Analysis 2.7 Differential Thermal Analysis 2.8 Solubility 2.9 Solvent Partitioning Data 2.10 Crystal Properties

3 . Synthesis

4 . Stability, Isomerization, Degradation

5 . Pharmacodynamic Studies


6 . 1 Elemental Analysis 6 . 2 Chromatographic Analysis

6.21 Thin Layer 6.22 Column

6.3 Direct Spectrophotometric Analysis

MINOCYCLINE 325

MINOCYCLINE HYDROCHLORIDE

1. Description


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-6- demethyl-6-deoxytetracycline hydrochloride.

OH

.HCL

CONHz

0 OH 0 OH

C23H2,NJO,.HCL MOL. Wt.: 493.94


Minocycline hydrochloride occurs as a yellow crystalline powder, somewhat bitter taste.

It is essentially odorless and has a


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,

In these cases it is not possible to uniquely most maxima represent a composite envelope of overlapping absorption peaks.

FIGURE 1

Infrared Spectrum of Minocycline HC1.2H20 i n KBr P e l l e t : Instrument: Ferkin-Elmer 2 1

FREQUENCY (CM-’)


MINOCYCLINE 327

a s s i g n maxima. Thus, t h e maximum a t about 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, toge ther wi th 1 2 hydroxy. The remainder of t he broad absorp t ion up t o 5.0 !-I i s composed of t h e hydrogen bonded phenol ic and e n o l i c hydroxy groups p lus t h e hydrogen atom on the protonated dimethylamino group. The maxima a t 6.07 1.1 is t h e carbonyl of t h e 2-carboxamido group, bu t t he broad maxima centered at about 6.25 1.1 is a composite of conjugated hydrogen bonded ketones, p lus t h e conjugated double bond systems p resen 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 phenol ic and e n o l i c hydroxyl groups p lus a c o n t r i b u t i o n from the 2-carboxamido group and t h e maxima a t about 8.2 1.1 i s composed of r e l a t i v e l y unbonded phenol ic hydroxy groups.

2 . 2 Nuclear Magnetic Resonance Spectrun’

The M.IR spectrum, Figure 2 , i n hexadeuterodimethyl- su l fox ide conta in ing te t ramethyl s i l a n e as i n t e r n a l s tandard i s a s i n g l e scan on a HA-100D Varian Spectrometer. s p e c t r a l assignments of Minocycline hydrochlor ide are shown i n Table I.

The

TABLE I

NMR S p e c t r a l Assignments of Minocycline Hydrochloride

c - NH2 It 0

C 1 o - OH

Chemical S h i f t s ( A )

2.60 S

2.94 S

4.34 S

7 . 4 1 d; J8,q = 8

6.83 d; J8,9 = 8 9.05 9.53 (2 broad s i n g l e t s )

11.30

s = s i n g l e t ; d = doublet ; J = coupling cons tan t i n Hz

MINOCYCLINE 329

2.3 Ultraviolet Spectrum

Martell et a12 in 1967 determined the ultra-violet properties of Minocycline. They reported -

in 0.1N HCL

in 0 . 1 N NaOH

X max 352 nm (log E 4.16) 263 nm (log 6 4.23)

243 nm (log E 4.38) X max 380 ~1 (log E 4-30]

2.4 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 Mino- cycline is available from Dr. R. T. Hargreaves, Lederle Labor- atories.


The following rotation was determinedl for Minocy- cline HC1.2H20 in 0.1N HC1:

Cal 25 - 166', conc. = 0.524

2.6 Thermogravimetric Analysis7 indicates that Mino- cyclineohydrochloride loses its water of hydrgtion between 75' and 150 and begins to decompose at about 175 .

2.7 Differential Thermal Analysis7 curves for Minocy- cline 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

0

w

2 m C H

0

2

hl

Ti

V

X

.. U

L)

a a

rn

rl

l-l

"

00

7

om

"

a

E

a

W r

v)

0

Ln N

(u

h .. m

0-

0

..

nlln

h

W

P

w

(I

0

J I

ou

I

n0

-

a

D

> I

w I

Y

J

u

L

0"

0

0

..I

CI

z

E

J

" m h

m

0

330

MlNOCYCLlNE 33 1

phenomenon. There are 16 p o s s i b l e i o n i c mic ros t ruc tu res f o r Minocycline. Thus, t he observed s o l u b i l i t y is gene ra l ly no t t h a t of a s i n g l e e n t i t y but r ep resen t s t h e sum of t he t o t a l of two o r mcre spec ie s i n a s o l u t i o n a t a given pH va lue . Minocycline, un l ike o the r a n t i b i o t i c s , con ta ins two amino groups which a r e respons ib le 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 ihydra t e i n va r ious so lven t s and of Minocycline n e u t r a l i n water are given i n Table I1 and Table I11 r e spec t ive ly .

TABLE I1

Aqueous S o l u b i l i t y of Minocycline a t 25OC.

Neut ra l

Hydrochloride

Dihydrochloride

pH 6.7

pH 3.9

m d m l

52

15

pH 0.8 >500

TABLE I11

S o l u b i l i t y of Minocycline Hydrochloride .2H7O i n

% Various Solvents a t 25'Cj

Solvent m d m l - w/v

Hexane 0.004 0.0004

Benzene

Chloroform

0.02

0.13

Ethyl Acetate 0.3 Methyl Ethyl Ketone 0.4

0.002

0.013

0.03

0.04

1-Oc t a n o l 0.5 0.05 Ace tone 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

Water 14

16

1 .4

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 va lues according t o Co la i zz i and Klink4 f o r t he apparent 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 water : n-octanol system a t var ious pH va lues are repor ted i n Table I V . 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 co inc ides wi th t h e i s o e l e c t r i c po in t of Minocycline.

The optimum pH va lue f o r t r a n s f e r i n t o t h e organic

TABLE I V

Apparent P a r t i t i o n Coef f i c i en t s (Octanol/Aqueous Buffer) of Minocycline Hydrochloride

2.1 3.9 5.6 6.6 8.5

0 0.051 1.11 1.48 0.36

2.10 Crys 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 hydrochlor ide is shown i n Table V.

M INOCYCLI N E 333

TABLE V

Powder X-Ray Diffraction Pattern of Minocycline HC15

d (Ao)* I / I O * *

12.0 0.15 7.05 1.00 6.60 0.04 5.70 0.08 5.20 0.07 4.95 0.09 4.73 0.09 4.45 0.01 4.28 0.06 4.00 0.04 3.82 0.15 3.68 0.50 3.56 0.45

3.26 0.40 3.03 0.04 2.86 0.05 2.73 0.02 2.67 0.02 2.60 0.01 2.44 0.06 2.31 0.02 2.25 0.02 2.13 0.02 2.06 0.01 1.96 0.01 1.91 0.01 1.85 0.03 1.72 0.02 1.52 0.01 1.20 0.02

3.43 0.02

* d = (interplanar distance) n X 2 sin 0, X = 1.539A0

** Based on highest intensity of 1.00 Radiation: Kal, and Ka2 Copper


3 . Synthes is

Previous syn thes 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-deoxy te t r acyc l ine2 . 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 undes i r ab le 9-n i t ro isomer involved ted ious procedures. La te ly , L. Bernardi and assoc i - a t e s 6 were a b l e t o block p o s i t i o n 9 w i th a t e r i a r y b u t y l group and thus s impl i fy 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 given i n Figure 4 .

6-demethyl-6-deoxytetracycline (I) was a lky la t ed t o g ive (11) wi th excess of t e r t i a r y b u t y l a l coho l and methane su l - f o n i c ac id . By adding fou r equ iva len t s of m02, compound (111) w a s obtained i n 76% y i e l d based on ( I ) . In te rmedia te compound (111) was c a t a l y t i c a l l y reduced over Pt02 t o g ive 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 then r educ t ive ly methylated t o (V). The l a s t s t e p involved the 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. This was accomplished by us ing t r i f luoromethane s u l f o n i c ac id wi th 63% y i e l d .

4 . S t a b i l i t y , I somer iza t ion , Degradation

I n t h e dry-powder s t a t e t he 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 yea r s when s t o r e d a t room temperature ( 2 5 C). Minocycline, l ack ing hydroxyl groups a t both C5 and c6 does not form t h e anhydro, iso, o r e p i compounds, which a r e the common degrada t ion compounds formed from o the 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 both 4-epimerization and ox ida t ive degrada t ion . Since 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-amino- phenol, i t is more s u s c e p t i b l e t o ox ida t ion than o t h e r t e t r a - cyc l ines .

0

S t a b i l i t y da t a f o r s o l u t i o n s of Minocycline a t va r ious pH va lues a r e summarized i n Table 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. than 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 tud ied .

These s o l u t i o n s were more s t a b l e

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 permit t h e p repa ra t ion of a p recons t i t u t ed 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, bes ides con- t r i b u t i n g t o increased s o l u b i l i t y of Minocycline n e u t r a l i n water , is a l so respons ib le f o r d i f f e r e n c e s i n physico-chemical

d

w

t t

335


and phys io log ica l p r o p e r t i e s . The i s o e l e c t r i c po in t of Minocycline is a f u l l pH u n i t h ighe r (pH 6 . 4 ) than 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 con- sequent ly has a p o t e n t i a l t h e r a p e u t i c s i g n i f i c a n c e . This p rope r ty 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, inc luding thy ro id , b r a i n and f a t t i s s u e .

PH

1.85

2 .5

4 . 2

5 . 2

6 .2

TABLE V I

A c t i v i t y Retained

Days S tored a t 2SoC

Minocycline So lu t ion S t a b i l i t y Data % I n i t i a l

0.5 1 1 . 5 2 3 4 7 8 9 11 14

96 9 4 9 1 22

97 95 93 8 1

99 96 98 95 90 9 1 90 87 84

98 98 98 96 92 89 85 8 1 72

98 95 93 89 7 6 72 64 53 37

5 . Pharmacodynamic S tud ie s

R. C . Kel ly and Assoc ia tes8 found t h a t t h e maximum serum concen t r a t ion of Minocycline was a t t a i n e d by t h e f i r s t sampl- i ng a t 1 hour and t h a t serum ha l f l i f e a f t e r o r a l admlnls t ra - 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 ighe r z w i t t e r i o n i c form which is predominant a t pH 6 . 6 , approximately one pH u n i t h ighe r than 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 igh ly l i p o p h y l i c t e t r a c y c l i n e has 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 ap id and h igh concent ra t ion of a n t i b i o t i c where recorded. 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, concen t r a t ions i n a l l t i s s u e d s t u d i e s were h ighe r than i n blood.

Okubo and

When t h e Minocycline was adminis te red t o p a t i e n t s be fo re su rge ry , a similar h igh t issue-blood r a t i o was found a f t e r

MINOCYCLINE 337

t h e organ was removed. c l i n e w a s found i n ga l lb l adde r , t hy ro id , duodenum and l iver .

The h ighes t accumulation of Minocy-

Minocycline i s metabolized t o i n a c t i v e subs tances t o a g r e a t e r ex ten t than o the r known t e t r a c y c l i n e s .


6 .1 Elemental Analysis 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


6.21 Thin Layer Chromatographic Analysis

Separa t ion and q u a n t i t a t i v e de te rmina t ion of Minocycline i n t h e presence of r e l a t e d minor components w a s achieved on diatomaceous e a r t h , used as suppor t ing phase. Plates were prepared by spreading i n t o a t h i n l a y e r a mixture of diatomaceous e a r t h , pH 6 EDTA b u f f e r , po lye thylene g lyco l 400 and g lyce r in . P l a t e s were developed wi th a so lven t con- s i s t i n g of a mixture of pH 6 EDTA b u f f e r and e t h y l ace t a t e - cyclohexane (9:2) . This system was previous ly used by P . P. AscionelO i n a sepa ra t ion of o the 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 spo t can be moved ha l f way on t h e p l a t e , thus g iv ing complete sepa ra t ion from t h e r e l a t e d compounds.

6.22 Column Chromatographic Analysis

Minocycline and r e l a t e d impur i t i e s were separa ted on an acid-solvent washed diatomaceous e a r t h column.11,12 Supporting phase was prepared by mixing t h e d ia - tomaceous e a r t h wi th 5% v /v polyethylene g lyco l 400 (PEG-400)- g lyce r ine mixture i n 0.lM EDTA pH 6 bu f fe r . r e l a t e d compounds were e l u t e d wi th s tepwise inc reas ing p o l a r i t y of t he chloroform-cyclohexane mixture and determined spec t rophotometr ica l ly a t 358 tun. 98-102% recovery of t he t o t a l s p e c t r a l va lue of t h e charge was obtained.

Minocycline and


6.3 Direc t Spectrophotometr ic Analysis

U. V. Absorption maximum of Minocycline a t 358 nm has been ex tens ive ly used f o r assay purposes , e s p e c i a l l y f o r reading of column e l u a t e s . The concent ra t ion of 1 6 micrograms p e r ml 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 has 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 concent ra t ion - abso rp t ion r e l a t i o n s h i p was achieved by Ace and J a f f e , 1 3 us ing pH 6.5 bu f fe r i n an ex t rac- t i o n of Minocycline. The f luorescence of t h e f i n a l product was read a t an e x c i t a t i o n wavelength of 380 run and an emission wavelength of 480 nm using a f i l t e r co lor imeter .

MI NOCYCLI N E 339

REFERENCES

1.

2.

3 .

4.

5 .

6.

7 .

8.

9.

10.

11.

12.

W. Fulmor, Leder le Labora tor ies , personal communication.

M. J. M a r t e l l , J. H. Boothe, J. Med. Chem.,x, 44 (1967).

W. C . Bar r inger , W. Shul tz , C. M. S ieger and R. A. Nash, Am. J. of Pharmacy, 146, 179 (1974).

J. L. Co la i zz i , P . R. Klink, J. Pharm. Sc i . , 58, 1184 (1969).

P . Monnikendam, Leder le Labora tor ies , personal communica t ion .

L. Bernardi , R. D e Cas t ig l ione , V. Colonna, P. Masi, I1 Farmaco, Ed. Sc. , 30 736 (1975).

L. M. Brancone, Leder le Labora tor ies , personal communica t ion .

R. G. Kel ly , L. A. Kanegis, Toxicol , Appl. Pharmacol., - 11, 171 (1967).

H. Okubo, Y. Fujimoto, Y . Okamoto, J. Tsukada, Jap. J. An t ib io t . , 22, 430 (1969).

P. P. Ascione, J . B. Zagar, and G . P. Chreklan, J. Pharm. S c i . , 56, 1393 (1967).

P. P. Ascione, Lederle Labora tor ies , personal communlca- t ion .

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, 12, 401 (1975)

NYSTATIN

Gerd W. Michel

342 GERD W. MlCHEL

TABLE OF CONTENTS

1.

2.

3.

4.

DESCRIPTION

1.1 Name, Formula, Molecular Weight, Elemental

1.2 Appearance, Color, Odor 1.3 Standards and Regulatory Status

Composition

PHYSICAL PROPERTIES

2.1

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

Crystal Properties

2.1.1 Optical Crystallographic Properties 2.1.2 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

BIOSYNTHESIS

METHODS OF MANUFACTURE

4.1 Historical 4.2 Microbiological Processes 4.3 Isolation and Purification Processes

NYSTATIN 343

TABLE OF CONTENTS (Cont'd)

5. STABILITY - DEGRADATION

5.1 Dry Thermal Degradation

5.1.1 Stability of Amorphous Product 5.1.2 Stability of Crystalline Product 5.1.3 5.1.4 Stability of Ointment Formulations

Stability of Solid Dosage Forms

5.2 Stability in Solution 5.3 Stability under Radiation 5.4 Microbial Degradation 5.5 Stabilization

6. METHODS OF ANALYSIS

6.1 6.2 6.3 6.4 6.5

6.6 6.7

6.8 6.9 6.10 6.11

Elemental Analysis Neutralization Equivalents Identification Tests color Reactions Direct Spectrophotometric Analysis

6.5.1 Fermentation Liquids and Products 6.5.2 Pharmaceutical Preparations 6.5.3 Other Applications

Colorimetric Analysis Chromatographic Analysis

6.7.1 Paper Chromatography 6.7.2 Thin-Layer Chromatography 6.7.3 Gas-Liquid Chromatography 6.7.4 High Performance Liquid Chromatography

Electrophoretic Analysis Polarographic Analysis Titrimetric Analysis Microbiological Methods

7. REFERENCES

8 . ACKNOWLEDGMENT

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 strepto- mycete 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 1 10-15.

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. Isolated in 1950 by Hazen and B r ~ w n ~ * - ~ l of the Division 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. 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: 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.

Initially called

Moronal, Mycomycin, Mycostatin,

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,

NYSTATI N 345

despite extensive efforts in a number of laboratorie~~~ I 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. structure of the aglycone portion of the molecule (nystatin- olide) 46, containing a diene and tetraene chromophore, has been deduced from the isolation of degradation products, Chong and R i ~ k a r d s ~ ~ have 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.

While the

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

17 COOH 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 re- resent mixtures of chemically closely related components56,

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 AlrA2 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.

8159.

The lack of uniformity between individual nystatin products generated under a wide variety of possible fermentation conditions161 27 ,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 chemothera- peutic 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.


Nystatin is a light yellow to yellow crystalline powder with a faint, characteristically musty odor; slightly hy- groscopic 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 Antibio- tic 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 Stan- dardization in 196373. 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

The reference material selected for

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.

348 GERD W. MlCHEL

The minimum allowable potency for commercial nystatin products was reviewed by the Food and Drug Administra- tion 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


2.1.1 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: para1 le 1 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. 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 hydra- tion.

A l l three forms are readily identified by their

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 A Type B

Squibb Res. Std. #MYNM-150-RP Squibb Res. Std. #MYNM-150-RP/H (Figure 1) (Figure 2)

d (8) * I/I,** v

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

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

0.27 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

0 *d = Interplanar distance (A), nh

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)

1/1 * * -0-

d (g) * 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

0.19 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 nX *d = Interplanar distance ( A ) , sin

**I/Io = Relative intensity (based on highest intensity of 1.00)

Radiation: K and K Copper C r l a2

Figure 1. X-Ray Powder Diffraction Pattern of Nystatin, Type A (Squibb Res. Std. #MYNM-150-RP) Instrument: Philips Norelco Diffractometer

w UI N

I 4 : I l I

1 I

Figure 2. X-Ray Powder Diffraction Pattern of Nystatin,

Type (Squibb Res. Std. #MYNM-150-RP/H) Instrument: Philips Norelco Diffractometer

w m w

.o 0

N d

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 . taken as a potassium bromide pellet (1.5 mg/300 mg KBr) was essentially identical to the one presented.

A spectrum of the same standard

Tentative assignments for some characteristic infrared absorption bands18153183-85 are listed in Table 111.

Table I11

Infrared Spectral Assignments for Nystatin (Squibb Res. Std. #MYNM-150-RPI Type A)

Frequency (cm-’) Vibrational ModeE6‘ 87

998

1065 1375 1448

1572 1705

3300-3500

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 ~ ~ 3 , A.O. 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 rela- tion 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.

4

a, rl

cv

355

Infrared Spectrum of Nystatin, Type A (Squibb Res. Std. #MYNM-150-W) Mineral Oil Mull Instrument: Perkin-Elmer Model 621


3500 2500 ZOO0 1800 1600 la00 1200 1OO0 800 600 200 FREQUENCY (CM’)

Figure 5. Infrared Spectrum of Nystatin, Type B (Squibb Res. Std. #MYNM-150-RP/H) Mineral Oil Mull Instrument: Perkin-Elmer Model 621

a, &

7

357

I n f r a r e d Spectrum of Nys ta t in , Type C (Squibb R e s . Std. #WSC-08982-FP) Mineral O i l Mull 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 comple- mented 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) Mu 1 t ipl ic i t y Assignment

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

Doublet Secondary Methyl Group Doublet Mu1 tip1 e t

I, 11 ,I

,I I, I,

I, II ,I

Methylene Proton I, II

I 1 11

0 , I,

Methine Proton (-CEO-)

Olef inic Proton

I 1 " (-CFOC=O)

Mu1 t iple t I, ,I

II I,

In addition, broad resonance occurs at 6 3.92 (NH2, OH, H20) which is exchanged with D2O91.

I 2 ) 1 1 ' 1 1 . 1 ' " " I " 4 1 1 ' ' I 1 " 1 ; 1 ' ' 1 , ' ' 1 ' ' , ' " ' ; " 1 ' ; ' I "; ' 1 ' ' , I ' I ' j " ' ' 1 1 ' I " , ' " I / , ' ' ' I : " ' 1 ; ' ' I 1 , " " L

Figure 7. NMR Spectrum of Nystatin (Squibb Lot #88645) Solvent : DMS0-d 6 Instrument: Varian Model XL-100-15

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 E nm (l%, 1 cm) -

280 (sh) 298 291 567 304 866 318 789

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 inf lection5r 32 83f is noted at 280 nm (El' = 298), and an additional band at 231 nm of lower absorp- tit??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

Source

Bolshakova e t a l . Brown and Hazen Doskochilova and G e s s Dutcher Dutcher e t a l . Dutcher e t a l . H a m i 1 t o n - M i l l e r Oroshnik and Mebane Oroshnik e t a l . Shenin e t a l . U m e z a w a Vining e t a l .

Ul t rav io le t Absorpt ion of N y s t a t i n

Reference

52 30 96 95 32 83 97 18a 12 56 89 1 3

Xhmax (nm)

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

2

c

k

a, -4

J

362

Ultraviolet Spectrum of Nystatin (Squibb Res. Std. #MYNM-150-=) Solvent: Methanol (1.076 mg/100 ml) Instrument: Cary 11 Spectrophotometer

NYSTATI N 363

Hayden et al . 5, 88. 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 corresponding absorbance maxima (#85

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 thorough- ly 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 cor- rected 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 Noone Yo0 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* 1 57-591 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-

200 280 360 440 520 600

Wavelength - nanometers

Figure 9. Fluorescence Spectra of Nystatin (Squibb Lot #88645) Solvent: Methanol Instrument: Aminco-Bowman SPF

NY STAT 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. 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.

The authors101 conclude that the

Other important features of the mass spectrum of per- TMS 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 cor- roborated 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 instru- mental conditions are givenlo'.


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 Solvent Reference

-100 25 AcOH 18a, 32 (C, not specified)

(C, not specified)

(C, not specified)

(C, not specified)

(C, not specified)

(C, not specified)

-ao 25 AcOH 83

-8O - AcOH 95

+21° 25 Pyridine 18a, 32, 83

+120 25 DMF 18a, 32

-70 25 0.1N HC1 in MeOH 18a, 32

Squibb Lot #a8645

+ 8.05 22.5 DMF

+21.04O 22.5 Pyridine (C = 1)

(C = 1)

94

94

2.8 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

NYSTAT I N 367

1650Ce3 , respectively, without melting by 25OoC.

Squibb Res. Std. #MYNM-150-RPI when heated on a Mett- ler 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 : Microtube (1.6-1.8 mm) /

Reference: Glass Beads Heating Rate : 15O~/min Temperature Scale: 50°C/in. AT : 1°C/in.

The respective thermogramsg4 , reproduced in Figure

Air Atmosphere

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 dis- cernible 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 l-

1 0 a

0 z W

TYPE A

% T V

TYPE B

TYPE C 7 153 'C

1 I I 1 1

50 100 150 200 250 Temperature, "C

Figure 10. DTA and TGA Thermograms of Nys ta t in (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 Thermogravi- metric 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 up to 13OoC -15 up to 2oooc

12.5 up to 13OoC -20 up to 185OC

4.0 up to 13OoC -12 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.

37 Solutions and suspension of nystatin in water 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 Me thano 1 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 ado3 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

NY STAT 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 (23 2 l0C) have been de- termined82 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. Indi- vidual solubility data are summarized in Table VII, and the corresponding solubility curves are presented in Figures 11 and 12.

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 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.

2'

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)

MeOH/H20 Gravi- Spectro- metric photom.

% Solvent Method Method (vol . /vol . I mg/ml u/ml* )

100 98 96 94 93 92 90 80 75 70 65 60 50 40 30 25 20 10

9.0440 9.6530 9.8440

9.4520

8.8080

2.4333

-

-

-

- - -

0.6520 - -

0.3240 - -

52 , 193 54,627 55,428

53,493

47,957

11 , 019

-

-

-

- - - 1 9 6 1 - - - - -

EtOH/H20

Gravi- Spectro- metric Method mg/ml

1.1240 1.0880 1.5160 2.0520

2.3680 2.3920 1.8560

1.7240 1.5960 1.5560 1.1480 0.7920 0.5720

0.3960 0.2480

-

-

-

photom. Method u/ml * 1

3800 4175 5910 9045

11,713 10,972

7284

6403 6463 5956 4322 2528 1191

450 356

-

-

-

* ) Based on a potency for Squibb Res. Std. #MYNM-150-W of 6190 u/mg (spectrophotometric assay, Section 6.5) .

0000000000

..

..

..

..

..

u o

u

40

rl

+I +

I m

373

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 (Squibb Res. S t d . #MYNM-150-RP) S o l v e n t Systems: Methanol/Mater, 23 + l0C

Ethanol/Water , 23 - +-l°C

W .A

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. More- over, 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 A2- component 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. 64r65 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 (A3) 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 ~ ~ ~ l ~ ~ conclude 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 on ly main constituent differ- entiating 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 pK2 (proton lost) = 8.89

Recently, Valentilg5 determined the ionization constants and the isoelectric point of nystatin in a ternary so 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, nystatin- Type 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 antibio- tics114 , 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 a1* 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,

n-propanolll6 , i ~ o p r o p a n o l l l 6 , ~ ~ ~ - ~ ~ ~ I 153 , methanol/ethanol (1 : 1) 116 , acetone33 I 1491 151 , 80% acetic acid/xylene150 and pyr idine154.

151-153, ethano1116,153, n-but~no~33136,114,116,145~147,

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 methan01~~,142,1~~, or 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 requir- ing 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 suscepti- bility 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-hexameta- phosphate. 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 ex- tent 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 38 1

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 10noval~~ 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 Na- metabisulfite) 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. 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.

The authors164 found

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. Benzoylper- oxide, 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 un- protected 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-hexameta- phosphate in aqueous isopropanol solution165 1 166 were reported to have superior stability, while being less susceptible to the deteriorating effect of humidity. 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.

The authors165 suggest

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. 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 investi- g 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 loss 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-

At either

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 6- month 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 cup- plate 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. 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.

In

Boudru and B ~ u i l l e t l ~ ~ examined 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 Va~ileval~~. 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 detec- table differences in the toxicity between irradiated and non- irradiated 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 Vondra~ek’~~ to several other antioxidants , in-

386 GERD W . MICHEL

cluding hydroquinone, 8-naphthol, propylgallate and 2,6-di- tert-butyl-o-cresol. - 6. METHODS OF 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 liter-

experimental e v i d e n ~ e ~ ~ , ~ ~ ~ lo’ 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.

aturel8a I 31 I 33 I 46 I 49 I 50 I 54 I 83 I 95 I 105 i 115. Among these , latest

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 re- serve, 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

Element H N Ref. - - C - -

% Theory 60.95 8.17 1.51 - (Calculated for C47H75N017)

% Found 58.86 8.97 1.7 32 58.50 8.57 1.6 32 58.42 8.18 1.66 83 58.58 8.28 1.62 83 58.42 8.18 1.6 95 58.22 8.21 1.51 105 58.21 8.26 1.75 105 58.86 8.21 1.64 180*)

*) Squibb Res. Std. #MYNM-150-RP

NYSTATI N 387

6.2 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 e ~ ~ and methanolg5). following neutralization equivalents were determined:

The

Neutralization Equivalents (NE)

Ref. - As Base As acid

956 955 , 956 955 -

922

950 950

- 32 83 95 181*)

* ) Squibb Res. Std. #MYNM-150-W

6.3 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.

Ref. - - Test Response

Benedi c t Positive 2 Carbazole Positive 2 , 32,126 Mo 1 is ch Positive (Faint) 2,31,32,83,126 Schif f Positive (Atypical) 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 I le4.

Fehling32,83,126f ferric chloride32r126t183, Millon32183t126, ninhydrinle3 , T01lens~~ r 83 126 , and 2 , 4-dinitrophenylhydrazine 83 reagents.

However, it does not give positive tests with biuretle3,

388 GERD W . MICHEL

6.4 Color Reactions

Seve a1 color reactions typical for nystatin have been reported’ (see tabulation below).

Reagent Color Ref.

Hydrochloric Acid Yellow 183 Phosphoric Acid Pink 183 Sulfuric Acid, Conc. Violet to Blue 32,126,183,184

FeC13-K3Fe (CN) Strong Blue 32,184 SbC13 in Chloroform Pink 184

-

to Black

(Carr-Price)

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 color- ation on addition of concentrated hydrochloric acid or trichloroacetic acid to an alcoholic solution of ny~tatinl~~.

Laubielg7 noted that a pink color is formed by heating an alcoholic solution of nystatin in the presence of re- sorcinol 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.

197 A related procedure, described by the same author 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

NYSTAT IN 389

in Section 6.6.


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 studies 97 I 1051 1-74 and in chemical transformations52 i 190-192.

185

the 170,

Although these methods were found by some authorsg6' to correlate acceptably with the biological activit of antibiotic , the majority of ~ t u d i e s l l , ~ ~ 83,96,97,185,168, 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 the difference i n ex t inc t ions a t 304.5 and 312 nm, is reported by Doskochilova and Gessg6. The method des- scribed i s claimed t o give r e s u l t s comparable t o those obtained by the biological assay, using an ac t id ione - re s i s t an t s t r a i n of Candida albicans (BUCAV 44) as test organism i n a p l a t e method of cu l t i va t ion . Sa t i s f ac to ry agreement between both spectrophotometric and biological methods w a s reported t o be maintained during the e n t i r e course of a fermentation. However, on prolonged fermentation beyond the attainment 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 deviate from each o the r , with the b io log ica l assay ind ica t ing a sharper decline i n a c t i v i t y of t he cu l tu re f l u i d than r e f l e c t - ed by the spectrophotometric method. The authors96 explain t h i s discrepancy with the l i k e l y decomposition of t he an t i - b i o t i c on extended fermentation, concurrent l o s s of bio- a c t i v i t y , but r e t en t ion during decomposition of the polyene chromophore responsible f o r the u l t r a v i o l e t absorption of ny- s t a t i n .

Alternate spectrophotometric assay procedures f o r the determination of nys t a t in , developed by Sherman e t al.186,187 , attempt t o account f o r the presence of ultraviolet-absorbing b a l l a s t substances i n fermentation l i qu ids and unpurified intermediates which otherwise tend t o a f f e c t the desired accu- racy of quan t i t a t ive assay methods based on ex t inc t ion measurements. The proposed d i f f e r e n t i a l methods, appl icable t o both broth and i so l a t ed product samples, involve the ext inc- t i o n measurement of nys t a t in solut ions ( i n methanol/dimethylsulfoxide mixtures) a t the absorption m a x i m u m i n the 302-306 nm range, plus the determination of the ex t inc t ion f o r the minima on e i t h e r s ide of the peak absorption, i . e . , near 295 and 312 nm, respectively. Detai ls of t he quan t i t a t ive procedures developed f o r the determination of nys t a t in broth and the pu r i ty of bulk product i n r e l a t i o n t o a standard sample a re out l ined below:

(a) Nystatin i n Broth

Procedure186 Measure 20 m l of well-mixed whole broth 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 deaerate the broth sample, spin f o r 5 min a t 2000 rpm i n a s u i t a b l e centr i fuge, and again mix the t e s t tube contents on a Vortex Mixer f o r 15-30 sec.

P ipe t t e 2 m l of the well-mixed sample i n t o a 100-ml volumetric f l a s k , add 75 m l of dimethyl-

NYSTATI N 39 1

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 What- man #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:

= Nystatin units/ml K

A = Absorbance at about 304 nm B = Absorbance at about 296 nm C = Absorbance at about 312 nm D = Dilution factor (2500) E = Potency of nystatin reference standard

K = Standardization factor determined with (unit s/mg )

nystatin reference standard by the procedure outlined below.

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 set- ting 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 =

B + C (A - 7) x D

Weight of standard in mg

A = Absorbance at about B = Absorbance at about C = Absorbance at about

304 nm 296 nm 312 nm

D = Dilution factor (500)

(b) Nystatin Products

187 Procedure 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 312 nm).

Calculation :

B + C ( A - - ) x D x E

K x Weight of sample in mg 2 = 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

K = Standardization factor determined with (units/mg)

nystatin reference standard by the same procedure as outlined above under (a) for nystatin in broth.

NYSTATIN 393

6.5.2 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. 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.

Ochab202

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. with a molar ratio of nystatin : titanium of 1:3 exhibits strong absorbance at 450 nm and its formation obeys Lambert- Beer'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.

The resulting complex

Chang et a1 . 205 have proposed a colorimetric method for the assay of nystatin, both as bulk material and in phar-

NY STAT I N 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. statin activity in creams, ointments and capsules, and was also employed in stability studies.

The procedure was applied to the measurement of ny-

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. tion of degradation products formed on storage of methanolic nystatin solutions in the presence of acid (pH 4 ) and alkali (pH 9).

The procedure has also been applied to the determina-


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 1 70 , 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 establish- ing 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 oly- enes is illustrated in several reviews16 70, 208-210 1 212 1 2y3 and individual studiesla3 1211 I 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

w (D U

Solvent System (See Table X)

H

I J K L

M

TABLE IX

Paper Chromatography Systems for Nystatin

Paper

Whatman No. 1 Not reported Whatman No. 1 Whatman No. 1 Arches No. 302 Whatman No. 1 Whatman No. 1 Arches No. 302 Schleicher & Schhl 2043b,

Schleicher & Schhl 2043b,

Whatman No. 1 Whatman No. 2 Whatman No. 1 Not reported Whatman No. 4

"hydrophobed"

" hydrophobed"

Development Method of Time (hrs) Detection 3-

(See Table X )

15-16 - 18

16 15-16

- 15-16 18 15

21

17 18-24 6-7 - -

1 2 3 1 4 5 1 4

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

617,8 Not reported

2 Not reported 2 0.40 1 0.82 ,O. 78 - Not reported 2 Not reported

Reference

126 , 215 65

221,222 126 , 215 218 177

126,215 218 217

217

211 220,221 126,215 219 65 , 67

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 authors2241 225 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 pH- chromatogram) 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 - mani- fests 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 o ~ ~ ~ recorded 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 co-~orkers~~ I 65 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 for Nystatin

Solvent System Adsorbent (See Table XII)

Method of

(See Table XII) Detection 3-

A A A €3

C D E F G H I I J K L M N 0 P

Q R

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

0.5 0.5 0.45 0.66 0.54 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)*

Reference

228 228 228 184 184

229-231 232 228 233 235 232

221,236 221,236 232 232 232 237 234 238 239

229-231

*Migration of nystatin relative to penicillin-G (1.0)

TABLE X I 1

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 : 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)

2 )

Methyl Ethyl Ketone/McIlvaine Buffer, pH 4.7/Ethanol (100:6.4:22) 0.025M - Phosphate Buffer (KH2P04-NaOH, pH 6.0), 0.5M - NaCl

TABLE XI1 (Cont'd.)


P 0 h)

Methods of Detection 1 2 3

8 9 10 11

12 13

Ultraviolet Light228,235i236 Bioautography vs. Candida albicans228 0.2% p-Dimethylaminobenzaldehyde Spray Reagent

0.5% Potassium Permanganate/O. 2% Bromophenol Blue Spray Reagent231 5% Potassium Permanganate Spray Rea ent (or H3P04)242

1% p-Dimethylaminobenzaldeh de/20% SbC13 Spray Reagent 235 (in EtOH, contg. HC1)

Ultraviolet Light (Fluorescence @ 350 nm) 235 0.02N Potassium Permanganate Spray Reagent221r236 Iodine/2,7-Dichlorof luorescein Spray Reagent237 Bioautography vs. Candida tropicalis SC 1674237 , or

Chlorine/o-Toluidine Spray Reagent243 Bioautography vs. Saccharomyces cerevisiae ATCC 9763237 239 1 240

(in H2SO4, contg. trace FeC13) 218,235

Charring with mineral acid (H2SO4) 233

Saccharomyces cerevisiae SC 160c123~ 2 3 9 p 240

NY STAT 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 r ~ ~ ~ and Kocy and Cole237.

N~ssbaumer*~~ 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 incom- plete 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. 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.

Some of the identified

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-di-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) , 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.

Finkle -- et al.244 developed a simple GLC

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 dis- tinguished 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 character- ize 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 Schaf fner2471248 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 instru- mentation 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 a ~ ~ ~ have 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 ti- trants, 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 Sec- tion 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 satura- tion 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 1 253 I 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 reviews511691 1701 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 intra- cellular constitwnts259, the analysis of released potassium ions260 and of yeast cell constitutents, specifically ninhydrin-positive mine products261; the latter method is an automated procedure.

Nystatin in animal feeds is measured by an a ar dif- De- fusion method following extraction with methan~l~~~,~'~.

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 micro- scale turbidimetric method is approx. 1 unit per ml.

408 GERD W. MICHEL

7. REFERENCES

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16. 17.

T.H. Sternberg and V.D. Newcomer, eds., Therapy of Fungus Diseases, An International Symposium, Little, Brown and Co., Boston/London 1955. S.A. Waksman and H.A. Lechevalier, The Actinomycetes, Volume 3, Antibiotics of Actinomycetes, The Williams and Wilkins Company, Baltimore, 1962. D. Perlman, in D.J.D. Hockenhull, ed., Progress in Industrial Microbiology, Volume 6, Heywood Books, London, 1967, p.3. J.D. Dutcher, in Kirk-Other, Encyclopedia of Chemical Technology, 2. Edition, Volume 16, Interscience Pub- lishers, New York, 1968, p.133. G.A. Brewer, Jr. and T.B. Platt, in F.D. Snell and C.L. Hilton, eds., Encyclopedia of Industrial Chemical Analysis, Volume 5, Interscience Publishers, New York/ London/Sydney, 1967, p.564. D. Perlman, in A. Burger, ed., Medicinal Chemistry, 3. Edition, Part I, Wiley-Interscience, New York, 1970, p.323. A.I. White, in C.O. Wilson, 0. Gisvold, and R.F. Doerge, eds., Textbook of Organic Medicinal and Pharmaceutical Chemistry, 6. Edition, J.B. Lippincott Company, Philadelphia/Toronto, 1971, p.377. Z. Rehacek, in J.C. Sylvester, ed. , Antimicrobial Agents and Chemotherapy - 1963, American Society for Microbiology, p.530. V. Sevcik, I. Malek, V. Musilek, and Z. RehAcek, Anti- biotica aus Actinomyceten, Fischer-Verlag, Jena, 1963. Y. Okami, R. Utahara, S. Nakamura, and H. Umezawa, J. Antibiot. (Japan) 2, 98 (1954). R. Utahara, Y. Okami, S. Nakamura, and H. Umezawa, J. Antibiot. (Japan) E, 120 (1954). W. Oroshnik, L.C. Vining, A.D. Mebane, and W.A. Taber, Science 121, 147 (1955). L. Vining, W.A. Taber, and F.J. Gregory, in H. Welch and F. Marti-Ibanez, eds., Antibiotics Annual 1954- u, Medical Encyclopedia, Inc., New York, 1955, p.980. R.A. Pledger and H. Lechevalier, in H. Welch and F. Marti-Ibanez, eds., Antibiotics Annual 1955-1956, Medical Encyclopedia, Inc., New York, 1956, p.249. S. Ball, C.J. Besell, and A. Mortimer, J. Gen. Micro- biol. =, 96 (1957). L.C. Vining, Hindustan Antibiotics Bull. 3, 37 (1960). A. Neelameghan, Hindustan Antibiotics Bull. 2, 131 (1960).

NYSTAT IN 409

18.

19. 20. 21.

22.

23. 24.

25.

26. 27. 28. 29.

30.

31.

32.

33.

34.

35.

36.

a. W. Oroshnik and A.D. Mebane, in L. Zechmeister, ed., Progress in the Chemistry of Organic Natural Products, Volume 21, Springer-Verlag, Vienna, 1963, p.17.

b. W. Keller-Schierlein, in L. Zechmeister, ed., Progress in the Chemistry of Organic Natural Products, Volume 30, Springer-Verlag, Vienna, 1973, p.360.

c. G. Hildick-Smith, in B.M. Kagin, ed., Antimicro- bial Therapy, W.B. Saunders Comp., Philadelphia/ London/Toronto, 1970, p.119.

S.A. Waksman, Advan. Appl. Microbiol. 2, 235 (1963). E. Drouhet, Antibiot. Chemother. (Basel) 11, 21 (1963). H. Umezawa, Recent Advances in Chemistry and Biochem- istry of Antibiotics, Microbial Chemistry Research Foundation, Tokyo, 1964, p.44. S.A. Waksman, H.A. Lechevalier, and C.P. Schaffner, Bull. World Health Org. 33, 219 (1965). J.O. Lampen, Symp. SOC. Gen. Microbiol. 16, 111 (1966). S.C. Kinsky, in D. Gottlieb and P.D. Shaw, eds., Anti- biotics, Volume I, Mechanism of Action, Springer- Verlag, New York, 1967, p.122. E. Drouhet, in G.E.W. Wolstenholme and R. Porter, eds., CIBA Found. Symp. Systemic Mycoses, Churchill, London, 1968, p.206. J.O. Lampen, her. J. Clin. Pathol. 52, 138 (1969). J.M.T. Hamilton-Miller, Bacteriol. Rev. 37, 166 (1973). E.L. Hazen and R. Brown, Science 112, 423 (1950). E.L. Hazen and R. Brown, Proc. SOC. Exptl. Biol. s! Med. - 76, 93 (1951). R. Brown and E.L. Hazen, in T.H. Steinberg and V.D. Newcomer, eds., Therapy of Fungus Diseases, An Inter- national Symposium, Little, Brown and Co., Boston, 1955, p.164. R. Brown and E.L. Hazen, Trans. N.Y. Acad. Sci., Ser. 111, 19, No. 5, 447 (1957). J.D. Dutcher, G. Boyack, and S. FOX, in H. Welch and F. Marti-Ibanm, eds., Antibiotics Annual 1953-1954, Medical Encyclopedia, Inc., New York, 1953, p.191. E.L. Hazen and R.F. Brown, U . S . Patent 2,797,183 (1957); C.A. 2: 14211b. British Patent 866,600 (Charles Pfizer and Co.), 1961; C.A. 56: 781f. K.V. G o and W.P. Cullen, Abstr. 134th Meeting Amer. Chem. SOC. 22-0-23-0 , 1958. K.V. Rao, W.S. Marsh, and A.L. Garretson, German Patent 1,052,065 (1959); C.A. 55: 18009b.

-

410 GERD W. MICHEL

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

The Merck Index, 8th Edition, Merck L Co., Inc., Rahway, N.J., 1968, p.753. M.E. Florey, The Clinical Application of Antibiotics, Volume IV, Erythromycin and Other Antibiotics, Oxford University Press, London, 1960, p.164. Martindale - The Extra Pharmacopoeia, 26th Edition (N.W. Blacow, ed.), The Pharmaceutical Press, London, 1972, p.778. a. The United States Dispensatory and Physicians'

Pharmacology, 26th Edition (A. Osol, R. Pratt, and M.D. Altschule, eds.,), J. Lippincott Comp., Philadelphia/Toronto, 1967, p.775.

(E.E.J. Marler, ed.), Excerpta Medica, Amsterdam, 1973, p.319.

Griffiths, ed.), United States Pharmacopeial Con- vention, Inc., Eockville, Md., 1972.

d. G. Binder, P. Munchow, and M. Thonke, Zbl. Pharm. 110, 833 (1971); C.A. 76: 6648h.

The United States Pharmacopeia, Nineteenth Revision , Mack Publishing Company, Easton, Pa., 1975, p.348.

b. Pharmacological and Chemical Synonyms, 5th Edition

c. USAN 10 and the USP Dictionary of Drug Names (M.C.

British Pharmacopoeia 1973, Her Majesty's Stationery Office, London, 1973, p.328; ibid., Appendix XIVA, p.Al03. J.D. Dutcher, M.B. Young, J.H. Sherman, W.E. Hibbits, and D.R. Walters, in H. Welch and F . Marti-Ibanez, eds., Antibiotics Annual 1956-1957, Medical Encyclo- pedia, Inc., New York, 1956, p.066. D.R. Walters, J.D. Dutcher, and 0. Wintersteiner, J. Amer. Chem. SOC. 2, 5076 (1957). M.H. von Saltza, J. Reid, J.D. Dutcher, and 0. Wintersteiner, J. Amer. Chem. SOC. 83, 2785 (1961). C. Djerassi, P.C. Seidel, J. Westley, A.J. Birch, C.W. Holzapfel, R.W. Rickards, J.D. Dutcher, and R. Thomas, Abstr. Papers, 142nd Meeting, Amer. Chem. SOC., Sept. 1962, p.5P. J.D. Dutcher, D.R. Walters, and 0. Wintersteiner, J. Org. Chem. 28, 995 (1963). M.H. von Saltza, J.D. Dutcher, J. Reid, and 0. Wintersteiner, J. Org. Chem. 28, 999 (1963). A.J. Birch, C.W. Holzapfel, R.W. Rickards, C. Djerassi, M. Suzuki, J.W. Westley, J.D. Dutcher, R. Thomas, Tetrahedron Letters, 1485 (1964). A.J. Birch, C.W. Holzapfel, R.W. Rickards, C. Djerassi, P.C. Seidel, M. Suzuki, J.W. Westley, and J.D. Dutcher, Tetrahedron Letters, 1491 (1964).

NY STAT I N 41 1

51,

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

w.D. Celmer, in G.L. Hobby, Antimicrobial Agents and Chemotherapy - 1965, American Society for Microbiology, p.144. L.O. Bolshakova, B.G. Belenky, and S.N. Soloview, Antibiotiki 11, 892 (1966). L.O. Bolshakova, B.G. Belenky, and E . I . Pokrovsky, Antibiotiki 12, 884 (1967). M. Ikeda, M. Suzuki, and C. Djerassi, Tetrahedron Letters, 3745 (1967). E. Borowski, W. Mechlinski, L. Falkowski, T. Ziminski, and J.D. Dutcher, Rozniki Chem. 41, 61 (1967). Y.D. Shenin, T.V. Kotienko, and O.N. Exzemplarov, Antibiotiki 13, 387 (1968). D.G. Manwaring, R.W. Rickards, and B.T. Golding, Tetrahedron Letters, 5319 (1969). C.N. Chong and R.W. Rickards, Tetrahedron Letters, 5145 (1970). E. Borowski, J. Zielinski, L. Falkowski, T. Ziminski, J. Golik, P. Kolodziejczyk, E. Jereczek, M. Gdulewicz, Y. Shenin, and T. Kotienko, Tetrahedron Letters, 685 (1971). C.N. Chong and R.W. Rickards, Tetrahedron Letters, 5053 (1972). F.E . Pansy, Wm. L. Parker, and N.S. Semenuk, in C.K. Cain, ed., Annual Reports in Medicinal Chemistry - - 1970, Academic Press, New York/London, 1970, p.129. W. Mechlinski, C.P. Schaffner, P. Ganis, and G. Avitabile, Tetrahedron Letters, 3873 (1970). E. Borowski, T. Zielinski, T. Ziminski, L. Falkowski, P. Kolodziejczyk, J. Golik, and E. Jereczek, Tetra- hedron Letters, 3909 (1970). N. Porowska, I,. Halski, 2. Plociennik, D. Kotiuszko, H. Morawska, 2. Kowszyk-Gindifer, and H. Bojarska- Dahlig, Rec. Trav. Chim. 2, 780 (1972). N. Porowska, L. Halski, Z. Plociennik, D. Kotiuszko, H. Morawska, Z. Kowszyk-Gindifer, and H. Bojarska- Dahlig, Adv. Antimicrob. Antineoplastic Chemother. L, 1031 (1972). D. Kotiuszko, K. Wituch, H. Morawska, and S. Siejko, Acta Microbiol. Pol. A3, - 135 (1971). J. Roszkowski, D. Kotiuszko, A. Rafalski, H. Morawska, and K. Raczynska-Bojanowska, Acta Microbiol. Pol. Bi, 9 (1972). W. Chojnowski, A. Zaremba, M. Jedrzejewska, and H. Cendrowska, Acta Polon. Pharm. 30, 81 (1973). W. Chojnowski, A. Zaremba, and B. Skwarko, Acta Polon. Pharm. 30, 223 (1973).

412 GERD W. MICHEL

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

I

2 . 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 Micro- bial Products, 1974. National Center for Antibiotic Analysis, FDA, Depart- ment 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, 141.101-

141.111, January 1, 1971, U.S. Government Printing Office, Washington, D.C.

130-146c, and Parts 147 to End, January 1, 1971, U.S. Government Printing Office, Washington, D.C.

21, Part 148k), U.S. Government Printing Office, Washington, D.C., 1971.

b. "Code of Federal Regulations", Title 21, Parts

c. Federal Register, Volume 36, p.7233-7238 (Title

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 Insti- tute 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.

84, R.M. Evans, The Chemistry of Antibiotics Used in

85. F.S. Parker, Applications of Infrared Spectroscopy in Medicine, Pergamon Press, London, 1965, p.170.

Biochemistry, Biology, and Medicine, Plenum Press, New York, 1971, p.400.

- cules, 2. Edition, John Wiley & Sons, Inc., N . Y . , 1958. 86. L.J. Bellamy, The Infra-red Spectra of Complex Mole-

NYSTATIN 413

87.

88.

89.

90.

91.

92.

93.

94.

95.

96. 97.

98.

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

R.M. Silverstein and G.C. Bassler, Spectrometric Iden- tification of Orqanic Compounds, 2. Edition, John Wiley & Sons, Inc., New York, 1967. A.L. Hayden, O.R. Sammul, G.B. Selzer, and J. Carol, J. Assoc. Offic. Agr. Chemists 45, 797 (1962). H. Umezawa, Index of Antibiotics from Actinomycetes, University of Tokyo Press, Tokyo, 1969, p.474. J.Z. Gougoutas, University of Minnesota, Personal Communication. A.I. Cohen and M.S. Puar, The Squibb Institute for Medical Research, Personal Communication. R.W. Rickards, R.M. Smith, and B.T. Golding, J. Antibiot. 23, 603 (1970). F. Raubitschek, R.F. Acker, and S.A. Waksman, Anti- biotics and Chemotherapy 2, 179 (1952). E. Fralick, The squibb Institute for Medical Research, Personal Communication. J.D. Dutcher, Monographs on Therapy, The Squibb Insti- tute for Medical Research, New Brunswick, N.J., 2, 87 (1957). D. Doskochilova and I. Gess, Antibiotiki 4, 48 (1959). J.M.T. Hamilton-Miller, J. Pharm. Pharmac. 25, 401 (1973). F. Schroeder, J.F. Holland, and L.L. Bieber, Biochem. 11, 3105 (1972). H. Kadin, The Squibb Institute for Medical Research, Personal Communication. M.M. Noone, Sadtler Research Laboratories, Inc., Philadelphia, Pa., Personal Communication. K.D. Haegele and D.M. Desiderio, Biomedical Mass Spec- trometry I, 20 (1974). H. Jacobson and V. Valenti, The Squibb Institute for Medical Research, Personal Communication. P.J. Weiss, M.L. Andrew, and W.W. Wriqht, Antibiot. Chemotherapy 1, 374 (1957). A.J. Keseleski and G.W. Michel, The Squibb Institute for Medical Research, Unpublished Data. D.M. Trakhtenberg, E.I. Rodionovskaya, Z.V. Gordina, L.I. Rostovtseva, G.I. Kleiner, and A.M. Nagle, Anti- biotiki 2 , 9 (1960). G.I. Kleiner and N.V. Ionova, Antibiotiki 1, 926 (1962). F. DGrsch, The Squibb Institute for Medical Research, Unpublished Data. J. Pluscec, The Squibb Institute for Medical Research, Unpublished data. M.L. Ray-Johnson, Squibb International Dev. Lab., Personal Communication.

-

414 GERD W. MICHEL

110.

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127.

128.

129.

130.

A. Albert and E.P. Serjeant, The Determination of Ionization Constants, 2. Edition, Chapman and Hall, Ltd., London, 1971, p.76. J.J. Kirschbaum, The Squibb Institute for Medical Research, Personal Communication. J . O . Lampen, E.R. Morgan, A. Slocum, and P. Arnow, J. Bacteriol. 78, 282 (1959). K. Kramarczyk and H. Berg, Abhandl. Deut. Akad. Wiss. Berlin (K1. Chem., Geol., Biol.) 1964, 23. H. Grisebach and W. Hofheinz, J. Roy. Inst. Chem. 1964, 332. A.J. Birch, in D. Gottlieb and P.D. Shaw, eds., Antibiotics, Volume 2, Springer-Verlag, New York, 1967, p.228. British Patent 714,189 (Research Corporation), 1954; C.A. 49: 4242i. A. Szentirmai, I. Horvath, and B. DOCZY, Hung. Patent 150,018 (1963); C.A. - 59: 9282f. J. Spizek, I. Malek, L. Dolezikova, M. Vondracek, and Z. Vanek, Folia Microbiol. (Prague) 10, 259 (1965). J. Spizek, I. Malek, J. Suchy, M. Vondracek, and Z. Vanek, Folia Microbiol. (Prague) 10, 263 (1965). Y.D. Shenin, V.M. Efimova, T.V. Kotenko, V.A. Tsyganov, and A.N. Egorenkova, Mikrobiologiya 38, 1027 (1969). G . I . Lavrentieva, R.A. Zhukova, L.N. Demchenko, and T.V. Kotenko, Antibiotiki 18, 692 (1973). N.M. Lyagina and G.C. Pestereva, Microbiologiya 32, 536 (1963). J-S. Tsai, C-C. Pao, S-Y. Wu, L-C. Shen, and P-Y. Kung, Sci. Sinica (Peking) 10, 436 (1961); C.A. - 56: 9208b. J-S. Tsai, C-C. Pao, S-Y. Wu, L-C. Shen, and P-Y. Kung, Yao Hsueh Hsueh Pao St 47 (1960); C.A. 56: 8842a. Z. Vanek and M. Vondracek, Antimicrobial Agents Chemother. 1965, 982. Y-S. Tsai, S-Y. Yu, L-C. Sheng, S-F. Lan,C-C. P'o,and P-Y. Kan, Antibiotiki 4, 131 (1959); C.A. 53: 22234i. E.G. Toropova, N.S. Egorov, and N.A. Pobedinsky, Antibiotiki 17, 398 (1972). F.D. Deindoerfer and J.M. West, J. Biochem. Microbiol. Technol. Eng. 2, 165 (1960). G. Liepius, Prod. Mikrobnogo Sin. , Akad. Nauk Latv. SSR, Onst. Mikrobiol. 1966, 203; C.A. - 67: 20571~. S. Cizek, J. France, and F. Ulrych, Czech. Patent 121,923 (1967); C.A. 68: 11716t.

-

-

-

-

NYSTATIN 41 5

131.

132.

133.

134. 135.

136.

137.

138. 139.

140.

141.

142.

143.

144.

145.

146.

147.

148.

149.

150.

151.

152.

L. Feuer , I. Inczefy , and S. I s t v a n , G e r . Offen. 2,063,030 (1971); C.A. 2: 1 5 2 6 8 5 ~ . A. Benda, M. Bucko, J. Dasek, J. Palkoska, R. Roubicek, and J. Z a j i c e k , Czech. P a t e n t 124,390 (1967); 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 (1960) . 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 (1961) . L.A. Popova and N.E. Stepanova, A n t i b i o t i k i 1, 868 (1962) . E.P. Yakovleva, E.S. Lantas , and E . I . I o f i n a , 4 t h S c i . Conf. Leningrad. R e s . I n s t . A n t i b i o t . , Leningrad, 194 (1965); B.A. 48: 92431 (1967).

M. Musilkova, F o l i a Microbio l . (Prague) 6, 175 (1961) . B.S. Nugumanov, E.G. Toropova, and N . S . Egorov, Anti- b i o t i k i =, 489 (1973) . I . Aburatami, K. K a s h i i , K. Minoura, K. Terai , Y. Imanshi, and T. Sugai , J . Osaka C i t y Med. Cent re 8, 91 (1959) . J. Vandeputte and W. Gold, U.S. P a t e n t 2 ,786,781 (1957); C.A. 51: 8388e. J. Vandeputte, U.S. P a t e n t 2,832,719 (1958); C.A. 52: 14095i. J . D . Dutcher and J. Vandeputte, U.S. P a t e n t 2,865,807 (1958); C.A. 53: 655033.

J . G . R e n e l l a , U.S. P a t e n t 3,517,100 (1970); C .A. 73: 86548b. R.C. E s s e , U.S. P a t e n t 3 ,517,101 (1970); C.A. - 73: 69838d. H. Mendelsohn, U.S. P a t e n t 3,509,255 (1970) ; C.A. 2: 28897d. Y . Okami, R. Utahara , S. Nakamura, and H. Umezawa, J. A n t i b i o t i c s ( J a p a n ) , Ser . A , z, 98 (1954) . M . Urks, C .D. Cerkes, and 2 . Severa , Med. Prom. S.S.S.R. - 14, 2 1 (1960); C . A . 55: 87549. J. Gyimesi, K. Magyar, B. Kasszan, and V. Senkar iuk , Hung. P a t e n t 149,132 (1962); C .A. 58: 5458g. E. Nevole, Czech. P a t e n t 110,992 (1964); C.A. - 61: 1 5 9 4 0 ~ . French P a t e n t 1 ,488,535 (E.R. Squibb & S o n s ) , 1967; C.A. 68: 9 8 6 4 3 ~ . J. Vandeputte and U.F. Nager, U.S. P a t e n t 3,332,844 (1967); C.A. - 67: 811299.

41 6 GERD W. MICHEL

153.

154.

155.

156.

157.

158.

159.

160.

161.

162.

163.

164.

165.

166.

167.

168.

169.

170.

171.

D.M. Trakhtenberg, E . I . Rodionovskaya, Z.V. Gordina, L . I . Rostovtseva, G . I . Kleiner, A.M. Nagle, and V. Lazdina, Med. Prom. S.S.S.R. 14, 18 (1960); C.A. 55: 2019d. G . I . K l e i n e r , V.Y. Lasdinya, and D.M. Trakhtenberg, U.S.S.R. P a t e n t 123,287 (1959). M. Matsuoka and K. Kitamura, Jap. P a t e n t 13,748 (1961); C.A. 56: 7445e.

G . I . K l e i n e r , N.V. Ionova, D.M. Trakhtenberg, and L . I . Rostovtseva, A n t i b i o t i k i 5, 200 (1961). V. Panes and V. Pecak, Czech. P a t e n t 106,428 (1963); C.A. 60: 1079f. V.S. z p e j e v , Czech. P a t e n t 116,958 (1965); C.A. 65: 167972. S. I s t v a n , Hung. P a t e n t 152,192 (1965); C.A. 63: 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 le ine r , E . I . Dangovet, D.M. Trakhtenberg, and P.A. K a l i n i n , U.S.S.R. P a t e n t 167,609 (1970); C.A. 73: 7231s. A.P. Bashkovich, Y.D. Shenin, T.V. KoGnko, V.Y. Raigorodskaya, 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, a im.-Farm. Zh. L, 31 (1967). S. Boteanu, S. Balliu-Mutu, E. Ai teanu , and 0. Ludu, Farmacia (Bucharest) 17, 681 (1969) . G . I . Kle iner and N.V. Ionova, A n t i b i o t i k i 5, 712 (1963) .

A.D. Kuzvkov, G.B. Lokshin, Y.V. Zhdanovich, and K.M. Khryascheva, A n t i b i o t i k i 11, 422 (1966) . G.B. Lokshin, Y.V. Zhdanovich, A.D. Kuzovkov, T . I . Volkova, V.Y. Shtamer, and G . I . Kleiner, A n t i b i o t i k i - 1 2 , 196 (1967) . G.B. Lokshin, Y.V. Zhdanovich, A.D. Kuzovkov, G . I . Kle iner , S.M. Chaikovskaya, V.Y. Shtamer, and T . I . Volkova, U.S.S.R. P a t e n t 179,429 (1966); C.A. 65: 2076a. A.E. Tebyakina, S.M. Chaikovskaya, and T.G. Venkina, A n t i b i o t i k i 6, 547 (1961) . A.E. Elkouly, N.A. Elsayed, and A.M. M o t a w i , Mfg. Chem. Aerosol N e w s , August 1973, p.37.

-

D.C. Grove and W.A. Randal l , Assay Methods o f A n t i - b i o t i c s - A Laboratory Manual ( A n t i b i o t i c s Monographs N o . 2 ) , Medical Encyclopedia , I n c . , N e w York, 1955, p.116. J . R . Gerke and M.E. Madigan, A n t i b i o t i c s Chemother. - 11, 227 (1961), I. Horvath and I. Koczka, Nature 203, 1305 (1964).

NYSTATI N 417

172.

173.

174.

175.

176.

177.

178.

179.

180.

181.

182.

183.

184. 185.

186.

187.

188.

189.

190.

191.

192.

193.

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,

J. TrAfouel, J. Cheymol, R. Paul, H. Phnau, G . Hagemann, J. Comar, R. Romain, M. Philippe, J. Vignalon, and A. Quevauviller, Therapie 14, 573

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).

D.C.r 1972.

(i959).

418 GERD W. MICHEL

194. 195.

196.

197.

198.

199.

200. 201. 202. 203. 204. 205.

206.

207. 208.

209.

210. 211.

212.

213.

214. 215.

216.

217.

218.

219.

K.V. Unterman, Antibiotiki 10, 867 (1965). V. Valenti, The Squibb Institute for Medical Research, Personal Communication. E.D. Etingov, G.V. Kholodova, V.O. Kul'bakh, and A.I. Karnatushkina, Antibiotiki 17, 301 (1972). H. Laubie, Bull. SOC. Pharm. Bordeaux 99, 3 (1960); C.A. 54: 17793d. 0. Szucs, Gy&gyszer&szet 2, 469 (1961); C.A. - 57: 4764i. B. Ozsoz, Turk Ijiyen Tecrubi Biyol. Dergisi 22, 59 (1962); C.A. 57: 9957d. W.H. Unterman, Rev. Chim. (Bucharest) 12, 415 (1961). W.H. Unterman, Rev. Chim. (Bucharest) 12, 504 (1961). S. Ochab, Diss. Pharm. 17, 323 (1965). W.H. Unterman, Rev. Chim. (Bucharest) 13, 618 (1962). L. Mazoi and K. Papay, Acta Pharm. Hung. 32, 59 (1962). J.C. Chang, A.B. Honig, A.T. Warren, and S. Levine, J. Pharm. Sci. 52, 536 (1963). M.M. Amer and A.A. Habib, Talanta 22, 605 (1975); see also GyAgyszer6szet 18, 472 (1974). B.Z. Chowdhry, Talanta 23, 79 (1976). V. Betina, in M. Lederer, ed., Chromatographic Reviews, Volume 7, Elsevier Publishing Company, Amsterdam/London/New York, 1965, p.119-178. 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. Z. RehAcek, Folia Microbiol. (Prague) 8, 228 (1963). 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. R. Fiigner and G. Bradler, Z. Allgem. Mikrobiol. 3, 173 (1963). V. Sevcik, M. Podojil, and A. Vrtiskovh, Cesk. Mikrobiol. 2, 175 (1957). V. Betina, J. Chromatogr. 78, 41 (1973). A. Ammann and D. Gottlieb, Appl. Microbiol. 3, 181 (1955). D.H. Peterson and L.M. Reineke, J. Amer. Chem. SOC. - 72, 3598 (1950). W.A. Ritschel and H. Lercher, Pharm. Ztg., Ver. Apotheker-Ztg. 106, 120 (1961); C.A. - 62: 397i. R. Paris and J.-P. Theallet, Ann. Pharm. Franc. 20, 436 (1962). K.V. Rao and W.P. Cullen, J. her. Chem. SOC. 82, 1127 (1960) .

-

-

NYSTAT I N 419

220.

2 2 1 .

2 2 2 .

223.

2 2 4 . 225.

226.

2 2 7 .

228 .

229.

230.

231. 232.

233.

234.

235. 236. 237.

238.

239.

240.

241 .

2 4 2 .

C . P . Schaf fner , I . D . Steinman, R . S . Safferman, and H. Lecheval ie r , i n H. Welch and F. Marti-Ibanez, eds . , Ant ib io t i c s Annual 1957-1958, Medical Encyclopedia, Inc . , New York, 1958, p.869. G.H. Wagman and M . J . Weinstein, Chromatography of A n t i b i o t i c s , Journa l of chromatography L ib ra ry - Volume 1, E l sev ie r S c i e n t i f i c Publ i sh ing 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 Chemo- therapy 9, 398 (1959). Y.M. Khokhlova, A.V. Puchnina, E.F. Oparysheva, L.M. Golovkina, and N.O. Blinov, Izv. Akad. Nauk SSSR, Ser . Bio l . 2, 433 (1966); C.A. - 65: 3668c. V . Bet ina and P. Nemec, Nature 187, 1111 (1960) . V . Bet ina and P . Nemec, Chem. Zves t i 15, 853 (1961); C .A. 58: 5453d. Y . V . Zhdanovich, G.B. Lokshin, and A.D. Kuzovkov, Khim.-Farm. Zh. I, 42 (1967); C.A. 68: 33147k. S.N. Li tvinenko, Lab. Delo 8, 39 (1962); C.A. 58: 2322d. A . Aszalos , S. Davis, and D. F r o s t , J. Chromatoqr. 37, 487 (1968). T. Ikekawa, F. Iwani, E . Aki ta , and H. Umezawa, J. An t ib io t . (Tokyo), Ser . A , 16, 56 (1963) . G. Zweig and J. Sherma, e d s . , Handbook of Chromato- graphy, Volume 1, CRC Press, The Chemical Rubber Co., Cleveland, 1972, pp.458, 459, 751. E. Akita and T. Ikekawa, J. Chromatogr. 12, 250 (1963) . S . Ochab, D i s s . Pharm. Pharmacol. 22, 351 (1970); C.A. 74: 797322. F. Tarqos and J. Metzqer, The Squibb I n s t i t u t e f o r Medical Research, Personal Communication. F. Tarqos, The Squibb I n s t i t u t e f o r Medical Research, Personal Communication. C. Mathis, Bul l . SOC. Chim. F r . 1973 (l), 93. P.-A. Nussbaumer, Pharm. A c t a Helv. 43, 462 (1968). 0. Kocy and N . Cole, The Squibb I n s t i t u t e f o r Medical Research, Personal Communication. J. Keiner, R. Hiittenrauch, and W. Poethke, Pharm. Zen t r a lha l l e 108, 525 (1969). M . H . J . Zuidweg, J . G . Oostendorp, and C . J . K . BOS, J. Chromatogr. 42, 552 (1969). E. Meyers and D.A. Smith, J. Chromatogr. 14, 129 (1964).

V.B . Korchagin, G.B. Lokshin, and V . I . Nirenberchik, A n t i b i o t i k i 11, 1047 (1966); C.A. 66: 40745r. N.O. Blinov and A.S. Khokhlov, A n t i b i o t i k i S , 751 (1963).

-

420 GERD W. MICHEL

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, Springer- Verlag, 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. Calm, 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 Inter- science Conf. on Antimicrobial Agents and Chemo- therapy, 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. Tech- nicon 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.

263.

264.

265.

266.

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). M.V. Bibikova et al. On the possibility of early identification of organisms producing polyenic antibiotics Antibiotiki 20, 675 (1975). E.D. Etingov et al. Ionization of acid-base groups of polyenic antibiotics in aqueous solutions Antibiotiki 20, 678 (1975). V.A. Weinstein et al. Studies on association of nystatin and amphotericin B in non-aqueous solvent systems Antibiotiki 20, 688 (1975) 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 DAISY B. WHIGAN

TABLE OF CONTENTS 1. Description

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

2 . 1 Spectra 2. Physical Properties

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

3. Synthesis 4. Stability-Degradation 5. Analysis of Intermediate Compound and

Hydrolysis Products 6 . Methods of Analysis

6 . 1 Identification Tests 6.2 Elemental Analysis 6.3 Spectrophotometric Analysis

6 . 3 1 Ultraviolet Spectrophotometric

6.32 Fluorescence Spectrophotometric Ana lys i s

Analysis 6.4 Titrimetric Procedures

6.41 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 425

6.52 With Sodium 1,2-Naphthoquinone-4- sul f onate

6.6 Chromatographic Procedures 6.61 Paper Chromatography 6.62 Thin-layer chromatography

7. Analysis of Hydrolysis Products in Body Fluids and Tissues

8. Serum Protein Binding 9. Drug Metabolism 10. References

426 DAISY B. WHIGAN

1. Description 1.1 Name, Formula, Molecular Weight

Proparacaine hydrochloride i s 2- (d ie thyl - amino)ethyl 3-amino-4-propoxybenzoate monohydro- chlor ide. Chemical Abs t rac ts l i s t i n g s a r e under t h e heading benzoic acid,3-amino-4-propoxy-2- (diethy1amino)ethyl e s t e r , monohydrochloride. The Chemical Abstracts Regis t ry Number i s 5875-06-9. It is a l s o known a s proxymetacaine hydrochloride. Common t r ade names a r e Ophthaine, Alcaine, and Ophthetic,

CH3CH2CH2- -0-CH2 CH2N (C2H5 ) 2

. H C 1 7

1 . 2 Appearance, Color, Odor Proparacaine hydrochloride i s a white o r

f a i n t buff c r y s t a l l i n e , odorless powder.

2. Physical Proper t ies 2 . 1 Spectra

2 . 1 1 In f r a red Spectra The in f r a red spectrum of propara-

caine hydrochloride compressed i n a potassium bromide p e l l e t i s shown i n Figure 1. The spectrum was obtained on a Perkin-Elmer Model 6 2 1 g ra t ing in f r a red spectrophotometer. The following assignments have been made fo 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 y cm'l A s s i q n m e n t 3420,3280 NH2 s t r e t c h 2700,2640 H C 1 1700 E s t e r G O 1610,1585,1510 Aromatic C=C 1295,1200 =C-0 (ester and

aromatic e t h e r )

PROPARACAI N 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

2 500

17 15 1630 1600 870

Assignment -NH2 group -NH~ group

ted amine

ted amine C=O Ester Phenyl ring Phenyl ring CH aromatic

N'H of trisubstitu-

N'H of trisubstitu-

The discrepancies in the spectral wavelengthsof the two interpretations could be attributed to calibration differences of the di f f eren t instruments used2.

WAVELENGTH (MICRONSI

FREQUENCY (CM’)

Figure 1. In f r a red Spectrum of Proparacaine Hydrochloride i n a Kl3r P e l l e t .

429 PROPARACAINE HYDROCHLORIDE

2.12 Nuclear Maqnetic Resonance S p e c t r a F igu re 2 shows the n u c l e a r magnet ic

resonance spectrum of p ropa raca ine 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 ime thy l su l fox ide . The spec t rum 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 ass ignments3 a r e r eco rded i n Table 1.

Table 1

H 3 C - CH2 -

HC 1

Proton P o s i t i o n

1 2 3 4 5 6 7

9 10 11

a

N+H

* ( N o . of Peaks)

1.27 ( t ) 3.17 (9) 3.44 ( t ) 4.61 ( t ) 7.32 (9) 7.40 (d) 6.90 (d) 5.00 (b) 4.00 (t) 1.75 ( m ) 1 . 0 0 ( t )

11.35 (b)

CH2 -

'CH2 - CH3

Coupling c o n s t a n t J (HZ)

7.0 7.0 6.0 6.0 9.0 9.0 1.0

6.0

6.5

---

---

---

* d = doub le t , 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

w % 3

DM% 8-25-76

P w 0

5 -5 - . . 7--7---*--- --7. I

I 1. I 4 5 6 7 8 * *t#l 9-72 GMJ

F i g u r e 2 . NMR Spectrum of P ropa raca ine Hydrochlor ide i n Deu te ra t ed Dimethylsu l f oxide.

PROPARACAINE HYDROCHLOR IDE 431

2.13 U l t r a v i o l e t S p e c t r a The u l t r a v i o l e t spectrum o f

p ropa raca ine hydroch lo r ide i n methanol , ca. 1 2 pg/ml, i s shown i n F i u r e 3 (1nstrument:Cary 1 5 ) . H e f f e r e n and co-workers' a t t r i b u t e d the fo l lowing 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 absorp- t i o n of s u b s t i t u t e d benzo ic 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 a t t a c h e d t o a r o m a t i c r i n g

Amino conjugated 300 w i t h ca rbonyl E t h e r s con juga ted 270 w i t h carbonyl

The u l t r a v i o l e t maxima obse rved f o r p ropa raca ine h y d r o c h l o r i d e a g r e e ve ry w e l l w i t h t h e above ass ignments . A l l t h r e e peaks a r e a l s o observed when e t h y l a l c o h o l 6 , wa te r5 , 7, and aqueous 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 ropa raca ine i s dependent on pH. 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 u l t r a v i o l e t a b s o r p t i o n of p ropa raca ine h y d r o c h l o r i d e w a s e x t e n s i v e l y s t u d i e d by Hefferen8. t h e a romat i c amine forms a p o s i t i v e l y charged 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 group i n resonance 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 of t h e s p e c t r a p r e s e n t e d by Hkfferen showed an i s o b e s t i c p o i n t a t 243 nm.

A t a c i d i c p H ,

E 0

k

'D h

432

Ultraviolet Spectrum of Proparacaine Hydrochloride So1vent:Methanol - 1nstrument:Cary 15

a, C

.rl

k

04

WI 0

k

JJ

m A

k

I

Ii d

433

P w P

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 spec t rum 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 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. T h i s w a s o b t a i n e d on a n A s s o c i a t e d E lec t r i ca l I n d u s t r i e s Model MS-902 M a s s Spec t romete r equipped 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 p a r e n t i o n , M+, o f t h e compound a t m / e 294 i s weak. The major i o n a t m / e 86 i s due t o t h e c l e a v a g e o f t h e bond beta t o t h e t e r t i a r y amine n i t r o g e n . 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 the f r a g m e n t a t i o n of amines. Mass spec t ra l a s s ignmen t s of prominent i o n s are 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.

m / e m / e

H3C- CH2-CH2- 0

m / e 178

99-H I-

f.i 195 +H

CH2 -1 m / e 222

~ ' 2 ~ 5

\C2H5 N

m / e 178 _I

m / e 295 (M+ + H )

m / e 136 + CH3CH = CH2

$0

0

I

hlISN31NI

3AIlt113tl

a, 5

.d

k

0

d

."u 0 k 5

sr m Q

) C

.d

rd U

rd k

rd a

0

k

PI

W 0

E 7

k

4J U

a, a

v)

111 111

2 c 0 -d

4J 7

d

0

ffl Q

) p: I

d

In

Q) k 7

tn -4

L4

435

436 DAISY 6. WHIGAN

2 . 1 5 Fluorescence Spectra Proparacaine hydrochloride e x h i b i t s

na t ive fluorescence4. I ts e x c i t a t i o n and f luorescence spec t ra i n methanol a s recorded on a Perkin- Elmer Fluorescence Spectrophotometer Model 204 a r e reproduced i n Figure 6. The fluorescence of proparacaine hydrochloride va r i e s with pH. I t is most in tense i n 0 .1N sodium hydroxide where a concentra- t i o n of 0.05 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 ac id , the fluorescence i s quenched. Fluorescence c h a r a c t e r i s t i c s of proparacaine hydrochloride i n a l imi ted list of so lvents a r e presented i n Table 2.

Table 2 Fluorescence Charac t e r i s t i c s of

Proparacaine Hydrochloride

Solvent Exci ta t ion Fluorescence

Water 3 16 460 Sodium Hydroxide,

0.1N 300 3 96 Phosphate B u f f e r ,

pH 7.0 316 454 Me thano 1 3 18 44 0

Maximum , nm Maximum, nm

I n 0.1g sodium hydroxide, t he re i s a l i n e a r r e l a t i o n s h i p between the f luorescence i n t e n s i t y and the concentrat ion of proparacaine hydrochloride up t o 5 pg per m l .


Figure 6. Excitation and Fluorescence Spectra of Proparacaine Hydrochloride 1nstrument:Perkin-Elmer Fluorescence

Solvent: Methanol Spectrophotometer Model 204

I I !

i . . .. , I

I I i I

, ; -

I

. -.

1

I I

. . I

I I

!

0

I d I d

' p i

/

/ i

I

i I I

I !

j I

I !

I

i ,

I

,

I I

i-

I

I

I I

I I

I

!

1

I

I

I

I

0 m m

I

I

~ . . 1

I

I .

. . . ,

0 N N

438 DAISY B. WHIGAN

2 . 2 c r y s t a l P r o p e r t i e s 2 . 2 1 C r y s t a l l i n i t y

P ropa raca ine 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 bromide'. n e e d l e s w i t h 5 - n i t r o b a r b i t u r i c ac id44 . Photomicro- g rap - , 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 potass ium permanganate w e r e t aken by Rich and Cha t t en lo .

It a l s o forms rosettes of long t h i c k

2.22 Polymorphism N o polymorphism has been r e p o r t e d

f o r p ropa raca ine hydroch lo r ide . However, Koehler and Feldmann'l sugges t ed t h e p o s s i b i l i t y of 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 d e r i v a t i v e .

2.23 D i f f e r e n t i a l Thermal Analysis(DTA) Jacobson12 conducted t h e d i f f e r -

e n t i a l t he rma l a n a l y s i s of p r o p a r a c a i n e hydroch lo r - i d e on a DuPOnt 900 Thermo-analyzer w i t h a t empera tu re r ise of 15O p e r minute. The thermogram o f p ropa raca ine h y d r o c h l o r i d e (Squibb House S tanda rd Lot 41519-003) showed a s h a r p endotherm a t 1 8 1 O C which cor responds t o t h e melt of t h e drug (See S e c t i o n 2.27 f o r Mel t ing Range).

2.24 Thermal Grav ime 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 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 conducted on a DuPont Thermogravimetric Analyzer Model 900.Working w i t h p r o p a r a c a i n e hydroch lo r ide , Squibb S tanda rd Lot 41519-003, Jacobson12 found no weight loss b e f o r e 150OC. 15O p e r minute under a n i t r o g e n sweep.

The compound w a s h e a t e d a t a r a t e of

2.25 D i f f e r e n t i a l Scanning Ca lo r ime t ry (DSC) Va1en t i l3 de te rmined t h e p u r i t y of

p ropa raca ine h y d r o c h l o r i d e by DSC. A scanning r a t e of 0.625 deg/min and a s e n s i t i v i t y o f 2 mil l ical /sec 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


was c a l c u l a t e d t o b e 99.94 mol pe rcen t . 2.26 X-Ray Powder D i f f r a c t i o n

of p ropa raca ine hydroch lo r ide was o b t a i n e d by Ochsl4 on a P h i l l i p s X-Ray Powder Di f f r ac tomete r , Type 120-101-11, a t a v o l t a g e of 35 kv and a c u r r e n t of 10 mA. Theosample w a s i r r a d i a t e d by a copper sou rce a t 1 .54 A . Data d e r i v e d from the spectrum ( F i g u r e 7 ) of p ropa raca ine hydroch lo r ide , Squibb Standard Lot 41519-003, a r e l i s t e d i n Table 3.

The x-ray powder d i f f r a c t i o n p a t t e r n

T a b l e 3 X-Ray Powder D i f f r a c t i o n P a t t e r n of

Proparaca ine 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

0 ** 1 ( 2 q * d' ( A ) I/IO***

7.04 10.02 11.29 12.74 13.59 15.63 16.22 17.33 17.84 19.62 21.32 23.36 24.47 25.15 26.42 27.02 27.36 29.31 33 .31 33.82 35.77

12.56 8 .83 7.84 6.95 6.52 5.67 5.46 5.12 4.97 4.52 4.17 3 .81 3.64 3.54 3.37 3.30 3.26 3.05 2.69 2.65 2 .51

0.663 0.460 0.176 0.197 1.000 0.212 0.140 0.178 0.357 0.518 0 .483 0.179 0.299 0.122 0.360 0.300 0.497 0.261 0.153 0.226 0.115

*Twice t h e anq le of i nc idence 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 s i n 8

0 = 1.539 A

R e l a t i v e i n t e n s i t y based on h i g h e s t i n t e n s i t y of 1.000.

***

PROPAR ACAl NE HY DROCH LOR I DE 44 1

2.27 Melting Range The melt ing range f o r U.S.P. pro-

paracaine hydrochlor ide i s s p e c i f i e d a s 178O t o 185OC. Clinton, -- e t . a l . repor ted a mel t ing range of 182-183.3OC. Squibb Standard proparacaine hydrochlor ide Lot 41519-003 gave a mel t ing range of 1820 t o 184OC. a melting range of 180' t o 182OC.

Monguzzi and c o - w ~ r k e r s ~ ~ repor ted

2.3 Solu t ion 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 Proparacaine Hydrochloride a t Room Temperature 17

Solvent S o l u b i l i t y (mq/ml) Water > 50 Dimethylsulfoxide 50 Chloroform 30 Ethanol 7 Benzene < 0 .1 Hexane (0.1 Ethyl Acetate (0.1 Ether (0.1

2.32 pKa Hef feren8 determined t h e apparent

d i s s o c i a t i o n constant of t h e aromatic amino group: +

R - NH3 R - NH2 + H+

Using a spectrophotometr ic method descr ibed by Flexser , Hammett, and Din t h e apparent pK' a i s 3.22 (Kh = 6.03 x lo-')

2.33 Phase S o l u b i l i t y Analysis The p u r i t y of proparacaine hydro-

ch lo r ide has been determined by phase s o l u b i l i t y ana lys i s6 . The a n a l y s i s i s c a r r i e d ou t by e q u i l i b r a t i o n i n absolu te e thanol a t 23OC f o r 24 hours , Proparacaine hydrochlor ide Lot N o . BR-1 assayed 99.8% pure by phase s o l u b i l i t y ana lys i s .

442 DAISY 6 . WHIGAN

3. x n t h e s i s

syn thes i zed19 by t h e sequence o f r e a c t i o n s shown i n F igu re 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 . T h i s i s e t h e r i f i e d w i t h n-propylbromide i n t h e p re sence of potass ium 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 thy1amino)e thy 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 ropa raca ine hydro- c h l o r i d e ( I V ) .

Proparaca ine h y d r o c h l o r i d e h a s been

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 ro-4- propoxybenzoic a c i d (11) by a l k y l a t i o n of 4- hydroxy-3-nitrobenzoic ac id w i t h p ropy l p- to luene- s u l f o n a t e i n xy lene s o l u t i o n . The f r e e a c i d i s produced by subsequent 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 es ter .

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-chloro- 3 -n i t robenzo ic a c i d by r e a c t i n g i t w i t h sodium n-propoxide i n d ime thy l su l fox ide s o l u t i o n .

The n i t r o group 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 conve r t ed t o t h e cor respond- i n g amino group by

15,20 a ) i r o n - h y d r o c h l o r i c a c i d r e d u c t i o n b) c a t a l y t i c hydrogenat ion w i t h pa l lad ium/

24 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 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

22,23 charcoa 1 c a t a l y s t

1 5

Proparaca ine 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, and from a b s o l u t e a l c o h o l - e t h y l acetate15.

e e w

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 Proparacaine H y d r o c h l o r i d e

STEP I ' H O D COOH + C H 3 C H 2 C H 2 B r , C H 3 C H 2 C H 2 0 0 COOH

S T E P 11: C H 3 C H 2 C H 2 0 C H 3 C H 2 C H 2 0 a C:OH

0 2 N I1 S T E P 111:

CH3 CH2 CH2 S O C l 2

CH3CH2Cz::@-OCH2CH2N ( C 2 H

D O H

0 2 N

S T E P IV:

I

CH3 CH2

CH3 CH2

CH3

5 ) 2 * H C 1

H?

H 2 N - IV

4. S tab i l i ty -Deqrada t ion

t u r e f o r a t l e a s t two years25. and exposure to a i r g . s t a b l e up t o a t l e a s t two years16 i n t h e absence of a i r . w i l l d i s c o l o r i n t h e presence of air2’.

c h l o r i c a c i d f o r 60 minu tes26 (Figure 9 ) .

Proparacaine hydrochlor ide i s chemically s t a b l e a s a s o l i d a t room tempera- The whi te c r y s t a l l i n e powder d i s c o l o r s o n hea t ing

Liquid formulat ions of proparacaine hydrochlor ide a r e However, s o l u t i o n s

Proparacaine hydrochlor ide undergoes hydro lys i s when b o i l e d i n 2N hydro-

COOH

+ (CH3CH2 ) 2NCH2 CH20H

B-diethylaminoethano 1 . QNH2

HC 1 Heat

OCH2 CH2 CH3

P P P

OCH2 CH2 CH3

Proparacaine 3-amino-4-propoxy- benzoic a c i d

Figure 9. Hydrolysis of Proparacaine

PROPARACAINE HYDROCHLOR ID€ 445

5. Analysis of Intermediate Compound and Hydrolys i s Produ c t s Traces of the n i t r o intermediate (I11 i n

Figure 8) i n proparacaine hydrochloride have been determined polarographical ly by Kocy45. and Northrup Electrochemograph type E equipped with a sa tura ted calomel e lec t rode and a dropping mercury e lec t rode was used. The e l e c t r o l y t e bu f fe r used i s pH 4.0 a c e t a t e buf fer containing 0.001; dodecyltrimethylammonium chlor ide (DTAC) a s a maxima suppressor. The "standard addi t ion 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 proparacaine hydrochloride. The n i t r o intermediate has an average reduction p o t e n t i a l of -0.37 v o l t s (vs . S . C . E . )

A Leeds

3-Amino-4-propoxy-benzoic ac id , a hydrolysis product of proparacaine, has been separated from proparacaine by l iqu id- l iqu id ex t rac t ion . The f r e e acid remains i n pH 6.8 buf fe r while proparacaine is ex t rac ted i n t o chloroform 27 . aqueous layer i s assayed spectrophotometr ical ly f o r 3-amino-4-propoxy-benzoic acid. When t h e pH of the aqueous layer i s lowered t o 4, t he f r e e ac id i s ex t rac ted i n t o chloroform26. Solut ions of t h e f r e e ac id were spot ted on s i l i c a g e l th in- layer p l a t e s and developed i n two separa te solvent 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: 10) . The pos i t ion of the f r e e ac id , r e l a t i v e t o caffeine, was 0.7 i n System I and 1.6 i n System 11.

The

Diethylaminoethanol, a l s o a hydrolysis product of proparacaine, has been determined i n plasma by a co lor imet r ic method with methyl orange . 28

446 DAISY 6. WHIGAN

6. 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 T e s t s

U.S.P. methods1 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 . I n f r a r e d s p e c t r o s c o p y ( S e c t i o n 2 . 1 1 ) may be used t o i d e n t i f y t h e drug. The pr imary a r o m a t i c amino g roup i s i d e n t i f i e d by r e a c t i n g w i t h aqueous sodium n i t r i t e , c o o l i n g t h e mix tu re , and t h e n adding a s o l u t i o n o f (3-naphthol i n sodium hydroxide . The s c a r l e t - r e d p r e c i p i t a t e formed does n o t d i s s o l v e upon a d d i t i o n

6 .62) and paper chromatography ( S e c t i o n 6 .61 ) have been u t i l i z e d f o r i d e n t i t y purposes . Photomicro- g raphs 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 (Tab le 4) and t h e d e t e r m i n a t i o n of t h e m e l t i n g r anges 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 r o v i d e f u r t h e r pa rame te r s f o r i d e n t i f i c a t i o n .

of a c e t o n e 1 . Thin - l aye r chromatography ( S e c t i o n

Tab le 4 Propa r a c a i n e D e r i v a t i v e s

D e r i v a t i v e Mel t inq Ranqe (OC)

F l a v i a n a t e 162.0-163.0 (dec ) C h l o r o p l a t i n a t e 195.5-198.5

Methiodide 145.0-147.5 P i c r a t e 122-124 Re inecka te 138.0-140.0 S typhna t e 151-158 Tet raphenyl - 143 -147

b o r a t e a 131.5-132.0

Refe rence 10 15 10 11 10 10 11

10

a 11 Polymorphism h a s been sugges t ed


6.2 Elemental A n a l y s i s (as C16H26N203-HC1)

Element % Theory % Repor ted Ref. 15 Ref.6

Carbon 58.08 58.04 Hydrogen 8.23 8 .11 Nit rogen 8.47 8. 56 8.54 C h l o r i n e 10.71 10.88 10.85

Chlo r ides may be de termined6 b y reac t ing t h e sample w i t h excess s i l v e r n i t r a t e i n t h e p re sence 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 po tass ium 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 s u l f a t e a s t h e i n d i c a t o r .

6 . 3 Spec t ropho tomet r i c A n a l y s i s 6 . 3 1 u l t r a v i o l e t Spec t ropho tomet r i c

A n a l y s i s S i n c e p ropa raca ine d i s p l a y s a h i g h

degree o f a b s 3 r p t i o n i n t h e 220 t o 320 nm range , u l t r a v i o l e t spec t roscopy ( S e c t i o n 2.13) p rov ides a convenient means €or i t s a s say . I n l o c a l a n e s t h e t i c fo rmula t ions , t h e p re sence o f some v a s o c o n s t r i c t o r a g e n t s , p r e s e r v a t i v e s , a n d s a l t s w i l l n o t i n t e r f e r e i f t h e s e m a t e r i a l s e i t h e r a ) d o n o t d i s p l a y a b s o r p t i o n i n t h i s a r e a o r 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 n e g l i g i b l e . I n more compl ica ted fo rmula t ions p n p a r a c a i n e has 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 by e x t r a c t i o n from an 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 ch loroform 11 .

I n t h e p re sence of s t r o n g a c i d s , t h e a romat i c amine forms a p o s i t i v e l y charged 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 of t h e amino group i n resonance i s n u l l i f i e d ( F i g u r e 4 ) . 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 de t e rmin ing propoxycaine i n t h e p re sence of proca ine30. t o t h e d e t e r m i n a t i o n o f p r o p a r a c a i n e i n t h e p re sence o f p roca ine .

The same phenomenon cou ld b e a p p l i e d

448 DAISY B. WHIGAN

6.32 F luo rescence 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 Although f l u o r o m e t r i c p rocedures

f o r t h e a s s a 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 have n o t been reported, t h e y shou ld be f e a s i b l e because 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 hydro- c h l o r i d e i n 0.1g sodium hydroxide i s s u f f i c i e n t l y s t r o n g ( S e c t i o n 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 ropa raca ine h y d r o c h l o r i d e can 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 acetous p e r c h l o r i c a c i d 29 .

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 been d e s c r i b e d f o r

p r o p ~ x y c a i n e ~ ~ which is an isomer o f p r o p a r a c a i n e . I n t h i s a s s a y , t h e pr imary a r o m a t i c amine undergoes d i a z o t i z a t i o n and t h e end-poin t i s de te rmined by s t a r c h - i o d i d e paper e x t e r n a l i n d i c a t o r . Ferrocyphen s o l u t i o n , which h a s been used a s an 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 , may be used 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 r e p o r t e d f o r p r o p a r a c a i n e , the p r e s e n c e o f a pr imary a r o m a t i c amino g roup i n p r o p a r a c a i n e s u g g e s t s a p p l i c a b i l i t y o f t h i s t i t r a t i o n .

46

6.43 Spec t ropho tomet r i c T i t r a t i o n w i t h N i t r o u s A c i d 31 I n t h i s t i t r a t i o n , abso rbance

measurements a re made d u r i n g t h e t i t r a t i o n o f t h e pr imary a romat i c amine w i t h n i t r o u s a c i d . The absorbance r e a d i n g s a r e dependent on the 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. I n p l o t t i n g t h e absorbances 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 and a f t e r r e a c t i o n comple t ion) i s t h e end-point . T h i s t i t r a t i o n h a s been a p p l i e d t o the d e t e r m i n a t i o n o f 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 pr imary 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


a p p l i c a b l e t o t h e d e t e r m i n a t i o n o f p ropa raca ine .

6 .5 Colorimetric Methods 6 .51 With Bra t ton-Marsha l l Reagent

The u t i l i t y of t h e Bra t ton - Marsha l l r e a g e n t i n t h e a n a l y s i s of pr imary a romat i c amines i s w e l l known. A p p l i c a t i o n of t h i s r e a g e n t t o t h e a n a l y s i s o f p ropa raca ine hydro- c h l o r i d e h a s been d e s c r i b e d by Poe t33 .

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 wa te r t o a 100 m l vo lumet r i c f l a s k c o n t a i n i n g abou t 4 mg o f p r o p a r a c a i n e hydro- 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 minutes then add 10 m l o f 0.5% ammonium su l f ama te . A f t e r 3 minutes add 10 m l of 0.1% Bra t ton-Marsha 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% propylene 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 wa te r and measure t h e absorbance a t 550 nm a g a i n s t a Reagent Blank.

6.52 With Sodium 1,2-Naphthoquinone-4- s u l f o n a t e I n t h i s a s s a y procedure , t h e

ye l low sodium 1,2-naphthoquinone-4-sulfonate, i n t h e p re sence o f a l k a l i , r e a c t s w i t h t h e pr imary amine t o y i e l d a h i g h l y c o l o r e d orange-red product . The excess ye l low r e a g e n t i s t h e n b l eached 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 w i t h a c e t a t e b u f f e r . T h i s procedure 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 e ~ ~ and 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 o f p ropa raca ine .

6 .6 Chromatographic Procedures 6 .6 1 Paper Chromatography

Koehler 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 loca l a n e s t h e t i c s i n c l u d i n g proparaca ine . The d rugs a re e x t r a c t e d from t h e i r dosage forms and t h e n s u b j e c t e d t o paper chromatographic a n a l y s i s u s i n g Whatman N o . 1 paper . I n t h e s o l v e n t system b u t y l a l coho1 :hydroch lo r i c

4 50 DAISY B. WHIGAN

ac id :wa te 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 system 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 locate t h e spots , t h e d r i e d s t r ips a r e e i t h e r viewed under an 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 modi f ied Dragendorff r e a g e n t ( a c i d i f i e d mix tu re of po tass ium i o d i d e , bismuth 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 po tass ium 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 rogeneous bases, C la rke9 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 paper N o . 1 i s p r e - t r e a t e d by d i p p i n g i n a 5% s o l u t i o n o f sodium dihydrogen c i t r a t e and d r y i n g a t 25OC f o r one hour . I n t h i s sys t em,p ropa raca ine h a s an Rf of 0.52.

6.62 Thin-Layer Chroma toq raphy Th in - l aye 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 p ropa ra - c a i n e . Using a s o l v e n t system of s t r o n g ammonium hydroxide solution:methanol(3:200), t h e Rf of p ropa raca ine i s 0.5g9 and i t s p o s i t i o n r e l a t i v e t o code ine i s 1.826. 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 sp ray . 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 by viewing under u l t r a v i o l e t l i g h t .

The s p o t s may b e l o c a t e d by

Local a n e s t h e t i c s have been ana lyzed u s i n g t h i n - l a y e r chromatography b y Fuwa and c o - ~ o r k e r 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 system, benzene:acetone:ammonium hydroxide(80:20:1) . 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) reagent.

S e p a r a t i o n o f t h e d rugs w a s

7. A n a l y s i s of Hydro lys i s P roduc t s i n Body F l u i d s and T i s s u e s Reed and Cravey26 r e p o r t e d the 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


and t i s s u e s . They prepared t u n g s t i c a c i d p r o t e i n - f ree f i l t r a t e s from blood, l i v e r , k idney , and 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 each 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 hydroxide 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 . Table 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 T i s s u e Concen t r a t ions o f Hydro lys i s

Product a s Equ iva len t Proparaca ine

Specimen mq/100 m l o r 100 g

Blood Bra in Lung L ive r Kidney U r i n e Stomach

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 chloroform. The chloroform e x t r a c t i s evapora ted 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 S e c t i o n 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 f luo rescence of p ropa raca ine hydroch lo r ide (Sec t ion 2 . 1 5 ) could provide a s e n s i t i v e t echn ique € 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 .

8. Serum P r o t e i n Bindin

of some drugs i n c l u d i n g p ropa raca ine h y d r o c h l o r i d e w i t h bovine serum p r o t e i n s . P ropa raca ine hydro- c h l o r i d e w a s d i s s o l v e d i n 5 m l o f serum and d i a l y z e d a t 4OC a g a i n s t a phosphate-ch lor ide b u f f e r of pH 7.4 f o r 48 hours . The c o n c e n t r a t i o n of proparaca ine i n t h e d i a l y z a t e was de te rmined by

Dastugue and 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

452 DAISY 6. WHIGAN

measurin t h e u l t r a v i o l e t absorbance a t 268 nm. Table 6 t abu la t e s t he amount of protein-bound drug depending on the concentrat ion of drug i n t he serum.

3%

Table 6 Serum Protein Binding of Proparacaine

Hydrochloride

D r u g Concentration ug/ml s e r u m % Bound D r u g

2 5 46.4 50 33 .6

100 26.4 2 00 2 1 . 9 300 19 .4 400 19.6

9. D r u q Metabolism

has been inves t iga ted by d i f f e r e n t workers37. 3 8 9 39,

47. f o r proparacaine hydrochloride i n the l i t e r a t u r e .

The pharmacology of proparacaine hydrochloride

There i s no evidence of blood l e v e l s t u d i e s

Proparacaine hydrochloride i s r ap id ly hydrolyzed by guinea pig l i v e r homogena tes4'. 3 7 O C i n t h e presence of 0.067M phosphate bu f fe r (pH 7 . 2 ) , 456 bmole of drug is hydrolyzed per gram of f r e sh t i s s u e per hour.

At

I n a s i n g l e case where a person purportedly inhaled about 500 mg of a white c r y s t a l l i n e ma te 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 ma te r i a l t o be proparacaine hydrochloride. Working with t h i s case, they reported t h e hydro lys is of proparacaine i n body f lu ids . This observat ion, s i m i l a r t o the hydro lys is of proparacaine when heated i n 2 g hydrochloric ac id (Figure 9), i s t y p i c a l of t he metabolic pathway found with o the r amino alcohol e s t e r type anesthet ics4l9 42. acce lera ted by enzymes i n t he l i v e r , o the r t i s s u e s ,

Hydrolysis i 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 o f t h e drug , Reed and Cravey found no p ropa raca ine i n samples of t h e b lood , b r a i n , l ung , and u r i n e . Some amounts o f t h e h y d r o l y s i s p roduc t , 3-amino-4- propoxy-benzoic a c i d , were found i n t h e b lood , b r a i n , lung, l i v e r , and kidney.

I n s t u d y i n g t h e f a t e of p r o c a i n e i n man, Brodie , L i e f , and Poet28 found t h a t some d ie thy laminoe thano l is e x c r e t e d i n t h e u r i n e w h i l e some of i t i s f u r t h e r metabol ized 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 thy laminoe thano l formed from t h e h y d r o l y s i s o f p ropa raca ine f o l l o w s t h e same f a t e i n man.

454 DAISY E. WHIGAN

10.

1. 2.

3.

4.

5. 6.

7.

0 .

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

2 1 .

R e f e r e n c e s

U n i t e d S t a t e s Pharmacopeia X I X , p 419. T o e p l i t z , B . , S q u i b b I n s t i t u t e , p e r s o n a l communicat ion. P u a r , M . , S q u i b b I n s t i t u t e , p e r s o n a l communica t ion . Whigan, D . , S q u i b b I n s t i t u t e , u n p u b l i s h e d o b s e r v a t i o n . S i e k , T . , J. F o r e n s i c S c i . , u , 1 9 3 ( 1 9 7 4 ) B r e w e r , G . , S q u i b b I n s t i t u t e , p e r s o n a l commu n i ca ti on. S u n s h i n e , I . , "Handbook o f A n a l y t i c a l T o x i c o l o g y " , The Chemical Rubber C o . , C l e v e l a n d , Ohio, 1969, pp. 212-281. H e f f e r e n , J . , K l e s s i g , R . , and D i e t z , C . , J. D e n t a l R e s . , u , 7 9 3 ( 1 9 6 3 ) . Clarke, E . G . , " I s o l a t i o n a n d I d e n t i f i c a t i o n o f Drugs" , The P h a r m a c e u t i c a l P r e s s , London, 1969. R i c h , N . a n d C h a t t e n , L. , J. Pharm. Sc i . , 54 995 ( 1 9 6 5 ) . Koehler, H. a n d Feldmann, E . , Anal.Chem., 3 2 , 2 8 ( 1 9 6 0 ) . J a c o b s o n , H . , S q u i b b I n s t i t u t e , p e r s o n a l communicat ion. V a l e n t i , V . , S q u i b b I n s t i t u t e , p e r s o n a l communicat ion. Ochs, Q . , S q u i b b I n s t i t u t e , p e r s o n a l communicat ion. C l i n t o n , R . , S a l v a d o r , U . , Laskowski , S . , a n d Wilson , M. , J . A m e r . Chem. SOC. , 7 4 , 5 9 2 ( 1 9 5 2 ) . L e r n e r , H . , S q u i b b I n s t i t u t e , p e r s o n a l communicat ion. D i c k c i u s , D . , S q u i b b I n s t i t u t e , p e r s o n a l communicat ion. F l e x s e r , L . , H a m m e t t , L . , a n d D i n g w a l l , A . , J. A m e r . Chem. SOC., 5 7 , 2 1 0 3 ( 1 9 3 5 ) McCredie, R . , S a u i b b I n s t i t u t e , p e r s o n a l communicat ion. P r i b y l , E . , S q u i b b I n s t i t u t e , p e r s o n a l communicat ion. W i l s o n , C . , G i s v o l d , O . , a n d Doerge, R . , "Textbook o f O r g a n i c , M e d i c i n a l a n d Pharmaceu-


22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

t i c a l Chemistry" , J. B. L ippincot t Co., P h i l a d e l p h i a , Toronto, S i x t h Ed. (1971) p. 667. P i f f e r i e , G . , Ger.Offen, 2,046, 620;C&.,=, 45962a (1972) . Monguzzi, R . , P inza, M., P i f f e r i e , G. , E u r . J. Med. Chem-Chim. Ther. , 53,214 (1974) . Buchi, J., S t r i n z i , E . , F l u r y , M., H i r t , R., Labhar t , P . , and Ragaz, L. , Helv.Chim.Acta,34, 1002 (1951) . Za tz , L. , Squibb I n s t i t u t e , p e r s o n a l communication. Reed, D. and Cravey, R . , J. F o r e n s i c S c i . ,=, 275 (1976) . Bickford , C. , Squibb I n s t i t u t e , p e r s o n a l communication. Brodie , B. , L i e f , P . , and P o e t , R . , JL Pharmacol. Exp.Ther., a, 359 (1948) . United S t a t e s Pharmacopeia X I X , F i r s t Supplement, p.42. Feldmann, E . , Mahler, W . , and Koehler , H . , J . A m e r . P h a r m . A s s . S c i . Ed. ,47,676 (1958). P r a t t , E. , J . A m e r . Pharm.Ass. , S c i . Ed. , 4 6 , 724 (1957) . Kel ly , C. i n "Encyclopedia Ind. Chem.Ana1. "

S n e l l , F. and H i l t o n C . , Ed., I n t e r s c i e n c e P u b l i s h e r s , N e w York, London, Sydney, 1967, V o l . 5,p.410. Poet, R . , Squibb I n s t i t u t e , p e r s o n a l commu n i c a ti on. Feldmann, E. , J . A m e r . P h a r m . A s s . S c i . Ed. ,@, 197 (1959) . Fuwa, T . , Kido, T . , and Tanaka, H. , Yakuzaiqaku, 24, 123 (1964)C.A. ,s, 15934g (1964) . Dastugue, G . , Ba t ide , P. , and Meunier, M., Therapie , 16, 804 (1961) . McIntyre, A . and S i e v e r s , R. , J. Pharmacol. Exp. Ther. , 63 ,369( 1938) . Adr i an i , J . , Zepernick, R . , Arens, J. , and Authement, E . , C l in . Pharmacol. Ther. ,?, 49 (1964) . McIntyre,A. , L e e , L . , Rasmussen, J. , Kuppinger, J. , S i e v e r s , R . , Nebraska S t a t e Med. J. , 35,100 (1950) .

4 56

40.

41.

42.

43. 44.

45.

46.

47. 48.

49.

DAISY 6. WHIGAN

L i v e t t , B. a n d L e e , R . , Biochem. Pharmacol . , - 17 ,385 (1968). Gray, C. and Geddes, I . , J .Pharm.Pharmacol . , - 6,89 (1954). K a l o w , W . , J. Pharmacol. Exp. Ther . ,104,122 (1952).

A d r i a n i , J . , C1in.Pharmacol .Ther . ,1,645 (1966). C h a t t e n , L . , C h a t t e n , V., J e f f e r y , D. a n d Uffelmann, S . , J.Pharm.Belq.,29,242(1974). Kocy, O . , Squ ibb I n s t i t u t e , p e r s o n a l commun i ca t i o n . S c h i l t , A . and S u t h e r l a n d , J . , A n a l . C h e m . , 3 6 , 1805, (1964). A d r i a n i , J . , Marque t t e Med. Rev. ,2,46 (1964). Funke, P., Squ ibb I n s t i t u t e , p e r s o n a l communication. Kisch ,B. , K o s t e r , H . , and S t r a u s s , E . , Exp.Med.and Surq . , L,51(1943).

Acknow ledqment

The a u t h o r g r a t e f u l l y acknowledges 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 .

PROPYLTHIOURAC IL

Hassari Y. A hod-Etiein

458 HASSAN Y . ABOUL-ENEIN

CONTENTS Analytical Profile - Propylthiouracil

1. Description 1.1 Nomenclature

1.11 Chemical Names 1.12 Generic Name 1.13 Trade Name

1.21 Emprical 1.22 Structural 1.23 Wiswesser Line Notation

1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Odor


1.2 Formulae


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

3. Synthesis 4. Stability, Decomposition Production and Metal

Comp 1 exe s 5. Metabolism 6. 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 459

1. Description

1.1 Nomenclature

1.11 Chemical Names 2-Thio-4-oxo-6-propyl-lI3-pyrimidine 2-Thio-6-propyl-lI3-pyrimidine-

1,2-Dihydro-6-propyl-2-thioxo-

4-Hydroxy-2-mercapto-6-propylpyri-

4-0xo-6-propyl-2-thio-lI2,3,4-tetra-

2,3-Dihydro-6-propyl-2-thioxo-4 (1H)-.

6-Propyl-2-thiouracil.

4 -one

pyrimidin-4-one

midine

hydropyr imid ine

pyr imidinone

1.12 Generic Name: Propylithouracil.

1.13 Trade Name: Propacil, Propycil, Prothyran, Procasil, Propyl-thyracil, Thyroestat 11.

1.2 Formulae

1.21 Emprical: C7 H10 N2 0s

1.22 Structural:

H Keto tautcawr en01 tautmer

1.23 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%.


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.



2.11 Crystallinity Propylthiouracil is a microcrystal-

line solid. Ashley (1) described a procedure for the preparation of distinctive crystals of propylthiouracil for the purpose of identification.

The crystals are prepared as follows:

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 microscopi- cally. 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. Nisi -- et a1 (3) described the elemental crystal structure €or the reaction product between propylthiouracil and formaldehyde under acidic condition, 8- propyl 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-B from alcohol

Fig. 1 : Photomicrograph of propylthiouracil crystals.


Table I shows the melting range of propylthiouracil reported in the literature

Table I

Reference 0 m.p. ,C

218-221 219-221 220 2 19

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.

(a) To a boiling saturated solution, add an 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. Propyl- thiouracil 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 inten- sive 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 ) . (m.p. 193 ) has been reported for propylthiouracil as a mean of identification of the drug (5).

p-Nitrobenzyl khio ether derivative

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. 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.

In

Galimberti et -- a1 (14) published a detailed study on the ultraviolet spectrophotometry of several derivatives. He reported that the replacement of an oxygen atom at C by sulfer caused 2

d 0

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c,

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.rl c

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464

.I

465

a, a

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466

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 thioamide- CONHCS - 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

o Q pH 7-8).

2.42 Infrared Spectrum The infrared spectrum of propyl-

thiouracil 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) Assignment

3120 NH Stretching imide

stretching

3020-2910 CHI CH2, CH3

2580 (weak since the SH stretching Keto form predominates)

1650 C=O imide carbonyls

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).


2.43 Nuclear Magnetic Resonance S pe c t r um

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 NMR spectrometer with TMS as the internal standard.

The following structural assignments have been made for Fig. 4 .

Chemical Shift ( b ) Triplet at 0.93

Assignment

-CH2CH2CH3

Multiplet centered at 1.60 Multiplet centered at 2.50 (Solvent protons at 2.63 for DMSO-d5).

Singlet at 5.66

Broad singlet at 12.6

-CH2CH2CH3

-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). --

I

4 69

P

51

142

1

170

0

9 H = 3 5

I

Fig. 5 : Mass Spectrum of propylthiouracil (EI).

m

0' I

K

I

U

r( s

i

u Q) a

rn

m

cn

471

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) A loss of CO to give an abundant ion at m/e 8 3 ,

(b) 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.

(c) A loss of HN=C-CHion to give ketene 3 7

m/e 41.

m/e 7 0 . (d) A loss of CH = C = 0 to give an ion

3 . Svnthesis

CH3 CH2 CH2 CO CH2 COOC H + H2N-CS-NH2 2 5

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 di- valent metals e C U + ~ , Pb+2, Cd+2, Ni+2 and Zn+2 but not with Fe $3; '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 re- garding their structures, stability constants, so 1 u b i 1 i t y an a 1 y se s and s p e c t r op ho t ome t r i c properties.

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) .

W

X u

to

m “

3

” xW

u

474

PROPYLTHIOURACIL 475

6. 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 phenolphtha- lein 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 was added and the solution was titrated with 0.1 N NaOH to a persist- ing 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

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

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.

ethanol as indicator. The method was adopted by

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 non-

aqueous 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 of colorimetric analyticalmthcds had been developed for the determination of propylthiouracil in pure form, pharmaceutical formulations, tablets and animal feeds.

(a) The use of Grote reagent:

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 3 x M.

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 modi- fiedvchristeinsen (47). The authors claimed that the method was suitable for the analysis of thiou- racild in very small amounts ( 2 0 0 p.p.m) in the presence of other biological substances.

Doden et a1 (44) had applied the --

the concentration range 0 .5 x lo-' to

Brueggeman and Schole (45) applied

(b) 2,6-Dichloroquinone Chloroimide reagent : McAllister and Howells (13) report-

ed a method for analysis of propylthiouracil in- tablets 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

( C ) Ruthenium c h l o r i d e color r e a c t i o n : Re inha rd t (48) d e s c r i b e d a co lor 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 imi la r compounds. I t w a s based on t h e reac- t i o n between t h e th iocarbamide l i n k a g e - CONHCS - and RuCl i n s t r o n g a c i d medium. The color devc- loped w a s measured a t 520 nm and compared w i t h a s t a n d a r d c a r v e .

( d ) Isopropylamine - c o b a l t acetate r e a g e n t : Ho l t and Mattson ( 4 9 ) deve loped a

colorimetric a s say f o r compounds c o n t a i n i n g t h e groups - CONHCO - and - CONHCS - , i n c l u d i n g propyl - t h i o u r a c i l . A c o l o r developed 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 sopropylamine 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 ch loroform) and c o b a l t a c e t a t e (made of 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 concen t r a - t i o n of 1 m c g / m l .

(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 ) pub l i shed 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 of t h iou - r a c i l and ana logs u s i n g hydroxylamine 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 re sence o f sodium b i c a r b o n a t e , bromine and phenol . The g r e e n i s h b l u e color developed w a s compared w i t h s t a n d a r d cu rve .

( f ) Potassium i o d a t e - a c e t i c a c i d c o l o r r e a c t i o n : A method based on t h e color deve-

loped by t h e r e a c t i o n of p r o p y l t h i o u r a c i l and i t s methyl ana log , w i th K I O and acet ic a c i d w a s used t o de te rmine t h e s e drug2 i n t a b l e t s ( 5 1 ) .

x ima te ly 50 mgs, i n 1 0 m l e t h a n o l and 30 m l of water, w e r e a l lowed t o s t a n d €or 30 minutes . The f i l t e r e d s o l u t i o n ( 0 . 5 m l ) , 5 m l of K I 0 3 (0 .5 w/v% s o l u t i o n ) , and 2 m l of acet ic a c i d were d i l u t e d w i t h water . The color a t 465 nm w a s measured a f t e r 8 0 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 minutes . A c a l i b r a t i o n cu rve w a s made f o r comparison.

Powdered t a b l e t s c o n t a i n i n g appro-


6 . 3 . Ultraviolet Spectrophotometric:

The alkaline solution of propylthiouracil shows two peaks, one at 234 nm and the other at 260 nm, the first maximum has a higher molar absorptivity. The ultraviolet absorption of propylthiouracil in ammoniacal solution at 234 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:

6 . 4 1 . 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 summari- zes the solvent systems and visualizing agents used

Table I1

Solvent System Visualising Reference agent

C6H6 : EtOH 16 : 6 RuC 1 5 5

AmOH : H 2 0 I vapor or 56 dicklorobemzapinone chloroimide and alkali

6 . 4 2 . Column Chromatography: Lindsay -- et a1 ( 3 2 , 3 3 ) separated

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 . 4 3 . Thin Layer Chromatography: Begliomini et _ _ a1 (58) described a

procedure for the seperation and identification of several anti thyro id drugs inc lud ing propylthiouracil

PROPYLTHIOURACIL 479

i n animal f e e d s and b i o l o g i c a l samples 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 system was a mix- t u r e of 50 m l ch loroform, 6 m l i sop ropono l , and 0 . 1 m l g l a c i a l a c e t i c a c i d . Amounts up t o 1 mcg were d e t e c t e d by t h i s method. P r o p y l t h i o u r a c i l showed Rfvalue of 0 . 8 1 whi le i t s methyl homologs moved s lower R f 0.65.

Other s o l v e n t systems 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 pub l i shed by Lindsay -- e t a1 (32 , 3 5 ) and summe- r i z e d i n Table 111.

Table I11

Solven t S y s t e m Developer

0 .05 M Ammonium a c e t a t e uv 1 M Ammonium a c e t a t e e t h a n o l

1 5 : 7 5 uv C6H6: i sop ropano l 6 : l uv Hexane : ace tone : e t h a n o l

60:20:2 uv Hexane : ace tone 3:l uv

6 . 4 4 . Gas Chromatographic Ana lys i s : Although a number of methods are

a v a i l a b l e f o r t h e 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 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 based on t h e p r o p e r t i e s of t h e s u l f h y d r y l groups (-SH). However, t h e same p r o p e r t i e s are a l so common t o t h e C=S and -S-S- groups .

F r a v o l i n i and Begl iomini ( 5 9 ) developed a s imple , r a p i d g a s chromatographic 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 animal 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 chromatographic sepa- 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 prepared acco rd ing t o Wheeler ( 6 0 ) . The b e s t r e s u l t s were ob ta ined wi th a g l a s s column c o n t a i n i n g Chro- mosorb was a s o l i d s u p p o r t and 3 % SE-30 polymer methyl s i l i c o n e as t h e l i q u i d phase. The procedure permited s imul taneous i d e n t i f i c a t i o n and de termina- t i o n of a v a r i e t y of t h y r o s t a t i c p roduc t s . A t y p i - c a l chromatogram i s shown i n F ig . ?


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 Retention times, Sec.

2-Thiouracil 5-Methyl-2-thiouracil 6-Methyl-2-thiouracil 6-Propyl-2-thiouracil 6-Phenyl-2-thiouracil

160 178 190 322 774

0

A s t

U D- u)

a

E

I I I

U 1 2 1 0 8 6 4 2 0 TIME, MINUTES

Fig. 7: Gas Chranatograph of thiouracils. A = 2-thouracil B = 5-methyl-2-thiouracil C = 6-methyl-2-thiouracil D = 6-propyl-2-thiouracil D = 6-pheny1-2-thiouracil.

HASSAN Y. ABOUL-ENEIN 482

1.

2.

3.

4.

5 .

6 .

7.

8.

9.

REFERENCES

M.G. Ashley, - - J. Pharm. Pharmacol., &lo1 (1953).

E. Kassau, -- Deut. Apoth. Ztg., 108, 424 (1968)

C. Nisi, M. Calligaris, S . Fabrissin and M. DeNardo, J. Org. Chem., 36, 602 (1971).

The United States Pharmacopeia XIV, Mack Print- ing Co., Easton, Pa. 1975.

- - - -

"Specifications for the Quality Control of Pharmaceutical Preparations" 2nd Ed., World Health Organization, Geneva, 1967, p. 502.

Merck Index, 8th edition, Merck b Co. Inc., Rahaway, N.J., p.878.

British Pharmacopeia, London Her Majesty's Stationary Office, 1973, p. 401.

"CRC Atlas of Spectral data and Physical constants of Organic Compounds" edited by J.G. Grasselli, CRC Press, Cleveland, Ohio, 1973, p. B 980.

M.M. Mosnier, French patent, 1, 012, 739 (1952); through C.A. 52, 4701 (1958). -

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, 8311(19=).

12. G. Nilsson, Sci. Rev. (Holland), 89, 86 (1957); (1957); through C.A. - 51, 8577 (1957).

Pharmacol., 4, 259 (1952).

- -

13. R.A. McAllister and K.W. Howells, J. Pharm. - -

14. P. Galimberti, V. Gerosa and M. Melandri, Ann. Chim. (Rome) , 48, 457 (1958)

15. B. Stanovnik and M. Tisler, Farm.Vestnik., 14, 129 (1963); through C.A. - 61, 6894 (1964).

PROPY LTH IOU RAClL 483

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 Chemica l C o . , Milwan- k e e l W i s c o n s i n , 1 9 7 0 , p . 9 9 4 G .

1 7 . 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 Labora- t 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 . R i c e , G . O . Dudek, M. B a r k e r , - - - J. Amer. Chem. - S O ~ . , 87, 4569 ( 1 9 6 5 ) .

1 9 . J. N i s h i w a k i , T e t r a h e d r o n , 2_2, 3117 ( 1 9 6 6 ) .

20. J. U l r i c h , R. T e o u l e , R. Massot, A . Cornu, Org. Mass. Spectrm. , 2 , 1183 ( 1 9 6 9 ) .

21. S.M. H e c h t , A.S. G u p t a , N.J. Leonard , Biochem. Biophys . A c t a , 1 8 2 , 4 4 4 ( 1 9 6 9 ) . - -

22. G.W. Anderson , I . F . Halvers tad t . W . H . M i l l e r , R . O . R o b l i n Jr. , J. Amer. Chem. SOC. , 6 7 , . 2197 ( 1 9 4 5 ) .

- ~- - -

23. J . D i c k , J. R i s t i c i , and L. Pod, Acad. Rep. P o p u l a r e Romine, Baza Cercetari S t i i n t . Timisoara, S t u d i i Cercetari.

24. S t i i n t e Chim., 8 , 233 ( 1 9 6 1 ) ; t h r o u g h C . A . - 5 8 , 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 , 893 ( 1 9 5 2 ) ; t h r o u g h C . A . - 5 0 , 7851 ( 1 9 5 6 ) .

2 6 . E . R . G a r r e t t and D . J . Weber, J. Pharm. S c i . , -- - - 59, 1 3 8 3 ( 1 9 7 0 ) .

27. E . R . G a r r e t t and D . J . Weber, J. Pharm. S c i . , -- - 6_0, 845 ( 1 9 7 1 ) .

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 , ( 1 9 7 2 ) .

M e t a b o l i s m , - 2 0 , 289

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 . , - 2 1 , 363 ( 1 9 7 2 ) .

484

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

HASSAN Y . ABOUL-ENEIN

D.S. Sitar and D.P. Thornhill, - J. Pharmacol.

R.H. Lindsay, J.B. Hill, K. Kelly and A. Vaughn,

Exp. Ther. 183, 440 (1972)

Endocrinology, 94, 1689 (1974)

- -

R.H. Lindsay, B.S. Hulsey and H.Y. Aboul-Enein, Biochem. Pharmacol., 24, 463 (1975) and references were citedtherein.

R.H. Lindsay, A. Vaughn, K. Kelly and P.V. Pboul - Enein, Biochem. Pharmacol., in press.

M.L. Desbarats - Schonbaum, L. Endrenyi, E. Koves, E. Schonbaum and E.A. Seller, Europ. - J. Pharmacol., 19, 104 (1972)

R.H. Lindsay, H.Y. Aboul-Enein, D. Morel, and S . Bowen, J. Pharm. Sci 63, 1383 (1974) - - - * I -

D.W. Alexander, V. Evans, A. MacAulay, T.F. Gallagher Jr., J. Londono, --- Brit.Med.J.,2, - 290 (1969).

G. Smith, J. Assoc. Office.=. Chemists, - 33, 196 (1955) .

A. Berggren and W. Kirsten, Farm. Revy, 50, 245 (1951); through C.A. - 45, 6348 (1951).

C.F. Abbott, - - J. Pharm. Pharmacol., - 5, 53 (1953).

H.

H.

H.

K.

W.

Wojahn and E. Wempe, Pharm. Zentralhalle, 92, - 124 (1953); through C.A. - 48, 3635 (1954)

Wojahn and E. Wempe, Arch. Pharm., 286, 344 (1953); through C.A. c11-1954). - Wojahn, Pharm. Acta Helv., 28, 336 (1953). --- - Backe-Hansen, Medd. Norsk. Farm. Selskap, - 17, 63 (1955); through C.A.0,113(1956). -

Doden, R. Kopf and H. Specker, Arch. exptl. Path. Pharmakol., 213, 467 (1951)hrough C.A. - 46, 8520 (1952).

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 ( 1 9 6 8 ) .

Provinciali., 13., 206 ( 1 9 6 2 ) ; through C.A. - 57., 1 5 2 4 1 h ( 1 9 6 2 ) .

46. F. Bucci and A.M. Cusmano, Boll. Lab. Chim. - - -

47. H. Christeinsen, - - - J. Biol. Chem., 160, 425 ( 1 9 4 5 ) .

48. F. Reinhardt, Z. Physiol. Chem., - 293, 268 ( 1 9 5 3 )

49. W.L. Holt and L.N. Mattson, Anal. Chem., 21, - - - 1 3 8 9 ( 1 9 4 9 ) .

50. W. Doden and R. Kopt, Arch. exptl. Path. u- makol. 213, 5 1 ( 1 9 5 1 ) ; through C.A. - 46, 383 d ( 1 9 5 2 ) .

51. S. Bruno, Boll. Chim. Farm., 102. , 478 ( 1 9 6 3 ) ; through-. - F 1 5 m a ( 1 9 6 3 ) .

576 ( 1 9 5 1 ) . 52. G. Smith, J. Assoc. Offic. Agr. Chemists., - 34, --

53. G. Smith, J. Assoc. Offic. Agr. Chemists., - 35, -~ 572 ( 1 9 F 2 ) .

54. "Official Method of Analysis of AOAC", W. Hor- witz editor, 11th ed., 1970., Association of Official Analytical Chemist, Washington D.C. 1970 , p . 691.

55. F. Reinhardt, Mikrochim. Acta, 219 ( 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 -- - 6, 1 3 3 ( 1 9 5 2 ) .

57. A.C. Shabica and E. Solook, Federation Proc., - 9, 314 ( 1 9 5 0 ) .

58. A. Begliomini, A. Fravolini, Arch. v e t . u., - 21, 63 ( 1 9 7 0 ) ; through C.A. - 73, 86595 g ( 1 9 7 0 ) .

59. A. Fravolini and A. Begliomini, J. Assoc. Offic. -- - Agr. Chemists, - 48, 908 ( 1 9 6 5 ) .


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 .

2.

3.

4.

5.

6 .

7.

8.

9.

Descr i p t i on

1 . 1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

Phys ica l Proper t ies

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 i ca 1 Rota t ion D i f f e r e n t i a l Scanning Ca lor imet ry Thermogravimetric Ana lys is S o l u b i l i t y Crys ta l Proper t ies

2.9.1 Crys ta l S t r u c t u r e 2 . 9 . 2 X-Ray D i f f r a c t i o n

Syn thes i s

Stabi li t y and Degradation

4.1 Sol i d Stabi li t y 4.2 S t a b i l i t y i n So lu t i on

Drug Metabo l ic Products

T o x i c i t y

Methods o f Analys is

7.1 7.2 7.3 7 . 4 7 . 5 7 .6 7 .7 7 .8 7 .9

Elemental Ana lys is I dent i f i ca t ion Tests Thi n-Layer Chroma tograph i c Ana lys is Spectrophotometric Ana lys is Co lo r ime t r i c Ana lys is Polarographic Ana lys is Cou lometr i c Analys is T i t r i m e t r i c Analys is Miscel laneous Methods of Analys is

Acknowledgements

References

SODIUM NITROPRUSSIDE 489

1 . D e s c r i p t i o n

1 . 1 Name, Formula, Mo lecu la r Weight

Sodium n i t r o p r u s s i d e i s d isodium p e n t a c y a n o n i t r o s y l - f e r r a t e (2 - ) d i h y d r a t e . I t i s a l s o known as sodium 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 fo rm o f t h e compound and i s assumed i n t h i s r e p o r t except where s p e c i f i e d as anhydrous.

C N OFeNa2.2H20 5 6

NC d ON

Sodium N i t r o p r u s s i d e

-2

. 2H20

Mol ecu 1 a r Weight : 297.95

1.2 Appearance, Co lo 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. Phys i ca l P r o p e r t i e s

2 .1 I n f r a r e d Spectrum

The i n f r a r e d spectrum o f sodium n i t r o p r u s s i d e i s p r e - sented i n F i g u r e 1 ( I ) . The spectrum was recorded on a Perk in-Elmer Model 621 G r a t i n g I n f r a r e d Spectro- photometer (Survey C p d i t i o n s ) . persed i n F l u o r o l u b e t o reco rd t h e spectrum i n the r e g i o n of 4000-1340 cm-l and i n m i n e r a l o i l f o r t h e r e g i o n of 1340-370 cm-l . Assignments f o r the bands i n F i g u r e 1 a r e l i s t e d i n Tab le I ( 1 ) . These assicln- ments a r e i n agreement w i t h thase r e p o r t e d i n the 1 i t e r a t u r e ( 2 - 4 ) .

The sample was d i s -

E

3

U 0

v)

al -u

0 .-

.- L

m I

%

C

-

33NV

lllWSN

VL

ll %

490

SOD I U M N IT ROP R USS I D E 49 1

TABLE I

Infrared Assignments for Sodium Nitroprusside

Band (cm-I)

3628

3547 2174

2144, 2157, 2162

1942

1614, 1618.5, 1624

662

65 1

49 1

46 1

418.5

Assignment

Asymmetric OH stretch

Symmetric OH stretch

-C:N axial stretch

-CzN radial stretch

N 4 stretch

OH bend i ng

Fe-N-tD linear bending

Fe-N stretch

Fe-C:N bending

Fe-C- axial stretch

Fe-CEN bending

492

2 . 2

2.3

2.4

2.5

2.6

RICHARD RUCK1

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 ) .

Ultraviolet/Visible Spectrum

The ultraviolet/visible spectrum of sodium nitroprusside (750 mg of sodium nitroprusside/lOO ml of water vs. 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).

Fluorescence Spectrum

Sodium nitroprusside exhibits no fluorescence in acidic, basic o r neutral media (14 ) .

Optical Rotation

A 0.6% (w/v) solution of sodium nitroprusside in water exhibited no optical rotation between 650 and 220 nm (15).

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


F I G U R E 2

N i t r o p r u s s i d e V i s i b l e A b s o r g t i o n Spect rum o f Sodiuni

494 RICHARD RUCK1

f i r s t endotherm (16) . endotherm corresponds t o a weight loss i n the TGA (Sect ion 2 .7 ) . Thermal ana lys i s o f sodium n i t r o - pruss ide has been repor ted i n the l i t e r a t u r e (17-19).

The temperature of each

2.7 Thermogravimetr i c Ana lys is

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 weight 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 we igh t loss f o r d ihyd ra te 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 weight ( t h e o r e t i c a l weight loss f o r cyanogen, (CN) i s 19.85% o f anhydrous sample weight ) (16). f i e iden- t i f i c a t i o n of the second weight loss as cyanogen i s specu la t i ve . Chamberlain and Greene (17,18), us ing dynamic gas e v o l u t i o n ana lys i s , have repor ted tha t the thermal decomposition of cyanon i t rosy l f e r r a t e s i nvo l ve the e v o l u t i o n of water , (CN) and NO. Ger i t i l e t a l . (19) and Mohai (20,21f have repor ted TGA data for sodium n i t r o p r u s s i d e .

2.8 Solubi 1 i t y

Approximate s o l u b i l i t y da ta obta ined for a sample o f sodium n i t r o p r u s s i d e a t 25°C a re l i s t e d i n Table I I (22) . E q u i l i b r a t i o n t ime was 20 hours f o r each system.

So lven t

SOD I U M N IT ROPR USSl D E

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

So lub i 1 i t y (mg/ml)

Water

95% Ethanol

Abso lu te Ethanol

Methanol

Acetone

D i e t h y l E t h e r

Ch lo ro fo rm

Benzene

l s o p r o p y l A l c o h o l

Hexane

E t h y l A c e t a t e

Normal S a l i n e

>200

1 . 1

5.0

100- 200

l n s o l u b

I n s o l ub

I nso lub

0.2

0.1

0.1

0.3

>200

49 5

2.9 C r y s t a l P r o p e r t i e s

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 occu rs as redd ish - brown (or ruby-red) c r y s t a l s ; anhydrous ( l y o p h i 1 i zed ) 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 orange, 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 tho rhomb ic w i t h space group G"-Pnnm. The n i t r o p r u s s i d e i o n 1 i e s or1 the m i r r o r p lane and has approx ima te l y C1,, symmetry. formula u n i t s o f t he type Na Fe (CN) N O ' 2H20.+ Thz c r y s t a l s t r u c t u r e i s compAsed of Na , F S ~ C N ) ~ N O ~ - , and H 0 u n i t s . T h e Fe(CN) N O ions occupy 5 i t e s o f C 5ym- m ~ t r y , ~ a n d the H 0 molecules occupy C s i t e s . The I igands a r e c o l i n e a r w i t h the

2b

The u n i t c e l l c o n t a i n s f o u r

2

2 5

1 2

496 RICHARD RUCK1

metal atom, which i s d isp laced s l i g h t l y i n the d i r e c t i o n o f the NO group from the p lane o f the f o u r pseudo-equivalent CN groups. Each sodium ion 1 i es a t the center o f a d i s t o r t e d octahedron composed of f ou r CN groups and two water molecules. These octahedra share edges i n such a way th$ t each CN group i s coord inated t o two Na ions, as i s each water molecule. The n i - t r o s o group i s coord inated on ly to Fe+2 (24). The water molecules are no t invo lved i n any s i g n i f i c a n t hydrogen bonding w i t h the Fe(CN) NO2- ion; they merely serve t o f i l l the ehpty space i n the l a t t i c e (2, 24, 26). Studies o f the i n f r a r e d (28, 29) and Mbssbauer (30) spec t ra i n d i c a t e a la rge amount o f back bonding between the metal and the n i t r o s y l l igand. Although the f o r - mal charge o f i r o n and n i t r o s y l i n the com- p l e x has been a mat te r of controversy, the general consensus appears t o be t h a t Fe and NO have formal chaKges o f +2 and + I , res- p e c t i v e l y (12, 24, 31-35).

2.9.2 X- Ray D i f f rac 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 russ ide 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 repor ted i n the l i t e r a t u r e us ing a molybdenum t a r g e t (37) .

I ns t rumen t a 1 Cond i ti ons

Instrument GE Model XRD-6 Spectrogon i -

Generator 50 KV, 12.5 mA 0

Tube Target Copper (Cu Km = 1.5418A) Opt ics 0.1" Detector s l i t

ometer

M.R. S o l l e r s l i t 3" Beam s 1 i t N i F i l t e r 4" Take-of f angle

Goniometer Scan a t 0.4" 20/minute Detector Sealed p ropor t i ona l counter

1 .75 KV ( f r o n t ) , 0 .95 KV ( r e a r ) . Pulse h e i g h t se lec to r E l 5


v o l t s , window out . Time constant = 2.5 seconds Range = 1000 c/sec f u l l

sca le Recorder Synchronized w i t h gonio-

Sample Ground a t room temperature meter a t l"/2.5 minutes

TABLE II I

Sodium N i t rop russ ide

11.60 15.61 19.00 21.65 23.08 27. I4

31.25 33.30 35.65

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

A d ( i n t e r p l a n a r d is tance) = n h / 2 s i n 0

*;': 1/10 = r e l a t i v e i n t e n s i t y (h ighes t

i n t e n s i t y = 100)

498 RICHARD RUCK1

3 . 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)

K4[Fe(CN)6].3H20 + 6 HNO + H2[(NO)Fe(CN) 5 ] + 4 KNO 3 + 3 +

NH NO + C02 4 3

H2[(NO)Fe(CN)5] + Na2C03 2 Na2[(NO)Fe(CN) 5 ].2H20 +

2 H20 + CO

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 photo- sensitive, undergoing rapid and numerous reactions, many of which are undefined. Literature descriptions o f the photodecomposition products of nitroprusside are, in some cases, contradictory.

In direct sunlight [Fe(CN) NO] ultimately yields Prussian blue, HCN and NO 144). 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:

2-

Kapatos et al. (45)

Na2[Fe(CN) 5 NO] + h v (X>300 nm) + Na[Fe"'Feli (CN) 61 (sodium salt of Prussian b l u e ) + NO + (CN) + HCN

2

SOD I U M N IT R OP R USS I D E 499

I n s o l u t i o n s b u f f e r e d a t pH 6, Wol fe and Swinehar t ( 4 6 ) have r e p o r t e d format i o n of t he pentacyanoaquo- f e r r a t e ( I I I ) , ag ree ing w i t h seve ra l o t h e r papers (47-50) :

[Fe(CN) + hy(X>jOOnm) -f [ F e l I I ( C N ) H O ] * - + NO 5 5 2

The pentacyanoaquof i - r a t e ( ',I$ undergoes r a p i d equ i - l i b r i u m w i t h [Fe 2 'lP(CN)lO]

Photoaquat ion t o y i e l d [Fe (CN) H O I 3 - and NO has been desc r ibed most f r e q u e n t l y a2 $he p r i m a r y pho- tochemical 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) found 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 :

( S O , 51 ) .

I I

+ NO + H20

++ [Fe' I (CN) 5H20]3- + 2H+ + NO;

The pentacyanoaquoferrate ( 1 I A-undergoes rap i d e q u i l i b r i u m w i t h [Fe2 (CN) ] (55 ) .

Pho to reduc t i on o f [Fe(CN) NO]2- t o [Fe(CN) aqueous 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 solu- t i o n upon s tand ing and exposure 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 ion (57, 58).

10

i n 5 ?he orange-

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 long as s i x months (11,59,60).

I n aqueous s o l u t i o n the 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 ide 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 substances t o form 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 pi-oducts (50, 52 , 6 1 - 7 1 ) .

Spec t ropho tomet r i c measurements have most o f bzen used t o determine 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 nc rease absorbance a t 350-395 nm w i t h deg rada t ion ( I 53 ,55 ,60 ) . Curce has developed a s t a b i 1 i t y

en t r o - i n ,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 the r than n i t r o p r u s s i d e w i t h az ide and measuring the r e s u l t i n g absorbance a t 560 nm ( 7 2 ) . Polaro- graphic s t a b i l i t y s tud ies (11, 73 ) have ind i ca ted t h a t the f i r s t two po la rograph ic waves (Sec t ion 7.6) decrease i n l i m i t i n g cu r ren t w i t h degradat ion, bu t spectrophotometry i s a much more s e n s i t i v e method f o r de tec t i ng photodegradation ( 1 1 ) .

5. Drug Metabol ic Products

When g iven in t ravenous ly , sodium n i t r o p r u s s i d e r a p i d l y lowers b lood pressure by pe r iphe ra l v a s o d i l a t a t i o n and reduc t ion i n pe r iphe ra l res is tance as a r e s u l t of a d i r e c t a c t i o n on the b lood vessel w a l l s , independent o f autonomic i nne rva t i on (74-78). Blood pressure can be main- ta ined a t any l e v e l depending on the 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 n i t r o s o (NO) group (57, 75-77, 79, 80) o f the n i t r o p r u s - s ide r a d i c a l and i s augmented i n bo th doqs and humans by autonomic gang l ion b lock ing agents (57,76).

The drug has an immediate e f fec t ,w i th des i red b lood 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 the i n f u s i o n , b lood pressure r a p i d l y r i s e s to prev ious l eve l s , u s u a l l y w i t h i n 1 t o 10 minutes (81-84). Th is evanescence o f the d rug ' s e f f e c t i s due t o rap id d e s t r u c t i o n of the a c t i v e n i t r o - p russ ide r a d i c a l which i s s low ly converted i n the body t o cyanide. Th is conversion i s a t t r i b u t e d t o the i n t e r - a c t i o n of the fe r rous i on 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 ry th rocy tes ( red b lood c e l l s ) and o the r t i ssues (57,76,79,85,86). I n v i v o and i n v i t r o s tud ies 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 contacted w i t h l i v e r (85) . whole blood, washed ery th rocy tes , plasma, and u r i n e (76,87-89). The re lease o f cyanide i s non-enzymatic (76,79,85,87), and i t s slow t i m e course prec ludes the reac t i on from being the 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 con- ver ted by the hepa t i c enzyme rhodanase ( t ranssu l fu rase ) t o th iocyanate (79,90). A small amount o f the th iocyanate i s ox id i zed back t o cyanide by a th iocyanate oxidase present in e ry th rocy tes (91,92) and perhaps a l s o by a reversa l of the rhodanase system (93). these two compounds to be i n dynamic equ t l t b r rum but t h a t the e q u i l i b r i u m 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. approx imate ly seven days w i t h normal renal f u n c t i o n (95) . A metabo l ic scheme (57) i s presented i n F igure 3.

Boxer and Rickards. (g$) found

The h a l f - l i f e f o r e x c r e t i o n o f th icyanate i s

m -----j

7-

U 0

m

C

.-

LL

'

c,

z

.- .- -0 0

v)

LJ

a) '>

U

Q)

x

c

Q)U

%A

.- 3

m

0 .-

Q)

c,

m

'I 'I I z

u

I c\l 0

z LA

u

a)

LL

Q) L

C

u .-

n

m

50 1

502 RICHARD RUCK1

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 long p e r i o d s does n o t s i g n i f i c a n t l y lower b l o o d p ressu re ; t h e e f f e c t s a r e s i m i l a r t o t h o 5 e o f t h i o c y a n a t c g i v e n o r a l l y ( 7 6 ) . S ince t h i o c y a n a t c accumulates i n b l o o d w i t h p ro longed n a t e o r cyan ide o f the d rug (57

6. T o x i c i t y

n f u s i o n o f sod may be respons 76,77) .

Sodium n i t r o p r u s s i d e has few s

um n i t r o p r u s s i d e , t h i o c y a - b l e f o r some l a t e r e f f e c t s

de e f f e c t s , none o f wh ich u s u a l l y r e q u i r e s d i s c o n t i n u a n c e o f therapy, p r o v i d e d t h a t dosage i s reasonable (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 hough t due p r i m a r i l y to f o r m a t i o n o f cya- 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 immediate 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 hypo tens ion , 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 depressor and t o x i c dosages as 1 : l O .

Cau t ion shou ld be e x e r c i s e d i n t r e a t m e n t w i t h sodium n i t r o p r u s s i d e s i n c e i t s immediate m e t a b o l i c p r o d u c t s a r e t h i o c y a n a t e and cyan ide ( S e c t i o n 5 ) . Pro longed t r e a t m e n t 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 impa i red (57,76,97). T o x i c 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 appe- t i t e (58,76). I n a p a t i e n t w i t h seve re r e n a l i n s u f f i c i - ency, long- term 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 by 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 up take 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 though 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 observed 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 pro longed, 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 ) . E l e v a t e d b l o o d cyanide l e v e l s i n v i vo . 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), b u t i n t h e v a s t m a j o r i t y o f cases the 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 cyan ide amounts were smal 1 and idel 1 below l e t h a l concen- t 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 cyan ide 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 imul taneous decrease i n plasma v i t a i i l i n B ( 9 9 ) , a l t h o u g h t h e r e were no adverse e f f e c t s on the

I2

SO D I U M N I TROPR USS I DE 503

p a t i e n t s . S ince t h e l i v e r serves as t h e main r e g u l a t o r y system o f cyanide 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 should 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 impai red 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 we igh 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 the 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 ove r 1-1/2 - 2 hours i s e q u i v a l e n t t o 320 mg/hour i n man, and 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 (101) . I n t ravenous LD has been determined t o be 8.4 2 0.3 mg/kg i n mice,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 approx ima te l y 5 mg/kg i n dogs (102). LD o r z ? l y and g r e a t e r than 2000 mg/kg t o p i c a l l y (103).

1 . 1 mg/kg

i n mice has been determined to be 48 2 2.9 mg/kg

7. Methods of A n a l y s i s

7.1 Elemental A n a l y s i s

An e lementa l a n a l y s i s o f a s tandard sample of 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 resen ted i n Tab le I V . Water was determined by K a r l F i s h e r

04 ) . t i t r a t i o n (

E 1 emen t a 1

E 1 emen t

C

H

N

"3

Fe

TABLE I V

A n a l y s i s o f Sodium N i t r o p r u s s i d e

% Theory % Found

20.14 20.12

1.34 1.40

28.21 29.68

15.44 14.98 18.74 18.72

12.09 12.03 H2°

7.2 I d e n k i f i c a t i o n Tes ts

The v i s i b l e a b s o r p t i o n spectrum ( S e c t i o n 2.3) i s s p e c i f i e d b y USP X I X as the 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

504

7 . 3

RICHARD RUCK1

t h e d rug .

For t h e dosage form, 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 dropwise a d d i t i o n o f sodium h y d r o x i d e T.S. t o produce 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) .

Thin-Layer Chromatographic 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 f rom i t s - m e t a b o l i t e s , t h i o c y a n a t e (SCN ) and cyan ide (CN ) , a r e l i s t e d i n T a b l e V ( 1 1 1 ) . 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 -wi th t h e method o f 0 . Wasc3wik e t al. ( 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 system 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 system ( 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 rom 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, r e s p e c t i v e l y ( 1 1 1 ) .

TABLE V

Thin-Layer Chromatographic Systems fo r Sodium N i t r o p r u s s i d e

S o l v e n t

Va 1 ues

- CN- N i t r o p r u s s i d e SCN-

- ! f

n-propanol :H20 (10:2) - - -- -- n -bu tano l :2N NH

(o rgan i c LhasJ) 0 0.20 0.71

n-propanol :H 0 (10: 1 ) 0 0.44 0.77

n-butano1:n-propanol :

( I : 1 )

2

d i b u t y l a m i n e ( 4 5 : 4 5 : 10) 0 0.95 0.85

7 .4 Spec t ropho tomet r i c A n a l y s i s

Sodium n i t r o p r u s s i d e may be analyzed spec t ropho to - m e t r i c a l l y by u t i l i z i n g the mo la 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 ) .


7.5 C o l o r i m e t r i c A n a l y s i s

Small amounts of n i t r o p r u s s i d e have been determined c o l o r i m e t r i c a l l y as t h e isophorone complex by measur- i 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 , lO-phenan th ro 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 Polarograph i c Ana l y s i s

Sodium n i t r o p r u s s i d e has been determined po la rog raph- i c a l l y by a number o f workers. A t t h e d r o p p i n g mer- c 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 f rom t h e n i n t h e l l k o v i c equa t ion , a r e independent o f the hydrogen i o n c o n c e n t r a t i o n i n t h e pH range 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 ( 3 1 , l l 6 , 1 1 7 ) . Zuman and Kabat (118,119) con f i rmed 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 reduc- t i ons , and deduced t h a t t h e t h i r d wave was a two- e l e c t r o n r e d u c t i o n , b u t cons ide red 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 t y p i c a l po larogram 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 about -0.33 v o l t s vs. Ag/AgCI re fe rence 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 fo rm (50 mg d r y - f i 1 l e d v i a l ) (105,120). Photodegra- d 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 by f o l lowing t h e decrease 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 Coulornet r ic A n a l y s i s

Cou lomet 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 rner- cu ry cathode 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 fou r faradays per mole o f e l e c t r o d e r e a c t i o n , respec- t i v e l y , w h i l e the p roduc ts o f r e d u c t i o n 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

F I G U R E 4 Polarogram of Sodium N i troprusside

SODIUM N ITROP RUSSl D E 50 7

( 1 2 1 , 1 2 2 ) . I t has a150 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 , probsb 1 y due to Loinpe t i ng background rcac- t i o n s ( 1 2 0 ) .

7 .8 T i t r i m e t r i c A n a l y s i s

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 the sample i n water 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 endpo in t i s 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 s i l v e r - s i l v e r c h l o r i d e e l e c t r o d e system. Each rnl o f 0.1N s i l v e r n i t r a t e i s equiva- l e n t t o 14.90 mg o f Na2TFe(CN) N0].2H20 (105) . 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 - con-rubber 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 (123) . T i t r i - m 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 desc r ibed 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 endpo in t , has been r e p o r t e d (125). A f t e r decomposi t ion 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 uo roch romic i n d i c a t o r .

7.9 M isce l l aneous Methods of A n a l y s i s

N i t r o p r u s s i d e has been determined 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 (126) , and by p re - c i p i t a t i o n o f n i c k e l hyd rox ide i n t h e r e a c l i o n o f n i c k e l cyan ide 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 than t h e former, b u t cyanide, f e r r i c y a n i d e , and l a r g e amounts o f f e r r o c y a n i d e w i 1 1 i n t e r f e r e ( 1 1 3 ) .

A m i c r o c r y s t a l t e s t , one i n which t h e p r e c i p i t a t e formed by t h e chemical r e a c t i o n between a substance and a reagent i s examined w i t h a microscope, has been r e p o r t e d f o r the detet-rni na t i o n o f sodi urn n i t r o p r u s s i d e ( 1 2 8 ) .

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 the i o n i c c o n c e n t r a t i o n (129).

508 RICHARD RUCK1

8. Acknow I edgiilen t-5

The a u t h o r w ishes t o acknowledge 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 Depar tment , and t h e Research Records O f f i c e o f Hoffrnann-La Roche I n c . i n t h e p r e p a r a t i o n o f t h i s a n a l y t i c a l p r o f i l e .

SOD I UM N ITROPR USSl DE

9. References

1 .

2.

3.

4.

5 -

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20. 21. 22 .

2 3 .

Waysek, E . , Hoffmann-La Roche Inc . , Personal Communication. Khanna, R.K. , Brown, C . W . and Jones, L.H., I n o r g . Chem. - 8, 2195 (1969). Bates J.B. and Khanna, R.K., Inorg. Chem. - 9 , 1376 (19701. Holzbecher, M. Knop, 0 . and Falk , M., Can. J . Chem. - 49, 1413 (1971) . P a l i a n i , G . , P o l e t t i , A. and Santucci, A., J S t r u c t . 8, 63 (1971). Krasser, W., Ber. Bunsenges. Phys. Chem. - 74, (1970). CA 73: 19964w, 1970. Tos i , L . , C .R . Acad. Sc i . , Ser. B. - 270, 688 CA - 72:138137t, 1970.

Mol.

476

1970).

509

Tosi , L . , Poulet, H. and Mathieu, J.P., C.R. Acad. Sc i . Pa r i s , Ser. Ai3. 110309k, 1969. Chang, F., Hoffmann-La Roche Inc., Personal Commun i ca t i on. Rubia, L., Hoffmann-La Roche Inc., Personal Commun i cat ion . Frank, M.J., Johnson, J.B. and Rubin, S.H., J. Pharm. Sci . 65, 44 (1976). Manoharan, P.T. and Gray, H.B., J. Amer. Chem. SOC. - 87, 3340 (1965). Swinehart, J.H. and Rock, P.A., Inorg. Chem. 5, 573 ( 1966). Winslow, W., Hoffmann-La Roche Inc. , Personal Commun i c a t ion. Toome, V., Hoffmann-La Roche Inc., Personal Commun i cat ion . Stewart, D . , Hoffmann-La Roche Inc., Personal Communication. Chamberlain, M.M. and Greene, A.F., J. Inorg . Nucl. Chem. 25, 1471 (1963). Greene, A .F .and Chamberlain, M.M., U.S. Dept. Cornm., O f f i c e Tech. Serv., AD 282, 397 (1962). CA 2: 10968d, 1963. G e n t i l , L.A., Olabe, J.A., Baron, E.J., and Aymonino, P.J., J. Therm. Anal. 7, 279 (1975) . Flohai, B., J. Therm. AKa1. 3 , 403 (1971). Mzjhai, B. , J. Inorg. Nucl. Them. 3 1 , 885 (1969). P h i 1 i p , C . , Hoffmann-La Roche Inc, Personal Communication. Scerbo, A . , Hoffrnann-La Roche Inc.,,Data on Fi l e .

- 2688, 249 (1969). CA - 70:

510 RICHARD RUCK1

24

25

20.

7 7 . 28.

29. 30.

31. 32. 33 - 34.

35. 36. 37.

38.

39.

40.

41.

42.

45.

46.

47.

48.

49. 50.

51. 52. 53.

Manohzran, P.T. and Hami l t on , W . C . , I n o r g . ihem. 2, 1043 (1963). Tos i . L . , C . R . Acad. S c i . P a r i s , Ser. AB e, 1313 (1967) . C A -_ 67:5890!+x, 1967. Cot ton . F.A., Monchanp, R.R. , Henry , R.J. and Young, R . C . , J. Ino rg . Nuc l . Chen. lo, 28 (1959). Brown, D.B., I n o r g . Chem. 16, 2582 (1975). H a b e r d i t z l , W . , S c h l e i n i t z , W.D. and B a r t e l , H .G. , 2 . N a t u r f o r s c h . E, 891 (1968). Folkesson, B., A c t a Chen. Scand., Ser . A . 28, 491 (1974). Costa, N.L., Danon, J. and X a v i e r , R.M. , J. Phys. Chem. Sol i d s 2, 1783 (1962). Masek. J . , I n o r g . Chim. A c t a Rev. 1, 101 (1969). B r o w , D . B . , I n o r g . Chim. Ac ta Rev. 5, 314 (1971). Swinehar t , J.H., Coord. Chem. Rev. 2. 385 (1967) . M i t r a , R.P. , Sharma, B.K. and M i t t a l , S . P . , J. Ino rg . N L I C ~ . Chern. 3, 3919 (1972). Garg, A . N . and Goel, P .S . , I n o r g . Chem. 10, 1344 (1971). N a c r i , F . , Hoffmann-La Roche Inc . , t ' e rson~ l Communication. ASTM, X-Ray Powder Data F i l e , Se ts 1-5 ( I n o r g a n i c ) , 1960, p. 43 . Ephraim, F . , I n o r g a n i c Chemis t ry , 6 t h Ed., I n t e r s c i e n c e , New York, N . Y . , 1954, p. 332. Braucr . G . , Handbuch der P r 8 p a r a t i v e n Anorganischen Chernie, 2nd Ed., Fe rd inand Enke, S t u t t g a r t , 1962, p. 1530. R m y , H. , T r e a t i s e on I n o r g a n i c Chemis t ry , E l s e v i e r , Amsterdam, 1956, p. 287. Johnson, J.B. and Frank, M.J., Hoffinann-La Roche Inc . , I n t e r n a l Repor t , May 7, 1973. Newrnark, H.L., Hoffmann-La Roche Inc . , I n t e r n a l Repor t , May 10, 1973. Johnson, J.B., t lof fmann-La Roche Inc . , Unpub l i shed Data . Gmel in 's Handbuch de r Anorgan ishen Chemie, I r o n 598, Ve r lay Chernie, Gmbh, B e r l i n , 1338, p. 903. Kapatos, L . , Voya tsak i s , E . and Yanoukakis, D., Chim. Chron ika 3, 72 (1958). CA 2 : 1 7 9 5 O f , 1958. Wol fe , S . K . and Sw inehar t , J .H . , I no rg . Chem. 14, 1045 (1975). Buxi:)n, C . V . , Da in ton , F.S. and K a l e c i n s k i , J . , I n t . J. R a d i a l . Phys. Chern. _I_, 87 (1969) . CA 71:34986g, 1969. Wolfe, S . K . , D i s s . Abst i - . I n t . 3, 31% (1974). CA g : 1 9 1 8 3 f , 1974. E n s c h w i l l e r , C . , C . R . Acad. S c i . , Ser . C 268, 692 (1969) . Espenson, J.H. and Wolcnuk. S . G . , I n o r y . Chem. !I, 2034

J ;Jse lsk i s , B . , J. Amer. Chen. S O C . 83 , 1082 ( 1 9 6 1 ) . B s u d i s c l i . O., Scicncr: IOS, 4 / 1 3 (l91+$)-. Lnclrin\l-.a, A. arid Cogo-lE, R . , Rocz. Chen. '17, 881 ( 1 9 7 3 ) . LA I;,: 120420q, 1973.

( 1772) .

SOD I U M N ITR OP R U S l O E 51 1

54.

55.

56. 57.

5 8 . 59.

60.

61.

62.

( ( 3 . 64. 65.

61.

67.

68.

69.

70.

71.

72 - 73.

74. 75. 76.

77.

78.

79. 80.

81.

M i t r a , R.P., Ja in , D . V . S . , Banerjee, A.K. and Ctiari , K . V . R . , J. Inorg. Nucl. Chem. 3, 1263 (1963). Enschwi I l e r , G . and Jorgensen, C.K., Chem. Phys. L e t t . - 5, 561 ( 1 9 7 0 ) . G r i f f i t h . W.P., Quart. Revs. 6, 188 (1962). Cacace, L. and Thomas, T . , Drug I n t e l . C l i n . Pharm. 4, 187 (1970). Tuzel, I.H., J. C l i n . Pharmacol. 14, 494 (1974). Anderson, R.A. and Rae, W., Aust. J. Pharm. Sci. [NS] 1, 45 (1972). Mar t in , 1. and Pa te l , J.A., Amer. J. Hosp. Pharm. 26, 51 (1969). Toma, H.E., Mal in , J.M. and Ciesbrecht. E., Inorg. Chem. - 1 2 , 2084 (1973). Mulvey, 0. and Waters, W.A., J. Chem. SOC., Dal ton Trans., 951 (1975). Masek, J. and Wendt, H., Inorg. Chim. Acta 3, 455 (1969). Dempir, J. and Masek, J., Inorg. C h i m . Acta 2, 402 (1968) Tarugi , N., Ann. Chim. Appl icata. 16, 407 (1926). CA 11: 855, 1927. Szrgceva, A.N., Sernenishin, D . I . and Mazepa, A.V., Zh. Neoiy. K h i m . E, 396 (1975). Ya ’ , im i r sk i i , K.B. e t a l . , Teor. Eksp. Khim. lo. 653 ( 1 3 7 4 ) . CA 82:49687m, 1975. B e i l i n g e r , J.R., L ind fo rs , K.L. and West, D.X., J. Inorg. Nucl. Chem. 32, 3837 (1970). Cambi, L., A t t i . Accad. Nazl. L i n c e i 14, 434 (1915). CA E : 1 3 5 1 , 1916. Rybkina, A.A. , Ref. Zh. Khim. 1969, Abstr . 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. 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 Stewart , 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. S, 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 rh lan t , R . C . , Tsagaris. T ’x and Robertson, R.J . , Amer. J. Card io l . 2, 51 ( I ) . G’iatia, S.K. and F r o h l i c h , ( I J73). Veriier, I . R . , Postgrad. Med. J. 0, 576 (1974). 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 2, 8011 (1968).

CA&:164344 j , 197.5.

. U . , Amer. Heart J. 5, 367

51 2 RICHARD RUCK1

82. 83.

84.

85. 66. 87.

88.

89.

30 * 91 - 92 - 93. 54.

95.

96. 97.

98. 99. 100.

101 *

102.

103.

104.

105.

106.

107. 108.

109.

hloraca, P. e t a l . , Anesthesiology 23, 193 (1962). Schiffmann, H. and Fuchs, P., Acta 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. H i l l , H .E. , Aust. Chem. I n s t . J . Proc. 9, 89 (1942). Michael is , L., J. B i o l . Chem. 86, 777 (1929). Smith, R.P. and Kruszyna, H., J. Pharmacol. Exp. Ther. - 191, 557 (1974). Vesey, C.J., Cole. P.V., L i n n e l l . J.C. and Wilson, J . , B r i t . Med. J. 2, 140 (1974). Vesey, C.J., Cole, P. and Simpson, P., B r i t . J. Anaesth. - 48, 268 (1976). Lang, K., Biochem. Z. 259, 243 (1333). Goldste in , F. and Rieders, F., Amer. J. Phys io l . 173, 287 (1953). Pines, K.L. and Crymble, M.M., Proc. SOC. Exp. B i o l . Hed. s, 160 (1952). Rosenthal, D., Fed. Proc. I-, 181 (1948). Boxer, G.E. and Rickards, J.C., Arch. Biochern. 39, 7 (1952). Dreisbach, R., Handbook o f Poisoning, 5 t h Ed., Lange, 1966, p. 298. Mani, H.K., B r i t . Med. J. 2, 407 (1971). Nourok. D.S., Glassock, R.J., Solomon, D.H. and Maxwel I , M .H., Amer. J. Med. Sci . 248, 129 (1964) . G i f f o r d , R., Med. C l i n . N. Am. %,TI (1961). Wilson, J . and Matthews, D.M., C l i n . Sc i . g, 1 (1966). Spiegel, H.E. and Kucera, V . , Hoffniann-La Roche Inc., 1rt::rnal Report, Nay I D , 1976 ( t o be pub l i shed i n C l i n . Chem. Acta) . McDowall, U . G . , Keaney, N.P., Turner, J.M.,Lane, J.R. anJ Okuda, Y. , B r i t . J. Anaesth. 5, 323 (1974). Pool, W. and Hane, D., Hoffmann-La Roche Inc.. I n t e r n a l Report, May I . 1973. Hane, D., Hoffniann-La Roche Inc. , I n t e r n a l Report, Scptember 8, 1975. Scheid l , F., Hoffmann-La Roche Inc. , Personal Communica- t i o n . Un i ted States Pharmacopeia X I X (2nd Suppl.), pp. 111-112 (1974) . Sas, F.E.R., Inform. Quini. Anal. 16, 179 (1962). CA 62: 1317e, 1965. Ohkuma, S., J. Pharm. SOC. Japan 2, 1101 (1952). S c a g l i a r i n i , G. and M a r l f o r t e , F., A t t i . Accad. L i n c e i 20, 4 1 (1934). CA 2 : 3 6 2 2 , 1935. G i r a l , J . , Anales S O C . Espan. F i s . Quim. 21, 236 (1923). CA x:300l, 1923.


110. Johar, G . S . and S i n > ? , J.P., Mikrochim. Acta 1974, 437

1 1 1 . Hobel, M. andRa i the lhuber , A., Arch. Pharmacol. 282

112. Waschwik, 0. e t a l . , Arch. f. Tox. 3, 52 (1967). 113. Jaselsk is , B. and Edwards, J.C., Anal. Chem. 2,

114. Buhl, F., Gregorowicz, 2 . and Kania, K., P r . Nauk. Uniw.

115. Ruhl, F., Kania, K. and Mikula, B., P r . Nauk. Uniw. Slask.

116. Ko l tho f f , I .M. and Toren, P.E., J. Amer. Chem. SOC. 75,

117. Masek, J. and Dempir, J., Co l l . Czech. Chem. Corn. 34, 118. Zuman, P. and Kabat, M., Co l l . Czech. Chem. C m . 19,

119. Zuman, P. and Kabat, M., Chem. L i s t y 5, 368 (1954). 120. Hamilton, C. and Moros, S., Hoffmann-La Roche Inc..

121. Lanza, P. and C o r b e l l i n i , A., A t t i Accad. Nazl. L i n c e i 2, 122. Lanza, P. and C o r b e l l i n i , A., A t t i Accad. Nazl. L i n c e i 13,

123. Siska, E. and Pungor, E., Ta lanta 19, 715 (1972). 1211. Tomicek, 0. and Kubik, J., Co l l . Czech. Chem. C m . 9,

125. Mart inez, F.B. and B a r r a l , A.M., Inform. Quim. Anal. 2,

126. Gusev, S.I. and Bei les, R.G., Zhur. Anal. Khim. 21, 219

127. Rose, P.H., Z. Anal. Chem. 143, 195 (1954).

128. Koles, J.E., Prog. Chem. Toxico l . 5, 293 (1974). 129. Voyatzdkis, E. and Jannakoudakis, D., Compt. Rend. 247,

(1974). CA 81:45039h, 1974.

(Suppl.) , R33 (1974).

381 (1960).

Slask. Katowicach 9, 9 (1970).

Katowicach 5, I I (1973). CA g :67758q , 1975.

1197 (1953).

727 (1969). CA Z:92619e, 1969.

673 (1954). CA 3 : 6 7 4 9 e , 1955.

I n t e r n a l Report, A p r i l 19, 1973.

65 (1953). CA ~ : 9 1 7 9 g , 1953.

Ji36 (1952).

377 (1937).

117 (1965). CA e : 5 7 5 8 , 1966.

(1952).

99, 1955. CA 9:

1721 (1958). CA 53: l0909, 1959.

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. D e s c r i p t i o n 1.1. N a m e , Formula, Molecu la r Weight 1 . 2 . Appearance , Colour, Odour, Taste

2 . 1 . I n f r a - r e d Spec t rum 2 . 2 . U l t r av io l e t Spec t rum 2.3. F l u o r e s c e n c e a n d Phosphorescence

S p e c t r a 2 . 4 . Mass Spec t rum 2.5. N . M . R . Spec t rum 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 Gravimetric 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 . 1 1 . S o l u b i l i t y

2 . P h y s i c a l P r o p e r t i e s

2 . 1 1 . 1 . I n Aqueous B u f f e r s and

2 . 1 1 . 2 . I n S o l v e n t s Ur ine

2 . 1 2 . D i s s o c i a t i o n C o n s t a n t 2 .13. P a r t i t i o n C o e f f i c i e n t s

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 S y n t h e s i s 3.2. P u r i f i c a t i o n

4 . 1 . Organ ic S a l t s 4 . 2 . Metal Complex S a l t s

4 . S a l t s

5 . Chemical S t a b i l i t y 5 .1 . 5 .2 . 5 .3 .

6 .1 . 6 . 2 . 6 .3 .

6 . Methods

6 . 4 .

H y d r o l y s i s P y r o l y s i s P h o t o l y s i s o f A n a l y s i s I d e n t i f i cat i o n E l e m e n t a l A n a l y s i s 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 . D i a z o m e t r i c 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 . A r g e n t o m e t r i c T i t r i m e t r y 6 .3 .5 . Complexometr ic T i t r i m e t r y 6 .3 .6 . Thermometr ic T i t r i m e t r y 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

SULPHAM E R A2 IN E 51 7

6 . 4 . 2 . U l t r av io l e t S p e c t r o s c o p i c

6 .4 .3 . C o l o r i m e t r i c Methods

6.5.1. High Performance L iqu id

6.5.2. Gas Chromatography 6.5.3. Thin Layer Chromatography 6.5.4. Paper Chromatography 6.5.5. Ion Exchange and P a r t i t i o n

Chromatography 6.5.6 E l e c t r o p h o r e s i s

6 . 6 . 1 . Po larography 6 . 6 . 2 . Ion S e l e c t i v e E l e c t r o d e s

M e t h o ds

6 .5 . Chromatographic Procedures

Chromatography

6 . 6 . E l ec t rochemica l Methods

6 . 7 . Bioassay 7. Es t ima t ion i n B i o l o g i c a l F l u i d s 8. Pharmacology

8.1. Metabolism 8.2. Absorption,Distribution,Excretion

8.2.1. I n Humans 8.2.2. I n Animals

8 .3 .1 . Acute T o x i c i t y 8 .3 .2 . Chronic T o x i c i t y 8 .3 .3 . C l i n i c a l T o x i c i t y

8 .3 . T o x i c i t y

9 . P r o t e i n Binding 10. Pharmacodynamics 11. Acknowledgements

R e f e r en ce s

518 RICHARD D. G. WOOLFENDEN

1. Desc-r ipt ion

1.1. N a m e , Formula, Molecular Weiqht 1 Gener ic names - Sulphamerazine;

Methylpyrimal; Sulphamethyld iaz ine .

Nomenclature - The f o l l o w i n g nomencla- t re i s used i n Chemical Abstracts: N - (4-methyl-2-pyrimidinyl) s u l p h a n i l - amide ; 4-amino-N- (4-methyl-2-pyrimidi- n y 1 ) be n zene s ul ph on a m i de . P

S t r u c t u r e

Chemical Abstracts R e g i s t r y N o . (127-79-7)

M o l . w t 264.30.

2

C11H12N402S

1 . 2 . Appearance, Colour , Odour, Taste

White or f a i n t l y y e l l o w i s h w h i t e c r y s t - a l l i n e powder which i s o d o u r l e s s b u t has a s l i g h t l y b i t t e r taste. I t i s s t a b l e i n a i r b u t s lowly da rkens on exposure t o l i g h t .

2 . 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 spec t rum o f sulphamera- z i g e (Squibb sample P083425) w a s record- e d i n K B r and i s shown i n F i g u r e 1. Assignments f o r t h e more i m p o r t a n t absorp t ign5bands are l i s t e d i n Table 1. '

I I 1 , , I ,lj, I I ! - I ? I , , v -- 15' j - * I - e

2000 1800 1600 1400 1200 1000 800 600 400 200 4000 3500 3000 2500 FREQUENCY (CM')

Fig. 1 Infra spectrum of sulphamerazine (KBr pellet)


TABLE 1

I n f r a r e d ass ignments f o r Sulphamerazine

Frequency ( c m - l )

3490 3380 2960 2870 1 6 30 1600) 15 70) 1500) 1325

1160 109 2

890 8 35

A s s i gnmen t

N H asymmetric s t r e t c h i n g . N H symmetric s t r e t c h i n g . CH asymmetr ic s t r e t c h i n g . CH3 symmetr ic s t r e t c h i n g . N H 2 s c i s s o r i n g . C = C s t r e t c h i n g , s k e l e t a l v i b r a t i o n s o f aromatic r i n g . S O 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 - i n g v i b r a t i o n . S O symmetric s t r e t c h i n g . Ar8matic 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 deforma- t i o n .

3

2 . 2 . U l t r a v i o l e t Spectrum

The u l t r a v i o l e t spec t rum of sulphamera- z i n e i n 0 1 M h y d r o c h l o r i c a c i d s o l u t i o n 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 a t 307 nm (F igure 2 ) . I n 0 . 1 M sodium hydroxide s o l u t i o n su lphameraz ine 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 sulphonamide f r a c t i o n of the molecule . The u l t r a v i o l e t spec t rum o sulphamerazine has been r eco rded 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 v a l u e s e v a l u a t e d f o r t h e aforemention&8msystems a r e g iven i n Table 2 .

3

6

I.

522

SULPH AMERAZI N E 523

TABLE 2

va lues € o r sulphamerazine i n v a r i o u s s o l v e n t systems E l c m

1% R e f e ren ce El c m So lven t Band (nm)

0 . 1 M H C 1 aqueous 2 4 3 5 79 7 6 2 5 3

3 0 7 2 00 3 0.1M NaOH aqueous 2 4 3 896 3

2 5 7 8 8 3 3 Water 2 43 875 6

2 5 7 82 2 6 95% Ethanol 2 7 1 835 6

2 . 3 . Fluorescence and Phosphorescence

N ’- Subs t i t U t e d s u 1 phon a m i de s c o n t a i n i n g a n -e l ec t ron d e f i c i e n t h e t e r o c y c l i c 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 . Sulphamerazine i s such a sulphonamide and i t s l a c k of f l u o r e s c e n c e h s been demonst ra ted by G i f f o r d and co-workers . The p resence o f t h e 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 quenching of f l u o r e s c e n c e o v e r t h e pH range 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 he t e rocyc - l i c su lphan i l amides and it 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 absorbed 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 . phosphorescence spec t rum 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 l e v e l i n t e a romat i c nuc leus . G i f f o r d and co-workers’ produced t h e phosphorescence p d emiss ion spectrum of sulphamerazine a t 7 7 K u s ing a Qaird-Atomic SF 100-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 phosphoroscope a t t a c h m e n t , t h e e x c i t a t i o n spec t rum showing a maximum a t 310nm ( A , ) and t h e emission spec t rum a maximum a t 412nm(Ap). The de layed luminescence l i f e t i m e ( T ) was 0 .8 seconds .

8

Sulphamerazine has been shown t o e x h i b i t a


2 . 4 . Mass Spectrum

The mass spec t rum of su lphameraz ine shown i n F igure 4 was o b t a i n e d on an AEI-MS 902 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 - t i o n i n t o t h e sou rce a t 90°C . The fragmen- t a t i o n p a t t e r n s which can be a s s i g n e d t o s b ~ ~ , more i m p o r t a n t i o n s are shown i n scheme I 11

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 su lphapyr imid ines and showed t h a t p r e f e r e n t i a l f r agmen ta t ion occu- r r e d t o e l i m i n a t e SO . The f r agmen ta t ion p a t t e r n s w e r e a t t r i b 6 t e d t o l o c a l i z a t i o n o f t h e cha rges on t h e he t e roa toms . The w o r k e r s cons ide red t h e peaks a t m / e = 2 0 0 and m / e = 1 9 9 a s ex t remely impor t an t co r re spond ing t o t h e removal of S O owing i o n s :

and S02H t o g i v e t h e f o l l - 2

m / e = 2 0 0 m / e = 1 9 9

2 .5 . N.M.R.Spectrum

Puar and Funkel’ have r eco rded t h e 6 0 MHz N . M. R. spec t rum o f su lphameraz ine i n d ime thy l su lphoxide - 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 ) . p r e s e n t e d i n Table 3 .

The n a t u r a l abundance 1 3 C magnet ic resonance spec t rum o f su lphameraz ine h a s been com- pa red wi th a sefjes of o t h e r sulphonamides by Chang and F l o s s . The s p e c t r a w e r e d e t e r - 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 echn ique . 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 sonance and s e l e c t i v e p r o t o n decoyg l ing t e c h n i q u e s as w e l l as by long-range C p ro ton c o u p l i n g p a t t e r n s .

The s t r u c t u r a l d a t a i s


TABLE 3 1 0 , l l NMR S p e c t r a l Assignments o f Sulphamerazine

Pro ton A s s i gnmen t

2 H p - s u b s t i t u t e d 2 H f benzene r i n g

p r o t o n s 3H C H 3 2 H NH2 1 H NH

s = s i n g l e t ;

Chemical J ( H z ) s h i f t , 6 (ppm)

6.57d 7 . 7 0 d

6.86d 8.30d

2 . 2 9 s 5 .95b , s

11.12b,s

9 .o 9 . 0

5 .O 5 . 0

d = d o u b l e t ; b = broad .

2 . 6 . Mel t ing Range

The m e l t i n g range quo ted i n t h e U.S.P. A m e l t i n g p o i n t of 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 rade sample of su lphameraz ine 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 Redc ro f t Thermal Analy- ser Model 671 a t a h e a t i n g r a t e o f 2OoC min- l , it w a s found t h a t U.S.P. g rade su lpha - merazine gave a s h a r p m e l t i n g endotherm a t 234OC 3 ( F i g u r e 6 ) . T h i s w a s r a p i d l y fo l low- e d by decomposi t ion . ( A H f ) e v a l u a t e d by Yang and G u i l l o r y w a s 8 .68 k . ca l .mo l - l , a t a f u s i o n f smpera tu re of 236OC whereas Sunwoo ancllEisen va lue o f 7.54 &.ca l .mo l , a t a f u s i o n temp- e 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 quo ted an e n t r o p y of f u s i o n o f 1 7 . 1 e . u . f o r su lphameraz ine .

The h e a t o f f u f i o n

q u o t e a

2.8. Thermal Grav ime t r i c A n a l y s i s

Hine The thermogravimetry of sulphamera h a s been s t u d i e d by Cook and Hi ldebrand . Sulphamerazine e x h i b i t e d n o we igh t loss up t o a t empera tu re of 26OoC, b u t between 26OoC and 396OC a r a p i d we igh t loss o c c u r r e d fo l lowed


by a less 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 , exhib i ted 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 - i n g p e r i o d . 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 dioxide would p r o b a b l y s p l i t o u t f r o m t h e s u l p h a m e r a z i n e m o l e c u l e i n a s i m i l a r 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 c h l o r i d e s .

2 .9 . X-ray D i f f r a c t i o n Ochs17 h a s recorded t h e X-ray powder

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 and 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 reported X-ray powder d i f f r a c t i o n da ta f o r s u l p h a m e r a z i n e .

TABLE 4 X-Ray Powder D i f f r a c t i o n Data of S u l p h a -

In te rp lanar 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 m e r a z i n e ( P 0 8 3 4 2 5 )

,. Q* 10.72

7.65 7 . 0 3 6 . 76 6 . 35 6.02 5 .46 5.14 4.72 4 .37 4 . 1 1 3.95 3.89 3.30 3.74 3.67 3 . 5 3 3.27 3.22 3.05 2.94 2 . 9 0 2 .76 2 , 3 8 * I n t e r p l a n a r d i s t a n c e d

1/10 0 .117 0 . 1 3 0 0 .949 0 . 3 1 5 0 . 1 9 9 0 . 8 8 5 0 . 6 3 6 0. 760 0 . 2 0 7 0 . 9 7 1 0 . 5 4 5 0 .432 0 . 3 2 2 0 . 2 5 7 0 . 3 0 7 1.000 0 . 3 2 4 0 . 2 0 7 0 . 2 8 6 0 .142 0 . 1 3 3 0 . 3 9 7 0 . 4 5 6 0 . 2 7 8

= n X - 2 s i n 0

u 8

8 8

a 8

8 6 ?


2 . 1 0 . Polymorphism

During e x t e n s i v e s t u d i e s on polymorphism i n sulphonamides 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 and G u i l l o r y l 4 found t h a t su lphameraz ine w a s among t h o s e sulphonamides i n which polymorphism cou ld n o t be d e t e c t e d .

2 . 1 1 . S o l u b i l i t y

2 . 1 1 . 1 . I n Aqueous B u f f e r s and Urine

The s o l u b i 1 i t y of sulphame ra z i n e i n aqueous 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 mainly been de termined i n aqueous b u f f - ers and u r i n e i n t h e approximate pH range 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 Table 5 a long w i t h t h o s e of t h e N'-acetyl d e r i v a t i v e .

TABLE 5

M e d i um

The S l u b i l i t y o f Sulphamerazine and -I-acetyl d e r i v a t i v e i n aqueous phosphate b u f f e r and u r i n e a t 3 7 O F

S o l u b i 1 i t y mg./ml.,

R e f e r en ce

M/30 Phosphate bu f fe r ,pH 6 . 1 Urine,pH 5.9 Urine,pH 6 . 9 Urine,pH 7.9

Sulpha- g - A c e t y l - m e ra z i n e s u l p hame r a -

z i n e 4 0 5 3 1 9

37 76 1 9 , 2 0 66 175 19 , 2 0

3 10 6 50 1 9 , 2 0

2 . 1 1 . 2 . I n S o l v e n t s

The approximate s o l u b i l i t i e s o f su lphameraz ine i n some s o l v e n t s are given i n Table 6 .

SU LPH AME 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

So lven t - S o l u b i 1 i t y Reference mg. / m l .

wa te r , 2 0 : ~ 1 6 Water,37 g 30 Water,100 C 3 30 1 . 5 N Aqgeous 2 9 0 NaOH,22 C Ethanol , 2 2 O C 3 30 I sopropanol , 2 2 C 1 7 4

6 6 6

2 1

6 2 2

2 .12 .Di s soc ia t ion Cons tan t

The d i s s o c i a t i o n of t h e pr imary a romat i c amine f u n c t i o n o f some sulphonamides has2kjeen s t u d i e d by Sa lvesen and Schroder-Nielson u s i n g spe c t r opho tome t r i c and p o t e n t iome t r i c methods. In 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 va lue r e p r e s e n t i n g t h e pr imary amine d i s s o c i a t i o n of sulphameraz ine w iven a s 2 . 2 9 . Ko izumi and co- workers" Zuoted a pKal va lue of 2 . 2 6 .

of a number of sulphonamides f r o m s o l u b i l i t y d a t a u s i n g t h e f o l l o w i n g r e l a t i o n s h i p .

Krebs and SpeakmanZ5 de termined t h e pK

s = s 0 (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 un ion i sed compound. e d a pK va lue of 6 . 9 5 ( S = 4 1 mg./lOOml.) f o r t h e a& s s o c i a t i o n of t h e s ulphonamide group of sulphamerazine i n a s o l u t i o n o f ion- i 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 jog ren and Or tenb lad o b t a i n e d a pKa v a l u e of 7 . 0 5 . Both t h e s e reports ass- ume8 t h a t t h e sulphonamides behaved as mono- b 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 value26has been confirmed by W i l l i and Meier o b t a i n e d a va lue 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 of 0.1.

ghese workers o b t a i n -

, who u s i n g a p o t e n t i o m g t r i c method,


2.13 P a r t i t i o n C o e f f i c i e n t s

During t h e i r s t u d i e s on some pharmacokine- t i c a s p e c t s of e r t a i n sulphonamides Koizumi and co-workers2' g e n e r a t e d p a r t i t i o n coef f i - c i e n t d a t a a t 37OC between an aqueous phase c o n t a i n i n g un ion i sed d rug and t h e s o l v e n t s carbon t e t r a c h l o r i d e I benzene , ch lo ro fo rm and isoamyl acetate. Suzuki and c o - ~ o r k e r s ~ ~ also g e n e r a t e d s i m i l a r d a t a u s i n g isoamyl a l c o h o l as $he o r g a n i c phase . The r e s u l t s f o r su lphameraz ine are g i v e n i n Table 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 su lphameraz ine

o 2 4 , 2 7 a t 37 C Organic Phase P a r t i t i o n C o e f f i c i e n t

CC14 0 . 0 2 2 0 . 2 0 2 2 . 4

'gH6 C H C l 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 S y n t h e s i s

Two pr imary s y n t h e t i c r o u t e s have been used t o p r e p a r e su lphameraz ine , t h e s e b e i n g v i a t h e r e a c t i o n between 2-amino-4-methyl- pyr imidine w i t h c e r t a i n d e r i v a t i v e s o f benz- enesu lphony l c h l o r i d e and a l s o by a condens- a t i o n p r o c e s s between su lphaguan id ine and c e r t a i n r i n g forming compounds.

e d su lphameraz ine by t h e a c t i o n of p-ace ta- midobenzene-sulphonyl c h l o r i d e on 2-amino- 4-methylpyrimidine i n a weakly b a s i sol- v e n t such 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 of su lphameraz ine . H y d r o l y t i c de- a 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 ach iev - 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 t h e v a r i o u s s teps i n v o l v e d i n t h e s y n t h e s i s a r e shown i n Scheme 2. The p - n i t r o d e r i v a - t i v e of benzenesulphonyl c h l o r i d e c o u l d a l s o

Roblin and co-workers28 f i r s t s y n t h e s i z -

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536

SULPHAMERAZIN E 537

be used 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 re- q u i r i n g a c a t a l y t i c r e d u c t i o n of t h e n i t r o group t o g ive t h e f i n a l p roduc t .

r i n g forming compounds were condensed w i t h su lphaguanid ine t o produce su lphameraz ine . T y p i c a l l y su lphaguan id ine has been condensed wi th ch orovinylmethyl ke tone 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 condensa t ion mechanism invo lved t h e removal of a molecule of w a t e r and a molecule 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 produc t . Other r i n g forming compounds which have been used i n c l u d e ace toace ta ldehyde ace- t a l s 3 0 , ace t a ldehyde m t h y l ace ta l s3 l1 and

In t h e second major method a number of

d ia lkylaminobutenynes 35 . 3.2. P u r i f i c a t i o n

Crude sulphamerazine i s u s u a l l y u r i f ied In one method31; t h e pH v i a i t s sodium s a l t .

of t h e medium w a s a d j u s t e d t o 10.5 by t h e ad- d i t i o n of calcium hydroxide . 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 added. D e - c o l o r i z a t i o n was then achieved 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 tempera tu re 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 ace t ic a c i d and t h e p r e c i p i t a t e d su lphameraz ine i s o l a t e d by f i l t r a t i o n . I f r e q u i r e d t h i s p roduc t could be r e c r y s t a l l i s e d from aqueous a l c o h o l o r benzene. A number of 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

Barry and P ~ e t z e r ~ ~ p repa red t h e c e t y l - m e t h y l ammonium s ulphamerazine d i h y d r a t e sa 1 t which w a s found to39ave a m e l t i n g p o i n t of 126OC. Schonhafer p repa red t h e d i e thy lami - noe thano l s a l t of sulphamerazine 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 repa red aqueous s o l u t i o n s of s t rep tomycin s u l p h a t e and bar ium o r ca lc ium s a l t s of c e r t a i n sulphonamides i n va ry ing 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 ep tomyc in c o n t a i n i n g 1 , 2 or 3 moles of sulphonamide.


The s t r ep tomyc in d i su lphameraz ine s a l t was found t o have a water s o l u b i l i t y of abou t 10%.

4 . 2 . Metal Complex S a l t s

Var ious complex salts o f sulphonamides w i t h m u l t i v a l e n t metals have been p r e p a r e d . Complex s a l t s of c o b a l t , su lphameraz in3gand e thylenediamine were prepatjed by Erdos t empera tu re o f less t h a n 5 C f o r 2 4 hour 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 p r e p a r e d t h e c o b a l t , n i c k e l , coppe r and z i n c complexes o f su lphameraz ine and found them t o be i n s o l u b l e i n water , a l c o h o l , e t h e r , ch loroform, ace tone and benzene.These complexes w e r e found t o be 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 hydroxide or ammonia. The molar r a t i o of su lphameraz ine t o metal w a s 2:l. L e e h a s s t u d i e d i n dep th t h e fo rma t ion o f copper com- p l e x e s of tile sulphonamides, 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 copper grjetate , t h e i r s e n s i t i v i t y t o micro-organisms , t h e 4 g e t e r - mina t ion of t h e i r s t a b i l i t y 4 $ o n s t a n t s , and t h e i r s t r u c t u r e ass ignments . The copper complex o f su lphameraz ine 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 sulphonamide wi th an aqueous s o l u t i o n o f c u p r i c ace- t a t e a t pH 7-9. The complex was i s o l a t e d as grey n e e d l e s , w a s less s e n s i t i v e t o microorganisms t h a n su lphameraz ine and had a s t a - b i l i t y c o n s t a n t of 9 .68 a t 2 5 C. t u r e o f t h e qomplex w a s de te rmined by i n f r a r e d spec t roscopy 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 from 7 . 6 2 ~ f o r su lphameraz ine t o 7 . 7 9 ~ i n t h e copper com- p lex . From t h e i n f r a r e d d a t a i t w a s deduced t h a t t h e copper c h e l a t e d between t h e S-0 group of t h e sulphonamides and a h e t e r o c y c l i c n i t r o g e n atom as f o l l o w s

a t a

6 TQe s t r u c -

SULPHAMERAZINE 539

5. Chemical S t a b i l i t y 5 .1 .Hydrolys is

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 hydro- l y s i s of some s u l p h a n i amidopyrimidines h a s been s t u d i e d by Zajac . The h y d r o l y s i s ra te was found t o fo l low f i r s t o r d e r k i n e t i c s i n each c a s e , t h e rate be ing dependent 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 tudy 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 methyl or methoxy groups w i t h i n t h e p y r i - midine nuc leus 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 of ghe su lphameraz ine hy- d r o l y t i c p r o c e s s a t 6 0 C (333OK) w a s found t o be 6 7 . 9 hours compared t o 9 4 . 7 hour s f o r s u l - phad iaz ine t h e p a r e n t su lphani lamidopyr imi- d i n e .

48

Auterhof f and Schmidt46 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 su lphani lamidopyr imi- d i n e s . 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 i n v e s t i g a t i o n s i d e n t i f i e d s u l p h a n i l i c a c i d , su lphan i l amide , 2-amino-4-methylpyrimidine and 2-hydroxy-4-methylpyrimidine as t h e decomposi t ion p roduc t s of su lphameraz ine .

5 . 2 . P y r o l y s i s

The pyro ly t i c de composi t ion of s u lphani - lamidopyrimidines w a s a l so s t u d i e d by Auter- ho f f and Schmidt46. 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. Yel lowish w h i t e sub- l imates appeared i n t h e upper p a r t o f t h e t e s t t u b e s which were subsequen t ly examined by TLC on Merck K i e s e l g e l F254 u s i n g n-butanol , acet ic a c i d , w a t e r (80 ,20 ,20) as s o l v e n t sys- t e m . Sulphamerazine ( R f 0 .59 ) w a s found to decompose t o 2-amino-4-methylpyrimidine ( R f 0 .48) i n 92% y i e l d .

5.3. P h o t o l y s i s

Nai to and M i ~ o g u c h i ~ ~ s t u d i e d t h e photo- l y t i c decomposi t ion of c e r t a i n s u l p h a d rugs and t h e i r benzoyl d e r i v a t i v e s i n aqueous 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 spec t ropho tomet r i c a s s a y method


showed t h a t about 50% of su lphameraz ine w a s decomposed ove r a p e r i o d of 8 hour s whereas t h e benzoyl d e r i v a t i v e w a s comple t e ly s t a b l e . The same samples s t o r e d i n t h e dark e x h i b i t e d no decomposi t ion .

6 . Methods of Ana lys i s 6 . 1 . I d e n t i f i c a t i o n

Two i d e n t i t y tests are g iven i n t h e U.S.P.XlX, one b e i n g an i n f r a r e d absorp- t i o n tes t and t h e o t h e r a microchemical t e s t . I n t h e l a t t e r method a sample of sulphamerazine i s suspended i n w a t e r and t h e suspens ion made a l k a l i n e w i t h sodium hydroxide . 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 reen p r e c i p i t a t e i s formed which t u r n s da rk g rey on s t a n d - i n g . Th i s t e s t has been s u c c e s s f u l l y used48t49to d e t e c t su lphameraz ine i n t h e p re sence of t h e r sulphonamides. Turczan and Medwickl’ have i n c l u d e d su lphameraz ine 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 of sulphonamides by N.M.R.spectroscopy .

6 . 2 . E l e m e n t a l Ana lys i s

The e l e m e n t a l composi t ion o f su lpha- merazine (Squibb b a t c h PO 83425) w a s ob- t a i n e d by Young50 w i t h t h e f o l l o w i n g re- s u l t s : -

Element % Theory % Found Carbon 49.98 50.08 Hydrogen 4.58 4.55 Ni t rogen 2 1 . 2 0 21.31 Sulphur oxygen

12.13 1 2 . 1 2 1 2 . 1 1 -

6.3. T i t r i m e t r i c Assay P rocedures

6 .3 .1 .Diazometr ic T i t r i m e t r y

Sulphamerazine may be t i t r a t e d i n s t r o n g l y a c i d s o l u t i o n w i t h a s t a n d a r d s o l u t i o n of sodium n i t r i t e , t h e end-po in t be ing d e t e c t e d w i t h an e x t e r n a l or i n t e r - n a l i n d i c a t o r , o r by an electrometric 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 LPHAM ERA2 IN E 541

t h e o f f i c i a l method of t h e U.S.P. X1X f o r sulphamerazine .

El-Sebai and c o - ~ o r k e r s ~ ~ have e v a l u a t - e d t h e sodium s a l t of 4- (benzy1amino)azo- benzene-4 ' - su lphonate 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 and c l a im t h a t i t p rov ides 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 ab le f o r 30 minutes . More a c c u r a t e r e s u l t s w e r e ob- t a i n e d than wi th an e x t e r n a l i n d i c a t o r such as s t a r c h i o d i d e paper . Other 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 used are cyanobis (1 , 10-phenan th ro l ine ) i r o n (II)f3 and t r o p a e o l i n 00 w i t h methylene 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 has been used f o r t h e de t e rmina t ion of su lphameraz ine i n t a b - l e t dosage forms wi thou t i n t e r f e r e n c e from e x c i p i e n t s such a s s t a r c h , lactose, calcium c a r b o n a t e , sodium b i c a r b o n a t e , magnesium s tearate s tear ic 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 powerful e l e c t r o n withdrawing s u l - phony1 group i n sulphonamides r e n d e r s t h e amide hydrogen atom a c i d i c so t h a t t h e s e drugs can be conven ien t ly t i t r a t e d w i t h a s u i t a g h e base 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 us ing sodium methoxide d i s s o l v e d i n a mixture o f benzene and methanol (3: 1) as titrant and thymol b l u e i n methanol 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 has a l s o been determined56 i n t e t r ame thy- 1 u re a w i t h t e t r a b u t y 1 ammon i um hydro x i de ( 0 . 1 M ) as titrant, t h e end-poin t b e i n g de- te rmined u s i n g e i t h e r po ten t iome t ry o r a thymol b lue i n d i c a t o r .

5 7 More r e c e n t l y Davis and co-workers e v a l u a t e d 3-methyl-2-oxazolidone as a s u i t - 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 sulphonamides 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 range c o n t r i b u t e d t o i t s o u t s t a n d i n g s o l v e n t

RICHARD D. G. WOOLFENDEN 542

6 .3 .3

p r o p e r t i e s . Te trabutylammonium hydroxide was used as t i t r a n t and p o t e n t i o m e t r y as end- poin t de t e c t i o n . B r o m o m e t r i c T i t r i m e t r y

The bromometric methods o f Wojahn and Conway59 are w e l l e s t a b l i s h e d and have been appl&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 a s s a y o f su lphameraz ine i n mix tu res w i t h o t h e r sulphonamides.

58

Recen t ly , 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 de t ec - t i o n of end-po in t i n he bromometric 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 su lphameraz ine , by a cou lomet r i c method i n v o l v i n g bromina- t i o n wi th 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 i n an aqueous 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 potass ium bromide. The end-po in 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 coulometric met&d w a s a l so adopted by Ebe 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-po in t .

A s p e c t r o p h o t o m e t r i c t i t r a t i o n w i t h bromide-bromate s o l u t i o n h a s been develop- ed63, t h e drug b e i n g d i s s o l v e d i n a m i x t u r e o f c o n c e n t r a t e d h y d r o c h l o r i c a c i d - 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 su lphameraz ine were r e p o r t e d as 98.43 - 0.58% w i t h brominat ion t i m e of 5 minutes .

6.3.4. Argentometr ic T i t r i m e t r y

The p r i n c i p l e of t h e a r g e n t o m e t r i c method i s t h a t some sulphonamides form in - soluble s i l v e r s a l t s . The sulphonamides are 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 s t a n d a r d s i l v e r n i t r a t e s o l u t i o n , t h e pre- c i p i t a t e removed by f i l t r a t i o n , and t h e e x c e s s s i l v e r n i t r a t e t i t r a t e d w i t h s t a n d - a 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 Reeder6I 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 de te rmina- t i o n of a mix tu re o f su lphameraz ine , su lpha - d i a z i n e and su lphamethaz ine .

SULPHAME RAZlNE 543

6.3.5.Complexometric T i t r i m e t r y

Abdine and S a ~ e d ~ ~ developed a complex- ome t r i c a s say f o r su lphameraz ine . 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 wi th e x c e s s copper s u l - pha te s o l u t i o n i n pH 6 b o r a t e b u f f e r . The excess copper w a s then de termined by t i t r a - t i o n w i t h t h e disodium s a l t of E.D.T.A.using 1- (2-pyr idylazo) -2-naphthol as i n d i c a t o r .

ed66 i n combined su lpha d rugs by p r e c i p i t a - t i o n wi th e x c e s s copper a c e t a t e fo l lowed by t h e d e t e r m i n a t i o n of t h e r e s i d u a l copper by complexing wi th E . D . T . A . The s e l e c t i v e p r e c i p i t a t i o n and complexometric a s say of mix tu res of su lphameraz ine , 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 .

Sulphamerazine has a l s o been e s t i m a t -

6.3.6.Thermometric T i t r i m e t r v

Bark and G r i m e 6 7 developed a thermometric assay f o r s e v e r a l sulphonamides i n - c l u d i n g sulphamerazine . The sulphamerazine was d i s s o l v e d i n the minimum volume of 0.1M aqueous sodium hydroxide 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 wi th s t a n d a r d s i l v e r n i t r a t e sol- u 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 re- s u l t i n g enthalpogram. E x c i p i e n t s such 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 of t h e a p p a r a t u s re- q 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 Bate68 ,6J .

Sulphonamides have a 1 so been de te rmin- e d by a c a t a l y t i c thermometr ic t i t r a t i o n t echn ique . The p r i n c i p l e of 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 wi th a base i n non-aqueous media us ing a c r y l o n i t r i l e as a thermometr ic i n d i c a t o r . Thus , a t t h e end- p 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 c a t a l y s e d a n i o n i c po lymer i za t ion w i t h a cor responding e v o l u t i o n of h e a t which i s measured. e d sulphamerazine by t h i s t echn ique u s i n g d i m e t hy 1 f o r m a m i de a s t h e n on - aq ue o us s o 1 ve n t and 0.1M o r 0.01M tetra-n-butylammonium

Greenhow and Spencer70 de termin-


hydroxide i n methanol - to luene or i sopropan- 01- to luene as titrant. The lower pract 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 be 0.0001 m.equiv. of drug . 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 re sence o f a c i d i c ex- c i p i e n t s .

6 .4 . Spec t ropho tomet r i c Assay P rocedures

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 been of 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 pub- l i c a t i o n s i n t h i s f i e l d . However, Dol insky7l de te rmined su lphameraz ine and s u l p h a d i a z i n e i n mix tu re by t h i s techn- i q u e u s i n g ca rbon 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 so de termined s u l - phamerazine i n mix tu re w i t h s u l p h a t h i a - z o l e u s i n g dimethylformamide as s o l v e n t .

6 . 4 . 2 . U l t r a v i o l e t 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 spec t ropho tomet ry h a s found some use i n t h e d e t e r m i n a t i o n of su lphameraz ine . S ince t h i s drug i s normal ly i n c o r p o r a t e d i n t o a double or t r i p l e sulphonamide 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 d e t e r m i n a t i o n i n t h e p re sence o e o r t w o o t h e r sulphonamides. 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 su lpha- merazine i n t h e p re sence 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 sepa ra - t i o n . Fol lowing t h e d e t e r m i n a t i o 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 colorimetric method d i r e c t u l t r a - v i o l e t spec t ropho tomet ry w a s used t o measure t h e q u a n t i t i e s of su lphameraz ine and 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 imul t aneous 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 Rapaport and Shakh d e t e r - mined su lphameraz ine i n f o r m u l a t i o n s w i t h o t h e r sulphonamides. The use of a com- p u t e r programming t echn ique 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 sulphamera-

SULPHAMERAZINE 545

z i n e has been d e s c r i b e d by Madsen and R ~ b e r t s o n ~ ~ .

6 .4 .3 . C o l o r i m e t r i c Methods

A number of colorimetric 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 sulphonamides which are a p p l i c a b l e t o su lphameraz ine . Probably t h e most w e l l known i s t h e B r a t t o n and Marsha l l meth- od77 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 pr imary 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 th sulphamic a c i d fo l lowed by coup l ing t h e d i a z o compound w i t h N- (1- naphthy1)-ethylenediamine. I n g e n e r a l 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 a s say of s m a l l a m

~ & , i P , i S , Q S chromatographic pro- s ulphon a m i t h i n l a y e r cedure .

1 w g a paper 78ybso;f

Other c o l o r i m e t r i c methods have been developed, b u t have n o t been as wide ly used a s t h e B r a t t o n ang4Marshal l proce- du re . Tulus and Guran developed a method f o r su lphameraz ine and o t h e r s u l - phonamides u s i n g t h e potass ium s a l t of 1,2-naphthoquinone -4-sulphonic a c i d as t h e coup l ing agen t . The use of d imethyl - aminobenzaldehyde f o r t h e q u a n t i t a t i v e a s say of sulphamerazine f o l l o w i n g pape r chromatographic s e p a r a t i o n has been s tud - i e d by L ~ i s e * ~ . t i o n f o r su lphameraz ine i n a t a b l e t dosage form u s i n g 9 -ch lo ro -ac r id ine h a s been developed by S tewar t and co-workers86 who found t h a t t h e r e s u l t s compared exce l l en 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 Marsha l l method.

A c o l o r i m e t r i c de te rmina-

6 . 5 . Chromatographic Procedures

6.5.1.High Performance L iqu id Chromatography

K r a m 8 7 q u a l i t a t i v e l y s t u d i e d t h e behaviour of some 2 1 sulphonamides by H.P.L.C. Using a s t a i n l e s s s teel column packed wi th s p h e r i c a l s i l i ceous p a r t i c l e s c o a t e d w i t h a s t r o n g an ion exchanger t h e


r e t e n t i o n t i m e s of t h e d rugs were estab- l i s h e d u s i n g a mobi le 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 levels 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 su lphameraz ine , s u l p h a d i a z i n e and su lphamethaz ine , t h e o f f - i c i a l t r i s u l p h a p y r i m i d i n e s .

A q u a n t i t a t i v e H.P .L .C . a s say 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 been r e p o r t e d by P o e t and u s i n g a "Zipax" SCX(DuPont) c a t i o n exchange column w i t h 0 .2M d isodium phosphate 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 chos- en as t h e i n t e r n a l s t a n d a r d . The recommended p r e s s u r e of 1000 p s i g produced a s c l v e n t f low r a t e of 0.7-0.8 ml./min. re- s u l t i n g i n a 15-20 minu te 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 re- p r e s e n t a t i v e l o t s o f t a b l e t f o r m u l a t i o n s and t w o suspens ion f o r m u l a t i o n s , t h e cal- 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 re- p l i c a t e i n j ec t ions r a n g i n g from 0.9 t o 4 . 0 % .

Westlie and c o - w ~ r k e r s ~ ~ have develop- e d a l i q u i d - s o l i d chromatographic a s s a y procedure which is a p p l i c a b l e t o t h e t r i - su lphapyr imid ines . A MicroPak Si -10 column w a s used i n c o n j u n c t i o n w i t h a mobile phase c o n s i s t i n g o f ch loroform, methanol , ammonia 25% (365, 75 , 10) f lowing a t a ra te of 0.73ml. /min. S u l p h a t h i a z o l e w a s i n c l u d e d as an i n t e r n a l s t a n d a r d .

A H.P.L.C. procedure f o r t h e s e p a r a - t i o n of a range of sulphonamides u t i l i s i n g s i l i c a ge l a s t h e column p i n g h a s been

t i o n was achieved on a 25cm. s t a i n l e s s s tee l column o f i n t e r n a l d i a m e t e r 4 m.m. packed 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 mobile phase c o n s i s t e d of a mix tu re o f cyc lohexane, 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 moni tored a t 260nm u s i n g a

d e s c r i b e d by Cobb and H i l l 8% . The s e p a r a -

SULPH AMERAZ INE 547

C e c i l CE 2 1 2 v a r i a b l e wavelength d e t e c t o r . I n i t i a l s e p a r a t i o n s w e r e o b t a i n e d u s i n g cyc 1 ohe xane -e thano 1 mix tu res of v a r i a b l e composi t ion and i t w a s found t h a t i n c r e a - s i n g the e t h a n o l c o n t e n t dec reased t h e observed r e t e n t i o n times. The a d d i t i o n 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 wi th- o u t a l t e r i n g r e s o l u t i o n . A t a f low r a t e of 2 m l . /min. t h e d e s c r i b e d mobile phase 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 use of h igh performance i o n p a i r p a r t i t i o n chromatography f o r t h e sepa ra - t i o n of sulphonamides has beenganves t i - g a t e d by Karger and co-workers . T h e i r e f f o r t s r e p r e s e n t e d a f e a s i b i l i t y s t u d y on t h e s e p a r a t i o n of 1 2 su lpha d rugs us- i n g a s i l i c a gel/CT (Reeve Angel) 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 coun te r ion ( 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 mobile phase of n-butanol , hexane (25,75 ) . Under t h e s e c o n d i t i o n s sulphamerazine w a s shown t o have a r e t e n t i o n t i m e of 13-14 minutes .

6 .5 .2 . Gas Chromatography

The main gas chromatographic method r e p o r t e d i n t h e l i t e r a t u r e f o r t h e d e t e r - mina t ion of su lphapyr imid ines invo lved an i n i t i a l h y d r o l y t i c s t e p , t h e r e s u l t i n g v o l a t i l e 2-aminopyrimidines b e i n g measur- e d . Turczangl developed such a method f o r q u a n t i t a t i v e l y a s s a y i n g t h e i n d i v i - d u a l sulphonamides, i n c l u d i n g sulphamer- a z i n e , i n t h e o f f i c i a l t r i s u l p h a p y r i m i - d i n e s . Concent ra ted 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 mixture h e a t - ed i n an oven a t 130 C f o r 1 hour . 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-dimethylpyr id ine 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 sepa ra - t e d a t 1 5 0 C on a column packed 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 achieved f o r t h e t r i- su lphapyr imid ines i n both s y n t h e t i c mix- t u r e s and s e v e r a l commercial t a b l e t p re- p a r a t i o n s .


Daung2 found t h a t t h e above method was un- s 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 su lpha - merazine i n p o u l t r y f e e d s a t l e v e l s rang- i n g between 0.002 and 0 .05%. The method ad- o p t e d r e q u i r e d t h e p r e p a r a t i o n o f a re la t - i 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 fo l lowed by an e x t r a c t i o n s t e p us ing e t h y l a c e t a t e . T h e r e s i d u e remaining 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 me thy la t ed w i t h d i azo - methane and then a c y l a t e d w i t h h e p t a f l uo ro - b u t y r i c anhydr ide . The a c y l d e r i v a t i v e s w e r e found t o be e a s & l y s e p a r a t e d on a 10% DC-200 column a t 230 C. De tec t ion w a s ach- i 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 used , 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 and sample peak h e i g h t s .

Roeder and S t u t h e g 3 developed a gas chromatographic method f o r t h e sulphonamides and t h e i r N4-ace ty l m e t a b o l i t e s i n b lood and u r i n e . The method w a s a p p l i c a b l e t o su lphameraz ine . The sulphonamides were e x t r a c t e d from t h e b lood and u r i n e samples and then me thy la t ed w i t h diazomethane. The methyl d e r i v a t i v e s were de termined 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 sulphonamides and 7% f o r t h e a c e t y l c o n j u g a t e s .

The s imul t aneous q u a l i t a t i v e a n a l y s i s of 1 4 s u l p h a drugs 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 gas l i q u i d chromatogra hy w e r e performed by Nose and co-workers 92A on s o l u t i o n s o f d ime thy l fo 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 ace tone . The d e r i v a t i v e s cou ld 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 w i t h a h i g h l y s e n s i t i v e r e sponse f o l l o w i n g 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 mesh) , 5% XE-60 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 and 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 su lphameraz ine v a r i e d between abou t 4 0 t o 80 minutes .

SU LPH AME RAZ I NE 549

6.5.3. Thin Layer Chromatography

A number of t h i n l a y e r chromatographic methods have been developed 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 of s u l - phamerazine and r e l a t e d s u l p h a d rugs . C e r - t a i n d e t a i l s of t h e s e methods are summaris- e d i n Table 9 and some spot l o c a t i n g reag- e n t s a r e g iven i n Table 11.

Bican-F i s t e r and Kajganovic8O reCOgniS- e 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 echn ique than pape 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 su lpha 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 suspen- s i o n s . Using a K i e s e l g e l G l a y e r combined wi th t h e s o l v e n t sys tem chloroform, meth- a 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 sulphamerazine , s u l p h a t h i a z o l e and su lpha- d i a z i n e was ach ieved . For t h e s e p a r a t i o n of c e r t a i n mixtures of su lphameraz ine , sulphacetamide , sulphamethazine and su lpha- d i a z i n e t h e s o l v e n t sys tem chloroform, meth- a 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 . Fol lowing e l u t i o n from t h e adso rben t t h e s e p a r a t e d sulphonamides were a t f i r s t a s sayed by a U.V. method b u t B ican -F i s t e r and Kajganovic found t h a t t h e K i e s e l g e l G gave a h igh c o n t r i b u t i o n t o t h e blank absorbance. They , t h e r e f o r e , a p p l i e d t h e Bra t ton and Marsha l l color imetr ic method, e x c e l l e n t r e c o v e r i e s be ing 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 error foq sulphamerazine ranged between - 3.2% t o - 4 . 1 % i n s y n t h e t i c mix- t u r e s wi th t h e o t h e r sulphonamides.

Brunner81 developed 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 p r e p a r a t i o n s c o n t a i n i n g su lphameraz ine , su l - phad iaz ine and sulphamethazine u s i n g s i l i c a g e l GF p l a t e s and a s o l v e n t system compris- i n g ch loroform, methanol , ammonia(30,12,1). Again t h e B r a t t o n and Marsha l l colorimetric method was used 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 tudy82 on t h e use 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 ranged from 0.76 t o 1 . 6 6 . The tr i-

TABLE 9

Adsorbent K i e s e l g e l G.

S i l i c a g e l G: impregnated w i t h f l u o r e s c e i n . Polyamide CM1011.

ln 8

P l a s t e r of P a r i s imp re gn a te d w i t h z,inc f e r r o c y a - n i d e .

S i l i c a g e l G i mpregn a te d w i t h sodium hydroxide.

Thin l a y e r chromatography o f su lphameraz ine

S o l v e n t System a)Chloroform,methanol

(90,lO). b ) Chlorof orm,methanol,

25 % ammonia ( 90,15 ,2 . 5 ) Chlo ro fo rm,e thano l , hep tane (1,1,1) c o n t a i n - i n g 1 . 2 % water.

a ) Chloroform, 95% e t h a n o l (90, lO).

b ) E t h y l a c e t a t e , 9 5 % e t h a - n o l ( 8 0 , 2 0 ) .

c) Water, 95% e t h a n o l (60 ,40) .

a ) 0.03M aqueous ace t ic a c i d .

b ) 1.74M aqueous acetic a c i d .

c ) 3.33M aqueous ace t ic a c i d .

a )Chloroform,methanol

b ) A c e t o n e , methanol ( 4 , 1 ) .

( 4 1).

U s e

- Q u a n t i t a t i v e a s s a y -Rf -

f o r t r i s u l p h a p y r i m i d i n e p r e p a r a t i o n s .

0 .57 I d e n t i t y tes t .

0.79 I d e n t i t y tes t .

0 .83 I t 11

0.59 11 II


0 .17 11 11

0 .38 11 II


0 . 6 1 II 11

- R e f .

80

9 4

95

9 6

9 7

Adsorbent

S i l i c a g e l G impregnated w i t h potass ium hydrogen s u l p h a t e . S i l i c a g e l G.

S i l i c a g e l G.

m ?

S i l i c a ge l .

S i l i c a g e l GF.

S i l i c a g e l GF.

TABLE 9 ( c o n t ' d ) Thin l a y e r chromatography of su lphameraz ine

S o l v e n t System

Chloroform,carbon te t - 0.34 r a c h l o r i d e , m e t h a n o l ( 7 , 2 I 1).

E t h y l acetate ,methanol 0 . 5 9 ( 9 8 1 ) .

a ) E t h y l a c e t a t e , m e t h a n o l , 0 . 4 7 25% ammonia(17,6,5).

b ) Pe t ro l eum e t h e r I ch lo ro - 0 . 6 7 form n - b u t a n o l ( l , l , l ) . Chloroform I methanol 0 . 2 9

E t h y l acetate ,methanol 0 . 6 3 ( 9 I l l . Chlorof o m I m e t h a n o l ammonia (30 ,12 ,l).

(95 1 5 ) -

-

Chloroform, methanol - ( 9 I 1 ) .

S i l i c a ge l H

sodium hydroxide. S i l i c a g e l G Acetone,n-heptane,meth- 0 .31 p r e c o a t e d p l a t e s ano1,28-30% ammonia,n- (Anal t ech ) . b u t a n o l ( 7 2 , 2 1 , 9 , 1 0 , 1 0 ) .

imp re gn a t e d w i t h

U s e -

Iden ti t y tes t .

I d e n t i t y tes t .

I d e n t i t y tes t . II II

Iden ti t y tes t .

I d e n t i t y t e s t . Q u a n t i t a t i v e a s s a y f o r t r i s u l p h a p y r i m i d i n e p r e p a r a t i o n s . Q u a n t i t a t i v e a s s a y 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 a s s a y i n an ima l t i s s u e s .

Ref.

97

-

97

98

98

99

81

100

a 3

C

d

TABLE 9 (con t ' d) Thin l a y e r chromatography of sulphamerazine

Adsorbent Solvent System

S i l i c a g e l G. pH 7 . 4 aqueous ve rona l a c e t a t e .

Polyamide 11. a)pH 7 . 4 aqueous ve rona l ace t a t e .

b ) pH 7 . 4 aqueous veronal a c e t a t e con ta in ing 10% acetone.

ammonium hydroxide (30,12,1).

S i l i c a g e l 6 0 a ) Chlorof o m , e t h a n o l (Merck precoated). ( 9 , l ) .

S i l i c a gel. Chloroform,methanol,

b)Chloroform,ethanol , ammonium hydroxide, ( 8 , 2 , 0 . 1 ) . Chloroform, e t h a n o l , dioxane, a c e t i c acid,

E thyl a c e t a t e ,dioxane, a c e t i c a c i d (8 ,2 ,0 .1 ) . ( 8 , l r 1 , 0 - 1 ) .

Ef -

-

-

-

0 . 3 3

0 . 2 0

0.49

0 . 4 6

Use - RM-structure a c t i v i t y c o r r e l a t i o n . R - s t r u c t u r e a c t i v i t y c o r r e l a t i o n . M

II

Ref.

10 1

101

Q u a n t i t a t i v e assay for 1 0 2 t r i s u l phapyrimi dine t a b l e t s and o r a l suspens ions Q u a n t i t a t i v e assay i n 1 2 8 human ur ine.

SU LPHAME RAZINE 553

su lphapyr imid ines have a l s o been assayed by a comb&d T.L.C. - i n s i t u d e n s i t o m e t r i c method .

One of t h e more common sulphonamide mix tu res used i n animal t h e r a p y c o n t a i n s sulphamerazine wi th su lphaqu inoxa l ine , s p h a t h i a z o l e , and su lphamethaz ine . C i e r i showed t h a t t h e su lphameraz ine , sulphameth- 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 of t h e s e mixtures were b e s t de te rmined by a t h i n l a y - e r method r a t h e r t han by t h e ga chromato- g r a p h i c method proposed by Dam” (reviewed i n s e c t i o n 6.5.2 . ) . Using s i l i c a g e l H impregnated w i t h sodium hydroxide and c h l o r - oform, methanol ( 9 0 , l O ) as t h e s o l v e n t sys - t e m C i e r i a s sayed t h e i s o l a t e d components by an 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 lowed t h e components t o be de te rmined 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

Thinlb2yer chromatography i s now t h e u. s . P . x1x o f f i c i a l method f o r t h e d e t e r - minat ion of sulphamerazine , s u l p h a d i a z i n e and sulphamethazine i n t r i s u l p h a p y r i m i d i n e tab le t s and ora l suspens ions having r e p l a c - e d t h e paper chromatographic method of t h e U.S.P.XVII1. The method i n v o l v e s t h e use of s i l i c a g e l as a d s o r b e n t combined wi th ch loroform, methanol , ammonium hydroxide (30 ,12 ,1 ) a s s o l v e n t system. The s e p a r a t e d su lphapyr imid ines a r e q u a n t i t a t i v e l y d e t e r - mined us ing t h e B r a t t o n and Marsha l l color- imetr ic procedure .

A t h i n l a y e r chromatographic sc reen - i n g method f o r t h e e s t i m a t i o n o f su lpha- merazine and o t h e r sulphonamide r e s i d u e s i n p o u l t r y t i s s u e s has been 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 of sulphonamide was found t o be about 2 pg or 0 . 0 4 p.p.m. u s i n g a 50g. sample. To determine 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. of a series 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 , then r e - i s o l a t e d and a s sayed by d i r e c t c o l o r i m e t r y and by t h e proposed 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-


6.5 .4 .

i e s of su lphameraz ine were r e s p e c t i v e l y 9 1 and 80%.

T.L.C. has a l s o been used f o r t h e es t i - mation of su lphameraz ine i n b i o l o g i c a l f l u i d s ( s e e sect ion 7 ) and f o r t h e examina- t i o n of su lphameraz ine decomposi t ion pro- d u c t s ( s e e s e c t i o n s 5 . 1 and 5 . 2 ) .

Paper Chromatography

Paper chromatography w a s o r i g i n a l l y us- e d e x t e n s i v e l y f o r t h e s e p a r a t i o n , i d e n t i - f i c a t i o n and q u a n t i t a t i v e a n a l y s i s of s u l - phonamide mix tu res . A number of a p p l i c a - t i o n s a r e summarized i n Table 10 and some s p o t l o c a t i o n a g e n t s are g iven i n Table 11.

Sulphamerazine has been q u a n t i t a t i v e l y de te rmined i n m i x t u r e s w i t h o f $ ~ r g ~ y l # y ~ i amides by a number of workers Most methods used 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 composi t ion of t h e mobile s o l v e n t system. The B r a t t o n and Marsha l l c o l o r i m e t r i c method has been ex- t e n s i v e l y used f o r t h e a t ' t t ' o f t h e i s o l a t e d components 79 ,18 ! i ,PO2-?0S ,P lP

6.5.5. Ion-Exchange and P a r t i t i o n Chromatography

Hutchins and Chr i s t i an113 a s sayed 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 1 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 sulphonamides on H -form,macropor- ous and ge l - type r e s i n s f o r a number o f wa te r -o rgan ic s o l v e n t m i x t u r e s . A mix tu re o f sulphabenzamide, su lphace tamide , su lpha - d i a z i n e , su lphameraz ine 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 40,52, 6 4 , 7 7 and 9 0 % d imethylsu lphoxide 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 chromatographic 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 sulphonamide r e s i d u e s i n mi lk . The recovery o f su lpha-

f541R-

TABLE 10

Pape r

Pape r Chromatography o f Sulphamerazine

-Rf Ascending o r S o l v e n t System D e s c e n d i n a

L

Whatman N o . 1 Ascending

Whatman No.1 -

m % Whatman N o . 1 Descending

imp re gn a te d w i t h 4 % aqueous po ta s s ium d i - hydrogen phosphate Whatman N o . 1 Descending

Whatman N o . 1 A s ce nd in g impregnated w i t h ace t o n e , f o r m a m i de ( 7 0 , 3 0 )

Butanol ,ammonia 0 . 3 w a t e r (40 ,10 ,50) .

a ) B u t a n o l , g l a c i a l acet ic 0.50 a c i d , w a t e r (50,15,60) .

b )Butanol ,ammonia ,water 0.34 (40 ,10 ,30) . Butanol s a t u r a t e d - w i t h w a t e r .

Butanol , 3% aqueous 0 . 2 9 ammonia ( u s e t h e or- g a n i c l a y e r ) . Chloroform,methyl - chloroform(55,S) .

U s e Ref. -

S e p a r a t i o n of 10 3 m e t a b o l i c pro- d u c t s from bio- l o g i ca 1 m a t e r i a Is. I den t i t y tes t . 1 0 4

I d e n t i t y test . 105

I d e n t i t y tes t 79 and q u a n t i t a t i v e as s a y . Q u a n t i t a t i v e 106 a s s a y f o r t r i- s u l p h a p y r i m i d i n e s i n t a b l e t s and or- a l s u s p e n s i o n s .

TABLE 10 (con t ' d)

Paper

Paper Chromatoqraphy of Sulphamerazine

Ascending o r De scendinu

Solvent Sy s tern - U s e Ref.

Whatman N o . 1 imp re gn a t e d wi th acetone, f ormamide (70,301.

Whatman N o . 1

Ln Ln m

Whatman No.1

Whatman No. 2

Ascending Methylene ch lo r ide . - Q u a n t i t a t i v e assay 107 , f o r t r i s u l p h a p y r i - 111 midines i n t a b l e t s and o r a l suspensions.

Descending Butano1,absolute 0.24 S t a b i l i t y assay. 1 0 9

Ascending 0.2N aqueous EDTA 0.87 I d e n t i t y t es t . 10 9

e t h a n o l , 2 N ammonia (10,2,4).

con ta in ing 2 0 % ammonia.

ac id ,wa te r (5,1,4). q u a n t i t a t i v e assay. C i r c u l a r Butano1,ace t ic - I d e n t i t y test and 110

TABLE 11

1.

2 .

3.

4 .

5 .

6 .

7 .

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 and p a p e r chromatography of su lphameraz ine

Spo t Colour R e f e r e n ce Reagent T .L .C . P .C . -

9 4 1 0 6 , 1 0 7 , U.V. (254n.m. ) - f l u o r e s c e n c e quenching . Dark b l u e - 108 ,111

E h r l i c h s r e a g e n t - 1 % dimethylamino- Y e l l o w . 95 ,97 ,98 79,105

e t h a n o l . B r a t t o n and M a r s h a l l r e a g e n t a ) I N H C 1 , Reddish- 94 ,96 , b ) 5% N a N O Z , C ) O . 1% N-1-naphthyl) - p u r p l e . 97,98 e t h y l e n e d l a m i n e d i h y d r o c h l o r i d e .

b l a c k .

benza ldehyde + 1-108 conc.HC1 i n 95% 1 0 9 , 1 1 0

Copper s u l p h a t e -1-5% CuS04. 5H20 i n w a t e r . Brown. 97,99 F l u o r e s c e i n - 1 % i n a c e t o n e , w a t e r ( 3 , 1 ) , Dark b lue - 97 f l uo rescence -quench ing a t 254 nm. b l ack . 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 Brown. methanol .

99

C e r i c s u l p h a t e - 2 % i n water c o n t a i n i n g Y e 1 lowish- 99

5% conc.H2S0 4 ' purp le .

1 1 2


meraz ine from mi lk w a s foundl& be 83% a t t h e 0.5 p.p.m. l e v e l . M i l l e r deve loped a p a r t i t i o n column chromatographic 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 1 y t r a n s f erre d i n ace tone t o t h e t o p of a po ta s s ium bicarbon- a te impregnated C e l i t e 545 column. Su l - phamethazine 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 w i t h 0 . 1 N aqueous potass ium b i c a r b o n a t e s o l u t i o n . Sulphamerazine w a s t h e n e l u t e d w i t h 2 0 % n- b 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 po tas s ium b i c a r b o n a t e and f i n a l l y sul- phad iaz ine w a s e l u t e d w i t h 4 0 % 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 sayed by u l t r a v i o l e t spec t ropho tomet ry . When t h e v a l i d i t y of 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 encoun te red wi th h igh 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 n- b u t a n o l and C e l i t e used. However, t h e over - 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 an over - a l l s t a n d a r d d e v i a t i o n of 2 .58%.

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 sulphonamides by p a r t i t i o n chromatography. One procedure has been a p p l i e d t o t h e sep- a r a t ion of su lphameraz ine , su lphamethaz ine and s u l p h a d i a z i n e by i o n - p a i r fo rma t ion w i t h t h e tetrabutylammonium i o n fo l lowed by sep- 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 su lphapyr imid ines 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 spec t ropho tomet ry .

Rader 1 1 7 a p p l i e d t h e concep t of ion-

6 . 5 . 6 . E l e c t r o p h o r e s i s

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 min., deve lope r p-dimethylaminobenzaldehyde) of s e v e r a l su lphonamif fs w a s s t u d i e d by K i n o s h i t a and co-workers a t v a r i o u s pH values 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 su lphameraz ine , su lphaguan id ine and s u l p p f g i a e i n e i n a t e r n a r y mix tu re . Garber has g e n e r a t e d

SULPHAMERAZINE 559

6 . 6 .

6 . 6 . 1 .

paper e l e c t r o p h o r e t i c m o b i l i t y d a t a f o r s e v e r a l sulphonamides, i n c l u d i n g su lpha- merazine, 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 .

E lec t rochemica l Methods Polarography

Using polarogrygby coupled w i t h micro- coulometry Okazaki s t u d i e d t h e e l e c t r o d e r e a c t i o n s of s e v e r a l su lphapyr imid ines . 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 ra z i n e w e re de termined , a l i n e a r p l o t be ing 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 of t h e drug 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 py r imid ine 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 behav iour of 2-aminopyrimidine. Okazaki a p p l i e d t h e method t o t h e d e t e r m i n a t i o n of su lpha - merazine i n t a b l e t s , i n j e c t a b l e s , sy rups and o in tments .

Woodson a p p l i e d t h e p r i n c i p l e s of d . c . and a . c . po larography ko t h e r e d u c t i o n of a number of pha rmaceu t i ca l s i n an a p r o t i c o r g a n i c s o l v e n t s y s t e m . Using a dropping 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 re- f e r e n c e t h e d . ~ . half-wave p o t e n t i a l of sulphamerazine 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 sys - t e m w a s found t o be - 1 . 9 5 ~ . The co responding d e t e c t i o n l i m i t w a s 1 x 10 moles/ l i t r e .

-5

The po la rograph ic behaviour of t h e S c h i f f base of syjghamerazine has been s t u d i e d by Donev . A l i n e a r response t o c o n c e n t r a t i o n was found and t h e method was subsequent ly a p p l i e d t o t h e d e t e r m i n a t i o n of sulphamerazine i n t h e b lood plasma and u r i n e of an imals 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 su lpha drugs us ing sulphamerazine and su lph i somid ine a s


examples. The e l e c t r o d e s e n s i n g sys tem con- s i s t e d of a l i q u i d membrane c o n t a i n i n g an i r o n (11) -ba thophenan th ro l ine 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 aga- i n s t s o l u t i o n s of sulpham r a z i n e ganging i n c o n c e n t r a t i o n between 10 and 10 M. High s e l e c t i v i t y w a s o b t a i n e d i n t h e p re sence o f u r e a , g l y c i n e , aminopyrine and p-aminobenzoic a c i d which are s u b s t a n c e s known t o i n t e r f e r e i n t h e u s u a l c o l o r i m e t r i c a n a l y s i s of s u l p h a drugs . In c o n t r a s t s m a l l amounts of 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 produced an a p p r e c i a b l e e f f e c t i n t h e measured p o t e n t i a l .

-5

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 sulphonamides , i n v o l v i n g measuring 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: deve loped by C a n t e l l i F o r t i and F r a c a s s o . A l i n e a r 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 ag- a i n s t i n h i b i t i o n zone d i ame te r a long w i t h a s e n s i t i v i t y of 6-50 ug./ml. of 1 8 su lphonamides t e s t e d su lphameraz ine was t h e f i f t h most a c t i v e .

S h i b a t a and co-workers 126 deve loped 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 sulphonamides , i n c l u d i n g su lphameraz ine ,us- i n g B a c i l l u s megaterium as t h e c h a l l e n g e organ i s m .

7 . - Es t ima t ion i n B i o l o g i c a l F l u i d s

Longene cker lo3 developed a pape r chroma- t 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 su lphameraz ine i n t h e plasma of ch ickens f e d w i t h a mix tu re of s u l p h a d i a z i n e , su lpha - merazine and s u l p h a t h i a z o l e . The b lood sample was drawn from 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 c o n t a i n i n g po ta s s ium o x a l a t e . Following c e n t r i f u g a t i o n t h e p l a - 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 hen developed by t h e a scend ing t echn ique u s i n g a n -bu tano l , ammonia,water ( 4 0 , 1 0 , 5 0 ) emulsion as t h e s o l v e n t sys tem. The s e p a r a t e d sulphonamides were t h e n loca t e d u s i n g p-dime t h y 1 aminoben zaldehyde re-

SULPH AM E RAZ IN E 561

agen t and u l t i m a t e l y de te rmined u s i n g t h e Bra t ton and Marsha l l coup l ing t echn ique . I n t h e case of b lood a n a l y s i s b e t t e r s epa r - a t i o n s were achieved when 0.1% of nona- e t h y l e n e g l y c o l monos teara te (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 deve lop ing so l - v e n t . For t h e a n a l y s i s o f u r i n e t h e add 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 paper chromato- g 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 su lphameraz ine i n ood and u r i n e has been developed by Sinha’”. The chromatograms were run i n a c i r c u l a r chromato- g r a p h i c chamber by bo th t h e c e n t r a l and l a t - e r a l f low p r o c e s s e s . The drug w a s l o c a t e d and e s t i m a t e d us ing p-dime t h y laminobenzal- 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 .

Ortengren and T r e i b e r 1 2 * have reviewed t h e v a r i o u s chromatographic 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 sulphonamides i n b i o l o g i c a l materials. An e x t e n s i o n of t h e i r r e p o r t d e s c r i b e d t h e q u a n t i t a t i v e a n a l y s i s of su lphonaqides ( i n c l u d i n g su lphameraz ine) 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. For a minimum drug c o n c e n t r a t i o n of 10 pg./ml. t h e sample was s p o t t e d d i r e c t l y on t o t h e p l a t e . B e l o w t h i s minimum c o n c e n t r a t i o n it w a s necessa ry t o s a t u r a t e t h e u r i n e sample wi th ammonium s u l p h a t e fo l lowed by e x t r a c - t i o n wi th e t h y l a c e t a t e . 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 then d i s s o l v e d i n a s m a l l amount of ace tone and s p o t t e d on 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 es- t i m a t e d us ing dens i tome t ry .

The s e p a r a t e d sulphonamide

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 sulphonamides and t h e sum o f t h e i r i n a c t i v e m e $38” i n body f l u i d s is g iven by R iede r . The Bra t ton and Marsha l l color imetr ic a s say was used f o r a l l q u a n t i t a t i v e measurements. The procedure 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 lood plasma, 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$: l-

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 ave been d e s c r i b e d by Hawking and Lawrence . ? 39

8. Pharmacology

8.1.Metabolism

Sulphameraz i n e undergoes t h r e e main types 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 be- i 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 and hydroxy- l a t i o n .

A c e t y l a t i o n i s t h e m o s t i m p o r t a n t o f 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 - a c e t y l d e r i v a t i v e . The p r o c e s s t a k e s p l a c e i n t h e l i v e r t o v a r y i n g d e g r e e s i n man, monkeys, m i c e , r a t s , r a b b i t s and most o t h e r a n i m a l s e x c e p t dogs. Var ious a s p e c t s of t h e metabol ism o f su lphameraz ine 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

e more t h e g a s t r o i n t e s t i n a l t r a c t f o l l o w i n g o r a l a d m i n i s t r a t i o n and h a s a tendency t y Murphy and co-worke rs r e p o r t e d c e r t a i n 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 su lphameraz ine 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 ec t a l a d m i n i s t r a t i o n t o humans.

r a p i d l y abso rbed t h a n 1 2 p p h a d i a z i n e 31: .

S t u d i e s on t h e d i s t r i b u t i o n of15ylpha- merazine have been d e s c r i b e d . I i r i de - monst ra ted t h e e x c r e t i o n o f su lphameraz ine 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 demonst ra ted t h e r a p i d t r a n s - p o r t of sulphamefg6ine a c r o s s t h e human p l a - c e n t a , and Boger s t u d i e d t h e e x t e n t t o which d i f f u s i o n of su lphameraz ine 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 b lood . A n e x t e n s i v e s t u d y of t h e c i r c u l a - t i o n of sulphonamides, i n c l u d i n g sulphamera- z i n e , i n t h e human organism has been r e p o r t -

SU LPHAM E R A 2 IN E 563

1 4 0 e d by A l l i n e . 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 of sulphamerazine 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 th n o r m a l l t f n a l f u n c t i o n has been conducted by E a r l e . Sulphamera- z ine 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 i n d i c a t i n g e x t e n s i v e r e a b s o r p t i o n v i a t h e r e n a l t u b u l e s and Binding 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 eabsorbed .

compared t h e re- Reinhold and co-workers n a l c l e a r a n c e s of su lphameraz ine and seve r - a l o t h e r sulphonamides i n man w i t h t h a t of i n u l i n (non-reabsorbed by t h e r e n a l t u b u l e s ) .

f45

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 su lpha - merazine i n m i c e , r a t s and monkey as been 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 be ing i n good agreement h those ob ta ined by Welch and co-workers i n g exper iments i n animal and human sub je - c t s .

F l o r e s t a n o and co-workers 144 compared t h e b lood c o n c e n t r a t i o n s produced i n dogs , swine and c a t t l e fo l lowing 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 seve r- a 1 o t h e r sulphonamides. The t i s s u e r e s i d u e d e p l e t i o n of su lphameraz ine i n sheep 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 co-

mons t ra ted t h e d i s t r i b u t i o n of sulphamera- z ine ( i n a t r i p l e sulphonamide m i x t u r e ) i n t h e b lood , lung and b r a i n of r a t s and rabb- i t s .

fol low- Y3k

workers . P r i o r t o t h i s work Lehr 146 de-

The mechanism of t h e r e n a l t u b u l a r ex- c 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 has beenlq jscussed by Despopoulos and Cal lahan .


8.3 . T o x i c i t y

8 .3 .1 .Acute T o x i c i t y

When given o r a l l y t o wh i t e m i c e as t h e sodium s a l t t h e LD of su lphameraz ine w a s about 2 .335/kg., a?? d e a t h s o c c u r r i n i t h i n 2 4 hour s . Schmidt 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 su lpha - merazine, su lphamethaz ine 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 su lphameraz ine i n mice w a s found t o be 3.3g./kg. a t a corresponding 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 co r re spond ing b lood level of 6 6 mgm.%.

8.3.2.Chronic T o x i c i t y

Welch and co-workers 135 have s t u d i e d t h e c h r o n i c t o x i c i t y of su lphameraz ine i n r a t s , dogs and monkeys. The compara t ive c h r o n i c t o x i c i t i e s of su lphameraz ine , su lpha - d i a z i n e and su lphamethaz ine p p j been r e p o r t - e d by Schmidt and co-workers .

8 . 3 . 3 . C l i n i c a l T o x i c i t y

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 observed d u r i n g t h e c l i n i c a l use of su lphameraz ine i n c l u d e r e n a l damage, acute l o i n p a i n , nausea 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$,a, thrombocytopenia , and psychos i s . Of a l l t h e s e m a n i f e s t a t - i o n s t h e problem o f r e n a l damage h a s r e c e i v - e d t h e g r e a t e s t a t t e n t i o n . The more common t y p e s of r e n a l damage r e s u l t e d f o l l o w i n g t h e d e p o s i t i o n o f d rug and/or d rug m e t a b o l i t e c r y s t a l s i n t h e k idney and u r i n e ( c r y s t a l l - u r i a ) . Sulphamerazine i t s e l f h a s been sj$yn f t3p roduce ren f48dfqnage i n bo th a n i m a l s

phonamides t h e i n c i d e n c e of r e n a l damage h a s been r e l a t e d t o t h e pg dependent s o l u b i l i t y o f t h e drug and i t s N - a c e t y l d e r i v a t i v e ( s e e s ec t ion 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 th t h e d rug h e l p e d t o oy5fcome t h e problem of c r y s t a l l u r i a b u t Lehr p o i n t e d o u t t h a t adequate a l k a l i z a t i o n canno t a lways be accomplished i n e v e r y p a t i e n t s i n c e i n

and humans , b u t as w i t h o t h e r s u l -

SULPHAMERAZ INE 565

c e r t a i n c a s e s such 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 - e d . 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 sulphonamide f o r m u l a t i o n s s 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 lood lev- e l s a t t a i n e d by su lphameraz ine and i t s de- g ree of b ind in y 5 f ~ plasma has been d i s c u s s - e d by G i l l i g a n . I n v i t r o expe r imen t s conducted w i t h pH 7.4 b lood plasma con ta in - i n g 10mgm.% of su lphameraz ine 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 rug wasl,€geely d i f f u s i b l e . Beyer and co-workers dur ing 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 sulphamerazine 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 pro- p o r t i o n bound t o plasma p r o t e i n w a s 36.5%. D i a l y s i s and e lectrgkjoresis were used by Dessi and B a r a t t i n i t o de te rmine t h e i n - t e r a c t i o n of su lphameraz ine wi th 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 degree of b i n d i n g were t h e degree of i o n i z a t i o n of t h e d rug and t h e pH of t h e medium. I n v i v o , su lphameraz ine was found t o be 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 1 t 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 an imal serums fo l lowed t h e F reund l i ch a d s o r p t i o n i so the rm. A r e l a t i o n between b i n d i n g cap- a 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 ac- t i o n w a s demonst ra ted .

The in te rdependence between t h e e l i - mina t ion by g lomeru la r f i l t r a t i o n and p l a s - ma p r o t e i n b ind ing of some sulphonamide was examined by Por twich and co-workers . P r o t e i n b i n d i n g w a s measured w i t h an u l t r a - c e n t r i f u g e and 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 b lockade . With su lphameraz ine , 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 idney e l i m i n a t i o n was found t o be dependent on t h e degree of pro- t e i n b ind ing .

959


Moriguchi and co-workers s t u d i e d t h e b i n d i n g of sulphonamides , i n c l u d i n g su lphameraz ine , t o bovine serum albumin de- m 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 c o n s t a n t , dec reased 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. I n a q 6 f x t e n s i o n o f t h i s work Wada and Moriguchi s p e c t roQho t o m e t- r i c a l l y e v a l u a t e d t h e b i n d i n g of N - a c e t y l sulphonamides fg2bov ine serum albumin .Agren and co-workers a l s o showed a c o r r e l a t i o n between pK,, pH and b i n d i n g t o human albumin i n v i t r o . The degree of b i n d i n g of su lpha - merazine p r e s e n t e d as a f u n c t i o n of 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 va lue 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 uncharged s p e c i e s .

The r e l a t i o n s h i p between s t r u c t u r e and b i n d i n g of sulphonamides t o bovine 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 probe 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 ayed an i m p o r t a n t role i n t h e b i n d i n g t o hydrophobic 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 pyr imidine r i n g of su lphameraz ine a p p a r e n t l y s i g n i f i c a n t l y i n c r e a s e s t h e b i n d i n g o f t h e drug t o albumin.

Other s t u d i e s on t h e b i n d i n g o f su lpha - mepg&gg8to p r o t e i n s have been r e p o r t - e d

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 sulphonamides th rough 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 of absorp- 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 9gen i n v e s t i g a t e d by Koizumi and 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 expe r imen t s i n which m a l e r a t s were o r a l l y dosed w i t h so l - u t i o n s of t h e d rug a t v a r i o u s pH v a l u e s . Sulphamerazine 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 ra te r e a c h i n g a maximum a t around pH 6-7 and t h e n f a l l i n g o f f under 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 un ion ized form w a s abso rbed predominant ly .

SULPHAMERAZINE 567

However, acco rd ing t o theo ry t h e pH a t which su lphameraz ine was complete- l y un ionized w a s c a l c u l a t e d t o be 4 . 7 . This d i sc repancy w a s a t t r i b u t e d t o cer- t 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 of 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 un ion ized form o f l sulphamerazine was f o r d t o be 0 . 0 7 h r . compared t o 0 . 0 9 h r . f o r su lphameraz ine . This 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 wi th t h e r e c i p r o c a l of t h e par - t i t i o n c o e f f i c i e n t de te rmined between isoamyl acetate and water s u g g e s t i n g t h a t t h e e lementary p r o c e s s e s of a b s o r p t i o n fo l lowed t h e model shown below.

drug a t __+ k 2 d r u g i n i n stomach + i n t e r f a c e plasma drug

-1

That t h e hydrophobic i n t e r a c t i o n between sulphonamides and t h e i n t e s t i n a l mem- b rane formed an impor t an t f a c t o r i n t h e i r abso rp t fgg was shown by Nogami and co- workers . A physico-chemical approach based on t h e a d s o r p t i o n of sulphonamides from pH 7 . 4 aqueous s o l u t i o n by carbon b lack was used a s a model. The expe r imen t s showed t h a t t h e i n t r o d u c t i o n of a methyl group i n t o t h e pyr imidine r i n g , a s i n t h e case o f su lphameraz ine , n o t on ly i n c r e a s e d t h e a d s o r p t i o n on t o carbon b l ack b u t a l s o i n c r e a s e d t h e b ind ing t o bovine serum a l - bumin and i n c r e a s e d t h e r a t e of absorp t ior ) from t h e r a t s m a l l i n t e s t i n e . A good corn 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 de- g r e e of a b s o r p t i o n and t h e p a r t i t i o n co- e f f i c i e n t i n n-butanol w a t e r .

August ine and Swarbrickl7O used a three-phase model c e l l employing an i s o - p e n t y l a c e t a t e l i q u i d l i p i d b a r r i e r t o t es t 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-subs t i tu ted h e t e r o c y c l i c sa lphonamides , i n c l u d i n g su lphameraz ine . 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 rates (de termined as a f u n c t i o n of pH), p a r t i t i o n c o e f f i c i e n t s i n i s o p e n t y l ace t a t e -aqueous b u f f e r , and i n v i v o gas t r i c , i n t e s t i n a l and rectal 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 ra te o f t r a n s p o r t occu r red 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 each d rug and t h a t it w a s relate8 t o t h e f r a c t i o n o f un ion ized drug.

The use of h igh performance 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 group of 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 , sulphamerazine and su lphamethaz ine w e r e ob- t 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 subsequen t ly 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-oc tanol -water ) ,pKa, and b i o l o g i c a l a c t i v i t y .

Taraszka and Forist l’* d i s c u s s e d such 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 connec t ion w i t h t h e a d m i n i s t r a - t 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 , su lphameraz ine and su lphamethaz ine . Two s imple 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 o f t w o d r u g s wi th d i f f e r e n t ra te c o n s t a n t s f o r absorp- t i o n and e l i m i n a t i o n t o o b t a i n average a sympto t i c serum l e v e l s o f each drug on mul- t i p l e dose a d m i n i s t r a t i o n and b ) t h e selec- t i o n of t h e r a t i o of t w o d r u g s w i t h d i f f e r e - n t ra te 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 mini- m 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 ex tended t o t h e t r i p l e s u l p h a s on t h e bas i s of s o l u b i l i t y and human blood data 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 , su lphameraz ine and su lpha- methazine r e s p e c t i v e l y .

SULPHAMERAZINE 569

11. Acknowledgements

The a u t h o r wishes t o thank Mr . J .E . F a i r b r o t h e r of E.R. Squibb and Sons L t d . , Moreton, England f o r h i s e d i t o r i a l ass- i 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 pro- f 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 manuscr ip t .

570 RICHARD D. G. VdaOLFENDEN

References

1.

2.

3.

4 .

5.

6.

7. 8 .

9. 10.

11.

12.

12.

14.

15.

16.

17.

1-8. 19.

20.

21.

22.

Merck Index, 8th ed. 1968, Merck and Co., RahOay, page 995. The United States Pharmacopeia XlX,page 478, (1975). R.D.G.Woolfenden, Squibb Private Communica- tion, (1976). M.F.Abde1-Wahab, S.A.El-Kinawy, N.A.Farid, Amina M.El-Shinnawy, Anal.Chem. - 38, 508-510, (1966). K.Nakanishi, Infrared Absorption Spectro- scopy - Practical. Holden-Day Inc., and Nankado Co. ,Ltd., (1962). E.G.C,Clarke, Isolation and Identification of Drugs. The Pharmaceutical Press (1969) . A.E.O.Marzys, Analyst, 86, 460-463, (1961). L.A.Gifford, W.P.Hayes, J.N.Miller, and D.Thorburn Burns, Anal. Chem. 46, 1010-1017, (1974). K.Bowden, Chem.Rev. 66, 119, (1966). M.S.Puar and P.T.Funke, The Squibb Institute for Medical Research. Private communication, (19’76). A.dambon, R.Guedj, D.Robert, J.Soyfer, and M.Azzaro, Bull.Soc.Chim.Fr.Part 2, 567-71, (1970). J.Turczan and T.Medwick, J.Pharm.Sci. - 61, 434-43, (1972). C.Chang and H.G.Floss, J.Med.Chem. - 18, 505-

S.S.Yang and J.K.Guillory, J.Pharm.Sci. - 61, 26-40, (1972). C.Sunwoo and H.Eisen, J.Pharm.Sci. - 60, 238- 244, (1971). L.E.Cook and D.A.Hildebrand, Thermochimica Acta 9 , 129-133, (1974). Q. Ochs, The Squibb Institute of Medical Research. Private communication, (1976) . D.H.Lennox, Anal.Chem. - 29, 1433-1435,(1957). A.R.Frisk, G.Hagerman, S.Helander, and B.Sjorgen, Gac.med. Uruguay 8, No.80, 6-7, 17, 19, No. 81, 9-10, 15-16, 23 (1947). B.Sjogren and B.Ortenblad, Acta Chem.Scand. - 1, 605-18, (1947). A.G.Onandia, E.Holz, and S.Holz, Acta Cient. Venezolana 6, 157-63 (1955). H.M.Burlage7 J.Am.Pharm.Assoc., Sci.Ed., - 37, 345, (1948).

509, (1975).

SULPHAMERAZINE 57 1

23.

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.

B. Salvesen and M. Schroder-Nielsen , Medd. Nor. Farm. Selsk. 32 , 87-96, (1971). T.Koizumi, Txrita, and K.Kakemi, Chem. Pharm.Bul1. 12 , 413-20, (1964). H. A. Krebs andJ. C. Speakman, Brit. Med. J. - 1 , 47 (1946). A.V.Willi and W.Meier, Helv.Chim.Acta - 39, 54-6, (1956). A. Suzuki , W. I. Higuchi, and N. F. H. Ho. , J.Pharm.Sci. 59, 651-659, (1970). R.O.Roblin, JZ.Williams, P.S.Winnek, and J.P.English, J.Am.Chem.Soc.62, - 2002-2005, (1940). T.H.Evans and R.W.Mills, U.S.Patent 2,693, 466, Nov.2, (1954). H.Mauss and H.Leuchs, German Patent 871,303, Mar.23, (1953). S. Ziemiauski, J.Orkiszewski, S.Rychter and M.Dalek, Polish Patent 60,639, Oct.20,(1970). N.A.Pogorzhe1' skaya , I. A . Maretina, and A. A. Petrov, 2h.Org.Khim. 5, 1179-83, (1969). H . R. Chipman , U . S . Patent 2 , 6 9 0 , 4 3 9, Sept .2 8 , (1954). C.Ch'en and S.Hsi, Yao Hsueh Hsueh Pao, - 13, 59-60, (1966). E.Kuh, L.H.Dhein, and R.H.Ebe1, U. S.Patent 2,417,939, Mar.25, (1947). R. H. Barry and B. Puetzer , J.Am. Pharm. Assoc. - 34, 244-5, (1945). F. Schonhofer, German Patent 812 , 342 ,Aug. 27, (1951). P . S. Winnek , U. S . Patent 2 , 620 , 3 36, Dec. 2 , (1952). J.Erdos, Bol.soc.quim. Peru 17, 3-9, (1951). Ts. I.Shakh, Ukrain.Khim. Zhur22, - 336-40, (1956). W.K.Lee, Yakhak Hoeji, - 1, 13-17, (1963). W.K.Lee, Ibid 9, 8-13, (1965). W.K.Lee, Ibid 9, 4-7, (1965). W.K.Lee, Ibid i3, 97-100, (1969). M.Zajac, Diss.Pharm.Pharmaco1. - 22, 455-64, (1970). H.Auterhoff and U.Schmidt, Dtsch-Apoth. Ztd. - 114, 1581-3(1974). S.Naito and S.Mizoguchi, Yakuzaigaku, - 18, 48-50, (1958). A.Goudswaard,Pharm.Weekblad. 95, 236,(1960). A. Goudswaard, Ibid. 95, 487-90, (1960) .


50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

6 0 . 61.

62.

63.

64. 65.

66.

67.

68. 69. 70.

71.

72.

73. 74. 75.

M.B.Young, The Squibb Institute for Medical Research. Private communication, (1976). A.I.El-Sebai, Y.A.Beltagy, and R.Soliman, Pharmazie 26, 615-16, (1971). F.Yoshikawz S.Yagi, and S.Iga, Eisei Shikenjo Hokoku 84, 17-19, (1966). A.C.C. Cavaco and-. F.Falcao, Rev.Port. Farm. - 16, 1-8, (1966). H. W.Conroy, J.Assoc.Of fic.Agr. Chemists. 38, 643-4, (1955). J. S . Faber, J. Pharm. and Pharmacol . - 6, 187-90, (1954). M.S.Greenberg, B.J.Baker, and J.A.Caruso, Anal.Chim.Acta 54, 159-61, (1971). G.M.Davis, J.E.Gphorn, and J.A.Caruso, J.Pharm.Sci. 63, 1136-1138, (1974). H.Wojahn, A r c K Pharm. 281, 124,193,289, (1943). H. S .Conway, J.Am. Pharm.Assoc. Sci. Ed. - 34,2 36 (1945). P.L. De Reeder, Anal.Chin.Acta 9, 314, (1953) . A.Ejima, J.Tokusawa, and M.Ishibashi, Yakugaku Zasshi 87, 769-77, (1967). S.Ebel and S.Kalb, Arch. Pharm. (Weinheim, Ger.) 307, 2-6, (1974). Through C.A.80, 12 4 8 0 8 r

S.P.Agarwa1, M.I.Walash, and M.I.Blake, J.Pharm.Sci. 61, 779-81, (1972). P.L.De Reeder7Anal.Chim.Acta 10, 413, (1954). H.Abdine and W.S.A. Sayed, J.PErm.Pharmaco1. - 14, 761-3, (1962). F.Said, M.M.Amer, and M.I.Walash, Bull.Fac. Pharm. Cairo Univ., 6, 199-215, (1967) Through C.A. 73, 485bOS. L.S.Bark and TK.Grime, Analyst,98,452-55, - (1973). L.S.Bark and P.Bate, Analyst, 96, 881, (1971). L.S.Bark and P.Bate, Ibid., 97,783, (1972). E . J. Greenhow and L .E. Spencer, Anal. Chem. - 47, 1384-1388, (1975). M. Dolinsky , J . Assoc. O f f . Agr . Chem. 34, 74 8- 6 3, (1951). T.N.Oi and K.Miyazaki, Yakugaku Zasshi 87, 875-877, (1967). Through IPA 4, 1263e. A.E.O. Marzys, Analyst, 86, 4x0-63, (1961). M.Zajac, Farmacja pol. 30, 43-8, (1974). L. 1.Rapaport and Ts.1. Shakh, Farm. Zh. (Kiev) - 21, 22-8, (1966).

SU LPH AMER A 2 INE 573

76.

77.

78.

79.

80.

81.

82. 82A.

83.

84.

85. 86.

87. 88.

89.

89A.

90.

91. 92.

93.

94.

9 4A.

95.

96.

97.

B.W.Madsen and J.S.Robertson, J.Pharm. Pharmacol. 26, 682-5, (1974). A.C.Bratton:nd E.K.Marshal1, J.Biol.Chem. - 128, 537, (1939). M.Th.van der Venne and J.B.T'Siobbe1, J.Pharm.Belg. 18, 557-68, (1963). A.K.Fowler, J.TMerrick, and B.E. Reidel, Can.Pharm.J., Sci. Sect., 91, 56-8,(1958). T.Bican-Fister and V. Kajganovic, J. Chroma- togr. 16, 503-9 (1964). C.A.Brunner, J.Assoc.Off.Anal.Chem.~, 194- 196, (1972). C.A.Brunner, Ibid., 56, 689-691, (1973). P.E.Flinn, J.ChromatG.Sci.,l3, - 580-582, (1975). W.F.Philips and J.E.Trafton, Ibid, - 58,44-47, (1975). R.Tulus and A.Guran, Istanbul Univ.Fen.Fak. Mecmuasi Ser. C., 28, 108-13, (1963) .Through C. A. 61,6 86 7h. M.Luise, Boll.Chim.Farm.108,223-9,(1969). - J.T.Stewart, A.B.Ray and W.B.Fackler, J.Pharn.Sci. 58, 1261-62,(1969). T.C.Kram, J.Pharm.Sci.,Gl, 254-56,(1972). R.B.Poet and H.H.Pu, J.Pharm. Sci. - 62,809-11, (1973). L. Wes tlie , B. Aaro and B . Salvesen, Medd. Nor. Farm.Selsk, 36, 121-28, (1974). P.H.Cobb andx.T.Hil1, J.Chromatog.,l23, - 444-47, (1976). B.L.Karger, S.C.Su, S.Marchese, and B.A. Persson, J.Chrom.Sci.,G, 678-683,(1974). J.W.Turczan, J.Pharm.Sci. 57, 142-44,(1968). R. J.Daun, Journal of the Az.A.C. ,=, 1277- 82, (1971). E.Roeder and W.Stuthe, Fresenius Z.Ana1. Chem. - 271, 281-83, (1974) .Through C.A. 82,51256~. S.Klein and B.T.Kho, J.Pharm.Sci.,51,966-969 - (1962). N.Nose, S.Kobayashi, A.Hirose and A.Watanabe, J.Chromatog. ,123, - 167-173, (1976). Y-T Lin, K-T Wang, and T.Yang, J.Chromatog. 20, 610-12, (1965). KG.Fogg and R.Wood, J.Chromatog., 20,613- 14, (1965). E.G.C.Clarke and D.J.Humphreys, J.Pharm. Pharmac.22, 845-47, (1970).


98.

99.

100.

101.

102.

103.

104. 105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

115.

116.

117.

118.

H.R.Klein and W.J.Mader, J.Pharm.Sci.60, - 448-50, (1971). M.I.Walash and S.P.Agarwa1, J.Pharm.Sci. - 61, 277-78, (1972). U.R.Cieri, Journal of A.O.A.C.,E,1475-79, (1973). G.L.Biagi, A.M.Barbaro, M.C.Guerra, G . Can tell i-For ti and 0. Gando If i, J.Chromatog. ,106, 349-55 , (1975). The United States Pharmacopeia, XlX, paqeS 527-28, (1975). W. H . Longenecker , Anal. Chem. , - 2 1,1402 - 5 , 119491. R.Robinson, Nature, - 168, 512, (1951). F.Abaffy and S.Kveder, Acta Pharm. Jugoslav, - 6, 207-9, (1956). Through C.A. 51 , 90833.. M.Maientha1, J. Carol, and F.K.Kunze, Journal of the A.O.A.C. ,GI 313-316, (1961). F.M.Kunze and L.Espinoza, Journal of the A.O.A.C. ,46, 899-901, (1963). H.R.RoberG, The Squibb Institute of Medical Research. Private communication (1976). C.Garber and E.M.Assem de Juarez,Rev. Assoc.Bioquim.Argent . r34,184-185, (1969). Through C.A. 73,913052. M.Luise, Boll.Chim.Farm. - 108, 223-9, (1969) . Through C.A. 71,53608b. The United States Pharmacopeia XVIII , page 760, (1970). S.Takagi, Japan J.Pharm.and Chem.22,145, - (1950). H.H.Hutchins and J.E.Christian, J.Am.Pharm. ASSOC. 42, 310-314, (1953). T.C.GilEr and D.J.Pietrzyk, Anal.Chem.43, - 1585-92, (1971). G.B.Selzer and D.Banes, Journal of the A.O.A.C. 46, 703-707 (1963). H.M.Mille5 Journal of the A.O.A.C. - 53, 1100-1102 I (1970) . B . R. Rader , J. Pharm. S ci . , - 6 2 , 114 8- 1150 , (1973). Y.Kinoshita, S.Moriyama and T.Shimizu, Japan, Analyst, 5 , 219-224, (1957). Through A.A. 5,692.

SULPHAMERAZINE 575

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.

C.Garber, Proanalysis, - 2, 16-21,(1969). Through C.A. 74,9124b. Y.Okazaki, Bunseki Kagaku, - 11, 1142-46, (1962). Y.Okazaki, Bunseki Kagaku, - 11, 1239-42, (1962). A.L.Woodson, Anal.Chem. ,42, 242-8, (1970). B. Donev, Nauchni Tr. , VisGh Veterinarnomed Inst. Sofia, 23, 431-8, (1973). N.Hazemoto, Nzamo, 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,Chemo- therapy (Tokyo) , 22, 1424-9, (1974). A.Sinha, J.Proc.Inst.Chem.(India), 3, 282- 4, (1959). B . Ortengren and L. R. Treiber, Res . Commun . Chem. Pathol. Pharmacol., 2, 339-57,(1974). J.Rieder, Chemotherapy, - 17, 1-21, (1972) . J.Rieder, Chemotherapy, 22, 84-87, (1976). F.Hawking and J.S.LawrenZ,The Sulphona- mides. 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.Ben- son and E.H.Shiels, J.Pharmaco1.77,357-911 - (194 3) . 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. Gru- endig, and W.Weigel,Gesundheitsw, 25,1900- 3, (1970). W.P.Boger, Antibiotic Med. and Clin.Therapy,

M.Allinne, Ann.pha rm. f r anc .4 ,56 -72 , (1946) . D.P.Earle, J.Clin.Investiga~ion,23,914-2Ol - (1944). J.G.Reinhold, J.Pharmacol.83, - 279-87, (1945). L.H.Schmidt, H.B.Hughes,E.A.Badger, and I.G.Schmidt, J . P h a r m a c o 1 . 8 1 , 1 7 - 4 2 , ( 1 9 4 4 ) . -

- 6, 32-40, (1959) -

576

144.

145.

146. 147.

148.

149.

150.

151. 152. 153. 154.

155.

RICHARD D. G. WOOLFENDEN

H.J.Florestano, M.E.Bahler, H.E. Blair, and G.R.Burch, N.Amer.Vet. , 34, 17-20, (1953). H.F.Righter, J.M.WorthiGton,and H.D.Mercer, J.Agr.Food Chem.,20, 876-78, (1972). D.Lehr,Brit.Med.J,2, 601-04,(1950). A.Despopoulos and PTX.Callahan, Am.J. Physiol. - 203, 19-26, (1962). J.K.Clark, H.F.Flippin, and F.D.Murphy, Am.J.Med.Sci. ,205,846-51(1943). - W.H.Hal1 and W.W.Spink, J.Am.Med.Assoc., 123, 125-31, (1943). H.F.Dowling, E.Dunnoff-Stanley, M.H.Lepper, and L.K.Sweet, J.Am.Med.Assoc. ,125, - 103-5, (1944). D.Lehr, J.Uro1. 55, 548, (1946). R.E.Irvine, Clin.J.,E, 319, (1950). A.L.Sahs, Neurology, 1, 394, (1951). G.Hagerman, Nord. MedT,g, 1223,(1944), - 241 1944, (1944). D.Lehr, Proc. Soc .Exptl .Biol .Med. , - 64 , 393-401 , (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. 5 7 , 13126e.

(1961) .Through C.A. 56,5336e.

Klin.Wochschr, - 41, 447-51(1963).Through 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 Pharmacol. Toxicol, Supp1.,29, - 48-56, (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.Leip- zig , Math-Naturwiss. Reihe , 17, 85-6 , (1968) .

166. R.Elofsson, S.O.Nilsson, and B.Kluczykowska, Acta Pharm. Suecica,8,465-74, (1971).

16 7. F . P . Me ye r , Ac ta B io 1. fie d . Ge r .2 8 I 2 1 - 5 , ( 19 7 2 ) .

15 8. W. Scholtan, Arzne imi tte 1-Forsch. 11 , 707-20 , 159. F-Portwich, H.Buettner, and K.Engelhardt,

-

SULPH AM E RAZ INE 577

168. J.W.Dunn,J.Pharm.Sci. ,c, 1575-7, (1973). 169. H.Nogami, T.Nagai, and S.Wada,Chem.Pharm.

170. M.A.Augustine and J.Swarbrick, J.Pharm.Sci.

171. D.Henry, J.H.Block, J.L.Anderson, and G.R.

172. M.J.Taraszka and A.O.=rist, J.Pharm.Sci.,

Bull. ,g, 348-52, (1970).

- 61, 1656-1658, (1972).

Carlson, J.Med.Chem. ,19, 619-626, (1976) . - 57, 1379-1383, (1968).

TRIAMCINOLONE HEXACETONIDE

VIadirnir Zbinovsky and Ge
orge P. Chrekian


CONTENTS

1. Description

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


2.1 Infrared Analysis 2 .2 Nuclear Magnetic Resonance Spectrum 2 .3 U1 traviolet Spectrum 2.4 Mass Spectrum 2.5 Optical Rotation 2 .6 Melting Point 2.7 Thermogravimetric Analysis 2.8 Differential Thermal Analysis 2.9 Solubility 2.10 Crystral Properties

3 . Synthesis

4 . Stability, Degradation

5 . Pharmacodynamic Studies


6 . 1 Elemental Analysis 6 .2 Direct Spectrophotometric Analysis 6 .3 Colorimetric Analysis 6.4 Polarographic Analysis 6 . 5 Chromatographic Analysis

6 . 5 1 Thin Layer 6 .52 Column

TR IAMCINOLONE HEXACETONIDE 58 1

Trlamclnolone Hexacetonlde

1. Description


Triamcinolone hexacetonlde is 9-Fluor0-118,16a,l7, 21-tetrahydoxypregna-l,4-dlene-3,2O-dlone cyclic 16,17-acetal with acetone 21-(3,3-dimethyl-butyrate). It I s also known as Pregna-l-4-diene-3,2O-dione, 21-(3,3-dlmethyl-1-oxobutoxy)-9- f luoro-ll-hydroxy-16,17-[ (1-methylethy1idene)bls (oxy) I-, (118, 16a)-.

*' CH*OCOCHzC(CH,)3 19 1

c3 0 H4 1 Fo7 MOL. Wt.: 532.65


White to cream colored, odorless crystalline powder.


2.1 Infrared Analysis]

The infrared spectrum of trlamclnolone hexacetonlde (Lederle House Standard No. 48550-115) I s presented In Figure


1. bands (CM-1) were assigned to triamcinolone hexacetonide:

The spectrum was taken in a KBr pellet. The following

a.

b. Characteristic for 20-one in the presence b f 21

c . Characteristic for c1, B unsaturated 3-One: 1664 d. Characteristic for double bond system, A-1; 4 :

e. Characteristic for C-0 stretching bands of 1 6 ;

f. Characteristic for Cis CH of the A - 1 , 4 system:

Characteristic for 21-OAc=0 in the presence of 20-one: 1745

OAc: 1715

1618, 1605

17 acetonide: 1078, 1063

890

2.2 Nuclear Magnetic Resonance Spectrum’

The NMR spectrum Figure 2 was obtained by dissolv- ing 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 HA- l 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 (Lederle House Standard No. 48550-115) is 238 nm, E 15,500.

FIGURE 1

Infrared Spectrum of Triamcinolone Hexacetonide in KBr Pellet; Instrument: Perkln - Elmer 21

FIGURE 2

Ln m P

NMR Spectrum of Triamcinolone Hexacetonide Containing Tetramethylsilane as Internal Standard. Instrument: HA-100D

TR I AMCINOLONE HEX ACETON I DE 585

1 TABLE I

NMR Spectral Assignments of Triamcinolone Hexacetonide

Protons at

C1

c2

c4

c11

c16

C18

c19

c2 1

c2 1

Acetonide Methyl

Acetonide Methyl

Side Chain at C ~ L

0 C CH2 a

Chemical Shift ( 5 )

7.29 d; J1,2 p 10.0

6.34 dd; J1,2 m 10.0, J2,4 2.0

6.14 m

4.41 m

5.03 m

0.97 S

1.58 8

4.86 d Jg em = 1 9

5.07 d ABq

1.24 s

1.44 S

2.34 S

1.08 S

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. fragment ions in the high mass region are observed at m/e 517

of CqHg0).

The major

(loss of CH3), 512 (loss of HF) 474 (loss of C3H6O), 459 (108s 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

1 0

. , . . . . . . . . . . . . . . . . . . . . . . . . . ( . ~ . . , . . . . , . ~ . , . . . . , , . I

5 0 t o o 1 5 0 900 250 300 350 L l o o 9 5 0 500 550

S P E C C 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 S T E P nRSS;l. I*8,S . 1 %

TnrAMClNOLONE HEXACETONIDE 587

and results from cleavage of the bond between C-17 and C-20 with loss of CeH1303. indicative of a CL-GYS conjugated dimone in the A ring.

Intense ions at m/e 122 and 121 are


The optical rotation was determinedl for triamcinolone hexacetonide in chloroform solution at conc. 1.13%.

[a] 25 + goo - + 2 D

2.6 Melting Point

The melting point of triamcinolone hexacetonide is 2 7 1 - 272' (decomposition).

2.7 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 melting- decomposition 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.


TABLE I1

SOLUBILITY OF TRIAMCINOLONE HEXACETONIDE AT 25OC.

Solvent

H20

% m d m l - WIV

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

Ethyl Acetate 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

Chloroform 172.6 17.260

2.10 Crys t a l P rope r t i e s

Triamcinolone hexacetonide does not 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 t r iamcinolone3 and triamcinolone d i a c e t a t e 4 , Mesley5 who inspected t r iamcinolone ace tonide by in f r a red spectroscopy was not a b l e t o demon- s 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 triamcinolone hexacetonide6 (Lederle House Standard No. 48550-115) is presented i n Table 111.

TRIAMCINOLONE HEXACETONIDE 589

TABLE I11

POWDER X-RAY DIFFRACTION PATTERN OF TRIAMCINOLONE HEXACETONIDE

d (Ao)*

15.70

13 .10

10.80

8.80

7.30

6.65

5.90

5.50

5.15

4.75

4.60

4.34

4.13

3.63

3.44

3.32

3.10

2.63

2.58

2.47

2.37

2.14

2.08

*d = (interplanar distance)

Relative Intensity**

0.06

0.13

0.10

0.03

0.07

0.04

1.00

0.01

0.17

0.15

0.01

0.05

0.05

0.07

0.03

0.02

0.12

0.05

0.01

0.03

0.05

0.01

0.02

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 VLADlMlR ZBINOVSKY AND GFORGF P. CHREKIAN

pre~iously~,~ 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 photo- lytic 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 a1 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 measur- able 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 C 1 4 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

FIGURE 4.

C H 2 0 - m i C = O I

H CH, I 1

CHI -0-C-C-C-CH3

I 0 " ' I H CH,

- - - 0, /CH3 o/c\

+ HCI


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.


6.1 Elemental Analysis for C30H41F07, Lederle House Standard No. 48550-1152

Found

C 67.65 67.79

H 7.76 7.60

F 3.57 3.62

Element % Theory -


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 due to hydrazone formation from A 1,4 -3 keto group.

The color is

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. triamcinolone hexacetonide was 30 ppm and a well defined peak could be obtained down to 3 ppm. 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.

The concentration of

Other parameters for the


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. determined spectrophotometrically at 238 w.

Compounds were eluted with methanol and quantitatively

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.


Fig. 5. Differential Pulse Polarogram of Triarncinolone Hexacetonide in 0.1M

Tetrabutylammonium Chloride buffer, pH 3.5

I -1.12

10.0cm

I 1 I I I

-0.80 -1.00 -1.20 -1.40 I I I I I 1

I I

POTENTIAL (VOLTS vs S.C.E.)

59 5 TR IAMCINOLONE HEXACETONIDE

1.

2 .

3 .

4.

5 .

6.

7.

8 .

9.

REFERENCES

W . Fulmor, Lederle Labora tor ies , personal communication.

L. M. Rrancone, Leder le Labora tor ies , personal communica- t ion .

G . Michel, K. Florey, Ana ly t i ca l P r o f i l e s of Drug Sub- s t ance , A, 380 (1972)

L. L. Smith and M. Halwer, J. Am. Pharm. ASSOC., Sc i . Ed., 48 348 (1959).

R. J. Mesley, Spectrochimica Acta, 22 889 (1966).

P . Monnikendam, Leder le Labora tor ies , personal communica- t i o n .

K. Florey, Ana ly t i ca l P r o f i l e s of Drug Substances, 1, 397 (1972).

S. Berns te in , R. H. Lenhard, W. S . Allen, 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. SOC. , 81, 1689 (1959).

M. Heller, S. S t o l a r and J. Berns te in , J. Org, Chm., - 26, 5044 (1961).

10. J. A. Morrison, Leder le Labora tor ies , p r i v a t e communica- t ion.

11. A. Michaleides, Lederle Labora tor ies , personal communica t ion .

1 2 . E . J. Umberger, Anal. Chem., 27, 768 (1955).

13. P. P. Ascione, Leder le Labora tor ies , personal communica- t i o n .

1 4 . W. E. Hamlin, T. Chuleki, R. H. Johnson and J. G . Wag- ne r , J. Am. Pharm. ASBOC., s, 253 (1963) and D. R. Burton and W . C . Taylor , J. Am. Chem. SOC. , 80, 244 (1958); J. Chem. SOC., 2500 (1958).

15. L. L. Smith, M. Marx, J. J. J. Gabardini, T. F o e l l , V. E. Origoni and J. J. Goodman, J. Am. Chem. SOC., 82, 4616 (1960).

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

Bendrof lumethiazide 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 chloroform- acetic acid (98+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 ,43 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;

Chloroquine Phosphate, 5, 61 Chlorprothixene, 2 ,63 Clidinium Bromide, 2, 145 Clnnazepam, 6 , 6 1 Clorazepate Dipotassium, 4 , 9 1 Cloxacillin Sodium, 4, 113 Cyclizine, 6 ,83 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,

Disulfiram, 4,168 Echothiophate Iodide, 3, 233 Ergotamine Tatrate, 6, 113 Erthromycin Estolate, 1, 101;2,573

4,517

173

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 ,523 Fluphenazine Hydrochloride, 2,263;

Gluthethimide, 5 , 139 Halothane, 1 , 119;2,573 Hydroxyprogesterone Caproate, 4, 209 lodipamide, 3, 333 Isocarboxazid, 2,295 Isoniazide, 6, l b3 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 ,225 Meperidine Hydrochloride, 1, 175 Meprobamate, 1 ,209; 4 , 5 19 Methadone Hydrochloride, 3, 365;4,519 Methaqualone, 4 ,245 , 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;

Nystatin, 6, 341 Oxazepam, 3,441

4,518

2 ,573

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;

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

4,5 19; 6,598

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;

Triamcinolone Diacetate, 1,423 Triamcinolone Hexacetonide, 6,579 Triclobisonium Chloride, 2,507 Triflupromazine Hydrochloride, 2,523;

Trimethaphan Camsylate, 3,545 Trimethobenzamide Hydrochloride, 2,55 1 Tropicamide, 3,565 Tybamate, 4,494 Vinblastine Sulfate, I , 443 Vincristine Sulfate, I, 463

4,520

4,520;5,557

A 8 7 C 8 0 9 € 0 F 1 G Z H 3 1 4 J 5

600

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