Analytical Profiles of Drug Substances and Excipients Volume 4 (Academic Press rapid manuscript reproduction)

Analytical Profiles of Drug Substances Volume 4 Edited b y

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

Contributing Editors

Norman W.Atwater Salvatore A. Fusari Glenn A. Brewer, Jr. Boen T. Kho Gerald J. Papariello Jack P. Comer Frederick Tishler

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

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

1975

EDITORIAL BOARD

Norman W. Atwater Olenn A. Brewer, Jr. Lester Chafetz Edward M. Cohen Jack P. Comer Klaus Florey Salvatore A. Fusari

*

David ElGuttman' Erik H. Jensen Boen T. Kho Arthur F. Michaelis Gerald J, Papariello Bernard 2. Senkowski Frederick Tishler

Until his death in 1974.

Academic Press Rapid Manuscript Reproduction

COPYRIGHT 0 1975, 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. 111 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 Cataloging in Publication Data Main entry under title: Analytical profiles of drug substances. Includes bibliographical references. Compiled under the auspices of the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences. 1. Drugs-Collected works. 2. Chemistry, Medical and pharmaceutical-Collected works. I. Florey, Klaus, 111. Academy of Pharmaceued. 11. Brewer, Glenn A. tical Sciences. Pharmaceutical Analysis and Control [DNLM : 1. Drugs- Analysis-Yearbooks. QV740 Section. AA1 ASS] 615'.1 70-187259 RM300.A5 6 lSBN 0-12-260804-6 (v.4)

PRINTED IN THE UNITED STATES OF AMERICA

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS N . W. Afwuter, Searle and Company, Chicago, Illinois J. I. Bodin, Carter-Wallace Inc., Cranbury, New Jersey

G. A . Brewer, Jr., The Squibb Institute for Medical Research, New Brunswick. New Jersey L. C h j e t z , Warner-Lambert Research Institute, Morris Plains, New Jersey E. M . Colien, Merck, Sharp and Dohme, West Point, Pennsylvania J. L. Cohen, School of Pharmacy, University of Southern California, Los Angeles, California

J. P. Comer, Eli Lilly and Company, Indianapolis, Indiana

L. F. Cullen, Wyeth Laboratories, Philadelphia, Pennsylvania R . D. Daley, Ayerst Laboratories, Rouses Point, New York N. J. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania

F. Eng, Parke, Davis and Company, Detroit, Michigan

K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey vii

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

S. A. Fusari, Parke, Davis and Company, Detroit, Michigan

D.E. Guttman, School of Pharmacy, University of Kentucky, Lexington, Kentucky

W. W.Holl, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

E. H. Jensen, The Upjohn Company, Kalamazoo, Michigan H. Kadin, The Squibb Institute for Medical Research, New Brunswick, New Jersey B. T. Kho, Ayerst Laboratories, Rouses Point, New York J. Kress, Carter-Wallace Inc., Cranbury, New Jersey

E. P. K.Lau, Searle and Company, Chicago, Illinois H. H. Lerner, The Squibb Institute for Medical Research, New Brunswick, New Jersey L. P. Marrelli, Eli Lilly and Company, Indianapolis, Indiana D. L. Mays, Bristol Laboratories, Syracuse, New York A. F. Michaelis, Sandoz Pharmaceuticals, East Hanover, New Jersey J. E. Moody, USV Pharmaceutical Corporation,Tuckahoe, New York

E. S. Moyer, Ortho Research Foundation, Raritan, New Jersey N. G. Nash, Ayerst Laboratories, Rouses Point, New York G. J. Papariello, Wyeth Laboratories, Philadelphia, Pennsylvania v iii

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

V. E. Papendick, Abbott Laboratories, North Chicago, Illinois

D. M. Patel, William H. Rorer Inc., Fort Washington, Pennsylvania R. B. Poet, The Squibb Institute for Medical Research, New Brunswick, New Jersey A . Post, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

J. A . Raihle, Abbott Laboratories, North Chicago, Illinois N. H.Reavey-Cantwell, William H. Rorer Inc., Fort Washington, Pennsylvania P. Reisberg, Carter-Wallace Inc., Cranbury, New Jersey R. E. Schirmer, Eli Lilly and Company, Indianapolis, Indiana A . P. Schroff. Ortho Research Foundation, Raritan, New Jersey

B. Z. Senkowski, Hoffmann-LaRoche, Inc., Nutley, New Jersey L. A . Silvieri, Wyeth Laboratories, Philadelphia, Pennsylvania A . M. Sopirak, Wyeth Laboratories, Philadelphia, Pennsylvania

J. L. Sutter, Searle and Company, Chicago, Illinois

D. Szulczewski, Parke, Davis and Company, Detroit, Michigan F. Tishler, Ciba-Geigy, Summit, New Jersey A . J. Visalli, William H. Rorer Inc., Fort Washington, Pennsylvania

J. J. Zalipsky, William H. Rorer Inc., Fort Washington, Pennsylvania A . F. Zappala, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania ix

PREFACE Although the official compendia list tests and limits for drug substances related to identity, purity, and strength, they normally d o not provide other physical or chemical data, nor d o they list methods of synthesis o r pathways of physical or biological degradation and metabolism. For drug substances important enough t o be accorded monographs in the official compendia such supplemental information should also be made readily available. To this end the Pharmaceutical Analysis and Control Section, Academy o f Pharmaceutical Sciences, has undertaken a cooperative venture t o compile and publish Analytical Profiles of Drug Substances in a series of volumes of which this is the fourth. The concept of Analytical Profiles is taking hold not only for compendial drugs but, increasingly, in the industrial research laboratories. Analytical Profiles are being prepared and periodically updated to provide physico-chemical 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 compendia] status. The cooperative spirit of our contributors had 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. This volume of Analytical Profiles is dedicated to the memory of David E. Guttman, an enthusiastic member of the Editorial Board until his tragic and untimely death in 1974.

Klaus Florey

xi

CEFAZOLIN

Alfred F. Zappala, Walter W . Holl, and Alex Post

ALFRED

F. ZAPPALA eta/.

Content s

1. D e s c r i p t i o n 2.

Physical Properties 2.1

I n f r a r e d Spectrum

2.2

Nuclear Magnetic Resonance Spectrum

2.3

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

2.4

Optical Rotation

2.5

Melting Range

2.6

D i f f e r e n t i a l Thermal A n a l y s i s

2.7

Solubility

2.8

pKa

2.9

Crystal Properties

3.

Synthesis

4.

Stability

5.

Drug Metabolic P r o d u c t s

6.

Methods of Analysis 6.1

Elemental A n a l y s i s

6.2

Non-Aqueous T i t r a t i o n of C e f a z o l i n

6.3

Non-Aqueous T i t r a t i o n of C e f a z o l i n Sodium

6.4

Thin-Layer Chromatography

6.5

Spectrophotometric

6.6

High P r e s s u r e Liquid Chromatographic Procedure

6.7

F e d e r a l R e g i s t e r Methods

-

2

UV Hydroxylamine Method

CEFAZOLIN

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

1.1 Name, Formula, Molecular Weight C e f a z o l i n is 3-[ [(5-Methyl-l,3,4-thiadiazol-2y l ) thio]methyl]8-oxo-7[2- (1H-tetrazol-1-yl) acetomido] 5-thia-1-azabicyclo [4.2.O]oct-2-ene-2-carboxylic acid. It a l s o e x i s t s as t h e sodium salt. P a r e n t e r a l p r o d u c t s a r e known as Ancef and Kefzol.

Mol. w t . C14H14NgOqS3 (Na, -HI

1.2

454.512 ( a c i d ) 476.495 ( s a l t )

Appearance, Color, Odor White t o s l i g h t l y o f f w h i t e , o d o r l e s s .

2.

Physical Properties 2.1

I n f r a r e d Spectrum

The i n f r a r e d spectrum of c e f a z o l i n is p r e s e n t e d i n Figure 1. The spectrum t a k e n was t h a t of a m i n e r a l oil d i s p e r s i o n of t h e s t a n d a r d u s i n g a Perkin-Elmer 4578 Grating I R Spectrophotometer. A l i s t of t h e assignments made f o r some of t h e c h a r a c t e r i s t i c bands is given i n Table I (1).

3

MICRONS 1 5

3 0

4 0

50

1500

1000

.O

7 0

S O

VO

10

11

I4

Ib I I 10

15 303540

DO

01 0 Y

01

L

P

0

03

::

04 05

10

4000

3500

3000

IS00

1600

1400

1100

1000

SO0

b00

WAVENUMBER 1CM-l)

Figure 1:

Infrared Spectrum of Cefazolin Reference Standard, mineral o i l dispersion. Instrument: Perkin-Elmer 4578

400

150

CEFAZOLIN

Table I

-

I R S p e c t r a l Assignments f o r C e f a z o l i n

Frequency (cm-’>

C h a r a c t e r i s t i c of

3280

-NH-

3140 3075

3

-N=N-

-C=N-

1

tetrazole ring

-OH, bonded, -COOH 2580 1770

>c=o

lactam

1715

>CEO

acid

1670

>c=o

amide I

1555

>c=o

amide I1

2.2

Nuclear Magnetic Resonance Spectrum The 60 MHz NMR spectrum of c e f a z o l i n p r e s e n t e d i n F i g u r e 2 was obtained i n t r i f l u o r o a c e t i c a c i d a t a c o n c e n t r a t i o n o f about 100 mg/ml and t e t r a m e t h y l s i l a n e as i n t e r n a l s t a n d a r d . The s p e c t r a l assignments are l i s t e d i n T a b l e I1 (1).

5

I

9

1

1

I

I

I

I

1

I

1

J

I

I

I

I

I

I

I

I

I

8

7

6

5

4

3

2

1

0 PPM

Figure 2:

NMR Spectrum of Cefazolip Reference Standard, i n TFA with TMS as internal standard. Instrument: JEOL Co., Model JNM-C-60H

Table I1 Chemical S h i f t (ppm)

-

N M R S p e c t r a l Assignments f o r Cefazolin

Multiplicity

C h a r a c t e r i s t i c of

I n t e g r a t i o n of of Protons

No.

3.11

s i n g 1e t

protons a t

1

3

3.85

singlet

protons a t

2

2

4.71

singlet

protons a t

3

2

5.40

doublet

protons a t

4

1

5.75

overlapping s i n g l e t & doublet

protons a t and

5 6

3

8.21

doublet

protons a t

7

1

proton a t 8 i s beyond 9 ppm; however, i t is masked by t h e s o l v e n t

ALFRED F. ZAPPALA eta/.

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 a b s o r p t i o n spectrum of c e f a z o l i n i n 0 . B NaHC03 is shown i n F i g u r e 3. When scanned between 350 a n z 220 nm, c e f a z o l i n e x h i b i t s a s i n g l e band w i t h a n a bs or pt i on maximum a t 270 272 nm ( E = 13,100).

-

2.4

Optical Ro tatio n The s p e c i f i c r o t a t i o n o f a 5% s o l u t i o n of c e f a z o l i n i n 0;1M_ NaHC03 when measured a t 25OC i n a 1 decimeter t u b e is -170 5 70.

2.5

Melting Range Ce f azo lin starts t o decompose a t about 190°C under USP c o n d i t i o n s f o r Class I s u b s t a n c e s (2). 2.6

D i f f e r e n t i a l Thermal An aly s is A d i f f e r e n t i a l th ermal a n a l y s i s w a s performed on c e f a z o l i n and t h e thermogram is p resen te d i n F ig u re 4. The t y p i c a l meltin g endotherm is a b s e n t and o n l y t h e decomposition exotherm a t about 205OC is p r e s e n t . 2.7

Solubility The approximate s o l u b i l i t i e s o b ta in e d f o r c e f a z o l i n a t rbbm temperature (25OC 5 l 0 C ) are l i s t e d i n Table 111. Table I11

- Approximate S o l u b i l i t i e s of

Solvent

Cefazolin

mg Cef a z o l i n / m l

ac e t one acetone:water ( 4 : l v:v) chlorof orm 95% e t h a n o l e t h y l acetate isobutylacetate isopropyl alcohol methanol methylene c h l o r i d e m e t hyl i sobut y lk eto n e sodium c h l o r i d e , h a l f -satu rated sodium c h l o r i d e , s a t u r a t e d water

8

5.7 21.2 0.02 1.1 0.24 0.05 0.21 1.7 0.02 0.25 0.83 0.44 1.1

CEFAZOLIN

pH Solubility Profile The pH solubility profile of cefazolin is presented in Figure 5. 2.71

2.8

pKa

The pKa is 2.15 determined spectrophotometrically. A pKa of 2.05 determined titrimetrically has been reported (3). 2.9

Crystal Properties Several crystal forms of sodium cefazolin have been reported (4). The pathways of conversion of one form to another are shown below.

4

gain of water

treat with methanol or ethanol (loss of water)

8-f o m 312 moles of water.

n-f orm

9

ALFRED F . ZAPPALAetal.

U.V. ABSORPTION SPECTRUM

0.9

COMPOUND: CEFAZOLIN CODE: REFERENCE STANDARD

0.8

CONCENTRATION: 0.0294 m g / m l SOLVENT: 0.7

0.1M NaHCOj

CELL PATH: 1 cm

0.6 Y

U

4 m w

0.5

0

2 4:

0.4

0.3

0.2

0.1

0.0

2 00

250 300 WAVELfNGTH nm

350

-

Figure 3:

W Absorption Spectrum of Cefazolin Reference Standard Instrument: Cary Model 14

10

h

SAMPLE: C E F A Z O L I N O R I G I N : REFERENCE STD. SIZE: M I C R O C A P I L L A R Y REF: GLASS BEADS P R O G R A M M O D E : HEAT RATE: 1 0 ° C / M I N ATM: NITROGEN

c c

0

1

I

1

50

100

150

I

200

250

300

350

1

I

400

450

T,"C

Figure 4 :

DTA of Cefazolin, Reference Standard Instrument:

DuPont 900

1

500

I In 0 0

I

o n

I

o In N

I

0

0 0 0

lU/

0

0 (Y

I

0

I

0

1

In

2 h

2

0

NllOZV333 s

12

G

N

rl rl 0

U

a

a w 0

w

a 4 rl Ll

0

w

h

c4

U

rl

l-4

rl

1

a

UY

0

I-l

X

m

n W

..a

u z 00

t-l

G

rl rl rl

0

91

rcI

5 h P

a 91

a

t-l (d

a

a

t-l

a

I

0=& I 0 I

o=u N

5I

fll

V

rf

o=v

I

0

+

!

o=vI

0

91

V

2-2

”9 II

I

N

P h

X V

(d

B G

rl rl

N (d

W

H

T m

74 1

cn

z-1”

*& w o

R

X

+

H

H H

13

ALFRED F. ZAPPALA e t a / .

4. S t a b i l i t y The s t a b i l i t y of Ancef v i a l s a f t e r r e c o n s t i t u t i o n w i t h water f o r i n j e c t i o n ( a ) , b a c t e r i o s t a t i c s w a t e r (b) , and normal s a l i n e s o l u t i o n (c) was determined ( 6 ) . The r e s u l t i n g s o l u t i o n s were analyzed by t h e UV hydroxylamine method ( S e c t i o n 6.5) i n i t i a l l y and a g a i n a f t e r s t o r a g e a t 50C f o r 96 hours. No d e g r a d a t i o n w a s observed. The p h y s i c a l and chemical s t a b i l i t y of c e f a z o l i n sodium in s e v e r a l i n t r a v e n o u s f l u i d s (Table I V ) w a s evaluated (7). The s o l u t i o n s were assayed by t h e UV hydroxylamine method ( S e c t i o n 6.5) i n i t i a l l y and p e r i o d i c a l l y up t o 72 hours. I n s p e c t i o n i n d i c a t e d no apparent p h y s i c a l change. The chemical s t a b i l i t y of t h e s o l u t i o n s can b e s e e n from t h e d a t a i n T a b l e I V . Lyophilized c e f a z o l i n sodium is s t a b l e € o r a t least two y e a r s i n t h e d r y s t a t e a t room temperature. A comparison of t h e chemical (8) and m i c r o b i o l o g i c a l (9) assay methods is i l l u s t r a t e d i n Table V.

14

CEFAZOLIN

Table I V

- S t a b i l i t y of

Cefazolin Sodium i n S e l e c t e d Intravenous S o l u t i o n s *

Time in hrs.

- - r0 24 48 -

- 'C 72 24 72

-

10% d e x t r o s e i n H20

100

101

98.2

95.8

104

99.1

5% d e x t r o s e i n H20

100

98.3

98.2

94.5

99.4

99.7

5% d e x t r o s e w i t h

100

98.3

96.6

94.4

98.6

97.8

5%d e x t r o s e i n 100 Ringer's i n j e c t i o r

101

100

96.3

100

93.2

5% d e x t r o s e i n 0 . 9 % NaCl

100

100

99.4

98.5

102

97.4

5% dextrose i n 0.45% NaCl

100

100

100

96.0

9 8 . 0 100

10%d e x t r o s e i n 0.9% NaCl

100

100

97.7

97.7

99.4

99.7

0.9% NaCl

100

101

99.7

98.9

105

100

l a c t a t e d Ringer lnje c t i o n

100

103

98.9

36.9

LO1

39.4

linger injection

too

102

101

36.9

LOO

99.7

lactated Ringer

kExpressed as a percent of i n i t i a l concentration

15

ALFRED

Table V

-

F. ZAPPALA er a/.

Comparison of Chemical and Microbiological Met hod s

*

Months

1

3

6

9

Chem. assay

100

101

97.3

Micro. assay

99.4

103

97.5

12

18

24

99.0

96.0

99.0

100

98.2

97.0

101

98.0

*Expressed as a p e r c e n t of i n i t i a l c o n c e n t r a t i o n .

5.

Drug Metabolic Products S t u d i e s conducted t h u s f a r i n d i c a t e t h e r e i s very l i t t l e b i o t r a n s f o r m a t i o n of p a r e n t e r a l l y administered c e f a z o l i n sodium i n t h e body. Between 94 98% i s excreted i n t h e u r i n e unchanged. Only trace amounts of m e t a b o l i t e s have been seen b u t n o t i d e n t i f i e d (10).

-

6.

Methods of Analysis 6.1

Elemental Analysis The r e s u l t s from an elemental a n a l y s i s of c e f a z o l i n r e f e r e n c e standard are l i s t e d i n Table V I Table V I Element

Theory

% Found

C

37.00

36.75

H

3.10

3.29

N

24.65

24.44

S

21.16

21.31

0

14.09

14.23

(by d i f f e r e n c e ) 16

.

CEFAZOLIN

6.2

Non-Aqueous T i t r a t i o n of C e f a z o l i n Reagents (1) Dimethylsulf oxide (DMSO) 0.05N (2) Tetrabutylamonium hydroxide (TBAH) i n 9 :1 benzene:methanol; t h i s s o l u t i o n is s t a n d a r d i z e d a g a i n s t benzoic a c i d ( N a t i o n a l Bureau of S t a n d a r d s ) .

-

Procedure An a c c u r a t e l y weighed sample of about 200 mgs of c e f a z o l i n is d i s s o l v e d i n 70 - 80 m l of DMSO. The r e s u l t i n g s o l u t i o n is t i t r a t e d p o t e n t i o m e t r i c a l l y w i t h standard 0.05E TBAH u s i n g a glass-calomel e l e c t r o d e p a i r o r combination e l e c t r o d e . Each m i l l i l i t e r of 0.05N TBAH is e q u i v a l e n t t o 0.02273 g of c e f a z o l i n .

6.3

Non-Aqueous T i t r a t i o n of C e f a z o l i n Sodium An a c c u r a t e l y weighed sample of about 200 mgs of c e f a z o l i n sodium is d i s s o l v e d i n 70 80 m l of dimethyls u l f o x i d e (DMSO). The r e s u l t i n g s o l u t i o n is t i t r a t e d p o t e n t i o m e t r i c a l l y w i t h s t a n d a r d 0.05E a c e t o u s p e r c h l o r i c a c i d u s i n g a glass-calomel e l e c t r o d e p a i r o r combination e l e c t r o d e . Each m i l l i l i t e r of 0.05g p e r c h l o r i c a c i d is e q u i v a l e n t t o 0.02383 g of c e f a z o l i n sodium.

-

6.4

Thin-Layer Chromatography The following t h i n - l a y e r method may be used f o r t h e q u a l i t a t i v e p u r i t y e v a l u a t i o n of c e f a z o l i n and i t s sodium s a l t . About 50 and 100 micrograms of c e f a z o l i n , d i s s o l v e d i n a 4 : l m i x t u r e of acetone:water, are s p o t t e d two cm from t h e edge of a S i l i c a G e l GF p l a t e . The p l a t e is placed i n a s u i t a b l e chromatographic chamber l i n e d w i t h f i l t e r paper s a t u r a t e d w i t h t h e developing s o l v e n t ( e t h y l a c e t a t e :acetone :acetic a c i d :water, 5 :2 :1:1 ) and allowed t o e q u i l i b r a t e f o r t e n minutes. The s o l v e n t is t h e n allowed t o r i s e t o a l i n e drawn a c r o s s t h e p l a t e 1 0 cm from t h e o r i g i n . The p l a t e is removed from t h e chamber and allowed t o a i r d r y i n a fume hood u n t i l s o l v e n t vapors a r e no longer d e t e c t a b l e . The developed chromatogram may b e v i s u a l i z e d under u l t r a v i o l e t l i g h t (254 and 365 nm), exposure t o i o d i n e vapors, and s p r a y i n g w i t h potassium permanganate. Cefazolin h a s an Rf v a l u e of about 0.45.

,

17

ALFRED F . ZAPPALA e t a/.

6.5

Spectrophotometric-UV Hydroxylamine Method Reagents (1) 0.5 Molar sodium b i c a r b o n a t e (2) Acetate B u f f e r - Equal volumes of 0 . g a c e t i c a c i d and 0 . g sodium acetate are mixed t o g e t h e r and t h e r e s u l t i n g s o l u t i o n is a d j u s t e d t o pH 4.0. 86.5 g of sodium (3) A l k a l i n e Sodium Acetate hydroxide and 1 0 . 3 g o f sodium acetate are d i s s o l v e d i n s u f f i c i e n t water t o make 1000 m l . (4) Hydroxylamine S o l u t i o n One volume of 5M hydroxylamine h y d r o c h l o r i d e i s mixed w i t h two volumes z f a l k a l i n e sodium acetate and t h r e e volumes of water.

-

-

Procedure An a c c u r a t e l y weighed sample of approximately

50 mg is d i s s o l v e d i n 5.0 m l of 0.5E sodium b i c a r b o n a t e and d i l u t e d t o 1000 m l w i t h water. F i v e m l a l i q u o t s of t h i s s o l u t i o n are t r a n s f e r r e d t o each of two 100 m l v o l u m e t r i c f l a s k s . To one f l a s k is added 5.0 r n l of hydroxylamine s o l u t i o n . The f l a s k I s s w i r l e d and allowed t o s t a n d f o r 45 minutes, a f t e r which b o t h s o l u t i o n s are d i l u t e d t o 100 m l w i t h acetate b u f f e r . Two a l i q u o t s of a standard s o l u t i o n of c e f a z o l i n are t r e a t e d i n t h e same manner. The u l t r a v i o l e t a b s o r p t i o n spectrum of t h e unreacted s o l u t i o n is recorded v e r s u s t h a t of t h e r e a c t e d s o l u t i o n i n t h e r e f e r e n c e c e l l from 350 t o 240 nm i n 1 cm c e l l s . The c a l c u l a t i o n of t h e p u r i t y of t h e sample is accomplished by comparison of t h e absorbance d i f f e r e n c e between 270 nm and 340 nm f o r t h e sample t o t h a t of t h e s t a n d a r d . T h i s procedure h a s a l s o been automated (8). 6.6

High P r e s s u r e Liquid Chromatographic Procedure Reagents ( 1 ) Mobile Phase: 0.02 Molar monobasic sodium phosphate a d j u s t e d t o pH 6.2 2 0 . 1 w i t h 1l sodium hydroxide. (2) Standard S o l u t i o n : Approximately 20 mg of r e f e r e n c e s t a n d a r d is a c c u r a t e l y weighed i n t o a 50 m l volumetric f l a s k and d i s s o l v e d i n and d i l u t e d t o volume w i t h 0.05M - sodium b i c a r b o n a t e .

18

CEFAZOLIN

I n s t r u m e n t a l Conditions Column Packing: Strong anion exchange r e s i n Column Diameter: 2.1 m I . D . Column Length: 1 m Column Temperature: Ambient Column P r e s s u r e : 1000 p s i g Flow Rate: 0.5 m l p e r minute Detector: U.V., 254 nm Procedure

An a c c u r a t e l y weighed sample of approximately 20 mg is d i s s o l v e d i n and d i l u t e d t o 50 m l w i t h 0.05g sodium b i c a r b o n a t e . D u p l i c a t e 20 p 1 a l i q u o t s of t h e s t a n d a r d and sample s o l u t i o n s are i n j e c t e d . The r e t e n t i o n time f o r c e f a z o l i n is approximately 20 minutes. The c a l c u l a t i o n of t h e p u r i t y of t h e sample is accomplished by comparison of t h e average peak h e i g h t of t h e sample s o l u t i o n t o t h a t of t h e s t a n d a r d s o l u t i o n . I n s t r u m e n t a l c o n d i t i o n s may r e q u i r e m o d i f i c a t i o n s w i t h o t h e r HPLC u n i t s and d i f f e r e n t l o t s of column packing. "Federal R e g i s t e r " 38:31505-31509, 1973 A d d i t i o n a l methods l i s t e d in t h e F e d e r a l R e g i s t e r are (1) m i c r o b i o l o g i c a l a g a r d i f f u s i o n a s s a y , (2) iodometric assay, and (3) hydroxylamine c o l o r i m e t r i c assay. 6.7

19

ALFRED F. ZAPPALA eta/.

7. References 1.

R. J. Warren, SmithKline Corp., Personal Communication

2.

U.S.P. XVIII, p. 935

3.

Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan, Personal Communication

4.

J. of Antibiotics, Vol. XXIII, NO. 3, 131 (1970)

5.

J. Hill and H. Winicov, SmithKline Corp., Personal Communication

6.

L. Ravin and E. Rattie, SmithKline Corp., Personal Communication

7.

L. Ravin and E. Rattie, SmithKline Corp., Personal Comunication

8.

W. W. Holl et al, SmithKline Corp., Personal Comunication, to be published

9. 10,

-

Antimicrobial Agents & Chemotherapy, Vol. 5 , - 227 (1974)

NO. 3, 223

J. of Antibiotics, Vol. XXV, NO. 2, 86 (1972)

20

-

93

136

CEPHALEXIN

Louis P.MarreUi

LOUIS P. MARRELLI

TABLE OF CONTENTS Page 1. Description 1.1 Name: Cephalexin 1.2 Formula and Molecular Weight 1.3 Isomers 1.4 Hydrates 1.5 Appearance 2. Physical Properties 2.1 Spectra 2.11 Infrared Spectrum 2.12 Nuclear Magnetic Resonance Spectrum 2.13 Ultraviolet Absorbance 2.2 Crystal Properties 2.21 X-Ray Powder Diffraction 2.22 Differential Thermal Analysis 2.3 Solubility 2.4 Dissociation Constant 2.5 Optical Rotatory Dispersion 3 . Cephalexin Stability 4. Synthesis 5. Methods of Analysis 5.1 Identification Tests 5.2 Quantitative Methods 5.21 Titration 5.22 Colorimetric Determination 5.23 Thin Layer Chromatography 5.24 Paper Chromatography 5.25 Column Chromatography 5.26 Electrophoresis 5.27 Microbiological Assays 5.3 Assay Methods for Intermediates and Imp uritie8 5.31 7-Aminodesacetoxycephalosporanic Acid (7-ADCA) 5.32 Phenylglycine 6. Protein Binding 7. Pharmacokinetics 8. References

22

3 3 3 4 4

4 4 4 4 b

7 7 7 7 7 10 10 10 10 11 11 11 11 12 14 15 15 16 16 16 16 17 18 18 20

CEPHALEXIN

1.

Description 1.1

Name: Cephalexin Chemical Abstracts

designates cephalexin as 5-thia1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid, 7-( 2-amino2-ph eny1-ac etamido) -3-methyl-8-ox0 Ce halexin monohydrate is also known as 5-thia-1azabicyclo f4.2.01 oct-2-ene-2-carboxylic acid, 7-[(aIUinophenylacetyl) amino]-3-methyl-8-oxo monohydrate' * 7-( D-2amino-2-phenylacetamido) -3-me thyl-3-cephem-4-carboxylic acid monohydrat3 * and 7-( D-cr-aminophenylacetamido)-3-methyl-3cephem-4-carboxylic acid monohydrate'

.

1.2

Formula and Molecular Weight

c16

365.41

17 N304S'H20

23

LOUIS P. MARRELLI

1.3 Isomers The s y n t h e s i s of t h e L epimer o f cephalexin has been r e p o r t e d The D-isomer e x h i b i t s considerably more b i o l o g i c a l a c t i v i t y than t h e L-isomer. P e n i c i l l i n s derived from D-cr-amino a c i d s a l s o show more b i o l o g i c a l a c t i v i t y than t h e i r L-epimerg ' 6 .

.

1.4

Hydrates P f e i f f e r e t . a1.' provided x-ray powder d i f f r a c t i o n d a t a f o r the monohFdrac and dihydrate of cephlexin. Cephal e x i n w a s found t o c r y s t a l l i z e from aqueous s o l u t i o n s a t room temperature as t h e dihydrate b u t converted t o t h e monohydrate when t h e r e l a t i v e humidity was below 70%. Refer t o Section 2.21.

1.5

Appearance Cephalexin i s a white t o cream-colored c r y s t a l l i n e powder, having a c h a r a c t e r i s t i c odor. 2.

Physical P r o p e r t i e s 2.1

Spectra

I n f r a r e d Spectrum The i n f r a r e d spectrum of cephalexin monohydrate recorded as a potassium bromide d i & i s presented i n Figure 1. I n t e r p r e t a t i o n of t h e spectrum is given i n Table 18. Changes i n the B-lactam carbonyl s t r e t c h i n g region (1760 cm-l) can i n d i c a t e opening of the 6-lactam ring. Morin' and coworkers have shown a r e l a t i o n s h i p between t h e B-lactam carbonyl s t r e t c h i n g frequency and b i o l o g i c a l a c t i v i t y . The importance of t h i s s t r e t c h i n g frequency has been discussed i n a recent reviewlo. 2.11

Nuclear Magnetic Resonance Spectrum Figure 2 shows the proton magnetic resonance spectrum of cephalexin monohydrate. The solvent used was deuterium oxide containing a small amount of t r i f l u o r o a c e t i c a c i d t o enhance s o l u b i l i t y . 3-( Trimethylsilyl) -propanes u l f o n i c acid, sodium s a l t w a s added as t h e i n t e r n a l r e ference. "he spectrum was recorded on a Varian "60-A i n s t r u ment. The assignment of t h e spectrum i s shown i n Table IIe A most c h a r a c t e r i s t i c region of t h e NMR spectrum i s t h a t o r i g i n a t i n g from the two 6-lactam r i n g protons, H(6) and 2.12

.

H(7)

24

4000

3600

3200

2800

2400

2000

1800

1600

1400

1200

1000

800

WAVENUMBER CM-'

FIGURE 1. Infrared spectrum of cephalexin monohydrate (potassium bromide disc).

600

400

7.0

5 .O

4.0 3.0 2.0 1.0 PPM (a) FIGURE 2. NMR spectrum of cephalexin monohydrate (020 + trifluoroacetic acid). 8.0

6.0

0

TABLE I

Infrared Spectrum of Cephalexin Monohydrate

-1

Wavelength (cm )

- 3000 (series of broad bands)

3500

2600 (broad)

Assignment OH from H 0 and amide NH stretch 2

+

Y3

1760

@-lactam c

1690

h i d e c = o stretch

(1600 [very broad] (1400

C-0

1550 (unresolved) 820

- 690

0

-

=

o stretch

(carboxylate stretching)

h i d e I1 band Mainly skelectal vibrations including out-of-plane aromatic hydrogen bending, characteristic of monosubstituted aromatic ring

TABLE I1

Proton Magnetic Resonance Spectrum Peak Assignments p .p .m. (6)

Relative Intensity

2.07

Mu1t ip 1icity

Assignment

3

singlet

CH3

(3)

3.30

2

quartet (AB)

CH*

(2)

4.85

-

singlet

HOD

(solvent)

4.97

1

doublet (J=4Hz)

H

(6)

5.34

1

singlet

H

(benzyl)

5.67

1

doublet (J=4HZ)

H

(7)

7.60

5

sing let

‘SH5

CEPHALEXI N

2.13

U l t r a v i o l e t Absorbance An aqueous s o l u t i o n of cephalexin e i b i t s a Pcm W absorption maximum a t 262 nm (Figure 3 ) . The El% reported f o r cephalexin (on an anhydrous b a s i s ) was 23611 The u l t r a v i o l e t absorbance of cephalexin a6 w e l l as o t h e r cephalosporins has been a t t r i b u t e d t o t h e 0 = C-N-C = Cchromophore of t h e ring1*. Chou” u t i l i z e d t h e W absorption a t 262 nm t o determine t h e cephalexin content of s o l u t i o n f r a c t i o n s i s o l a t e d from human urine.

.

2.2

C r y s t a l Properties 2.21

X-Ray Powder Diffraction Cephalexin was found t o occur i n s e v e r a l solvated c r y s t a l forms, and o f t e n i n widely varying mixtures of these f o r m d 4 . Some of t h e solvated c r y s t a l forms prepared were the dihydrate, monohydrate, d i a c e t o n i t r i l a t e , formamid a t e , methanolate, and a c e t o n i t r i l e hydrate. X-ray powder d i f f r a c t i o n data f o r cephalexin monohydrate i s presented i n Table 111. 2.22

D i f f e r e n t i a l Thermal Analysis D i f f e r e n t i a l thermal analysis’ of cephalexin monohydrate was conducted on a W o n t Model 950 thermal analyzer i n a nitrogen atmosphere. A heating r a t e of 2OoC p e r minute w a s u t i l i z e d . An endotherm w a s noted at 123OC i n d i c a t i n g the loss of water, and an exotherm of 203OC i n d i c a t i n g decomposition. 2.3

Solubility The s o l u b i l i t y of cephalexin monohydrate i n t h e following solvents has been reported’ :

Mg. Cephalexin Monohydrate Per M l . Solvent

Solvent, 25OC.

Water Methanol N-oc tan01 Chloroform Ether

13-5 3- 4 0.03 4.01 4.01

Table I V r e l a t e s t h e s o l u b i l i t y of cephalexin monohydrate i n water as a function of pH.

29

LOUIS P. MARRELLI

2.0

1.5

1.0

O.!

0.0 I

I

I

250

300

monohydrate. 49 mcg per ml in H20

30

I

350

CEPHALEXIN

TABLE I11

of Cephalexin Monohydrate Norelco De Bye-Scherrer Camera

X-Ray Powder Diffraction Pattern

Radiation; Cu/Ni.

Cephalexin Monohydrate

d

1/11 0.40 1.00 0.30 0.20 0.50 0.50 0.20 0.40 1.00 0.50 0.40 0.40 0.60 0.60 0.70 0.70 0.80 0.30 0.60 0.60 0.40 0.60 0.40 0.20 0.40 0.10 0.30 0.15 0.30 0.30 0.10 0.05 0.02 0.05 0.05 0.05 0.10 0.05 0.02 0.02

-

-

15.15 11.85 11.00 9.36 8.55 7.86 6.89 5.98 5.39 4.97 4.76 4.57 4.39 4.22 4.00 3.86 3.60 3.46 3.24 3.10 2.98 2.90 2.81 2.73 2.68 2.63 2.47 2.41 2.31 2.25 2.12 2.09 2.01 1.93 1.87 1.85 1.82 1.72 1.66 1.62 31

TABLE IV

Solubility

PH

- pH Profile of Cephalexin in Water

(37°C)

Cephalexin Monohydrate mg I d .

Cephalexin Monohydrate mg Iml

.

. .

~

W

N

2.3

13

2.5

16

3 .O

.o

24

3.5

40

4.0

75

5.0

100

CEPHALEXIN

2.4

Dissociation Constant (pKa) The following d i s s o c i a t i o n constants were reported:

Solvent

rn

66% 66% DMF

h0

Carboxyl

Amino

5-2

7.3

4

5.3

7.3

17

7.1

Reference

18

2.5

Optical Rotatory Dispersion Optical r o t a t i o n has been used as an a u x i l i a r y method f o r the q u a n t i t a t i o n of cephalexir?. The s p e c i f i c r o t a t i o n [@ID reported f o r cephalexin, calculated on an anhydrous b a s i s , w a s +153O (C = 1.0 i n & 0)l 9 .

3.0

Cephalexin S t a b i l i t y The s t a b i l i t y of cephalexin i n solution i s dependent on pH, degrading r a p i d l y i n b a s i c media and remaining s t a b l e under mild a c i d i c conditions. No loss i n cephalexin a c t i v i t y occurred i n 72 hours a t 25OC i n the pH range from 3 t o 5. The r a t e of degradation found at pH 6 and pH 7 (25OC) was approximately 3% and 18%per day, r e s p e c t i v e l y 0 . With r e f r i g e r a t i o n , no appreciable loss occurs between pH 3 and pH 7 a f t e r 72 hours. I n U.S.P. hydrochloric a c i d b u f f e r (pH 1.21, cephalexin l o s t 5% a c t i v i t y i n 24 hours a t 37OC as compared t o a 45% loss i n phosphate b u f f e r at pH 6 . 9 l The a n t i b i o t i c r e t a i n s a c t i v i t y well i n serum and u r i n e as no loss i n a c t i v i t y was noted a f t e r storage a t -2OOC f o r 14 Cephalexin i n serum w a s found t o l o s e lo%, 5% and dayg2 75% a c t i v i t y , respectively, a f t e r storage a t 5'C, 25OC, and 37OC f o r 48 h o u r d l ' z z . Some organisms have been found t o produce a @-lact m a s e ( cephalosporinase) which can r a p i d l y degrade cephalexid' Degradation of cephalexin a l s o r e s u l t s from heat, s t r o n g alkali, strong a c i d s and u l t r a v i o l e t l i g h t (260 nm)

.

.

.

.

4.0

Synthesis Two synthetic r o u t e s of general a p p l i c a b i l i t y have been proposed f o r cephalexin'

.

The f i r s t method i s based on the cleavage of t h e acetoxyl group from cephaloglycid (I) by hydrogenation o r more s a t i s f a c t o r i l y , from N-t-butoxy-carbonylcephaloglycin (11) t o profuce the corresponding desacetoxy analogs V (cephalexin) and I11 as shown i n Figure 4, Scheme I. The t-

'

33

,

LOUIS P. MARRELLI

SCHEME I &lfON::@ !-

N

/

@:b.lii>

CHzOAc

N

C02H

/

COzH

I, R-H II. A=Boc

111. R-Boc

V. R-H

SCHEME I1

CO2H

COZH

V

IV

FIGURE 4. Synthasis routes for eaphelexin.

34

CH3

CEPHALEXIN

butoxy-carbonyl (BOC) group was removed from I11 with trifluoroacetic a c i d and the r e a c t i o n product was converted t o desacetoxycephaloglycin ( cephalexin, V) by treatment with Amberlite LA-1 r e s i n . The second method w a s s i m i l a r t o t h a t previously The nucleus, 7-aminodesused t o obtain cephaloglycid' acetoxycephalosporanic a c i d ( 7-ADCA)25 was prepared, acylated with BOC-protected D-phenylglycine employing a mixed anhydride synthesis, and then deblocked with t r i f l u o r o a c e t i c a c i d as shown i n Figure 4, Scheme 11.

.

5.0 Methods of Analysis 5.1 I d e n t i f i c a t i o n Tests Cephalexin may be i d e n t i f i e d by i n f r a r e d spectroscopy. B r i t i s h Pharmacopoeia' u t i l i z e s two charact e r i s t i c color r e a c t i o n s f o r i d e n t i t y . Thin l a y e r (Sec. 5.23), paper (Sec. 5.24), and column chromatography (Sec. 5.25) have been u t i l i z e d f o r i d e n t i t y purposes.

5.2

Q u a n t i t a t i v e Methods 5.21

--.T i t r a t i o n

The iodometric t i t r a t i o n procedure has The been used f o r the determination of c e p h a l e x i d 6 method i s based on the f a c t t h a t t h e i n t a c t cephalexin molecule does not consume iodine, whereas the alkali-hydrolysis product of cephalexin does. Alkaline hydrolysis of cephalexin r e s u l t s i n cleavage of t h e @-lactam ring. V a r i a t i o n s i n hydrolysis time, temperature, pH of t h e iodine solut i o n and concentration o f cephalexin present influence t h e consumption of iodine by t h e test solution. The method compares favorably t o t h e microbiological cylinder-plate method (Sec. 5.27) i n accuracy, and is much more rapid. Possible intermediates used i n the s y n t h e s i s such as 7-ADCA w i l l also respond t o the test.

'*'.

An automated iodometric assay has been used r e c e n t l y f o r t h e assay of cephalexin and formulations thereof4 The procedure incorporates a sample hydrolysis step N, at 37OC f o r 10 minutes followed by a 5-minute iodine consumption s t e p (pH 5.3-5.5, 37°C). Concentration of the sample i s r e l a t e d t o the decrease i n iodine color measured a t 350 MI. A reference standard i s run concurrently through the analyzer f o r comparative purposes. The automated system gives excellent l i n e a r i t y of response f o r t h e

!A;:(

35

LOUIS P. MARRELLI

-

recommended concentration range of cephalexin (0 1.5 mg. per m l . sample s o l u t i o n ) , with a l l standard curve p l o t s passing through t h e origin. The r e p r o d u c i b i l i t y of t h e method on t h e same sample o r standard s o l u t i o n on a given day i s generally b e t t e r than +l% r e l a t i v e standard deviation (RSD). Cephalexin can be titf;ated with p e r c h l o r i c a c i d in a g l a c i a l acetic acid m e d i d 9 . Crystal v i o l e t indicator (2% i n g l a c i a l a c e t i c acid) may be used t o determine t h e endpoint.

Moll and D6keio have reported using a formol t i t r a t i o n procedure f o r t h e determination of cephal e x i n , a m p i c i l l i n and r e l a t e d compounds. I n t h i s procedure 4 m l . of d i l u t e formaldehyde s o l u t i o n ( n e u t r a l i z e d t o the phenolphthalein end p o i n t ) is added t o 10 m l . of an aqueous s o l u t i o n containing 15.0 mg. of cephalexin. After 2 minutes t h e s o l u t i o n is t i t r a t e d with 0.02N sodium hydroxide. A p r e c i s i o n of +O.% RSD could be achieved i n t h e t i t r a t i o n o f cephalexin mozohydrate raw m a t e r i a l samples. Acidic compounds as well as amino a c i d s must not b e present as i m p u r i t i e s i n t h e sample. The formol t i t r a t i o n takes advantage of the r e a c t i o n between an amino a c i d and formaldehyde as a means of suppressing t h e b a s i c i t y o f the amino group and thus making p o s s i b l e t h e t i t r a t i o n of t h e acid. Colorimetric Determination Reaction with hydroxylamine has been u t i l i z e d f o r t h e colorimetric determination of cephaThe method i s based on t h e f a c t t h a t hydroxylexid’ ’32 lamine cleaves t h e 8-lactam r i n g (pH 7.0) t o form a hydroxamic a c i d which forms a colored complex with f e r r i c ion. Degradation products o r intermediates having an i n t a c t 8lactam r i n g r e a c t as well. 5.22

.

Kirschbaud’ has described a procedure f o r the colorimetric determination of t h e a n t i b i o t i c cephradine’ and r e l a t e d cephalosporins. An aqueous s o l u t i o n of t h e compound (1 t o 30 mcg. p e r ml.) i s r e a c t e d with sodium hydroxide, p a r t i a l l y n e u t r a l i z e d , and then r e a c t e d with 5*5’dithiobis-( 2-nitrobenzoic acid) resulting i n t h e formation of a y llow chromophore (412 nm.). The molar a b s o r p t i v i t y E x lo-$ reported f o r cephalexin when c a r r i e d through t h i s procedure w a s 1.29. The formation of the yellow chromophore w a s a t t r i b u t e d t o the presence of t h e R C q -CO-cephalo-

-

’Cephradine i s t h e generic name

f o r 7-[D-2-amino-2-(lq4c yclohexadienyl) ace tamido] desac e toxycephalosporanic acid

36

CEPHALEXIN

sporin nucleus, i n which R i s a mono-, di- or tri- enyl cyclohexyl ring. A s p e c i f i c colorimetric t e s t w a s developed f o r t h e determination of cephalosporin d e r i v a t i v e s having the following i n t a c t s i d e chain i n t h e 7- p o s i t i o n : R (3%-CO-cephalosporin nucleus, R being a heterocyclic o r aromatic r i n 8 ' . The D-phenylglycine d e r i v a t i v e s of both 7-ADCA (cephalexin) and 7-ACA ( cephaloglycin)2 respond well. These compounds (0.5 1.0 mg. per m l . i n %O) react with acetone and sodium hydroxide a t 1 0 0 ° C t o form c h a r a c t e r i s t i c A t the 1 mg. per ml. l e v e l , t h i s red chromophores (520 nm.). t e s t w i l l v i s u a l l y d i f f e r e n t i a t e cephalexin from cephradine.

-

-

Cephaloglycin is the generic name f o r 7-( D-a-aminophenylacetamido) cephalosporanic a c i d

31

LOUIS P. MARRELLI

5.23

Thin Layer Chromatography The following t h i n l a y e r chromatographic systems have been reported:

TABLE V Adsorbent

Solvent System

Ref.

Rf -

-

Silica G e l

Ace t o n i t r i l e / W a ter

0.67

13

Silica Gel

Ethyl Acetate/Acetone/Acetic Acid/ Water (5:2:2:1)

0.22

35

Cellulose Chromatogram Sheet

Butanol/Acetic Acid/ Water (3:l:l)

-

36

Sheet

Ethyl Acetate/Acetic A c i o a t e r (3:l:l)

-

36

Sheet

Acetonitrile/Ethyl Acet at e/Water ( 3 :1 :1)

-

36

Cellulose

Acetonitrilefiater (3:1)

0.50

37

Cellulose

Butanol/Acetic Acid/ 0.70 Water (3:l:l)

37

(3:l)

Cellulose is t h e p r e f e r r e d sorbent s i n c e i t i s i n e r t toward cephalexin. Additional t h i n l a y e r chromatography systems used f o r cephalexin and o t h e r cephalosporins have been t a b u l a t e d ? . Cephalexin may be detected by u l t r a v i o l e t absorbance and quenching, ninhydrin, i o d o p l a t i n a t e , a l k a l i n e permanganate, and phosphomolybdic a c i d sprays. Iodine detection and vanillin-phosphoric a c i d spray have a l s o been u t i l i z e d . O f t h e microorganisms used, Sarcina l u t e a i s p r e f e r r e d over B a c i l l u s s u b t i l i s o r Staphylococcus aureus.

-

38

CEPHALEXIN

5.24

Paper Chromatography The following paper chromatographic systems have been reported: Solvent Systems Butanol/Ac e t i c AcidJWate r (3:l:l)

Rf -

Ref. -

0.60

13, 36, 38

-

36

Ethyl Acetate/Acetic Acid/ Water (3:l:l)

Whatman No. 1*,untreated, was used with both solvent systems and the e q u i l i b r a t i n g solvent w a s t h e same as t h e developing solvent. Additional paper chromatographic systems f o r cephalexin and o t h e r cephalosporins have been t a b u l a t e d 8 . The butanol/acetic aciuwater (3 :l:l) system w i l l separate cephaloglycin from cephalexin, cephaloglycin being l e s s mobile. An acetonitrile/water (9:l) s y s t e d 9 using Whatman No. 3 paper buffered a t pH 5.0 (16 hour development) h a s been u t i l i z e d t o s e p a r a t e cephradine from cephalexin, cephradine being l e s s mobile. The developed chromatogram can be examined under u l t r a v i o l e t l i g h t , dipped i n ninhydrin, or bioautographed, using Sarcina lutea'o

.

5.25

Column Chromatography The Moore-Stein amino acid analyzer has been used f o r the determination of cephalexin i n u r i n e samplesb1 Beckman Custom Research Resin Type PA-35, packed t o a height of 9.0 cm. i n a water-jacketed column (0.9-cm. i.d. x 2'3-cm. length) w a s used f o r t h e separation. The u r i n e sample was d i l u t e d with an equal volume of sodium c i t r a t e buffer (pH 2.2) and 100 h applied t o t h e column. The e l u t i o n time for cephalexin was approximately 61 minutes. Excellent agreement was found between the analyzer method and the microbiological method (Section 5.27) on a s e r i e s of u r i n e samples tested. I n a d d i t i o n , t h i s technique has been u s e f u l i n determining low l e v e l s of 7-ADCA and phenylglycine i n cephalexin'l (Section 5.3). Determination of t h e l e s s a c t i v e ( b i o l o g i c a l l y ) L-isomer i n cephalexin by t h e Moore-Stein amino a c i d analyzer has been r e p o r t e 8 . Chou" used the anionic r e s i n , Bio-Rad AG2 x8 ( a c e t a t e form), t o i s o l a t e cephalexin from human urine.

.

-

*Whatman chromatography' paper, Reeve Angel, 9 Bridewell Place, C l i f t o n , New Jersey 39

LOUIS P. MARRELLI

5.26

Electrophoresis Paper e l e c t r o p h o r e s i s has been u t i l i z e d test f o r impurities by the B r i t i s h Pharmacopoeia' as present i n cephalexin (Section 5.3).

5.27

Microbiological Assays Microbiological assays f o r cephalexin have been discussed by Marrelli'*, Wick" Mann'' and Simmond' and are l i s t e d i n the Federal R e g i s t e r C 6 . B r i e f l y , the two p l a t e systems well s u i t e d f o r t h e determination of cephalexin i n pharmaceutical formulations a r e t h e cylinder p l a t e methods u t i l i z i n g S t a hylococcus aureus ( ATCC 6535) and Bacillus s u b t i l i s (A--e-ac range f o r both i s approximately 2.5 t o 20 mcg. of cephalexin p e r ml.'2 '" The B. s u b t i l i s p l a t e test h a s an advantage over t h e S. aureus p l a t e t e s t i n t h a t b e t t e r defined zones of inhTbition a r e obtained, thereby increasing t h e assay precision4 Since degradation product6 of cephalexin possess p r a c t i c a l l y no antimicrobial a c t i v i t y 2 r a p i d and p r e c i s e photometric microbiological assays a r e p o s s i b l e with cephalexin. The t e s t organism f o r the photometric assay i s Staphylococcus aureus 9144, 3 t o 3.5 hours being required f o r incubation. I n Antibiotic No. 3 Broth, t h e concentration range is 0.2 t o 2.0 mcg. p e r m l . of broth. I f t h e automated AUTOTU@ System is used, p r e c i s i o n i n the order o f 1 2 % i s possible". Cephalexin i n b i o l o g i c a l f l u i d s may be assayed by a Sarcina l u t e a p l a t e system. The concentration range f o r assay i s 0.2 t o 3.5 m~g./ml.~+

.

5.3

Methods f o r Intermediates and Impurities

5.31 7-Aminodesacetoxycephalosporanic Acid ( 7-ADCA) Cole'' u t i l i z e d the Moore-Stein amino a c i d analyzer f o r the determination of 7-ADCA i n cephalexin. Column s p e c i f i c a t i o n s were o u t l i n e d i n Section 5.25. Twenty-five milligrams of cephalexin sample were dissolved w i t h sodium c i t r a t e b u f f e r (pH 2.2) t o a t o t a l volume of 10.0 m l . and 1.0 m l . applied t o t h e column. The e l u t i o n time f o r 7-ADCA w a s approximately 45 minutes. 'The s e n s i t i v i t y of the assay w a s 0.B 7-AKA.

A colorimetric procedure was developed by M ~ ~ r r e l l iwhich '~ permitted t h e d i r e c t determination of The prolow l e v e l s of 7 - A K A i n cephalexin (0.4 1.5%). cedure i s based on t h e i n t e r a c t i o n of 7-ADCA with ninhydrin under c o n t r o l l e d conditions t o produce a s p e c i f i c chromophore

-

40

CEPHALEXIN

.

( A max. a t 480 nm.) Compounds having an a-amino group adjacent t o a B-lactam r i n g respond i n general t o t h e t e s t . B r i t i s h Pharmacopoeia' u t i l i z e d a paper e l e c t r o phoresis technique which estimated t h e 7-ADCA content i n cephalexin a t the l% l e v e l . The b u f f e r s o l u t i o n s p e c i f i e d i n the t e s t consisted of a mixture of 5 m l . of formic acid, 25 m l . of g l a c i a l a c e t i c a c i d , 30 m l . of acetone and water t o a t o t a l volume o f 1000 m l . A 2.0 pl. a l i q u o t of the cephalexin sample s o l u t i o n (5.0% w/v i n 0.5N HC1) along with the s p e c i f i e d amounts of reference standards and markers were applied t o the paper. The voltage was adjusted t o about 20 v o l t s per cm. of paper and e l e c t r o p h o r e s i s w a s allowed t o proceed u n t i l the c r y s t a l v i o l e t spot moved 9 cm. from the base l i n e . Both ninhydrin spray and W were used f o r detection purposes.

5.32

Phenylglycine The ChromatonraDhic Drocedures o u t l i n e d i n Section 5.31 f o r the determination o f 7 - m A i n cephal e x i n have been concurrently used for the determination of phenylglycine i n cephalexin. The e l u t i o n time f o r phenylglycine i n the amino a c i d analyzer assay was approximately '56 minutes. The s e n s i t i v i t y of the assay w a s 0.01%. The paper electrophoresis technique permitted estimation of phenylglycine a t the 1%l e v e l . Hussepe has u t i l i z e d a chromatographic procedure f o r phenylglycine similar t o t h a t of the amino a c i d analyzer assay but incorporating a Fluramm detection system'. I

t

&

Hoffman-LaRoche Inc., Nutley, New J e r s e y

41

LOUIS P. MARRELLI

6.

P r o t e i n Binding Various values have been reported f o r t h e percentage of cephalexin bound t o serum protein. Wicp' concluded t h a t serum i n a c t i v a t i o n o r p r o t e i n binding of cephalexin i s low. Addition of serum t o broth medium did n o t a f f e c t -.i n -v .- i t r o minimal i n h i b i t o r y concentration determinations. Cephalexin assays i n pH 7 b u f f e r and human serum r e s u l t e d i n i d e n t i c a l standard curves when 6-mm d i s c s were satur a t e d with s o l u t i o n s and t e s t e d by a Sarcina l u t e a microb i o l o g i c a l assay. U t i l i z i n g a similar method, G r i f f i t h and Black!) found t h a t p r o t e i n binding of cephalexin i n human berum was % a t concentrations above 1.0 bg./ml. Naumann and Fedde9O a l s o found and 4196 a t 0.2 yg./ml. t h a t the amount o f cephalexin bound t o serum p r o t e i n s varied with t h e cephalexin concentration. Using an u l t r a f i l t r a t i o n method, Kind e t . al.51 estimated t h e serum binding as being 15%. O v a l r q h a n and Muggletor?2 obtained a value of 43% by u t i l i z a t i o n of t h e u l t r a f i l t r a t i o n technique.

7.

Pharmacokinetics Oral doses of cephalexin are r a p i d l y absorbed by animals and man, r e s u l t i n r a t h e r high blood serum l e v e l s , and a r e excreted unchanged i n t h e urine. Wick?* found t h a t a 20-mg./kg. o r a l dose of cephalexin i n mice gave Wells e t . al.5' a blood serum l e v e l of 18 pg./ml. reported a similar value (17 Fg./ml.) with a 10 mg./kg. o r a l dose i n dog. The r a p i d i t y of o r a l absorption was demonstrated by t h e f a c t t h a t a n t i b a c t e r i a l a c t i v i t y i n t h e serum was t h e same within 1.5 hours a f t e r o r a l or intramuscular administration; t h e r e a f t e r , the l e v e l s were higher f o r t h e o r a l dose. I n man, a f t e r a 500-mg. dose t h e mean of t h e peak a n t i b i o t i c a c t i v i t i e s found '5' 'J and by s e v e r a l i n v e s t i g a t o r s w a s 15 pg./ml.' u s u a l l y occurred a t 1-1.2 hours a f t e r treatment. The compound is almost completely absorbed from the upper small i n t e s t i n e i n both m a n and animals6'J6. I n addition, t h e a n t i b i o t i c i s excreted unchanged i n u r i n e with almost 100% r e c o ~ e r f ' ~ ' 5 9 . A meal j u s t p r i o r t o treatment r e s u l t e d i n lower blood l e v e l s and increased t h e time required for peak t i t e ? ' 6 0

--

*

"'

42

.

CEPHALEXIN

Kirby et. a1.61 c a l c u l a t e d the serum h a l f - l i f e of intravenously administered cephalexin as 36 minutes. Kabins et. al.62 and Naumann and FeddeSO c a l c u l a t e d the serum h a l f - l i f e a f t e r o r a l dosage a s 54 minutes. al.5 found t h a t probenecid increased the Thornhill et. peak serum concentration by 50%. but Meyers e t . al.55 found a l e s s e r e f f e c t . Linquist --e t . a1.6sexamined the disappearance of cephalexin from the blood s e r a of aneph41.0 r i c p a t i e n t s . H a l f - l i f e values ranged from 23.5 hours with a mean of 31 hours, c l e a r l y demonstrating t h e dependence upon the kidney f o r excretion.

-

--

43

LOUIS P. MARRELLI

8. References

1. The United States Pharmacopoeia, XIX, Proof p. 2122. 2. Federal Register, 21CFR148w.6. 3. British Pharmacopoeia, 1973, p. 87. 4. Ryan, C.W., Simon, R.L., and Van Heyningen, E.M., J. Med. Chem., l2, 310 (1969). 5. Doyle, F.P., Fosker, G.R., Naylor, J.H.C., and Smith, H., J. Chem. SOC., 1440 (1962). 6. Analytical Profiles of Drug Substances, Vol. 2 (K. Florey, ed.) p.4, Academic Press, New York and London, 1973. 7. Pfeiffer, R.R., Yang, K . S . and Tucker, M.A., J. Phann. Sci., 59, 1809 (1970). 8. Underbrink, C.D., Eli Lilly Analytical Development, Unpublished Data. 9. Morin, R.B., Jackson, B.G., Mueller, R.A., Lavagnino, E.R., Scanlon, W.B., and Andrews,, S.L., J. her. Chem. SOC., 91, 1401 (1969). 10. Flynn, E.H., ed., Cephalosporins and Penicillins. Chemistry and Biology, Academic Press, New York and London (1972), p. 315. 11. Flynn, op. cit., p. 631. 12. Chawette, R.R., et. al., J. Am. Chem. SOC., 84, 3401 (1962). 13. Chou, T.S., J. Med. Chem., l2, 925 (1969). 14. Pfeiffer, R.R., Eli Lilly Analytical Development, Personal Communication, (1969). 15. Cole, T.E. , Eli Lilly Analytical Development, Personal Communication, (1968). 16. Pfeiffer, R.R., Eli Lilly Analytical Development, Personal Communication, (1970). 17. Flynn, op. cit., p. 310. 18. Hargrove, W.W. , Eli Lilly and Company, Personal Communication, (1967). 19. Flynn, op. cit., p . 633. 20. Winely, C.L., Eli Lilly Analytical Development Laboratories, Unpublished Data. 21. Simmons, R. J. , Anal. Microbiol., 11. , 193 (1972). 22. Wick, W.E., Appl. Microbiol., l5, 765 (1967). 23. Ott, J.L., and Godzeski, C.W., Antimicrob. Ag. Chemother. 1966, 75 (1967). 24. Spencer, J.L., Flynn, E.H., Roeske, R.W., Siu, F.Y., and Chauvette, R.R., J. Med. Chem., 2, 746 (1966). 25. Stedman, R.J., Swered, K., Hoover, J.R.E., J. Med. * .Chem 7, 117 (1964). 26. Federal Register, 21CFR141.506. 44

CEPHALEXIN

27. British Pharmacopoeia, p. 88 (1973). 28. Stevenson, C.E., and Bechtel, L.D. (1971) Publication submitted for review in J. Pharm. Sci. 29. Marrelli, L.P., Eli Lilly and Company, Personal Cmunication (1967). , -~ 30. Moll, F., and Dzker, H., Arch. Phann., Berl., 305 (7), 548 (1972). 31. Federal Register, 21CFR141.507. 32. Plynn, op. cit., p. 615. 33. Kirschbaum, J., J. Pharm. Sci., 63, 923 (1974). 34. Marrelli, L.P., J. Pharm. Sci., 6 l , 1647 (1972). 35. Thomas, P.N., Eli Lilly and Company, Personal Cmunication (1972). 36. Sullivan, H.R., Billings, R.E., and McMahon, R.E., J. Antibio., 22, 195 (1969). 37. Flynn, op. cit., p. 621. 38. Flynn, op. cit., p. 620. 39. Marrelli, L.P., Eli Lilly and Company, Personal Cmunication (1972). 40. Miller, R.P., Antibiot. and Chemother., 12, 689 (1962). 41. Flynn, op. cit., pp. 629, 680. 42. Flynn, op. cit., p. 610. 43. Flynn, op. cit., p. 497. 207 (1972). 44. Mann, J.M. , Anal. Microbiol., Simmons, R.J., Anal. Microbiol., s, 193 (1972) 45. 46. Federal Register, CFR148~6. 47. Kuzel, N.R. and Kavanagh, F.W., J. Phann. Sci., 60, 767 (1971). 48. Hussey, R.L., Eli Lilly Analytical Development, Personal Communication (1974). 49. Griffith, R.S. and Black, H.R., Postgrad. Med. J., 47, February Suppl. , 32 (1971). 50. Kumann, P. and Fedder, J., Int. J. Clin. Pharmacol., Suppl., 2, 6 (1970). 51. Kind, A.C., Kestle, D.G., Standiford, H.C. and Kirby, W.M.M., Antimicrob. Ag. Chemother., 405 (1968). 52. Flynn, op. cit., p. 438. 53. Wells, J.S., Froman, R.O., Gibson, W.R., Owen, N.V., and Anderson, R.C., Antimicrob. Ag. Chemother., 489 (1968). 54. Kunin, C.M., and Finkelberg, Z . , Ann, Inst. Med., 72, 349 (1970). 55. Eyers, B.R., Kaplan, K., and Weinstein, L., Clin. Pharmacol. Ther., 10, 810 (1969). 56. Muggleton, P.W., O'Callaghan, C.H., Foord, R.O., Kirby, S.M., and Ryan, D.M., Antimicrob. Ag.

a,

45

LOUIS P. MARRELLI

57. 58. 59. 60. 61. 62. 63.

Chemother., 353 (1968). Perkins, R.L., Apicella, M.A., Lee, I., Cuppage, F.E., and Saslaw, S., J. Lab. Clin. Med., 7 l , 75 (1968). Thornhill, T . S . , Levison, M.E., Johnson, W.D., and Kaye, D., Appl. Microbiol., l7, 457 (1969). Gower, P.E. and Dash, C.H., Br. J. Pharmac., 37, 738 (1969). O'Callaghan, C.H., Footill, J.P.R., and Robinson, W.D., J. Pharm. Pharmac., 23, 50 (1971). Kirby, W.M.M., de Maine, J.B., and Serrill, W.S., Postgrad. Med. J., 47, February Suppl., 46 (1971). Kabins, S.A., Kelner, B., Walton, E., and Goldstein, E., her. J. Med. Sci., 259, 133 (1970). Linquist, J.A., Siddiqui, J.Y., and Smith, I.M., New Engl. J. Med., 283, 720 (1970).

ACKNOWLEDGEMENTS The author wishes to express his indebtedness to Dr. C. L. Winely for his contribution of the sections on microbiological assays, protein binding and pharmacokinetics.

46

CHLORAMPHENICOL

Dale Szulczewski and Fred Eng

D A L E SZULCZEWSKI A N D FRED ENG

CONTENTS

1. Description 1.1 Nomenclature 1.11 Chemical Names 1.12 Generic Names 1.13 Trade Names 1.2 Formulae 1.21 Empirical 1.22 Structural and Stereochemical 1.3 Molecular Weight 1.4 Elemental Composition 1.5 General 2. Physical Properties 2.1 Crystal Properties 2.11 Crystallinity 2.12 X-Ray Diffraction 2.13 Melting 2.131 Range 2.132 As criteria of acceptability 2.133 In relation to purity determinationdifferential scanning calorimetry 2.2 Solubility 2.21 Single Solvents 2.22 In mixed solvents or as a result of complexation 2.23 pH Effect 2.3 Distribution 2.4 Spectral Properties 2.41 Ultraviolet 2.42 Infrared 2.43 Nuclear Magnetic Resonance 2.44 Optical Rotation 2.45 Mass Spectrum 3 . Synthesis 4. Stability and Decomposition Products 4.1 Crystalline solid and solid dosage forms 4.2 In solution 4.3 In presence of microorganisms 5. Metabolism 6. Methods of Analysis 6.1 Colorimetric 6.11 For identification 6.12 Quantitative Analysis 48

CHLORAMPHENICOL

CONTENTS (Cont ' d) 6.2 6.3 6.4 6.5 6.6

Polarographic Spectrophotometric Titrimetric Microbiological Chromatographic

49

DALE SZULCZEWSKI AND FRED ENG

1. Description 1.1 Nomenclature 1.11 Chemical Names a. Dg-(-)-threo-2 ,2-dich1oro-N- [B-hydroxya- (hydroxymethyl)-p-nitrophenethyl]acetamide b. Dg-(-)-threo-l-(~-nitrophenyl)-2- (2,2dich1oroacetamido)-l,3 propanediol

.

1.12 Generic Name Chloramphenicol 1.13 Trade Names Chloromycetin (The Merck Index' other trade names.)

lists 45

1.2 Formulae 1.21 Empirical C11H12C12N205 1.22

Structural and Stereochemical CH20H

I

C12CHCONHCc

r l ~ D

-(or Ls)-threo-l-(p nitrophenyl)-2-(2,2dichloroacetamid )-1,3 propanediol9 I3

I )Lc

*OH

bI

or

N02

(lR, 2R) l-(E-nitrophenyl)-2-(2,2dich1oroacetamido)1,3-propanediol

1.3 Molecular Weight 323.14

1.4 Elenental Composition C-40.88; H-3.74; C1-21.95; N-8.67; 0-24.76

50

CH LORAMPHENICOL

1.5 General3 Fine, white to grayish white or yellowish white, needle-like crystals or elongated plates. Its solutions are practically neutral to litmus. 2. Physical Properties 2.1 Crystal Properties 2.11

Crystallinity Chloramvhenicol is a crystalline solid. A typical photomicrograph4 of chloramphenicol is shown in Figure 1. 2.12 X-ray Diffraction Analytical X-ray diffraction powder data5 indexed using single crystal diffraction data6 for chloramphenicol follows: Radiation. CuKct(A1.5418);

Filter. Ni.

I/I, Diffractometer. System. Orthorhombic. Space Gr0up.C222~(DX) a, 17.6A: b, 7.35A; C, 22.38. 2. 8. Ect.

1.519; nuB. 1.601; EY. 1.668.

Sign. Negative. Measured Density.

51

2V. 7 8 O . 1.49.

DALE SZULCZEWSKI AND FRED ENG

Fig. 1

Photomicrograph of Chloramphenicol. 52

CHLORAMPHEN ICOL

2.12

X-ray Diffraction (continued)

Powder data for sample of chloramphenicol. (Chloromycetin sample from Parke, Davis & Company, Detroit, Lot I1 573326.) dA

20, deg. C u m

11.08 8.76 8.15 6.87 5.64

7.98' 10.10 10.85 12.89 15.72

12 1 32 64 57

4.98 4.68 4.47 4.37 4.28

17.81 18.98 19.85 20.30 20.74

32 61 57 79 100

113 204 311 400 401,114

3.95 3.88 3.70 3.66 3.61

22.50 22.91 24.05 24.31 24.63

11 11 18 64

205 313 115,006 020 021

3.44 3.38 3.34 3.28 3.23

25.93 26.37 26.68 27.17 27.61

54 7 7 7 18

404

3.16 3.13 3.05 2.97 2.92

28.25 28.55 29.25 30.05 30.64

4 br* 510,315 10 br* 511,405 7 024 7 207 600,513 79

2.89 2.87 2.82

30.90 50 31.19 14 31.69 89 plus other lines

*

-1 111

4

broad 53

h k-&-

002 200 201 202 203

220 221 023 222

601 316,117 406,602,025

DALE SZULCZEWSKI AND FRED ENG

2.12

X-ray D i f f r a c t i o n (continued)

Further information regarding confirmation of t h e chemical s t r u c t u r e of chloramphenicol through analysis of x-ray d i f f r a c t i o n p a t t e r n s obtained on t e a n t i b i o t i c and i t s bromo analog are given by Dunitz

k.

2.13

Melting 2.131

Range Bartz7 determined the melting range of chloramphenicol t o be 149.7 150.7 'C (corrected).

-

As a c r i t e r i a of a c c e p t a b i l i t y The United S t a t e s Food and Drug Administration8 and the U.S.P.3 both specify a melting range of 151k2'C. 2.132

2.133

I n r e l a t i o n t o p u r i t y determination Information p e r t a i n i n g t o t h e p u r i t y of chloramphenicol can be obtained through i n t e r p r e t a t i o n of t h e thermograms obtained v i a D i f f e r e n t i a l Scanning Colorimetry. A t y p i c a l thermogramg obtained on chloramphenicol follows as Figure 2. 2.2

Solubility

2.21 Merck Index1:

Single Solvents The following d a t a are a b s t r a c t e d from the

S o l u b i l i t y a t 25' i n water = 2.5 mg./rnl.; i n propylene glyc o l = 150.8 mg./ml. Very s o l u b l e i n methanol, ethanol, butanol, e t h y l a c e t a t e , acetone. F a i r l y s o l u b l e i n e t h e r ; i n s o l u b l e i n benzene, p e t r . e t h e r , vegetable o i l s . Solub i l i t y i n 50% acetamide soln. about 5%. S o l u b i l i t i e g determined by Weiss e t a l . , A n t i b i o t i c s & Chemotherapy 7,374 Water 4.4; methanol 720; (1957) i n mg./ml. a t about 28': ethanol >20; isopropanol >20; isoamyl alcohol 17.3; cyclohexane 0.13; benzene 0.26; toluene 0.145; p e t r . e t h e r 0.085; isooctane 0.022; carbon t e t r a c h l o r i d e 0.295; e t h y l a c e t a t e >20; isoamyl a c e t a t e >20; acetone >20; methyl e t h y l ketone >20; e t h e r >20; ethylene c h l o r i d e 2 . 3 ; dioxane >20; chloroform 1.95; carbon d i s u l f i d e 0.35; pyridine >20; formamide >20; ethylene glycol >20; benzyl alcohol 14.6. 54

CHLORAMPHENICOL PARKE D A V I S LOT H700171 mg

4.421

4 millicalories f u l l scale 1.25"/min % P U R I T Y = 99.863

FIG. 2

-

THERMOGRAM AND P U R I T Y DETERMINATION OF CHLORAMPHENICOL.

DALE SZULCZEWSKI A N D F R E D ENG

I n mixed s o l v e n t s o r as a r e s u l t of complexation The s o l u b i l i t y p r o f i l e s f o r chloramphenicol i n s e v e r a l aqueous solvent mixtures were determined by Negoro and Associates". H i s r e s u l t s are summarized i n Figure 3. 2.22

Kostenbauderll determined the s o l u b i l i t y of t h i s a n t i b i o t i c i n aqueous s o l u t i o n s of N, N , N ' , "-tetramethylphthalamides as p a r t of a study on t h e complexing p r o p e r t i e s of these amides. Results obtained indicated a moderate influence on s o l u b i l i t y as shown i n Figure 4. Aqueous s o l v e n t s containing 5% Tween 20-80 i n c r e a s e t h e water s o l u b i l i t y of chloramphenicol approximately 3 fold12. The s o l u b i l i t y of chloramphenicol i n serum and u r i n e i s approximately t h e same as i t is i n wa t e r l 3 . The s o l u b i l i t y of chloramphenicol i n water is increased by a d d i t i o n of boraxl4. This s o l u b i l i t y inc r e a s e i s explained on t h e b a s i s of t h e formation of a 1:2 complex between the b o r a t e i o n and t h e a n t i b i o t i c . Res u l t s are summarized i n Table 1.

Table 114 S o l u b i l i t y of Chloramphenicol i n Borax Solutions Molar Borax Concentration

0 0.0001 0.001 0.005 0.01 0.02 0.05 0.11 0.125 0.15

Solubility

PH

%

of Solution

0.375 0.391 0.438 0.614 0.732 1.23 2.14 3.46 3.67 3.87

4.70 7.15 8.65 8.65 8.65 8.65 8.70 8.90 8.90 9.00

56

CHLORAMPHENICOL

401

h

0,

\

F

v

> I-

-Tm

301

3

.J

0

v)

20(

101

~~

50

1 10

CONCENTRATION OF PURE SOLVENT ( w t . %)

I. II. I I I. IV. V.

VI.

Dimethylacetamide, 3 1 ° C Acetone, O°C Methanol Ethanol N-Propanol Propylene Glycol

FIG. 3

10

-

SOLUBILITY CURVES OF CHLORAMPHENICOL IN MIXED AQUEOUS SOLVENTS 57

$1

-

h.

X

1/M

9

'9 f 1031R~WdWWYOlW3

58

CHLORAMPHENICOL

pH E f f e c t Since chloramphenicol i s an e s s e n t i a l l y n e u t r a l compound, changes i n pH (over t h e pH r e g i o n 3 t o 9 ) do n o t r e s u l t i n s i g n i f i c a n t changes i n s o l u b i l i t y . The solubility t h e a n t i b i o t i c i s i n c r e a s e d i n p r e s e n c e of s t r o n g acidPf due t o p r o t o n a t i o n of t h e weakly b a s i c amido nitrogen. 2.23

2.3

Distribution As expected, t h e d i s t r i b u t i o n of chloramphenicol between water and an immiscible s o l v e n t i s n o t markedly pH dependent. The p e r c e n t of t o t a l chloramphenicol found i n t h e aqueous phase a f t e r d i s t r i b u t i o n between e q u a l volumes of water and immiscible o r g a n i c s o l v e n t s i s t a b u l a t e d i n Table 27,16. Table 2 % Chloramphenicol i n Aqueous Phase Immiscible Solvent Cyclohexanone n-Butanol E t h y l acetate

Methyl i s o b u t y l ketone N i trobenzene Nitromethane Ethyl e t h e r Chloroform Benzene Petroleum e t h e r Ethylene d i c h l o r i d e

% -

Ref.

8 8 3 8 38.0 17.0 20 82 93 96 50.0

7 7 16

7 7 7 16 16 16 16

7

Brunzell16 d i s c u s s e s t h e u t i l i t y of s i m p l e e x t r a c t i o n techniques i n t h e a n a l y s i s of f o r m u l a t i o n s c o n t a i n i n g t h i s a n t i b i o t i c . The same a u t h o r provides e x t r a c t i o n methods f o r a n a l y s i s of t h e drug i n t h e presence of hydrol y t i c decomposition products. 2.4

Spectral Properties 2.41

Ultraviolet Chloramphenicol i n s o l u t i o n a b s o r b s u l t r a v i o l e t r a d i a t i o n over a broad range t o produce a spectrum with a maximum near 278 nm and a minimum n e a r 240 nm ( s e e Fig. 5 f o r a t y p i c a l spectrum). 59

D A L E SZULCZEWSKI A N D FRED ENG

I.

nl 0

2 4

m U

0 VI

m 4

0.

240

280

280 278

300

320

340

380

WAVELENGTH (nrn)

FIG. 5

-

ULTRAVIOLET SPECTRUM OF CHLORAHPHENICOL I N WATER.

60

380

400

CHLORAMPHENICOL

As was e s t a b l i s h e d by Vandenbelt

7,17

, this a b s o r p t i o n i s due t o t h e p-nitrophenyl chromophore and prov i d e s both a d i s t i n g u i s h i n g c h a r a c t e r i s t i c of t h e a n t i b i o t i c and a u s e f u l method f o r a n a l y s i s . An u l t r a v i l e t s p e c i f i c a t i o n i s i n c l u d e d i n b o t h t h e F e d e r a l Register8 and t h e U.S.P.3 as a c r i t e r i a of acceptability.

The u l t r a v i o l e t spectrum of chloramphenicol i n aqueous s o l v e n t s is n o t s i g n i f i c a n t l y i n f l u e n c e d by changes i n pH. Aqueous Borate b u f f e r s (pH 9.0) p e r t u r b t h e spectrum, s h i f t i n g t h e maximum from 278 nm t o 284 nm as t h e r e s u l t of complex formation. 2.42

Infrared The i n f r a r e d spectrum of chloramphenicol (KBr d i s p e r s i o n ) i s shown i n F i g u r e 6. F u r t h e r i n f o r m a t i o n with regard t o c o r r e l a t i o n of f u n c t i o n a l groups o i n f r a r e d a b s o r p t i o n maxima i s given by Suzuki and Shindo" who determined i n f r a r e d s p e c t r a of racemic e r y t h r o and t h r e o isomers of t h e a n t i b i o t i c as w e l l as t h e s p e c t r a of r e l a t e d compounds. These a u t h o r s o b t a i n e d evidence f o r i n t r a molecular hydrogen bonding from i n f r a r e d s p e c t r a determined on d i l u t e s o l u t i o n s . Assignments f o r t h e accompanying spectrum follow: Functional Group

Wavenumber cm-1

bonded OH, NH

3340, 3260

amide p o r t i o n of 2,2-dichloracetamide moiety amide I amide I1 n i t r o group ( n i t r o PhenYl) hydroxyl

1697 1568 1530, 1358 1068

61

WAVENUMBER (cm-1) 3000

4000

1Tw

1000

1400

1600

111

1100

118)

im

a*

900

in

1

J

4

C

I

7

1

8

10

WAVELENGTH (microns) FIG. 6

-

INFRARED SPECTRA OF CHLORAMPHENICOL, KBr PELLET.

11

12

13

14

CHLORAMPHENICOL

2.43

Nuclear Magnetic Resonance

A typical nuclear magnetic resonance spectrum

of chloramphenicol in deuterated acetone is given in Fig. 7. Further information concerning the interpretation of the NMR spectra of chloramphenicol and its diastereoisomer, the L(+)-erythro isomer, can be obtained from Jardetsky's careful studylg concerning the conformation of chloramphenicol in solution. Jardetsky's chemical shift assignments of the chloramphenicol protons were determined from spectra in deuterated acetone of chloramphenicol samples lyophilized from D20 and H20. Jardetsky's assignments are as follows (with respect to benzene as external standard with 60-megacycle NMR) Proton( s ) Associated with cps -46.5 (center of H,B2 quartet) R ' -CHC12

+32.6 +97.2 4-156.2 +177.5 +187.7

R1 I

H N - R ~ ~ ~

-29.3

C1 OH

+99.6

C3OH

+151.0 63

(Water)

b-

(Acetone)

3.0 I

I

I

I

I

I

FIG.

LO I I

6.0

7.0

I

7 - NMR SPECTRUM OF CHLORAMPHENICOL, PARKE DAVIS LOT H 7 0 0 1 7 1 I N OEUTERATED ACETONE CONTAINING TRIMETHYLSILANE AS INTERNAL STANDARD.

Lo

I

I

1

CHLORAMPHENICOL

2.44

Optical Rotation I n the chloramphenicol series c o n s i s t i n g of four diastereoisomers, therapeutic a n t i b i o t i c a c t i v i t y res i d e s i n the Dg-threo (or I R , 2R) isomer. S p e c i f i c a t i o n s on s p e c i f i c r o t a t i o n as a c r i t e r i t of a c c e p t a b i l i t y are 3 contained i n t h e Federal Register as c i t e d by t h e U.S.P.

.

The Food and Drug Administration8 has establ i s h e d the following s p e c i f i c a t i o n : "Its (chloramphenicol' s) s p e c i f i c r o t a t i o n i n absolute ethanol a t 2OoC i s +20 2 1.5' and a t 25' is +18.5 - 1.5O." (NaD O r 589 nm) The s p e c i f i c rotation2' (C=5%, ethanol, 25OC) of chloramphenicol a t some other wave lengths follows:

578

+19.8"

546

+23.8'

436

+59.7O

Both magnitude and s i g n of o p t i c a l r o t a t i o n are solvent dependent, i.e., i n ethanol the a n t i b i o t i c i s dextrorotatory while i n e t h y l a c e t a t e i t is levorotatory. Circular dichroism measurements on t h e four chloramphenicol isomers have been made and are recorded i n the literature21. Analysis of the a n t i b i o t i c i n combinat i o n with sulfonamides w a s accomplished by polarimetric means22.

Mass Spectra The mass s p e c t r a of chloramphenicol obtained by conventional e l e c t r o n jmpact i o n i z a t i o n does not e x h i b i t a parent peak. BrunnGe and associates23 obtained the m a s s spectrum of chloramphenicol using a combined f i e l d ionizat i o d e l e c t r o n impact ion source. A parent peak of only 5% height i s obtained (see Fig. 8). The base peak is a t m / e 152 and is a t t r i b u t e d t o fragment I. 2.45

65

DALE SZULCZEWSKI AND FRED ENG

40'

I70

20 *

oi-, 60

. - . , I00

.

.

. A . .. I50

, 200

.

.

.

, . 250

300

340 m/e

F I G . 823

-

MASS SPECTRA OF CHLORAMPHENICOL - (a) ELECTRON IMPACT I O N I Z A T I O N (b) FIELD IONIZATIOW

66

CH LORAMPHENICOL

NH-COCHC1, OZN-@-f

3.

:i21 H

f-

I CH-CH,OH

1;;

Production and S y n t h e s i s

Chloramphenicol w a s o r i g i n a l l y produced by i s o l a t i n g t h e a n t i b i o t i c from c u l t u r e s of Streptomyces Venezualae 24925. A f t e r i t w a s demonstrated t h a t s y n t h e s i s of t h e a n t i b i o t i c was p o s s i b l e 2 6 s e v e r a l s y n t h e t i c p r o c e s s e s were developed f o r manufacture. A f l o w diagram f o r one such p r o c e s s follows: hexamethylenetetramine > 0 2 N o C O C H 2 M 2 0 2 N B C H 2 B r

-

-

67

DALE SZULCZEWSKI AND F R E D ENG

Detailed information on various steps in this synthesis are found in references 27-40. Other synthetic schemes are contained in references 41-47. Processes for conversion of erythro-p-nitrophenyl-2-amino-1,3-propanediol, produced as a by-product, to the desired threo isomer generally proceed via oxazoline formation and are detailed in references 48-54. Resolution of the threo-(p-nitrophenyl)-2-amino1,3-propanediol roduced can be accomplished by convenA tional means5 5 9 5 % or by fractional cry~tallization~~. rather complete review of the synthetic chemistry of chloramphenicol is a~ailable~~. This review also documents structure-activity relationships in the chloramphenicol series.

4. Stability and Decomposition Products 4.1 Crystalline solid and solid dosage forms

Chloramphenicol in the solid state as a bulk drug or present in solid dosage forms is a very stable antibiotic. Reasonable precautions taken to prevent excessive exposure to light or moisture are adequate to prevent significant decomposition over an extended period. 4.2

In Solution The stability of chloramphenicol in aqueous solution is governed by the rate at which hydrolytic processes occur. The two rimary routes of decomposition have been determined15,59,%0,61to be (a) amide hydrolysis with the formation of l-(p-nitrophenyl)-2-amino-1,3-propanediol %o~N(() ~ - C - C H ~ O H H

HN-C-CHCla

NH,

II

+ CHC l2COzH

0

and (b) hydrolysis of covalent chlorine of the dichloroacetamide moiety

H

OH

HN+-CHCl, 11

0

68

+ 2HC1

CHLORAMPHENICOL

The hydrolytic cleavage of t h e amide linkage5' i s the major cause of chloramphenicol breakdown and is the only s i g n i f i cant r o u t e of degradation i n s o l u t i o n s below pH 7. The r a t e of amide hydrolysis i s independent of pH over t h e pH region 2 t o 6 and independent of the i o n i c s t r e n g t h of t h e medium. Studies involving phosphate, acetate, and c i t r a t e b u f f e r s i n d i c a t e t h a t the amide hydrolysis i s general acidbase catalyzed6'. The hydrolysis of covalent-bound c h l o r i n e is i n s i g n i f i c a n t a t pH values below 6 but i n c r e a s e dramatically as pH increases. This increase i s a t t r i b u t e d t o hydroxyl i o n catalysis59. Numerous secondary r e a c t i o n s can occur which g i v e rise t o a v a r i e t y of decomposition products. Among these secondary r e a c t i o n s are those associated with subsequent hydrolysis of dichloroace t i c acid'' and oxidat ion-reduction r e a c t i o n s which involve t h e n i t r o gsoup as oxidant and the s i d e chain ( p a r t i c u l a r l y t h e aminodiol s i d e chain of the primary hyd r o l y s i s product) as reductant. Products i s o l a t e d from p a r t i a l l y o r completely decomposed chloramphenicol solut i o n s exposed t o a v a r i e t y of conditions are given i n Table 3. The presence of borate b u f f e r s has been shown t o i n c r e a s e the aqueous s t a b i l i t y of chloramphenicol. B r u n ~ e l l ~ ~ studied the s t a b i l i t y of 0.5% chloramphenicol ophthalmic s o l u t i o n s i n pH 7.4 b o r a t e buffer and i n unbuffered aqueous s o l u t i o n s . The r e s u l t s i n d i c a t e t h a t t h e a n t i b i o t i c i s more s t a b l e i n the presence of h i s b u f f e r than i n i t s absence. Heward and associates"studied the s t a b i t y of t h e borate-buffered chloramphenicol eye drops BPCi!!j The r e s u l t s obtained are l i s t e d below.

.

Temper a t u r e OC

Rate Constants k hrs-1

Calculated L l O L

115 110 30 20 4

0.2188 0.7413 0.1153 0.3589 0.4592

29 minutes 85 minutes 38 days 4 months 31 months

x 10:'

x 10 x lo-' x lo-'

69

DALE SZULCZEWSKI A N D FRED ENG

Table 3 Decomposition Products of Chloramphenicol

No.

Compound

1.

Environmental Conditions

Ref. -

Acidic o r b a s i c aqueous solution

M12 2.

C~,CHCO~H

3.

o,N@HO

17

11

Aqueous s o l u t i o n , ambient temperature.

62 9 63 9 64

H

OH

4 . H2N @--;-CH

OH 2

I

Aqueous s o l u t i o n , ambient temperature

-CH3

HN-C

.

62

II

0

65

5

R,, =Rz ‘CHz

6.

%=R2=C02H

II

7.

%

11

8.

Rl = Rz = OH

OH

Aqueous a l k a l i n e s o l u t i o n , high temperature.

= Rz = CHO

65

II

70

CHLORAMPHENICOL

Decomposition Products ofChloramphenico1 (Continued)

= C0,H;

9.

10.

5

11.

R,, = OH

12.

€$ = OH R2

R2 = OH

65

= C02H; Ra = CH,OH

II

11

R2 = CH20H = CHO

II

13. 02N@C0,H

Aqueous solution after exposure to light.

66

0 14

HOzC @“r;-@Co,H

Aqueous solution after exposure to light.

15. HC1

Aqueous solution; high temperature.

71

It

DALE SZULCZEWSKI AND FRED ENG

James and Leach70,14suggested that complexation between the antibiotic and borate ion is responsible for the increased stability of chloramphenicol in this buffer system. In the presence of microorganisms Smith'l studied the decomposition of chloramphenicol in the presence of various microorganisms. He defined the five routes by which chloramphenicol could undergo degradation. These possibilities are summarized in scheme 4.3

1.

5. Metabolism and Pharmacokinetics Biochemical changes which occur during metabolism of chloramphenicol were determined by Glazko and associate^^^. Isolation and identification of metabolites found in various body fluids after administration of this antibiotic indicates that its metabolism can occur by routes shown in scheme 2. Other pertinent information in this regard is to be found in references 73-76. The absorption characteristics of chloramphenicol from oral dosage forms were determined by means of blood level measurements and urinary excretion measurements of chloramphenicol and its rnetab~lites~~. Investigators concluded that absorption occurs mainly by simple diffusion mechanisms in the intestinal tract with minimal absorption from the ~ t ~ ~ n and a ~ that h ~the ~ degree s ~ ~of absorption was influenced b pharmaceutical factors involved in capsule formulation7is.

6. Methods of Analysis 6.1

Colorimetry

6.11 Qualitative e following color 88action is published in the U . S . P . W', and the B.P. 1968 as part of an identification scheme. Dissolve 10 mg. of chloramphenicol in a mixture of 1 ml of diluted alcohol and 3 m l of dilute calcium chloride T . S . (1 in 10). Add 50 mg of zinc dust, and heat on a steam bath for 10 minutes. Decant the clear, supernatant liquid into a test tube, and add 100 mg of anhydrous sodium acetate and 2 drops of benzoyl chloride. Shake the 12

/

Na?:-fHNH2 COMl P-aminophenyl serine

/

? If'

acid I

I

t\

\

iI '

NH~&-FH-NH,

cn20n

I-p-aminophenyl-2-dichl~ro-

acetamido-1.3-prapanediol

N H 2 ~ C O O HI P-amlnobcnroic

I

NH&-CMl&HCl2 CW20H

I I

r'

I-p-aninophenyl-2-..inoI.3-propanedIol

o-amino-8-hydroxy-paminonronioohenone . .

*-

Ilntermed i a t e r )

( I ntermd iates)

/

I

SCHEME I

-

ethanolamine, a m n i a (Carbon d i o x i d e . formaldehyde)

.'

H l C R O B l A L DECOMPOSITION PATHWAYS

OF CHLORAHPHENICOL.

POLV MERS

( C L U C U R O ~ IDATION)

1

I

I

(HYDROLYSIS)

SCHEME 2

-

PATHWAYS I N THE METABOLIC DISPOSITION OF CHLDRAHPHENICOL

CH LOR AMPHEN ICOL

m i x t u r e f o r l m i n u t e , add 0.5 m l of f e r r i c c h l o r i d e T.S. and, i f n e c e s s a r y , d i l u t e d h y d r o c h l o r i c a c i d t o produce a c l e a r s o l u t i o n : a r e d v i o l e t t o p u r p l e c o l o r i s produced. The formation of a yellow c o l o r by h e a t i n g t h e a n t i b i o t i c w i t h c o n c e n t r a t e d sodium hydrox Qf;8gerved i n t h i s c a p a c i t y i n t h e t h i r d supplement of DAB6 6.12

Quantitative Analysis 6.121 Reduction of N i t r o Group followed by D i a z o t i z a t i o n and Couplinv This approach t o a n a l y s i s of c h l o r amphenicol was among t h e f i r s t used € o r d e t e r m i n a t i o n of t h e a n t i b i o t i c i n body f l u i d s 8 3 . 1 zko's o r i g i n a l procedure w a s modified s e v e r a l t i m e ~ ~ ~ ~ r~e g~a rw d it ot ht h e reducing a g e n t used f o r r e d u c t i o n t o the corresponding a r y l m i n e . Since t h e n i t r o group, t h e r e a c t i v e moiety, e x i s t s i n decomposition p r o d u c t s and m e t a b o l i t e s as w e l l as i n chloramphenicol, t h i s procedure i s n o t s p e c i f i c when d i r e c t l y a p p l i e d . S p e c i f i c i t y i s imparted by i n c l u d i n g a s e p a r a t i o n s t e p p r i o r t o a n a l y s i s . The procedure i n v o l v e s r e d u c i n g t h e n i t r o group t o an m i n e followed by d i a z o t i z a t i o n w i t h a c i d i c n i t r o u s a c i d and c o u p l i n g w i t h N-(1naphthy1)-ethylenediamine d i h y d r o c h l o r i d e . 6.122

Hydroxamic Acid Method A h i g h e r degree of s p e c i f i c i t y i s imparted t o c o l o r i m e t r i c a n a l y s i s by u s i n g t h e hydroxamic a c i d r e a c t i o n . I n t h i s method chloramphenicol i s h e a t e d w i t h hydroxylamine under b a s i c c o n d i t i o n s t o form a hydroxamic a c i d which t h e n complexed w i t h f e r r i c i o n t o produce a r e d c o l o r This approach h a s a l s o been used f o r a n a l y s i s of chloramphenicol estersg7. It should be noted t h a t t h e p r i n c i p a l h y d r o l y s i s product of chloramphenicol does n o t y i e l d s i g n i f i c a n t c o l o r under t h e c o n d i t i o n s of t h i s test.

$8

.

6.123

I s o n i c o t i n i c Acid Hydrazide Procedure This procedure, as developed by Kakemi88, h a s been s t u d i e d i n some depthg9,90,91. The b a s i s of t h e a s s a y i s t h e development of a yellow c o l o r which r e s u l t s from mixing chloramphenicol w i t h i s o n i c o t i n i c a c i d h y d r a z i d e and sodium hydroxide i n aqueous solut i o n . The procedure i s s i m p l e and can b e performed

75

DALE SZULCZEWSKI AND FRED ENG

r e l a t i v e l y rapidly. Other a n t i b i o t i c s and t h e s u c c i n a t e and p a l m i t a t e esters of chloramphenicol have been found not t o i n t e r f e r e with t h e assay procedure. 6.124

Miscellaneous Other c o l o r i m e t r i c procedures have evolved f o r a n a l y s i s of chloramphen 01which depend on r e a c t i o n of acetoneg2 o r l-naphthol with t h e a n t i b i o t i c under b a s i c conditions. The chemistry of these methods i s not defined and, although convenient, they o f f e r l i t t l e advantage over those previously discussed.

4s

6.2

Polarographic Analysis The presence of t h e nitrophenyl group makes i t possible t o u t i l i z e d i f f e r e n t instrumental techniques f o r d e t e c t i o n and a n a l y s i s of t h i s a n t i b i o t i c . Methods which depend on t h e p-nitrophenyl group a r e not s e l e c t i v e unless preceded by a s e p a r a t i o n s t e p o r accompanied by independent a n a l y s i s t o give assurance t h a t the sample being analyzed contains only chloramphenicol and i s not a mixture of the a n t i b i o t i c and degradation o r metab o l i c products. Among t h e more convenient instrumental approaches t o a n a l y s i s of t h i s a n t i b i o t i c i s polarography. It i s less s u s c e p t i b l e t o i n t e r f e r e n c e from o t h e r materials than i s , f o r example, u l t r a v i o l e t spectroscopy, b u t , as discussed previously, could not be considered a s p e c i f i c procedure without modification. A d e t a i l e d study of t h e polaro raphic behavior of chloramphenicol w a s reported by Fossdal 84 The a n t i b i o t i c undergoes a 4-electron reduction a t t h e dropping mercury e l e c t r o d e producing a well-defined diffusion-controlled polarographic wave of a n a l y t i c a l u t i l i t y . Results obtained indicated t h a t chloramphenicol could be determined over t h e range 0.3 t o 60 mcg/ml. Previous s t u d i e ~ ~ ~ - ~ ~ reported a p p l i c a t i o n of polarography t o a n a l y s i s of chloramphenicol i n pharmaceutical preparations.

.

The n a t u r e of polarography i m p a r t s a degree of s e l e c t i v i t y t o the assay of chloramphenicol. 2- (2,2dichloracetamido)-3-hydroxy-4'-nitropropiophenone, a poss i b l e t o x i c contaminant i n s y n t h e t i c chloramphenicol w a s

76

CHLORAMPHENICOL

determined by direct polarographic measurementloo. In this case the reduction potential of the impurity is sufficiently different from that of the drug's to permit direct instrumental analysis 6.3

Spectrophotometric Because both spectrophotometric and polarographic methods depend on the existence of the p-nitrophenyl group, they are both subject to the same specificity considerations. Quantitative determination via direct ultraviolet measurement is not a specific analytical method since decomposition products absorb over the same region As previously mentioned, an ultraviolet procedure is official as a method to determine the potency of chloramphenicol'

i'e''2.

.

Ultraviolet spectroscopy has been extensively applied to chloramphenicol determination in methods involving separation prior to quantitation43~44~47,63. It has also been employed to determine the antibiotic in pharmaceutical preparationsa0. 6.4 Titrimetric Methods Titrimetric methods have been developed for analysis of chloramphenicol. Such procedures are dependent on only a limited portion of the molecule, i.e., the nitro group in the p-nitro-benzene portion or the covalent chloride contained in the dichloracetamido moiety and hence would not be selective unless metabolic or degradative processes yielded products not containing these functional groups. Procedures utilizing covalent chloride involve converting the covalently-bound chloride to its ionic form. The ionic chloride is then determined by argentometric titration103,104. Titrimetry of chloramphenicol using the nitro group has two variations both of which require reduction to an arylamine. Reduction of the nitro group with excess titanium chloride followed by determination of the excess reagent by back titration with ferric ammonium sulfate constitutes the titanometric methodlo5.

77

DALE SZULCZEWSKI AND FRED ENG

The bromatometric methodlo5 consists of a Zn-HC1 reduction to form the arylamine. The arylamine is then determined by bromination in the presence of excess bromine followed by iodometric determination of the excess bromine.

6.5 Microbiological Microbiological procedures have been developed for to analysis of dosage assay of chloramphenicol and appl forms, body fluids, and bulk drugf". Although time consuming, these methods are as accurate as physicochemical tests, provide analytical sensitivity equal to or surpassing many, and have the advantage of being directly related to use. Since chloramphenicol's decomposition products or metabolites do not possess significant antibiotic activity, only intact chloramphenicol is measured providing that no other antibiotics or chemotherapeutic agents (i.e., a fixed combination dosage form, supplemental therapy) exist. Although the basic microbiological assay procedures may be subject to this kind of interference, the problem may be obviated by selective inactivation of the interfering antibiotics by using a microorganism sensitive for chloramphenicol but resistent to the interfering antibiotic, by including a separation scheme as part of the assay sequence, or by compensating for the presence of the interfering antibiotic by adding it to each solution of chloramphenicol used for the s tandard response curve1°7. In the case of chloramphenicol, two basic microbiological methods are in general use viz Cylinder Plate and Turbidimetric

.

The Cylinder Plate Method for assay of chloramphenicol is an agar diffusion procedure using Sarcina lutea ATOC 9341 as the test organism. The response of the assay is produced by solutions of chloramphenicol in 1% phosphate buffer pH 6 diffusing through an agar layer uniformly inoculated with the test organism. To accomplish this response, stainless steel cylinders ( 8 mm o.d., 6 mm i.d.,lO mm long) are placed on the seeded agar surface and filled with the chloramphenicol solutions, and then inclubated overnight at 32OC. The responses that are produced are clear circular zones of inhibited growth around the 78

CHLORAMPHENICOL

cylinder on the agar surface otherwise totally covered with heavy growth. The dose-response relationship is a linear one in restricted limits of concentration when the dose is expressed logrithmically and the response arithmetically. In order to determine the concentration of an unknown, a reference standard must be used for comparison on each petri dishlo*. Two assay designs are commonly employed using the cylinder plate technique: the single dilution-standard curve design and a three by three (or 2 x 2) factorial designlog. The first assay design is the official method of the F.D.A. 'lo. The second has the advantage of being able to compare the parallelism of the dose response line of the standard and the unknown. The turbidimetric method determines the concentration of chloramphenicol by measuring the turbidity that is produced by the actively growing test organism in a series of test tubes containing chloramphenicol and inoculated liquid culture medium. The test is incubated in a 37OC water bath for 2 to 5 hours. After the desired incubation, the growth is stopped by the addition of formaldehyde or other appropriate means, and the responses are read in terms of absorbance on a suitable photoelectric colorimeter or spectrophotometer. By comparing the turbidity of the unknown to that of the refer nce standard, the potency of chloramphenicol is found1lP

.

Like the cylinder plate assay, the assay design may vary. The single dilution-standard curve design and 3 x 3 (or 2 x 2) factorial assay are commonly usedlog. Several different organisms have been used for the turbidimetric assay of chloramphenicol. Escherichia & ATCC 10536 is the organism used in the official method of the F.D.A. The dose-response line, log of concentration vs. response, produced by the organism is ear with a limited range of chloramphenicol concentration'". Shigella sonnei ATCC 11060 has been used for the turbidimetric assay of chloramphenicol. Because it is sensitive to lower concentrations of chloramphenicol than the plate assay, it is useful in determining chloramphenicol levels in blood serum and other clinical specimens. The dose-response line 79

DALE SZULCZEWSKI AND FRED ENG

obtained with t h i s organism i s n o t l i n e a r over the wide range of concentration f o r which i t can be used. @bacterium tumefaciens (Parke Davis c u l t u r e No. 05057) has a l s o been used i n place of 2. sonnei when a nonpathogenic organism is necessary. However, i t is not as sensitive as S -sonnei t o low levels of chloramphenicollo8.

.

I n general t u r b i d i m e t r i c techniques are f a s t e r and more e a s i l y adapted t o automated techniques. 6.6

Chromatographic 6.61

Paper The chromatographic behavior of chloramphen i c o l and r e l a t e d compounds l i k e l y t o be encountered i n metabolic s t u d i e s o r involved i n enzymatic and chemical degradation work w a s e s t a b l i s h e d by Smith112. Whatman No. .1 p a p e r was used together with a mobile phase c o n s i s t i n g of water s a t u r a t e d n-butanol containing 2.5% a c e t i c a c i d . Several reagents were used t o d e t e c t v a r i o u s compounds a f t e r chromatography. These included p-dimethylaminobenzaldehyde (arylamines), reduction with stannous c h l o r i d e , followed by p-dimethylaminobenzaldehyde (aromatic n i t r o compounds), Ninhydrin ( a l i p h a t i c amino compounds, ammonia c a l s i l v e r n i t r a t e [Formyl or Carbonyl groups]). Table 4 l i s t s Rf values and the response t o v a r i o u s d e t e c t i o n reagents. 6.62

Thin Layer Several t h i n l a y e r chromatographic systems have been developed t o study t h e v a r i o u s a s p e c t s of chloramphenicol chemistry. The procedures described h e r e have been used f o r t h e s e p a r a t i o n and i d e n t i f i c a t i o n of chloramphenicol d e r i v a t i v e s , decomposition products, and synt h e t i c intermediates. Lin113 achieved s e p a r a t i o n of chloramphenicol , chloramphenicol palmitate, and chloramphenicol s u c c i n a t e i n two solvent systems using polyamide t h i n l a y e r p l a t e s . The 4 values reported are: Solvent A 0.35 0.95 0.25

Chloramphenicol Chloramphenicol Palmitate Chloramphenicol Succinate

80

Solvent B 0.80 0.90 0.72

l

1

l

1

l

1

+

+

IS31 HOeN

IS32 ‘WN‘ON6V I

I

I

I

I

IS31 3 N l O l Z N 3 B +

I

+

+

IS31 N I W A H N I N +

u

n

e a e

U

e

*I P

C 1

l

B N

+

+

l

l

1

I

-

C

n

a

m I

I

N I

I

.- .-e 4J

-

n .- e

c

:

I .0 t 0

I

c

l

+

W Y

L

- e-

a > . r I m

.-Ec r

+

IS31 3NIW 1AUV

I

I

+

U

P)

n

t

.-e-

z c a

a 0)

I

.-

+

n

IS31 O U l N

N

ri ri

w il

br

m

v

I

N I

I

l

+ 1

l I

+

1

+

1

I

I

+

I

+

1

+

1

1

+

1

1 +

1 I

1

1

1

+

1

I

I I

I

I

I

I

I

I

I

I

I

I

I

+

I

+

+

I

z

*

e

ee

0)c u m c o

Y0)

V

.-E

0

c z

V

0

I

Y 0)

P N

U

E

4 . 8

a

D

0

a 4 -cb N

N l C

N U

N .

-.-2

s on m .- 0 b b c u .a .- L5 .n L 0 an

>. r I

0

e

z

I

*

C I

Y

e

a

c

.-

2r

r;,

n

+

I

+

+

I

I

0

3

5 n

!I

n

E

+

+

QJ

U

+

+

V

L 0

Y

n

2 I

e

+

V

w

U

U

a

U

0

.- - cz - .- 5

n

u

n

n

.-e .t .- h

9

+

I -

-

81

DALE SZULCZEWSKI A N D F R E D ENG

Solvent A Solvent B

-

n-Butanol-CHC13-acetic

- n-Butanol-water-acetic

a c i d (10:90:0.5) acid (82:18:0.5)

Schlederer114 accomplished a comparable s e p a r a t i o n using s i l i c a g e l G p l a t e s and CHC13:MeOH ( 9 : l ) as developer. The separation and i d e n t i f i c a t i o n of decomposition products of chloramphenicol by t h i n l a y e r has been done mostly on s i l i c a g e l p l a t e s using a v a r i e t y of s o l v e n t systems. T a ~ h a r m eused ~ ~ s i l i c a g e l G p l a t e s and two-dimensional chromatography t o achieve separation. The Rf values he obtained are l i s t e d as follows:

1st Solvent 2nd Solvent 0.98 ( s t r e a k ) 0.91 0.10-0.15 0.80-0.81 0.72 0.89-0.91

p-nitrobenzaldehyde p-nitrobenzoic acid Chloramphenicol l-(~-nitrophenyl)-2amino-ly3-propanediol 1st Solvent 2nd Solvent

-

0.08-0.10

0.35

e t h y l acetate ( s a t u r a t e d with water) n-Butanol ( s a t u r a t e d with 2.5% a c e t i c acid)

Shih63 66 used f i v e binary developers with s i l i c a g e l p l a t e s t o d e t e c t and i d e n t i f y s e v e r a l secondary decomposit i o n products as w e l l as t h e azoxy compound formed from photolysis (see Table 3 ) . Aromatic decomposition products (see stability-decomposit i o n ) a r i s i n g from chloramphenicol a f t e r heating i n alkal i n e s o l u t i o n were detected by Knabe65 using s i l i c a g e l G p l a t e s . The r e s u l t s he obtained are a s follows:

82

CHLORAMPHENICOL

Compound

Rf

Developer

4,4'-azodiphenol

0.25 Methylene Chloride-Ether g:1

e-n t rophenol

0.55

II

4,4 -azoxyd i pheno I

0.21

II

e-n trosophenol e-[ ( hydroxypheny1)-

0.30

fe-[( hydroxypheny1)benza 1 dehyde azofazo benzyl alcohol

II

0.15

II

0.45

II

4,41-azodibenzaldehyde 0.92 Chloroform-Ether 9:l

e- [ (a-hyd roxy-e-to1 y 1 ) azo] benza 1 dehyde

0.43

II

4,4'-azodibenzylalcohol 0.03

II

Thin layer chromatography has been used as part of a quantitative scheme of analysis. SchwarmlOl used silica gel HF254 thin layer plates to separate chloramphenicol from decomposition products. Once separation was accomplished, the intact antibiotic was desorbed and photometrically determined. CHC13-Isopropanol 4:l was used as developer. The same approach was used by Kassem115 for analysis of intact drug. In this case, silica gel plates were used with CHC13-MeOH, 85:15, as mobile phase. Libsovar116 developed thin layer chromatographic systems €or uae in monitoring the classical synthesis of chloramphenicol. For this purpose, Aluinina plates were used with binary developers of benzene-ethanol (2.5 20% ethanol).

-

6.63

Partition (Column) Intact chloramphenicol can be determined in the presence of decomposition products by using a partition c01umn''~. The column is prepared with a silicic acid-

83

DALE SZULCZEWSKI AND F R E D ENG

water as internal phase. Chloroform followed by 10% ethylacetate in chloroform serve as eluents, the eluent being monitored spectrophotometrically at 278 nm. This analytical procedure was validated by direct comparison with microbiological assay before being used to study the kinetics of degradation of chloramphenicol.

6.64 Gas Li uid d v e l o p e d a gas chromatographic procedure for analysis of chloramphenicol in the presence of structurally related compounds likely to be present in the growth medium or cell free extract of cultures of 2. venezuelae. The procedure involved chromatography of these compounds after conversion to their corresponding trimethylsilyl derivatives. The column temperature was programmed over the region 11O-26O0C and hydrogen flame ionization was used for detection. A chromatogram of a synthetic mixture of these compounds is included as Fig. 9. Resnick adapted Shaw's procedure to analysis of chloramphenicol in serumg0. In brief, this procedure involves extraction of chloramphenicol from serum with amyl acetate, derivitization using Tri-Si1 reagent followed by chromatography using Qf-1 on silanized Gas Chrom P. The sensitivity of the method was adequate for determination of chloramphenicol in the region of 0.6 mcg/ml. Y a m a m ~ t o ~ ~ ~ reported that chloramphenicol can be determined in body fluids and pharmaceutical preparations by gas chromatography without prior derivatization. Two per cent diethyleneglycol succinate on PVP modified Anakrom was used with a column temperature of 195OC. Davies"' developed a gas chromatographic method for the determination of small amounts of the ortho and meta nitro isomers of chloramphenicol as possible impurities in chloramphenicol.

84

DERIVATIVES OF 2 - 1 PHENYL-2-AUlNO-I.3PROPANEDIOL

4,IO

Com-

6

3,

m

2

v,

10

I

1.

15

R

pound

2

3

4

5 6

7 8

9

H H

n

CH ,co

OUCCO CH2FC0

(cr,)L:nco CH;CI LO CHF,CO CHCllCO

12

NO2 CH,O NOi. NO

13

NO2

CH,BrCO

CH,S@, NO: NO, NO NO

cncl,co

10

I1

14

15 16 17 18

20

H NH; NO 2 NO 2 NO 2 NO2 NO : NO

R‘

CF,CO

cnci2co C C I ,co C H B r C l CO CnBr,CO CBr,CO

25

TIWE IN UINUTES

FIG.

9

118

-

GAS CHROHAT3GRAM OF C H L O R A n P H E N l C O L AND STRUCTURALLY R E L A T E D COYFOUNDS.

-

DALE SZULCZEWSKI AND FRED ENG

References 1. The Merck Index, 8th ed., Merck and Co., Inc., Rahway, N.J. (1968), p. 233. 2. W. H. Hartung and J. Andrako, J. Pharm. Sci., 50, 805 (1951). 3. The United States Pharmacopeia, 18th revision, Mack Publishing Co., Easton, Pa. (1970). 4. J. Krc, Parke Davis, personal communication. 5. J. Krc, Parke Davis, personal comunication. 6. J. D. Dunitz, J. Am. Chem. SOC., 74, 955 (1952). 7. Q. R. Bartz, J. Biol. Chem., 172, 445 (1948). 8. Federal Register, 146 d. 301, July (1961). 9. C. Johnston, Parke Davis, personal communication. 10. H. Negoro, et al., Annual Report, Takanime Research Institute, 9, 77 (1957). 11. H. Kostenbauder and T. Higuchi, J. Am. Pharm. Assoc., Sci. Ed., 45, 518 (1956). 12. E. Regdon-Kiss, Pharmazie, 18,755 (1963). 13. M. Suzuki, J. Antibiot., Ser. B., 2 323 (1961). 14. K. James and R. Leach, J. Pharm. Pharmacol., 22, 612 (1970). 15. T. Higuchi and A. Marcus, J. her. Pharm. Ass., Sci. Ed., 43, 530 (1954). 16. A. Brunzell, J. Pharm. Pharmacol., 8, 329 (1956). 17. M. Rebstock, H. Crooks, J. Controulis, and Q. Bartz, J. her. Chem. SOC., 71, 2458 (1949). 18. M. Suzuki and H. Shindo, Yakugaku Zasshi, 76, 927 (1956). 19. 0. Jardetsky, J. Biol. Chem., 238, 2498 (1963). 20. D. Szulczewski, unpublished information. 21. L. A. Mitscher, F. Kautz, and J. Lapidus, Can. J. Chem., 47, 1957 (1969). 22. S. C. Ray, Ann. Biochem. Exp. Med., 23, 411 (1963). 23. C. Brunnee, G. Kappus, and K. H. Maurer, Z . Anal. Chem., 232 17 (1967). 24. U. S. Pat. 2,438,871 25. U. S. Pat. 2,483,892 26. J. Controulis, M. Rebstock, H. Crooks, J. her. Chem. SOC., 71, 2463 (1949). 27. L. M. Long, H. D. Troutman, J. her. Chem. SOC., 71, 2469 (1949). 28. ibid 71, 2473, (1949). 29. ibid 73, 481 (1951). 86

CHLORAMPHENICOL

30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

i b i d 73, 542 (1951). U. S . P a t . 2,681,364 U. S. P a t . 2,687,434 U. S. P a t . 2,562,107 U. S. P a t . 2,515,239 U. S. P a t . 2,515,240 U. S. P a t . 2,515,241 U. S. P a t . 2,546,762 U. S. P a t . 2,483,885 U. S. P a t . 2,692,897 U. S. P a t . 2,677,704 U. S. P a t . 2,538,763 U. S. P a t . 2,515,377 U. S. P a t . 2,686,788 U. S. P a t . 2,751,413 U. S. P a t . 2,543,957 U. S. P a t . 2,483,884 U. S. P a t . 2,699,451 G. Moersch, D. Hylander, J. h e r . Chem. SOC., 76, 1703 (1954). 49. S. Igiguma, Yakugaku Z a s s h i , 75, 673 (1952) 50. M. Myamoto, i b i d , 72, 673 (1952) 51. U. S. P a t . 2,562,113 52. U. S. P a t . 2.718,520 53. U. S. P a t . 2,807,645 54. U. S. P a t . 2,562,114 55. U. S. P a t . 2,734,919 56. U. S. P a t . 2,727,063 57. U. S. P a t . 2,586,661 58. M. Suzuki, J. A n t i b i o t . , Ser. B., 14,323 (1961). 59. T. Higuchi and C. Bias, J. Amer. Pharm. Ass., S c i . Ed., 42, 707 (1953). 60. T. Higuchi, A. Marcus, and C. Bias, i b i d , 43, 129 (1954). 61. C. Trolle-Lassen, Arch. Pharm. Chemi., 60, 689 (1953). 62. R. Saba, D. Monnier, and F. R. Khalil, Pharm. Acta. Helv. , 42, 335. 63. K. K. Shih, J. Pharm. S c i . , 60, 786 (1971). 64. J. Lacharme and D. Netien, Bull. Trav. SOC. Pharm. Lyon. 4, 122 (1964). 65. J. Knabe and R. K r a u t e r , Arch. Pharm., (1962) 190. 66. I. K. S h i h , J. Pharm. S c i . , 60, 1889 (1971). 67. A. B r u n z e l l , Sv. Farm. T i d s k r . , 61, 129 (1957).

87

D A L E SZULCZEWSKI A N D F R E D E N G

68. 69. 70.

71. 72. 73. 74. 75. 76.

77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94.

M. Heward, D. A. Norton, and S. M. Rivers, Pharm. J., 386 (1970). B r i t i s h Pharmaceutical Codex 1968 London, The Pharmac e u t i c a l P r e s s , 1968. K. C. James and R. H. Leach, Pharm. J., 204, 472 (1970) G. Smith and C. Worrel, Arch. Biochem., 28, 232 (1950) A. Glazko, Antimicrob. Ag. Chemother., (1966) 655. W. A. D i l l , E. M. Thompson, R. A. F i s k i n , and A. J. Glazko, Nature, 185, 535 (1960). A. Glazko, W. A. D i l l , A. Kazenko, L. M. Wolf, and H. E. Carnes, A n t i b i o t . Chermother., 8, 516 (1968). A. J. Glazko, W. A. D i l l , and L. M. Wolf, J. Pharmacol. Exp. Ther., 104,452 (1952). A. J. Glazko, L. M. Wolf, W. A. D i l l , and A. C. B r a t t o n , Jr., i b i d , 96, 445 (1949). A. J. Glazko, A. W. Kinkel, W. C. Alegnani, and E. L. Holmes, C l i n . Pharmacol. T h e r . , 2, 472-483(1968) A. J. Aguiar, L. M. Wheeler, S. F u s a r i , and J . Zelmer, J . Pharm. S c i . , 57, 1555 (1968).

204,

United S t a t e s Pharmacopeia XVI. 1960 B r i t i s h Pharmacopeia 1968. Deutsche Apotheker. 5 D b l l , Arzeim. Forsch., 5, 97 (1955). A. Glazko, L. Wolf, and W. D i l l , Arch. Biochem., 23, 411 (1949) S. P. Bessman and S. Stevens, J. Lab. C l i n . Med., 35, 1 2 7 (1950). J. Levine and H. Fishback, A n t i b i o t . Chemother., 1, 59 (1951). T. H. Aihara, H. Machida, and Y. Yoneda, J. Pharm. SOC. J a p . , 77, 1318 (1957). M. S. Karawya and M. G. Ghourab, J. Pharm. S c i . , 2, 1331 (1970). K. Kakemi, T. A r i t o , and S. Ohasaki, Yakugaku Z a s s h i , 82, 342 (1962). D. Hughes and L. K. Diamond, Science, 144,296 (1964) G. L. Resnick, D. Corbin, and D. H. Sandberg, Anal. Chem., 38, 582 (1966) R. C. Shah, P. V. Raman, and P. V. Sheth, I n d i a n J. Pharm., 30, 68 (1968). F. M. Freeman, Analyst, 80, 299 (1956). D. Masterson, J. Pharm. S c i . , 57, 306 (1968). K. Fossdal and E. Jacobson, Anal. Chim. Acta., 56, 105 (1971)

88

CHLORAMPHENICOL

G. B. Hess, Anal. Chem., 22, 649 (1950). C. G. Macros, Chem. Chron. A , , 32 104 (1967). C. Russo, I. Cruceanu, D. Monciu, and V. Barcaru, 11. Farmaco, 20, 22 (1965). 98. C. Russu, I. Cruceanu, and V. Barcaru, Pharmazie 2, 799 (1963). 99. A. F. Summa, J. Pharm. S c i . , 54, 442 (1963). 100. P Zuman, "Organic Polarographic Analysis", MacMillan Co., New York, 1964, p. 186. 101. E. Schwarm, C. Dabner, J. Wilson, and M. Boghosian, J. Pharm. S c i . 55, 744 (1966). 102. T. Higuchi, A. D. Marcus, and C. D. Bias, J. Amer. Pharm. A s s . , S c i . Ed., 43, 135 (1959). 103. W. Awe and H. Stohlman, Arch. Pharm., 289, 61, 276 (1956). 104. M. Hadicke and G. Schmid, Pharm. Z e n t r a l h . , 95, 387 (1956). 105. W. Awe and H. Stohlman, Arzeim. Forsch., 7 (81, 495 (1959). 106. D. C. Grove and W. A. Randall, "Assay Methods of Antib i o t i c s : A Laboratory Manual", 238 pp Medical Encyclopedia, I n c . , New York, 1955 107. B. Arret, M. R. Woodard, D. M. Wintermere, and A. Kirschbaum, A n t i b i o t . Chemother., 1 545 (1957). 108. R. Hans, M. G a l b r a i t h , and W. C. Alegnani, "Analyt i c a l Microbiology," F. Kavanagh, Ed., Academic P r e s s , 1963, p 271-281. 109. United S t a t e s Pharmacopeia XVIII, 1970, pp 857-864, Antibiotics-Microbial Assays. 110. F.D.A. Regulations T i t l e 2 1 Sec. 141.110. 111. F.D.A. Regulations T i t l e 2 1 Sec. 141.111. 112. G. N. Smith and C. S. Worrel, Arch. Biochem., 28, 1 (1950) 113. Y. T. Lin and K. T. Wang, J. Chromatogr., 21, 158 (1966). 114. E. S c h l e d e r e r , Cosm. Pharma., 2, 1 7 (1966). 115. M. A. Kassem and A. A. Kassem, Pharm. Ztg., 48, 1972 (1966). 116. J. Lebsovar, Cesk. Farm. 11,73 (1962). 117. T. Higuchi, C. Bias, and A. Marcus, J. Amer. Pharm. Ass. , S c i . Ed., 43, 135 (1954). 118. P. D. Shaw, Anal. Chem., 3 5 , 1580 (1963). 119. M. Yamamoto, S. I g u c h i , and T. Aoyama, Chem. Pharm. Bull. , 15, 123 (1967). 95. 96. 97.

.

.

89

DALE SZULCZEWSKI AND FRED ENG

120.

V. Davies, Parke Davis, personal communication.

ACKNOWLEDGMENT

The authors express appreciation to Mrs. Pat Greenwood of the Microbiology Department at Parke, Davis & Company for assistance in preparing a portion of this profile.

90

CLORAZEPATE DIPOTASSIUM

James A . Railile and Victor E. Papendick

JAMES A. RAIHLE AND VICTOR

E. PAPENDICK

Contents Analytical Profile 1.

Description 1.1 1.2

2.

- Clorazepate Dipotassium

Name, Formula, Molecular Weight Appearance, Color, Odor

Physical Properties 2.1 2.2 2.3 2.4

Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Raman Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Solubility 2.9 2.10 Crystal Properties 2.11 Dissociation Constant 2.12 Fluorescence 2.13 Hygroscopic Behavior

3.

Synthesis

4. Stability

-

Degradation

5.

Drug Metabolic Products and Pharmacokinetics

6.

Methods of Analysis 6.1 6.2

6.3

6.4 6.5 6.6

Elemental Analysis Phase Solubility Analysis Chromatographic Analysis 6.31 Thin Layer Chromatographic Analysis 6.32 Gas Liquid Chromatography Direct Spectrophotometric Analysis Colorimetric Analysis Non-Aqueous Titration

7, References

92

CH LORAZEPATE DI POTASSI UM

1.

Description

1.1

Name, Formula, Molecular Weight Clorazepate dipotassium is 7-chloro-1,3-dihydro2-oxo-5-phenyl-1H-l,4-benzodiazepine-3-carboxylic acid, monopotassium salt, monopotassium hydroxide.

0

11

H

0

\ C=N

CI

C16H1 1°4N2C1K2

II

CHCOK. KOH

Clorazepate Dipotassium Molecular Weight 408.93

1.2

Appearance, Color, Odor Off white to pale yellow, fine crystalline powder which is practically odorless. 2.

Physical Properties 2.1

Infrared Spectrum The infrared spectrum of clorazepate dipotassium is presented in Figure i. The spectrum was measured in the solid state as a mull in mineral oil. The following bands (cm-1) have been assigned for Figure 1. (1) a. 3530 cm-l characteristic for hydroxyl b. 1610 cm-1 characteristic skeletal stretching modes of the aromatic ring c. 1560 cm-1 characteristic C=O stretching mode of the carboxyl salt 2.2

Nuclear Magnetic Resonance Spectrum (NMEl) The NMR spectrum shown in Figure 2 was obtained by dissolving 50 mg of clorazepate dipotassium in 0.5 ml of D20 containing tetramethylsilane as an internal reference. Only the aromatic protons between 7.0 and 7.6 ppm are visible. (2) 93

4c

P

Figure 2 NUCLEAR MAGNETIC RESONANCE SPECTRUM OF CLORAZEPATE DIPOTASSIUM

I . . 8.0

. , . . . .

I I I I . . . . I . . . . I I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. _. ,. .. 7 .O

6.0

5.0

CCM(

d

)

4.0

3.0

2.0

I .o

. .

JAMES A. RAIHLE AND VICTOR E. PAPENDICK

2.3

U l t r a v i o l e t Spectrum C l o r a z e p a t e d i p o t a s s i u m when scanned between 400 and 200 nm i n 0.03% aqueous potassium c a r b o n a t e e x h i b i t s a maximum a t 230 nm a s shown i n F i g u r e 3 , (c = 35,000) chara c t e r i s t i c of benzodiazepines. 2.4

Mass Spectrum The mass spectrum shown i n F i g u r e 4 was o b t a i n e d u s i n g an Associated E l e c t r i c a l I n d u s t r i e s Model MS-902 Mass Spectrometer w i t h a n i o n i z i n g energy o f 50 eV and a tempera t u r e of 185°C. C l o r a z e p a t e d i p o t a s s i u m y i e l d s a spectrum w i t h t h e base peak a t m / e 270 a t t r i b u t e d t o t h e decarboxyl a t i o n of t h e a c i d s a l t . Subsequent fragments, Table I and F i g u r e 5, r e f l e c t t h e l o s s of p a r t s of t h e seven membered ring o r chlorine.(3) The mass spectrum p a r a l l e l s t h a t r e p o r t e d f o r diazepam. (4) 2.5

Raman Spectrum The Raman spectrum of c l o r a z e p a t e d i p o t a s s i u m , a s shown i n F i g u r e 6 , was o b t a i n e d i n t h e s o l i d s t a t e on a Ramalog Spectrophotometer a t Spex I n d u s t r i e s . The followi n g bands (cm-l) have been a s s i g n e d f o r F i g u r e 6 . ( 1 ) a. 1595 cm-l s k e l e t a l s t r e t c h i n g mode of a r o m a t i c r i n g C=N s t r e t c h i n g v i b r a t i o n o f t h e h e t e r o b. 1565 cm-l cyclic ring c. 1495 cm'l s k e l e t a l s t r e t c h i n g mode o f a r o m a t i c r i n g

2.6

Optical Activity C l o r a z e p a t e d i p o t a s s i u m e x h i b i t s no o p t i c a l ac-

tivity.

2.7

Melting Range C l o r a z e p a t e d i p o t a s s i u m does n o t have a d e f i n i t e m e l t i n g range. Typical b e h a v i o r when t h e m a t e r i a l i s slowl y h e a t e d i n a g l a s s c a p i l l a r y t u b e may be d e s c r i b e d i n t h e following manner: d i s c o l o r a t i o n b e g i n s a t about 215"C, s h r i n k i n g i s observed t o b e g i n between 225OC t o 235°C w i t h t o t a l decomposition o c c u r i n g between 235°C and 295°C.

2.8

D i f f e r e n t i a l Thermal A n a l y s i s (DTA) The DTA curve o b t a i n e d on a DuPont Model 900 Anal y z e r a s shown i n F i g u r e 7 confirms t h e observed m e l t i n g c h a r a c t e r i s t i c s d e s c r i b e d i n s e c t i o n 2.7.

96

CH LORAZEPATE D IPOTASS IUM

FIGURE 3. ULTRAVIOLET SPECTRUM O F CLO R A 2 EPATE DIPOTASSI UM

I

I

1

250 300 WAVELENGTH (nm)

97

350

Figure 4 MASS SPECTRUM OF CLORAZEPATE DIPOTASSIUM

W

00

20

40

60

80

100

120

140

160

180

ZOO

220

240

260

280

vl

m

UI

d

d

r

o 0

0

o

N

o

0

l

0

r

0

o

0

l

0

r

rl

l

0

0

r

rl

0

l

rl

d

0

I4

m

rl

I-

rl

co

N

m

N

ul

d

rl rl

N

0 rl

rl

rl

rl

03 ul

m *

m *

co m

00

I-

Ih

I-

0

a\

0 I-

h rl

I-

rl

I-

0

h

m

rl

\o

0

m h

*

rl

h

m

0

h

m

m

rl

m

m N

N

2

*0

d

m I4

ul

N

m

0

03

I-

rl

rl

0

N

IN

U 0

Irl

ul 0

ul

Id

h

0

m

d

4

* 2

0

N I-

m N

2 4 *

N

ul

0

m

2

co

rn

0 rl

rn

rl

rl

*co0 m

\D N

u l

I-

*0

rl

hl

N

N

* 3

99

CH LORAZEPATE DlPOTASSlUM

FIGURE 5. FRAGMENTATION PATHWAYS OF CLORAZEPATE DIPOTASSII M

-

0

Probably on the probe

CI

McLafferty Rearrangement Before or After Ion Formation

m/e 314

..

not observed

co, m/e 44 Bare Peak

/-".

lC1

m h 270

*

m/e 235

m/e 242

rFk

m/e 214

/-co

\C-

m/e 179''

100

L

-3

-3

101

OOL

m OW

m 009

OOL

008

006

0001

001I

OOL I OOEl

oopl

0091

OOLL

FIGURE 7. DIFFERENTIAL THERMAL ANALYSIS CURVE OF CLORAZEPATE DIPOTASSIUM

c

0

h)

O

m

0

25

100 175 250 325 TEMPERATURE; DEGREES CENTIGRADE

400

CHLORAZEPATE DIPOTASSIUM

2.9

Solubility Approximate s o l u b i l i t y d a t a o b t a i n e d a t room t e m p e r a t u r e a r e given i n t h e f o l l o w i n g t a b l e : S o l u b i l i t y (mg/ml)

Solvent Water Absolute Ethanol Chloroform Ether Acetone Benzene Isopropanol Methylene D i c h l o r i d e

> 100 < 200 0.6 0.5 0.5 0.5 0.5 0.7 < 0.1

< < c <

2.10

Crystal Properties The X-ray powder d i f f r a c t i o n p a t t e r n o f c l o r a z e p a t e d i p o t a s s i u m was determined by v i s u a l o b s e r v a t i o n o f a f i l m o b t a i n e d w i t h a 143.2 mm Debye-Scherrer Powder Camera. An Enraf-Nonius D i f f r a c t i s 601 G e n e r a t o r ; 38 KV and 18 MA w i t h n i c k e l f i l t e r e d copper r a d i a t i o n ; A = 1.5418, were employed. (5)

2.11

D i s s o c i a t i o n Constant Attempts t o measure t h e pKa of t h e carboxyl group by t i t r a t i o n i n water w i t h h y d r o c h l o r i c a c i d were unsucc e s s f u l . Only t h e potassium hydroxide which i s l i b e r a t e d on d i s s o l v i n g c l o r a z e p a t e d i p o t a s s i u m i n w a t e r i s t i t r a t e d . (6)

2.12

Fluorescence C l o r a z e p a t e d i p o t a s s i u m does n o t e x h i b i t f l u o r e s c e n t p r o p e r t i e s i n an aqueous s o l u t i o n , however, i t does e x h i b i t f l u o r e s c e n c e a t 508 nm when e x c i t e d a t 388 nm i n 9 -N a l c o h o l i c s u l f u r i c a c i d . ( 7 ) 2.13

Hygroscopic Behavior Clorazepate d i p o t a s s i u m was n o t hygroscopic when exposed t o a r e l a t i v e humidity of 30%-40% f o r 4% months.

3.

Synthesis Clorazepate d i p o t a s s i u m may be prepared by t h e r e a c t i o n scheme shown i n F i g u r e 8 w i t h t h e r e a c t i o n of (2-amino-5chloropheny1)phenyl methane imine and d i e t h y l 2-amino malo n a t e t o form e t h y l 7-chloro-1,3-dihydro-2-oxo-5-phenyl-lH103

JAMES A. RAIHLE AND VICTOR E. PAPENDICK

TABLE I1 X-Ray Powder Diffraction Pattern d-Spacings and Intensities dA 17.5 8.6 7.7 7.0 6.18 5.7 5.05 4.82 4.35 4.20 4.11 4.05 3.95 3.80 3.66 3.52 3.48 3.40 3.30 3.25 3.14 3.06

-111 1

-III 1

100 10 25 10 20 20 30 75 10 5 1 1 5 5 25 40 1 10 60 65 1 15

2.99 2.93 2.88 2.82 2.78 2.67 2.62 2.57 2.46 2.42 2.27 2.17 2.13 2.07 2.03 1.99 1.97 1.93 1.87 1.81 1.78 1.71

104

5 1

2 10 3 5 5 1 10 2 1

5 2 1 1 1 1 1 1 1 1 1

CH LORAZEPATE DlPOTASSl UM

FIGURE 8. SYNTHESIS OF CLORAZEPATE DIPOTASSIUM //O C-OCzH, I

H,N-CH CI

I

C=NH

(2-Amino-5-chlorophenyl) Diethyl-2-aminomalonate phenyl methane imine

Ethyl 7-cidoro-l,3-dihydro-2-oxo5-phenyl-1H-1,4-benzodiazpine-3car boxylate

Potassium Hydroxide

/p \ CHC02K CI

Clorazepate Dipotassium 105

*

KOH

JAMES A. RAIHLE AND VICTOR E . PAPENDICK

lY4-benzodiazepine-2-carboxylate. This intermediate is then converted to the drug substance with alcoholic potassium hydroxide. (8)

4. Stability

-

Degradation The hydrolysis products for clorazepate dipotassium are shown in Figure 9. The final hydrolysis product is the same as that reported for the acid hydrolysis of clordiazepoxide and the major metabolite of diazepam.(4) The kinetics of the decomposition of clorazepate dipotassium in buffered aqueous solution at different temperatures was studied over the pH range of 2-11. (9) The extent of degradation was determined by a dichloromethane extraction and spectrophotometric measurement. Ring opening was negligible under the conditions of pH and temperature used. The degradation to N-desmethyl diazepam is first order with respect to clorazepate dipotassium. The relationship log, (Ct/Co) = Kt was verified where Ct is the concentration of clorazepate dipotassium at time t, Co is the concentration of clorazepate dipotassium at time 0, and k is the reaction rate constant. Plots of loge (Ct/Co) as a function of time t were linear. The slopes of these plots give the reaction rate constants k. Some rate constants, sec-1, at different pH and temperature values are shown in Table 111. The data shows that in the degradation of clorazepate dipotassium the reaction rate constant, k, increases with temperature. The activation energy of the degradation reaction, Ea (obtained from the slope of the plot of loge k as a function of 1/T where T is the absolute temperature), is about 20.3 kilocalories/mole and is independent of pH.

5.

Drug Metabolic Products and Pharmacokinetics The major metabolites of clorazepate dipotassium are shown in Figure 10. The drug substance is decarboxylated in acidic media to N-desmethyl diazepam, which can undergo hydroxylation to oxazepam and conjugation in the urine to the glucuronide. (10) Some clorazepate dipotassium (2-6%) is excreted into the urine unmetabolized.(ll) Acid hydro1 ysis of the drug substance and its metabolites affords 2amino-5-chlorobenzophenone. Analytical procedures have been published for the metabolites using gas chromatography (12), thin layer chromatography ( 7 ) , high pressure 106

TABLE I11

Rate Constant vs pH f o r C l o r a z e p a t e Dipotassium t oc+

PH

f

c 0

4

2.0 3.0 4.0 5.0 6.0 6.5 7.0 7.5 8.0 8.5 9.5 11.0

2 2" -

3. 60X10'3 3.40~10-~ 1. 16X10-3 2.17~10-4 2.23xlO-5 3.9ox10-6 2. 33X10-6 1. 05X10'6

---

1. 05X10'6 9.10~10-7 8.64XlO-7

27.5" 6. 46X10-3 5.96~10-~ 1.98~10'~ 4.10~10-4

---

------- --------

37" 1. 93X10'2 1.42xlO-2 6.33~10'~ 1.14~10-3 1. 18X10'4 8. 33X10'5 2.14xlO-5 1.32~10'5 1. 27X10'5 5. 61X10'6 4. 83X10'6 4. 55X10m6

JAMES A. RAIHLE AND VICTOR E. PAPENDICK

FIGURE 9. DEGRADATION PATHWAYS OF CLO RAZEPAT E D IPOTASSI UM

Clorazepate Dipotassium 0 NH-C,

II

CHZ

+

KzCO,

CI

N-Desmethyl Diazepam

CI

a:b II

0

II

+

H2NCHZCOH

-

0

2-Amino-5-chlorobentophenone 108

Glycine

FIGURE 10 METABOLIC PATHWAYS 0 F CLOR AZEPATE D IPOTASSI UM 0 -

II

H

CHCOK CI

C= N

Clorazepate Dipotassium

KOH

-

Strong AcidHydrolysis

CI

aNH' H,NCHZCOOH

c=o

0 I

2-Amino-5-chlorobenzophenone + Glycine

c

0 \D

0 0

H

II

\

Hydroxylation CI

CI

CHOH

C=N

C=N

N-Desmethyl Diazepam

Oxazepam

JAMES A. RAIHLE AND VICTOR E. PAPENDICK

liquid chromatography (13) and colorimetry.(l4)

6. Methods of Analysis 6.1

Elemental Analysis

E1ement Carbon Hydrogen NF trogen Chlorine Potas sium

*

% Theory

Typical Result Reported for Lot 849-648

46.99 2.71 6.85 8.67 19.12

46.82 2.65 6.95 8.85 19.48, 19.34*

Values determined by atomic absorption analysis

6.2

Phase Solubility Analysis Data is not available due to the instability of clorazepate dipotassium in the solvent systems screened. 6.3

Chromatographic Analysis 6.31

Thin Layer Chromatography Clorazepate dipotassium is partially degraded in numerous systems to N-desmethyl diazepam, therefore, thin layer chromatography is not considered a reliable indicator of purity. The drug substance is readily identified by ultraviolet light on Silica Gel GF254 in the system methano1:acetone (1:l). Clorazepate dipotassium has an Rf value of 0.15 while N-desmethyl diazepam has an Rf value of 0.90. LaFargue, et a1 (7) differentiated clorazepate dipotassium from other 1,4-benzodiazepines in urine and gastric fluid by hydrolysis to 2-amino-5-chlorobenzophenone and subsequent thin layer chromatography on aluminum oxide in the system benzene:chloroform (3:l). The Rf value of the hydrolysis product is 0.55. If benzophenone is used as a reference standard, the relative Rf value of 2-amino-5-chlorobenzophenone is 0.79.

110

CH LORAZEPATE DiPOTASSl UM

Clorazepate may be directly extracted from gastric fluid as N-desmethyl diazepam and identified on aluminum oxide in the system ch1oroform:ethanol (29:l). The absolute Rf is reported as 0.39 or 0.48 relative to a diazepam reference standard.(7) 6.32

Gas Chromatography Clorazepate dipotassium can not be directly chromatographed, however, gas chromatography is readily applied to the metabolites and acid hydrolysis products.(7, 12) LaFargue (15) has employed 3% OV-17 (methylphenylsilicone) on Gas Chrom Q to separate 2-amino-5-chlorobenzophenone from the acid hydrolysis products of five major benzodiazepins.

6.4

Direct Spectrophotometric Analysis Direct spectrophotometric analysis of clorazepate dipotassium is applicable provided significant quantities of interfering contaminants are not present. The drug substance may be examined directly in an aqueous carbonate buffered media at 230 nm ( 6 = 35,000) or indirectly in alcoholic sulfuric acid by ultraviolet absorption (16) at 388 nm or fluorescence (7) with an excitation maximum of 388 nm and an emission maximum at 508 nm. The degradation products may be quantitated in the drug substance by a solid-liquid extraction into dichloromethane. The solvent is separated from the drug and evaporated. The residue is redissolved in alcohol and compared to N-desmethyl diazepam at 230 nm. 6.5

Colorimetric Analysis Clorazepate dipotassium and its metabolites may be determined by the Bratton-Marshall reaction after hydolysis to 2-amino-5-chlorobenzophenone.(l4) 6.6

Non-Aqueous Titration Clorazepate dipotassium may be potentiometrically titrated in glacial acetic acid using perchloric acid in glacial acetic acid and glass-calomel (0.1 LiC104 in HOAc) electrodes. Each ml of 0.1 fi HC1O4 is equal to 136.31 mg of clorazepate dipotassium.

111

JAMES A. RAIHLE AND VICTOR E. PAPENDICK

7.

References 1. 2.

3.

4.

5.

6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16.

Washburn, W., Abbott Laboratories, Personal Communication. Egan, R., Abbott Laboratories , Personal Comunication. Mueller, S . , Abbott Laboratories, Personal Communication. MacDonald, A., Michaelis, A. R., and Senkowski, B. Z., "Analytical Profiles of Drug Substances," Vol. I, K. Florey, Ed., Academic Press, New York, (1972). Quick, J . , Abbott Laboratories, Personal Communication. Wimer, D. C., Abbott Laboratories, Personal Communica tion. LaFargue, P., Meunier, J., and Lemontey, Y. , J. Chromatog., 62, 423, (1971). Schmitt, J., (Establissements Clin-Byla, 4306CB) U. S. Patent 3,516,988. Raveux, R., and Briot, M. , Chem. Therap., 4 , 3 0 3 , (1969). Beyer, K. , Deut. Apoth. Ztg., 111, 1503, (1971). LaFargue, P., Pont, P., and Meunier, J . , & Pharm. Fran., 28, 343, (1970). Viala, A . , Cano, J. P., and Angeletti-Philippe, A . , J. Eur. Toxicol. , 2, 109, (1971). Scott, C. G. , and Bomer, P. , J. Chrom. Sci. , 8 , 446, (1970). Gros, P., and Raveux, R., Chim. Ther., 4 , 312, (1969). Ibid 11, 28, 477, (1970). Laguleau, J . , Crockett, R., and Mesnard, P., Bull. SOC. Pharm. Bordeaux, 110, 10, (1971).

112

CLOXACILLIN SODIUM

David L. Mays

D A V I D L. MAYS

TABLE OF CONTENTS 1.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance

2.

Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectra 2.4 Mass Spectra 2.5 Cryst a1 Properties 2.6 Melting Range 2.7 Thermal Analysis 2.8 Solubility 2.9 Ionization Constant, pKa 2.10 Optical Rotation

3.

Synthesis and Purification

4.

Stability

5.

Methods of Analysis 5.1 Analysis of Impurities 5.2 Identification Tests 5.3 Quantitative Analysis 5.31 Volumetric Methods 5.32 Colorimetric Methods 5.33 Polarography 5.34 Gas Chromatography 5.35 Infrared Spectroscopy 5.36 Optical Rotation 5.37 Biological Methods 5.38 Automated Methods 5.4 Thin-layer Chromatography

6.

Protein Binding

7.

Metabolism

8.

References

114

CLOXACILLIN SODIUM

1.

Description 1.1 Name, Formula, Molecular Weight

Sodium cloxacillin is found in Chemical Abstracts under 4-Thia-1-azabicyclo [3.2.0] heptane-2carboxylic acid, 6-[[[3-(2-chlorophenyl)-S-methy1-4isoxazolyl] carbonyl] amin0]-3,3-dimethyl-7-0~0-, monosodium salt (1). It is more comonly known as 3-0chlorophenyl-5-methyl-4-isoxazolyl penicillin sodium salt (2).

Molecular Weight 457.89

C H C1N3Na05S 19 17 1.2 Auuearance

Sodium cloxa illin is a white, odorles crystalline powder (3). 2.

Physical Properties 2.1 Infrared Spectra

Infrared absorption frequencies were reported for oil suspensions of cloxacillin and other penicillins (3a). An infrared spectrogram of sodium cloxacillin monohydrate obtained on a Perkin-Elmer Model 21 Spectrophotometer is included in the compilation of Wayland and Weiss (4). A spectrogram of Bristol Laboratories Primary Reference Standard recorded as a potassium bromide disk using a Beckman Model IR9

115

Figure 1

Infrared absorption spectrum of sodium cloxacillin monohydrate

CLOXACILLIN SODIUM

Spectrophotometer is shown in Figure 1. Characteristic absorption frequencies (cm-l) are as follows: a. H20: 3519 b. N-H stretching band: 3370 c. beta-lactam carbonyl: 1770 d. secondary amide carbonyl: 1669 e. aromatic ring: 1619 f. carboxylate carbonyl: 1604 Within the carbonyl stretching region, the beta-lactam frequency is most characteristic of penicillins. Opening of the beta-lactam can be indicated by changes in this part of the spectrum. 2.2

Nuclear Magnetic Resonance Spectra

Proton nuclear magnetic resonance spectra for a number of penicillins were reported by Pek and coworkers (5) and the chemical shifts useful for identification were tabulated. An nmr spectrogram of Bristol Laboratories Primary Reference Standard sodium cloxacillin monohydrate as obtained on a Varian HA-100 spectrometer is shown in Figure 2. Proton resonance lines were measured in D20 solution with deuterated sodium trimethylsilyl propionate as the internal reference. Structural assignments are as follows: Chemical Shift, (Coupling Colrstant, Hz)

Assignment aromatic 6-H 5-H

3-H 5-CH3 (isoxazole) 2-B-CH3 2-a-CH3 2.3

7.50 5.62 5.46 4.81 2.63 1.43 1.39

m d (5-4.0) d (J=4.0) s s s s

Ultraviolet Spectra

The ultraviolet spectrum of an aqueous solution of sodium cloxacillin monohydrate obtained on a Cary-14 spectrophotometer is shown in Figure 3. An absorption maximum at 194 nm and a weak shoulder with

I17

Figure 2

NMR spectrum of sodium cloxacillin monohydrate

CLOXACILLIN SODIUM

Figure 3 Ultraviolet absorption spectrum of sodium cloxacillin monohydrate

119

DAVID

L. M A Y S

maxima at 267 and 273 nm are observed. The effect of halogen substitution on the phenyl ring of 5-methyl-3phenyl isoxazole has been discussed by Doyle (6). 2.4

Mass Spectra

With few differences the mass spectrometric behavior of cloxacillin methyl ester (Figure 4, Table I) follows the fragmentation pattern deduced by Richter and Biemann (7) from high resolution mass measurements. These fragmentations have previously been discussed in this series (8,9). The presence of the isoxazole ring in the acyl moiety results in diagnostically useful peaks at m/e 43, 178, 193, and 220. M/e 220 is presumed to result from cleavage of the amide bond. The origin of the other three peaks follows from the discussion of the mass spectra of isoxazoles by Ohashi and coworkers (10). 2.5

Crystal Properties

Sodium cloxacillin is a microcrystalline powder exhibiting birefringence and extinction positions under a light polarizing microscope (11-13). 2.6

Melting Range

Sodium cloxacillin melts with decomposition at 170' (6,ll). Cloxacillin free acid melts with decomposition at 126-127' (14). 2.7

Thermal Analysis

Differential thermal analysis curves of sodium cloxacillin monohydrate and cloxacillin free acid were recorded over a range of 0 to 250' on a Perkin Elmer Differential Scanning Calorimeter Model DSC-1B (15). No significant transitions were observed with cloxacillin free acid; decomposition appeared to occur over several broad temperature ranges. With sodium cloxacillin, broad endothermic transitions with peak temperatures at 176' and 193' were recorded. Dehydration probably occurred during the first transition, since no loss of water was observed at lower temperature. 120

Figure 4 Mass spectrum of cloxacillin methyl ester

TABLE I Low Resolution Mass Spectrum of Cloxacillin Methyl Ester

h)

w

m/ e 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2s

0

1

2 .07

3 .05

.98 .72 1.02 .47 1.10 .2S .20 1.63 .34 .07 .28 .54 4.55 .21 .21 10.68 .05 .12 .05 9.27 .07 .07

.36 3.25 1.19 .38 .77 1.12 .ll .54 1.99 .07 .08 .45 .72 .08 .40 1.40 .05 .42 .05 1.24 .03 .15

.26 2.20 .SO .36 .34 1.25 .07 1.29 .90 .07 .03 3.05 1.71 .16 .22 .18 .18 .12 .10 3.41 .05 .21

.18 48.29 2.85 .54 .88 1.82 .10 .44 2.15 1.14 .05 .49 .31 -23 .65 .08 2.57 .06 2.93 .47 .23 .13

-07

4 .23 .06 .36 2.20 .94 .25 1.41 .61 .26 .20 14.35 .42 .05 1.24 .28 .21 100.00 .08 .36 .06 .45 .21 2.28

5 .10 .10 -24 2.52 3.09 .24 4.96 .88 .31 .08 2.00 .63 .08 .15 .20 .16 9.59 .23 .85 .10 .13 .23 .41 .06

6 .03 .62 1.36 .43 .73 .25 .90 1.15 1.16 .08 1.06 1.02 .12 .21 .24 .08 5.24 .55 .13 .10 .07 .05 .76 .05

7 .16 2.28 .24 .80 .35 .60 1.20 3.58 .72 .08 .23 .68 1.38 .07

.ll

.05 1.54 .20 .10 .06 .10 .24 .20 .48

8

9

4.59 .52 .13 .60 1.01 .21 .78 .37 .15 .07 1.37 .72 .07 .48 .08 30.89 .18 .07 .06

1.86 2.44 .18 6.31 1.01 .ll .56 1.56 .21 .07 .31 1.14 .23 .13 .20 3.41 .10 .32 .04 .31 .05 .03 .21

.I1

.08 .07 .14

m/ e 26 27 28 29 30

0

.05 .18

1 13.30 .03

2 2.11 -07

3 4.63 .07

TABLE I (cont.) 4 5 .70 .07

ZA

+ h,

37 38 39 40 41

.29

-08

.ll

7

8

9

2.11

.44 -07

.76 - 50

.16 -.11

.63

.12

.03

.10

.ll

1.71

.31

.32

31 32 33

6

~~

.ll

.02 -09

-

43

44 45

.35

Base Peak:

.60

-16

.05

m / e 174 = 100.00% Relative Abundance

1.44

D A V I D L. M A Y S

2.8

Solubilitv

Sodium cloxacillin is very soluble in cold water (11,12,16). The ratio of sodium cloxacillin concentration in chloroform versus pH 6 buffer solution was determined to be 0.118 (17). The solubility of penicillin salts in nonpolar solvents is significantly increased by the presence of a small amount of water, even water of hydration (18). The tabulation of sodium cloxacillin solubility shown below is taken from Marsh and Weiss (19).

So1vent

sdulu;;f ty

Water

> 20

Methanol Ethano 1

> 20 > 20

Isopropanol Isoamyl alcohol Cyclohexane

9.158 5.865 0.028

Benzene Petroleum ether I sooctane Carbon tetrachloride Ethyl acetate

0.044 0.0 0.0 0.010 0.598

Isoamyl acetate Acetone

0.421 2.723

2.9

Methyl ethyl ketone Diethyl ether Ethylene chloride 1,4 -Dioxane Chloroform Carbon disulfide Pyridine Formamide Ethylene glycol Propylene glycol Dimethyl su1foxide 0.1 N NaOH 0.1 N HC1

1.771 0.086 0.260 4.224 1.820 0.062 20 20 20 20

> > > > >

20

> 20

4.526

Ionization Constant, pKa

Budgaard and Ilver (20) reported an apparent pKa of 2.68 i 0.05 at 3SoC., determined by measuring the pH of a partially neutralized 0.0025 M solution of sodium cloxacillin. Rapson and Bird (21) obFained replicate apparent pK values of 2.73 i 0.04 and 2.70 f 0.03 at 25OC. by titrating 0.0025 M*sodium cloxacillin solutions.

124

CLOXACILLIN SODIUM

2.10

Optical Rotation

Cloxacillin has 3 asymmetric carbon atoms and is strongly dextrorotatory. Specific rotation values in the literature are shown below: Cloxacillin sodium salt sodium salt free acid 3.

Temp.

Conc. and Solvent

+163O 20° +159O 20° +122O 20°

1% in water 1% in water 1% in acetone

Ref. 6,11,12 14 14

Synthesis and Purification

Cloxacillin is a semisynthetic beta-lactam antibiotic prepared by acylation of 6-amino penicillanic acid with 3-o-chlorophenyl-5-methyl isoxazolyl chloride (6,22, 23). The route of preparation of isoxazole acid chlorides has been described by Doyle (6). More recently, the conversion of Penicillin G to other penicillins without isolation of 6-amino penicillanic acid has been reported (24-26). A method of purification by deionized water elution through a G-25 Sephadex column has been described (27). Recrystallization of the sodium salt may be accomplished by methyl isobutyl ketone extraction of the free acid from an acidic aqueous solution and precipitation with sodium salts of carboxylic acids.

4.

Stability

The general pattern of penicillin degradation has been described in several reviews (18, 28). Sodium cloxacillin is stable in water for one week at SOC. The half-life in a solution of 50% aqueous alcohol at pH 1.3 and 35OC. was reported to be 160 minutes, approximately the same as Penicillin V (12, 18). At a concentration of 4 mg/ml in water or saline at pH 5.5 to 6.0, cloxacillin loses 15-25% of its biological activity in 7 hours (29). The rates of hydrolysis of cloxacillin and other penicillins were studied by Kinget and Schwartz (30). By following the amount of acid formed, they showed that 125

D A V I D L. MAYS

alkaline hydrolysis was more rapid for cloxacillin than other penicillins tested. The presence of aminoalkyl catechols doubled the rate of hydrolysis at pH 8 but reduced the rate at pH 6.5. The rate of cloxacillin hydrolysis was also increased above pH 8 by the presence of carbohydrates (31) and at neutral pH by aminoglycosides (32) * The kinetics of cloxacillin degradation in aqueous solution has been studied as a function of pit, buffer species, ionic strength, and temperature (20). Decomposition appears first order with respect to sodium cloxacillin content at any given pH. A rate-ofdegradation versus pH profile over a range of pH from 1 to 11 showed that decomposition is accelerated as the pH is moved above or below 6. The rate of hydrolysis is significantly affected by the type of buffer ions. Citrate buffers show the least catalytic effect and phosphate show the greatest. Degradation by enzymes has been studied extensively. Cloxacillin is resistant to enzymes which catalyze hydrolysis of the beta-lactam to produce the corresponding penicilloic acid (penicillinase or beta-lactamase enzymes). Citri and Zyk (33) studied the effect of different penicillin side chains on penicillinase activity. They showed the rate of cloxacillin inactivation by penicillinase (penicillin amidohydrolase EC 3.5.2.6) to be about 3% the rate of penicillin G inactivation. Smith and coworkers (34) investigated the stability of some penicillins to penicillinase. The initial rate of hydrolysis of cloxacillin was 0.3% and 1.1% of penicillin G hydrolysis by the penicillinases produced by B. cereus and Staph. aureus, respectively. Chapman (35) used the infrared absorption of the betalactam at 5.6 II to observe penicillin inactivation by beta-lactamase. Cloxacillin was more stable to staphylococci beta-lactamase than to coliform betalactamase. Combined treatment of penicillin amidase (enzymes which deacylate penicillins to 6-amino penicillanic acid) and beta-lactamase caused considerable inactivation of cloxacillin in 2 hours.

126

CLOXACILLIN SODIUM

5.

Methods of Analysis 5.1

Analysis of Impurities

The penicillenic acid of cloxacillin and other thiol-containing products such as penamaldic acid and penicillamine can be determined by reaction of the free thiol group with Ellman's reagent (36, 36a). The bright yellow anion formed is measured at the 412 nm absorption maximum. Other thiols and reducing agents interfere. If present in sufficient quantity, penicillenic acid may be measured directly by the natural absorbance at 337 nm (36a). The penicilloic acid of cloxacillin has been separated from cloxacillin by thin-layer chromatography (37). The penicilloic acids (and other iodine consuming substances) of some penicillins have been estimated by direct consumption of iodine (36a, 38). 5.2

Identification Tests

Sodium cloxacillin is identified by the infrared absorption spectrum and by the penicillin characteristic purple color formed upon treatment with chromotropic acid in sulfuric acid at 150°C. (38). In dosage forms, cloxacillin has been identified by the infrared absorption spectrum after extraction from aqueous phosphoric acid solution into chloroform and evaporation to a concentration of 2 mg/ml (39)* Weiss and coworkers (17) identified penicillins in dosage forms by determining the amount of penicillin partitioned between pH 6 buffer and organic solvent. 5.3

Quantitative Methods 5.31

Volumetric Methods

Iodine is not consumed by penicillins but is consumed by the hydrolysis products. The 127

DAVID L. MAYS

difference in iodine consumption before and after alkaline hydrolysis is used as a standard method of determining cloxacillin content (13). Hydrolysis with a measured excess of alkali generates an additional carboxyl group in the penicilloic acid. Back titration of the excess alkali with hydrochloric acid is used for determination of cloxacillin content. (38) 5.32

Colorimetric Methods

Cloxacillin has been determined by measurement of the side chain absorbance at 275 nm (39a). Penicillins have been determined by measurement of the absorption maximum near 340 nm produced by acid degradation to the corresponding penicillenic acids (38, 40-44). Formation of the penicillenic acid is catalyzed by copper and other metals and by imidazole (44). Penicillins have long been determined by reaction with hydroxylamine. The hydroxamic acids generated produce a red-colored chelate with iron (111). Details of the procedure are available in the Federal Register (13). Automated versions of the method have also been used (45). 5.33

Polarography

Penicillins in serum were determined by polarographic techniques (46) The analysis required about two hours. Cloxacillin was determined at levels of 5 vg/ml in sulfuric acid solution.

.

5.34

Gas Chromatography

Organic acid side chains produced by vigorous alkaline hydrolysis of penicillins were converted to methyl esters for gas chromatographic separation on a 3.5% SE-30 column (47). Intact penicil 1 ins were gas chromatographed as the corresponding methyl esters on a 2% fluoro-silicone phase by Martin and coworkers (48). Separation of the trimethylsilyl esters of several penicillins on a 2% OV-17 column was 128

CLOXACILLIN SODIUM

reported by Hishta, et. al. (49). Cloxacillin was separated from several other penicillins and quantitation was indicated by reproducibility of response factors on reference samples. 5.35

Infrared Spectroscopy

The infrared absorption due to the betalactam has been used to quantitate penicillins after extraction into a suitable solvent (39). The cloxacillin beta-lactam band near 1760 cm-1 was used to measure cloxacillin inactivation by beta-lactamase as differentiated from amidase (35). For this work solutions were lyophilized in the presence of potassium bromide and infrared absorption measurements were made from the solid disks. 5.36

Optical Rotation

The change in optical rotation upon treatment with penicillinase has been used to quantitate penicillins (50). Penicillins which are more susceptible to penicillinase can be determined in the presence of resistant penicillins. Ampicillin and cloxacillin were determined in combination. 5.37

Biological Methods

The cylinder-plate agar diffusion method is the official microbiological method of determination (13). Staph aureus (ATCC 6538P) is the organism of choice (13, 51). Cloxacillin has been microbiologically determined in the presence of ampicillin using agar impregnated with penicillinase to destroy the ampicillin activity (52) or after ion exchange separation on a column of IRA-402 (53). Cloxacillin is routinely measured by the turbidimetric method using Staph aureus FDA-209P (ATCC 6538P) or Staph aureus BL-A9596 (54).

A microbiological paper disk procedure has been described for measuring cloxacillin and other antibiotics in as little as 10 p 1 of plasma (55). Hooke and Ball (56) used an agar plate method and Sarcina lutea NCIB 8553 to measure cloxacillin at levels below 10 pg/ml in milk. 129

DAVID L. MAYS

In a test to detect trace levels in milk, penicillin inhibits growth of Streptococcus thermophilus B.C., which otherwise causes the dye 2,3,5-triphenyltetrazolium chloride, to turn from colorless to red (57). Other antibiotics interfere. 5.38

Automated Methods

Several chemical methods for penicillin determination have been automated, including the hydroxylamine method (45, 58-60), the iodometric method (61-63), the penicillenic acid method (64), and a colorimetric method based on enzyme deacylation to 6-amino penicillenic acid and detection with p-dimethylaminobenzaldehyde (65). Cloxacillin, specifically, is mentioned in the hydroxylamine (45) and penicillenic acid (64) automated procedures. 5.4

Thin-layer Chromatography

Reviews on chromatography (66) and analysis (67) of antibiotics are available. Cloxacillin has been chromatographed on paper (68), kieselguhr G (68), cellulose (69-71), polyamide (72), silica gel G (37, 70, 73-75), commercial silica gel plates (76, 77) and silica gel G predeveloped with silicone (5% DC 200 in ether) (78-80). Cloxacillin has been separated from other penicillins (37, 66, 75, 77-80), and from impurities (37), from cephalosporins (79), from other antibiotics (70, 74), and from constituents of body fluids (76, 77). Chromatography has been used to study solvent partitioning (78) and structure-activity relationships (79, 80). Visualization spray reagents used include iodine-azide solution followed by aqueous starch to give white spots on blue purple (37, 68, 74); 10% acetic acid in acetone followed by starch-iodine to give white spots on blue (69); ammoniated copper sulfate (73); 0.5% bromine solution (72) ; 0.25% fluorescein (72) ; ferric chloride and potassium ferricyanide with sulfuric acid to give blue spots on green (70, 71); alkaline potassium permanganate and heat to give yellow spots on 130

CLOXACILLIN SODIUM

pink (78-80); chloroplatinic acid with potassium iodide in acetone to give white spots on red-purple (75); and alkaline silver nitrate (68). Bacillus subtilis ATCC 6633 impregnated in agar has been used to detect penicillins by observation of the zone of inhibition after contact with the plates (76, 77). 6.

Protein Binding

The reversible binding of protein to penicillins (81i and drugs in general (82), has been reviewed. Schwartz's penicillin review (28) includes a section on reversible and irreversible protein binding. Schwartz postulated that the high local concentration of penicillin reversibly bound to protein accelerates aminolysis, particularly in the case of cloxacillin which is highly bound to serum protein. Batchelor (83) also presented evidence that penicillins, including cloxacillin, can become irreversibly bound to protein. Several workers have measured the proportion of cloxacillin reversibly bound t o serum protein. Values of 94% (29), 95% and 96% (84), 85% (85) and 62% (86) have been reported. Rolinson and Sutherland (87) made a thorough study of the degree of binding of several penicillins as a function of concentration of penicillin and protein, of sera from different animal species, and of temperature. Binding was shown to be essentially reversible and competitive with other drugs. Cloxacillin at 50 pg/ml (considered approximate blood level from a normal dose) was 94% bound to human serum. Affinity for Sephadex gel of a number of penicillins was studied as a function of protein binding (88). 7.

Metabolism

After oral administration of 100 mg/kg to rats, 18.7% of the cloxacillin was recovered in the urine after 24 hours, and 10.2% was recovered in the bile. Paper chromatographic examination of the urine indicated small amounts of two unidentified metabolites (12). Cloxacillin was reported to be approximately 10% metabolized in man (16, 89). One unidentified metabolite was found which 131

DAVID L. MAYS

had biological activity similar to the parent compound. The rate of elimination of cloxacillin from young men given oral doses was studied. (90) 8.

References

1. 2. 3.

Chem. Abs. Index Guide 76 Jan-June (1972). J. h e r . Med. Ass. 185 (8), 656 (1963). United States P h a m z p e i a XVIII, Mack Publishing Co., Eaton, Pa., 1970, p. 620. Rudzit, E. A., et. al., Antibiotiki 17 ( l l ) , 978981 (1972). Wayland, L., and Weiss, P. J., J. Ass. Offic. Anal. Chem., 48 (S), 965-972 (1965). Pek, G.%. , et. al., Izv. Akad. Nauk SSSR, Ser. Khim. 1968, (lo), 2213-22 through Chem. Abs. 70 :282=( 1969) Doyle, F. P., et. al., J. Chem. SOC. 5838-5845 (1963). Richter, W., and Biemann, K., Monatsh. Chem. 95, 766-778 (1964). Dunham, J. M., Analytical Profiles of Drug Substances, Vol. 1, K. Florey, ed., Academic Press, New York, New York (1973) p. 258. Ivashkiv, E., Analytical Profiles of Drug Substances, Vol. 2, K. Florey, ed., Academic Press, New York, New York (1973), pp. 12-13. Ohashi, M., et. al., Org. Mass Spectrom. 2, 195-207 (1969). The Merck Index, 8th ed., Merck and Co., Inc., Rahway, N. J., p. 271. Nayler, J. H. C., et. al., Nature (London) 195, 1264-1267 (1962). Code of Federal Regulations, Title 21, April 1973 Revision, Chapter I, Section 149j.l. Koenig, R., et. al., Hung. Patent 151,377 (May 23, 1964) through Chem. Abs. 61 :5658f (1964). Marr, T. R., Bristol Laboratories, personal comunication. Knudsen, E. T., et. al., Lancet 2, 632-634 (1962). Weiss, P. J., et. al., J. Ass. OFfic. Anal. Chem, 50 (6), 1294-1297 (1967). 60 (4), Hou, J. P., and Poole, J. W., J. Pharm. Sci. 503-532 (1971).

3a. 4. 5. 6.

7. 8. 9. 10.

11.

12. 13. 14. 15. 16.

17.

18.

.

-

132

CLOXACILLIN SODIUM

19. 20. 21. 22.

23. 24.

25. 26. 27. 28.

29. 30. 31.

32.

33 34. 35. 36. 36a. 37. 38. 39.

Marsh, J . R., and Weiss, P. J., J. Ass. Offic. Anal. Chem. 50 (2), 457-462 (1967). BundgaGd, H . , and Ilver, K., Dan. Tidsskr., Farm. 44, - 365-380 (1970). Rapson, H. D. C., and Bird, A. E., J. Pharm. Pharmacol 15 (Suppl .I 222T-231T (1963). Axerio, P., Farm. Nueva 28 (3181, 315-320 (1963). through Chem. Abs. 59:15269c (1963). Doyle, F. P., and Naylor, J. H. C . , U.S. Patent 2,996,501 (August 15, 1961). Fosker, G. R . , e t . a l . , J. Chem. SOC. C., 1917-1919 (1971). Heuser, L. J . , F r . Patent 1,596,495 (July 31, 1970) through Chem. Abs. 74:141781 (1971). Jinnosuke, A., e t . a l . , Ger. Patent 1,943,667 (May 14, 1970) through Chem. Abs. 73:45505s (1970). Feinberg, J . G . , and Weston, R. D., B r i t . Patent 1,131,741 (October 23, 1968) through Chem. Abs. 70: 14410r (1969). Schwartz, M. A . , J. Pharm. Sci 58 ( 6 ) , 643-661 (1969). 5 , 26-34 (1964). S i d e l l , S., Clin. Pharmacol. Ther. Kinget, R. D., and Schwartz, M. A . , J. Pham. Sci. 58 (9), 1102-1105 (1969). Simberkoff, M. S., e t . a l . , N. Engl. J. Med. 283 ( 3 ) , 116-119 (1970). Lynn, B., and Jones, A., Advances i n Antimicrobial and Antineoplastic Chemotherapy, Vol. I/2, University Park Press, Baltimore, Md., (1972) pp. 701-705. C i t r i , N., and Zyk, N . , Biochim. Biophys. Acta 99, 427-441 (1965). Smith, J . T., e t . a l . , Nature 195, 1300-1301 (1962). Chapman, J. J . , et. a l . , J. G e r M i c r o b i o l . 36, 215-223 (1964). Vermeij, P . , Pharm. Weekbl. 107, 249-259 (1972). Seitzinger, Ing. R. W. Th., Pharm. Weekbl. 108, 961968 (1973). Vandamme, E. J . , and Voets, J. P., J. Chromatog. 71, 141-148 (1972). B r i t i s h Pharmacopeia, Her Majesty's Stationery Office, London, 1973, pp. 81, 117. Coclers, L., et. a l . , J. Pharm. Belg. 24, 475-491 (1969).

.

-

-

-

-

-

133

DAVID L. MAYS

39a. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

51. 52. 53. 54. 55.

56. 57. 58. 59.

Davidson, A. G . , and Stenlake, J. B., J . Phann. Pharmacol. 25, Suppl., 156P-157P (1973). H e r r i o t t , R T M . , J. Biol. Chem. 164, 725-736 (1946). Weaver, W. J . , and Reschke, R. F Y J . Phann. S c i . 52 ( 4 ) , 362-364 (1963). Saccani, F., and P i t r o l o , G . , Boll. Chim. Farm, 108, 29-33 (1969). Yasuda, T., and Shimada, S., J. Antibiot. 24 (S), 290-293 (1971). Bundgaard, H., and Ilver, K . , J . Pharm. Pharmacol. 24, 790-794 (1972). Lane, J. R . , and Weiss, P. J . , Presented at t h e Technicon Symposium, "Automation i n Anal. Chem.", New York, New York, October 17, 1966. Benner, E. J . , Presented a t t h e 10th Interscience Conference on Antimicrobial Agents and Chemotheraphy Chicago, October 18-21, 1970. 13 (12), Kawai, S., and Hashiba, S., Bunseki Kagaku 1223-1226 (1964). Martin, J. B., et. al., presented a t t h e 17th Annual Pittsburgh Conference on Applied Spectroscopy, February 21, 1966. Hishta, C . , e t . a l . , Anal. Chem. 43 (11) , 1530-1532. (1971) Rasmussen, C. E., and Higuchi, T., J. Pharm. Sci. 60 ( l l ) , 1608-1616 (1971). Arret, B., e t . a l . , J. Phann. S c i . 60 ( l l ) , 16891694 (1971). Sabath, L. D., e t . a l . , Appl. Microbiol. 15 (3), 468-470 (1967). 108 (12), Saccani, F., e t . a l . , Boll, Chim. Farm. 777-780 (1969) Tylec, F . , and Kianka, J . , B r i s t o l Laboratories, p r i v a t e communication. 4, 150-157 J a l l i n g , B . , e t . a l . , P h a n a c o l . Clin. (1972). Hooke, E. J., and B a l l , G. M., J. Appl. Bacteriol. 26 (2), 216-218 (1963). S c a r l e t t , C. A., Agriculture (London) 73 (9), 423426 (1966). Avanzini, F., e t . a l . , Automation i n Analytical Chemistry, 1966, Vol. 11, Mediad, Inc., White Plains, New York, pp. 31-34. Niedermeyer, A. O., e t . a l . , Anal. Chem. 32, 664-666

-

-

-

-

-

.

-

.

-

134

CLOXACILLIN SODIUM

60. 61. 62. 63. 64. 65. 66.

67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80.

.

(1960) Stevenson, C, E., Automation in Analytical Chemistry 1969, Vol. 11, Medlad, Inc., White Plains, New York, pp. 251-256. Bomstein, J., et. al., Ann. N. Y. Acad. Sci. 130, 589-595 (1965) Ferrari, A , , et. al., Anal. Chem. 31 (lo), 1710-1717 (1959). Russo-Alessi, F. M., et. al., Ann. N. Y. Acad. Sci. 87, 822-829 (1960). Celletti, P., et. al., Farmaco, Ed. Prat. 27 (12), 688-698 (1972) through Analyt Abs. 25 :42771973) Evans, W. G., et. al., Automation in Analytical Chemistry, 1969, Vol. 11, Mediad, Inc., White Plains, New York, pp. 257-259. Chromatography of Antibiotics, Wagman, C. H., and Weinstein, J. J., Elsevier Sci. Pub., New York (1973), pp. 140-146. Schmitt, J. P., and Mathis, C., Pharma. Int., Engl. Ed., pp. 17-28 Mar. (1970). Hellberg, H., J. Ass. Offic. Anal, Chem. 51 (3), 522-556 (1968) Bird, A. E., and Marshall, A. C., J. Chromatog. 63, 313-319 (1971). McGilvery, I., and Strickland, R. D., J. Pharm. Sci. 56, 77-79 (1967). Wayland, L. G., and Weiss, P. J., J. Pharm. Sci. 57 ( S ) , 806-810 (1968). Wang, J. T., et. al., Tai-Wan K'o Hsueh, 24 (1-2) 19-20 (1970) through Chem. Abs. 74:115942r(1971). Guven, D. C., and Ari, A., Eczacilik Bul. 13 (2), 20-23 (1971). Saccani, F., Boll. Chem. Farm. 106, 625-628 (1967). Pokorny, M., et. al., J. Chroma=. 77, 457-460 (1973). Nishida, M., et. al., Nippon Kagaku Ryohogakukai Zasshi 17 (lo), 1973-1976 (1969) through Chem. Abs. 73: 127313 (1970) Murakawa, T., J. Antibiot. 23 (S), 250-251 (1970). 41, 371-379 Biagi, G. L., et. al., J. Chromatog. (1969) Biagi, G. L., et. al., J. Chromatog. 51, 548-552 (1970). 13 (3), 511-516 Biagi, G. L., et. al. J. Med. Chem. -

-

.

-

-

.

.

-

.

-

-

.

.

135

DAVID L. MAYS

81. 82. 83. 84. 85. 86. 87. 88.

89. 90.

.

(1970) Scholtan, W., Antibiot. Chemother. (Basel) 14 5393 (1968). Meyer, M. C., and Guttman, D. E . , J. Pharm. S c i . 57 (6). 895-918 (1968). 206, 362-364 Batchelor, F. -R., et. a l . , Nature (1965). Kunin, C. M., Antimicrob. Ag. Chemother., 1025-1034 (1965) S i d e l l , S., e t . a l . , Arch. Intern. Med. 112, 21-28 (1963). Kato, Y . , Nippon Kagaku Ryohogakukai Zasshi 13 ( 2 ) , 89-93 (1965) through Chem. Abs. 64 :7214e ( 1 9 z ) . Rolinson, G . N . , and Sutherland, R., B r i t . J. Pharmacol. 25, 638-650 (1965). Murakawa, T Z J. Antibiot. 23 ( l o ) , 481-487 (1970). Lynn, B., Antibiot. ChemothG. 13, 125-226. (1965) Kislak, J. W., e t . a l . , h e r . J . Med. S c i . 249 ( 6 ) , 636-646 (1965)

.

-

.

.

I appreciate t h e h e l p of Mr. N. Muhammad, Dr. R. D. Brown, and M r . T. R. Marr of t h e Chemical Control Department of B r i s t o l Laboratories f o r a s s i s t a n c e i n obtaining and i n t e r p r e t i n g t h e spectrograms and thermal a n a l y s i s d a t a used i n t h i s p r o f i l e .

136

DIATRIZOIC ACID

Hyam Henry Lerner

HYAM H. LERNER

TABLE OF CONTENTS 1. Description 1.1 Name , Formula, Molecular Weight 1.2 Appearance, Color, Odor 2. Physical Properties 2.1

Spectra 2.11 2.12 2.13 2.14

2.2

Crystal Properties 2.21 2.22 2.23 2.24 2.25

2.3

Differential Thermal Analysis Thermal Gravimetric Analysis X-Ray Powder Diffraction Water of Crystallization Melting Range

Solution Data 2.31 2.32 2.33 2.34 2.35 2.36 2.37 2.38

3.

Infrared Nuclear (Proton) Magnetic Resonance Ultraviolet Mass Spectrometry

Solubility pKa pH Osmotic Properties and Ionic Strength Index of Refraction Specific Gravity Freezing Point Depression Viscosity

Manufacturing Procedures 3.1 3.2

Synthesis Purification

4. Stability 5.

Separation Techniques and Analysis for Impurities

138

DlATRlZOlC ACID

5.1 5.2 5.3 5.4 5.5

Free Amino Compounds Free Iodine and Free Halide Complexometric Methods of Separation Countercurrent Distribution Phase Solubility Analysis

6. Methods of Analysis 6.1 6.2 6.3 6.4 6.5 6.6

Elemental Analysis Identification Tests Direct Spectrophotometric Analysis Polarography Organically Bound Iodine Chromatography 6.61 6.62 6.63

Paper Chromatography Thin Layer Chromatography Electrochromatography

6.7 Titrimetric Methods of Analysis 6.8 Radiometric Methods of Analysis 6.81 X-Ray 6-82 6- Particle Dispersion 7.

Pharmacology 7.1 7.2

Drug Metabolism Toxicity

8. References

139

HYAM H. LERNER

1.

Description 1.1

Name. Formula. Molecular Weight

-

Diatrizoic Acid is 3,5- diacetamido 2,4,6 triiodbenzoic acid. Chemical Abstracts listings are under the heading benzoic acid, 3,5 diacetamido 2,4,6 triiodo-.

-

-

-

Among the generic and trivial names for this c m pound are urografin acid. Common trade names include Renografin and Hypaque.

Mol. Wt. 613.928 1.2

Appearance, Color, Odor

Diatrizoic Acid is a white, odorless and tasteless crystalline powder. The sodium salt is colorlese and odorits ta te reported as both weakly salty and slightly bitter8 The methylglucamine (meglumine) salt is colorless, odorless and has a slight sweetish taste1

.

2.

.

Phvsical ProDerties 2.1

Spectra 2.11

Infrared

The infrared spectrum in Fig. 1 was obtained on a Perkin-Elmer Model 21 infrared spectrophotmeter, from a mineral oil dispersion, The following spectral assigmnents were made by Toeplitz5

.

140

e

f

F i g . 1 I n f r a r e d Spectrum o f D i a t r i z o i c Acid

HYAM H. LERNER

Wavelength -1

-cm E

Assignment N-H stretch Acid C=O Amide C=O Secondary amide and aromatic C=C stretch

3.35 5.88 6.02 6.60

2985 1700 1661

1515

This spectrum is in basic fgreement with a published spectrum for sodium diatrizoate

.

2.12

Nuclear (Proton) Magnetic Resonance

The NMU spectrum of diatrizoic acid shown in Fig. 2, was determined on a Varian XL-100 NMR spectrometer at ambient probe temperature (ca.31"). The sample was dissolved in deuterated dimethylsulfoxide, containing tetramethylsilane as an internal reference, Spectral assignments are recorded in Table I2

.

TABLE I NMR Spectral Assignments

No, of Protons

Chemical Shift (ppm,b) 2.02

6

(4

Assignment -CH3 0

9.88

1 (s)

-!-OH

9.96

2 (s)

-m-L

0

s = singlet m = multiplet

2.13

Ultraviolet

The following ultraviolet spectral data have been reported for diatrizoic acid:

142

i

143

1

HYAM H. LERNER

E -

Form -

Free Acid Sodium Salt Free Acid Free Acid Free Acid Free Acid

0.13 NaOH 238 Ethanol 239 Ethanol 238 Methanol 238 .01g Methanolic 238 NaOH .01g Methanolic 238 HC1

Reference

521 525 589 539 538

32,000 33,400 36,200 33,100 33,000

1 4 12 32 32

531

32,600

32

Although none of the references explicity state it, it appears the above results are reported on an "as is" basis, except for reference 12, which appears to be on the dried basis. Purkiss et.a1.26, reported that the absorbance of sodium diatrizoate is due to the presence of the acetamido group and is not dependent on iodine content. Removal of iodine from the compound did not affect absorbance at 238 m. Neudert and R ~ p k e ~however, ~, determined the molecular extinction coefficients (€) of related iodinated compounds at their peaks. They reported the for 1,3,5,-triiodobenzene at 232 nm,,is 36,600 and that for 2, 4,6-triiodobenzoic acid at 238 run, to be 33,600. They also reported the extinction of 3-acetamidobenzoic acid at 243 tnn to be 13,500. Therefore it seems reasonable to suggest that the strong ultraviolet absorption maximum of diatrizoic acid at 238 nm, is due, to a large extent, to iodine. 2.14

Mass Spectrometry

No molecular ion is observed for diatrizoic Weak peaks at m/e 569, corresponding to the loss of -COOH at m/e 572, for the loss of -COCH2, from the unobserved molecular ion, do occur. Major fragment ions at m/e 487 and 486 occur, due to loss of I and HI, respectively, Loss of CH3CO from the m/e 486 fragment results in a peak at m/e 443 and loss of a second CH3CO results in the 3 observed peak at m/e 400 acid.

.

2.2

Crystal Properties 2.21

Differential Thermal Analysis

144

DlATRlZOlC ACID

Valenti34 determined the DTA of diatrizoic acid on a DuPont 900 Thermoanalyzer at a temperature rise of 15" per minute. The thermogram showed endotherms at 168 and 321", exotherms at 220" (small) and 330", and a shoulder at 306". Kabadi35 reprecipitated diatrizoic acid from alkaline solution and dried it at roan temperature, under vacuum. He reported DTA data under the same conditions referred to above as follows: endotherms at 119" (large), 157" (small), 316" broad; exotherm at 234"; shoulder at 29$. The moisture content of this specimen was <0.4%. The work of Kabadi35 indicates that other polymorphic forms of diatrizoic acid exist. 2.22

Thermal Gravimetric Analysis

Valenti34 determined the TGA of diatrizoic acid on a DuPont Thermogravimetric Analyzer, The compound was heated at a rate of 15" per minute under nitrogen sweep. Weight loss of 5% was observed, with all volatile material driven off before 140". 2.23

X-Ray Powder Diffraction

Ochs13 obtained the X-ray powder diffraction spectrum of diatrizoic acid on a Phillips X-Ray Powder Diffractometer, at a voltage of 45 kv and a current of 15 ma. 0 The sample was irradiated by a copper source at 1.54A. Data are recorded in Table 2. 2.24

Water of Crystallization Langecker et.al. reported that the diatri-

zoic acid crystal can contain up to 2 moles of water of

crystallization.

145

HYAM H. LERNER

TABLE I1 X-Ray Powder D i f f r a c t i o n P a t t e r n of D i a t r i z o i c Acid Squibb Lot 1987

&

Relative Intensity**

*&d

0.23 0.40 0.19 0.34 0.51 0.19 0.15 0.20 0.31 1.00 0.43 0.32 0.49 0.69 0.19 0.19 0.16 0.53 0.43 0.53 0.29 0.17

13.2 9.10 7.20 6.50 5.66 5.50 5.00 4.50 4.42 4.28 4.18 4.02 3.90 3.75 3.68 3.60 3.48 3.44 3.36 3.30 3.23 3.17

* **

Relative Intensity** 0.45 0.49 0.71 0.23 0.27 0.21 0.13 0.11 0.19 0.16 0.24 0.27 0.21 0.12 0.19 0.19 0.17 0.16 0.15 0.17 0.13 0.15 0.16

3.13 3.12 3.07 3.00 2.98 2.93 2.88 2.68 2.60 2.56 2.54 2.50 2.46 2.37 2.28 2.24 2.18 2.16 2.13 2.09 1.93 1.89 1.87

nn

d = (interplanar distance) = 2 s i n 0 where? = 1.5398 Based on h i g h e s t i n t e n s i t y of 1.00

146

DlATRlZOlC ACID

2.25

Melting Range

Diverse values have been reported in the literature for the melting range of diatrizoic acid. They are shown in the table below. A possible explanation for the wide diversity in reported values may be that different (unidentified) polymorph forms were used in the work. Melting Range

--

>300

6 1

from 260

Decompositon, I2 Vapors reported

>340

Crystal form: needles

11

Using KSfler Microblock (Reichert)

12

260" 2.3

Ref. -

Remarks

-

290

Solution Data 2.31

Solubility

The data in Table I11 were reported for the solubility of diatrizoic acid, at room temperature. TABLE I11

-

SOLUBILITY DATA Solubility (mg/lOO ml)

Solvent Acetone Benzene Chlorofo m Ethanol Ether Hexane Me thano1 Propylene Glycol Water at 25" Water at 50" Water at 90" 0.1N Sodium hyaroxide

Ref. 1

Ref. 12

-


110 700
0.1 1,018 0.3

-

2,400 -

7,205

100 150 270

-

147

Ref. 34

HYAM H. LERNER

.

Langecker e t a 1 .l r e p o r t e d the s o l u b i l i t y a t 20" of t h e sodium s a l t of d i a t r i z o i c a c i d t o be 60 g1100 m l i n w a t e r and t h a t ol t h e methylglucamine s a l t t o be 89g/100 m l , Drug Standards r e p o r t e d t h a t t h e sodium s a l t i s f r e e l y s o l u b l e i n w a t e r and dimethylformamide and v e r y s l i g h t l y s o l u b l e i n chloroform and e t h e r . The s o l u b i l i t y i n e t h a n o l was r e p o r t e d t o b e 2 g/100. 2.32

pKa The pKa of d i a t r i z o i c a c i d i s 3.4

2.33

.

1

pH

R e pH of a 1%s u s p e n s i o n of t h e f r e e a c i d i n water i s 2.1 The pH of a 50% s o l u t i o n of t h e sodium Langecker e t . a l . l r e p o r t e d t h e s a l t i s between 7 and 94 pH of a s o l u t i o n of t h e sodium s a l t t o b e ' 7 . 3 and t h a t of t h e meglumine s a l t t o be 6.0.

.

2.34

.

Osmotic P r o p e r t i e s

and I o n i c S t r e n g t h

Berdalen et.a1.36 determined t h e molal osmot i c c o e f f i c i e n t s and i o n i c s t r e n g t h of some commercial p r e p a r a t i o n s of d i a t r i z o a t e . 2.35

I n d e x of R e f r a c t i o n

25 The r e f r a c t i v e i n d e x (n ) of sodium d i a t r i z o a t e i n w a t e r , a t 25", i s g i v e n i n Ta l e I V .

g

2.36

Specific Gravity

.'

Langecker e t , a 1 reported the s p e c i f i c g r a v i t i e s (p) of w a t e r s o l u t i o n s of sodium d i a t r i z o a t e a t 25". Data are recorded i n Table I V . 2.37

Freezing Point Depression

The f r e e z i n g p o i n t d e p r e s s i o n (-AT) of v a r i o u s c o n c e n t r a t i o n s of sodium d i a t r i z o a t e were r e p o r t e d by Langecker e t . a l . l , and a r e r e p o r t e d i n Table I V .

148

DlATRlZOlC ACID

2.38

Viscosity 59 Schmid studied the viscosity of 0.75PJ aqueous solutions of diatrizoate at pH 7, and 20". He reported viscosities by Rheomat 16 and Ostwald viscometers for sodium diatrizoate (3.29 cps), meglumine diatrizoate (6.19 cps), and tris-(hydroxymethy1)-aminomethane diatrizoate (4.76 cps.). TABLE IV

Physicochemical Data of Sodium Diatrizoate

0.0 4.74 14.20 28.40 37.90 47.40

3.

25

Mo 1arity

nD

0.0 0.074 0.223

0.997 1.338 1.353 1.376 1.392 1.407

0.446 0.595 0.744

-

1.025 1.085 1.175 1.235 1.295

0.27 0.78 1.70 2.38 3.20

Manufacturing Procedures 3.1

Synthesis

Diatrizoic acid may be prepared from 3,5-dinitrobenzoic acijlOby catalytic reduction with e.g. Raney nickel in methanol or palladium (on carbon support)", to yield the 3,5 diaminobenzoic acid. Reaction of dilute hydrochloric acid solutions of the latter compound with potasaturn iodochloride or iodine monochloride yields the 3,5-diamino2,4,6-triidobenzoic acid7y10y11. Acylation of this compound is accomplished by dissolving it in acetic anhydride, heating and then acidifying with concentrated sulfuric acid, added dropwise. After a brief exothermic reaction the solution is heated on a steam bath and cooled in ice6 Alternatively 3,s-diacetamidobenzoic acid can be iodinated in pH 5-6 methanolic solution with odine monochloride, at 8OoC, to yield the triiodo compound .'1S'',' (See Fig. 3 for stepwise synthetic route)

.

Davidson28 acetylated the diamino compound with acetic H3 anhydride to obtain tritiated diatrizoic acid.

149

HYAM H. LERNER

Figure 3 Synthetic Route for Diatrizoic Acid '

OOH

NO A

CH3-

3.2

T

e

COOH

N

H

b

Z

H2

-NH

Purification

Crude diatrizoic acid may be purified in any of the following ways: The precipitate is dissolved in dilute ammonia or sodium h droxide, charcoaled and reprecipitated by acidification6,g, 10,ll. Recrystallization from a 50% aqueous dimethylformamide solution with charcoaling6

.

Filtration of a solution in dilute sodiumhykoxide containing sodium hydrosulfite and charcoal. The filtrate is passed through a medium basic anion exchange resin (in the OH form) The eluate is acidified to reprecipitate the pure product3

.

4.

Stability

Diatrizoic acid is chemically stable at room temperature. Extrapolation o€ the activation energy and frequency constant data, between 70" and 100" to room temperature, indicates decomposition of the N-acyl bond to be 0.1% in 50 years37 , Evolution of iodine at 90" from diatrizoic acid in solution at pH 9, is less than 3% in 75 hours. The under iodinated and free amino presence of impuritie,s compounds, enhance degradation37

.

150

DlATRlZOlC ACID

Commercial preparations of diatrizoic acid neutralized with meglumine, and containing sodium citrate (3.2 mg/ml) and disodium edetate (0.4 mg/ml), show no significant loss of potency after storage for 5 years, at room temperature?? 5.

Separation Technique and Analvsis for ImDuritiea

Many of the chromatographic procedures described in Section 6.6 are used to separate, detect, and estimate impurities in diatrizoic acid, In addition, the following methods have been described in the literature to detect and quantify impurities and decomposition products, and to isolate pure diatrizoic acid. 5.1

Free Amino Compounds

Hartmann and determined free m i n o compounds of iodinated contrast agents by a kinetic method based on the greater reactivity of these impurities with elemental bromine in acetic acid. Under these conditions, the free amino compound quantitatively splits off iodine, which is oxidized to iodate by the bromine. After destruction of the excess bromine, the iodate is reduced to iodine and titrated with thiosulfate, to a starch endpoint, Free iodides in the sample also react with bromine and give positive results, by this procedure. Hoevel-Kestermann and Muhlemann12 and Hartmann and R t i ~ k edescribed ~~ a Bratton-Marshall colorimetric reaction to quantitate free amino compound impurities.

5.2 Free Iodine and Free Halide G ~ n d detected a ~ ~ free iodine in diatrizoic acid by dispersal in water, filtering and adding starch to the filtrate. A blue coloration indicates the presence of free iodine. An alternate p r o ~ e d u r e ~is, ~to~ acidify the filtrate, add chloroform and sodium nitrite and observe for reddish coloration in the lower chloroform layer. Free halide was detected38 by acidification of a water suspension filtrate of the drug substance with nitric acid, addition of silver nitrate and observing for the presence of opalescence or precipitation.

151

HYAM H. LERNER

Poet3' quantitated free halide coulometrically, with an Aminco-Cotl.ove Chloride Titrator. Hartmann and quantitated free halide in contrast media by potentiometric titration with 0.001E silver nitrate, under a protective cover of nitrogen. 5.3 (=analexometric Mpthod o f SeDaration Hentrich and Pfeifer40 described methods for the precipitation of ten contrast agents as their metallic salts or metallic complex salts. Diatrizoic acid can be precipitated quantitatively by cadmium sulfate with thiourea, copper sulfate with thiourea, silver nitrate, cadmium sulfate with pyridine and copper sulfate with pyridine. Chelatometric methods are also described for the titration of excess precipitantj after separation of the precipitated salt by filtration. The molecular formulae, weights, melting range and equivalent weight of diatrizoic acid precipitated by 1 ml of 0.1gsolution of the precipitant, for the salts and complexes, are given in Table V below. TABLE V

40

Metallic Salt and Metallic Complexes of Diatrizoic Acid

. Weight Precipitant A " 3

Mol. Formula of Salt or Complex Mol. Wt.

Melting Range

to 1ml qf 0.m Precipitant

720.8

281-284

0.0614 g

1,642.7

226-227

0.0307 g

[Cd(C6H5N)4]* 1,656.6 (C11H813N204) 2

288-290

0.0307 g

C11H813N204Ag

CdSO4{Cd [(NH2) 2CSl$ Thiourea (CllH813N204)2 CdSO4-

CuSO4f2u[(NH2)2CS$ Thiourea 11H813N204

-

ine

828.7

[cU( C6H5N) 21 1,447.5 (C11H813N204)2

152

245

0.0614

266-267

0.0307

DlATRlZOlC ACID

5.4

Countercurr-

Strickler et.a1.42 separated diatrizoic acid and other contrast agents from sera by countercurrent distribution. They used a solvent system composed of E-butanol: dil. aqueous ammonia (l:$. Both a 30-tube manual procedure and a 200-tube automatic procedure are described. Evaluation of other methods e.g. separation by ion exchange on Dowex 1 column (not successful) and 2-dimensional paper chromatography, are also discussed.

-

5.5

Phase Solubilitv Analvsig

K a l l ~ sreported ~~ on the use of phase solubility analysis to determine the purity of diatrizoic acid. He used a solvent system composed of acetone: ethanol (17:3), with equilibration over a 23 hour period at 25". The extrapolated solubility in this system is 3.5 mg per g. 6. Methods of Analysis

6.1

Elemental Analysis

The elemental analysis6' to the anhydrous basis. Element

% Theoretical

21.52 1.478 62.01 4.562 10.42 6.2

results were calculated % Reported60

21.98 1.78 61.69 4.26 10.29 (CALC)

Identification Tests

Infrared (section 2.11), paper chromatography (section 6.61), and thin-layer chromatography (section 6.62) have been used to identify diatrizoic acid. The evolution of intense violet fumes of liberated iodine can be observed b heating a sample of diatrizoic acid over an open flame 12,

.rs,

153

H Y A M H. LERNER

Identification of the amide funcionality can be made by first ascertaining the absence of free amino groups (section 5.1) and then cleaving the acyl group, by refluxing in base, and repeating the Bratton-Marshall reaction12

.

The pH (section 2.33) of a water suspension and the ability to titrate a suspension with base, can be used to identify the presence of the carboxylic acid12. 6.3

Direct SDectroDhotometric Analysis

Purkiss et.a1.26, determined diatrizoic acid directly in urine and heparinized protein-free plasma at the 238 nm maximum. Samples taken from the subject prior to administration of diatrizoic acid were used as blanks. Dilution with water decreased blank interference. Hoevel-Kestermann and Muhlmann12 used spectrophotometric analysis at the 238 run peak to determine solubility in various solvents.

6.4

Polarography

Kabasakalian and Mc G 1 0 t t e n ~studied ~ 10 iodinated radiocontrast agents polarographically in ethanolwater (1:l) solutions between pH 1.3 and 10.5. The effects of pH, temperature and sample concentration on wave form and height are examined and the analytical possibilities of polarography as a quantitative procedure for diatrizoic acid are discussed. Diatrizoic acid in phospha e buffer, f pH 8.9, exhibits two waves: -E$ at 0.90 and -E% at 1.22. 6.5

Ornanicallv Bound Iodine

Hoevel Kestermann and M"uhlemann12 reviewed three methods for liberating organically bound iodine in contrast agents: A. Parr bomb (fusion with sodium peroxide); B. catalytic dehalogenation with sodium borohydride; and C. reductive dehalogenation with zinc-sodium hydroxide. They recommend method B because of its simplicity, short assay time and high recision. This method was originally proposed by Egli48 . wantitation is accomplished by potentiometric titration with silver nitrate.

154

DlATRlZOlC ACID

Yakatan and TuckermanLC3reviewed four methods for decomposing iodinated contrast agents to liberate organically bound iodine: A. Parr bomb (fusion with sodium peroxide); B. alkaline permanganate reduction; C. Zinc-sodium hydroxide reduction; and D. oxygen flask (Schoniger) combustion. Method D is recommended as a general technique for all iodinated organic compounds because of its high reproducibility, simplicity, and rapidity of analysis (20 min. per test). For compounds that have all iodine atoms ortho or para to the electronegative carboxylic acid on the diatrizoic acid, method C, zinc-sodium aromatic ring s. hydroxide reduction, is recommended. In method C, the iodine is reduced under reflux conditions and replaced by hydrogen, generated by the reaction of powdered zinc with sodium hydroxide. The iodide thus formed, is determined by titration with silver nitrate in acidic solution, in the presence of the absorption indicator, tetrabromophenolphtalein ethyl ester4

.

Ates and hall4 decomposed diatrizoic acid with alkaline permanganate. After decoloration of the permanganate with sodium nitrite and acid, they titrated the liberated iodine with thiosulfate. Zak and B ~ y l e used ~ ~ chloric acid as an oxidizing digestion reagent to wet ash iodine-containing organic compounds. The iodine present in this solution as iodate may then be determined by titrimetry, spectrophotometry or by polarography. Strauss and Erhardt46 determined diatrizoic acid directly, in urine, by splitting off the organic iodine, followed by thiosulfate determination. They boiled urine samples with sulfuric acid at 150" for 5 minutes, cooled the sample, added potassium iodide, and titrated.

6.6

Chromatographic Analysis 6.61

Paper Chromatography

Many systems have been described in the literature for separation and detection of diatrizoic acid. These are summarized in Table VI. The drug substance may be quantitated from the developed chromatogram by elution 155

HYAM H. L E R N E R

in aqueous solution and subsequent spectrophotometric analysis by densitometry, or by other suitable means. Pileggi et.al.17, described a paper chromatographic precedure for separation of 19 or anic iodide compounds from blood serum. Ates and Amall' describe systemg4for separation of 8 contrast agents. B1 fox et.al. - , and Turula'' described 2 dimensional paper chromatographic procedures for separation of various iodinated contrast agents and iodinated proteins, from bile and serum. Key To Table VI, Solvent Systems

I Ethanol: 25% ammonia (ratio of solvents not given). 0.25u sodium citrate, pH 8.2. 4% ammonia (3:l). I11 =-butanol: IV toluene: ethanol: formic acid (10:3:3). alcohol: 2g ammonia (1:l) V =.-amyl VI water: formic acid (5:l) VII n-butanol: 95% ethanol: water (4:1:5); upper phase used for development. VIII n-butanol: 1J ammonium hydroxide: ethanol USP (5: 2:9 IX water: _n-butanol: ethanol USP (5:4: 1) ; upper phase used for development. X a-butanol: dioxane: ammonia (ratios not reported). I1

-

Methods of Detection A. Spray with 10% ceric sulfate and 5" sodium arsenite,

both prepared in

lE sulfuric

.

acid1.k

Detection B. Compound assayed was labeled with I ? accomplished by autoradiography with Kodak Radiography film. C. Viewed under ultraviolet light,

D. Spray with mixture of ceric ammonium sulfate and arsenious acid, followed by spraying with 0.5% solution of methylene blue18

.

E. Spray on both sides with a reagent containing 5 parts of a 2.7% solution of FeCl '6H 0 in 2N HC1, 5 parts of a 3.5% solution of K3$e(Ci)G and 1 part 156

TABLE VI

Paper Chromatographic Systems f o r S e p a r a t i n g D i a t r i z o i c A c i d S o l v e n t System

1 I1 I1 I1 I11 IV V VI VI I VIII IX X

Paper Whatman No. 3 Whatman AE 30 DE 20 Whatman No. 1 Whatman No. 3 Whatman No. 3 Whatman No. 3 Whatman No. 3 Whatman No. 1 Whatman No. 1 Whatman No. 4 Not r e p o r t e d

Method of Development descending ascending ascending ascending descending ascending descending descending descending descending descending descending

Method of Detection A, C B B B D E

E E C C C B

FS Not r e p o r t e d 0.76 0.66 0.73 0.57 Not r e p o r t e d 0.00 1.00 0.27 Not r e p o r t e d Not r e p o r t e d Not r e p o r t e d

Reference

14 16 16 16 17 19 19,24 19 21 22

23 24

HYAM H. LERNER

of an acified 5% NaAs02. The color is allowed to develop for 15 minutes, between glass plates, the paper wasked with water and dried with a current of warm air, Spraying and development are carried out in a dim-lit or dark room20

.

6.62 Thin-Layer Chromatography Thin-layer chromatographic methods found suitable for the separation and detection of diatrizoic acid, are summarized in Table VII. Some of the references cited, present sample preparation techniques and methods for eluting the drug from the developed plate, for quantitation by other means. Hollingsworth et.al., separated iodoamino acids and related compounds on cellulose plates with a solvent system composed of E . - b u t a n o l : 2N ammonia: chloroform (376:70:60). They did not report this technique for diatrizoic acid, however, it seems reasonable to expect that this system will separate iodinated contrast agents. Stahl and Pfeifle2’ reported on 6 systems to separate 17 iodinated contrast agents and Hoevel-Kestermann and Muhlemann12 separated 8 contrast agents, with a single system.

6.63

Electrochrmatography

Da~nenberg’~separated diatrieoic acid on Whatman No. 3 paper by high voltage paper electrophoresis. He used a pyridine: acetic acid water (100:10:890), pH 6 buffer, at a potential gradient of 45 v/cm, for 1 hour. Diatrizoic acid in this system has a mobility of 3 cm11800 vlhr. Turula” used a pH 8.6 diethylbarbiturate buffer system, Whatman Nos. 1 and 3 paper, a potential gradient of 6 v/cm, for 17 hours, to separate iodinated contrast agents. Gopal16 separated a number of iodinated compounds including diatrizoic acid, by paper electrophoresis on Whatman AE 30, Whatman No. 1 and DE 20 (W and R Balston Ltd) paper. Electrophoresis was accomplished in .025u sodium citrate buffer, at a potential gradient of 7 vlcm, 158

DlATRlZOlC ACID

Key To Table VII Solvent System I ethanol: ammonia, 25% (2:l) 11 benzene: acetone: formic acid (5:5:1) 111 ethylacetate: isopropanol: ammonia, 25% (11:7:4) IV acetone: isopropanol: ammonia, 25% ( V isopropanol: ammonia 25% (4:l) VI acetic acid: chloroform (1:19) VII ethylacetate: methanol: diethylamine (5:4:2) VIII chloroform: methanol: pyridine (17:1:2) IX methanol: chloroform: ammonia (10:20:2) X methanol: chloroform: ammonia (4:5:2) Plate Silica gel G (Merck) B Silica gel HF 254-366 (Merck) C Silica gel GF (Brinkman)

A

Detection System a.

Spray with 1:l solution of 10% ceric sulfate and 5% sodium aresenite, both in 1g sulfuric acid15.

b.

Autoradiography of H3 labeled diatrizoic acid.

c.

Ultraviolet light,

d.

Spray with 50% acetic acid, followed by irradiation at 254 nm for 10 min., to give blue violet spots.

159

TABLE VII Thin Layer Systems for Separation and Detection of Diatrizoic Acid Solvent System

I I1 I11 IV V

Plate A, J3 A

A, B

VI

A A A

VII VIII IX

A C

A

Detection System

Rf -

Reference

Not reported Not reported 0.12 0.30 0.37 0.40

.37

.oo

0.45

14 28 12,29 29 29 29 29 29 30

DlATRlZOlC ACID

in 1 hour. 6.7

Titrimetric Methods of Analysis

Sodium diatrizoate dissolved in dimethylformamide can be litrated potentiometrically with perchloric acid in dioxane

.

Although no references appear in the literature, it seems reasonable that a simple acid-base titration to a phenolphtalein endpoint, of diatrizoic acid in aqueous methanolic solution, would be successful.

6.8 Radiometric Methods of Analysis 6.81

X-Ray

Holynaka and Jankiewiczb8 used X-ray fluorescence and absorption techniques to determine iodine in various commercial contrast agent preparations. In the Xray fluorescence work, excitatiggn of the sample was accomplished by irradiation from a ' ~ m source, of 5-mci activity. The energy of excitation was 60 keV. Fluorescence of the K series of iodine (28.5 keV) was measured in a scintillation counter having a 6-mm thick NaI/Tl crystal. The characteristic radiation of iodine was separated by means of a single-channel, pulse-amplitude analyzer covering the total width of the K-peak iodine. Measurement time was 1 min. A calibration curve was made using HI03 as a standard. A 241Am source at 60 keV was used in the Xray absorption procedure. The detector was a scintillation counter equipped with a 6-mm thick NaI/T1 crystal. Absorption measurements were made by means of a single-channel, pulse-amplitude analyzer in the energy channel of 5keV, covering 60 keV. Calibration cutges were prepared, using H103 as a standard, The authors preferred the X-ray fluorescence technique because of its higher precision and accuracy. Schiller and Synek" used radiometric methods to determine iodine in various preparations of contrast agents. They used a measuring probe with a GM coun-

161

HYAM H. LERNER

t e r and scaler t o determine r e f l e c t e d r a d i a t i o n .

Buersch e t .al.'O determined t h e continuous spectrum of X-rays on aqueous s o l u t i o n s of d i a t r i z o i c a c i d . They r e p o r t e d t h a t Beer's l a w w i t h r e s p e c t t o l i n e a r a b s o r p t i o n , h o l d s , provided t h e e x c i t a t i o n beam i s s u f f i c i e n t l y monochromatic. The a p p l i c a t i o n t o b i o l o g i c a l systems i s a l s o discussed. 6.82

8- P a r t i c l e D i s p e r s i o n

Mikolajek et.al.51 used t h e method of r e t r o r a d e d i s p e r s i o n of b e t a p a r t i c l e s t o a s s a y c o n t r a s t media, "'Tl with about 3-pCi a c t i v i t y , d e p o s i t e d on a r i n g was used as t h e s o u r c e of r a d i a t i o n . A c a l i b r a t i o n showing t h e number of s c a t t e r e d e l e c t r o n s vs c o n c e n t r a t i o n w a s e s t a b l i s h e d . R e s u l t s by t h i s method a r e i n good agreement w i t h d e t e r m i n a t i o n s made by more c o n v e n t i o n a l methods.

7.

F'harmacolonv 7.1

Drug Metabolism

D i a t r i z o i c a c i d i s e x c r e t e d r a p i d l y and un hanged 1 r e l y through t h e kidneys by glomular f i l t r a t i o n 28,46,53,

5"'-

Tauxe et.alS3 s u g g e s t t h a t t h e r e i s a minor d e g r e e of plasma p r o t e i n b i n d i n g , i n man. Woodruff and MalvinS6 i n f e r r e d about 5-10% p r o t e i n b i n d i n g of d i a t r i z o a t e , b u t d i d not p r e s e n t d a t a . Stokes et.a1.57 r e p o r t e d t h a t 50% d i a t r i z o a t e was p r o t e i n bound, b u t b i n d i n g was e a s i l y reversed. No r e p o r t s were uncovered i n t h e l i t e r a t u r e sugg e s t i n g m e t a b o l i t e s of d i a t r i z o i c a c i d o r c o n c e n t r a t i o n of d i a t r i z o i c a c i d i n organs of man, o t h e r t h a n t h e kidney.

7.2

Toxicity

!a

Langecker et.al.' r e p o r t e d t h e L 0 of d i a t r i z o i c a c i d i n t h e r a t , t o b e 14.7 gfkg. Miller reported the LD50 of d i a t r i z o i c a c i d t o be about 10.1 gfkg, i n t h e mouse, SchmidS9 r e p o r t e d t h e LD50 o f sodium d i a t r i z o a t e t o b e 14.0 gfkg i n t h e mouse and 11.4 g f k g i n t h e rat. ?he LD50 of meglumine d i a t r i z o a t e was r e p o r t e d as 14.7 g l k g i n t h e rat59.

162

DlATRlZOlC ACID

Acknowledpement The author acknowledges with gratitude the cooperation of Ms. Cheryl Pernell for her patience in the preparation and correction of this manuscript. 8. References 1. Langecker, H., A. Harwart and K. Junkmann, Exp. Pathol. Pharmakol., 222, 584 (1954).

a.

2.

Puar, M., Squibb Institute, personal communication.

3.

Funke, P., Squibb Institute, personal ccnnmunication

4. Drug Standards, 25, 84 (1957). 5. Toeplitz, B., Squibb Institute, personal conrmunication. 6. Larsen, A., C. Moore, J, Sprague, B. Cloke, J. Moss and J. Hoppe, J. Am. Chem. SOC., 78, 3210 (1956).

7. Lugens, J.,

g . ,2,2836

(1896).

8. Lenkowski, P., T. Dzieciak, H. Cieslik and M. Drzejszczak, Polish Patent 62,290 (1967) ; -** Abstr 75, 151549 l~(1971).

m.

-

9. Kulhanek, J., J. Chromik, K. Skaba and K. Mejstrick, Czech. Patent 134,140 (1969); Chem. Abstr., 53288 i (1971).

74,

10.

Cassebaum, H. and K. Dierbach, German (East) Patent 43,994 (1965); Chem. Abstr., 64, 17494 (1966).

11. British Patent, assigned to Mallinckrodt Chemical Works, No. 782,313 (1957); Chem. Abstr., 52, 2916b (1958). 12.

Hoevel-Kestermann, H., and H. Miihlemann, Pharm. 47, 394 (1972).

Acta. Helv - 0 ’ -

163

HYAM H. LERNER

13. 14.

Ochs, Q.,

Squibb I n s t i t u t e , p e r s o n a l canmunication.

Ates, O.,

and H. Amal, Istambul Univ. E c z a c i l i k Fak. (1966).

m.,2, 82

.

Bull. SOC. Chim. b i o l . , 37, 89

15.

L i s s i t z k y , S., (1955)

16.

Gopal, N., (1966).

17.

P i l e g g i , v., R. Henry, M. Segalove and G. H a m i l l , Clin. Chem., 8, 647 (1962).

18.

Mandl, R. and R. Block, Arch. Biochem. Biophys, 25 (1959).

19.

Turula, K.,

20.

Gmelin, R. and A. V i r t a n e n , Acta Chem. Scand., l3, 1469 (1959).

21.

Brewer, G. tion.

22.

Soh, T., cation.

23.

Chow, L., cat ion.

24.

Blaufox, M., D. Sanderson, W. Tauxe. K. Wakim, A. Orvis and C. Owens, h e r . J. P h y s i o l . , 204, 536 (1963).

25.

J.

Appl. R a d i a t i o n I s o t o p e s ,

l7, 75

81,

Acta Endocrinol. 48, 31 (1965).

, Squibb

I n s t i t u t e , p e r s o n a l connnunica-

Squibb Q u a l i t y C o n t r o l , p e r s o n a l c a m u n i Squibb Q u a l i t y C o n t r o l , p e r s o n a l c m u n i -

Kabasakalian, P., and J. Mc G l o t t e n , Anal. Chem., 513 (1959)

31, -

26.

P u r k i s s , P., R. Lane, W. C a t t e l l , I. Fry, and A. Spencer, I n v e s t . Radiol., 3, 271 (1968).

27.

Dennenberg, T., (1966).

Acta Med. Scand. Suppl,

164

442,

17

DlATRlZOlC ACID

28. Davidson, A., Invest. Radiol.,

1,271

(1966).

29. Stahl, E. and J. Pfeifle, 2. Anal. Chem., (1964)

.

200,

377

30. Jacob, ?,.,Squibb Quality Control, personal communication. 31. Hollingsworth, D., M. Dillard and P. Bondy, J. Lab. Clin. , 62, 346 (1963).

- e.

32.

Willis, S., Squibb Institute, personal cmunication.

33.

Neudert, W., and H. Rapke, Chem. Ber., 87, 659 (1954).

34.

Valenti, V., Squibb Institute, personal communication.

35.

Kabadi, B., Squibb Quality Control, personal communicat ion.

36.

Boerdalen, B., H. Wang and H. Hotterman, Invest. (1970); Chem. Abstr., 7 4 , 123391 E (1971).

-RadioL, - 5, 595

z.

232,

37.

Hartmann, E. and H. RGpke, 268 (1967).

38.

Gonda, H., Squibb Quality Control, personal communication

39.

Poet, R., Squibb Institute, personal communication.

Anal, Chem.,

.

40. Hentrich, K. and S. Pfeifer, Pharmazie, 21, 296 (1966).

41.

Targos, F., Squibb Institute, personal communication.

42.

Strickler, H., E. Saier, E. Kelvington, J. Kempic, E. Campbell and R. Grauer, 2. endocrin. Metabol., 24, 15 (1964).

m.

165

HYAM H. LERNER

55,

43.

Yakatan, G. and M. Tuckerman, J. Pharm. S c i . , 532 (1966).

44.

Zak, B. and A. Boyle, J. h e r . Pharm. Assn., ed., 4 l , 260 (1952).

45.

E g l i , R.,

46.

87,

47.

K a l l o s , P., Squibb Q u a l i t y C o n t r o l , p e r s o n a l communication.

48.

Holynska, B. and J. Jankiewicz, Chem. Anal, (Warsaw), 14,219 (1969); Chem. Abstr., 7 l , 24774 -X (1969).

49.

S c h i l l e r , P., and J. Synek, Cesk. Farm., 15, 508 (1966); Chem. Abstr., 66, 79647 k (1967).

50

Buersch, J., R. Johs and P, Heintzen, F o r t s c h r . Geb. Roentgenstr. Nuklearmed. , 112, 259 (1970) ; Chem. Abstr., 72, 126781 k (1970).

51.

Mikolajek, E., J. Kormicki and Z. Kowalczyk, Pharm., Pharmacol, l8, 523 (1966); Chem. Abstr. 64, 14905 2 (1967).

g.

Anal. Chem.,

247,

sci.

39 (1969).

S t r a u s s , I., F. E r h a r d t and K. May, F o r t s c h r . Med., 1296 (1969).

w.

-

52.

Z a t z , L., Squibb Q u a l i t y C o n t r o l , p e r s o n a l communication,

53.

Tauxe W., M. Burbank, F. Maher and J. Hunt, Mayo C l i n i c , 39, 761 (1964).

54.

78,

55.

m o e f e l , P., Ann. Rev. Pharmacol.,

56.

Woodruff, M. and R. Malvin, J. Urol., (1960)

57.

tokes J J Conklin i 7 , 636 (€462)’.

e.

Mc Chesney, E. and J. Hoppe, h e r . J. Roentgen., 137 (1957).

166

2, 321

(1965).

84, 677

and H. Huntley, J. Urol.,

DlATRlZOlC ACID

58.

Miller, M., Squibb Institute, personal conrmunication.

59.

Schmid, H., Pharm.ActaHelv., 46, 134 (1971); Abstr. , 75, 3915 m (1971).

60.

Alicino, J., Squibb Institute, personal cmmunication.

167

m.

DISULFIRAM

Norris G. Nash and Raymond D.Daley

DISULFIRAM

CONTENTS 1.

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

2.

Physical Properties 2 . 1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectra 2 . 4 Mass Spectra 2.5 Differential Thermal Analysis 2.6 Solubility 2.7 Crystal Properties 2.8 Melting Point

3.

Synthesis

4.

Stability-Degradation

5.

Drug Metabolic Products

6.

Methods of Analysis 6 . 1 Identification Tests 6 . 2 Elemental Analysis 6.3 Colorimetric Methods 6 . 4 Titration Methods 6 . 5 Infrared Spectroscopy 6 . 6 Proton Magnetic Resonan 6.7 Polarography 6.8 Gravimetric Methods 6 . 9 Chromatography

7.

References

169

troscopy

NORRIS G. NASH AND RAYMOND D. DALEY

1.

DESCRIPTION

1.1 Name, Formula, Molecular Weipht The Chemical Abstracts name f o r d i s u l f i r a m is tetraethylthioperoxydicarbonic diamide, s t a r t i n g with Volume 76. Previously the Chemical Abstracts name was bis(diethylthiocarbamoy1)disulf ide. The CAS Registry No. Other common names a r e tetraethylthiuram is [ 97-77-81. d i s u l f i d e and TTD. S

CH CH 3 '\N-;-S-S-C-N CH,CH~'

"

/

CH CH

2

3

\CH~CH~

Mol. W t . :

296.54

1.2 Appearance, Color, Odor Disulfiram is a l i g h t yellow odorless c r y s t a l l i n e powder.

2.

PHYSICAL PROPERTIES

2.1 Infrared Spectra The i n f r a r e d spectrum of d i s u l f i r a m (Figure 1) was obtained with a Beckman IR-12 spectrophotometer. The spe t r u m was run i n three s e c t i o n s : ( a ) from 200 t o 650 cm-I as a m n e r a l o i l m u l l on polyethylene; (b) from 500 t o 1365 cm-' a s a mineral o i l m u l l between potassium bromide p l a t e s ; ( c ) from 1360 t o 4000 cm-l a s a perfluorinated o i l m u l l between potassium bromide p l a t e s . The spectrum matches those published elsewhere (1,2). Some of the absorption bands can be assigned t o the a l k y l groups: 2980 t o 2870 cm'l, C-H s t r e t c h i n g ; 1460 cm", C-H bending; 1378 cm'l, methyl C-H bending (symmetrical). The assignment of bands t o the various v i b r a t i o n s of the remainder of the molecule is complicated by the extensive coupling of v i b r a t i o n s of the N-C-S bonds and the apparent conjugation of the n i t r o g e n unshared e l e c t r o n p a i r with the C=S bond (2,3). The 1500 cm'l band has been assigned t o a C-N s t r e t c h i n g v i b r a t i o n (3) and the 1000 cm'l band t o a C=S s t r e t c h i n g v i b r a t i o n (9). Other suggested assignments ( 5 ) a r e : 926 crn'l, N-C-S; 976 cm'l,

8

C=S;

1149 and 1206 cm'l,

170

N-C-S;

II

1274 cm-l, N-C.

Figure 1.

Infrared spectrum of disulfiram, mineral oil and perfluorinated oil mulls.

NORRIS G.NASH AND RAYMOND D. DALEY

2.2 Nuclear Magnetic Resonance Spectra The proton magnetic resonance spectrum of disulfiram shown in Figure 2 was run in CDCl-j, on a Varian A-60A 60 MHz NMR spectrometer, with a tetramethylsilane reference (6). The sweep width is 0 to 500 Hz. The triplet at 1.40 pprn (J=7.3 Hz) is assigned to the methyl protons, while the quartet at 4.06 ppm (J=7.3 Hz) is assigned to the methylene protons. The small bands at 2 27 Hz of each Line are spinning sidebands (6). The temperature dependence of the NMR spectrum of disulfiram and similar compounds has been investigated (78). 2.3 Ultraviolet Spectra Figure 3 shows the ultraviolet absorption spectrum of disulfiram, run on a Cary Model 14 spectrophotometer. The spectrum was obtained at two concentrations, 100 ,g per ml and 10 u g per ml, in methanol solution, in 1 cm cells. 2.4 Mass Spectra The low resolution mass spectrum of disulfiram is shown in Figure 4. This spectrum was obtained on an LKB 9000s mass spectrometer, with an ionizatio voltage of 70 eV, source temperature 25OoC (6). This spectrum is similar to one reported and interpreted by Madsen et a1 (9); their suggested fragmentation scheme s shown in Figure 5.

2.5 Differential Thermal Analysis The only thermal event in the differential thermal analysis curve of this compound is the melting endotherm at 7OoC (10). Solubility The solubility of disulfiram at room temperature is as follows:2.6

172

Figure 2.

NMR spectrum of d i s u l f i r a m , C X 1 3 s o l u t i o n .

'-4

0.9

,

O.*1

.

w e

4

P

$

0.4

6

m 0

m

m 6

0.3 0.2i 0.1 -

',

',,

'\

'/

\ L 1 '

0.0--

Figure 3.

-c

Ultraviolet spectrum of disulfiram, 100 u g per ml and 10 ,,g per ml, in methanol, 1 cm cells.

> 100-

-

Ic

4

m

?W!

80-

f-

z - 60w

2 40-

I-r

I50

200

MASSICHARGE RAT 10

Figure 4.

Mar8 spectrum of disulfiram.

2 50

300

N O R R I S G. N A S H AND R A Y M O N D D. D A L E Y

C H 25\

C2H5 H

I

/

+

N:

C2H5

(88)

H

Figure 5.

Mass spectrometric fragmentation (9).

176

DlSULFlRAM

Solvent

Approximate S o l u b i l i t y , mg/ml

Me t hano 1

25

Ethanol (95%)

22

Ace tone

440

Water

0.3

Chloroform

740

Ether

80

Benzene

560

Petroleum e t h e r

2.9

2.7 Crystal Properties Grabar and McCrone reported the c r y s t a l p r o p e r t i e s of d i s u l f i r a m (11). The c r y s t a l system is-monociinic, with a x i a l r a t i o s a:b:c of 0.870 : 1 : 0.545, i n l e r f a c i a l angles ( 0 1 1 ~o i l ) = 47.5', p r o f i l e angle ( 0 1 1 ~011 i n 100 plane) = 123', and beta angle = 126'. The r e f r a c t i v e in0.005, @=1,67+ 0.01, dices (5893 A, 25'C) a r e cu=1.590 and y=1.740 0.005. The o p t i c a x i a l angle is 2V-= 84 2 5', dispersion v > r , o p t i c a x i a l plane 010, s i g n of double r e f r a c t i o n negative. The molecular r e f r a c t i o n is 84.2 (calculated), 85.0 (observed). Some a d d i t i o n a l information regarding c r y s t a l h a b i t , fusion behavior, and xray d i f f r a c t i o n is a l s o given i n t h i s paper. No polymorp h i s m was observed. Crabar and McCrone (11) a l s o reported x-ray d i f f r a c t i o n data f o r disulfiram. They found the c e l l dimensions t o be a313.844, b=15.90A, and c=8,66A, with four formula weights per c e l l . The d-spacings and r e l a t i v e int e n s i t i e s of the p r i n c i p a l d i f f r a c t i o n l i n e s a r e tabulated. Karle e t a 1 (12) made a complete c r y s t a l s t r u c t u r e a n a l y s i s . The molecule contains two planar S

+

+

I1

-S-C-N,

,c-

C-

groups, nearly perpendicular t o each other. The configur a t i o n about the nitrogen atoms is planar, not pyramidal. The space group i s P21/c, with four molecules i n the u n i t c e l l . Their u n i t c e l l parameters were a = l l . l l 2 .02, b=15.90 2 .03, c=8.66 + .02, 8=92' 42' 15'. The u n i t

-+

-

177

NORRIS G. NASH AND RAYMOND D. DALEY

TABLE I

X-RAY POWDER DIFFRACTION PATTERN OF DISULFIRAM

d (A) 9.11 7.94 7.59 6.36 6.15 5.55 5.24 5.11 4.77 4.55 4.50 4.40 4.32 4.21 4.16 4.08 3.96 3.83 3.79 3.73 3.62 3.54 3.46 3.41 3.34 3.28 3.23

d(A)

I/I1 LO

3.18 3.17 3.08 3.05 3.00 2.91 2.83 2.80 2.77 2.70 2.65 2.61 2.56 2.52 2.48 2.44 2.38 2.33 2.29 2.26 2.22 2.20 2.15 2.06 1.88 1.71

8 27

100 37 9 14 49 7 21 11 1 12 9 60 8 13 3 4 1 27 1 19

Ll 7 8 5

178

I/I1 23 23 5 6 16 10 3 4 12 4 3 2 2 10 8

5 3 3 4 4 10 5

7 5 4 2

DlSULF IRAM

c e l l reported by Grabar and McCrone can be mathematically transformed i n t o t h i s one. The x-ray powder d i f f r a c t i o n p a t t e r n f o r d i s u l firam is shown i n Table I. This p a t t e r n was obtained on a Norelco x-ray diffractometer w i t h n i c k e l - f i l t e r e d copper Kcr radiation. This p a t t e r n c l o s e l y resembles t h a t r e p o r t ed by Grabar and McCrone (11). 2.8 Melting Point The following melting p a i n t s have been reported: 67-71'C 70.5

68.4 74 69-70

72 69.5-70 3.

SYNTHESIS

Disulfiram has been prepared i n a v a r i e t y of ways. The usual method involves oxidation, by various means, of diethyldithiocarbamic a c i d , i t s sodium s a l t , or some o t h e r metal s a l t , t o give the d i s u l f i r a m product d i r e c t l y . Oxidants used include hydrogen peroxide (18, 19, 2 0 , 72), sodium hypochlorite ( 2 1 , 2 2 ) , chlorine o r bromine ( 1 3 , 15, 2 3 ) , sodium n i t r i t e and hydrochloric a c i d ( 2 4 , 731, POtassium ferricyanide (14), a i r ( 2 5 ) , and amonium persulf a t e (16). An a l t e r n a t i v e method is the r e a c t i o n of d i ethylamine monosulfide with carbon d i s u l f i d e (17). These r e a c t i o n s are shown i n Figure 6. 4.

STABILITY-DEGRADATION

No r e p o r t s of s t a b i l i t y s t u d i e s or of the format i o n of degradation products were found i n the l i t e r a t u r e , except f o r metabolic products (Section 5 ) . Several unid e n t i f i e d degradation products a r e formed i f an a l k a l i n e s o l u t i o n of d i s u l f i r a m i n methanol is heated t o 50°C f o r 2 hours, however, and a t least one unidentified product is formed when a n a c i d i c methanol s o l u t i o n is heated s i m i l a r l y (26).

179

X

rn

v ) - yI zN n N

X

3:

W

u u

W

N v)

+

0

i5 N

N

X 0 W

X 0

w

.

r(

N

W

n

X X u

W

+

cn-y I

v)

N

N

+

h

X

- l %

u N

X U

v I

v) I

N

v

X U

Pl

U

X

hl n hl

!2

+

v)

U

rl

U

XW u

X U

N

n

N

+

F

v):u VJ

XN

N n

I

?

I

v):u zN n hl

X

X*

U

U

W

h)

. N

180

N h

N .. v)

u

+

N

m X

N n

X

N

v)

W

u

+ +

N

h

X

W

X U

u

W

0)

VI

.r(

R

s

v)

. 9

DlSULF I RAM

5.

DRUG METABOLIC PRODUCTS

The reported metabolic products of disulfiram in various species are as follows: Metabolite

Species

Die thyldithiocarbamic Acid

Man Rat Rabbit Cattle Mice

Carbon Disulfide

Man Rat Rabbit Cattle Guinea Pig

N,N-Diethyldithiocarbamoyl1-thio-l3-D-glucopyranosiduronic Acid

Man Rat

Diethyldithiocarbamic Acid, Mice Methyl Ester

(36 1 (31932) (34)

Hydrogen Sulfide

Sulfate

Rat

(31)

Gessner and Jakubowski have proposed the metabolic scheme in Figure 7 ( 3 4 ) . 6.

METHODS OF ANALYSIS

6.1 Identification Tests Disulfiram is easily identified by the physical properties described in section 2 above. Where identification of disulfiram in formulations is necessary, i t can be extracted by solvents such as carbon disulfide. If identification of very small amounts is necessary, the colorimetric tests described in section 6 . 3 or polarog-

181

CH3CHZJ, ,N-C CH3CH2

CH CH 2;~-~>CH3CH2

‘s-s

‘\\ / ,C-N,

CH2-CH3 CH -CH3 2

CH3-CH

CH3-CH2,

S 2XN-C4 + -. CH3-CH2 ’ ‘SH

1

+ cs2

CH3CH2.N-C 4 CH3CH2 ’ \

CH3-CH2

,

I

A-‘

Other Metabolites

F i g u r e 7.

A

\

s-EH- (cH&1,-dHcmH

\ \ \

S

\

\

\

22 ..

CH3-CH2. 4S P C \ CH3-CH2 OH

4S

,N-C

\

\

HSCH~+

\ \ Y

=

s04 HCHO

-

--a

Glucuronic Acid Conjugate

Metabolic products of d i s u l f i r a m (34).

DISULFIRAM

raphy (section 6.7) may be useful.

6.2 Elemental Analysis The elemental composition of d i s u l f i r a m is as f o l -

lows : 7. Theory

Element

40.50 6.80 9.45 43.25

Carbon Hydrogen Nitrogen Sulfur

6.3 Colorimetric Methods The b a s i s f o r many of the colorimetric methods f o r disulfiram i s the formation of highly colored metal-diethyldithiocarbamate complexes. Very s e n s i t i v e procedures have been developed f o r use with a v a r i e t y of types of samples. Copper is the most frequently used complexing metal. Domar e t a 1 (27) developed a c o l o r i m e t r i c method f o r disulfiram i n excreta samples, based on the r e a c t i o n with cuprous iodide t o form the copper-diethyldithiocarbamate complex. Disulfiram was e x t r a c t e d with carbon t e t rachloride. The e x t r a c t was t r e a t e d with cuprous iodide t o y i e l d a brown-yellow complex. Diethyldithiocarbamic a c i d , a d i s u l f i r a m metabolite, was determined by t r e a t i n g the carbon t e t r a c h l o r i d e extracted aqueous phase with cup r i c s u l f a t e i n a buffer s o l u t i o n , followed by carbon t e t rachloride e x t r a c t i o n . Domar's method was adapted t o the determination of metabolites i n blood and urine by s e v e r a l i n v e s t i g a t o r s (29,37,38,39). Pa tzch (40) subs t i tuted cup r i c chloride f o r the cuprous iodide used by Domar. Parquot and Mercier (41) determined d i s u l f i r a m i n f a t samples a t the 5 t o 50 u g per g level. The d i s u l f i r a m was extracted i n t o petroleum e t h e r and reduced t o d i e t h y l d i thiocarbamate. The copper-dithiocarbamate complex was formed by treatment with a cupric sulfate-hydroquinone reagent, extracted i n t o chloroform, and determined colorime t r i ca 1l y Egorova and Trukhina (42) determined d i s u l f i r a m i n suppositories by carbon t e t r a c h l o r i d e e x t r a c t i o n , r e a c t i o n with cuprous iodide, and colorimetric measurement a g a i n s t a blank containing no cuprous iodide.

.

183

NORRIS G. NASH AND RAYMOND D. DALEY

Vignoli and Cristau (43) determined disulfiram by reduction of phosphomolybdotungstate in alkaline solution, to form a blue color. Fried et a1 (44) adapted the disulfiram identification color reaction in Clarke's manual of drug identification (45) to develop a quantitative procedure. Reaction of disulfiram with ethanol and sodium cyanide produces a blue color. The response is linear in the range 2 to 20 u g disulfiram per ml. Diethyldithiocarbamate and carbon disulfide do not interfere. Kofman and Arzamastsev (46) and Piotrowska (47) reported determining disulf iram by combustion by the oxygen flask method. The sulfur dioxide produced is bound as a nonvolatile complex by sodium tetrachloromercurate. The complex is determined colorimetrically by reaction with formaldehyde and pararosaniline in hydrochloric acid solution. 6.4 Titration Methods Disulfiram is not sufficientlv basic to be titrated with acid even in non-aqueous media. However, several titration methods based on complex formation or oxidation have been reported. Ferreira (48) titrated an acidified carbon tetrachloride solution, containing from 15 to 25 mg of disulfiram, with a 0.15 bromate-bromide solution. The following reaction is given.

s,

s

(C H ) -N-C-S-S-C-N-(C H )

2 5 2

26 HBr

2 5 2

+ 13

Br2

+ 4 H2S04 + 2 HCOOH + 2

+ 20

H20

(C2H5)2NH

Analyses of the reaction mixture for sulfate and diethylamine, as well as the amount of bromine consumed, support the stoichiometry given above. Varga (49) determined disulfiram in the drug substance and tablets by oxidation with excess 0.1l ceric sulfate and determination of the excess reagent by back titration with 0.1E ferrous sulfate. Sandri (50) oxidized disulfiram with an excess of periodic acid; after 30 minutes the excess periodic acid was determined volumetrically by addition of potassium iodide and titration of the iodine formed.

184

DlSULFlRAM

Roth ( 5 1 ) determined disulfiram i n crude products and fungicide formulations a f t e r conversion t o carbon dis u l f i d e . The sample was refluxed i n formic acid and the carbon d i s u l f i d e evolved was trapped i n an a l k a l i n e cadmium a c e t a t e solution. The cadmium a c e t a t e s o l u t i o n was neut r a l i z e d and t i t r a t e d with 0.1E methanolic iodine t o a starch-iodine end point, Bayer and Posgay (52,53) determined t h a t the addit i o n of mercuric a c e t a t e t o a g l a c i a l a c e t i c acid s o l u t i o n of disulfiram formed a complex t h a t consumed 2 moles of perchloric acid per mole of disulfiram. The sample was t i t r a t e d t o a gentian v i o l e t end point with perchloric acid i n g l a c i a l a c e t i c acid. Bukreev e t a 1 (54) treated disulfiram with 0.1E n i c k e l s u l f a t e i n an alcohol-ammonia solution. After 1 hour the excess nickel ion was t i t r a t e d with 0.lE t e t r a sodium edetate. The accuracy of the method was s t a t e d t o be within 2 0.6%. Li and Li ( 5 5 ) t r e a t e d a dioxane solut i o n of tetramethylthiuram d i s u l f i d e with an excess of s i l v e r n i t r a t e s o l u t i o n , separated the p r e c i p i t a t e , and determined the excess silver ion by t i t r a t i n g with thiocyanate solution. Two moles of s i l v e r reacted with 1 mole of tetramethylthiuram d i s u l f i d e . Fournier e t a 1 (56) determined disulfiram metabol i t e s and other compounds with C-S bonds i n urine samples by the c a t a l y t i c e f f e c t of the metabolites on the r e a c t i o n of iodine with iodazide. Scalicka ( 5 7 ) used sodium azide instead of iodazide t o determine d i s u l f iram metabolites by the c a t a l y t i c e f f e c t ; q u a n t i t a t i o n was by t i t r a t i o n of excess sodium azide with sodium arsenate a f t e r the r e a c t i o n was comp l e ted Stefek e t a 1 ( 5 8 ) determined the nitrogen content by a Kjeldahl method. The ammonia was collected i n a 2% boric acid s o l u t i o n , cooled i n an i c e - s a l t mixture, and t i t r a t e d with 0.05t-J hydrochloric acid. The standard devia t i o n f o r the determination of nitrogen was l e s s than 0.2%.

.

6 . 5 Infrared Spectroscopy Salvesen e t a 1 ( 5 9 ) determined disulfiram i n tab-

l e t s by infrared spectroscopy. The s t r e n g t h was d e t e r mined i n a carbon t e t r a c h l o r i d e s o l u t i o n using the absorpt i o n bands a t 7.41, 8.32, 9.92, and 10.31 microns with a r e l a t i v e e r r o r of 2%. The band a t 818 cm” (carbon d i s u l f i d e s o l u t i o n )

185

NORRIS G. NASH AND RAYMOND 0.DALEY

can a l s o be used. Diethyldithiocarbamic a c i d has no abs o r p t i o n bands near 818 cm'l. 6.6 Proton Magnetic Resonance Spectroscopy Sheinin e t a1 ( 6 0 ) determined d i s u l f i r a m i n the

drug substance and t a b l e t s by proton magnetic resonance spectroscopy. The average r e s u l t s on a t a b l e t composite were 100.8 2 1.4% of claim by magnetic resonance spectroscopy and 100.7 0.4% by a c o l o r i m e t r i c procedure (61).

-+

6 . 7 Polarography

Belitskaya (62) analyzed d i s u l f irarn i n rubber samples by micropolarographic procedure a t a 0.003M l e v e l using the wave a t -2.5V i n an ammonia-ammonium c h l o r i d e buffer. Taylor (63) used cathode-ray polarography t o make the determination i n the 1.5 t o 15 ppm range on rubber samples; a t t h i s l e v e l a l i n e a r response was obtained. Porter e t a 1 ( 6 4 ) used cathode-ray polarography on urine samples and increased the s e n s i t i v i t y and s p e c i f i c i t y by the a d d i t i o n of copper t o the sample. The wave f o r t h i uram a t -0.5V was replaced by a wave a t -0.65V and t h a t of the excess copper a t -0.23V. The method gave 85% recovery on urine samples a t the 0.5 ,,g per m l l e v e l and 95% recovery on samples e x t r a c t e d w i t h chloroform t o give a 2 , g per m l s o l u t i o n . Prue e t a 1 (65) used pulse polarography f o r determining d i s u l f iram i n t a b l e t samples. The pulse polarographic method i s more s e n s i t i v e than d.c. polarography and the peak is e a s i e r t o i d e n t i f y and q u a n t i t a t e . The use of an aqueous-ethanol a c e t a t e b u f f e r r a t h e r than an aqueous-e thanol a m o n i a b u f f e r e l i m i n a t e s the i n t e r f e r e n c e of diethyldithiocarbamate, increasing the s p e c i f i c i t y of the method. 6.8 Gravimetric Methods

Three papers describe oxidation of d i s u l f iram i n - various ways t o produce s u l f a t e , determined gravimetrica l l y as barium s u l f a t e . F e r r e i r a ( 4 8 ) used bromine, Wojahn and Wempe ( 6 6 ) used s t r o n g a l k a l i , and Parrak ( 6 7 ) used n i t r i c a c i d in the presence of amonium vanadate. 6.9 Chromatography Parker and Berriman (68) developed a s e p a r a t i o n system u t i l i z i n g s i l i c a g e l - C e l i t e adsorbents and 4

186

DlSULFlRAM

binary solvent mixtures to separate 32 rubber additives, one of which was disulfiram. S t r o m e (32) developed a method for the separation of proteins, diethyldithiocarbamate, and disulfiram in diluted plasma, liver homogenate, and diluted urine using a Sephadex G-25 gel filtration column. Goeckeritz (69) used paper chromatography to separate disulfiram from 4 other antimycotic drugs using paper impregnated with one of the following: (a) heptane; (b) heptane-xylene (1:l); (c) cyclohexane; (d) methanol-ethano1 (1:l); (el acetone with dimethylformamide; (f) paraffin. As stationary solvents acetone or hexane was used. For detection, iodoazide or mercury fluorescein was used. Gessner and Jakubowski (34) used descending paper chromatography on Whatman No. 1 or No. 4 paper to separate disulfiram metabolites. The following systems were employed: (a) n-hexane on paper impregnated with 50% dimethylformamide solution in ethanol; (b) cyclohexane-nbutanol (1:l) on paper impregnated with 507. ethanol solution of dimethylformamide. Detection was by observation of quenching spots under ultraviolet light. Farago (70) chromatographed biological extracts on silica gel G plates. The Rf values for disulfirarn were (a) 0.82, using methanol-ace tone-triethanolamine (1:1 : 0.03); and (b) 0.77, using ethylene dichloride-benzene-88% formic acid-H20. Palladium chloride was used to detect disulfiram. The unsprayed spot can be eluted from the silica gel with chloroform-ethanol (2:l) and the absorbance determined at 272 nanometers Drost and Reith (71) chromatographed biological extracts on Kieselgel G plates. Two solvent systems were used: (a) toluene-ethyl acetate-diethylamine (7:2:1), and (b) cyclohexane-diethylamine (9:l). The spots were detected by ultraviolet light irradiation or by spraying with a mixture of 3 m l 107. H2PtCl6, 100 ml 6% KI, and 47 ml of water. Unsprayed spots were scraped off and extracted with 1,2-dinitrochloroethane; the solution was evaporated and the residue taken up in methanol for ultraviolet determination.

.

187

NORRIS G. NASH AND RAYMOND D. DALEY

7.

REFERENCES

I.

C. J. Pouchert, "The Aldrich Library of Infrared Spectra", Aldrich Chemical Company, Milwaukee, Wisconsin, 1970, spectrum 369D. 2. A. T. Pilipenko and N. V. Mel'nikova, Zh. Neorg. Khim. 14 ( 7 ) , 1843-6 (1969); C.A. 71, 7 5 8 1 5 ~ . 3. L. J. C l l a m y , "Advances i n I n f r a r e d Group Frequencies", Methuen & Co. Ltd., London, 1968, pp. 212-214. 4. H. C. Brinkhoff and A. M. Grotens, Rec. Trav. Chim. Pays-Bas 90 ( 3 1 , 252-7 (1971). 5. M. L. Shankaranarayana and C. C. P a t e l , Spectrochim. Acta 21 (11, 95-103 (1965). 6. G. S c h i l l i n g y A y e r s t Research Laboratories, p r i va t e communicat ion. 7. H. C. Brinkhoff, A. M. Grotens, and J . J. Steggerda, Rec. Trav. Chim. Pays-Bas 89 ( l ) , 1117 (1970). 8. A. M. Grotens and F. W. P i j p e r s , Rec. Trav. Chim. Pays-Bas 92 (5), 619-27 (1973). 9. J. 0, Madsen, S.-0. Lawesson, A. M. Duffield, and C. D j e r r a s i , J. Org. Chem. 32, 2054-8 (1967). 10. C. E. Orzech, Ayerst Laboratories Lnc., p r i v a t e communication. 11. D. J. Grabar and W. C. McCrone, Anal. Chem. 22 (4), 620-1 (1950). 12. I. L. Karle, J. A. E s t l i n , and K. Britts, Acta Crystallogr. (2), 273-80 (1967). 13. R e H. Cooper, U.S, Patent No. 2,375,083, May 1, 1945; C.A. 40, 1875. 14. R. R o t h s t e i n a n d K. Binovic, Rec. Trav. Chim. Pays-Bas 73, 561-2 (1954). 15. M. M. Miville, Ger. Patent No. 1,023,030, Jan. 23, 1958; C.A. 54, 17274~. 16. B. L. R i c h a z s , Belg. Patent No. 648,878, Sept. 30, 1964; C.A. 63, 14714a. 17. E. S. Blake, J. Am. Chem. SOC. 65, 1267-9 (1943). 18. H. S. Adams and L. Meuser, U.S. Patent No. 1,782, 111; C.A. 25, 303. No. 281,099, Nov. 6, 1962; C.A. 59, 19. Span. Pate; 73 78a. 20. J. P. Zumbrunn, Fr. Patent No. 2,038,575, Jan. 8, 1971; C.A. 75, 87658r.

-

188

DlSULF I RAM

21.

22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

33. 34. 35. 36. 37.

38. 39. 40. 41 42.

P a t e n t No. 1,796,977; C.A. 25, 2598. S. L. Kemichrom, Span. P a t e n t No. 354,918, NOV. 16, 1969; C.A. 72, 110847a. J. C. Counts, R. T. Nelson, and W. R. T r u t n a , U.S. P a t e n t No. 2,777,878, Jan. 15, 1957; C.A. 2,968li. J. L. Eaton, U.S. P a t e n t No. 2,464,799, Mar. 22, 1949; C.A. 43, 4690h. W. L. Cox and A. Gaydash, Fr. P a t e n t No. 1,322,579, Mar. 29, 1963; C.A. 59, 9812. G. L. Boyden, Ayerst L a b o r a t o r i e s Inc., p r i v a t e communication. G. D o m a r , A. Eredga, and H, Linderholm, Acta Chem. Scand. 3, 1441-2 (1949); C.A. 44, 10025f. L. E l d j y r n , Scand. J. Clin. Lab. Invest. 2, 202-8 (1950); C.A. 45, 6298c. H. Linderholm and K. Berg, Scand. J. C l i n . Lab. I n v e s t . 3 , 96-102 (1951); C.A. 45, 10286e. R. FischTr and H. Brantner, Naturwissenschaften 50, 551 (1963). J. H. StrOmme, Biochem. J.-92, 25P (1964). J. H. Stromme, Biochem. Pharmacol. 2,393-410 (1965). J. H. Stromme, Biochem. Pharmacol. 15, 287-97 (1966). T. Gessner and M. Jakubowski, Biochem. Pharmacol. 21, 219-30 (1972). E. Merlevede and J. P e t e r s , Arch. Belg. Med. SOC., Hyg., Med. Trav. Med. Leg. 23, 513-51 (1965); C.A. 64, 1 8 2 2 4 ~ . J. Kaslander, Biochim. Biophys. Acta 71, 730-2 (1963). K. J. D i v a t i a , C. H. Hine, and T. N. Burbridge, J. Lab. Clin. Med. 2, 974-82 (1952). S. L. Tompsett, Acta Pharmacol. Toxicol. 2, 20-2 (1964). G. Bors and N. I o a n i d , Farmacia (Bucharest) 2, 593-a (1965); C.A. 64, 8 7 8 4 ~ H. Patzsch, Deut. Apoth. Ztg. 94, 1284-6 (1954); C.A. 53, 202291. C. P a q u o t and J. Mercier, Rev. Franc. Corps Gras 6 , 695-9 (1959); C.A. 54, 6158g. N. M. Egorova and V. I. Trukhina, Nauch. T r . Perm. Farm. I n s t . 4, 96-7 (1971); C.A. 79, 149338e. G. C. B a i l e y , U.S.

-

-

-

-

189

NORRIS G. NASH AND RAYMOND D. DALEY

43. 44. 45

.

46. 47. 48. 49.

50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60 61, 62. 63.

L. Vignoli and B. Cristau, Congr. SOC. Pharm. Fr. C. R., 9th, 221-8 (1957); C.A. 53, 20690f. R. Fried, A. N. Masoud, and M. Francis, J. Pharm. Sci. 62, 1368-9 (1973). E. G . C . Clarke, Editor, " I s o l a t i o n and I d e n t i f i c a t i o n of Drugs", The Pharmaceutical Press, London, England, 1969, p. 319. M. D. Kofman and A. P. Arzamastsev, Farm. Zh. (Kiev) 26, 52-6 (1971); C.A. 75, 91338d. A. P i o t G s k a , Diss. Pharm. Pharnucol. 24, 93-7 (1972); C.A. 76, 158430~. P. C. F e r r e i r a , Arquiv. biol. (Sao Paulo) 34, 103-5 (1950) E. Varga, A c t a Pharm. Hung. 28, 38-43 (1958); C.A. 53, 2541bc. G , Sandri, A t t i Accad. Sci. Ferrara 35, 17-24 (1957-58); C.A. 54, 18160d. H. Roth, Angew. Chem. 73, 167-9 (1961); C.A. 55, 13161e. I. Bayer and Mrs. G. Posgay, Acta Pharm. Hung. 2, 43-50 (1961); C.A. 56, 7431f. I. Bayer and E. Posgay, Pharm. Zentralh. 100, 65-71 (1961); C.A. 55, 14819a. A. I. Bukreev, V. V. Hinakova, and V. F. Soinikova, Med. Prom. SSSR l8, 32-3 (1964). M. L i and K. L i , Chung-Shan Ta Hsueh Hsueh Pao-Tzu Jan K'o Hsueh, 1959 (3), 26-8; C.A. 56, 6667. E. Fournier, L. P e t i t and A. Lecorsier, J. Eur. Toxicol. 2, 337-40 (1971); C.A. 76, 149631h. B. Scalicka, Prac. Lek l9, 408-12 (1967); C.A. 68, 58318e. S. Stefak, M. Blesova, and M. Zahradnicek, Cesk. Farm. 21, 64-6 (1972); C.A. 77, 39330~. B. S a l K s e n , L. Domange, J. Ann. Pharm. Fr. 13, 208-15 (1955); C.A. 49, 11959a. E. B. Sheinin, W. R. Benson and M. M. Smith, Jr., J. Ass. Offic, Anal. Chem. 56, 124-7 (1973). "Official Methods of Analysis", 1970, 11th edition, Association of O f f i c i a l Analytical Chemists, Washington, D. C., s e c t i o n 29.148. R. M. Belitskaya, Kauch. Rezina l9, 53-5 (1960); C.A. 55, 4025e. A. F. Taylor, Talanta ll, 894-6 (1964).

-

xy,

-

190

DlSULFlRAM

64 65. 66.

S. P o r t e r and A. Williams, J. 24, 144-145P (1972) D. G. Prue, C. R. Warner, and B. G.

Pharm. Pharmacol.

T. Kho, J. Pharm. Sci. 61, 249-51 (1972). H. Wozhn and E. Wempe, Arch. Pharm. (Weinheim) 285, 375-82 (1952); C.A. 47, 12126g. V. Parrak, Farmacia 22, 19-22 (1953); C.A. 49, 5776g. C. A. Parker and J. M. Berrimn, Trans. Inst. Rubber Ind. 28, 279-96 (1952); C.A. 47, 36021. D. Coeckeritz, Pharmazie 2,668-74 (1966); C.A. 66, 98510b. A. Farago, Arch. Toxikol. 22, 396-9 (1967); C.A. 67, 79553x0 R. H. Drost and J . F. Reith, Pharm. Weekbl. 105, 1129-38 (1970); C.A. 74, 23165. A. D. Cumnings and H. E. Siamons, Ind. Eng. Chem. c 20, 1173-6 (1928). J. Giral and C. Soler, Rev. SOC. Quim. Mex. 2, 167-70 (1958); C.A. 53, 11220d. 7

67. 68. 69. 70.

71. 72. 73.

-

-

ACKNOWLEDGMENTS The w r i t e r s wish t o thank Dr. B. T. Kho f o r h i s r e view of the manuscript, Dr. G. S c h i l l i n g of Ayerst Research Laboratories f o r h i s NMR and mass s p e c t r a l data, the library s t a f f f o r t h e i r l i t e r a t u r e search, and Mrs. B. Juneau f o r typing the p r o f i l e

.

191

ESTRADIOL VALERATE

Klaus Florey

ESTRADIOL VALERATE

CONTENTS 1.

Description 1.1 Name, Fornula, Ylolecular 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 Rotation 2.6 Melting Range 2.7 Solubility 2.8 Crystal Properties

3.

Synthesis

4.

Stability

5.

Drug Metabolic Products

6.

Methods of Analysis 6 . 1 Elemental Analysis 6.2 Spectrophotometric Analysis 6.3 Spectrofluorometric Analysis 6.4 Colorimetric Analysis 6.5 Chromatographic Analysis 6.51 Paper 6.52 Thin-Layer 6.53 Gas - Liquid

7.

Identification and Determination in Body Fluids and Tissues

8.

References

-

Degradation

193

KLAUS FLOREY

1.

Description 1.1 Name, Formula, Molecular Weight Estradiol Valerate is estra-l,3,5 (10) triene-3,17p-diol-17-valerate (pentanoate).

Mol. Wt. 356.51

c2 3H3203

1.2

Appearance, Color,Odor White, odorless, crystalline powder.

2.

Physical Properties 2.1 Infrared Spectrum The infrared spectrum of estradiol valeratel is presented in Figure 1. 2.2

Nuclear Magnetic Resonance Spectrum The NMR spectrum is presented in Figure 2. It was obtained on a 60 MHz spectrometer in deuterochloroform containing tetramethylsilane as an internal reference The following proton assignments were made2 :

194

Protons at

Chemical Shift T

C- 1H

2.86

C-2H

3.38 (quartet)

1H, 2H = 9 ; 2 H y 4 H =2.5

C-4H

3.43

2 H y 4 H = 2.5;4H, 6H =1

C-

18H

(doublet)

Coupling Constants T (in Hz)

(multiplet)

1Hy2H = 9

9.19 (singlet)

r \D

VI

C-17aH

5.25

(triplet)

ester - CH3

9.09 (triplet)

6.5

phenolic-OH

4.94 (singlet)

Concentration dependent

16H, 17H = 7.5

; 4

FREQUENCY (CM')

c

W

Q\

WAVELENGTH (MICRONS)

Figure 1.

Infrared Spectrum of Estradiol Valerate in Mineral Mull. Instrument: Perkin-Elmer 621.

1_

T

I

F i g u r e 2.

I I

NMR Spectrum of E s t r a d i o l V a l e r a t e i n D e u t e r a t e d Chloroform. Perkin-Elmer R-12B.

KLAUS FLOREY

2.3

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

Xmax

281 nm E1% 613 1c m

Fmax 281 nm; I m a x 287 nm;

= 20906 = 1800 ( s m a l l peak)

6

2.4

Mass Spectrum The low r e s o l u t i o n mass spectrum, shown i n F i g u r e 3 , was o b t a i n e d on an A E I MS-902 s p e c t r o m e t e r equipped w i t h a frequency modulated analog r e c o r d e r 2

.

I t demonstrates the e x p e c t e d M+ o f m/e 356. There i s a l s o t h e M+ o f an a c i d homolog a t m / e 370 p r e s e n t a s a minor component. T h e h i g h mass fragment i o n a t m / e 271 o c c u r s by the c l e a v a g e of t h e a c y l p o r t i o n of t h e e s t e r a t t h e C-17 p o s i t i o n . There i s complement fragment i o n a t m/e 8 5 t h a t r e p r e s e n t s t h e o t h e r p o r t i o n of t h e molecule. The i o n s a t m/e 253-255 r e p r e s e n t t h e c l e a v a g e of t h e e n t i r e C-17 moiety. Because i t i s an a r o m a t i c s t e r o i d , there a r e a series of s t r o n g fragment i o n s w i t h t h e p r o g r e s s i v e loss of f i r s t D-ring carbons, t h e n C-ring carbons and f i n a l l y B-ring c a r b o n s , The m/e 1 0 7 i o n c o n t a i n s t h e A-ring and t h e C-6 carbon. Phenols can a l s o e l i m i n a t e t h e oxygen Thus a second s e r i e s by e x p l u s i o n of CO o r HCO. of p r o g r e s s i v e loss o f carbons can o c c u r b u t does n o t produce a s many i o n s . The assignment o f some o f t h e d i a g n o s t i c i o n s i s d e p i c t e d below.

198

57 H2 -CH2 -CH2 -CH3

HO (+1H)107

1 hlhl

c h) 0

6

0

3hl IZI

2hl lhl

MASSKHARGE

F i g u r e 3.

Low R e s o l u t i o n Mass Spectra of E s t r a d i o l V a l e r a t e .

I n s t r u m e n t : AE1-902.

ESTRADIOL VALERATE

2.5

Rotation [a& + 44O (dioxane)3

2.6

Melting Ranqe Like many steroids, Estradiol Valerate does not melt sharply. The following melting range temperatures (OC) were reported: 144-1454 (from methanol-water) 156 146-1486 1477 142-1442 The thermomicroscopic6 and fusion properties5 of estradiol valerate have also been described. 2.7

Solubility Practically insoluble in water: soluble in caster oil, in methanol, in benzylbenzoate and in dioxane; sparingly soluble in sesame oil and in peanut oil8

.

2.8

Crystal Properties No polymorphism was found7. Isomorphic mixed crystals were formed with estradiol propionate5 The powder X-ray diffraction pattern is presented in Table 1’.

.

20 1

KLAUS FLOREY

Table I Powder X-ray D i f f r a c t i o n P a t t e r n of E s t r a d i o l V a l e r a t e

3.50 3. 3 1 3.22 3.14 3.02 2.96 2. 32

0.06 0.60 0.21 0.24 0.51 1.00 0.46 0.34 0.32 0.13

11.5 10.0 6.95 6.21 5.97 4.98 4.68 4.12 3.96 3.83

*d = i n t e r p l a n a r d i s t a n c e

0.19 0.34 0.08 0.07 0.09 0.08 0.05

na

2 sin@ = 1.539A;

**

R a d i a t i o n : K a l and K a l Copper

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 1.00

3.

Synthesis The s y n t h e s i s of e s t r a d i o l v a l e r a t e w a s f i r s t d e s c r i b e d b y Miescher and S c h o l z 4 by m e t h a n l y s i s of e s t r a d i o l 3 , 1 7 - d i v a l e r a t e .

202

0

II

OC- (CH2)3-CH3

Alternative1 the 3-benzoate has been removed selectively with sodium borohydride 2 7

.

Stability - Degradation Estradiol Valerate is very stable as a solid. In solution under certain conditions, particularily alkaline, saponification to valeric acid and estradiol can occur.

4.

KLAUS FLOREY

5.

Druq M e t a b o l i c P r o d u c t s An i n c r e a s e i n b l o o d and u r i n e e s t r o g e n

l e v e l s was n o t e d a f t e r a d m i n i s t r a t i o n o f e s t r a d i o l v a l e r a t e t o o v a r e c t o m i z e d lo and postmenopausalUwomen. N o m e t a b o l i t e s have been i d e n t i f i e d so f a r , a l t h o u g h e s t r a d i o l v a l e r a t e most l i k e l y f o l l o w s t h e pathway of e s t r a d i o l metabolism. 6.

Methods of A n a l y s i s 6.1 Elemental Analysis % Calc. C 77.49 H 9.05 0 13.46

% Found 4 77.54;77.42 9.00: 9.20

--

6.2

Spectrophotometric Analysis The U.V. a b s o r p t i o n a t 2 8 2 nm can be u s e d t o d e t e r m i n e e s t r a d i o l v a l e r a t e i n sesame o i l a f t e r e x t r a c t i o n w i t h 80% m e t h a n o l , however, w i t h an a c c u r a c y o f n o t b e t t e r t h a n 10% b e c a u s e o f i n t e r f e r e n c e from t h e o i l 1 5 . In the compendia1 a s s a y t h e a b s o r b a n c e o f an a l k a l i n e a s s a y s o l u t i o n a t 300 nm i s d e t e r m i n e d a g a i n s t an a c i d i c a s s a y p r e p a r a t i o n 8 . 6.3

Spectroflurometric Analysis A q u a n t i t a t i v e f l u o r o m e t r i c method f o r

determination of e s t r a d i o l v a l e r a t e i n o i l has been d e s c r i b e d 1 6 . A f t e r column c l e a n up t h e s t e r o i d concentration i n ethanol is determined i n a s p e c t r o f l u o r o m e t e r ( E x c i t a t i o n wave l e n g t h : 285 nm: F l u o r e s c e n c e maximum ca. 328 nm). 6.4

Colorimetric Analysis E s t r a d i o l v a l e r a t e responds t o colorimetric t e s t s used f o r e s t r o g e n s such a s t h e phenol r e a g e n t ( F o l i n - C i o c a l t e a u reagent,phosphomolybdic p h o s p h o t u n g s t i c a c i d ) i r o n - p h e n o l solut i o n 1 7 a n d t h e I t t r i c h m o d i f i c a t i o n o f t h e Kober

*,

204

t 4 0

reaction (see section 7). 6.5 Chromatographic Analysis 6.51 Paper The quantitative determination of estradiol valerate by paper chromatography has been described18. After spotting the paper strip strips were impregnated with 20% diethylene glycol monoethylether(Carbitol) in chloroform and developed for 3 hours with methylcyclohexane saturated with diethylene glycol monoethyl ether. The steroid is located on a guidestrip by spraying with Folin-Ciocalteau reagent, eluted with ethanol and quantitated fluorometrically. (Activation wave length at 280 nm, fluorescence at 310 nm). In this system estradiol stays at the origin. 6.52 Thin-Layer Thin-layer chromatographic systems have been tabulated in Table 11. Table I1

- -

Solvent S y s tem Ref. Ref. Absorbant Magnesium silicate Benzene-ethanol(9:l) 0.52 19 Magnesium silicate Chloroform 0.62 19 Stationery phase:mineral oil Silica gel G 0.64 20 Mobile phase:50% acetic acid Silica gel G Stationery phase:propylene glycol 0.60 22 Mobile phase:cyclohexane-pet. ether (1:l) Stationery phase:tetraethylene glycol Silica gel G -24 Mobile phase:xylene Detection systems. Phosphoric acid (1:5)spray (green color);conc. sulfuric acid, p-toluenesulfonic acid.

1

3

KLAUS FLOREY

Quantitative determination i n harmaceutical p r e p a r a t i o n h a s b e e n d e s c r i b e d5 3 , The t e t r a e t h y l e n e g l y c o l - x y l e n e system24 s e p a r a t e s e s t r a d i o l v a l e r a t e from e s t r a d i o l a n d i s t h e b a s i s f o r a compendia1 l i m i t t e s t f o r f r e e estradiol. 6.53

Gas-Liquid E s t r a d i o l v a l e r a t e can b e d e t e r m i n e d q u a n t i t a t i v e l y b y g a s chromatography u s i n g a 3% OV-17 on 80-100 mesh V a r a p o r t 30 column a t a column t e m p e r a t u r e o f 260025 A l t e r n a t e l y a 3% J X R ( m e t h y l s i l i c o n e po1ymer)on s i l a n i z e d 100-200 mesh Gas C h r o m P column a t a column t e m p e r a t u r e of 230° c a n be u s e d f o r t h e tetramethylsilyl derivate26.

.

I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n Body F l u i d s and T i s s u e s . A method12 h a s b e e n d e v e l o p e d t o d e t e r m i n e e x t r e m e l y low c o n c e n t r a t i o n o f t o t a l e s t r o g e n s i n b o v i n e t i s s u e s , making u s e o f a s h o r t e n e d v e r s i o n o f t h e e x t r a c t i o n p r o c e d u r e of G o l d z i e h e r 1 3 , f o l l o w e d by f l u o r o m e t r i c r e a d o u t u s i n g t h e I t t r i c h 1 4 m o d i f i c a t i o n of t h e Kober reaction. 7.

206

ESTRADIOL VALERATE

8. 1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

References T o e p l i t z , The S q u i b b I n s t i t u t e , P e r s o n a l Communication. A. I. Cohen, The S q u i b b I n s t i t u t e , P e r s o n a l Communication. N. H. Coy a n d C. M. F a i r c h i l d , The S q u i b b I n s t i t u t e , P e r s o n a l Communication. K. Miescher a n d C. S c h o l z , H e l v . Chim.Acta 20,1237(1937): U.S.Patent, 2,233,035 (1941). J. P. C r i s l e r , N. F. W i t t a n d M. H. C r i s l e r , Microchimica A c t a 1962,317. M. K u h n e r t - B r a n d s t a t t e r , E. J u n g e r a n d A . Kof l e r , Microchem. J. 2 , 1 0 5 ( 1 9 6 5 ) M. B r a n d s t a t t e r - K u h n e r t a n d E. J u n g e r , Microchimica Acta 1964,238. U.S.P. X V I I I Q. Ochs, The S q u i b b I n s t i t u t e , P e r s o n a l Communication. G. I t t r i c h a n d P. P o t t s , A b h a n d l . D e u t . Akad. W i s s , B e r l i n , K1.Med. 1 9 6 5 , 5 6 ; C.A. 64,11501 4 . (1966). R. K a i s e r , Symp. D e u t . G e r . E n d o k r i n o l . 8 , 2 2 7 ( 1 9 6 2 ) : C.A. 6 5 , 7 5 6 5 h ( 1 9 6 6 ) . H. K a d i n , The S q u i b b I n s t i t u t e , P e r s o n a l Communication. J. W. Goldzieher, R. A. B a k e r a n d E . C . R i h a , J. C l i n . E n d o c r . 2 l , 6 2 ( 1 9 6 1 ) . G. I t t r i c h , A c t a E n d o c r . 3 5 , 3 4 ( 1 9 6 0 ) . N. H. Coy a n d C. M. F a i r c h i l d , The S q u i b b I n s t i t u t e , P e r s o n a l Communication. Th. J a m e s , J o u r n a l of t h e AOAC 5 4 , 1 1 9 2 ( 1 9 7 1 ) : i b i d . 56,86 (1973). B r i t i s h P h a r m a c o p o e i a 1973 p. 330. H. R. R o b e r t s a n d M. R. S i i n o , J. P h a r m . S c i . 52,370 (1963). V. S c h w a r z , P h a r m a z i e 1 8 , 1 2 2 ( 1 9 6 3 ) . D. S o n a n i n i a n d J. A n k e r , Pharm. A c t a . H e l v . 42,54 (1967). B.

.

-

-

-

207

KLAUS FLOREY

21. 2 2.

23. 24. 25. 26.

27.

T. D i a m a n s t e i n and K. Lorcher, J. Anal.Chem. 191,429(1962). A. Vanden B u l c k e , Pharm. T i j d s c h r . B e l g . 4 6 , 2 2 1 (1969) C.A. 7 2 , 1 0 3 7 9 0 y ( 1 9 7 0 ) . N. A r i , T u r k H i j . Tecr. B i y o l . Derg. 2 9 , 2 0 0 (1969) : C. A. 73,4856933 ( 1 9 7 0 ) . H. K l e i n and S. Hays, Drug S t a n d a r d s L a b o r a t o r y , P e r s o n a l Communication. Th. James and B. R a d e r , F.D.A. B y - l i n e s 4, 1 6 1( 1 9 7 2 ) . G. C a v i n a , G. M o r e t t i and P. S i n i s c a l c h i , J. Chromatog. 4 7 , 1 8 6 ( 1 9 7 0 ) . K. T s u n e d a , J. Yamada, K. Yasuda and H . M o r i , C h e m . Pharm. Bull. (Tokyo) ll,510 ( 1 9 6 3 ) , J a p a n P a t e n t 2 0 , 1 6 3 ( 1 9 6 4 ) :C. A. 62,1048421 (1965).

-

L i t e r a t u r e s u r v e y e d t h r o u g h December 1972. The h e l p o f H. Gonda and A . Mohr 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 i s g r a t e f u l l y acknowledged.

208

HYDROXYPROGESTERONE CAPROATE

Klaus Florey

KLAUS FLOREY

CONTENTS 1. 2.

3. 4.

5. 6.

7.

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 Spectra 2.5 Rotation 2.6 Melting Range 2.7 Solubility 2.8 Crystal Properties Synthesis Stability - Degradation Drug Metabolic Products Methods of Analysis 6 . 1 Elemental Analysis 6.2 Spectrophotometric Analysis 6.3 Spectrofluorometric Analysis 6.4 Polarimetric Analysis 6.5 Polarographic Analysis 6.6 Chromatographic Analysis 6.61 Paper 6.62 Thin-Layer References

210

H Y DROXYPROGESTE RONE CAPROATE

1.

Description 1.1 Name, Formula, Molecular Weight Hydroxyprogesterone caproate is 17-~-(1-oxohexyl)oxy~-4pregnene-3,20-dione; also 17-hydroxypregn-4-one-3,2O-dione hexanoate

# 20

'iH3 c=o OCOCH2 (CH2)3CH3

0

Mol.Wt. : 428.62

C27H4004 1.2

Appearance, Color, Odor White or creamy white odorless crystalline powder. 2.

Physical Properties 2.1 Infrared Spectrum The infrared spectrum of hydroxyprogesterone caproate is presented in figure 1.1 2.2 Nuclear Maqnetic Resonance Spectrum The NMR spectrum is presented in figure 2. It was obtained on a 60 MHz NMR spectrometer in deuterochloroform containing tetramethylsilane as an internal reference. The following proton assignments were made: 6 Protons at Chemical Shift

c-4 C-18 c-19 c-21 Ester methyl

4.20 singlet 9.30 singlet 8.80 singlet 7.96 singlet 9.08 multiplet, coupling constant 6.5 Hz

21 1

Figure 1.

Infrared Spectrum of Hydroxyprogesterone Caproate in mineral oil mull. 1nstrument:Perkin Elmer 621.

F i g u r e 2.

NMR Spectrum o f Hydroxyprogesterone C a p r o a t e i n D e u t e r a t e d Chloroform. I n s t r u m e n t :Perkin-E l m e r R-12B.

KLAUS FLOREY

2.3

U l t r a v i o l e t Spectrum I m a x 241 nm;E = 170008

2.4

Mass Spectrum The l o w r e s o l u t i o n mass spectrum, shown i n f i g u r e 3, w a s o b t a i n e d on an A E I MS-902 s p e c t r o m e t e r equipped w i t h a f r e q u e n c y modulated analog t a p e r e c o r d e r . 6 I t d e m o n s t r a t e s t h e e x p e c t e d M+ of m/e 428. The m/e 385 i o n r e p r e s e n t s t h e loss of t h e C-17 a c e t y l group o r C3H7 from t h e a c y l a t e , probably t h e former i s t h e predominant pathway. The loss of t h e C-17 a c y l a t e g i v e s r i s e t o t h e m/e 330 i o n and t h e m/e 312, 313 i o n s . Prominent i o n s a t m/e 7 1 and m/e 99 correspond t o t h e a c y l a t e p o r t i o n of t h e molecule. The m/e 287 i o n i s shown below. The m / e 269 i o n can be envisoned a s t h e d e h y d r a t e d i o n of t h e m / e 287 ion. A s e r i e s of fragment i o n s occur from t h e p carbons r o g r e s sbi vu et , loss of D-ring, C-ring and B-ring

&

m/e 269

0

m / e 287 while present, a r e n o t p a r t i c u l a r l y in ten s e enough t o i n d i c a t e t h e p r e s e n c e of t h e A4-3-keto group. However, t h e i o n s a t m/e 121-124 a r e p r e s e n t and have t h e compositions e x p e c t e d f o r t h e A-ring group. The assignment o f some of t h e d i a g n o s t i c i o n s i s d e p i c t e d below. 6

-

214

I

215

0

m

m

0 k pc Q)

u C 0

0)

k 4J 10 Q)

F

h

0 LI pc X

0 LI

h

a 0

X w C

LI

Q)

4J

m

4J

o(

&

F

0 W

Figure 3.

0 0 T ( 0

Low Resolution Mass Spectrum of Hydroxyprogesterone Caproate. Instrument : AE1 MS-902.

HYDROXYPROGESTERONE CAPROATE

2.5

Rotation

DJ;”= +

61’

( c = 1, i n c h l o r o f o r m ) 2

2.6

M e l t i n q Ranqe L i k e many s t e r o i d s , h y d r o x y p r o g e s t e r o n e c a p r o a t e d o e s n o t m e l t s h a r p l y , and t h e m e l t i n g p o i n t depends on t h e r a t e of h e a t i n g 7 . The f o l l o w i n g m e l t i n g r a n g e t e m p e r a t u r e s (OC) were r e p o r t e d . 117-1228 120.0-121.09 115- 1183 119-1212 120.1-121.910 The t h e n n o m i c r o s c o p i c 8 and f u s i o n p r o p e r t i e s 1 1 of h y d r o x y p r o g e s t e r o n e c a p r o a t e h a v e a l s o been d e s c r i b e d . 2.7

Solubility Insoluble i n waterlo 10 50 mg/ml i n e t h y l e t h e r 1 . 2 mg/ml i n b e n z e n e l o 25-29 mg/ml i n sesame o i l 2 350-400 mg/ml i n l e v u l i n i c a c i d butyl ester2 S o l u b l e i n a m i x t u r e of b e n z y l 10 b e n z o a t e and sesame o r c a s t o r o i l

217

.

KLAUS FLOREY

Crystal Properties Dense needles from isopropanol2 The powder X-ray diffraction pattern is presented in Table I. 2.8

.

TABLE I Powder X-ray Diffraction Pattern of Hydroxyprogesterone Caproa te2

**

(2:

I/I 1

12.2 9.1 7.15 6.90 6.63 6.35 5.80 5.47 5.28 5.08

0.26 0.11 0.31 0.10 0.13 0.37 1.00 0.42

d

*d

0.21 0.28

4.64 4.53 4.05 4.00 3.88 3.80 3.70 3.62 3.30 2.88

0.21 0.33 0.09 0.11 0.10 0.11 0.15 0.11 0.08 0.09

-

= interplanar distance n b 2 sin0 0

A= 1.539A; Radiation; K 1 and K Copper ** Relative intensity based on highest intensity of 1.00.

218

3.

& -:fl Synthesis

p 3

c=o

e=0

OH

anhydride

CO (CH2)4CH3

0

c!

\D

Hydroxyprogesterone caproate is synthesized by acylation of 17a-hydroxyprogesterone (cf.4) with caproic acid in the presence of p-toluene sulfonic acid3. Variations of this basic method have also been It can also be prepared by oxidation of the corresponding reported’. 5-prengnen-17-01-3-one caproate2. 4. Stability - Degradation Hydroxyprogesterone caproate is very stable a s a solid. The 17a-esterlinkeage issterically hindered and therefore quite stable, but can be saponified under forcing conditions. In solution photolykic degradation of the A-ring is possible, when exposed to ultraviolet light or ordinary fluorescent laboratory lighting (cf. 12).

KLAUS FLOREY

5.

Drug Metabolic Products Excretion and tissue distribution of labeled hydroxyprogesterone caproate was studied in ratsl3, steers14 and pregnant women 16 Langeckerl5 was not able to recover unchanged hydroxyprogesterone caproate in amounts exceeding 0.1%after administration to man. Plotzl6 incubated rat liver homogenates with 4-14Clabeled hydroxyprogesterone caproate under anaerobic conditions and recovered allopregnane38, 17a-diol-20-one-17a-caproate in 25% yield and pregnane-3@, 17a-diol-20-one-17a-caproate in 2% yield, together with some unchanged starting material. That the caproic acid ester was not removed attest to the stability of the bond (see section 4) and there is evidence that metabolites in urine also still carry the ester group. Plotz also found that in homogenate of human placenta the 20-keto group was not reduced under conditions which reduce the 20-keto group of progesterone.

.

6. Methods of Analysis 6.1 Elemental Analysis Calc. % C 75.66 H 9.41 0 14.93

6.2

Spectrophotometric Analysis The compendia1 assay of hydroxyprogesterone caproate is based on the U.V. absorption maximum at 240 nm17. 6.3

Spectrofluorometric Analysis Fluorescence was used for detection in biological f luids15.

220

HYDROXYPROGESTERONE CAPROATE

6.4

P o l a r i m e t r i c Analysis Rotation has been used t o determine hydroxyprogesterone c a p r o a t e i n o i l vehicles18. 6.5

Polarographic Analysis The h a l f wave p o t e n t i a l (E-1/2 v e r s u s s t a n d a r d calomel e l e c t r o d e ) was determined a s -1.65 v o l t s i n 1M l i t h i u m c h l o r i d e i n 95% methanol due t o t h e r e d u c t i o n of t h e a, B uns a t u r a t e d ketone22. The d i f f u s i o n c u r r e n t c o n s t a n t ( I d ) was found t o be 5.27 f 3%. G.6

Chromatoqraphic Analysis 6.61 Paper The 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 and s e p a r a t i o n from t h e - f r e e s t e r o i d of hydroxyprogesterone c a p r o a t e by paper chromatography i n o i l y veh4cles has been d e s c r i b e d by Roberts and Florey19. A f t e r s p o t t i n g , t h e paper s t r i p s were impregnated w i t h 30% d i e t h y l e n e g l y c o l monoethyl e t h e r ( C a r b i t o 1 ) i n chloroform and developed f o r 4 hours with methylcyclohexane s a t u r a t e d with d i e t h y l e n e g l y c o l monoethyl e t h e r . A f t e r l o c a t i n g of t h e s t e r o i d s p o t s w i t h a f l u o r e s c e n t paddle, t h e s p o t s a r e e l u t e d and r e a c t e d w i t h i s o n i c o t i n i c a c i d hydrazide i n methanol c o n t a i n i n g a l s o h y d r o c h l o r i c a c i d . The absorbance of t h e yellow hydrazone i s r e a d a t 415 nm. 6.62

Thin-Layer Thin-layer chromatographic systems have been compiled i n Table 11.

22 1

Table I1 Absorbent Magnesium silicate Magnesium silicate Magnesium silicate Magnesium silicate Magnesium silicate Magnesium s i1icate Magnesium silicate Silica gel Silica gel Silica gel Alumina

Solvent System Benzene-Ethanol (98:2) Benzene-Ethanol (9:1) Chloroform Chloroform-Ethanol (98 :2) Chloroform-Ethanol (96:4) Benzene-Dioxane (2:l) Ether-Ethanol (98:2) Benzene-Acetone (4:1) Benzene-Methanol (9: 1) Pet. ether, Benzene, Acetic acid, Water (67:33:85:15) Benzene-Acetone (4:1)

Rf -

Ref.

0.35 0.61 0.44 0.53 0.60 0.71 0.83 0.67 0.61 0.44

20

0.61

20 20 20 20 20 20

21 21 21

21

Detection system: U.V. light; sulfuric acid (dark brown): sulfuric-acetic acid (yellow red); vanillin-sulfuric acid (dark yellow)

HYDROXYPROGESTERONE CAPROATE

7.

References

1. B. T o e p l i t z , The S q u i b b I n s t i t u t e , P e r s o n a l Communication. 2. E. K a s p a r , K. H. Pawlowski, K a r l Junkmann and M a r t i n Schenk, U . S . P a t e n t 2,753,360 (1956). 3. V. M. B a k s h i and Y. K. Hamied, I n d . J. Chem. 2 ,294 ( 1964) 4. L. F. F i e s e r and M. F i e s e r , " S t e r o i d s " R e i n h o l d P u b l i s h i n g Corp. 1959. 5. B r i t . P a t e n t 848,881 ( C h e m . A b s t r . 57,2348g) ; J a p a n P a t e n t 3574(62 ) (C.A. 58,91896); Belg. P a t e n t 661,975 (C.A. 65,5510) ; U. S. S. R. P a t e n t 210,858 (C.A. B Y 9 6 , 9 9 8 e ) . 6. A. I. Cohen, The S q u i b b I n s t i t u t e , P e r s o n a l Communication. 7. I. Gyenes and A. L a s z l o , Magyar Kem. F o l y o i r a t 67, 360 (1961) (C . A . 56,149506, 1962). 8. M. K u h n e r t - B r a n d s t a t t e r , E. J u n g e r and A. K o f l e r , Microchem. J. 9,105 (1965). 9. K. Junkmann, Arch. e x p e r . P a t h . u. Pharmakol. 223,244 (1954). 10. G. A. B r e w e r , The S q u i b b I n s t i t u t e , P e r s o n a l

-

.

-

Communication

.

11. J. P . C r i s l e r , N. F. W i t t a n d M. H . C r i s l e r , M i c r o c h i m i c a Acta 1962,317. 1 2 . D. R. B a r t o n and W. C. T a y l o r , J.Am.Chem.Soc. 80,244 (1958) ;J. Chem.Soc. 1958,2500. 13. H. K i e s l i n g and1 A . E l m q u i s t , Acta. E n d o c r i n o l . 28,502 (1958). 14. F.X. G a s s n e r , R. P. M a r t i n , W. Shimoda and J. W. Algeo, F e r t i l i t y and S t e r i l i t y l l , 4 9 (1960). 1 5 . H. L a n g e c k e r , A. H a r w a r t a n d K. Junkmann, Arch. e x p t l . P a t h o l . Pharmacol. 225,309 (1955). 16. M. Wiener, C. I. Lupu a n d E. J. P l o t z , A c t a . E n d o c r i n o l . 3 6 , 5 1 1 (1961).

-

-

223

KLAUS FLOREY

1 7 . U.S.P. XVIII. 18. V. Gerosa and M. Melandri, Ann.Chim. ( R o m e ) 4 7 , 1 3 8 8 , (C.A. 5 2 , 7 6 2 0 ( 1 9 5 8 ) ) 1 9 . H. R. Roberts a n d K. F l o r e y , J . P h a r m . S c i . 51,794 (1962). 20. 7 S c h w a r z , P h a r m a z i e 1 8 , 1 2 2 (1963). 21. S. Hara a n d K. Mibe, Chem.Pharm.Bul1. 15,1036 (1967). 22. N. H. Coy a n d C. M. F a i r c h i l d , T h e S q u i b b I n s t i t u t e , P e r s o n a l Communication. 23. Q. Ochs, T h e S q u i b b I n s t i t u t e , P e r s o n a l

.

Communication. L i t e r a t u r e s u r v e y e d through December 1 9 7 2 .

Gonda and A. Mohr 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 i s g r a t e f u l l y acknowledged.

The h e l p of H.

224

ISOSORBIDE DINITRATE

Luciano A . Silvieri and Nicholas J . DeAngelis

L U C I A N 0 A. SlLVlERl AND NICHOLAS J. DeANGELIS

CONTENTS 1. 2.

3. 4. 5. 6. 7.

8.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Physica1 Properties 2 . 1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectrum 2 . 4 Mass Spectra 2.5 Optical Rotation 2 . 6 Melting Range 2.7 Differential Thermal Analysis 2.8 Solubility 2.9 Crystal Properties S ynthesis Stability and Degradation Metabolism Identification Aesay Methods 7.1 Elemental Analysis 7.2 Colorimetric Analysis 7.3 Infrared Analysis 7.4 Gas Liquid Chromatographic Analysis 7.5 Polarographic Analysis 7.6 Automated Analysis 7.7 Radiochemical Analysis 7.8 Thin Layer Chromatographic Analysis 7 . 9 Paper Chromatographic Analysis References

226

ISOSORBIDE DINITRATE

1.

Description 1.1

Name, Formula, Molecular Weight

Isosorbide d i n i t r a t e i s designated by t h e f o l lowin chemical names: 1,4: 3,6-dianhydro-~-glucitol d i n i t r a t e ; 1,4:3,6-dianhydrosorbitol 2,5-dinitrate1; d i n i t r o sorbidel. I n Chemical Abstracts t h e compound i s l i s t e d under the heading (Glucitol:1,4:3,6-dianhydro, " d i n i t r a t e , Dtl). Some of the commonly used t r a d e o r t r i v i a l names are: I s o r d i l , Isorbid, Vascardin, and Carvanil.

f

1.2

" 4

Appearance, Color, Odor

Isosorbide d i n i t r a t e i s a white, odorless, cryst a l l i n e powder. However, d i l u t e d isosorbide d i n i t r a t e , which i s a d r y mixture of isosorbide d i n i t r a t e w i t h lact o s e , rnannitol, o r o t h e r i n e r t e x c i p i e n t s t o permit s a f e handling, occurs as an ivory-white powder. The mixture u s u a l l y contains about 25% of isosorbide d i n i t r a t e .

2.

Physical Properties

2.1

Infrared Spectra

The i n f r a r e d (1.R.) spectrum of isosorbide d i n i t r a t e 4 6 is given i n Figure 1.2 The I.R. spectrum was obtained i n a KBr p e l l e t and i s i d e n t i c a l t o t h a t pubAssignment of some of t h e s i g l i s h e d by Sammul et.al.3 n i f i c a n t absorption bands i s given i n Table I. Table I Infrared Spectral Assignments of Isosorbide D i n i t r a t e Frequency (cm.-l> Vibration Mode Reference 2950-2850 a l i p h a t i c C-H s t r e t c h i n g 48 47 1665 and 1635 asymnetric N 4 s t r e t c h i n g 1460 methyl ene s c i s soring 48 vibration 1285-1270 symmetrical N 4 s t r e t c h i n g 47 rVllO0 asymnetrical C-0-C s t r e t c h i n g 48 0-N4 s t r e t c h i n g 47 865 221

Figure 1

-

I.R. Spectrum of Isosorbide Dinitrate, Lot No. 5123 V 6/1. Instrument: Perkin Elmer Model 21.

0.25% KBr P e l l e t

-

ISOSORBIDE DINITRATE

Hayward et.al .4 reported t h e n i t r a t o and hydroxyl s t r e t c h i n g bands of n i t r a t e e s t e r s of 1,4:3,6-dianhydroh e x i t o l s , including isosorbide d i n i t r a t e , i n d i l u t e solut i o n s of benzene, a c e t o n i t r i l e , chloroform, isopropyl e t h e r , methylal, pyridine, and carbon t e t r a c h l o r i d e .

2.2

Nuclear Magnetic Resonance Spectra

The nuclear magnetic resonance (N.M.R-) spectrum (Figure 2) w a s obtained by preparing a s a t u r a t e d s o l u t i o n of isosorbide d i n i t r a t e 4 6 i n deutero chloroform containing tetramethylsilane as i n t e r n a l r e f e r e n ~ e . ~There a r e no exchangeable protons. The NMR proton s p e c t r a l assignments a r e given i n Table 11. Table I1 NMR Spectral Assignments of Isosorbide D i n i t r a t e Chemical S h i f t (ppm.1 Pro ton 3.9-4.2 -0-%

Hopton and Thomas6 have reported on NMR s t u d i e s of isosorbide which e l u c i d a t e the conformation of t h i s molecule. Their findings i n d i c a t e t h e conformation i s a composite of t h e envelope and half-chair forms. 2.3

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

Isosorbide d i n i t r a t e gives no eak maxima i n t h e but does absorb wavelength region of 400 nm. t o 220 run.’, U.V. l i g h t . The absorption begins a t about 290 nm. and continually increases as the wavelength decreases. I n U.S.P. alcohol a t a concentration of approximately 0.1 mg./ m l . an absorbance of 0.1 i s obtained a t 50 run., and an absorbance of 1.5 i s obtained a t 220 nm.

3

2.4

Mass Spectra

The mass s p e c t r a of isosorbide d i n i t r a t e 4 6 were obtained’ by d i r e c t i n s e r t i o n of the sample i n t o an MS-902 double focusing mass spectrometer modified with a SRI dual C I E I source. Both low r e s o l u t i o n e l e c t r o n empact (EI) and chemical i o n i z a t i o n (CI) mass s p e c t r a were obtained. For t h e E I spectrum the ionizing e l e c t r o n beam energy w a s 70 eV and f o r the C I spectrum the beam energy was 500 eV. The mass spectrum assignments’ of t h e prominent 229

I

N

W

0

Figure 2

- NMR Spectrum of

Isosorbide Dinitrate, Lot No. 5123 V 6/1. Solvent: deutere chloroform, internal standard: tetramethylsilane. Instrument: Varian A-60.

ISOSORBIDE DINITRATE

ions under b a h E I and C I conditions a r e given i n Table 111.

&I

23 7 190 144 127

Table I11 Mass Spectral Assignments of Isosorbide D i n i t r a t e Chemical Ionization Electron Im act R.I.(%> m/e R.I. % S ecies 15 2362.5 M+ 24 W-HNQ 143 75 M-HN4, N 4 MIP-HNQ, N Q 126 60 100 M-HNC+, HNQ M-HNOZ, CHON4 27 N 4 114 100 MH+-HN%, H 100 25 M - H N 4 , WCHONQ 2.5

O p t i c a l Rotation

The s p e c i f i c rotati0nL4];~ o f ' isosorbide d i n i t r a t e46 w a s determined 7 t o be +137O i n U.S.P. alcohol a t a concen1 t r a t i o n of 3 mg./ml. The Merck Index r e p o r t s anb];5 of +135O. Jackson and Hayward" and Goldberg" +134O. 2.6

fpund thelo(]:O

t o be +141°

Melting Range

There a r e two ranges reported i n t h e l i t e r a t u r e f o r the melt of isosorbide d i n i t r a t e . The Merck Index1, Wiggins12, Goldberg", and Haywa et.al.8, reporf3values of 70°C.-7L0C. J ckson and Hayward", Forman et.al. and Hayward et.al.' report .5OC.-52OC. We found a value of 70°C.-71.50C., Class f o r isosorbide d i n i t ~ - a t e . ~This ~ d i f f e r e n c e i n melting point may be a r e s u l t of polymorphism. The melting temperature range does n o t change s i g n i f i c a n t l y with v a r i a t i o n s i n heating r a t e of from 1 t o 5OC./rnin.

,

IJ',

2.7

D i f f e r e n t i a l Thermal Analysis

The d i f f e r e n t i a l thermal a n a l y s i s (DTA) curve of isosorbide d i n i t r a t e 4 6 run from room temperature t o t h e melting point e x h i b i t s no endotherms o r exotherme o t h e r than t h a t associated with t h e m e l t . The DTA curve7 run on a DuPont 900 DTA using a micro c e l l and a heating r a t e of 5'C./min. i s shown i n Figure 3. 2.8

Solubility

The following s o l u b i l i t i e s have been determined f o r isosorbide d i n i t r a t e a t room temperature. 15 U.S.P. Solvent Authors Merck Index' 0.5 mg./ml. sparingly soluble very s l i g h t l y water Soluble f r e e l y soluble very s o l u b l e 7 1 g./ml. acetone f r e e l y soluble sparingly 24 mg./ml. alcohol soluble

-

<

23 1

h)

W

h)

Figure 3

-

DTA Spectrum of Isosorbide D i n i t r a t e , Lot N o . 5123 V 6/1, heating r a t e SO/min. Instrument: DuPont 900 DTA.

ISOSORBIDE DIN ITRATE

Solvent ether chloroform hexane 2.9

Authors -m ' g-m /. .l

Merck Index1 f r e e l y soluble

330 mg./ml.

U.S.P.

15

f r e e l y soluble

< 1 mg./ml.

Crystal Properties

The X-ray powder d i f f r a c t i o n p a t t e r n of isosorbide d i n i t r z ~ t eobtained ~~ 7 with a P h i l i p s diffractometer16 using CuKa r a i a t i o n i s shown i n Figure 4. The c a l c u l a t e d d spacings' f o r the d i f f r a c t i o n p a t t e r n shown a r e given i n Table I V . Table I V X-Ray Powder D i f f r a c t i o n P a t t g r n f o r Isosorbide D i n i t r a t e

28 (degrees) 10.0 16.1 17.3 19.5 20.2 21.1 23.2 23.8 25.1 25.6 2772 28.9 29.3 31.4 32.6 33.2 34.4 35.8 41.1

d (A) 8.85 5.50 5.13 4.55 4.40 4.21 3.83 3.74 3.55 3.48 3.28 3.09 3.05 2.849 2.747 2.698 2.607 2.508 2.196

I/Io 40 25 100 3 95 1 2 9 32 20 2 3 2 5 5 2 9 3 12

d(interp1anar distance) = nh 2sin4 1/10 = r e l a t i v e i n t e n s i t y (based on highest i n t e n s i t y = 100) 3.

Synthesis

Isosorbide d i n i t r a t e has been prepared by routes u t i l i z i n g both L-sorbose and D-glucitol ( s o r b i t o l ) . I n one s y n t h e t i c rout:,() isosorbide w a s prepared from L-aorbose by themethod of Wiggine,17 This w a r accompltshed by hydrogenation of L-sorbose followed by dehydration t o o b t a i n isosorbide. The n i t r a t e e s t e r of isosorbide w a s then prepared by t h e method of Forman et.al.18 i n a y i e l d of about 50%; t h e y i e l d w a s increased t o 85-907- by n i t r a t i o n i n

233

N

w

p.

Figure 4

- X-Ray

Diffraction Pattern of Isosorbide Dinitrate, Lot No. 5123 V 6/1. Radiation: CuKa Instrument: Norelco p h i l i p s Diffractometer.

ISOSORBI DE DIN ITRATE

a c e t i c anhydride-ni t r i c a c i d - a c e t i c acid mixture. I n another synthesis,19 shown i n Figure 5, i s o s o r b i d e was prepared by dehydration of D-glucitol ( s o r b i t o l ) . Isosorbide d i n i t r a t e was subsequently obtained by t r e a t i n g Concentrated i s o s o r b i d e with differentnitratingagents. n i t r i c acid (98%) gave a y i e l d of 83%, a mixture of n i t r i c acid and s u l f u r i c acid (30 and 60% r e s p e c t i v e l y ) gave a y i e l d of 65%. Isosorbide dinitrate-C14 w a s prepared b anhydrization of s o r b i t o l using p-toluene s u l f o n i c acid.28 The r e s u l t i n g i s o s o r b i d e was n i t r a t e d using 97% n i t r i c acid. 4.

S t a b i l i t y and Degradation

The s t a b i l i t y of i s o s o r b i d e d i n i t r a t e , i n t h e s o l i d form, has been studied a t various temperatures. It w a s found t o be s t a b l e a t a temperature of 45OC. f o r a period of 1 2 months and a t room temperature f o r a period of 60 months. Z1. I n a c i d i c medium, under vigorous h y d r o l y t i c conditions, t s o s o r b i d e d i n i t r a t e degrades i n a stepwise manner forming 2-mononltrate and 5-mononitrate as i n t e r mediates with t h e f i n a l products being i s o s o r b i d e and 25% decomposition22 inorganic nitrate.21 For example, r e e u l t s from heating i s o s o r b i d e d i n i t r a t e i n 1 N hydroc h l o r i c acid a t 100OC. f o r one hour. Decomposition i n base i s somewhat more r a p i d , i.e., a 1 N sodium hydroxide s o l u t i o n of i s o s o r b i d e d i n i t r a when heated a t 10o°C. f o r one hour, decomposes about 4 X

55 .

*Jackson and Hayward” s t u d i e d t h e decomposition of i s o s o r b i d e d i n i t r a t e i n p y r i d i n e , and found t h a t a slow decomposition t o a polymer, n i t r o g e n oxides, and pyridinium n i t r a t e took place when s o l u t i o n s were heated above 50° C. 5.

Metabolism

reasoned t h a t i s o s o r b i d e d i n i t r a t e i s completely metabolized i n man and i n t h e dog s i n c e no unchanged drug was d e t e c t e d i n urine. L e s s than 1%of t h e dose w a s recovered as 5-isosorbide mononitrate and 2-isoeorbide mononitrate and t h e r e f o r e he postulated t h a t t h e removal of t h e n i t r a t e groups i s a stepwise process ending with t h e completely d e n i t r a t e d i s o s o r b i d e as t h e major metabolite. Sherber et.al.2L demonstrated that 86% of i s o s o r b i d e d i n i t r a t e , intravenously administered, i s c l e a r e d from 235

CH20H t4

w

al

I I

HCOH

HCOH

I

0

HOCH HCOH

I

0

1

98%HN03 OR MIXTURE OF HNO~AND

HCOH

I

Lr,, HCONOZ

I

H2S04

CH2OH

I SOSORBIDE

D-GLUC ITOL (SORB ITOL) Figure 5

o

-A

I SOSORBI DE DINITRATE

Synthetic Route for Isosorbide Dinitrate

ISOSORBIDE DINITRATE

r a b b i t blood w i t h i n 90 seconds. I n a l a t e r study, Reed et.al.20 found t h a t a f t e r o r a l doses of i s o s o r b i d e d i n i t r a t a n e a r l y a l l o f the d r u g , w i t h carbon s k e l e t o n i n t a c t , was e x c r e t e d i n t h e u r i n e of dogs d u r i n g t h e f i r s t 24 hours. Some 20 t o 30% o f t h e carbon s k e l e t o n of i s o s o r b i d e d i n i t r a t e w a s e x c r e t e d as n e u t r a l m a t e r i a l , p r i n c i p a l l y as i s o s o r b i d e , 2-isosorbide mononitrate and 5 - i s o s o r b i d e mononitrate and t h e e t h e r monoglucuronide of i s o s o r b i d e . Sisenwine and Rue1iusZ5 made a complete i n v e s t i g a t i o n of t h e plasma c o n c e n t r a t i o n and u r i n a r y e x c r e t i o n o f i s o s o r b i d e d i n i t r a t e and i t s m e t a b o l i t e s i n dogs. They found t h a t t h e i n i t i a l b i o t r a n s f o r m a t i o n occurs by d e n i t r a t i o n of t h e two isomeric mononitrates, 2 - i s o s o r b i d e mononitrate and 5i s o s o r b i d e mononitrate. A s t h e mononitrate e s t e r s d i s a p p e a r a small amount o f i s o s o r b i d e appears, probably stemming more from h y d r o l y s i s of t h e more l a b i l e n i t r a t e group i n 2-isosorb i d e mononitrate than from t h e s t e r i c a l l y hindered n i t r a t e group i n 5 - i s o s o r b i d e mononi trate. F u r t h e r t r a n s f o r m a t i o n o f t h e i s o s o r b i d e molecule does n o t take p l a c e as demonstrat e d by t h e f i n d i n g t h a t o n l y unchanged i s o s o r b i d e i s e x c r e t e d a f t e r a d m i n i s t r a t i o n of i ~ o s 0 r b i d e - C t~o ~dogs. The d i s a p pearance o f 5 - i s o s o r b i d e mononitrate from plasma i s probably caused by t r a n s f o r m a t i o n o f t h e molecule t o g l u c u r o n i d e and o t h e r conjugates. The i s o s o r b i d e glucuronide found i n u r i n e i s a m e t a b o l i t e formed from t h e 5 - i s o s o r b i d e mononitrate glucuronide and n o t from i s o s o r b i d e , s i n c e t h e l a t t e r does not undergo t r a n s f o r m a t i o n o r conjugation. A p o s t u l a t e d b i o t r a n s f o r m a t i o n scheme i s i l l u s t r a t e d i n Figure 6. These i n v e s t i g a t o r s d i d n o t f i n d any 2-isosorbide_qononi t r a t e i n dog2grine c o n t r a r y t o t h e f i n d i n g s of Dietzz’ and Reed et. a1

-. 6.

Identification

I s o s o r b i d e d i n i t r a t e can b e i d e n t i f i e d by v i r t u e of i t s c h a r a c t e r i s t i c I R and X-ray s p e c t r a ( s e e 2.l-and 2.9).

7.

Assay Methods

H N 7.2

3.41 11.86

Determined 30.39 3.24

5,46

11.77

C o l o r i m e t r i c Analysis The most comon procedures f o r c o l o r i m e t r i c a n a l y s i s 237

(-yJ-d(yj 1 2 - ISMN

OH

ON02

2 - ISMN GLUCURONIDE

OG I

IS GLUCURONIDES

5

- ISMN

1 OG I

5

- ISMN GLUCURONIDE

ISDN = ISOSORBIDE DINITRATE ISMN: ISOSORBIDE MONONITRATE IS

Figure 6

-

=

ISOSORBIDE

Metabolic Pathways of Isosorbide Dinitrate

ISOSORBI DE DINITRATE

of n i t r a t e e s t e r s involves a l k a l i n e hydrolysis and c o l o r i metric measurement of l i b e r a t e d n i t r i t e . This b a s i c procedure has been used by i n v e s t i g a t o r s f o r a t l e a s t t h e l a s t 40 years .26 For isosorbide d i n i t r a t e more d i r e c t methods have been used. Most of these involve r e a c t i o n with t h e n i t r a t e group.

Reed et.al.*O measured isosorbide d i n i t r a t e by r e a c t i n g i t with 1%2,6-xylenol i n g l a c i a l a c e t i c acid i n the presence of 80% (w/v> s u l f u r i c acid f o r one-half hour t o form nitroxylenol. The nitroxylenol w a s extracted from the aqueous r e a c t i o n mixture using chloroform and then reextracted i n t o sodium hydroxide t o form t h e yellow sodium s a l t whose absorbance w a s measured a t 420 run. This method i s extremely s e n s i t i v e . Jackson and Hayward” reacted isosorbide d i n i t r a t e with 70% (v/v) s u l f u r i c acid and a 1%s o l u t i o n of 3,4-dimethylphenol f o r 30-60 minutes a t 3OOC. Water w a s then added and t h e s o l u t i o n d i s t i l l e d c o l l e c t i n g t h e d i s t i l l a t e i n a f l a s k containing a s o l u t i o n of 2% sodium hydroxide. The absorbance of t h e r e s u l t i n g s o l u t i o n w a s then measured a t 435 nm. Nagese et.a1.27 determined isosorbide d i n i t r a t e i n pharmaceuticals by two c o l o r i m e t r i c methods. One w a s based on t h e Griess-Romijin reagent and t h e o t h e r on t h e phenold i s u l f o n i c acid reagent. The phenoldisulfonic acid o l o r i m e t r i c method f o r determining mannitol h e x a n i t r a t e 28-51 was adapted32 f o r measuring isosorbide d i n i t r a t e in. t a b l e t s and i n j e c t a b l e s . In t h i s method a sample containing 10 mg. of isosorbide d i n i t r a t e i s extracted from an a c i d i c s o l u t i o n w i t h a t o t a l of 100 m l . of e t h e r o r chloroform. An a l i q u o t (4 ml.) of t h e e x t r a c t i s then evaporated t o dryness and 1 m l . of g l a c i a l a c e t i c acid and 2.0 m l . of phen. ’ ’ ’ a r e added. A f t e r 15 minutes t h with ammonium hydroxide and i t s _ _ .,a nm. I n t h i s procedure it i s not necessary t o use isosorbide d i n i t r a t e as a standard, any of t h e inorganic n i t r a t e s , such as sodium o r potassium n i t r a t e , can be u t i l i z e d . a

__

A method used f o r determining n i t r a t e 3 3 has been adopted34 t o study s o l u t i o n s of isosorbide d i n i t r a t e . This method involves hydrolyzing isoeorbide d i n i t r a t e with sodium hydroxide t o form n i t r i t e ions. The n i t r i t e i s then diazot i z e d with p-nitroaniline t o form a diazonium i o n which when coupled with azulene i n t h e presence of perchloric acid e x h i b i t s an absorbance maximum a t 515 run. 239

LUCIAN0 A. SlLVlERl AND NICHOLAS J. DeANGELlS

7.3

I n f r a r e d Analysis

Infrared spectropho om r i c procedures used f o r t h e estimation of n i t r a t e e s t e r s 5 5 9 4 6 have been adopted32 f o r determining isosorbide d i n i t r a t e i n t a b l e t s and i n j e c t a b l e s . The method c o n s i s t s o f simply e x t r a c t i n g isosorbide d i n i t r a t e from i t s dosage fo with chloroform and reading t h e absorbance a t 1650 cm.-'in a sodium c h l o r i d e c e l l . Carbon t e t r a c h l o r i d e can be s u b s t i t u t e d f o r chloroform i n t h i s procedure. 7 7.4

Gas Liquid Chromatographic A n a l y s i s

The colorimetric and I R methods mentioned above a r e v i r t u a l l y non-specific. This i s a disadvantage e s p e c i a l l y i n cases where mixtures of e s t e r s are present due t o p a r t i a l d e n i t r a t i o n of t h e parent e s t e r . Gas l i q u i d chromatographic methods overcame t h i s d i f f i c u l t y , , provide adequate separat i o n and, a t t h e same t i m e , maintain s e n s i t i v i t y of detection. Sherber et.a1.24 determined i s o s o r b i d e d i n i t r a t e i n r a b b i t blood by e x t r a c t i n g i t i n e t h y l a c e t a t e and i n j e c t i n g t h e e x t r a c t without f u r t h e r p u r i f i c a t i o n i n a 6 f t . column packed with 3.8% SE-30 on Gas Chrom P. The system w a s operated isothermally under t h e following conditions: oven temperature, l l O ° C . , f l a s h h e a t e r , 130.C., d e t e c t o r , 190.C.; nitrogen flow r a t e , 55 ml./min. These same i n v e s t i g a t o r s a l s o used a 3% XE-60 on Gas Chrom Q column with an oven temperature of 15OOC.; f l a s h h e a t e r a t 16OOC.; flame ionizat i o n d e t e c t o r temperature a t 18OOC.; and nitrogen flow r a t e The i n t e r n a l standard used was m-dinitroa t 55 ml./min. benzene. The s e n s i t i v i t y of t h i s method i s such t h a t as l i t t l e as 0.01 pg. can be detected with a flame i o n i z a t i o n detector. Davideon et.a1.38 used a gas chromatographic method f o r s e p a r a t i o n and q u a n t i t a t i o n of isosorbide d i n i t r a t e i n t h e presence of o t h e r organic n i t r a t e e s t e r s of common therapeutic use. They used two columns: one packed with 1%SE-30 on trimethylchlorosilylated Chromsorb P and t h e o t h e r packed with 1%Dexsil 300 GC on dimethylchlorosilylated Chromosorb W. The i n j e c t i o n p o r t and d e t e c t o r s were maintained a t 200OC. with nitrogen as t h e c a r r i e r gas. The columns were programned a t a r a t e of 10°/min. with an i n i t i a l s e t t i n g of 65OC.

A mixture of isosorbide d i n i t r a t e and i t s two monon i t r a t e s w a s assayed by gas chromatography using columns packed with e i t h e r 3% XE-60 o r 3.5% QF-1 on G a s Chrom Q. 39 240

ISOSOR B IDE DIN ITRATE

Both columns were operated isothermally, t h e QF-1 a t 110OC. and t h e XE-60 a t 1 5 O O C . The i n j e c t i o n p o r t was maintained a t 160OC. Good separation of t h e d i f f e r e n t n i t r a t e s w a s obtained on the QF-1 column. The XE-60 column was less s a t i s f a c t o r y as 5-isosorbide mononitrate overlapped w i t h isosorbide d i n i t r a t e . The minimum amount of isosorbide d i n i t r a t e t h a t could be determined was 0.5 pg. with a flame i o n i z a t i o n d e t e c t o r and 8 ng. i t h an e l e c t r o n capture detector. These same investigators4' have subsequently determined isosorbide d i n i t r a t e i n human plasma on t h e QF-1 column. I s o i d i d e d i n i t r a t e was used as the i n t e r n a l standard. Another gas chromatographic method f o r t h e d e t e r mination of isosorbide d i n i t r a t e i n t a b l e t s 2 2 uses a 5 f t . x 1/8 in. s t a i n l e s s s t e e l column packed with 1%OV-17 on Diatoport S , 80/100 mesh operated a t 155OC. The isosorbide d i n i t r a t e i s extracted from an a c i d i c s o l u t i o n with chloroform, and e t h y l pentadecanoate i s used as t h e i n t e r n a l standard. This method w i l l separate isosorbide d i n i t r a t e from isosorbide and the two mononitrates. 7.5

Polarographic Analysis

A 4 r l a r o g r a p h i c methzg used f o r determining glyceryl trinitrate has been adopted f o r assaying isosorbide d i n i t r a t e i n i n j e c t a b l e 8 and t a b l e t s . This method involves t h e reduction of the n i t r a t e e s t e r a t t h e dropping mercury electrode i n a 0.5 N t e t r a m e t h y l m n i u m hydroxide aqueous solut i o n buffered a t pH 8.7 with an ammonium b u f f e r (0.5 N i n NH,,Cl and NH,,OH). The reduction wave has a half-wave potent i a l of -0.78V. N i t r a t e and n i t r i t e ions do not i n t e r f e r e . 7.6

Automated Analysis

The phenoldisulfonic acid r e a c t i o n (7.2) has been u t i l i z e d f o r determining isosorbide d i n i t r a t e i n t a b l e t s using an automated p r o ~ e d u r e . 4 ~ Glacial a c e t i c acid i s used as t h e e x t r a c t i o n solvent.

7.7

Radiochemical Analysis Isosorbide dinitrate-CI4 w a s analvzed 20925 in a

l i q u i d s c i n t i l l a t i o n s ectrometer under conditions approp r i a t e f o r counting C. P4 7.8

Thin Layer Chromatographic Analysis

Several TLC s y s t e m f o r t h e separation of isosorbide d i n i t r a t e from i t s metabolites and similar s t r u c t u r e compoun s have been reported i n t h e l i t e r a t u r e . I n one method2' the sample was spotted on chromatographic p l a t e s

24 1

L U C I A N 0 A. SlLVlERl AND NICHOLAS J. DeANGELlS

coated with s i l i c a g e l (-25 mm) and were developed by ascending techniques with two s o l v e n t systems, one a benzene: ethyl a c e t a t e ( 1 : l ) mixture, and t h e second a 2-propanol: ammonium hydroxide (4:1 ) mixture. For v i s u a l i z a t i o n t h e p l a t e s were sprayed with 1%diphenylamine i n methanol solut i o n and exposed t o u l t r a v i o l e t l i g h t f o r 5 minutes. Diphenylbenzadium i n s u l f u r i c acid has a l s o been used as a spray reagent.24 The l a t t e r reagent w as found t o b e 10 times more s e n s i t i v e than diphenylamine f o r n i t r a t e edters. The Rf f o r isosorbide d i n i t r a t e i s .68 i n t h e f i r s t solvent system and .77 i n t h e second. Both of t h e s e systems separated i s o s o r bide d i n i t r a t e from i t s two mononitrates. Other i n v e s t i g a t o r s using these same solvent systems v i s u a l i z e d i s o s o r b i d e with a metaperiodate-permanganate spray. 25 Another TLC method44 uses as an adsorbent a mixture Chromatoof s i l i c i c acid and p l a s t e r of P a r i s (70:30 w/w). grams were developed with mixtures of anhydrous s o l v e n t s i n the following volume r a t i o : benzene-petroleum e t h e r (1: 1 ) ; ether-petroleum e t h e r (3: 39) ; ether-benzene (1: 19). A 1% s o l u t i o n of diphenylamine i n 95% ethanol, followed by expos u r e t o a shortwave u l t r a v i o l e t l i g h t f o r 10 minutes w a s used f o r detection.

21

Isosorbide d i n i t r a t e w a s separated from i s o s o r bide and i t s two mononitrates on p l a t e s coated with S i l i c a Gel G by using a mixture of carbon t e t r a c h l o r i d e and acetone (8:2) as the solvent. 7.9

Paper Chromatographic Analysis

Jackson and Hayward21 developed s e v e r a l methanolhydrocarbon solvent systems u s e f u l f o r paper chromatography of isosorbide d i n i t r a t e . For v i s u a l i z a t i o n t h e paper w a s sprayed with 1%alcoholic diphenylamine and exposed t o W l i g h t f o r 5 t o 10 minutes.

242

ISOSORBIDE DINITRATE

8.

References 1. 2.

Merck Index, 8 t h Ed., Merck and Co., Inc.(1968). Benjamin K. A y i , Wyeth Laboratories, Personal Communication. 3. 0. R. Sammul, W. L. Brannon, and A. L. Hayden, J. A s s Off. Agr. Chem., 47 (51, 918(1964). 4. L. D. Hayward, D. J. Livingstone, M. Jackson, and V. M. Csizmadia, Can. J. Chem.,*, 2191(1967). 5. Bruce Hoffmann, Wyeth Laboratories, Personal Communication. 6. F. J. Hopton and G. H. S. Thomas, Can. J. Chm., 47, 2395 (1969). c 7. N. J. DeAngelis, Wyeth Laboratories, Unpublished r e s u lts 8. L. D. Hayward, R. A. Kitchen, and D. J. Livingstone, Can. J. Chem.. 40, 434(1962). 9. Charles Kuhlman, Wyeth Laboratories, Personal Communication. 10. M. Jackson and L. D. Hayward, Can. J. Cham, 38 496(1960). 11. L. Goldberg, Acta Physiol. Scand., IS, 173(1948); C.A. 42, 5564d. 12. L. F. Wiggins, Advances i n Carbohydrate Chem., 5, 206 (1950). 13. S. E. Forman, C. J. Carr, and J. C. Krantz, J. Am. Pharm. h s o c . , 30, 132(1941). 14. USP XVIII, Mack P r i n t i n g Co., P. 935(1970). 15. USP XIX, i n print. 16. North American P h i l i p s Company, Mount Vernon, New York. 17. L. F. Wiggins, J. Chem. SOC., 4, (1945). 18. S. E. Forman, C. J. Carr, and J. C. Krantz, J. Am. Pharm. ASSOC., 30, 132(1941). 19. P. M. Kochergin and R. M. Titkova, Med. Prom.S,S.S.R. 13, NO. 8, 18-20(1959). 20. D. E. Reed, J. F. May, L. G. Hart, and D. H. McCurdy, Arch. I n t . Pharmacodyn. , 318-336(1971). 21. J. Rutgers, Wyeth Laboratories, unpublished r e s u l t s . 22. K. Dilloway, Wyeth Laboratories, unpublished r e s u l t s . 23. A. J. Dietz Jr., Biochem. Pharmacol., 2 (121, 24478(1967). 24. D. A. Sherber, M. Marcus, and S. Kleinberg, Biochem. Pharmacol. , l9, (2) , 607-12(1970). 25. S. F. Sisenwine and H. W. Rueliue, J. Pharmacol. Exp. Ther., 1 7 6 ( 2 ) , 296-301(1971). 26. L. A. Crandall, C. D. Leake, A. S. Leovenhart, and C. W. Muehlberger, J. Pharmacol. Exp. Ther., 2, 283

.

-

191,

-

243

LUCIAN0 A. SlLVlERl AND NICHOLAS J. DeANGELlS

(1929) Y. Nagase, Y. Kanoya, A. Sugiyama, and H. Haruhiko, Yakaguaku Zasshi, 8 5 ( 2 ) , 119-25(1965). 28. E. Sornoff, J. A s s . Off. Agr. Chem., 38, 637(1955). 39, 630(1956). 29. 9- Ibid 40, 815(1957). 30. 9- Ibid 31. "Official Methods of Analysis of the Association of O f f i c i a l Agricultural Chemists", 10th Ed., 605(1965). 32. D. Gutekunst, Wyeth Laboratories, unpublished r e s u l t s . 33. E. E. Garcia, Anal. Chem., 39, 1605(1967). 34. J. Poole, Wyeth Laboratories, unpublished r e s u l t s . 35. Jonas Carol, J. Ass. Off. Agr. Chem., 42, 468(1959). Ibid , 43, 259(1960). 36. 2 37. D. Woo, J. K. C. Yen, and P. Sofronas, Anal. Chem., 45(12), 2144-45(1973). 38. I. W. F. Davidson. F. J. Dicarlo. and E. I. Szabo, J. Chromatogr., S?, 345-52(1071): 64 39. M. T. Rossell and M. G. Bogaert, J. Chromatogr., -' 364-7(1972). M. T. Rossell and M. G. Bogaert, J. Pharm. Sci., 62 40 (51, 754(1973). 41. B. C. Flann, J. Phann. Sci., 58(11), 122(1969). 42. J. Bathish, Wyeth Laboratories, unpublished r e s u l t s . 43. C. Davis, Wyeth Laboratories, unpublished r e s u l t s . ,440 L. D. Hayward, R. A. Kitchen, and D. J. Livingstone, Can. J. Chem., 40, 434-40(1962). 45. M. Jackson and L. D. Hayward, ,J. Chromatogr.,>, 166-69(1961) 46. Lot No. 5123 V 6/1, Wyeth Laboratories. 47. R. D. Guthrie and H. Spedding, J. Chem. S O C . , (1960). 48. L. Bellamy, "The I n f r a r e d Spectra of Complex Molecules," 2nd Ed., J. Wiley and Sons, Inc., New York, N. Y., 1964.

27.

-

-

-

-

953

244

METHAQUALONE

Dahyabhai M . Patel, Anthony J. Visalli, Jerome J. Zalipsky and Nelson H. Reavey-Cantwell

DAHYABHAI M. PATELetal.

CONTENTS Analytical P r o f i l e

- Methaqualone

1.

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

2.

Physical Properties 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2 . 3 UI t r a v l o l e t Absorptlon Spectrum 2.4 F1 uorescence Spectrum 2.5 Mass Spectrum 2.6 M e l t i n g Range 2.7 O p t i c a l R o t a t i o n 2.8 D i f f e r e n t i a l Scanning Calorimetry 2.9 S o l u b i l i t y 2.10 Crystal P r o p e r t i e s 2.11 D i s s o c i a t i o n Constant

3.

Synthesis

4.

Stabi 1 ity-Degradat ion

5.

Metabolism

6.

Methods o f Analysis Elemental Analysts Phase S o l u b i l i t y Analysis Nonaqueous T i t r i m e t r l c Analysis Color imeiir i c Anal ys is Spectrophotometric Analysis Spectrof 1 uorometr l c Anal ys i s Gas Chromatographic Analysis Thin Layer Chrornatographlc Analysls

6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8

7.

References

8.

Ac know1edgemen t s

246

METHAQUALONE

1.

Description

1.1

Name, Formula, Molecular U e i g h t Methaqua 1 one i s 2-methyl -3-o-to1 y 1-4

zol i none

16"1 4N20 1.2

Molecular Weight:

bH)-qu ina-

250.30

Appearance, Color, Odor

Methaqualone occurs as a w h i t e , odorless, c r y s t a l l i n e powder.

2.

Physical P r o p e r t i e s 2.1

I n f r a r e d Spectrum (IR)

The i n f r a r e d spectrum o f methaqualone i s presented i n Figure 1 (1). The spectrum was o b t a i n e d from a KBr p e l l e t c o n s i s t i n g o f a 0.5% d i s p e r s i o n o f methaqualone i n KBr. The assigned p r i n c i p a l a b s o r p t i o n bands a r e l i s t e d i n Table I ( 1 ) . Table

I

P r i n c i p a l Bands i n t h e I R Spectrum o f Methaqualone Wave1 eng t h ( 0 ' ' )

Ass ignmen t

3049, 2994 and 2907 1672 1603, 1595 and 1471 1340 and 1326 778, 760, 720 and 703

CH s t r e t c h i n g Amido carbonyl s t r e t c h i n g Aromatic s k e l e t a l v i b r a t i o n Aromatic C-N s t r e t c h i n g CH deformation o f aromatic r i n g

247

METHAQUALONE

Nuclear Magnetic Resonance Spectrum (NMR)

2.2

The N M R spectrum of methaqualone shown in Figure 2 was obta ned by dissolving 30 mg o f methaqualone in 0.5 ml of CCl4 containing tetramethylsilane as the internal reference. The assigned proton signals are listed in Table I1 (1). Table I1

NMR Absorption Signals of Methaqualone No. o f Protons

Chemical Shift (ppm)

Signal Appearance

203 i none

(Quinar i ng)

3

2.06

s ing 1 et

CH

(Tolyl)

3

2.15

s i ng 1 et

Aromatic Protons

8

Group

CH

3

2.3

7-8.4

multiplet

U1 trav iolet Absorption Spectra (UV)

Methaqualone exhibits characteristic ultraviolet absorption properties in different solvents. These characteristic properties were established using a Cary Model 14 Spectrophotometer in 1 cm cells. The U V Spectra are presented in Figure 3 and the relevant UV absorption data are listed in Table I11 (1). Table I11 UV Absorption Characteristics o f Methaqualone

Solvent

X max. nm X min. nm a

at

x

max.

(€1

Methanol

225.5 265

214.0 249.0

140.9 (35.26 X 103) 38.0 ( 9.51 X 103)

Ethanol (95%)

225.5

214.0

139.6 (34.94 X

249

lo3)

i ,

I

250

I

METHAQUALONE

Figure 3 Ultraviolet Absorption Spectra of Methaqualone C .a

A : Methaqualone In methanol

( I :200,000)

.0

B : Methaqualone i n ethanol

(I

:zoo ,000)

(95%)

C : Methaqualone in 1 N H C l ( I :200,000)

.7

.6

"

y.5

a m w

Y) 0

a m

.4

.3

.2

.I

',/--I 200

I

I 220

I

I

1

I 260

240 NANOMETERS

25 1

1

I 280

I

I 300

DAHYABHAI M. PATELetal.

265.0

249.0

37.7 ( 9.43 X 103)

H C l (1 N)

235.0 -270

215.5 258.5

133.0 (33.42 X lo3) 30.8 ( 7.71 X 103)

H C l (0.1 N)

234.5 -270

215.5 257.5

133.4 (33.39 X lo3) 31.5 ( 7.88 X 103)

2.4

Fluorescence Spectrum

Methaqualone does n o t e x h i b i t any n a t i v e f l u o r e s cence. However, t h e reduced d e r i v a t i v e t e t r a h y d r o q u i n a z o l i n o n e e x h i b i t s an emission maximum a t 450 nm w i t h e x c i t a t i o n a t 345 nm i n methanol (2). 2.5

Mass Spectrum

The mass spectrum o f methaqualone i n t h e form o f a bar graph i s presented i n F i g u r e 4. The spectrum was obtained on a, H i t a c h i - P e r k i n Elmer Model RMU-6D Mass Spectrometer u s i n g t h e d i r e c t i n l e t probe w i t h sample temperature a t 60°C. The e l e c t r o n energy was 70 ev and t h e a c c e l e r a t i n g v o l t a g e was 1700 v o l t s . The c h a r a c t e r i s t i c fragmentation p a t t e r n i s presented i n Table IV ( 1 ) . Table IV Fragmentation P a t t e r n Produced i n Mass S p e c t r a l Examination o f Methaqualone Mass (m/e) 250 235

145 144 132

91

76 65 50

41

Intensity Strong Very Strong Weak Weak Med ium Strong Med iurn Med i um Med ium Very Weak

252

Fragment Ion Molecular i o n (M) M-CH M-C t$N M - [ $ H + CH3]

M-C7$4L

M-C H N20 C H9-$H 7 7 2 C7H7-C2 C 6H4 C2 H

-

a

C9H1 0N-C7H7

Figure 4 Mabs Spectrum of Hethaqualone

I00

90

80

70

2 60 z 0

-

50

== Y

;40

4 z Y

30

20

10

c 20

40

60

80

100

120

140 We

160

180

200

220

260

DAHYABHAI M. PATEL et a/.

Med i um

39 2.6

Me1 t i ng Range

The m e l t i n g range o f methaqualone i s dependent X V I I I Class 1 on the r a t e o f heating, Uslng the U.S.P. procedure, methaqualone me1 t s between 114"-117"C ( 1 ) . 2.7

Optical Rotation

Methaqualone e x h i b i t s no o p t i c a l a c t i v i t y when analyzed a t a s e r i e s o f mercury and sodium s p e c t r a l l l n e s (1). 2.8

D i f f e r e n t i a l Scanning Calorimetry (DSC)

The DSC thermogram o f methaqualone i s shown I n Figure 5. A m e l t i n g endotherm i s observed a t 385"K(l12OC) The A Hf w i t h the temperature programing a t lO"C/minute. was found t o be 5.55 Cal/mole C1). 2.9

Solubility

The e q u i l i b r um s o l u b i l i t y data obtained on methaqualone a t 23°C s l i s t e d i n Table V (1). Table V Methaqualone Solubi 1 i t y So 1vent

Water Methanol E t ha no1 I sopropanol Ether Chloroform Benzene Acetonitrile Petroleum Ether (B.P.

S o l u b i l i t y . ( g / l O O ml) 0.03

16.58 12.53

4.76 3-71

>4 0

28.00

>4 0 30-60")

254

0.33

a,

0

C

3

m

c

CT (D

a,

U

Jz

x rc

E

0

E

0

ul

E

a, Jz

I-

u In n

255

0 0 W C

0 w

3 XJ

?

Y

DAHYABHAI M. PATEL et a/.

2.10

Crystal Properties

The X-ray powder d i f f r a c t i o n p a t t e r n o f methaqualone was obtained on a P h i l l i p s scanning d i f f r a c t o m e t e r u s i n g copper r a d i a t i o n coupled w i t h N i c k e l f i l t e r . The d i f f r a c t i o n p a t t e r n i s presented I n Table VI ( 1 ) . Table VL X-ray D i f f r a c t i o n P a t t e r n o f Methaqualone d (A1)*

15

9.4

8.2

25 25

7.76 7.30

50

65

6.95

6.10

25

75

5.53 4.76 4.13

100

30 20 30 30

4.00

3.75 3.66 3.53 3.39

15

60

15 15

3.24

3.17

3.12 3-05

*

**

30 25

d = (Interplanar distance)

nX 2 Sin 8

1/l0 = 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 o f 1 .OO)

2.11 D i s s o c i a t i o n Constant The pKa o f methaqualone by t h e s p e c t r o p h o t o m e t r i c method a t i o n i c s t r e n g t h ~1 = 0.1 was found t o be 2.54 ( I ) .

256

METHAQUALONE

3.

Synthesis

Methaqualone i s synthesized by t h e r e a c t i o n scheme shown i n F i g u r e 6. A n t h r a n i l i c a c i d is reacted w i t h acetyl chloride t o y i e l d n-acetylanthranilic acid. This i n t e r m e d i a t e product is condensed with o - t o l u i d i n e i n t h e presence o f PC13 t o y i e l d methaqualone (3).

4.

S t a b i l ity-Degradation

On r e f l u x i n g methaqualone w i t h 1.0, 0.1 N NaOH and 1.2 N H C I , the p r i n c i p a l degradation products found a r e a n t h r a n i l i c a c i d , o - t o l u i d i n e and a c e t i c a c i d . In a d d i t i o n , 2-aminobenzo-o-toluidide was found on r e f l u x l n g methaqualone w i t h 1.0 and 0.1 N NaOH. Also, n - a c e t y l a n t h r a n i l i c a c i d was found as a degradation product on r e f l u x i n g methaqualone w i t h 0.1 N NaOH. Under these s t r i n g e n t c o n d i t i o n s , a low y i e l d o f degradation products was obtained; thereby i n d i c a t i n g good s t a b i l i t y o f t h e drug under normal c o n d i t i o n s (4). The degradation scheme i s presented i n F i g u r e 7.

5.

Metabol i s m

Methaqualone (MTQ) and i t s h y d r o c h l o r i d e sa t i s rapi d l y absorbed (Ka = 0.82 h r ' l ) from t h e stomach and duodenum 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 o f t a b l e o r capsule dosage forms. MTQ e x i s t s i n blood p r i m a r i l y i n t h e plasma phase bound t o serum albumin. Peak serum l e v e l s o f 3-4 kg/ml a r e seen 1 - 1 1/2 hours a f t e r a 300 mg dose. The serum e l i m i n a t i o n curve i s b i e x p o n e n t i a l w i t h a d i s t r i b u t i o n a l phase (a = 0.97 h r - ' ) and an e l i m i n a t i o n The p r i n c i p l e t i s s u e s o f d i s t r i phase (p = 0.036 h r ' l ) . b u t i o n a r e l i p i d t i s s u e , l i v e r and kidney. MTQ i s metabolized by h e p a t i c microsomal oxidoreductases. H y d r o x y l a t i o n of 2 and 2' methyl s u b s t i t u e n t s , t o l y l sidechain and quinazolene nucleus, g i v e s r i s e t o t h e 0-g 1 ucuron i d e conj u pr i nc ip l e monohydroxy metabol i t e s g a t i o n and 0-methylation occur p r i o r t o e x c r e t i o n o f m e t a b o l i t e s i n b i l e o r u r i n e . Ring opening i n v o l v i n g f o r m a t i o n o f N-1-oxide and 2-nitrobenzo-o-toluidide i s a minor metabol i t i c path. B i l l a r y e x c r e t i o n and e n t e r o -

.

257

Figure

6

Synthesis o f Ilethaqualone

Anthranil ic Acid

n-acetyl anthranil ic acid

Methaqualone o-to1 u i d i ne

o

+

M

I

-

+ I 0

-

=

N

u

p u

o I +

x

6:

M 259

Y

U

.-

c

Y

-03 -.-u u .-C I P , "I O

c

N

mI

E

~

mI

U

2u :oc " uz .- .e U

U

a

c

DAHYABHAI M. PATEL et a/.

hepatic r e c i r c u l a t i o n o f metabolites occur. A t a therap e u t i c dose o f 75 t o 300 mg i n man, MTQ i s completely biotransforrned and l i t t l e , i f any, unchanged drug appears i n u r i n e (5).

6.

Methods o f Analysis

6.1

Elemental Analvsls

The r e s u l t s from the elemental a n a l y s i s a r e l i s t e d i n Table VII ( 1 ) . Table VII Elemental Analysis o f Methaqualone Element C

H N

6.2

9: Theory

76 77 5.64 11.19

4; Found

76-50

5.74 10.89

Phase S o l u b i l i t y Analysis

Phase s o l u b i l i t y a n a l y s i s was c a r r i e d o u t using n-hexane-absolute alcohol (145:5 v/v) as a s o l v e n t system. The r e s u l t s o f the a n a l y s i s a r e shown i n Figure 8 along w i t h the l i s t i n g s of the c o n d i t i o n s under which t h e a n a l y s i s was performed (1).

6.3

Nonaqueous

T i t r i m e t r i c Analysis

The nonaqueous t i t r a t i o n i s the o f f i c i a l method o f a n a l y s i s i n B.P. f o r t h e b u l k methaqualone. An a c c u r a t e l y weighed 0.5 g sample Is dissolved i n 80 m l o f g l a c i a l a c e t i c acid, a few drops o f c r y s t a l v i o l e t T.S. i s added and the s o l u t i o n i s t i t r a t e d t o an emerald green end p o i n t against p r e v i o u s l y standardized 0.1 N HClO4 i n King and Perry (7) reported t h e g l a c i a l a c e t i c a c i d (6). a p p l i c a t i o n o f the nonaqueous t i t r i m e t r i c method f o r assay ing met haqua 1 one tab1 e t s

.

260

L Nn

\

N 0

In

-

I N q A l O S 6/3lfllOS 40 6m

261

0

-

:NOIlISOdW03 N O l l f l l O S

f In

f 0

m Ln

0 m

N In

N 0

In

-

0

-

In

0

DAHYABHAI M. PATEL e t a / .

6.4

Colorimetric Ana 1 ys is

Nakano et al. (8) reported a colorimetric assay method for methaqualone in microgram quantities based upon diazotization of the diazotizable amines formed by acld hydrolysis of methaqualone. Pirl et al. (9) developed assay methods for methaqualone in biological specimens as low as 0.3 Kg/ml. The method entails isolation of methaqualone from the biological specimen and condensing with p-dimethylaminobenzaldehyde in alkaline media. The resulting chromophore is measured at 503 nm in weak acid.

6.5 Spectrophotometric Analysis Akagi Masuo et al. (10) described two methods for assaying methaqualone in blological f l u i d s and tissues. In the first method, methaqualone is extracted from alkalinized biologlcal material with hexane, the solvent i s evaporated and the residue after dissolving in dil HCl i s measured at 234 nm. The second method eliminates the normal biological blank by geometric treatment of the absorbances at three wavelengths. These methods are specific for methaqualone in that they do not include degradation products of methaqualone.

6.6 Spectrof 1 uoromet r ic Anal ys 1 s A fluorimetrlc method of assaying therapeutic plasma levels of methaqualone has been reported by Brown, S. S. et al. (2). The method i s based on reducing methaqualone to its dlhydro-derivatlve using lithium borohydride as a n effective reducing agent. The resulting dihydro-methaqualone derivative exhibits emission maximum at 450 nm at the activation maximum o f 345 nm.

6.7 Gas Chromatographic Analysis There are several reports regarding the gas chromatographic analysis of methaqualone in biological fluids (1, 10, 11, 12, 13). These methods can be applled for formulation analysis, evaluation of quality, and characterization of methaqualone. Table VIII summarizes the gas chromatographic systems wl t h references.

262

METHAQUALONE

Tab1 e VIII Gas Chromatographic Systems Used for Methaqualone Ana ysis Note:

All systems listed used flame onization detectors.

Reference

Col umn

Carrier Gas

Column Internal Temp."C Standard

5

X 4 m., I.D., N2 glass tubing, 10% 45 ml/ SE-30 on Chromosorb min WAW(DMCS), 100-120 mesh

240"

Diphen- 2.17 hyd ram i ne

4 ft. X 2 mm., I.D.,

240"

Codeine

0.53

4 ft.

X 4 m., I.D., N2 glass t u b i n g , 3.8% 45 ml/ UC-Wg8 on Chromosorb rnin W(H.P.), 80-100 mesh

2300

Codeine

0.56

He 2 ft. X 4 mm., I.D., glass tubing, 3% XE- 50 ml/ rnin 60 on Gas Chrom Q, 100-120 mesh

180"

Butyl

2.81

7 ft. X 4 mm., I.D., Np glass t u b i n g , 3% 55 ml/ Cyclohexanedimethan- rnin 01 succinate 85-100 mesh

200"

Buto2.14 barb i tone

N2 2 ft. X 4 mm., I.D., glass tubing, 2.5% 40 ml/ SE-30 on Chromosorb min G, 80-100 mesh

200"

Codeine 0.42

2-m. X 3 mm., I.D.,

190"

Bu tobarbital 0.24

6 ft.

N2

glass tubing, 3% 30 ml/ OV-17 on Chromosorb min W(H.P.), 80-100 mesh

N2

glass tubing, 4% SE- 30 ml/ 30 and 6% QF-1 on min

263

S tea rate

DAHYABHAI M. PATEL er a/.

Chromosorb WAW (DMCS) , 80-100 mesh

*

Pentobarbital Phenobarbital

0.30 0.69

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

6.8

T h i n Layer Chromatoqraphic A n a l y s i s

There a r e several TLC systems r e p o r t e d i n t h e l i t e r a t u r e which a r e l i s t e d i n Table IX. These systems a r e used f o r d e t e c t i n g t h e a s s o c i a t e d I m p u r i t i e s i n methaqualone, i d e n t i f i c a t i o n o f methaqualone or c h a r a c t e r i z a t i o n o f methaqualone and i t s m e t a b o l i c products. Table IX T h i n Layer Chromatographtc Systems f o r Methaqua 1one Ana 1 ys is Reference

Support

Solvent System

Application

6

Kieselghur G

Anaesthetic Ether saturated w i t h water

Detection o f a s s o c i a t e d impurities

I4

S i l i c a Gel G

Chloroform-acetone l d e n t f i c a t i o n

14

S i l i c a Gel G

I sopropanolldent f i c a t ion c h 1 o r o f orm-25% ammonium hydroxide (45 :45 :10)

14

S i l i c a Gel G

Petroleum e t h e r (B.P. 50-75") P y r i d i n e (75:15)

15

S i l i c a Gel G

Chloroform-acetone Separation o f -25% ammonium hy- methaqualone d r o x i d e (50:50:2) from t h e metabol i t e s

264

Identification

METHAQUALONE

15

Silica Gel G

Cyclohexane-chloro-Separation of form-d i ethyl ami ne methaqualone (7:2:1) from the metabol i tes

15

Silica Gel G

Propanol-ethyl aceta te-25% ammon i um hyd rox i de (6:2:2)

265

Sepa ra t ion of methaqualone from the metabol i tes

DAHYABHAI M. PATELetal.

7.

References

1.

2.

A n a l y t i c a l and Physical Chemistry Department contribution, Research D i v i s i o n * , W i l l i a m H. Rorer, Inc., F o r t Washington, Pa. Brown, S. S. and Smart, G. A,, 466-468 (1969).

J. Pharm. Pharmacol.,

21,

3.

nthese De La MethylHerbert, M. and Plchat, L., 2, 0-Tolyl-3, Quinazolone-4 "C-2, Report CEA No. 1302, Centre D'Etudes Nucleaires De Saclay, Service De Documentation, B o l t e p o s t a l e n02-Gif-sur-Yvette, S.-et-0 (1959).

4.

Zalipsky, J., Patel, D. M. and Reavey-Cantwell, N. H., H y d r o l y t i c Degradation o f Methaqualone, submitted t o J. Pharm. Sci. f o r pub1 i c a t i o n .

5.

Smyth, R. D., Research D i v i s i o n , W i l l i a m H. Rorer, Inc., Personal Comnunication.

6.

B r i t i s h Pharmacopoeia, 296 (1972).

7.

King, E. E. and Perry, A. R.,

-7,

8.

Nakano, M.,

9.

P i r l , Joerg, N., (1972).

10.

J. Ass. Pub. Anal.

55-59 (1969). Yakuzaigaku,

22, 267-269

(1962).

e t a l . Anal. Chem. 44, 1675-1676

Douglas, J. F. and Shahinian, S., J. Pharm. Sci.,

62,

835-836 (1973).

42,

39-44 (1969).

11.

Berry, D. J., J. Chromatog.,

12.

F i n k l e , B. S., Cherry, E. J. and Taylor, D. M., J. Chromatog. Sci 9, 393-414 (1971).

13.

Hatch, R. C.,

14.

Stahl, E., Thin Layer Chromatography, 2nd E d i t i o n , Spring-Verlag Berlin-Heideberg, New York, 539.

.,

Am. J. Vet. Res.,

266

33,

203-207 (1972).

METHAQUALONE

15.

H i r t z , Jean L., A n a l y t i c a l M e t a b o l i c Chemistry o f Drugs, Marcel Dekker, I n c . , New York, 242’243. ;:Mass spectrum and X-ray d i f f r a c t i o n a n a l y s i s was performed by Morgan Schaffer, Inc. and Walter C. McCrone Associates, r e s p e c t i v e l y .

8.

Acknowledgements

The authors wish t o thank a l l members o f t h e A n a l y t i c a l and Physical Chemistry Department, Research D i v i s i o n , W i l l i a m H. Rorer, Inc. f o r t h e i r h e l p and cooperation. Also, special thanks a r e due t o M r s . Sandy Landis f o r her v a l u a b l e s e c r e t a r i a l h e l p i n p r e p a r i n g t h i s monograph.

267

NORETHINDRONE

Arvin P. Shroff and Ernest S. Moycr

NORETHINDRONE

1,

DESCRIPTION NAME, FORMULA, MOLECULAR WEIGHT 1 . 2 APPEARANCE, COLOR, ODOR 1.1

2,

PHYSICAL PROPERTIES 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

INFRARED SPECTRUM NUCLEAR MAGNETIC RESONANCE SPECTRUM MASS SPECTRUM ULTRAVIOLET ABSORPTION SPECTRUM MELTING RANGE DIFFERENTIAL SCANNING CALORIMETRY SOLUBILITY OPTICAL ROTATION

3.

SYNTHESIS

4,

METABOLISM

5.

METHODS OF ANALYSIS 5 ,1 5.2 5.3 5.4 5.5 5.6

5.7

6.

ELEMENTAL ANALYSIS PHASE SOLUBILITY THIN-LAYER CHROMATOGRAPHIC ANALYSIS VAPOR PHASE CHROMATOGRAPHY SPECTROPHOTOMETRIC ANALYSIS COLORIMETRIC ANALYSIS A. BLUE TETRAZOLIUM B. ISONICOTINIC ACID HYDRAZIDE TITRIMETRIC ANALYSIS

REFERENCES

269

ARVlN P. SCHROFF AND ERNESTS. MOYER

1. Description 1.1 Name, Formula, Molecular Weight Norethindrone is 17a-ethinyl-17fl-hydroxy-4estren-3-one. It is also known by the following chemical names. a. 17-Hydroxy-19-nor-17a-pregn-4-en-2O-yn-3-one b. 19-Nor-17a-ethynyltestosterone C. 17a-Ethinyl-19-nortestosterone d . 19-Nor-17a-ethyny1-17f3-hydroxy-4-androsten-3-one e. 19-Nor-17a-ethynylandrosten-17fl-ol-3-one f, 17a-Ethynyl-19-nortestosterone

g. Anhydrohydroxynorprogesterone h. 19-Norethisterone i. Norpregneninolone OH

Molecular Wt. 298.41

C20H2602

1.2 Appearance, Color, Odor White to creamy white, odorless, nonhygroscopic, crystalline powder. 2. Physical Properties 2.1

Infrared Spectrum

The infrared absorption spectrum of norethindrone is presented in Figure 1. The spectrum was taken in a KBr pellet with a Beckman IR-8 Spectrophotometer. Some of the absorption assignments are given in Table 1.l

270

Figure 1.

Infrared spectrum o f Norethindrone

A R V l N P. SCHROFF A N D E R N E S T S . MOYER

TABLE I INFRARED ASSIGNMENTS FOR NORETHINDRONE

-x

Intensity*

Vibrational Assignments OH s t r e t c h i n g

2.95

3400

v,sh,b

2.98

3300

s,sh

C zCH stretching

3.05

3220

m,sh

O l e f i n i c C-H s t r e t c h i n g

3.4(3.5)

2970 (2840)

m,sh

A l i p h a t i c C-H s t r e t c h i n g

6.1

1650

s ,sh

C=O s t r e t c h i n g

6.3

1590

m,sh

C=C s t r e t c h i n g

6.7(7.5)

1490 (1340)

m,sh

C-H deformation

6.92

1445

m,sh

C-H2 bending

7.1

1390

m,sh

CH3 deformation

8.7

1135

m,sh

OH bending

9.42

1060

s,sh

C-0 deformation

11.29

885

m,b

14.65

680

m,b

OH wagging O l e f i n i c C-H wagging

* s = s t r o n g , m = medium, v = v a r i a b l e , b = broad, sh = shoulder

272

NORETHINDRONE

Mesley2 assigned the following bands (cm-l) to norethindrone. a. Absorption associated with 176-hydroxyl group: 1065, 1132. b.

Characteristic absorption due to 4-ene-3-keto function: 1416, 1337, 1269, 1211, 1199, 1018, 960, 890, 764, 685.

These assi ments as well as those made by Djerassi3 and others&"essentially agree with absorption peaks and shoulders presented in the spectrum, Figure I. 2.2 Nuclear Magnetic Resonance Spectrum Cross, Landis, and Murphy5 reported the chemical shift of the C18 angular methyl group of norethindrone in various solvents. They observed the chemical shifts at 54.5 cps in d-CHC13, 54.5 cps in CHCl3, 47.5 cps in d6-DMS0 and 53.5 cps in d7-DMF. A reference NMR spectrum of norethindrone recorded on a Varian A-60 Spectrometer is presented in Figure 11. An 8% solution in deuterated chloroform containing tetramethylsilane as an internal reference was used. Characteristic chemical shifts are observed at 5.87 ppm (C4 Vinyl); 2.37 ppm (C-17 OH); 2.57 ppm (C-17 C :CH); and 0.90 ppm (C-18 CH3). 2.3 Mass Spectrum A low resolution mass spectrum of norethindrone on a magnetic instrument (Atlas CH-4) was reported by De Jongh and co-workers? Major fragments were observed at m/e 110, 215, and 231 representing the following fragmentation pattern. ?H , C ECH

&

231

n v

0

110 273

215

.--

, ~ " " " ' - ' ~ ' . . - ' . . . ' ~ . ." .- " . ~ . . . . ~ . ' . . ~ . . . . ~ . . . . 80

1 . . . . 1 . . . . 1 .

70

b0

50

F i g u r e 11.

M

"'

40

30

20

NMR S p e c t r u m o f N o r e t h i n d r o n e

10

0

E m

275

a, E 0

k E

a .I+

c k

a,

c,

0 z 0

+I

5 k

a,

u

c,

a v1

v) v)

3! a,

H H H

k 9 M

.I+

c4

ARVlN P.SCHROFF AND ERNESTS. MOYER

In Figure 111, t h e mass spectrum o f norethindrone obtained on a quadrupole instrument [Finnigan Model 1015) i s depicted. The r e l a t i v e i n t e n s i t i e s of t h e major i o n s a r e as follows:

2.4

m/e

%I -

55

76.63

67

74.94

79

95.36

91

100.00

105

59.15

110

75.78

215

44.77

231

48.35

298

44.63

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

The E ( l % , lcm) value a t 240 nm reported with t h e f i r s t synthesis of norethindrone3 was l a t e r corrected. Different E ( l % , lcm) values have been suggested from a low of 525 t o a high of 590. Many of t h e s e preparations contain foreign r e l a t e d s t e r o i d s which a f f e c t t h e E ( I % , 1cm) value.8 Nielseng reported a value of 560 which was confirmed l a t e r by Bastowl who isomerized norethynodrel t o norethindrone. We have determinedl by t h i n - l a y e r chromatographic s t u d i e s t h a t norethindrone, no matter how it i s manufactured, can be p u r i f i e d chemically by first converting t o t h e enol ether12 followed by h y d r o l y s i s , The u l t r a v i o l e t curve’ shown i n Figure I V was obtained on a U.S.P. reference standard material. I t s E ( l % , lcm) a t 240 run was 575.

276

NORETHINDRONE

9 0

7 6 W

0

Z

U m

5

Q

9m

a

4

3

2 1

0

2 3

Figure I V

I

I

220 240 260 280 WAVELENGTH ( n m r

300

U l t r a v i o l e t Spectrum of Norethindrone 277

A R V l N P. SCHROFF AND ERNESTS. MOYER

2.5

Melting Range

The melting point a l s o varied according to d i f f e r e n t i n v e s t i g a t o r s . For example, Djerassi e t a13 reported a melting p o i n t of 203-204'C, whereas Nielseng found a range of 199-208'C with decomposition, Kofler e t a l l 3 on t h e o t h e r hand, suggested a melting p o i n t In our experience, most of t h e range of 202-207'C. batches had a melting range of 3'C and a l l melted between 202-208'C (Class I A U . S . P . ) f 4

--

--

2.6

D i f f e r e n t i a l Scanning Calorimetry

Figure V shows t h e DSC thermogram of norethindrone. A Perkin-Elmer model DSC-1B employing a heating r a t e of lO'C/minute was used. The d i f f e r e n c e between t h e "extrapolated onset" and t h e "peakt' f o r reference standard U,S .P. norethindrone was 2 . S o C , The "extrapolated onset" and t h e "peak" a r e defined as follows: a.

"Extrapolated Onset": The temperature corresponding t o t h e i n t e r s e c t i o n of t h e extrapolation of t h e b a s e l i n e and t h e longest, sharpest l i n e s e c t i o n of t h e low temperature s i d e of t h e peak. :

b. 2.7

The temperature of r e v e r s a l .

Solubility

Nielsen9 r e p o r t s t h e s o l u b i l i t i e s of norethindrone t o be: almost insoluble i n water, 1 p a r t i n 80 p a r t s ethanol, 1 p a r t i n 4 0 0 . p a r t s e t h e r , and 1 p a r t i n 15 p a r t s chloroform. A p l o t of t h e s o l u b i l i t i e s of norethindrone a s a function of temperature f o r eleven common organic solvents i s i l l u s t r a t e d i n Figure V I . *

278

NORETHINDRONE

7

EXTRAPOLATED ONSET

0 x w n

I

a Y

0

n Z W

I

170

180

1

1

190 200

I

210

22b

ARVlN P. SCHROFF AND ERNEST S.MOYER

Figure V I

S o l u b i l i t y of Norethindrone i n Organic S o l v e n t s

280

NORETHINDRONE

2.8

Optical Rotation The following r o t a t i o n s have been reported:

3.

Ia1D

-25"

(Chloroform)

['ID

-24'

(Chloroform)

['ID

-31.7'

(Chloroform)

[a],

-33'

(Chloroform)

Synthesis

D j e r a s s i , Miramontes , Rosenkranz and Sondheimer3 f i r s t reported t h e synthesis (Scheme I ) of norethindrone Chromium t r i o x i d e oxidation from 19-nortestosterone I . afforded estr-4-ene-3,17-dione 11, Reaction of t h e dione with ethyl orthoformate under c a r e f u l l y c o n t r o l l e d conditions r e s u l t e d i n s e l e c t i v e enol e t h e r formation a t C-3, Ethynylation followed by cleavage of t h e enol e t h e r with aqueous mineral acid gave norethindrone.

Alternate methods using e s t r a d i o l 3-methyl e t h e r o r estrone tetrahydropyranyl e t h e r have a l s o been described.l63l7 I n a recent patent1* the preparation of 17aethynyl-4-androstene-17f3,19f3-diol-3-onewas reported i n d e t a i l . I t was mentioned t h a t t h e 19-hydroxy compound can be converted t o the 19-nor d e r i v a t i v e with a strong base The general as reported by Barber -e t a l l 9 and Meyer?O scheme o f preparing norethindrone by t h i s method i s outlined i n Scheme 11. 4.

Metabolism

Tissue d i s t r i b u t i o n of t r i t i a t e d norethindrone i n They r a t s was studied by Watanabe and co-workers?l observed no s e l e c t i v e uptake by any t i s s u e up t o f o u r hours a f t e r administration. Matsuyoshi22 incubated norethindrone with rat l i v e r and kidney homogenate and obtained dihydro and tetrahydro d e r i v a t i v e s of norethindrone. 281

A R V l N P. SCHROFF AND ERNESTS. MOYER

&-&

0

0

I

'I

IV Yicherne 1

I

Ill Synthetic Pathway to Norethindrone

282

NORETHINDRONE

&--’”

H

Scheme I1

Alternate Synthetic Pathway to Norethindrone

283

ARVlN P. SCHROFF AND ERNESTS. MOYER

The metabolic fate of norethindrone in humans has been studied by several investigators.23-39 Kamyab and c o - ~ o r k e r sadministered ~~ lk-norethindrone intravenously to seven women and found the radioactivity excreted mainly in the urine. In the first twenty-four hours 32.1%, and in five days 53.9% of the activity given appeared in the urine. Layne and cow o r k e r ~found ~ ~ a similar excretion pattern with tritiated norethindrone; namely, 50.4% in five days after oral dose and 70.2% after intravenous administration. A radioactivity excretion of only 33% in five days was reported by M ~ r a t aafter ~ ~ oral administration of 100 mg of 14C or 3H labeled norethindrone to postmenopausal women. et a130 observed the urinary radioactivity Kamyab -excretion in rabbits to be comparable to that found in humans. The amount of radioactivity in the conjugated and unconjugated states was measured31 in plasma after intravenous administratibn. Substantial fractions of the administered dose were present in the circulation for up to forty-eight hours after injection. The unconjugated activity in the plasma decreased rapidly to less than 0.4% of the dose per liter of plasma after twelve hours whereas after thirty minutes, a considerable amount of activity was in the form of conjugated metabolites. Gerhards and c o - ~ o r k e r sstudied ~~ plasma radioactivity as well as excretion in urine and feces after p.0. administration of 20 mg of 14C-norethindrone of 0 . 2 5 mg 3H-norethindrone to males. The radioactive substances in plasma were characterized as: 17a-ethynyl58-estrane-3~,17B-diol;17a-ethynyl-58-estrane-38,178-diol and 17a-ethynyl-5a-estrane-3~,178-diol. Earlier, a number of i n ~ e s t i g a t o r s ~ 3 ~ ~ ~ ~ ~ ~ J had reported excretion of 17a-ethynyl-178-estradiol after oral administration of norethindrone. These observations, however, were not supported by -in vitro investigations. As an explanation of this discrepancy, B r e ~ e r ~ ~ recently suggested that the increased estrogen content in -in vivo experiments probably is due to artifacts produced during work-up of the urine. Recent work of Sti1lwe11 and co-workers substantiated this observation. They found no ethynyl estradiol in urine after administration of norethindrone to a female. J 3 2 >

36

284

NORETHINDRONE

All norethindrone metabolites characterized from humans and rabbits possess the 17a-ethynyl group. One exception to this was the in vitro experiments of Palmer.37 He identified 4-estrene-3,17-dione as a metabolite from norethindrone. The overall metabolic pathway of norethindrone is illustrated in Scheme 111. 5.

Methods of Analysis 5.1 Elemental Analyses

The elemental analyses obtained on USP reference standard norethindrone and that reported by Djerassi and co-workers3 are as follows: Element

% Theory

Ref. Std.

Reported3

C

80.49

80.69

80.83

H

8.79

8.64

8.80

5.2

Phase Solubility

The phase solubility analysis of norethindrone was carried out using ethyl acetate as the solvent. Calculations as per USP XVIII were performed on a timesharing computer using an in-house program. The results are illustrated in Figure VII.40 5.3 Thin-Layer Chromatographic Analysis A TLC procedure for separating norethindrone from possible impurities is described in the USP (XVIII)?l One hundred micrograms of the norethindrone sample and one microgram each of four possible impurities (19-norandrostendione, norethynodrel, log-hydroxy-norethynyltestosterone and 106-hydroxy-norandrostendione) are spotted in individual lanes on a silica gel plate. The TLC plate is developed to a height of 17.5 cm by ascending chromatography employing a solvent system of 95:s chloroform-methanol. After air drying the plate, it is sprayed with a mixture of 1:3 H2S04 and dehydrated alcohol. The plate is heated in an oven at 105'C for five minutes and the intensities of the various foreign related

285

Scheme 111.

Norethindrone and i t s major metabolites

NORETHINDRONE

X CQORDINATES 3.8489063 8 . 6 4 9 2 7 14 1 1 51 38223 15.8089375 20.6400349 25.5679YSt:

I

30.0288913 3 4 - 410872V 42.2361355 5’1 * 8 5 6 0 5 19

Y CQORDJMATES 4.0180889 8 - 75336YO 9.3M7186 9.2340285 9.2dTU611 Y. 4029951 9.401 9 4 9 0 9.6795672 9.6282336 Y. 6Y19881

................................................. HBH K A N Y vOIIJTS TO

SLOPE

Pt F I T T E D ? 8

O.GlU19193

=

PERCENT P U K I l Y =

Y INTERCEPT

=

9.158532656

98.V80807304

I

Y

I

0

1

0

1

0 ,

O t .

I

*

t

+

t

*

*

*

t

4

0

F i g u r e VII

Phase S o l u b i l i t y o f Norethindrone

287

ARVlN P. SCHROFF AND ERNESTS. MOYER

s t e r o i d spots a r e compared v i s u a l l y under long-wavelength u l t r a v i o l e t l i g h t with the i n t e n s i t i e s produced by t h e corresponding s t e r o i d s i n t h e norethindrone sample. No more than two impurities t o t a l i n g a maximum of two percent a r e allowed. This TLC procedure t a k e s i n t o consideration only one s y n t h e t i c method which produces t h e above mentioned impurities. The USP Subcommittee # 6 r e c e n t l y recognized t h i s and proposed another TLC procedure which w i l l accommodate o t h e r methods of s y n t h e s i s .42 Thin-layer chromatography was r e c e n t l y reported43 f o r q u a n t i t a t i n g norethindrone i n t a b l e t s . Here a s i n g l e t a b l e t is e x t r a c t e d , t r e a t e d with i n t e r n a l standard s o l u t i o n (norethynodrel) and spotted (10-20 pg) on a s i l i c a g e l GF p l a t e . After development with 4 : l benzene-ethylacetate, t h e p l a t e i s a i r d r i e d and scanned a t 250 nm with a Schoeffel double-beam spectrodensitometer. Quantitation i s achieved by comparing t h e norethindrone t o i n t e r n a l standard r a t i o (area) with t h a t of a standard obtained i n t h e same manner. Thin-layer chromatogra hy has a l s o been used t o i d e n t i f y norethindrone i n An a l i q u o t o f a chloroform e x t r a c t i s s p o t t e d and t h e p l a t e developed using cyclohexane-ethylacetate (1:l) as t h e solvent system. After drying a t 100°C f o r t e n minutes, t h e p l a t e i s sprayed with antimony t r i c h l o r i d e i n chloroform. Norethindrone has an Rf o f 0.47 and a v i o l e t c o l o r i n daylight and red c o l o r under u l t r a v i o l e t l i g h t .

tablet^!^

5.4

Vapor Phase Chromatography

Oxidation of norethindrone t o 19-norandrostendione was observed45 on metal columns. Lodge46 s u b s t i t u t e d a g l a s s column packed with 5% QF-1 on Diatoport S and developed a s i n g l e t a b l e t assay method. A t a b l e t containing 1-5 mg of norethindrone i s dissolved i n acetone containing pregnenolone a c e t a t e as an i n t e r n a l standard and an a l i q u o t i s i n j e c t e d d i r e c t l y i n t o t h e g l a s s column operated isothermally a t 20OoC.

288

NORETHINDRONE

5.5

Spectrophotometric Analysis

K e a reported ~ ~ ~ an ultraviolet method for the quantitative analysis of norethindrone in tablets. Here, powdered tablets equivalent to 6 mg of norethindrone were extracted with 100 ml of chloroform and a 20 ml aliquot evaporated to dryness. The residue is redissolved in 100 ml methanol, its absorbance measured at 240 nm and the norethindrone content calculated. The accuracy of the method has been claimed to be better than 5 % . A modification of the above procedure has been included in the NF XII147 for determining content uniformity of norethindrone tablets. 5.6 Colorimetric Analysis A number of colorimetric methods have been used to detect and determine the quantity of norethindrone.

A. Blue Tetrazolium The reaction of blue tetrazolium with various steroids has been studied thoroughly by Meyer and Lindberg ,4 8 They noted that A4-3-ketosteroids (devoid of an a-ketol function) react with blue tetrazolium and reduce it to form a reddish-purple colored formazan. This reactivity is considerably enhanced in the case of 19-norsteroids and may result from a complex oxidative mechanism resulting in aromatization of the A-ring of these steroids. Smith and c o - w ~ r k e r sextended ~~ this concept for the analysis of norethindrone in tablets. They extracted the progestin with a chlorinated solvent mixture and developed the color by adding blue tetrazolium and tetramethylammonium hydroxide. The reaction is allowed to proceed in absence of light for sixty minutes at room temperature. Quantitation is achieved by measuring the color intensities of the standard and sample preparations at 530 run.

289

ARVlN P. SCHROFF AND ERNESTS. MOYER

B.

Isonicotinic Acid Hydrazide

Reaction of A4-3-ketosteroids with isonicotinic acid hydrazide (isoniazid) produces a ellow hydrazone which can be used for quantitation.50 8 5T* 52 Barth14 used this reaction in automating the col imetric quantitation of norethindrone in tablets, and Wu85,54 used the manual approach for the AOAC collaborative study on norethindrone, Recently,55 the Food and Drug Administration Laboratory published an automatic method which is capable of performing single tablet assays. 5.7

Titrimetric Analysis

a potentioThe British P h a r m a ~ o p e i arecommends ~~ metric titration method for the assay of norethindrone. Here a tetrahydrofuran solution of norethindrone, which also contains silver nitrate, is titrated with sodium hydroxide solution and the end point is determined potentiometrically.

Rizk and c o - ~ o r k e r smodified ~~ the method so that 30 mg of norethindrone can be easily titrated as compared to 200 mg required for the Britith Pharmacopeia method.

290

NONETHINDRONE

6. References

1. C. J. Shaw, Ortho Research Foundation, personal communication. 2. R. J. Mesley, Spectrochimica Acta 22, 889 (1966). 3. C . Djerassi, L. Miramontes, G. Rosenkranz, and F. Sondheimer, J. A!I?. Chem. SOC. 76, 4092 (1954). 4. W. Neudert and H. Ropke, "Atlas ofsteroid Spectra", Springer-Verlag, Inc., New York, 1965 Spectrum #770. 5. A. D. Cross, P. W. Landis and J. W, Murphy, Steroids 5 , 655 (1965). 6. D. C. DeJFngh, J. D. Hribar, P. Littleton, K. Fotherby, R. W. A. Rees, S. Shrader, T. J. Foell, and H. Smith, Steroids 11,649 (1968). 7. A. Zaffaroni, personal communication, 1961. 8. A. D. Mebane, Ortho Research Foundation, personal communication. 9. L. S. Nielsen, Arch. Pharm. Chemi. 74,78 (1967). 10. R. A. Bastow, J. Pharm. Pharmac. 19, 41 (1967). 11. A. P. Shroff and G. Karmas, unpublished data. 12. H. J. Ringold, C. Djerassi, and A. Bowers, U.S. Patent 3,138,589. 13. M. Kuhnert-Brandstatter, E. Junger and A. Kofler, Microchem. J. 9 , 105 (1965). 14. H. Barth, Ortho Research Foundation, personal communication. 15 The Merck Index 8th Edition, Merck and Co., Inc., Rahway, N.J. 1968, p.748 16. F. B. Colton, U. S. Patents 2,655,518 (1952), 2,691,028 (1953) , and 2,725,378 (1955). 17. C. Gandolfi and P. deRuggieri, Gazz. Chim. Ital. 94, 675 (1964). Bowers, Canadian Patent 746,499. 18. 19. G. W. Barber and M. Ehrenstein, J. Org. Chem. 2 0 , 1253 (1955). 20. A . S. Meyer, Experientia 11, 99 (1955). 21. H. Watanabe, N. N. Saha a g D. S. Layne, Steroids 11, 97 (1968). 22. K. Matsuyoshi, Folia Endocr. Jap. 43, 91 (1967). 23. H. Breuer, U. Dardenne, and W. Nocke, Acta. Endocr. 33, 10 (1960. 24. H. Breuec Lancet 11, 615 (1970). a

29 1

ARVlN P.SCHROFF AND ERNESTS. MOYER

25. 26. 27. 28. 29. 30. 31. 32. 33.

34. 35. 36. 37. 38. 39. 40. 41.

42. 43. 44. 45.

46. 47. 48.

J. B. Brown and H. A, F. B l a i r , Proc. Roy. SOC. Med. 53, 433 (1960). K. F o z e r b y , S. Kamyab, P. L i t t l e t o n , and K. J . Dennis, Acta Endocr. Suppl. 2,136 (1966). E. Gerhards, W. Hecker, H. Hitze, B. Nieuweboer, and 0. Bellmann, Acta Endocr. 68, 219 (1971). S. I s h i h a r a , F o l i a Endocr. Jap. g , , , 5 5 (1966). R. Kaiser and H. Stecher, Arch. Gynak. 194,146 (1960). S. Kamyab, P. L i t t l e t o n , and K. Fotherby, J . Endocr. 39, 423 (1967). S. Kamyab, K. Fotherby, and A. Klopper, J. Endocr. 41, 263 (1968). H. Langecker, Acta Endocr. 37, 14 (1961). C . Lauritzen and W. D. Lehmann, Arch. Gyngk. 204, 212 (1967). D . S. Layne, T. Golab, K. Arai, and G . Pincus, Biochem. Pharmac. 12, 905 (1963). S. Murata, Folia Endocr. Jap. 43, 1083 (1967). H. Okada, M. Amatsu, S. Ishihara, and G . Tokuda, Acta Endocr. 46, 31 (1964). K. H. P a l m e r , J . F. Feierabend, B. Baggett, and M. E . Wall, J. Pharmac. Exp. Ther. 167, 217 (1969). C . A. Paulsen, Metabolism 14 ,313 ( 1 9 6 5 ) . W. G . S t i l l w e l l , E . C . Horzng, M. G . Horning, R. N. S t i l l w e l l , and Z . Z l a t k i s , J . S t e r . Biochem. 3, 699 (1972). R. E. Huettemann, Ortho Research Foundation , personal communication, U. S. Pharmacopeia, 18th Rev., Mack Publishing Co. , Easton, Pa. 1970, p.453. K. Florey, U.S.P. Sub-Committee #6, personal communication. A. P. Shroff and C. J . Shaw, J. Chromatog. S c i . 10, 509 (1972). G . R. Keay, Analyst 93, 28 (1968). 5, Facts and Methods, F G M S c i e n t i f i c Research (3) 1964. B, A. Lodge, Can. J . Pharm. S c i . 5, 74 (1970). National Formulary, 13th Edirion, Mack Publishing Co. , Easton, Pa. , 1970, p.488. A. S. Meyer and M. C . Lindberg, Anal. Chem. 27, 813 (1955).

-

292

NORETHINDRONE

49. 50. 51. 52. 53. 54. 55. 56. 57.

R , V. Smith, T. H. Hassall, and S. C . Liu, J. Ass, O f f , Anal. Chem. 53, 1089 (1970). L. L. Smith and T. F o e l l , Anal. Chem. 31, 102 (1959). E . J . Umberger, Anal. Chem. 27, 768 (1955). A. E r c o l i , L. deGiuseppe and P. deRuggiere, Farm. Sci. Tec. (Pavia) 7, 170 (1952). J . Wu, J. Ass. O f f . AnalTChem. 53, 831 (1970). J . Wu, J. Ass, O f f , Anal. Chem. 54, 617 (1971). L. K. Thornton, Public Health Service (FDA) Drug Auto Analysis Manual, Method # 2 2 , April 1972, B r i t i s h Pharmacopoeia, 1973, University P r i n t i n g House, Cambridge 1973, p.323. M. R i z k , J . J. Vallon, and A. Badinand, Anal. Chim. Acta 65, 220 (1973).

-

293

NORGESTREL

Andrew M . Sopirak and Leo F. Cullen

NORGESTREL

CONTENTS

1.

2.

3. 4. 5. 6.

7.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 U l t r a v i o l e t Spectra 2.4 Mass Spectra 2.5 Melting Range 2.6 D i f f e r e n t i a l Thermal Analysis 2.7 S o l u b i l i t y 2.8 C r y s t a l Properties 2.9 Optical Rotation Synthesis S t a b i l i t y and Degradation Drug Metabolic Products Methods of Analysis 6.1 Elemental Analysis 6.2 U 1 t r a v i o l e t S pec t r o pho t ometri c Anal ye i 8 6.3 Colorimetric Analysis 6.31 I s o n i c o t i n i c Acid Hydrazide 6.32 Dinitrophenylhydrazine 6.33 Blue Tetrazolium 6.34 2 , 6 - D i - s - b u t y l - p - c r e s o l 6.4 Fluorometric Analysis 6.5 T i t r i m e t r i c Analysis 6.6 Polarographic Analysis 6.7 Chromatographic Analysis 6.71 Thin-Layer Chromatography 6.72 Gas Chromatography 6.73 Column Chromatography References

295

ANDREW M. SOPIRAK AND LEO F. CULLEN

1.

Description 1.1

Name, Formula, Molecular Weight

Norgestrel i s designated a s &-13-ethyl-l7&-ethynyl17-hydroxygon-4-en-3-one and is l i s t e d i n t h e s u b j e c t i n d i c e s of Chemical Abstracts under the heading: dl-13-ethyl-17hydroxy-18,19-dinor-l7&-pregn-4-en-20-yn-3~ne.

'21H28'2 1.2

Appearance, Color, Odor

Norgestrel i s a white t o off-white, p r a c t i c a l l y odorless, c r y s t a l l i n e powder. 2.

Physical Properties 2.1

I n f r a r e d Spectra

An i n f r a r e d absorption spectrum of a potassium bromide dispersion of n o r g e s t r e l (Wyeth Reference Standard m a t e r i a l , Lot #C-10484) i s presented i n Figure 1. This spectrum agrees with a published spectrum1 The s p e c t r a l band assignments a r e l i s t e d i n Table I.

.

-

~~

~~

-~ ~

a. Table I Infrared S p e c t r a l Assignments of Norgestrel Frequency Vibration Mode Reference _ _ _(cm.-1) 3350 OH s t r e t c h i n g 2 3270 Acetylenic C-H s t r e t c h i n g A l i p h a t i c C-H s t r e t c h i n g 2940 and 2865 Conjugated C=O s t r e t c h i n g 1655 1615 C=C s t r e t c h i n g Methyl C-H asymmetrical bending 1448 OH i n plane bending 1420 and 1335 1360 Methyl C-H synnnetrical bending 1065 Alcoholic C-0 S t r e t c h i n g Acetylenic C-H bending 700 670 AOY4.Z

-

296

WAVILINGIH IMICROhlSl

w

\D

4

3aa

IWO WAVfNUMBfR ICM ‘I

Figure 1

-

I . R . Spectrum of Norgestrel (Wyeth Reference Standard Material, 1% KBr Pellet Lot #C-10484)

-

M

ANDREW M. SOPIRAK AND LEO F. CULLEN

2.2

Nuclear Magnetic Resonance

The 60 MH, nuclear magnetic resonance (NMR) spectrum, shown i n Figure 2, w a s obtained by d i s s o l v i n g norgest r e l (Wyeth Reference Standard material, Lot tC-10484) i n deuterochloroform containing t e t r a m e t h y l s i l a n e as an i n t e r n a l reference. The only exchangeable proton i s t h e hydrogen associated w i t h oxygen a t p o s i t i o n 17. The NMR proton spect r a l assignments a r e l i s t e d i n Table 11. Table I1 0

5.86

-&cH=c’

2.61

-WE

si ng 1e t

2.31

OH

elnglet

1.02 2.3

-

-

\

-% -5

singlet

triplet

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

4 Fernandez and Noceda reported a Amax. of 240 nm. f o r n o r g e s t r e l i n absolute methanol ( a = 56.0). Norgestrel i n 95% ethanol (Wyeth Reference Standard m a t e r i a l (Lot #C10484) when scanned between 360 tun. and 200 nm. exhibited a Amax. a t 240 run. ( a = 54.6). This spectrum i n 95% ethanol is ehown i n Figure 3. 2.4

Mass Spectra

The mass spectrum of n o r g e s t r e l (Wyeth Reference Standard m a t e r i a l , Lot 8C-10484) w a s obtained by d i r e c t i n s e r t i o n of t h e sample i n t o an MS-902 double focusing, high r e s o l u t i o n mass spectrometer. The sample was run a t 200°C. and 1.0 x t o r r with t h e i o n i z a t i o n e l e c t r o n beam energy a t 70 eV.. The high r e s o l u t i o n d a t a were compiled and tabul a t e d with the aid of an on-line PDP-8 D i g i t a l Computer. Results a r e presented as a b a r graph i n Figure 4 and t h e high r e s o l u t i o n mass s p c t r u m assignments o f prominent ions a r e given i n Table 111.’ This s p e c t i s i n agreement with t h a t presented by DeJongh et.a?

298

am

Im,

m

ro

r..

f0 a0

4rJ

m

a CPS

. . . . , _ .m _ ._. _ _ , _ . .... . . , . _ _im.. . . . . . . . . . . . . . . . . . . . . .a.CPS .

t d W W

Figure 2

- NMR Spectrum of Norgestrel (Wyeth Reference 10484) - Solvent: deuterochloroform

Standard Material, Lot #C-

W

0 0

0 WAVELENGTH lnml

Figure 3

-

Ultraviolet Spectrum of Norgestrel (Wyeth Reference Standard Material, Solvent: 95% ethanol Lot #C-10484)

-

3 2 OH

MOL. WT. 312

C17H250 245

229 110 229

I

20

-

C19H2302 283

-CzHj

+-I

0

320

m/eFigure 4

- Mass

Spectrum of Norgestrel (Wyeth Reference Standard Material, Lot #C- 10484)

ANDREW M.SOPIRAK AND LEO F. CULLEN

Table I11 High Resolution Mass S p e c t r a l Assignments of Norgestrel Measured Mass Calculated Mass Formula 312.2082 312.2088 '21H28' 2 284.18 12 284.1775 '1gH24'2 283.1698 283.1698 1 ' SH23' 2 245.1902 245.1905 '1?25' 229.1596 229.1592 c16H220 110.0730 110.0731 7H100 Norgestrel gives a molecular i o n a t nJ" 312 as i t s base peak. The f i r s t prominent fragment occurs a t 297 which corresponds t o the l o s s of t h e methyl r a d i c a l , followed by e 283 which r e p r e s e n t s cleavage t h e more i n t e n s e peak a t A peak a t 284 i s of t h e 13-ethyl r a d i c a l &C2H5.). observed from t h e l o s s of C2H4.

dz

d=

Ions of 4 2 245 and d ' 229 a r e explained by the cleavage of the D-ring with hydrogen transfer.6 The i n t e n s e fragment i n t h e spectrum a t d~ 110 i s i n d i c a t i v e of t h e remaining A-ring molecular fragment w i t h t h e formula C+IloO. 2.5

Melting Range

The following melting temperature range has been observed on n o r g e s t r e l (Wyeth Reference Standard m a t e r i a l , Lot #C-10484) employing t h e U.S.P. XVIII, Class I conditions: 207-210°C.8 Short et.al. r e p o r t s a melting range temperat u r e of 2 0 6 - 2 0 7 ° C . 1 T m e l t i n g temperature range does n o t change s i g n i f i c a n t l y with v a r i a t i o n s i n heating rates from 1 t o SO~./min. 2.6

D i f f e r e n t i a l Thermal Analysis

The d i f f e r e n t i a l thermal a n a l y s i s (DTA) curve of n o r g e e t r e l (Wyeth Reference Standard material, Lot 8C-10484) obtained from room temperature t o t h e m e l t i n g point a t a heating r a t e of 20°C./min. i s presented i n Figure 5. A sharp endothermic change observed a t 209OC. corresponds t o t h e m e l t of t h e drug. 2.7

Solubility

4

The f o l owing s o l u b i l i t y d a t a were obtained a t room temperaturet

302

w

0

W

T. OC (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES)

Figure 5

- DTA

Spectrm of Norgestrel (Wyeth Reference Standard Material,

LOt #C- 10484)

ANDREW M. SOPIRAK A N D LEO F. CULLEN

chloroform acetone 95% ethanol benzene ether water 2.8

111 mg./ml. 15 mg./ml. 13 mg./ml. 7 mg./ml. 2 mg./ml. l e s s than .01 mg./ml.

C r y s t a l Properties

The X-ray powder d i f f r a c t i o n p a t t e r n of n o r g e e t r e l (Wyeth Reference Standard m a t e r i a l , Lot 8C-10484) obtained with a P h i l i p s diffractometer a t a scan rate of 1°/min. using CuKz r a d i a t i o n i s shown i n Figure 6. The c a l c u l a t e d d-spacings for the d i f f r a c t i o n p a t t e r n a r e presented i n Table I V . The c r y s t a l has orthorhombic synnnetry wizh space group P212121. U n i t ~ $ 1 8parameters are a = 20.673A, b = 12.9791, and c = 6.567A. Table I V X-Ray Powder D i f f r a c t i o n P a t t e r n f o r Norgestrel

28

d (8)

I/Io

10.95 13.9 14.2 14.5 15.2 15.8 17.4 18.0 18.9 19.8 21.2 21.8 23.3 24.9 25.3 26.0 26.3 27.6 29.05 30.4 31.8 34.7

8.08 6.37 6.24 6.11 5.83 5.61 5.09 4.79 4.69 4.48 4.19 4.075 3.817 3.575 3.520 3.428 3.389 3.232 3.073 2.940 2.814

0.02 1.00 0.53 0.81 0.20 0.72 0.17 0.06 0.06 0.42 0.04 0.05 0.19 0.24 0.12 0.09 0.09 0.07 0.07 0.10 0.10 0.07

d = (interplanar distance

1/10 = r e l a t i v e i n t e n s i t y (based on highest i n t e n s i t y of

1.001 3 04

I00

100

100

PO

90

90

80

80

80

70

70

70

60

60

60

50

50

50

40

40

40

30

30

w

0 vI

30

20

10

b

10

10

Figure 6

14

- X-Ray

18

22

2 8

10

26

M

34

3a

Powder Diffraction Pattern of Norgestrel (Wyeth Reference Standard Material, Lot fC-10484)

ANDREW

2.9

M.SOPIRAK AND LEO F. CULLEN

Optical Rotation

d l - n o r g e s t r e l i s a racemate and t h e r e f o r e e x h i b i t s no o p t i c a l r o t a t i o n . A s o l u t i o n of 500 mg. of n o r g e s t r e l i n 10 m l . of chloroform has a reading of Oo 2 0.05O i n a 1 decimeter c e l l a t 25OC. with sodium D light.9

3.

Synthesis

The b a s i c s y n t h e t i c f o r n o r g e s t r e l has been developed by Hughes and Smith and i s presented i n Figure 7. The r i n g formation i s accomplished by t h e base catalyzed condens a t i o n of 6-(m-me thoxyphenyl) -hex-l-ene-3-one (I) with 2-ethylcyclopentan-l,3-dione (11) t o y i e l d t h e t r i o n e (111). Cyclodehydration of (111) with p-toluenesulfonic acid forms gonapentaene-17-one (IV). An a l t e r n a t e s y n t h e t i c r o u t e f o r t h i s r i n g s t r u c t u r e (IV) has been described by Hughes et.al.12 and i s a l s o presented i n Figure 7. I n t h i s r o u t e t h e gonapentaene-17-one (IV) i s prepared by t h e metha n o l i c hydrochloric acid c y c l i z a t i o n of t h e eeco-oestratetraene-14,17-dione (IIIA) prepared from t h e base catalyzed condensation of 2-ethylcyclopentan-l,3-dione (IIA) with t e t r a l o l CIA). C a t a l y t i c hydrogenation of (IV) y i e l d s t h e corresponding gonatetraene-17-one (V) and subsequently converted i n t o t h e e s t r a d i o l d e r i v a t i v e (VII) by reduction i n i t i a l l y with sodium borohydride followed by potassium-ammoniat e t r a h drofuran. Conversion of (VII) by t h e Wilds and NelsonP3 modification of t h e Birch reduction14 gives t h e 1,4-dihydro d e r i v a t i v e (VIII) An Oppenauer oxidation15 produces t h e 17-ketone (1x1, which is then ethynylated, giving t h e enol-ether intermediate (XI. Treatment of (XI with methanolic hydrochloric acid y i e l d s n o r g e s t r e l (XI). A s i m i l a r syn h e t i c route i s presented i n a review a r t i c l e by K l i m s t r a . 16

€83

.

4.

S t a b i l i t y and Degradation Norgestrel i s extremely s t a b l e i n t h e s o l i d s t a t e when exposed t o long term accelerated thermal and photochemical conditions. No decomposition has been observed f o r t h i s compound when s t o r e d f o r periods of 5 years a t room temperat u r e , 1 year a t 45OC., 1 month a t 75OC. and 1 month under d i r e c t W light.17 Decomposition has been reported under severe hydrolytic and more accelerated photolytic and t h e r mal conditions.18 Studying s t e r o i d s s t r u c t u r a l l y r e l a t e d t o n o r g e s t r e l , Savard et.al.19, demonstrated t h a t exposure o f 04-3-ketos t e r o i d s t o u l t r a v i o l e t l i g h t produced s a t u r a t i o n of t h e 4,5 double bond and/or migration of t h e double bond from t h a t 306

NORGESTREL

KOH

I

CH30

IA

OH-

CH30H

C H30

IIA

1 lllA

CaC03

Pd

CH30 V

NaBH,

I

CH30H

continued Figure 7

- Synthetic Routes for Norgestrel 307

....

ANDREW M. SOPIRAK A N D LEO F. CULLEN

cn30&On

y

3

>

do

c n30

VI

Figure 7

VII

- Synthetic Routes €or Norgeetrel

308

(concluded)

NORGESTREL

p o s i t i n i n conjugation with t h e 3-ketone. Subsequent work with ~ ‘ - 3 - k e t o progestational s t e r o i s n pharmaceutical systems under photolytic conditions 28*2f have f u r t h e r supported degradation of s t e r o i d a l A-ring as described by Savard et.al. However, t h e s p e c i f i c decomposition products f o r the progestin, n o r g e s t r e l , have not been reported i n the l i t e r a t u r e .

5.

Drug Metabolic Products

Administration of racemic n o r g e s t r e l t o humans has been shown by mass spectroscopy andother analytical techniques t o y i e l d a range of metabolites. The major metabolic product has been i d e n t i f i e d as tetrahydronorgestrel, 13$-ethyl-1706 ethyn 1-5P-gonan-35 17$-diol; i.e., n o r g e s t r e l reduced a t theAe-3-keto group i n the A-ring.6 By o p t i c a l r o t a t o r y dispersion s t u d i e s , t h i s metabolite w a s shown t o be e n t i r e l y t h e o p t i c a l l y pure d-isomer22. Other metabolites have been i d e n t i f i e d as 13$-ethyl-l7e~-ethynyl-5$-gonan-3$, d i o l and mono-hydroxylated d e r i v a t i v e s of norgestrel. 4 3 3 4

6.

Methods of Analysis 6.1

Elemental Analysis

The following d a t a were obtained on n o r g e s t r e l (Wyeth Reference Standard m a t e r i a l , Lot #C-10484). E 1ement C

H

% Theory 80.73% 9.03%

6.2

% Determined 80.66% 8.92%

U l t r a v i o l e t Spectrophotometric Analysis A 4 -3-ketosteroids produce a c h a r a c t e r i s t i c chromophore s stem with maximum absorption between 230 nm. and 270 The u l t r a v i o l e t absorption maximum of n o r g e s t r e l a t about 240 nm. i n 85% ethanol i n water (v/v) has been u t i l i z e d f o r t h e analysis of the s t e r o i d i n t a b l e t formulat i o n s containing estrogens.26 In t h i s procedure a sample equivalent t o about 0.5 mg. n o r g e s t r e l i s s t i r r e d i n 85% ethanol i n water (v/v>. This s o l u t i o n i s d i l u t e d t o o b t a i n a f i n a l concentration of about 10 pg./ml. i n t h e 85% ethanol. Following c e n t r i f u g a t i o n t o remove insoluble excipient materi a l , the absorbance i s spectrophotometrically measured and compared with a standard s o l u t i o n of norgestrel. Norgestrel has been i s o l a t e d from i n t e r f e r i n g formulation components by an e x t r a c t i o n of the s t e r o i d with organic solvents (viz., chloroform and methylene c h l o r i d e ) from an a c i d i c aqueous solution.

309

ANDREW M. SOPIRAK A N D L E O F. C U L L E N

A modified v e r s i o n of an automatic analyzer system procedure f o r a relatedA4-3-ketosteroid27 has been applied t o t h e q u a n t i t a t i v e a n a l y s i s of norgestre1.l’ This completely automated technique i s capable o f d i s i n t e g r a t i n g a whole t a b l e t , dissolving the active constituent, f i l t e r i n g it, dilut i n g a portion of t h e c l e a r f i l t r a t e t o a d e s i r e d volume and o b t a i n i n g a complete u l t r a v i o l e t absorption spec trum. The accuracy of t h e automated method i s comparable t o t h a t of t h e manual spectrophotometric method f o r n o r g e s t r e l .

The absorption c h a r a c t e r i s t i c s e x h i b i t e d by norg e s t r e l i n ethanol s o l u t i o n a t about 240 nm. i s due t o t h e conjugated c h a r a c t e r of t h e OC, p-unsaturated carbonyl group i n t h e A-ring. Any a l t e r a t i o n i n t h e conjugated c h a r a c t e r of t h i s s y s t e m by prolonged h e a t i n g a t extreme temperatures o r by prolonged i r r a d i a t i o n w i t h a s t r o n g u l t r a v i o l e t l i g h t source w i l l r e s u l t i n a corresponding l o s s i n u l t r a v i o l e t a b s o r p t i v i t y a t 240 nm. Consequently, t h i s method w i l l measure i n t a c t n o r g e s t r e l i n t h e presence of i t s photochemi c a l and t h e ma1 degradation products and i s , thus, s t a b i l i t y i n d i c a t i n g 2E

.

Reactions of t h e 3-keto group of p r o g e s t a t i o n a l s t e r o i d s with t h e connnon carbonyl reagents have provided t h e b a s i s f o r s e v e r a l o t h e r spectrophotometric a n a l y t i c a l These techniques can b e applied t o t h e a n a l y s i s of methods. n o r g e s t r e l . Evans and G i l l i a m observed t h a t t h e absorption maxima of t h e thiosemicarbazones of s a t u r a t e d and oC, p-unsatur a t e d ketones could be determined i n t h e presence of excess thiosemicarbazide.28 Bush29 and Talbot et.al.30 have ap l i e d t h i s technique t o t h e determination of a s e r i e s of A -3-ketosteroids which y i e l d thiosemicarbazones w i t h absorption maxima a t 299 t o 301 nm.

e

Gtlr6g3l developed a procedure f o r t h e determinai s based on t h e C l a i s e n cont i o n 0 € ~ ~ - 3 - k e t o s t e r o i d which s densation of t h e a c t i v e methylene groups of k e t o s t e r o i d s w i t h d i e t h y l o x a l a t e l e a d i n g t o spectrophotometrically a c t i v e glyoxalyl d e r i v a t i v e s . The development of t h e chromophore was c a r r i e d o u t a t room temperature i n a mixture of t e r t i a r y butanol and cyclohexane i n t h e presence of sodium t e r t i a r y butoxide while t h e absorbance w a s measured i n modera t e l y a c i d i c ethanol. The method i s s u i t a b l e f o r t h e chara c t e r i z a t i o n and q u a n t i t a t i v e determination of n o r g e s t r e l which produces absorption maxima a t about 244 run. and 318

nm.17

310

NORGESTREL

S a l i c y l o y l hydrazide r e a c t s with A4-3-ketoeteroids t o form c h a r a c t e r i s t i c hydrazones which absorb s t r o n g l y i n t h e ~ l t r a v i o l e tand ~ ~m a be used f o r t h e a n a l y t i c a l d e t e r mination of n o r g e s t r e l . 17 Giirijg developed a simple and r a p i d u l t r a v i o l e t spectrophotometric method f o r t h e d e t e r m i n a t i n of A4-3The method k e t o s t e r o i d s i n pharmaceutical i s based on t h e reduction of t h e C-3 carbonyl group with sodium borohydride, followed by t h e determination of t h e decrease of absorbance due t o t h e reduction ae measured by d i f f e r e n t i a l spectrophotometry. This procedure i s a p p l i cable t o t h e a n a l y s i s of n o r g e s t r e l i n t a b l e t formulations.1 7 E t h i n y l e s t r a d i o l and common i n a c t i v e t a b l e t components do not i n t e r f e r e with t h e determination.

preparation^.^'

6.3

Colorimetric Analysis 6.31

I s o n i c o t i n i c Acid Hydrazide

Norgestrel can be assayed u t i l i z i n g t h e wellknown i soni cot i n i c acicj4hydr azid e (INH) c o l o r i m e t r i c method described by Umberger. The chemistry of t h i s technique i s based on t h e formation of t h e i s o n i c o t i n y l hydrazone of n o r g e s t r e l from t h e r e a c t i o n of t h e s t e r o i d with INH reagent i n absolute alcohol a c i d i f i e d w i t h hydrochloric acid. The A 4-3-keto group r e a c t s q u a n t i t a t i v e l y a t room temperat u r e i n l e s s than 1 hour. A s t a b l e colored s p e c i e s r e s u l t s which allows measurement of i t s absorption a t about 380 nm. I n t h e a n a l y s i s of t a b l e t formulations, n o r g e s t r e l i s extracted with chloroform from an a c i d i c aqueous suspension of t h e t a b l e t s , INH reagent i s added t o an a l i q u o t of t h e e x t r a c t and t h e r e s u l t a n t yellow s o l u t i o n i s spectrophotometrically measured. Estrogens E i z . , 1 7 a e t h i n y l e s t r a d i o l and 17.C e t h i n y l estradiol-3-methyl e t h e r ( m e s t r a n o l x and i n a c t i v e components t y p i c a l l y found i n o r a l estrogen-progestin combil o r developn a t i o n dosage forms do not c o n t r i b u t e t o t h ment; t h u s , do n o t i n t e r f e r e i n t h e assay. 28,

-

%

The absorption c h a r a c t e r i s t i c s exhibited by n o r g e s t r e l i n t h i s c o l o r i m e t r i c method a r e due t o t h e conjugated c h a r a c t e r of t h e s t e r o i d A - r i n g w i t h t h e formed hydrazone. Thus, t h i s method w i l l measure i n t a c t norgest r e l i n t h e presence of i t s photochemical and thermal degrad a t i o n products f o r reasons analogous t o those d e s c r i ed f o r t h e u l t r a v i o l e t spectrophotometric procedure.17, 28 Consequently, t h e procedure i s a l s o s t a b i l i t y - i n d i c a t i n g .

311

ANDREW M. SOPIRAK A N D LEO F. CULLEN

Russo-Aleei describes a completely automated INH reagent procedure f o r the content uniformity t e s t i n g of c o r t i c o s t e r o i d t a b l e t formulation^.^^ This s e n s i t i v e , accur a t e , and r a p i d automatic analyzer procedure has been adapted t o the analysis n o r g e s t r e l i n o r a l contraceptive t a b l e t formulations.

~5

6.32

Dinitrophenylhydrazine

S t e r o i d s with an K , p-unsaturated carbonyl group were observed by Djerassi and Ryan t o produce 2,4dinitrophenylhydrazones i t h absorption maxima a t 390 nm. i n chloroform ~ o l u t i o n . ~ ’ Gornall and M a ~ D o n a l dhave ~~ described a c o l o r i m e t r i c procedure, a p p l i c a b l e t o norgest r e l , i n which t h e pale yellow c o l o r of the hydrazone d e r i v a t i v e i s converted t o a s t a b l e , deep red resonating quinoidal i o n species i n a l k a l i n e s ~ l u t i o n . ~ ’ The r e a c t i o n with n o r g e s t r e l i s complete i n l e s s than 10 minutes and t h e r e s u l t i n g colored species i s spectrophotometrically measured a t about 450 nm. I n a r e l a t e d study, Nishina et.al.40 r e p o r t t h a t A 4-3-ketosteroids w i l l condense w i t h p-nitrophenylhydrazine t o yield t h e corresponding hydrazone, which produces a reddish-purple species i n an a l k a l i n e s o l u t i o n of dimethylformamide. Norgestrel produces a Amax a t 540 run. 6.33

Blue Tetrazolium

The u t i l i t y of t h e blue tetrazolium reagent i n t h e anal s i s o f 4 - k e t o l and non-ketol s t e r o i d s i well d o ~ u m e n t e d . z ~Meyer ~ ~ ~ and Lindberg43 noted t h a t ~‘-3-ketos t e r o i d s , devoid of an c - k e t o l function, r e a c t with blue tetrazolium i n t h e presence of a s t r o n g organic base t o form the reddish-purple difomazan d e r i v a t i v e with a Amax a t 530 nm. Application of t h e t e t r a z o l i u m technique t o the s e l e c t i v e a n a l y s i s of n o r g e s t r e l i n anovulatory t a b l e t formulations containing estrogens has been described by Smith et.a1.44 This c o l o r i m e t r i c r e a c t i o n has been adapted t o an automatic analyzer procedure f o t h e a n a l y s i s of n o r g e s t r e l i n biopharmaceutical s ys tem s. 45

6.34

2,6-Di-tert-butyl-p-cresol

A c o l o r r e a c t i o n i s obtained by the a c t i o n of 2,6-$&;~frt-butyl-p-cresol on n o r g e s t r e l i n an a l k a l i n e medium. The concentration of n o r g e s t r e l i n t h e r e s u l t i n g colored s o l u t i o n can be determined spectrophotometr i c a l l y i n t h e v i s i b l e region.

312

NORGESTREL

6.4

Fluorometric Analysis

Cullen et.al.18 describe a s e n s i t i v e procedure, based on a s u l f u r i c acid-induced fluorescence, f o r t h e analysis of norgestrel i n t a b l e t s of low dosage, i.e., 1575 pg. per t a b l e t . Fluorogen formation, which i s e f f e c t e d with an 85% s u l f u r i c acid reagent, i s measured a t an emiss i o n h a x of 550 nm. with an e x c i t a t i o n k a x of 418 nm. S p e c i f i c i t y of t h e method with respect t o the a n a l y s i s of i n t a c t n o r g e s t r e l i n the presence o f i t s photochemical and thermal degradation products w a s demonstrated by comparison t o q u a n t i t a t i v e thin-layer chromatography assay values. This procedure has been completely automated t o permit rapid content uniformity t e s t i n g of the dosage form. The automatic analyzer procedure i s capable of analyzing 15 samples per hour with a r e l a t i v e standard deviation of 2 1.4% a t t h e 50 pg. n o r g e s t r e l per t a b l e t level. Short and coworkers ” 48 applied fluorometry t o the determination of n o r g e s t r e l i n b i o l o g i c a l f l u i d s . Norg e s t r e l i s extracted from serum with methylene c h l o r i d e and subsequently re-extracted i n t o an 80% s u l f u r i c acid i n ethanol reagent which produces an i n t e n s e fluorescence. The r e s u l t i n g f luorogen i s measured a t an emission Amax of 520 nm. and e x c i t a t i o n k a x of 460 nm. This method can be applied t o the analysis of n o r g e s t r e l s o l u t i o n s containing 0.02 pg. per m l . Bush and Sandberg4’ noted t h a t paper chromatograms sprayed with sodium hydroxide developed an orange-yellow fluorescence s p e c i f i c for&-3-ketosteroids under W i r r a d i ation. Subsequently, Abelson and Bond$’ found t h a t potassium --butoxide could produce the a l k a l i n e fluorogenic r e a c t i o n , and i s s u i t a b l e f o r q u a n t i t a t i v e measurement of n o r g e s t r e l i n t h e 0.1-10.0 pg. per m l . l e v e l i n e t h a n o l i c solu t i on. 17

6.5

T i t r i m e t r i c Analysis

The e t h i n y l group of norgeetrel r e a c t s s t o i c h i o m e t r i c a l l y with s i l v e r n i t r a t e i n tetrahydrofuran. The n i t r i c acid produced can be t i t r a t e d w i t h 0.1 N sodium hydroxide t o a poten iometric endpoint using a glass-calomel electrode system. 51,52

313

ANDREW M. SOPIRAK A N D LEO F. CULLEN

6.6

Polarographic Analysis

Norgestrel e x h i b i t s a well-defined cathodic wave a t t h e dropping mercury e l e c t r o d e i n a l k a l i n e isopropanol. The halfwave p o t e n t i a l of n o r g e s t r e l i n t h i s system i s about - 1 . 5 ~ and t h e d i f f u s i o n c u r r e n t i s l i n e a r with concentration over t h e range of to M.9 6.7

Chromatographic Analysis

Chromatography i s used q u a l i t a t i v e l y 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 l y f o r t h e determination of p u r i t y and s t a b i l i t y of norgeetrel. 6.71

Thin-Layer Chromatography

The various e l u a n t and adsorbent s y s tems used €or thin-layer chromatography of n o r g e s t r e l are given i n Table V. The v i s u a l i z a t i o n techniques used f o r d e t e c t i o n of n o r g e s t r e l a r e a l s o included i n Table V.

6.72

Gas Chromatography

Norgestrel has been d i r e c t l y chromatographed on a 4 f t . x f inch g l a s s column packed with 0.5% QF-1 on Chromosorb G (80/100 mesh) u t i l i z i n g a flame i o n i z a t i o n detector.55 A column temperature of 215OC. i s employed with helium a t 40 ml./min. as the carrier gas. This technique has been applied t o the s e l e c t i v e a n a l y s i s o f norgest r e l a t t h e 0.25 t o 1.0 mg. dosage l e v e l i n anovulatory t a b l e t formulations. I n t h e a n a l y s i s n o r g e s t r e l i s extracted with chloroform from an a c i d i c aqueous auspeneion of the t a b l e t s , the chloroform e x t r a c t i s concentrated by evaporat i o n and subsequently chromatographed using c h o l e s t e r o l as the i n t e r n a l standard. 6.73

Column Chromatography

T h e q u a n t i t a t i v e a n a l y s i s of n o r g e s t r e l and s t r u c t u r a l l y r e l a t e d progestational s t e r o i d s i n o r a l l y admini s t e r e d anovulatory formulations hae been described u t i l i z i n g a gel f i l t r a t i o n column technique and u l t r a v i o l e t spectrome t r ~ A . ~s y n t h e t i c p l y s a c c h a r i d e (Sephadex LH-20) column with methanol-water (17:3) as t h e e l u a n t permits t h e quantit a t i v e separation of n o r g e s t r e l from formulation components. Estrogens (viz., e t h i n y l e s t r a d i o l , mestranol, e s t r a d i o l , and e s t r a d i o l benzoate) and comnon t a b l e t e x c i p i e n t m a t e r i a l s were shown not t o i n t e r f e r e i n t h i s assay procedure. 314

NORGESTREL

Table V Thin-Layer Chromatographic System for Norgestrel Solvent Visualization Technique Rf Reference System Adsorbent 1, 2 0.37 53 Silica Gel GF A B Silica Gel GF 1; 2 0.45 53 C 1, 2 0.59 53 Silica Gel GF D 1, 2 0.53 53 Silica Gel GF E 1, 2 0.47 54 Silica Gel G impregnated with DuPont Lumines cent Chemical 609 F Silica Gel G 1, 2 0.46 54 impregnated with DuPont Luminescent Chemical 609 G Silica Gel G 3 0.54 26 Silica Gel G 4 0.73 1 H MN Silica Gel 1, 2 18 I

-

-

--

G-HR/W

A B C D E F G H

I 1 2 3

4

Solvent Systems Benzene:methanol (95: 5) Benzene:acetone (80:20) Chloroform:methanol (90: 10) Methylene ch1oride:me thanol: water (150: 9! 0.5) Petroleum ether: ethyl ether (10: 90) Petroleum ether: ethyl ether:diethylamine (4 0 : 60: 10) Chloroform: alcohol, U.S. P. (96: 4 ) N-hexanet ethyl acetate (1: 3) Benzene:chloroform (8:2) Visualization Techniques Ultraviolet light Sulfuric Acid Spray Reagent 10% Ethanolic Phosphomolybdic Acid Spray Reagent Antimony Trichloride Spray Reagent

315

ANDREW M. SOPIRAK AND LEO F. CULLEN

7.

References

1

2 3.

4. 5. 6.

7. 8. 9. 10.

P. M. Short, E. T. Abbe and C. T. Rhodes, Can. J. Pharm. S c i . , A, 8(1969). L. Bellarny, I I T h e I n f r a r e d Spectra of Complex Molecules," 2nd Ed., J. Wiley and Sons, Inc., New York, N. Y., 1964, Chapter 6. R. M. S i l v e r s t e i n and G. C. Bassler, "Spectrometric I d e n t i f i c a t i o n of Organic CompoundeYf12nd Ed. J. Wiley and Sons, Inc., New York, N. Y., 1967, Chapter 3. A. A. Fernandez and V. T. Noceda, J. Pharm. Sci., 58, 740(1969). T. Chang and C. Kuhlman, Wyeth Laboratories, Inc., Personal Communication. D. C. DeJongh, J. D. H r i b a r , P. L i t t l e t o n , K. Fotherby, R. W. A. Reee, S. Shrader, T. J. F o e l l and H. Smith, S t e r o i d s , 2, 649(1968). W n i t ed S t a t e s Phannacopei a," 18th Revision , Mack Publishing Co., Easton, Pa., 1970, pp. 935-936. N. DeAngelis , Wyeth Laboratories, Inc., Personal Comnunicat ion. M. B. Freeman, Wyeth Laboratories, Inc., Personal Communication. H. Smith, G. A. Hughes, G. H. Douglas, G. R. Wendt, G. C. Buzby, R. A. Edgren, J. F i s h e r , T. F o e l l , B. Gadsby, D. Hartley, D. Herbst, A. B. A. Jansen, K. Ledig, B. J. McLoughlin, J. McMenamin, T. W. Pattieon, P. C. P h i l l i p s , R. Rees, J. S i d d a l l , J. Siuda, L. L. Smith, J. Tokolics and D. H. P. Watson, J. Chem. SOC., 4472(1964). V. Petrow, Chem. Rev., 70, 713(1970). G. H. Hughes and H. Smith, U. S. Patent 3,478,106 (1969) : i . S . Patent 3,442;920(1969). A. L. Wilds and N. A.-Nelson, J. Amer. Chem. SOC., -975 5360(1953). A. J. Birch and S. M. Mukherji, Nature, 163, 766 (1949). R. V. Oppenauer, Org. Syn., 2,18(1941). P. D. Klimstra, J. h e r . &am. Educ., 630(1970). L. F. Cullen, Wyeth Laboratories, Inc., Personal Communic a t ion L. F. Cullen, J. G. Rutgers, P. A. Luccheei and G. J. Papariello, J. Pharm. Sci., 57, 1857(1968). K. Savard, H. W. Wotiz, P. Marcus and H. M. Lemon, J. Amer. Chem. SOC., 75, 6327(1953).

,

-

1964,

11. 12.

13. 14. 15. 16. 17. 18. 19.

10,

.

316

NORGESTREL

20.

21.

22. 23 24. 25. 26.

27. 28 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

R. E. Graham, P. A. Williams and C. T. Kenner, J. Pharm. Sci., !By 1152(1970). W. E. Hamlin, T. Chulski, R. H. Johnson and J. G. Wagner, J. Amer. Pharm. A s s . , S c i . Ed., 253(1960). K. Fotherby and C. A. Keenan, Acta Endocrinol. Suppl. -9138 83(1969). P. L i t t l e t o n and K. Fotherby, Acta Endocrinol. Suppl. 119, 162(1967). S. Kamyab, K. Fotherby and G. Wilson, J. Biochem., -9103 14(1967). T. Higuchi and E. Brochmann-Hanssen, I1Pharmaceutical Analysis,11 I n t e r s c i e n c e P u b l i s h e r s , Inc., New York, N. Y., 1961, Chapter IV. G. J. P a p a r i e l l o , Wyeth L a b o r a t o r i e s , Inc. Personal Communication. A. J. Khoury and L. J. C a l i , Ann. N. Y. Acad. Sci., 153, 456(1968). L. K. Evans and A. E. G i l l i a m , J. Chem. SOC., 565(1943). I. E. Bush, Fed. Proc., 186(1953). N. B. Talbot. S. Ulick. A. Koupreianow and A. 301 Zygmuntowicz, J. Clin. Endocrinol. Metab., (1955). S. Gorog, Anal. Chem., 42, 560(1970). 292(1959). P. S. Chen Jr., Anal. Chem., S. Gerog, J. Pharm. S c i . , 57, 1737(1968). E. J. Umberger, Anal. Chem., 27, 768(1955). Y. P. John, J. A s s . Offic. Anal. Chem., 53, 83l(1970). F. M. Russo-Alesi, Ann. N. Y. Acad. S c i , , 511 (1968). C. D j e r a s s i and E. Ryan, J. h e r . Chem. SOC., lOOO(1949). A. G. Gornall and M. P. MacDonald, J. Biol. Chem., 201, 279(1953). F. D. Chattaway and G. R. Clemo,,J. Chem. SOC., 3041(1923). T. Nishina, Y. Sakai and M. Kimura, S t e r o i d s , 4, 255(1964). W. J. Mader and R. R. Buck, Anal. Chem., 24,. 666 (1952). J. E. Sinsheimer and E. F, Salim, Anal. Chem., 37, 566(1965). A. S. Meyer and M. C. Lindberg, Anal, Chem., 27, 813(1955). R. V. Smith, T. H. Hassall and S. C. Liv, ,J. A s s . Offic. Anal. Chem., 53, 1089(1970). P. M. S h o r t and C. T. Rhodes, S t e r o i d s , 6 .l, 217(1970).

2,

-

,

-

1943,

2,

15,

2,

153,

71,

-

1923,

317

ANDREW

46. 47. 48. 49

.

50. 51 52. 53.

54. 55.

M.SOPIRAK AND

LEO F. CULLEN

2,

E. P. Schultz and J. D. Neuss, Anal. Chem., 1662(1957). E. P. Schultz, M. A. Diaz and L. M. Guerrero, J. Pharm. Sci., 53, 1119(1964). M. P. Short and C. T. Rhodes, Can. J. Pharm. Sci., 26(19731_ I. E. Bush and A. A. Sandberg, J. Biol. Chem., 205, 783(1953). D. Abelson and P. K. Bondy, Arch. Biochem. Biophys., -' 57 208(1955). "United S t a t e s Phanacopei a," 1 8 t h Revis ion, Mack Publishing Co., Easton, Pa., 1970, pp. 454-455. J. G. Rutgers, Wyeth Laboratories, Inc., Personal Communication. M. B. Simard and B. A. Lodge, J. Chromatogr., 51, 517(1970). G. Moretti. G. Cavina, P. P a c i o t t i and P. Siniscalchi, I1 Farmaco; 27, 537(1972). K. Dilloway, Wyeth Laboratories, Inc., Personal Communication.

S,

The L i t e r a t u r e Search was conducted up t o July, 1974.

318

PHENFORMIN HYDROCHLORIDE

Joseph E. Moody

JOSEPH E. MOODY

CONTENTS Analytical P r o f i l e

- Phenfonnin Hydrochloride

1, Description 1.1 N a m e , Formula, Molecular Weight 1.2 Appearance, Color, Odor 2.

Physical P r o p e r t i e s 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 Mass Spectrum 2.5 Melting Range 2.6 Solubility 2.7 Distribution Coefficients 2.8 I o n i z a t i o n Constant pKa 2.9 D i f f e r e n t i a l Scanninq Calorimetry 2.10 Thennogravimetric Analysis

,

3.

Synthesis

4,

Stability

5.

Metabolism

6.

Methods of Analysis E l e m e n t a l Analysis 6.1 6.2 Ultraviolet 6.3 Fluorometric 6.4 Colorimetric 6.5 Chromatographic 6.51 Paper 6.52 Thin Layer 6.53 P a p e r E l e c t r o p h o r e s i s 6.54 Gas-Liquid

7.

Rsferences

320

PHENFORMIN HYDROCHLORIDE

1.

Description

1.1 N a m e , Formula, Molecular Weight P h e n f o d n hydrochloride is named i n Chemical Abstracts as 1-phenethylbiguanide hydrochloride. Other names occasionally used are N ’ - P -phenethylformamidinyliminourea hydrochloride and DBI.

CH2-CH2-NH-C-NH-C-NH2. I1 II N-H

HC1

N-H

Mol. W t .

C10H16N5C1

9

241.73

Appearance, Color, Odor Phenfonnin hydrochloride i s a white o r p r a c t i c a l l y white, o d o r l e s s , c r y s t a l l i n e powder. 1.2

2.

Physical 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 of p h e n f o d n hydrochloride (USP Standard) i s shown i n f i g u r e 1. The spectrum w a s obt a i n e d on a Perkin-Elmer 621 Spectrophotometer from a KBr The s t r u c t u r a l assignments have been c o r r e l a t e d pellet, with t h e following band frequencies. Frequency (cm’l)

3400 3200 1660

Ass iqnment

- 3250

-

N-H

stretching

Amine s a l t N-H s t r e t c h i n g

3100

- 1610

;C=N

(a-P-unsaturated)

1590

-

1510

Amine s a l t bending

756

&

695

Phenyl (monosubstitutedl

Nuclear Magnetic Resonance Spectrum Phenformin hydrochloride displayed t h e NMR spectrum shown i n f i g u r e 2. The sample w a s d i s s o l v e d i n dimethyl sulfoxide-d6. The spectrum was measured on a Varian A-60 NMR Spectrometer with TMS as t h e i n t e r n a l reference. The follcrwi n g s t r u c t u r a l assignments have been made f o r f i g u r e 2. 2.2

321

W

?

N t 4

Figure 1.

Infrared Spectrum of Phenformin Hydrochloride (USV House Standard batch 22574) taken i n KBr Disc. Instrument: Perkin E l m e r 621.

PHE NFORMl N HYDROCHLORIDE

Chemical S h i f t ( 6 1

Assignment

2.90 and 3.35

-C;rCErNH-

7.05

Exchangeable

7.30

Aromatic

U l t r a v i o l e t Spectrum S h a p i r o (1) r e p o r t e d t h e a b s o r p t i o n s p e c t r a f o r phenformin hydrochloride. The s p e c t r a are shown i n T a b l e 1. Samples were measured i n a c i d i c , n e u t r a l and a l k a l i n e medium a s w e l l a s i n methanol. 2.3

TABLE 1

U l t r a v i o l e t S p e c t r a of Phenformin Hydrochloride Solvent

X max (nm)

E x 10-3

H 20

233

14.5

1~10'~NHCl

233

11.2

1~10'~NHCl

233

14,s

1x1 '0 'NN aOH

2 32

12.7

1 N NaOH

methanol

225

- 228

2 34

12,o 17.7

Mass Spectrum Phenformin hydrochloride w a s found t o be u n s t a b l e under electron impact-mass spectrometry c o n d i t i o n s . The major fragments peaks were observed a t m/e 205, 146, 104, and 101 ( 2 ) . The molecular ion d i d n o t appear. 2.4

2.5 176-178'

Melting Range Phenformin h y d r o c h l o r i d e h a s a m e l t i n g p o i n t of

(1).

323

w

w P

F i g u r e 2.

NMR Spectrum of Phenformin Hydrochloride (USV House Standard batch 22574) in deuterated DMSO. Instrument: V a r i a n A-60.

PHENFORMIN HYDROCHLORIDE

2.6

Solubility The s o l u b i l i t y of p h e n f o d n hydrochloride was determined a t room temperature i n t h e following s o l v e n t s .

mg/ml

Solvent

S o l v e n t < 0.02 m q / d

Water

83

Chloroform

Ethanol Abs.

34

Acetone

Ethanol 959

64

E t h y l Acetate

Methanol

225

Isopropanol

Ether

Hexane

3.8

D i m e thy1f ormamide

415

2.7

Distribution Coefficients The d i s t r i b u t i o n c o e f f i c i e n t s , P, of p h e n f o d n hydrochloride were determined i n t h e oil/water systems, A and B. The values a r e shown below as a function of sodium hydroxide concentration ( 3 ) . The p a r t i t i o n i n g of t h e drug i n t o t h e organic phase i n c r e a s e s w i t h higher pH levels. (NaOH)

(NaOH)

pB

0.32

0.20

1.92

0.24

0.57

0.80

2.37

0.64

System A = methylene chloride/water System B

-

(111)

chloroform t-amylalcohol/water

2.8

I o n i z a t i o n Constant, p K a P h e n f o d n i s a s t r o n g l y b a s i c substance and consequently e x i s t s i n d i and mono i o n i c f o r m . Ray (4) has reported t h e i o n i z a t i o n c o n s t a n t s , pKa' 11.8 and pKa" 2.7 a t 32'. I n our l a b o r a t o r y the apparent pKa" 3.1 w a s measured a t 25' by p o t e n t i o m e t r i c t i t r a t i o n . The second i o n i z a t i o n c o n s t a n t , pKa' could n o t be r e l i a b l y measured

,

,

325

-

- -

JOSEPH E . MOODY

i n aqueous m e d i u m by our method. However, Garrett (3) has c a l c u l a t e d an approximate pKa' of 13.0 from plots of the rec i p r o c a l s of the apparent p a r t i t i o n c o e f f i c i e n t s a g a i n s t t h e hydrogerl ion concentration. 2.9

D i f f e r e n t i a l Scanning Calorimetry The thermal s t a b i l i t y of phenformin hydrochloride w a s determined on a Perkin-Elmer Model DSC-1B Thermal Analyzer ( 2 ) . The sample is stable when heated t o loo below i t s melting p o i n t for 30 minutes. A m e l t endotherm w a s observed a t 175 179O. However, phenformin hydrochloride i s unstable on h e a t i n g above i t s melting p o i n t . The following data shows t h e h e a t of fusion and melting p o i n t of p h e n f o d n hydrochloride as determined by DSC Analysis on a sample of 99.8% p u r i t y

-

.

- 39.8 cal/gram 9600 cal/mole Melting Range - 175 - 179OC

Heat of Fusion

Melting P o i n t

- 176.5OC

Thermogravimetric Analysis Phenformin hydrochloride i s weight stable through i t s melting point. At-temperatures above 230°, the sample loses approximately 30% of i t s i n i t i a l weight. The thermal a n a l y s i s d a t a was determined on a Perkin-Elmer TGS Thermalanalyzer (2). 2.10

3.

Synthesis Phenformin hydrochloride can be prepared by the a d d i t i o n of 2-phenylethylamine hydrochloride t o dicyanodiamine a t l5Oo (1). Phenformin hydrochloride i s then r e c r y s t a l l i z e d from the r e a c t i o n mixture with isopropanol.

Stability Phenformin can be recovered as t h e d i a c i d s a l t a f t e r prologned h e a t i n g with 3 and 6N hydrochloric acid, 50% s u l f u r i c acid o r polyphosphoric acid. Phenformin hydrochloride i s s t a b l e i n d i l u t e sodium hydroxide a t room temperature. However, t h e compound hydrolyzes i n s t r o n g sodium hydroxide 4.

326

PHENFORMIN HYDROCHLORIDE

t o p-phenethylguanidine and small amounts o f P-phenethyiurea and p-phenethylamine (1). 5.

Metabolism Wick (5) i d e n t i f i e d t h e major metabolites of phenformin hydrochloride i n t h e e x c r e t e d u r i n e o f male rats as p-hydroxy p - phenethylbiguanide and i t s glucuronide conjugate.

-

Beckmann (6) d i s c o v e r e d t h a t m a n also m e t a b o l i z e s phenformin hydrochloride t o p-hydroxyphenformin. Nearly 33% o f t h e dose was e x c r e t e d as t h i s m e t a b o l i t e . H a l l (7) s t u d i e d t h e metabolism o f phenfonnin w i t h an i n v i t r o l i v e r system. The b i o t r a n s f o r m a t i o n t o p-hydroxphenformin w a s i n h i b i t e d by a known m e t a b o l i c i i b i t o r . T h i s evidence and t h e t i s s u e d i s t r i b u t i o n o f Cl'labeled phenformin s u g g e s t t h a t t h e l i v e r is t h e major s i t e of metabolism.

-6.

Methods o f Analysis 6.1

Elemental Analysis Element

%

Theory

0 Reported

C

49.7

49.6

H

6.7

6.7

N

29.0

29.3

c1

14.6

14.3

6.2

Ultraviolet Phenformin hydrochloride can be analyzed by measuring i t s absorbance i n water a t 233 nm ( 8 ) . 6.3

Fluorometric Bailey (9) treated s o l u t i o n s o f phenformin hydroc h l o r i d e with a l k a l i n e ninhydrin and measured t h e f l u o r e s cence of t h e phenformin-ninhydrin complex a t 518 nm on a Turner fluorometer. The f l u o r e s c e n t complex showed e x c i t a t i o n peaks a t 304 and 392 nm. The a c t i v a t i o n monochrometer w a s s e t a t 390 nm. This method can detect 0.025 pg/ml of phenformin.

321

JOSEPH E . MOODY

Colorimetric Methods Phenformin hydrochloric forms an e x t r a c t a b l e ( 1 1 1 ) i o n p a i r with bromthymol blue. Garrett (10) found t h a t the i o n p a i r i s e x t r a c t e d q u a n t i t a t i v e l y i n methylene c h l o r i d e from aqueous s o l u t i o n . - The absorbance of t h e ion p a i r was then measured i n methylene c h l o r i d e a t 413 nm. Garrett discovered a more s e n s i t i v e procedure by t r e a t i n g t h e phenfodn-bromthymol blue i o n pair with excess tetrabutylammonium hydroxide t o form a ( 2 r l ) tetrabutylammniumbromthymol blue ion pair. The absorbance of t h i s (211) i o n pair w a s determined a t 630 nm. This l a t t e r method w a s applied t o t h e a n a l y s i s of phenformin i n human urine. Minor i n t e r f e r e n c e from n i c o t i n e i n some u r i n e samples was observed. 6.4

Shepherd and McDonald (11) developed a c o l o r i m e t r i c assay based on t h e c o l o r r e a c t i o n of phenfonnin hydroc h l o r i d e with t h e 1-naphthol-diacetyl reagent. The q u a n t i t y of phenformin was determined from t h e absorbance of the colored s o l u t i o n a t 565 nm. Beer's Law w a s obeyed up t o a concentration of 20 pg of phenfonnin per m l of s o l u t i o n . This method was applied t o t h e a n a l y s i s of phenformin i n b i o l o g i c a l f l u i d s (12). The U. S. Pharmacopeia (13) des c r i b e s t h i s c o l o r i m e t r i c procedure. To achieve t h e s p e c i f i c i t y required o f modern a n a l y t i c a l methods, t h e s e c o l o r i m e t r i c procedures should be coupled with a suitable chromatographic s e p a r a t i o n technique. 6.5

Chromatographic Methods Phenformin hydrochloride can be determined by t h e following chromatographic methods. 6.51

Paper Chromatography The following d a t a show t h e s o l v e n t system and response f a c t o r s , (Rf) f o r p h e n f o d n hydrochloride. For t h e q u a l i t a t i v e d e t e c t i o n of phenformin, t h e chromatograms can be sprayed with the l o c a t i o n reagents. pentacyanoaquoferriate (PCF) Sakaguchi, and 1-naphthol-diacetyl reagents (14, 15). 1-Naphthol-diacetyl reagent i s the most s e n s i t i v e , with a d e t e c t i o n l i m i t a t i o n of 0 -05 pg phenfonnin.

.

328

PHENFORMIN HYDROCHLORIDE

-

Solvent system Acetone :water

Rf

1:1

Reference

0.76

9,16

Isopropanol :water: ammonium hydroxide 40 t 9 t 6

0.73

16

Pyridinerbutanoltwater

0.79

16

Ethyl a c e t a t e rethanolrwater 6r3tl

0.76

13,16

Butanol :water: ammonium hydroxide 4 t 5 :1

0.49

16

Butanol s a t u r a t e d with water

0.43

16

1:l:i

6.52

Thin Layer Chromatography A TLC system was developed by Bailey (9) f o r phenformin hydrochloride on s i l i c a - g e l GF-g l a s s p l a t e s . The TLC plates were e l u t e d w i t h t h e upper layer of e q u i l i b r a t e d n-butanolracetic acid:H20 The migration rate w a s approximately (40:5 :55) 14 cm i n 5 hours w i t h an Rf o f 0.45.

.

Phenformin hydrochloride is q u a n t i t a t i v e l y determined i n our l a b o r a t o r y on c e l l u l o s e p l a t e s which are pre-washed i n t h e developing s o l v e n t , isopropanol:HZO:acetic a c i d , 80120:s. After development i n t h e above s o l v e n t system, phenformin hydrochloride i s e x t r a c t e d i n methanol and t h e absorbance of t h e s o l u t i o n determined a t 233 nm. 6.53

Paper E l e c t r o p h o r e s i s Bailey (9) h a s developed an e l e c t r o p h o r e s i s method using Whatman N o . 3 paper a t 400 v o l t s . With lN acetic a c i d , phenformin showed a migration rate of 8.25 c m i n 1 hour. Phenformin w a s e x t r a c t e d from t h e paper chromatogram and determined by U.V. spectroscopy i n t h e usual manner.

6.54

Gas Liquid Chromatography Wickramasinghe and Shaw (17) have reported t h e thermal i n s t a b i l i t y of phenformin hydrochloride t o gas chromatographic conditions. A s i n g l e peak of r e t e n t i o n t i m e about 22 minutes was observed on a g l a s s s p i r a l column (122 cm long) packed with 0.5% Carbowax 20M on g l a s s beads (80-100 mesh) a t 329

JOSEPH E. MOODY

a column oven temperature of 225O. The analysis of the s i n g l e GC peak on a LKB-9000 gas chromatographmass spectrometer showed nrominent GC-MS s i s n a l s a t m/e 230, 139, 110, 91 and68. This GC-MS data is consistent with the s-triazine structure shown below.

330

PHENFORMIN HYDROCHLORIDE

7.

References

1.

S.L. S h a p i r o , V.A. P a r r i n o and L. Freedman, J. Am. Chem. SOC., E l , 2220 (1959).

2.

F. T i s c h l e r , Ciba-Geigy Pharm. Corp., comunication.

3.

E.R.

Sci -.I

Garrett, J. Tsau, and P.H.

-

personal

H i n d e r l i n g , J. Pharm.

61, 1411 (1972).

-

4.

P. Ray, Chem, Rev., 61, 313-359

5.

P.J. Murphy and A.N. (1968).

6.

R.

7.

H. Hall, G. Ramachander and J.M. G l a s s m a n , Ann. N.Y. Acad. S c i . -11968 148, 6 0 1 - 6 1 1 ~ Chem. Abstr. 69, 65872e (1968).

8.

U.

9.

R.E.

B a i l e y , C l i n . Biochem., f,23-31 (1970).

10.

E.R.

Garrett and J. Tsau, J. Pharm. SCi.,

(1961)

Wick, J. Pharm. S c i .

, 57,

1125

148,

Beckmann, Ann. N.Y. Acad. S c i . , 820-832 (1968) t Chem. Abstr. 69, 50656p (1968).

,

-

,

S. Pharmacopeia X V I I I , p. 488.

61, 1404

(1972). H.G. Shepherd, Jr., and H.J. Chem., 496 (1958).

McDonald, C l i n i c a l

11.

- A,

12.

L. Freedman, M. B l i t z , 3%. Gunsberg, and S. Zak, J. Lab. Clin. Med., 58, 662-666 (1961).

13.

U. S. Pharmacopeia X V I I I , p. 487.

14.

R. E. B a i l e y and D. A. Durfee, J. Chromatogr. 546 (1964).

15.

I. Smith "Chromatographic and E l e c t r o p h o r e t i c Techniques" I n t e r s c i e n c e , New York (1960) p. 226.

16.

,

,

J. L. Faymon, C. J. S t e w a r t and A.

-,

4,

, l.6,

378-381 (1962).

331

N.

Wick, Appl.

JOSEPH E. MOODY

17.

J. Wickramasinghe and S. R. Shaw, J. Chromatogr.,

-

71, 265-273 (1972).

332

PROCAINAMIDE HYDROCHLORIDE

Raymond B. Poet and Harold Kadin

RAYMOND 6. POET AND HAROLD KADIN

TABLE OF CONTENTS 1.

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

2.

Physical Properties 2.1

Spectral Properties 2.11 2.12 2.13 2.14 2.15

2.2

Crystal Properties 2.21 2.22 2.23 2.24 2.25

2.3

Infrared Spectra Nuclear (Proton) Magnetic Resonance Spectra Ultraviolet Spectra Mass Spectra Fluorescence

Differential Thermal Analysis Differential Scanning Calorimetry Melting Range X-Ray Powder Diffraction Hygroscopicity

Solution Data 2.31 2.32 2.33 2.34

Solubility pKa Surface Chemistry Complex Formation

3.

Synthesis

4.

Stability

5.

Analysis of Impurities, Degradation Products and Residual Intermediates

334

PROCAl NAMl DE HYDROCHLORIDE

6.

Analytical Tests and Methods 6.1 6.2 6.3 6.4

Elemental Analysis Identification Tests Spectrophotometric Methods Isolation and Chromatographic Methods 6.41 6.42

Solvent Extraction Paper and Thin-Layer Chromatography 6.43 Gas Chromatography 6.44 Ion-Exchange Chromatography 6.5 6.6 6.7

Spectrophotofluorometric Methods Titrimetric Methods Microbiological Methods

7.

Analysis in Biologic Fluids and Tissues

8.

References

335

RAYMOND 6. POET AND HAROLD RADlN

1.

Description 1.1 Name, Formula, Molecular Weight

Procainamide hydrochloride is benzamide, ~-amino-N-~2-diethylamino)ethy~monohydrochloride with Chem. Abstr. registry number 614-3 9-1. Among the generic and trivial names for this compound are procaine amide hydrochloride, amidoprocain and procainamide. Common trade names are Pronestyl, Procamide, Procardyl, Novocamid, Novocainamid and Supicaine Amide. H2N

@

C13H21N30'HCl

PH2CH3 !-NHCH~CHZN HC1 'CH2CH3 Molecular Weight 271.8

1.2 Appearance, Color,Odor Procainamide hydrochloride is a white-to-tan, odorless crystalline powder. 2.

Physical Properties 2.1 Spectral Properties 2.11 Infrared Spectra

Samples of procainamide hydrochloride were dispersed in a potassium bromide pellet or in mineral oil to obtain the infrared spectra given in Figures la and lb from a The Perkin-Elmer Model 21 prism instrument12 potassium bromide and mineral oil spectra are essentially identical.

.

336

W W

4

Figure l a .

I n f r a r e d Spectrum of Procainamide H y d r o c h l o r i d e from K B r p e l l e t . I n s t r u m e n t : P. E. Model 2 1 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 .

w w

Do

F i g u r e lb.

I n f r a r e d S p e c t r u m o f P r o c a i n a m i d e H y d r o c h l o r i d e from mineral o i l mull. I n s t r u m e n t : P.E. Model 2 1 I n f r a r e d S p e c t r o p h o tome t e r .

PROCAINAMIDE HYDROCHLORIDE

S t r u c t u r a l l y s i g n i f i c a n t bands w e r e i n t e r p r e t e d b y T o e p l i t z l 2 a s f o l l o w s (see Figures l a and l b ) . Wave l e n q t h ( c m - l ) Interpretation NH2 and NH 3389,3279,3205 2564,2457 HC 1 1639 Amide C=O 1538 S e c o n d a r y Amide NH2 a n d A r o m a t i c C=C 1600 1511 A r o m a t i c C=C 840 Para-Substituted Aromatic 2.12

Nuclear (Proton) Magnetic Resonance S p e c t r a

Cohen59 o b t a i n e d b o t h 60- a n d 100-MHz s p e c t r a of p r o c a i n a m i d e and i t s h y d r o chloride. The 6O-MHZ NMR s p e c t r a were o b t a i n e d on a V a r i a n A s s o c i a t e s A-60 s p e c t r o m e t e r of CD30D s o l u t i o n s 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 an i n t e r n a l reference. The 100-MHz NMR s p e c t r a were o b t a i n e d on a V a r i a n A s s o c i a t e s XL-100 s p e c t r o meter d e u t e r i u m - l o c k e d t o t h e s o l v e n t (CDC13, CD30D, o r d g - p y r i d i n e ) c o n t a i n i n g t e t r a m e t h y l s i l a n e a s i n t e r n a l reference. Because procainamide hydrochloride is not soluble i n chlorinated h y d r o c a r b o n s , t h e 60-MHZ NMR s p e c t r u m was obtained i n tetradeuteromethanol (Figure 2 ) : t h i s s p e c t r u m i s compared w i t h t h a t o f t h e f r e e b a s e i n t h e same s o l v e n t ( F i g u r e 3 ) . A l t h o u g h t h e s p e c t r a a r e q u a l i t a t i v e l y t h e same, t h e s p e c t r u m of t h e h y d r o c h l o r i d e s a l t d i f f e r s because of protonation a t t h e alkylamine s i t e , r e s u l t i n g i n a g r e a t d o w n f i e l d s h i f t of t h e p r o t o n s on t h e m e t h y l e n e g r o u p s d i r e c t l y a t t a c h e d t o t h e a l i p h a t i c amine n i t r o g e n a n d a

339

w

P

0

Figure 2.

6O-MHZ NMR Spectrum of Procainamide Hydrochloride in Tetradeuteromethanol. Instrument: Varian Associates A-60 Spectrometer. Tetramethylsilane as Internal Standard.

Figure 3.

60-MHz NMR Spectrum of Procainamide Base in Tetradeuteromethanol. Instrument: Varian Associates A-60 Spectrometer. Tetramethylsilane as Internal Standard.

T a b l e 1. NMR S p e c t r a l A s s i g n m e n t s

43

a 0

TT

n

H2

0

or

Or

I1 D

C-N-CH2-

D2*

CH 2-

@ O

@

/H

C ' H2

2-CH 3 -CH3

8

C h e m i c a l S h i f t (6) ppm (No. o f Peaks***, C o u p l i n g C o n s t a n t ( J )i n Hz) 60-MHz S p e c t r u m ( F i g u r e 2 ) ( F i g u r e 3) ~ssignment H C 1 S a l t Base i n o f Prot o n i n HOCG3and HOCD3 a n d P o s i ti o n DOCD3 DOCD3 1.33 (t, 7.3) 1 . 2 2 ( t , 7.3) ( 9 , 7 . 3 ) 2 . 6 1 (9, 7 . 3 ) 3 . 2 9 Q * %3.35 (m) *'L2.60 (m) 0 * 3.47 ( t , 9 ) *3 . 7 1 ( t , 7 ) 7.70 (d, 8.6) 7.65 (d, 8 . 6 ) @ c

P N

0

CD

@

6.71

I

(d, 8 . 6 )

6.68

(d, 8.6)

Table 1, c o n t i n u e d . .

..

Assignment of Proton Position

(Figure 4 ) Base in HOCD3 and DOCD3 1.08(t, 7 ) 2 . 6 1 (9,7 ) ** 2 . 6 6 (m) ** 3 . 4 8 (m, 8 ) 7.58 (d, 8 . 5 ) 6 . 6 5 (a, 8 . 5 )

-

(Figure 5 ) Base in CDC13 1.04 2.57 2.62 3.46 7.59 6.59 6.79 4.04

(Figure 6 ) Base in Perdeuteropyridine

(t, 7 ) (9, 7 ) (m)

0.95

(t, 7 )

2.48 2.67

(t, 7)

(m) (d, 8 ) (d, 8 ) (Broad) (Broad)

3.71

(9,

7.13

(d, 8 ) (d, 8 ) (Broad) (Broad)

5.93 6.79 4.04

(q, 7) 7)

*Hydrogens not distinguishable. **Hydrogens are distinguishable. ***No. of peaks is designated t = t r i p l e t , d = doublet, q = quartet, and m = multiplet.

".

I

I

w

P

P

10

I

<-

Figure 4.

-

2

3

4

?.,

,,.

, 9 ,

, ? ,

P

100-MHz NMR Spectrum of Procainamide Base in Tetradeu teromethano 1. Instrument : Varian Associate s XL- 100 Spectrometer, Deuterium-Locked to the Solvent Containing Tetramethylsilane as Internal Reference.

.. &

I

I

nb

,k

i 1

w

P

VI

Figure 5.

100-MHz NMR Spectrum of Procainamide Base in Deuterochloroform. Instrument: Varian Associates XL-100 Spectrometer, Deuterium-Locked to the Solvent Containing Tetramethylsilane as Internal Reference.

W

P

m

F i g u r e 6.

100-MHz NMR S p e c t r u m of P r o c a i n a r n i d e B a s e i n P e r d e u t e r o p y r i d i n e . I n s t r u m e n t : V a r i a n A s s o c i a t e s XL-100 S p e c t r o m e t e r , D e u t e r i u m Locked t o t h e S o l v e n t 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 Reference.

PROCAl NAMl DE HYDROCHLORIDE

s l i g h t d o w n f i e l d s h i f t of t h e p r o t o n s on t h e @-carbons. T h a t t h e s i t e of p r o t o n a t i o n i s n o t on t h e a r o m a t i c amine i s d e m o n s t r a t e d b y t h e s i m i l a r i t y of t h e a r o m a t i c p r o t o n r e s o n a n c e s o f t h e two compounds. Protonation of t h e aromatic amine would have r e s u l t e d i n a l a r g e d o w n f i e l d s h i f t of t h e o r t h o p r o t o n s and a small d o w n f i e l d s h i f t of t h e meta p r o t o n s , b e c a u s e t h e -NH3 + g r o u p i s n o t a s s t r o n g an e l e c t r o n - d o n a t i n g g r o u p a s i s t h e -NH2group. The a s s i g n m e n t s of t h e chemical s h i f t s shown i n T a b l e 1 a r e g i v e n i n D e l t a v a l u e s ( 6 ) and t h e c o u p l i n g c o n s t a n t s , J, a r e g i v e n i n Hz w i t h t - t r i p l e t , d - d o u b l e t , q - q u a r t e t and m - m u l t i p l e t . I t i s e v i d e n t t h a t t h e 100-MHz spectrum o f t h e b a s e i n CD30D y i e l d s a d d i t i o n a l r e s o l u t i o n ( F i g u r e 4 and T a b l e 1). The 100-MHz NMR spectrum of procainamide b a s e ( F i g u r e 5) shows t h e amine and amide p r o t o n s . There are a d d i t i o n a l l i n e s f o r t h e methylene p r o t o n s a t t a c h e d t o t h e amide n i t r o g e n due t o c o u p l i n g of t h e amide p r o t o n , which i s Ja7Hz. The a s s i g n m e n t s a r e shown i n T a b l e 1. When t h e 100-MHz NMR spectrum o f p r o c a i n a m i d e b a s e i s obtained i n perdeuteropyridine, considerable changes i n c h e m i c a l s h i f t s o c c u r t h a t r e s u l t i n separation of a l l t h e proton resonances ( F i g u r e 6 ) . The a s s i g n m e n t o f t h e s p e c t r u m i s d e p i c t e d i n T a b l e 1.

2.13

Ultraviolet Spectra

D ~ n h a mhad ~ ~ indicated t h a t the u l t r a v i o l e t s p e c t r a of p r o c a i n a m i d e h y d r o c h l o r i d e s h o u l d b e measured i n 0.1g ammonium h y d r o x i d e i n e i t h e r methanol o r e t h a n o l . S p e c t r a f o r t h e compound d i s s o l v e d i n e i t h e r s o l v e n t a l o n e can have v a r i a b l e w a v e l e n g t h s of maximum a b s o r b a n c e , 341

RAYMOND 6 . POET AND HAROLD KADIN

a s indicated by t h e following s p e c t r a l d a t a o b t a i n e d w i t h a Cary 1 5 S p e c t r o p h o t o m e t e r . Solvent MeOH c o n t a i n i n g

0.13 NH40H

max (nm) 282 282 282

E (l%,

1 cm) 667 670 676 ( N o t e )

MeOH

2 91

6 94

MeOH

291

690

- HC1

293 2 93

68 2

EtOH,

95%

286

656

EtOH,

95%

286

6 54

282

6 58

282

654

MeOH c o n t a i n i n g N HC1

95% containing 0.1N NH4OH

EtOH,

3 99

N o t e : The s p e c t r u m o f t h i s s o l u t i o n , d e t e r m i n e d a g a i n 5 h r . l a t e r , showed max = 282 nm, E (l%,l c m ) = 670.

The d a t a i n d i c a t e t h a t t h e wavel e n g t h of maximum a b s o r b a n c e i s pH d e p e n d e n t . Both m e t h a n o l i c and e t h a n o l i c s o l u t i o n s of proc a i n a m i d e h y d r o c h l o r i d e h a d a % max of 2 8 2 nm i n 0.1N ammonium h y d r o x i d e , a l t h o u g h t h e E ( l % , l c m ) v a r i e d between t h e t w o s o l v e n t s . A d d i t i o n of a c i d t o t h e methanolic s o l u t i o n s h i f t s the maximum t o 293 nm. S p e c t r a of t h e compound d i s s o l v e d i n e i t h e r s o l v e n t a l o n e c a n show h m a x v a l u e s t h a t a r e q u i t e v a r i a b l e , d e p e n d i n g on t h e p o s s i b l e c o n t r i b u t i o n of s l i g h t r e s i d u a l a c i d o r 348

PROCAlNAMl DE HYDROCHLORIDE

b a s e on t h e g l a s s w a r e o r from i m p u r i t i e s i n t h e b a t c h of s o l v e n t used t o make t h e s o l u t i o n s and measurements. When N hydrochloric acid i n methanol i s used a s s o l v e n t , t h e r e i s a s l i g h t d e c l i n e ( a b o u t 1.5%) i n a b s o r b a n c e , w i t h a 2-nm upward s h i f t i n ~ m a x ,r e l a t i v e t o t h e a b s o r b a n c e i n methanol a l o n e . However, when t h e a c i d concentration is increased t o 3, t h e a b s o r b a n c e d e c l i n e s a p p r e c i a b l y ( a b o u t 42%), r e l a t i v e t o a b s o r b a n c e i n methanol a l o n e . T h i s d e c l i n e h a s been a s c r i b e d t o p r o t o n a t i o n of t h e aromatic-NH2 group55, 65. The e f f e c t s of t h e s u b s t i t u t i o n of amino and c a r b o n y l g r o u p s on t h e s h i f t o f t h e a b s o r p t i o n o f t h e benzene u l t r a v i o l e t chromophore The u s e of u l t r a v i o l e t have been discussed51. s p e c t r o p h o t o m e t r y f o r d e t e r m i n a t i o n of r o c a i n amide h y d r o c h l o r i d e h a s been d e s c r i b e d 5 9 5 5

P

(see Section 6.3). 2.14

Mass S p e c t r a

C0hen5’ h a s r e p o r t e d t h e f o l l o w i n g c o n c e r n i n g t h e mass s p e c t r a o f p r o cainamide h y d r o c h l o r i d e . The l o w - r e s o l u t i o n mass spectrum (see F i g u r e 7 ) i s dominated by t h e m / e 86 i o n ( b a s e peak) r e s u l t i n g from t h e c l e a v a g e o f t h e bond b e t a t o t h e t e r t i a r y amine n i t r o g e n . T h i s cleavage i s a n t i c i p a t e d i n t h e fragmentation of amines. The M+ of t h e compound i s o b s e r v e d a t m / e 235. H i g h - r e s o l u t i o n peak matching, T a b l e 2 , and h i g h - r e s o l u t i o n d a t a a c q u i s i t i o n , T a b l e 3, were employed t o d e t e r m i n e t h e c o m p o s i t i o n o f e a c h fragment i o n . Assignment o f t h e fragmentat i o n i s shown b e l o w 3 ~ 5 .

349

RAYMOND B. POET AND HAROLD K A D l N

only

The, low-and high-resolution mass spectra were obtained on an AEI MS-902 Spectrometer equipped with a frequency-modulated analog tape recorder The low-resolution mass spectrum was processed on a Digital Equipment Corporation PDP-11 computer using Squibb programs62, while the high-resolubion mass spectrum was processed by W ~ m a c kon ~~ an EAI computer. Peak-matched data were obtained from the AEI MS-902 spectrometer equipped with a wide-ratio accessory.

350

PROCAINAM I DE HYDROCHLORIDE

RELATIVE INTENSITY

ru m u LJl

m 0

ru

.-LN

LN

n

0 0

i

rl ru nl I F

I

u

a u3

m ru Lhl ul I m m

nl

LN

m

m

-

m

0

ru

LN

m

e,

I

m

PERCENT TOTAL I O Y I Z A T I O N

LJl

39

Low Resolution Mass Spectrum of Procainamide Hydrochloride. Instrument: AEI MS-902 Spectrometer Equipped with Frequency Modulated Tape Recorder;Spectrum Processed on Digital Equipment Corporation PDP-11 Computer

Fig. 7

351

RAYMOND 6 . POET AND HAROLD KADlN

Table 2 H i g h - R e s o l u t i o n Peak-Match D a t a of P r o c a i n a m i d e

Found

M+ 235.1685 206.1293 205.1215 189.1266 188.1188 174.1031 163.0871 148.0678 136.0627 120.0451 99.1041 92.0507 86.0963 71.0735 70.0653

Ca l c d .

C H N -

235.1685 206.1293 205.1215 189.1266 188,1188 174.1031 163.0872 148.0637 136.0637 120.0449 99.1048 92.0500 86.0970 71.0735 70.0657

13 11 11 11 11 10 9 8 7 7 6 6 5 4 4

352

21 16 15 15 14 12 11 8 8 6 13 6 12 9 8

3 3 3 3 3 3 2 2

2 1 1 1

1

1 1

0 -

1 1 1 0 0 0

1 1 1 1 0 0 0 0 0

PROCAINAMIDE HYDRDCHLORIDE

Table 3 High-Resolution Mass Spectrum of Procainamidea Rel. Found c/c13 Calcd. H N 0 Int. 13/0 21 3 1 4 235.1685 M+ 235.1667 13/0 233.1529 19 3 1 4 233.1530 11 189.1266 15 3 0 4 189.1241

---

188.1137 187.1151 164 149.0705 148.0700 137.0646 136.0597 121.0479 120.0458 119.0368 100.1078 99.1031 93.0554 92.0488 91.0421 87.0974 86.0944 72.0799 71.0732 70.0662 65.0395 58.0687 57.0625 56.0501

a

188.1188

11

149.0715 148.0637 137.0715 136.0637 121.0482 120.0449 119.0371 100.1064 99.1048 93.0533 92.0500 91.0422 87.1003 86.0970 72.0813 71.0735 70.0657 65.0391 58.0657 57.0578 56.0500

8 8 7 7 6/1 7 7 5/1 6 5/ 1 6 6 4/1 5 4 4 4 5 3 3 3

14

3

9 8 9 8

2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1

6

6 5 13 13 6 6 5 12 12 10 9 8 5 8 7 6

0

1 1 5 1 4 1 5 1 3 1 7 1 12 1 141 1 2 0 20 0 242 0 6 0 57 0 8 0 78 0 999 0 22 0 25 0 13 0 55 0 90 0 11 0 36

High-Resolution FM analog tape processed by Dr. J. Womack, E A I , Princeton, N . J .

353

RAYMOND E. POET AND HAROLD KADIN

A s i m i l a r m a s s - s p e c t r a l fragment a t i o n p a t t e r n was r e p o r t e d b y A t k i n s o n e t a139. These a u t h o r s commented t h a t t h e most p r o m i n e n t i o n ( m / e 8 6 i o n ) r e s u l t s from t h e a b i l i t y o f t h e f r e e e l e c t r o n p a i r o n t h e t e r t i a r y amine n i t r o g e n atom t o s t a b i l i z e a p o s i t i v e c h a r g e on t h e a d j a c e n t c a r b o n atom. A t k i n s o n e t a 1 . 3 9 a s c r i b e d t h e m / e 99 f r a g m e n t t o a M c L a f f e r t y rearrangement66.

2.15

Fluorescence Procainamide h a s been r e p o r t e d 1 9

t o e x h i b i t maximum f l u o r e s c e n c e a t 385 nm, w i t h maximum a c t i v a t i o n a t 295 nm, a t pH 11. The s p e c t r o p h o t o f l u o r o m e t e r u s e d employs a xenon a r c source e m i t t i n g a continuum from 200 t o 800 nm a s t h e a c t i v a t i n g l i g h t source. This instrument y i e l d s spectra uncorrected f o r the non-linear i n t e n s i t y of e m i s s i o n o f t h e xenon a r c and t h e uneven p h o t o m u l t i p l i e r r e s p o n s e o v e r t h i s continuum. T h i s may e x p l a i n the same maximum a c t i v a t i o n a t 295 nm, b u t a d i f f e r e n t f l u o r e s c e n c e maximum a t 360 nm, a t pH 11,t h a t was r e p o r t e d b y Koch-Weser 8

.

R e f e r t o s e c t i o n s 6 . 5 and 7 . 0 f o r a p p l i c a t i o n s of t h i s p h y s i c a l p r o p e r t y . 2.2

Crystal Properties 2 . 2 1 D i f f e r e n t i a l Thermal A n a l y s i s D i f f e r e n t i a l thermal a n a l y s i s

(DTA) of p r o c a i n a m i d e h y d r o c h l o r i d e b y V a l e n t i 3 0 y i e l d e d a s h a r p endotherm a t 169OC. A DuPont

900 Thermoanalyzer programmed f o r a t e m p e r a t u r e r i s e o f 15O p e r min produced t h e thermogram o f F i g u r e 8.

354

w

Figure 8.

Differential Thermogram of Procainamide Hydrochloride Instrument: DuPont 900 Thermoanalyzer, lSO/min

RAYMOND B. POET AND HAROLD KADIN

2.22

D i f f e r e n t i a l Scanninq C a l o r i m e t r y (D. S. C. )

Valenti3' D.S.C. p u r i t y o f t h e same h y d r o c h l o r i d e t o be 99.94 P e r k i n E l m e r D.S.C. Model 2.23

a l s o r e p o r t e d the l o t of procainamide mole p e r c e n t . A 1B was u s e d .

M e l t i n g Range

The m e l t i n g r a n g e f o r U.S.P. procainamide h y d r o c h l o r i d e i s s p e c i f i e d a s 1 6 5 . 0 t o 169.0°C1. The s a m p l e o f p r o c a i n a m i d e h y d r o c h l o r i d e u s e d f o r most of t h e s t u d i e s rep o r t e d h e r e i n h a s a U.S.P. m e l t i n g r a n g e b e t w e e n 1 6 8 . 4 and 169.2OC. T h i s narrow, high-melting r a n g e s i g n i f i e s e x c e l l e n t p u r i t y , which i s c o n f i r m e d b y t h e s h a r p 169OC D . T . A . endotherm and t h e 99.94% D.S.C. p u r i t y ( S e c t i o n s 2 . 2 1 a n d 2.22). Various p r e p a r a t i o n s of procainamide h y d r o c h l o r i d e a r e reported t o h a v e the following melting ranges:

356

PROCAINAMIDE HYDROCHLORIDE

Melting Range (OC) 165-9 165-7

164 167 166-7

Author ( s ) K. K. M. Y. S. Y. G. H. M. V.

E. 166-6.5

Z.

M. “A. A.

167-8

165-8 165-8 165-8 2.24

E. J. M. K. P. Y. M. X-Ray

Iwaya and Yoshida Yamazaki, Kitagawa, H i r a k i , and Tsukamoto Ghielmetti Najer and Joannic Hack and Koppovs Led6chowski , Bogucka, Ledbchowski , and Chimiak Pavlovsk a, Lukac and Borovicka Radushkevich Egli Tashika and Kuranari Powder D i f f r a c t i o n

Ref. N o . 98 85

99 86 87 89

100

72 93 84

The X-Ray powder d i f f r a c t i o n of procainamide h y d r o c h l o r i d e was o b t a i n e d by 0cl-1~ a t~ a~ v o l t a g e of 25 kv and a c u r r e n t of 1 0 ma, u t i l i z i n g a P h i l l i p s X-Ray Powder Diffractometer. The sample was i r r a d i a t e d by a copper s o u r c e a t 1 . 5 4 A O . Data d e r i v e d from t h e spectrum a r e l i s t e d i n Table 4; t h e i n t e n s i t y of each peak i n t h e spectrum i s shown r e l a t i v e t o t h e i n c i d e n t r a d i a t i o n (Io), a s 1/10 x 100, a t twice t h e a n g l e of i n c i d e n c e o r r e f l e c t i o n of the r a d i a t i o n ( 2 0 ) for t h e d i s t a n c e , d , between t h e atomic p l a n e s i n t h e c r y s t a l .

357

RAYMOND B. POET AND HAROLD KADIN

X-Ray

1

Table 4 Powder D i f f r a c t i o n P a t t e r n o f Procainamide Hydrochloride Instrument: P h i l l i p s X-Ray Powder Dif f r a c t o m e t e r

*

8.30 25.26 7.40 12.63 6.43 7.37 5.76 10.53 5.40 14.74 5.26 100.00 4.82 14.74 4.52 40.00 4.30 27.37 4.10 30.53 4.01 90.53 3.92 66.32 3.74 49.47 3.48 52.63 3.34 18.95 3.23 8.42 3.14 18.95 2.98 28.42 2.95 14.74 2.86 8.42 2.77 20.00 2. 50 10.53 2.36 7.37 *d ( i n t e r p l a n a r d i s t a n c e ) =

** ***

***

1/10 x loo**

dl (Ao)

I (20) 24 12 07 10 14 95 14 38 26 29 86 63 47 50 18 08 18 27 14 08 19 10 07

n A

2 sin 8

= 1.539A0

Based on h i g h e s t i n t e n s i t y o f 100. Twice t h e a n g l e o f i n c i d e n c e o r r e f l e c t i o n .

358

PROCAINAMIDE HYDROCHLORIOE

2.25

Hygroscopicity

WaltOn6’ r e p o r t e d t h a t s o l i d procainamide h y d r o c h l o r i d e is r e a d i l y converted t o a s o l u t i o n i n a n a t m o s p h e r e o f 65% r e l a t i v e humidity. 2.3

S o l u t i o n Data 2.31 S o l u b i l i t y

Valenti3O r e p o r t e d t h e f o l l o w i n g s o l u b i l i t i e s (U. S. P. d e f i n i t i o n ) o f p r o c a i n a m i d e h y d r o c h l o r i d e i n v a r i o u s s o l v e n t s , a t room temperature. P a r t s of S o l v e n t / O n e Solvent P a r t of S o l u t e Solubility less t h a n 1 very soluble Water 0.1g Hydroless than 1 very soluble c h l o r i c acid 0 . 1 g Sodium hydroxide

very soluble

less t h a n 1

95% E t h a n o l

soluble

1 0 t o 30

Propy l e n e Glycol

sparingly soluble

30 t o 100

Chloroform

slightly soluble

100 t o 1000

Acetone

slightly soluble

100 t o 1000

Ether

insoluble

more t h a n 10000

Benzene

insoluble

more t h a n 10000

Hexane

i n so l u b 1e

more t h a n 10000

359

RAYMOND 6 . POET AND HAROLD K A D I N

The s o l u b i l i t y of procainamide h y d r o c h l o r i d e i n a c e t o n e a t 32OC was r e p o r t e d t o be 3.43 mg p e r g of solvent". R a d u ~ h k e v i c hr~e ~ ported the s o l u b i l i t y of procainamide h y d r o c h l o r i d e i n a b s o l u t e e t h a n o l t o be 6% a t 20° and 70% a t t h e Procainamide hydrob o i l i n g p o i n t of ethanol. c h l o r i d e was r e p o r t e d t o be i n s o l u b l e i n 1 , 2 - d i chloroethane o r i n d i e t h y l ether72. 2.32

pKa

The pKa o f procainamide hydroc h l o r i d e , determined by a t i t r i m e t r i c procedure, was 9.24 ( 2 0.10)77. The pKa r e f e r s t o t h e following type of d i s s o c i a t i o n : R-NH-3' 2.33

R-NH2

+

H+

S u r f a c e Chemistry

The d i e l e c t r i c p o t e n t i a l s and s u r f a c e t e n s i o n s of s o l u t i o n s o f p r o c a i n e and o f procainamide h y d r o c h l o r i d e were compared o v e r a wide pH range97. A t pH 1 0 . 3 and 1 0 . 5 t h e following d a t a on d i e l e c t r i c p o t e n t i a l were obtained : C o n c e n t r a t i o n and Druq Ell D i e l e c t r i c P o t e n t i a l (mv) 0.0066PJ P r o c a i n e 10.5 477 - Procainamide 10.3 268 0.02M Although t h e s o l u t i o n of procainamide was t h r e e times a s c o n c e n t r a t e d a s t h a t o f p r o c a i n e , t h e d i e l e c t r i c p o t e n t i a l of t h e former was less. A t lower pH's, t h e d i e l e c t r i c p o t e n t i a l o f t h e procainamide s o l u t i o n was a l s o l e s s than t h a t o f procaine, although t h e d i f f e r e n c e w a s n o t a s 360

PROCAINAMIDE HYDROCHLORIDE

pronounced a s a t pH 10.3 and 10.5. Similarly, the surface tension f o r t h e procainamide s o l u t i o n was less t h a n t h a t f o r p r o c a i n e , over t h e pH range t e s t e d . The a u t h o r s g 7 concluded t h a t t h e t o x i c i t y of procainamide i s less t h a n t h a t o f p r o c a i n e because procainamide i s l e s s s u r f a c e active. 2.34

Complex Formation

Complex formation by p r o c a i n e and procainamide w i t h adenosine t r i p h o s p h a t e (ATP) i n aqueous s o l u t i o n was s t u d i e d by o p t i c a l r o t a t i o n and NMR methodsg6. These s t u d i e s i n d i c a t e d t h a t hydrophobic s t a c k i n g o f t h e a r o m a t i c i t y , r a t Q e r than h o r i z o n t a l hydrogen bonding, i s i m p l i c a t e d i n t h e formation o f t h e s e complexes. A t pH 7.0, ATP b i n d s one drug molecule t i g h t l y o r two molecules weakly. The formation c o n s t a n t s f o r t i g h t l y bound complexes a s determined by b o t h o p t i c a l r o t a t i o n and NMR measurements, were i n good agreement. The weak-association c o n s t a n t s were o b t a i n e d by t r i a l - a n d - e r r o r procedure. The complex-formation c o n s t a n t s were : ATP Complex

Strenqth Tight Weak

Procaine 1430 f 38Og-l 1.1 f 0.7x105g-2

Procainamide 3.60 f 90M-l 4.7 f 2.8F103g-2

According t o Thymm e t a l . 96 t h e a c t i v i t y of t h e s e drugs on n e r v e membranes may be a s c r i b e d t o complexing w i t h membrane-bound ATP.

361

RAYMOND 6 . POET AND HAROLD KADIN

3.

Synthesis

E-Nitrobenzoic a c i d i s c o n v e r t e d t o t h e a c i d c h l o r i d e by r e a c t i o n w i t h s u l f o n y l c h l o r i d e . E-Nitrobenzoyl 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 diethylaminoethylamine. The r e s u l t a n t n i t r o condensation p r o d u c t i s t h e n t r e a t e d w i t h h y d r o c h l o r i c a c i d and reduced t o y i e l d t h e f i n a l product85.

-@ *

COOH + S 0 C l 2

02N + NH CH CH N2;:5; 2 2 2 HC 1

red.

’

02N e

c o c 1

- -

CONHCH,CII,N

0,N

2 5

H2N

-+

tI -@CONHCH2CH2N. ,c c2H5 2 5

*

,C H 7 5 ‘C,tIs

-

HC 1

A l t e r n a t i v e s y n t h e s e s of procainamide have been d e s c r i b e d by s e v e r a l workers72,86-90,95,98. P r e p a r a t i o n of procainamide by e l e c t r o l y t i c r e d u c t i o n of N- ( 2 - d i e t h y l a m i n o e t h y l ) -E-nitrobenzamide h a s a l s o been r e p o r t e d 8 4 . The s y n t h e s i s of 14C-procainamide hydroc h l o r i d e , r e p o r t e d by E g l i g 3 , u t i l i z e d E-aminobenzoic a c i d l a b e l l e d i n t h e c a r b o x y l carbon. The s y n t h e s i s o u t l i n e d above was followed. A p u r i t y a n a l y s i s was performed by r a d i o s c a n n i n g a t h i n - l a y e r chromatogram o f t h e f i n a l p r o d u c t , u s i n g t h e s u p p o r t and s o l v e n t system I1 of F a i r b r o t h e r and Shand68as d e s c r i b e d i n s e c t i o n 6.42. The f i n a l p r o d u c t c o n t a i n e d 4.83 yCi/mg, w i t h l e s s t h a n 0.5% of r a d i o c h e m i c a l i m p u r i t i e s . E g l i g 3 r e p o r t e d t h a t i t was p o s s i b l e t o m a i n t a i n t h e p r o d u c t a t o n l y 0.5% i m p u r i t y b y keeping t h e s p e c i f i c a c t i v i t y a s low a s p o s s i b l e and growing t h e c r y s t a l s a s l a r g e a s p o s s i b l e ; t h i s procedure a p p a r e n t l y minimizes t h e s u r f a c e

362

PROCAINAMIDE HYDROCHLORIDE

oxidation that is common with labelled compounds.

4. Stability Bulk samples of procainamide hydrochloride were stored at 25 and 5OoC for as long as 1 year. Samples stored at these temperatures showed no significant differences in intravenous or oral toxicity in mice. The purity, measured by titration with perchloric acid, ranged from 98.9-100.1% over the period of one year. Procainamide hydrochloride bulk appeared to be stable under the conditions employed91,92. 5.

Analysis of Impurities, Degradation Products and Residual Intermediates

N- (2-diethylaminoethyl)-p-nitrobenzamide, an intermediate in the synthesiz of procainamide, has been determined polarographically73. The pH 4.0 buffer contained 250 ml of 1M sodium acetate, 750 ml of 1M acetic acid, 7.46 g of potassium chloride and 530 mg of dodecyltrimethyl ammonium chloride. A dropping-mercury electrolysis cell was used in which the average half-wave reduction potential (vs. Hg) was found to be -0.405 volts. Use of a gravimetric method69 has also been reported, in which the nitrobenzamide compound was extracted into ether from alkaline solution; the solvent was removed by evaporation and the residue was dried and weighed.

The determination of pnitrobenzoic acid, a possible process contaminant, was accomplished gravimetrically69 after extraction from acid solution into ether and removal of the solvent by evaporation. The stability of p-nitrobenzoic acid can be monitored by thin-laye? chromatography83 on silica gel plates (with indicator) with 363

RAYMOND 6. POET AND HAROLD KADlN

a s o l v e n t system p r e p a r e d by mixing 10 m l s w a t e r w i t h 20 m l s of U.S.P. e t h a n o l t h e n d i l u t i n g t o 1000 m l s w i t h methyl i s o b u t y l k e t o n e . C o n c e n t r a t i o n s of N,N-diethylethylenediamine ( a p r e c u r s o r diamine) i n t h e range o f 0.01-0.3% have been determined i n procainamide hydroc h l o r i d e by a c o l o r i m e t r i c method7' by Whigan I n t h i s t e s t , based upon a r e p o r t by and Kadin. J. Bartos94, a 50-mg sample of procainamide h y d r o c h l o r i d e i s d i s s o l v e d i n dimethylformamide and r e a c t e d w i t h a 0.1% s o l u t i o n of a s c o r b i c a c i d , d i s s o l v e d i n t h e same s o l v e n t , by h e a t i n g t h e mixture f o r 10 min i n a boiling-water bath. The absorbance of t h e c o l o r e d r e a c t i o n p r o d u c t i s measured a t 530 nm. The c o l o r r e a c t i o n between t h e diamine and a s c o r b i c a c i d i s enchanced by t h e p r e s e n c e of procainamide. A s i m i l a r enhancement by p a m i n o b e n z o i c a c i d (PABA) had been observed. T h e r e f o r e , 30 mg of PABA were added t o b o t h t h e r e a g e n t b l a n k and t h e diamine s t a n d a r d t o c o r r e c t f o r t h i s enhancement. A s l i t t l e a s 0.1% N,N-diethylethylenediamine i n procainamide c o u l d a l s o be determined q u a n t i t a t i v e l y by paper e l e c t r o p h o r e s i s and densitometry7'. Samples and s t a n d a r d s w e r e s p o t t e d 1 2 c m from t h e a n o d i c edge of 1 2 x 36 c m Whatman 3MM paper. E l e c t r o p h o r e s i s i n a pyridinium-formate b u f f e r was c a r r i e d o u t f o r 30 m i n a t a v o l t a g e The s h e e t was a i r d r i e d , g r a d i e n t of 1 0 V/cm. sprayed w i t h a n i n h y d r i n r e a g e n t c o n t a i n i n g sym-collidine, and developed f o r 3 h r . The c o l o r e d s p o t s were e v a l u a t e d b y d e n s i t o m e t r y .

E-Aminobenzoic acid i n s o l u t i o n s of procainamide h a s been detected by c i r c u l a r paper chromatography60. A f t e r development w i t h ammonia-saturated b u t a n o l , t h e paper was d r i e d 364

PROCAlNAMl DE HYDROCHLORIDE

and s p r a y e d w i t h sodium n i t r i t e i n 5% hydroc h l o r i c a c i d , f o l l o w e d by 1 - n a p h t h o l and e t h a n o l i c potassium h y d r o x i d e , Procainamide and i t s c l e a v a g e p r o d u c t g i v e r e d c i r c u l a r zones. p-Aminobenzoic a c i d , a p o t e n t i a l c l e a v a g e p r o d u c t , h a s been s e p a r a t e d by e l e c t r o p h o r e s i s on paper78, e l u t e d i n d i s t i l l e d water, t h e n d e t e r m i n e d c o u l o m e t r i c a l l y . p-Aminobenzoic a c i d may be s e p a r a t e d from p r o c a i n a m i d e i n an ext r a c t i o n system79. The a l k a l i n i z e d sample i s e x t r a c t e d w i t h c h l o r o f o r m and t h e E- aminobenzoic a c i d remaining i n t h e aqueous phase a s t h e sodium s a l t can be d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y (Section 6.3). The p h a s e - s o l u b i l i t y of p r o c a i n a m i d e i n a c e t o n e a t 23 and 32OC h a s been used t o d e t e r m i n e t h e p u r i t y of t h e compound 52980. P u r i t y was a l s o e v a l u a t e d by D i f f e r e n t i a l Scanning C a l o r i m e t r y (Section 2.22). 6.

A n a l y t i c a l T e s t s and Methods 6 . 1 Elemental A n a l y s i s

Element C H N 0

c1 6.2

Theory 57.45% 8.16% 15.46% 5.89% 13.04%

Reported64 57.45 8.16 15.46 5.88 13.05

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

A t e s t b a s e d o n t h e p r e p a r a t i o n of b e n z o y l procainamide and t h e d e t e r m i n a t i o n of i t s m e l t i n g p o i n t i s d e s c r i b e d i n t h e U.S.P. 1

.

3 65

RAYMOND 6. POET AND HAROLD KADlN

Clarke has reported results for microcrystals and Vitalit s color testsll. Crystalreaction identification tests have been described58. A number of spot-plate tests to l7 9 identify procainamide have been reported133 18. Identification has been achieved by paper and test-tube chromatography,16, as well as by gas chromatography41. Color reactions with barbituric acid and 2-thiobarbituric acid have been reported33. Infrared spectroscopy (Section 2. ll), spectrophotometry (Section 6.3), paper and thinlayer chromatography (Section 6.42), spectrophotofluorometry (Section 6.5) and gas chromatography (Section 6.43) provide alternate methods for purposes of identification. 6.3

Spectrophotometric Methods

Procainamide and its decomposition product E-aminobenzoic acid have been determined by measuring their ultraviolet absorption55 (see Section 2.13). The color developed upon the reaction of procainamide with vanillin in acid solution has been used to measure its c o n ~ e n t r a t i o n ~ ~ j ~ ~ , 54. A reaction with thyme camphor after diazotization has also been reported49. Reaction with sulphonphthalein dyes50 has been used to give a colored product for measurement. Refer to Section 7 for spectrophotometric methods applicable to the determination of procainamide in biologic fluids and tissues.

366

PROCAINAMIDE HYDROCHLORIDE

6.4

I s o l a t i o n and C h r o m a t o g r a p h i c Methods 6.41 Solvent Extraction Procainamide h a s been i s o l a t e d by s o l v e n t e x t r a c t i o n (see S e c t i o n s 5 . 0 and 7 . 0 ) for s u b s e q u e n t d e t e r m i n a t i o n b y a v a r i e t y of methods (see S e c t i o n s 6 . 4 3 , 6 . 6 and 7 ) . 6.42

P a p e r and Thin-Layer Chroma tography

Procainamide h a s been assayed by p a p e r c h r o m a t o g r a p h i c method^^^,^^, 60. R o b e r t s 4 7 u s i n g S&S 597 f i l t e r p a p e r , d e v e l o p e d chromatograms f o r 1 6 h r i n t h e s o l v e n t - r i c h u p p e r p h a s e of a s o l v e n t s y s t e m composed o f e q u a l volumes of i s o b u t a n o l and 0.05N p h o s p h o r i c a c i d . The z o n e s on t h e d e v e l o p e d chromatograms were l o c a t e d b y t h e i r u l t r a v i o l e t absorbance. A number o f t h i n - l a y e r chromatog r a p h i c s y s t e m s h a v e b e e n u s e d t o examine proc a i n a m i d e u a l i t a t i v e l 20, 2 1 22, ~ 38 and q u a n t i t a t i v e l y2 2 , 3 2 9 4 0 , 6 8 , 8 2 9 g 3 . The q u a n t i t a t i v e TLC s y s terns369 82 c a n s e p a r a t e p r o c a i n a m i d e from s u c h known i m p u r i t i e s a s p a m i n o b e n z o i c a c i d a n d p n i t r o b e n z o i c acid. The most u s e f u l t h i n - l a y e r systems are given i n t h e n e x t t a b l e .

6.43

G a s Chromatography

Gas c h r o m a t o g r a p h y h a s b e e n u s e d t o s e p a r a t e and q u a n t i t a t e procainamide ( s e e S e c t i o n 7 . 0 ) , a s w e l l a s f o r i d e n t i f i c a t i o n of t h e compound (see S e c t i o n 6 . 2 ) .

367

Thin-Layer Chromatographic Systems

W

QI W

Ref. 38

Support Silica Gel GF250

Solvent Composition Benzene:Ethyl Acetate:Ethanol: Ammonium Hydroxide (15:15:5:1)

36,38,40

Silica Gel GF250

Benzene:Ethyl Acetate:Methanol: Ammonium Hydroxide(160:80:160:1)

82

Kieselgel GF254 (Merck) Benzene:Ethyl Acetate:Methanol: Ammonium Hydroxide (20:10 :20 :1)

93,68

Silica Gel QlF250 (Quantum Industries)

I. Benzene:Ammonium Hydroxide: 1,4-Dioxane (10:5:80) 1I.Isopropanol:Chloroform:Ammonium Hydroxide (45:45 :5)

PROCAINAMIDE HYDROCHLORIDE

6.44

I o n Exchange Chromatography

The u s e of ion-exchange chromatography f o r t h e s e p a r a t i o n of procainamide from m i x t u r e s h a s been r e p o r t e d 24,29,74,75 6.5

Spectrophotofluorometric Methods

F l u o r e s c e n c e o f procainamide i n t h e u l t r a v i o l e t h a s been r e p o r t e d 19 It exhibited an u n c o r r e c t e d a c t i v a t i o n maximum a t 295 nm,with an u n c o r r e c t e d f l u o r e s c e n c e maximum t o 385 nm a t pH 11. The minimal 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 1 p,g/ml.

.

The c o n c e n t r a t i o n of procainamide i n b i o l o g i c f l u i d s h a s been measured s p e c t r o p h o t o f l u o r o m e t r i c a l l y 8 2 4 3 (see S e c t i o n 7 . 0 ) . 6.6

T i t r i m e t r i c Methods

Procainamide h a s been determined by t i t r a t i o n w i t h IC126. The most g e n e r a l l y u s e f u l methods f o r t h e d e t e r m i n a t i o n o f procainamide have b e e n t h o s e i n v o l v i n g t i t r a t i o n w i t h n i t r i t e 37,42 2 3 J 2 5 J 2 7 9 2 8 J 4 2 and w i t h p e r c h l o r i c a c i d KadinG1 r e c e n t l y r e p o r t e d t h a t procainamide, a s w e l l a s some o t h e r a r o m a t i c amines, r e a d i l y undergo p a r t i a l a c e t y l a t i o n i n g l a c i a l a c e t i c a c i d c o n t a i n i n g a c e t i c anhydride. The a c e t y l a t e d a r o m a t i c amines a r e n o t t i t r a t a b l e w i t h a c e t o u s p e r c h l o r i c a c i d . The a d d i t i o n of a c e t i c anhydride t o g l a c i a l a c e t i c a c i d i s commonly employed t o remove water when t h e a c e t o u s p e r c h l o r i c a c i d i s c o n s t i t u t e d . Indeed, q u i t e s a t i s f a c t o r y t i t r a t i o n s may be o b t a i n e d when t h e a c e t i c a n h y d r i d e i s n o t added t o t h e a c e t o u s p e r c h l o r i c a c i d . However, r e a g e n t g r a d e 369

RAYMOND 6. POET AND HAROLD K A D I N

g l a c i a l a c e t i c a c i d may a l r e a d y c o n t a i n o f f e n d i n g a c e t i c anhydride. KadinG1 h a s shown t h a t a c e t i c a n h y d r i d e i n t e r f e r e n c e can b e e l i m i n a t e d by p r i o r r e a c t i o n of t h e t i t r a t i o n s o l v e n t w i t h an aromat i c amine. He t i t r a t e d an a l i q u o t o f s o l v e n t , which had been c l e a r e d o f a c e t i c a n h y d r i d e through r e a c t i o n w i t h t h e a r o m a t i c amine benzocaine, t o n e u t r a l i t y j u s t p r i o r t o d i s s o l v i n g t h e procainamide sample f o r t i t r a t i o n . However, he s t r o n g l y recommended abandonment o f t h i s r a t h e r cumbersome nonaqueous t i t r a t i o n i n f a v o r of a s i m p l e r , more s e l e c t i v e n i t r i t e t i t r a t i o n of procainamide u s i n g t h e s t a b l e i n t e r n a l i n d i c a t o r ferrocyphen. Procainamide, i s o l a t e d by e x t r a c t i o n i n t o chloroform from ammoniacal aqueous s o l u t i o n , was d i s s o l v e d i n d i l u t e h y d r o c h l o r i c a c i d a f t e r removal of t h e s o l v e n t by e v a p o r a t i o n . Procainamide was t i t r a t e d w i t h 0 . 1 g sodium n i t r i t e by o x i d a t i o n - r e d u c t i o n p o t e n t i o m e t r y , u s i n g platinum electrodes3’. The d i r e c t conductometric t i t r a t i o n of procainamide h y d r o c h l o r i d e w i t h sodium hydroxide h a s been d e s c r i b e d 3 4 . The u s u a l change i n t h e s l o p e o f t h e conductance response w i t h volume of t h e sodium h y d r o x i d e t i t r a n t o c c u r s a f t e r t h e n e u t r a l i z a t i o n of t h e amine h y d r o c h l o r i d e . I n t e r s e c t i o n of t h e two l i n e a r p l o t s , r e p r e s e n t i n g t h e changes i n conductance b e f o r e and a f t e r n e u t r a l i z a t i o n , then a c c u r a t e l y d e l i n e a t e s the endpoint. 6.7

M i c r o b i o l o g i c a l Methods

A microbiological assay of procainamide w i t h A c e t o b a c t e r suboxydans h a s been r e p o r t e d 32

.

370

PROCAl NAMl DE HYDROCHLORIDE

7.

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 and T i s s u e s

C o n c e n t r a t i o n s of procainamide i n b i o l o g i c a l media have been measured c o l o r i m e t r i c a l l y a f t e r d i a z o t i z a t i o n and coupling t o t h e Bratton-Marshall r e a g e n t ( N (1-naphthyl) - e t h y l e n e d i a m i n e ) t o form a c o l o r e d product. These methods have involved p r o t e i n p r e c i p i t a t i ~ on r ~s o~l v e n t e x t r a c t i o n 2 4 3 9 4 6 9 56 t o i s o l a t e procainamide p r i o r t o t h e c o l o r i m e t r i c measurement. J

C o n c e n t r a t i o n s of procainamide i n blood and u r i n e were e s t i m a t e d a f t e r r e a c t i o n of t h e drug o form a w i t h 4 - d i r n e t h y l a m i n o ~ i n n a m a l d e h y d e ~t ~ c o l o r e d product. A r a p i d procedure f o r measuring plasma procainamide c o n c e n t r a t i o n s by g a s chromatography h a s been r e p o r t e d by Atkinson e t a139. They used a 2 m x 2 mm i . d . g l a s s c o i l packed w i t h 0.2% OV-17 on a 100/120 mesh g l a s s bead (Corning GLC1 1 0 ) . The temperature o f t h e i n j e c t o r p o r t was 23OoC, t h a t of t h e column 225OC and of t h e ) flame i o n i z a t i o n d e t e c t o r , 2400C. A flow r a t e of 30 m l / m i n N 2 c a r r i e r g a s and H2 was used. The a i r - f l o w r a t e f o r t h e d e t e c t o r was 300 m l / m i n . Procainamide was e x t r a c t e d from a l k a l i n e s o l u t i o n i n t o methylene c h l o r i d e , w i t h p-amino-N- (2dipropyl-aminoethy1)benzamidine HC1 a s t h e i n t e r n a l s t a n d a r d . The r e s i d u e r e s u l t i n g from t h e e v a p o r a t i o n of t h i s methylene c h l o r i d e s o l u t i o n was d i s s o l v e d i n e t h y l a c e t a t e and i n j e c t e d i n t o t h e chromatograph.

The d e t e r m i n a t i o n o f procainamide i n serum8t 43by use of i t s f l u o r e s c e n t p r o p e r t i e s h a s provided an a d d i t i o n a l t o o l . Procainamide was e x t r a c t e d from a l k a l i n i z e d s a l t - s a t u r a t e d serum i n t o benzene c o n t a i n i n g 1.5% i s o p e n t y l a l c o h o l . 371

RAYMOND 6. POET AND HAROLD K A D I N

I t was acid. pH had sodium

re-extracted i n t o d i l u t e hydrochloric I t s f l u o r e s c e n c e was measured a f t e r t h e been a d j u s t e d t o 11 by t h e a d d i t i o n o f hydroxide.

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 N-acetylprocainamide, a m e t a b o l i t e of procainamide, h a s been r e p o r t e d by Poet44. In t h i s method2 Y ~ N-acetylprocainamide ~ , is e x t r a c t e d from a l k a l i z e d serum i n t o benzene c o n t a i n i n g 1.5% isoamyl a l c o h o l . I t i s t h e n r e - e x t r a c t e d from t h e s o l v e n t i n t o 13-hydroc h l o r i c acid. An a l i q u o t is heated i n a b o i l i n g - w a t e r b a t h t o h y d r o l y z e any N-acetylprocainamide p r e s e n t . N-acetylprocainamide i s then determined a s t h e d i f f e r e n c e i n procainamide c o n t e n t o f t h e sample e x t r a c t b e f o r e and a f t e r h y d r o l y s i s , a s measured by t h e r e p o r t e d c o l o r i m e t r i c method2 5

ireviou .

The metabolism and d i s t r i b u t i o n of procainamide h y d r o c h l o r i d e i n man and dog was s t u d i e d by Mark e t a12. They r e p o r t e d t h a t 50-60% of a s i n g l e i n t r a v e n o u s dose a d m i n i s t e r e d t o man was e x c r e t e d unchanged i n t h e u r i n e ; t h a t about 2-10% was accounted f o r a s p a m i n o b e n z o i c a c i d ; and t h a t t h e drug d i d n o t accumulate when given i n r e p e a t e d o r a l doses. Procainamide c o n c e n t r a t i o n s i n t h e s e s t u d i e s w e r e measured a f t e r i s o l a t i o n of t h e drug from a l k a l i z e d b i o l o g i c a l m a t e r i a l by e x t r a c t i o n i n t o benzene, augmented by s a t u r a t i o n of t h e aqueous phase w i t h sodium c h l o r i d e . The drug was r e t u r n e d t o d i l u t e a c i d , d i a z o t i z e d , and coupled w i t h N(1-naphthy1)ethylene diamine. The r e s u l t i n g c o l o r e d dye was determined s p e c t r o p h o t o m e t r i c a l l y (see Section 7.0). The s p e c i f i c i t y of t h e method was demonstrated by t h e c a l c u l a t i o n of comparative d i s t r i b u t i o n r a t i o s 6 . 312

PROCAINAMIDE HYDROCHLORIDE

Dreyfuss e t a l . 3 9 4 s t u d i e d t h e b i o t r a n s f o r mation of procainamide h y d r o c h l o r i d e i n r h e s u s monkey, man, and dog, u s i n g I 4 C - l a b e l l e d d r u g . I n t h e dog, 50-67% o f t h e 14C a c t i v i t y e x c r e t e d i n t h e u r i n e was unchanged p r o c a i n a m i d e ; f o u r m e t a b o l i t e s were r e c o g n i z e d . I n monkey, 22-49”/0 of t h e 1 4 C a c t i v i t y was e x c r e t e d i n t h e u r i n e as unchanged p r o c a i n a m i d e ; t w o metabolites w e r e r e c o g n i z e d . N-acetyl p r o c a i n a m i d e , i s o l a t e d and i d e n t i f i e d ‘ b y a c o m b i n a t i o n o f TLC, NMR and mass s p e c t r o m e t r y , was a major m e t a b o l i t e e x c r e t e d by monkey: a l t h o u g h p r e s e n t i n man t h i s metabolite d i d n o t a p p e a r t o o c c u r i n dog. The c o m b i n a t i o n of these a n a l y t i c a l techniques helped t o provide e s s e n t i a l d a t a i n t h e s e m e t a b o l i c s t u d i e s . These s t u d i e s a l s o s u g g e s t e d t h a t compounds c o n t a i n i n g r e a d i l y a c e t y l a t e d a r o m a t i c amino g r o u p s s h o u l d be s t u d i e d i n p r i m a t e s r a t h e r t h a n i n d o g s , b u t p r e f e r a b l y i n b o t h species. Weily and Genton‘ have r e p o r t e d on t h e r o u t e s and mechanism o f p r o c a i n a m i d e e x c r e t i o n : t h e e f f e c t o f u r i n e pH; and t h e e f f e c t s o f f l o w r a t e and t h e p r e s e n c e o f r e n a l o r h e p a t i c impairment on t h e plasma h a l f time and on t h e r e n a l and h e p a t i c c l e a r a n c e and e x c r e t i o n of procainamide. The p h a r m a c o k i n e t i c s o f p r o c a i n a m i d e hydroc h l o r i d e i n man h a s b e e n s t u d i e d . e x t e n s i v e l y 7 ’ 8, 9. Koch-Weser d e m o n s t r a t e d t h a t plasma c o n c e n t r a t i o n s between 4 and 8 mg/L a r e found a f t e r t h e u s u a l l y e f f e c t i v e t h e r a p e u t i c doses. H e showed t h a t knowledge o f plasma c o n c e n t r a t i o n s i s h e l p f u l i n e s t a b l i s h i n g t h e optimal dosage i n i n d i v i d u a l p a t i e n t s . Procainamide c o n c e n t r a t i o n s were d e t e r m i n e d s p e c t r o p h o t o m et r i c a l l y 8 and s p e c t r o p h o t o f l u o r o m e t r i c a l l y 8 b y methods t h a t a r e w e l l within the capability of h o s p i t a l laboratories (see S e c t i o n 7 ) .

373

RAYMOND 6 . POET AND HAROLD KADIN

A recent report'' indicated that N-acetyl procainamide was detected in the plasma of two patients receiving procainamide. This compound was identified by a combination of TLC, gas chromatography and mass spectrometry. This metabolite was demonstrated to have antiarrhythmic activity in mice. The report concluded that the observed anti-arrhythmic activity of procainamide in man may be due, in part, to its conversion to N-acetylprocainamide (see also Section 7). 8.

References 1.

United States Pharmacopeia, XVIII, pgs. 8,541 (1970).

2.

L. C. Mark, H. J. Kayden, J. M. Steele, J. R. Cooper, I. Berlin, E. A. Rovenstine, and B. B. Brodie, J. Pharmacol. Exp. Ther., 5-15 (1951).

z,

3.

J. Dreyfuss, J. T. Bigger, A. I. Cohen and E. C. Schreiber, Clin. Pharmacol. Ther. 13, 366-71 (1972).

-4.

J. Dreyfuss, J. J. Ross, and E. C. Schreiber, Arzneim-Forsch. ,=(7), 948-51 (1971).

5.

A.

6.

I. Cohen,Squibb Institute, Personal Communication.

H. S. weily and E. Genton, Arch. Intern. Med 130, 366-69 (1972).

- 0 9

7.

-

J. Koch-Weser, Ann. N.Y. Acad. Sci. , 370-82 (1971).

179,

374

PROCAINAMIDE HYDROCHLORIDE

8.

J. Koch-Weser and S. W. K l e i n , J. A m e r . Med. A s s . , 2 1 5 , 1 4 5 4 - 6 0 ( 1 9 7 1 ) .

9.

E. G. G i a r d i n a , R. H. H e i s s e n b u t t e l and J. T. B i g g e r , C i r c u l a t i o n , 42, Supp. 3,

156 ( 1 9 7 0 ) .

10. 11.

E . D r a y e r and M. M. R e i d e n b e r g , P h a r m a c o l o q i s t , l5, 154 ( 1 9 7 3 ) .

D.

E.

-8 ,

H. Coy and B. T o e p l i t z , S q u i b b I n s t i t u t e , P e r s o n a l Communication.

12.

N.

13.

P.

14.

C. C l a r k e , J. Pharm. P h a r m a c o l . , 202-6 ( 1 9 5 6 ) .

G.

cooper, Pharm.

J.,

177,

495-6(1956).

F i s c h e r and N. O t t e r b e c k , S c i . Pharm., 76-8(1958) ,Chem.Abstr., 5 3 , 4 6 5 7 g (1959).

R.

26,

/

15.

16.

Med. Prom.S.S.S.R., L5 No. 8 , 4 3 - 5 ( 1 9 6 1 ) , C h e m . A b s t r . , E , 2512c ( 1 9 6 2 ) .

G.

I. Luk-Yanchikova,

L. 0. K i r i c h e n k o , Farm. Zh. ( k i e r ) , 1 7 ( 1 5 ) , 68-72 ( 1 9 6 2 ) ,Chem. A b s t r . , 6 l , 4155e (1964).

17.

L.

18.

N. M.

M.

-

A t h e r d e n , Pharm. J . , 195, 1 1 5 (1965).

Y a v o r s k i i , V. S. Koval and S t a r u s h c h e n k o , Farm. Zh. ( K i e v ) , 3 ( 5 ) , 31-7(1965) ( U k r a i n ) , Chem. , A b s t r . , 64, 9512f ( 1 9 6 6 ) . P. M.'

19.

S. U d e n f r i e n d , D. E. Duggan, B.M. V a s t a , J. Pharmacol. Exp. T h e r . , 120, 26-32(1957) 375

RAYMOND 6. POET AND HAROLD KADIN

20.

J.

21.

H. A m a l , E. G u r s u and S. D e m i r , E. Symp. Chromatoqr. E l e c t r o p h o r e s i s , 5 t h 1968 (Pub. 1969) 441-6 (FR) Ann.ArborHumphrey S c i . Pub. I n c . , Ann Arbor, Mich., Chem. A b s t r . , 72, 2 8 1 9 1 ( 1 9 7 0 ) .

22.

K. Macek and J. Vecerkova, Pharmazie, 2 0 ( l o ) , 605-16 (1965) (GER) , Chem. A b s t r . , 6 4 , 527c ( 1 9 6 6 ) .

A. F r e s e n , Pharm. Weekbl., 659-78 (1967)

.

102

(281,

23. L. N. Guseva, N. I . V e s t f a l and M. I. Kuleshova, F a r m a t s i y a [Moscow), 16 ( 3 ) , 46-51 (1967) ( R u s s . ) , Chem. A b s t r . , 67, 67668 ( 1 9 6 7 ) . 24. G. A . Vaisman and M. M. YampollSkaya, Aptechn. Delo., l l ( 5 ) , 38-41 ( 1 9 6 2 ) , Chem. A b s t r . , 60, 10476b (1964)

.

25. R. V a s i l i e v , A. Cosmin, M. Mangu and I. Burnea, Rev. Chim. ( B u c h a r ) , 1 3 , 2 3 9 ( 1 9 6 2 ) . , Chem.Abstr. 16752a ( 1 9 6 2 ) .

,=,

26. Y a . I. Kadyrov and 0. Alimkhanov, Uzbeksk. Khim. Zh., 2 (l), 31-3(1965) ( R u s s . ) , Chem. A b s t r . , 63, 2852a ( 1 9 6 5 ) . 27. W. Wisniewski and T. K i n d l i k , A c t a . P o l . Pharm., 25 (l), 55-8(1968) ( P o l . ) , Chem. A b s t r . , 6 8 , 107930 ( 1 9 6 8 ) .

28. W. Wisniewski and T. K i n d l i k , Acta. Pharm., 26 ( 1 1 , 39-43(1969) ( P o l . 1, Chem. A b s t r . , 7 0 , 109201 ( 1 9 6 9 ) .

376

Pol.

PROCAINAMIDE HYDROCHLORIDE

29

.

S. Yaskina a n d I. Nguyen-Bah, Aptechn. D e l o . , 1 2 ( 1 ) , 7 6 - 7 ( 1 9 6 3 ) , Chem. A b s t r . , 6 l , 131319 ( 1 9 6 4 ) .

D.

30. V. V a l e n t i , S q u i b b I n s t i t u t e , P e r s o n a l Communication. 31.

W i s n i e w s k i and T . K i n d l i k , A c t a . Pol. Pharm., 23 ( 6 ) , 517-22 ( 1 9 6 6 ) ( P o l . , Anal. A b s t r . , IS, 1 0 3 9 ( 1 9 6 8 ) .

w.

32. A. Rappe, S. Baur and G. Mauquoy, Ann. Pharm. F r . , =(11), 655-62 (1969) ( F r . ) , Chem. A b s t r . , 73, 28977 ( 1 9 7 0 ) . 33. G. I. Kudymov, A. A . K i s e l e v a a n d M. V. Mokrouz, T r . Permsk. Farm. I n s t . , N o . 3, 107-9 ( 1 9 6 9 ) (Russ. ) , Chem. A b s t r . , 75, 40532 ( 1 9 7 1 ) . 34. B. P. ArtAmonov and S. L. M a i o f i s , F a r m a t s i y a (Moscow), 20 ( l ) ,3 6 - 4 1 ( 1 9 7 1 ) ( R u s s . ) , Chem. A b s t r . , 75, 4 0 5 3 5 ( 1 9 7 1 ) . 35. Q. Ochs, S q u i b b I n s t i t u t e , P e r s o n a l Communication. 36. P. Taylo? and H. R o b e r t s , S q u i b b I n s t i t u t e , P e r s o n a 1 Communication. 37. G. H a r t , S q u i b b Q u a l i t y C o n t r o l , P e r s o n a l Communication.

38. P. T a y l o r and H. Roberts, S q u i b b I n s t i t u t e , P e r s o n a l Communication. 39. A. J. A t k i n s o n , M. P a r k e r and J. S t r o n g , C l i n . Chem., l 3 ( 7 ) , 643-46 ( 1 9 7 2 ) .

377

RAYMOND 6. POET AND HAROLD K A D I N

40.

H. R. R o b e r t s and P . T a y l o r , S q u i b b I n s t i t u t e , P e r s o n a l Communication.

41.

B. S. F i n k l e , E. J. C h e r r y a n d D. M . T a y l o r , J. Chromatogr. S c i . , 9,393 (1971).

42.

H. Kadin and E. J. J e n k i n s , S q u i b b I n s t i t u t e , P e r s o n a l Communication.

43.

J. Koch-Weser, S . W. K l e i n , L. L. Foo-Canto, J. A. K a s t o r a n d R. W. D e s a n c t i s , N. Eng. J. Med., 281, 1253-60 (1969).

44. R . B. P o e t , S q u i b b I n s t i t u t e , Communication.

Personal

45.

S. B e l l e t , S. Am. J. Med.,

Zeeman and S. A . l3, 145-157 ( 1 9 5 2 ) .

46.

R . B. P o e t and J. Kowald, S q u i b b I n s t i t u t e , P e r s o n a l Communication.

47.

H. R. Roberts, s q u i b b I n s t i t u t e , P e r s o n a l communication,

48.

G.

E.

Hirsh,

/

I . Luk-Yanchikova and V. N. B e r n s h t e i n , U.S.S.R. P a t e n t N o . 141, 4 8 6 (16,10,61), Anal. A b s t r . , 9, 2492 ( 1 9 6 2 ) .

49. W. Wisniewski and T. K i n d l i k , D i s s . Pharm. Pharmacol., 18 ( 5 ) , 529-32 ( 1 9 6 6 ) ( P o l . ) , Chem. A b s t r . , 67, 14908 ( 1 9 6 7 ) . 50. M. H o r i o k a , Yakuqaku Z a s s h i , 77, 200-6 ( 1 9 5 7 ) , Chem. A b s t r . , 51, 8 3 6 7 b ( 1 9 5 7 ) .

378

PROCAINAMIDE HYDROCHLORIDE

51.

J.

Kracmar a n d J. Kracmarova, Cesk.Farm. ( 3 ) , 121-9 ( 1 9 6 6 ) (Czech. ) , Chem. A b s t r . , 65, 3 6 6 8 b ( 1 9 6 6 ) .

15

52. D. D i c k s i u s , S q u i b b I n s t i t u t e , P e r s o n a l Commun i c a t i o n . /

53. G. I. Luk-Yanchikova a n d V. N. B e r n s h t e i n , P e r e d o v y e . Metody. K h i m . Tekhnol, i Kontrolya. P r o i z . S b ., 257-60 ( 1 9 6 4 ) ( R u s s . ) , Chem. A b s t r . , 6 2 , 1297733 ( 1 9 6 5 )

.

/

54. V. N. B e r n s h t e i n a n d G . I . Luk-Yanchikova Uch. Zap. P y a t i g o r . Gos. F a r m a t s e v t . I n s t . , 5 , 165-9 ( 1 9 6 1 ) , Chem. A b s t r . 2592g ( 1 5 6 3 ) .

,=,

55. M. D. D e n i s o v , Farm, Zh. ( K i e v ) , 1 9 ( 3 ) , 60-5 ( 1 9 6 4 ) ( U k r a i n ) , Chem. Abstr.64 -9 3286a ( 1 9 6 6 ) . 56. G. P i t e l a n d T. L u c e , Ann. Pharm. F r a n c . , 23 (ll), 6 7 3 - 8 1 ( 1 9 6 5 ) ( F r . ) , Chem. A b s t r . 6 4 , 14775a ( 1 9 6 6 ) .

-

57. J. M. Dunham, S q u i b b I n s t i t u t e , P e r s o n a l Communication. 58. T. Van d e r Wegen, Pharm. Weekbl., 173-197 ( 1 9 6 8 ) .

103,

59. A . Cohen, S q u i b b I n s t i t u t e , P e r s o n a l Communication. 60.

z,

J.

Z i m m e r , Krankenhaus-Apotheker, 2 6 ( 1 9 7 5 ) , Chem. A b s t r . , 5 2 , 8 4 6 3 d ( 1 9 5 8 ) .

379

RAYMOND B. POET AND HAROLD KADlN

61. H. Kadin, J. Pharm. Sci., 6_3, 919(1974). 62. P. J. Black and A. I. Cohen, paper presented at Twentieth Annual Congress on Mass Spectrometry and Allied Topic, Dallas, Texas (6/4-9/72). 63. J. Womack, E.A.I., Princeton, N.J., Personal communication. 64. J. Alicino and J. Hydro, Squibb Institute, Personal Communication,

65. E. Feldman, J. Am. Pharm. Assoc. Sci.Ed. 47, 676 (1958). 66. K. Biemann, "Mass Spectrometry: Organic Chemical Applications", McGraw-Hill Book Co., Inc., New York(1962). 67. R. Walton, Squibb Institute, Personal Communication. 68. J. Fairbrother and S. Shand, Squibb International Development Laboratories, Personal Communication. 69, G. Brewer, Squibb Institute, Personal Communica tion.

70. D. Whigan and H. Kadin, Squibb Institute, Personal Communication. 71. 0. Kocy, Squibb Institute, Personal Communication. 72. K. Radushkevich, Mater. Obmenu. Peredovym. Opytom.Nauch.Dostizhen. Khim-Farm.Prom. ,No.1,22-6 (1958),Chem. 380

PROCAINAMIDE HYDROCHLORIDE

Abstr.,

54, 173163. ( 1 9 6 0 ) .

73. 0. Kocy, S q u i b b I n s t i t u t e , P e r s o n a l Communication. 74. G. L. S t a r o b i n e t s a n d I . P. Koka, V e s t s i . Akad. Navuk. B e l a r u s . SSR S e r . Khim. Navuk., ( 1 9 6 7 ) (4) 16-21 ( R u s s . ) , C h e m . A b s t r . , 68, 8 9 8 3 9 ( 1 9 6 8 ) . 75. G. L. S t r o b i n e t s a n d S. D. K u l l k i n a , V e s t s i . Akad. Navuk. B e l a r u s . SSR. K h i m . Navuk., ( 1 9 7 1 ) ( 2 ) 46-50 ( R u s s . ) , Chem. A b s t r . , 75, 25276 ( 1 9 7 1 ) . 76. B. B r o d i e and S . U d e n f r i e n d , J. B i o l . 4 Chem , 1 5 8 , 705 ( 1 9 4 5 ) .

-

77. H. J a c o b s o n a n d C. S c h a e f e r , S q u i b b I n s t i t u t e , P e r s o n a l Communication. 78. K. K a l i n o w s k i a n d 2. Z w i e r z c h o w s k i , A c t a . P o l o n . Pharm., 15, 175-8 (19581, Chem. A b s t r . , 52, 17617b ( 1 9 5 8 ) . 79. M. D o l l i v e r , S q u i b b I n s t i t u t e , P e r s o n a l Communication. 8 0 . D. D i c k s i u s , S q u i b b I n s t i t u t e , P e r s o n a l Communication.

81. C. K. P a r e k h , U.S. P a t e n t 3 , 7 1 6 , 3 3 6 (C1,23/230B; G O h ) , 1 3 Feb. 1 9 7 3 , Chem. A b s t r . , 79, 1 5 5 6 3 ( 1 9 7 3 ) . 8 2 . J. F a i r b r o t h e r , S q u i b b I n t e r n a t i o n a l Development L a b o r a t o r y , P e r s o n a l Communication.

381

RAYMOND 6 . POET AND HAROLD K A D I N

83. T. Soh, Squibb Quality Control, Personal Communication. 84. Y. Tashika and M. Kuranari, J. Pharm. SOC. Japan, 73, 1069-71 (1953), Chem. Abstr., 48, 12027g (1954). 85. M. Yamazaki, Y. Kitagawa, S. Hiraki and Y. Tsukamoto, J. Pharm. SOC. Japan, 73, 294-7 (1953), Chem.Abstr. , 48,2003i (1954). 86. H. Najer and M. Joannic, Ann. Pharm. Franc, l3, 556-9(1955), Chem. Abstr., 50, 9331d (1956). /

-

87. V. Hach and E. Koppova, Cesk. Farm., 582-3 (1956), Chem. Abstr., 51, 8693c (1957).

I,

88. K. Iwaya and K. Yoshida, Ann. Rep. Shionogi. Res. Lab. , #2, 40-1 (1952), Chem. Abstr., 5 l , 10424h (1957). 89. Z. Ledgchowski, M. Bogucka, A. Led6chowski and A. Chimiak, Przem. Chem., 38, 91-2 (1959), C x m . Abstr., 53, 1 9 G 3 d (1959).

-

90. T. Shen, L. Yu, C. Chin, I. Wang and W. Luh, Yao Hsueh T' ung. Pao,, 8,319-21 (1960), Chem. Abstr. ,59, 269911 (1963).

91. M. Blaich and A. Restivo, Squibb Institute, Personal Communication. 92. M. Blaich and A. Restivo, Squibb Institute, Personal Communication. 93. P. Egli, Squibb Institute, Personal 382

PROCAlNAMl DE HYDROCHLORIDE

Communication. 94. J. B a r t o s , Ann. Pharm., (1964).

F r a n c , 22, 383

95. J . O r l o s k i , S q u i b b I n s t i t u t e , P e r s o n a l Communication. 96. P. T. Thymm, R. L u c h i , H. L. Conn., J. Pharmacol. Exp. T h e r . , 164, 239-251 (1968). 97. J . Kruk, Zesz, Nauk. Uniw. J a g i e l l o n . P r . Chem., N o , 1 2 , 111-14(1967) (Eng. ) Chem. A b s t r . , 69, 89883 ( 1 9 6 8 ) .

,

98. K. Iwaya and K. Y o s h i d a , J a p a n , 4325 ( ' 5 2 ) , O c t . 2 2 , Chem. A b s t r . , 48, 5 2 1 8 i (1954)

.

99. G. G h i e l m e t t i , Farmaco ( P a v i a ) Ed. S c i . 9 , 384-6 ( 1 9 5 4 ) , Chem. A b s t r . , 49, 7521a ( 1 9 5 5 ) . 100. E. P a v l o v s k a , J. Lukac a n d M. B o r o v i c k a , C z e c h . , 114, 447(C1. C 0 7 c ) A p r i l 15, ( 1 9 6 5 ) , Chem. A b s t r . , 64, 6575c ( 1 9 6 6 ) .

383

RESERPINE

Roger E. Schirmer

RESERPINE

COWENTS 1. Descript ion 2. Physical Properties 2.1 Melting Point 2.2 Electronic Spectra 2.2.1 Ultraviolet Absorption Spectrum 2.2.2 Optical Activity 2.2.3 Fluorescence and Phosphorescence 2.3 Infrared Spectrum 2.4 Nuclear Magnetic Resonance Spectrum 2.5 Crystal Form and X-Ray Powder Pattern 2.6 Solubility and Partition Coefficients 3. Degradation of Reserpine 3.1 Chemistry of Reserpine Degradation 3.1.1 Hydrolysis 3.1.2 Epimerization 3.1.3 Oxidation 3.2 Degradation of Reserpine in Pharmaceutical Dosage Forms 4. Metabolism of Reserpine 5. Identification of Reserpine 6. Elemental Analysis 7. Chromatographic Methods of Analysis 7.1 Thin Layer Chromatography 7.2 Paper Chromatography 7.3 Column Chromatography 7.4 Electrophoresis 7.5 Countercurrent Distribution 8. Titrimetric Determination 9. Electrochemical Analysis 10. Spectrophotometric Analysis 10.1 Infrared 10.2 Ultraviolet 10.3 Colorimetric 10.4 Ion-Pair Extraction with Spectrophotometric Detection 10.5 Fluorescence 11. Analysis of Reserpine in Biological Systems

385

ROGER E . SCHIRMER

1. Description Reserpine is 1,2-didehydro-2,7-dihydro-ll,l7a-dimethoxy3f3,20a-Yohimban-l6f3-~arboxylic acid methyl ester, 18B-trimethoxybenzoate ester.

Reserpine is generally obtained by extraction of the roots of certain species of Rauwolfia (Apocynaceae), prin5igjally R. Serpentina and R. Vomitoria, or by eyntheIt occurs as a white or pale buff to slightly sis. yellow, odorless, crystalline substance. It is weakly basic, with a pK of 6.6.

-

2.

Physical Properties 2.1

Melting Point 1 Reserping melts at 264-265°C with decomposition. u Hochstein &. have reported that reserpine melts at 284-286°C in an evacuated tube, and that the apparent melting point is less dependent on heating rate when measured under vacum. The melting pqints of several salts of reserpine are listed in Table 1, and melting points for several stereoisomers have been included in Table 2.

s.

386

RESERPINE

Table 1 Melting P o i n t s of Several Reserpine S a l t s 7 Salt

hydrochloride sulfate

E m p i r i c a l Formula C33H40N2040

HCll H 2 0

c33H40N209' H2S04

perchlorate

C3 H40N209* HC 1O4

nitrate

C33H40N209'm03

oxalate

C33H40N209*

methylbromide

C

maleat e picrate

Melting Point* 224" 242-244" 238-23 9"

235 "

C2H204- 1/2H20

206"

H N 0 *CH3Br 33 40 2 9

271-272"

3 3 H4ON 2 '9'

c4H404

226-227"

H2

183 186"

'3 3 H40N2'9'

387

-

Table 2 Sodim D-line Rotations for Reserpine and Several of Its Configurational Isomers Cornpound reserpine

Isomer*

Melting Point "C

Conditions

-

U D

Ref -

264-265

-118"

CHCl , 23°C pyriaine, 26°C d imethylforamide, 26°C dilute acetic acid, 25°C Dioxane, 25°C CHC13 CHC13

798

-164" -168"

-100"

16a 16-epireserpine 18a 18-epireserpine 16-epi-17-epi-reserpine 16a,178 (neoreserpine) 3- isoreserpine 3a 182 18-epiisoreserpine

- 125"

180 141-145

+ 44" + 38"

-

+ 29"

163 170 152-165 245-248

-164"

CHC13 CHC13 CHC13

7

7

16 16

1 10 11 899 10

W h e entries in this column give the configuration o f the carbons which differ from the configuration in reserpine.

RESERPINE

2.2

Electronic Spectra 2.2.1

Ultraviolet Absorption Spectrum The ultraviolet spectra of indole alkaloids generally consist of three bands, one band in the region of 225 nm and two bands, often unresolved, in the region of 280 run. These bands have been assumed to have the same origin as the low enfsgy transitions of benzene, and so thp3 benzene nomenclature is used for the indole chromophore. As shown in Figure 1, all thfee bafds are distinct in the spectrum of reserpine. lThe F a c A band maximum occurs at 216 p" (€=35,700), the La t A band at4267 nm (€=15,700) and The absorption the I,,* A band at 296 nm (€=9,660). maxima and extinctions are essentially identical in chloroform and methanol. 2.2.2

Optical Activity Carbons 3 , 15, 16, 17, 18, and 20 of reserpine are asymmetric, resulting in 64 poseible configurational isomers. Reserpine itself ha rotation of -118" The D-line (sodium D line) at 23°C in chloroform.'18 rotations for reserpine and several of its isomers are listed in Table 2. The circular dichroism (CD) and magnetic circularlgichroism (MCD) spectra of reserpine have been reported and are reproduced in Figure 2. The magnetic circular dichroism is of special analytical interest because the sign pattern can be used to distinguish indole alkaloids from alkaloids bearing the indoline or oxindole chromophores: chromophores show a positive effect at low wavetransition) and negative effects at higher wavetransition), whereas indole alkaloids have fects at low wavelength and positive effects at higher wavelength. 2.2.3

Fluorescence and Phosphorescence The absorption band at 296 nm in resgz&;19 gives rise to strong fluorescent emission at 375 nm. In water, the fluorescence intensity is maximum at low pH: 17 a spectrum in water at pH 1,O is reproduced in Figure 3 . Fluorescence is typical of compounds closely related to reserpine, including alkaloids which occur naturally along with reserpine and degradation products of reserpine infra). The fluorescence characteristics of several of these are summarized in Table 3. In reserpine, deserpidine, and rescinnamine, it has been shown that light energy absorbed by the indole c9tjoypphore is emitted from the trimethoxybenzoate The intramolecular singlet-singlet excichromophore. '

(e

389

ROGER E. SCHIRMER

5

4

* b

P

3

X W

2

1

200 Figure 1.

250

300 Clm

350

400

The Ultraviolet Absorption Spectrum of Reserpine

390

RESERPINE

20 c3

I

0

v

10

x W

I

I

250

300

0

Yigure 2. Magnetic Circular Dichroism (-), Circular Dichroism (----), and Absorption Spectra (.....) of Reserpine in Methanol

391

loo

1

90 80 -

70 -

I

200 250 Figure 3.

300

350

400 450 500 550 600 W AVELENGTH-MILLIMICRONS

650 700

Fluorescence Spectrum of Reserpine in Water at pH 1.

750

800

RESERPINE

tation transfer occurs with 100% efficiency, and iyolves (v,TT*) state to the transfer from the indole S (TT,TT*) state of the trimetioxybenzoate acceptor. T e 22 energy transfer appears to occur bF3an exchange mechanism rather than a resonance mechanism.

3

Table 3 Fluorescence Characteristics o Compounds Related to Reserpine €9 Excitation Emission Maximum Maximum Reserpine 280 360 5 10 3-is0 Reserpine 390 Dehydroreserpine perchlorate 390 510 Tetradehydroreserpine chloride 340 440 440 Rescinnamine 310 Rescinnamine N-oxide 310 340 Deserpidine 280 360 Reserpine N-oxide 280 360 Methyl Reserpate 300 360 Reserpinine 300 360 Methyl-0- (35-dimethoxy-4hydroxybenzoyl reserpate) 300 360 Syrosingopine 300 360 Syrosingopine N-oxide 290 350 Trimethoxybenzoic Acid 280 360 Trimethoxycinnamid Acid 300 400 Indole 280 340 Hannaline HC1 390 490 Compound

Relative Sensitivity 6 18 75

50 2 2-3 15 8 19 2 8

4

6 2 <1 36 150

Phosphozgscent emission from reserpine occurs at about 460 nm. 2.3

Infrared Spectrum The infrared spectrZ-96 reserpine has been reported by several workers. The spectrum in chloroform is reproduced in Figure 4, and the assignments of several bands in the spectrum are as follows: 3480 cm" 2840-3039 ~m-1 1732 cmI1 1713 cm

>N-H stretch >C-H stretch >C=O stretch, acetyl group >C=O stretch, trimethoxybenzoate group 393

101

90

80

u

40

k 30 20 10 0

~

1

1

1

1

~

1

1

1

1

1

1

1

~

~

~

~

4001 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 ll00 1800 1700 1601 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400

WAVENUMBER CM-'

Figure 4 .

The Infrared Spectrum of Reserpine i n Chloroform

~

~

'

RESERPINE

At one time Wenkert3O suggested that the presence of t o or more bands or distinct shoulders between 2700-2900 cm on the low wave number side of the major band at about 2900 cm-; indicated an a hydrogen on carbon 3, whereas the absence of these bandf8indicated a p hydrogen on carbon 3. It has since been shown that these bands are associated with axial hydrogens at C-3, and are therefore indicative of the conformation rather than the configuration of the molecule.

-Tfr

Nuclear Magnetic Resonance Spectrum The n.m.r. spectra of methyl 3-isoreserpate,methyl reserpate, methyl neoreserpate, and the 3',4',5'-trimethoxybenzoate esters of eacb of these compounds have been reported by Rosen and Shoolery. The 60 MH2 spectrum of reserpine is reproduced in Figure 5, and the assignments of several of the resonances in the spectrum are given in Table 4. As axial protons are generally found at higher fields in the n.m.r. spectrum than the corresponding equatorial protons, the resonances of the C-3 and C-18 protons may be used to distinguish reserpine from isoreserpine or neoreserpine. In the most stable conformation of reserpine, the C-3 proton is equatorial and the C-18 proton is axial. In 3-isoreserpine,however, the C-3 proton is axial and is shifted to high enough field that it is obscured by the -WH3 resonances: the axial C-18 resonance is essentially unchanged. In neoreserpine the C-3 proton is again axial (therefore not discernable in the spectrum) and the C-18 proton is equatorial and therefore shifted to lower field than in reserpine (5.83 p.p.m. compared with 5.05 p.p.m., respectively). 2.4

395

I

I

I

I

I

I

I

8.0

7.0

6.0

5.0

4.0

3.0

2.0

PPM Figure 5.

I

1.o

I

0

I61

The 60 MHz NMR Spectrum of Reserpine in CDC13.

See Table 5 for Assignments.

RESERPINE

Table 4 Assignment of the N.M.R. Spectrum of Reserpine in CDC13 Functional Group

NH Indole aromatic H ((3-9,C-lO,C-12) Trimethoxybenzoic acid aromatic H C-18 H C-3 H Trimethoxybenzoic acid, -OCH3 (2-11, -OCH3

@.p.m.

Chemical shift from tetramethylsilane)

7.85 p.p.m. (broad singlet) Two groups of lines at about 6.7 and 7.3 p.p.m. 7.34 p.p.m. (sharp singlet) 5.05 p.p.m. multiplet 4.43 p.p.m.

multiplet

3.92 p.p.m.

singlet

3.79 p.p.m.

singlet

3.79 p.p.m. 3.46 p.p.m.

singlet singlet

0

C-16, -C-OC% C-17, -OCH3

Crystal Form and X-Ray Powder Pattern CrystaJiographic data on reserpine has been reThe crystals are monoclinic with axial ported by Rose, They occur ratios, a:b:c, of 1.654:1:1.537 and B = 115'8'. as blades or needles elongated parallel to b and lying on the orthopinacoid 100 or basal pinacoid 001 The crystals sometimes show the clinodome 011 and the hemipyramid: blades are generally terminated by the clinopinacoid 010 TQe density of the crystals wag determined to be 1.298 g/cm by flotation and 1.293 g/cm by x-ray. The refractive indexes of the crystal at 25°C for 5893A radiation are as follows: ~ 1 . 5 1 ,p=1.568 + 0.002 andY=1.687 2 0.004. The unit Sell contsins two molegules and has dimensions A=14.57AI b=8.8lA, and c=13.5A. The powder pattern is summar ed in Table 5. Hamilto$ has reported that reserpine is one of the largest acentric molecules whose structure has been determined by direct x-ray techniques, but he did not report the details of this study. 2.5

.

391

.

ROGER E. SCHIRMER

Table 5 31 X-Ray Powder P a t t e r n of Reserpine

-d

-I/I 1

hkl -

d (calculated)

13.14 12.22 7.44 7.19 5.73 5.34 5.02 4.78 4.49 4.21 4.18 3.71 3.56 3.47

0.40 0.40

100 001 101 011 111 210 012 102 103 112 021 202 401 103

13.11 12.18 7.48 7.14 5.70 5.26 5.01 4. 80 4.45 4.21 4.15 3.74 3.57 3.48

0.20 1.oo 0.40 0.40 0.40 0.60 0.80 0 -40 0.20 0.40 0 -40 0.20

2.6

S o l u b i l i t y and P a r t i t i o n C o e f f i c i e n t s Reserpine i s only soluble t o t h e extent of 6 mcg/ml i n 0.1 N HC1 at 37"C, and i t s s o l u b i l i t y i n aqueous solvents decreases a t high pH. Reserpine i s much more soluble i n less polar solvents, with a s o l u b i l i t y of about 0.56 mcg/ml 13 ethanol and 167 mg/ml i n chloroform a t room temperature, D i s t r i b u t i o n c o e f f i c i e n t s f o r r e s e r p i n e and seve a1 r e l a t e d a l k a l o i d s have been reported by Hochstein e t . a l . and are reproduced i n Table 6.

E

Table 6 D i s t r i b u t i o n C o e f f i c i e n t s f o r Reserpine 6 and Related Alkaloids Solvent system Compound Reserpine Yohimbine Rauwol sc i n e Ajmalicine Heterophyllin Serpentine A jmaline

398

A

B

C

9.0 9.0 9.0 11.0 20.0 0.01 0.01

0.01 0.01 0.01 0.01 0.01 11-13 11-13

1.5 0.7 8.0 8.0 7 1

8.0

RESERPINE

system

A

Solvent 1 benzene

B

Solvent 2 .05M pH 7 phosphate buffer CHC13

50% CH OH in H20

3 n-butano1

C

15% CH3COOH in H20

Distribution Coefficient = conc. solvent lfconc. solvent 2 3. Degradation of Reserpine 3.1

Chemistry of Reserpine Degradation 3.1.1

Hydrolysis The reserpine molecule contains two ester groups, both of which are susceptible to hydrolysis, as shown in Figure 6. Hydrolysis of the trirnethoxybenzoate ester group occurs2~~sg,ygre rapidly than hydrolysis of the methyl ester group. As with all esters, the hydrolysis of reserpine is catalyzed by both acid and b e with basic catalysis generally being most efficient," Although the full pH profile of reserpine hydrolysis has apparently not been studied, one would expect the rate of hydrolysis to be minimum in the pH range of 3-4. 3.1.2

Epimerization Although reserpine has six asymmetric carbons, only C-3 is inverted easily enough for isomerization to be a significant route of degradation. Epimerization of reserpine t 3-gsoreserpine occurs gsincipally in strong acid solution.'6-3 Gaskell and Joule have shown that epimerization under these conditions is initiated by protonation of C-2 with subsequent ring opening to give an intermediate in which C-3 is planar and properly oriente 99 for e icient reclosure of the system to 3-isoreserpine. Bayer has reported that epimerization of reserpine in solution in chloroform is a380 promoted by heat and light. Hakkesteegt has reported that the ratio of epimerization to hydrolysis was 2.2, 5.6 and 8.1 for solutions of reserpine at pH 1.3, 2.2 and 3.0, respectively, after storage of the solutions for 48 hours at 100°C.

66

399

OCH3

OPlMERlZATlON [C-3)

HYOROLYSIS. ACID OR BASE

3-ISORESERPINE

P 0

0

METHYL RESERPATE

18-(3.4,5-TRIMETHOXYBENZOYl) RESERPIC ACID

3 ,I-DIDEHYDRORESERPIWE

RESERPIC ACID

Figure 6 .

Principle Routes for Degradation of Reserpine

RESERPINE

3.1.3

Oxidation Oxidation of reserpine may be induced either photolytically or chemically, and it is usuallglthe major route of degradation for reserpine. Ljungberg found that photo-oxidation of reserpine first produces a yellow substance with yellow-green fluorescence, but that further photolysis gives rise to a reddish brown solution with blue fluorescence (due to lumireserpine) which eventuphly retunes through red and orange to yellow. Banes s. first suggested that the initial oxidation product was 3,4 didehydrore$5rpine, and this was later confirmed by Kzgbs and Futscher. Lunireserpine was shown by Hakkesteegt to be 3,4,5,6-tetradehydroreserpine, a resul which has bee reconfirmed recently by Wright and Tang.65 Hakke~teegt'~ has suggested that some of the non-fluorescent brown and yellow products formed during oxidative degradation of reserpine are polymers. The oxidations of reserpine are also summarized in Figure 6. The oxidation of reserpine is rapid in the presence of 1' ht and oxygen. The oxidation is also catalyzed by acid38 , and by certain metal ions. The efficiency of several metal ions in catalyzing the oxidation of 46 reserpine decreases in the following order: Cu Mn Fe Al.

e.

*

3.2

Degradation of Reserpine in Pharmaceutical Dosage Forms Hydrolysis of4reserpine appears to be very unlikely in solid dosage forms , and there apparently have been no reports in the literature of significant levels of hydrolysis in reserpine izdectables or elixirs. Bayer has reported that reserpine does undergo epimerization in both solution and crystalline pure drug under the influence of heat and light, but did not provide any tqformation on the extent of conversion observed. WeisFogh studied the degradation of a 0.25% reserpine solution at pH 2.5 under the influence of sunlight. After three months at room temperature, he found that the reserpine content had decreased to 70% of initial, and that there was 5% 3,4-didehydroreserpine present. Weis-Fogh felt that the remaining 25% of the reserpine was present largely as 3-isoreserpine, but this conclusion was probably incorrect because his assay method would have measured most of the 0 unable reserpine along with the reserpine. Hakkesteegt3 P was to find 3-isoreserpine in crystalline reserpine or in reserpine tablets from one to seven years old and estimated from his kinetic studies that it would take 26 years for 10% of the reserpine in a pH 3.0 solution to be lost through

-

401

ROGER E. SCHIRMER

cornbivsd hydrolysis and epimerization at 20°C. Wright and Tang found evidence of 3-isoreserpine in several commercial brands of reserpine tablets by TLC, but it may be concluded that epimerization is not likely to be a significant route of degradation for reserpine in most pharmaceutical preparations. Oxidation appears to be the most importag3 route of degradation for reserpine in formulations, Banes examined several reserpine injections, elixirs and tablets. All injections and elixirs examined contained significant levels of oxidation products, whereas only one of the eight lots of tablets exaytned showed any evidence of these products. Haddesteegt found levels of oxidation products as high as 35% in one lot 85 reserpine tablets, and more recently, Wright and Tang found that 3,4-didehydroreserpine was present in all commercially available reserpine tablets that they examined (3,4,5,6-tetradehydroreserpine was not detected in any of them).

4. Metabolism of Reserpine The distribution, excretio~ a - ~ ~ ~ 6 ~ ~ 5 ~ ~ ~ ~ , rat54,63 havZglqp6ftudied ig2the mous89 ,62 ' , monkey , rabbit Y Y gljinea Pig , and Busycon canaliculatum. man The met&43sm of reserpine is qualitatively similar in all species , with oxidative demethylat&;59f6fjhe 4 position of the 3,4,5-trimethoxybenzoyl group &rlgglysis of the trimethoxybenzoate ester linkage 4 9 5 3 y being the major identified routes of metabolism metabolic products therefore4&&$j ,$&y#1 re erpat so Y 3,4,5-trimethoxybenzoic acid and its glucuronide or sulfate Sgnjugates67 syringoyl methyl reserpate54, and syringic acid. Sheppard has made special note of the absence of 3-isoreserpine, reserpine N-oxide, didehydroreserpine, and tetradehydroreserpine in guinea pig brain at doses which produced measurable quantities of unmetabolized reserpine in these tissues. ¶

,?Be

5.

Identification of Reserpine Reserpine can be identified by its NMR, IR, and W spectra and by melting point (See section 2). Reserpine forms a rose-pink color upon addition of about 1 mg. to a few tenths of a 1-2% solution of vanillin in7g"f: the color A green will deepen upon standing or gentle heating. color is produced when 0.5 mg, of reserpine is treated with 5 mg dimethylaminobenzaldehyde, 0.2 ml glacial acetic acid, and 0.2 ml of sulfuric acid: the color will change from green 402

~ ~ ~

RESERPINE

to red upon addition of 1 ml of glacial acetic acid.77 1 mg of reserpine treated with 0.1% solution of sodium molybdate in sulfuric acid produces7;) yellow color which turns to blue If 1 mg of reserpine i s diswithin about two minutes. solved in 0.5 ml of dilute acetic acid and 5 drops of sodium chlorigg test solution are added, a white precipitate forms. A yellow color accompanied by greenish fluorescence develops in a solution of 1 mg reserpine in 5 ml of chloroform upon addition of 5 m17gf a 10% solution of trichloroacetic acid in chloroform. Identi cation of reserpine by fonnatio of crystalline derivatives , formation of eut 80 S€% mixtures , and by use of a variety of other color tests have been reported.

54

6.

Elemental Analysis Carbon 65.12% Hydrogen 6.62% Nitrogen 4.60% Oxygen 23.66%

7. Chromatographic Methods of Analysis 7.1

Thin Layer Chromatography A variety of thin layer systems have -een developed for reserpine and a number of these are summarized in Table 7. Reagents used for detection and identification of reserpine on the plate are summarized in Table 8 . Thin layer systems have also been described in references 102-111. Quantitation of reserpine following thin layer chromatography is described in references 50, 92, 96-101, 104, 107 and ,110-111. 7.2 Paper Chromatography Several paper chromatography systems for reserpine are summarized in Table 10 and methods for visualizing reserpine on the paper are summarized in Table 11. Paper chromatography systems for f57erpine are also discussed in references 119-127. Becker has applied elatography to reserpine, elatography being the technique in which a sample is spotted on the paper, treated with an appropriate reagent, and the reaction products are then separated chromatographically. Quantitation of reserpine by ultraviolet and infrared spectrophotometry, colorimetry, titration, and polarography after isolation by paper chromatography are discussed in references 116 and 125.

403

Table 7 Thin Layer Chromatography Systems for Reserpine Solvent System

P

0

P

Sorbent

Rf

Application and Comnents

1.

CHC13:Me2CO:NH40H, 80:20:1

Silica gel-gypsum plates pretreated at 120°C for 30 minutes

2.

CHC13:Me2C0, 85:15

Alumina

0.60

Separation from other 88, 90, alkaloids of R. serpentina 94

3.

CHC13 :EtOH:Me2C0 ,

Alumina

0.89

Separation from other alkaloids of R. serpentina 88, 90

Silica Gel G

0.72

Separation of Rauwolfia and opium alkaloids

Silica Gel G

0.80

Separation of Rauwolfia alkaloids

90, 91

6. Cyclohexane:CHC13: FT2NH, 5:4:1

Silica Gel G

0.25

Separation of Rauwolfia alkaloids

50, 9092

7.

Alumina G

0.35

Separation of Rauwolfia alkaloids

90, 91

Silica Gel G, 0.1N NaOH impregnated

0.69

Separation of Rauwolfia alkaloids

90, 91

90:5:5 4. CHC13:Me2CO:Et2NH, 5.

5:4:1 CHC13:Et2NH, 9:1

Cyc1ohexane:CHCl3’ 3:7 and 0.05% Et2NH

8. Methanol

Separation from other tranquilizers

Reference 87

89-91

P 0

Solvent System

Sorbent

Rf -

Application and Coments

Reference

9. Heptane:Me-CO-Et, 1:l in atmosphere of annnonia

Cellulose, formamide impregnated

0.59

Separation of Rauwolfia alkaloids

90, 93

10.

Heptane:Me-CO-Et: MeOH, 60:30:10

Whatman SG 41 Silica Gel

0.25

Separation of 10 Rauwolfia alkaloids

95

11.

Heptane:Me-CO-Et: MeOH, 60:30:10

Kieselgel G (Silica Gel)

0.32

Separation of 10 Rauwolfia alkaloids

95

12.

Heptane:Me-CO-Et: MeOH, 60:30:10

Aluminum oxide G

0.63

Separation of 10 Rauwolfia alkaloids

95

13.

Heptane:Me-CO-Et:nBuOH, 60:30:10

Whatman SG 41 (Silica Gel)

0.47

Separation of 10 Rauwolfia alkaloids

95

14. Heptane:Me-CO-Et:n-

Kieselgel G (Silica Gel)

0.52

Separation of 10 Rauwolfia alkaloids

95

15. Heptane:Me-CO-Et:nBuOH, 60:30:10

Aluminum oxide G

0.78

Separation of 10 Rauwolfia alkaloids

95

Whatman SG 41 (Silica Gel)

0.07

Separation of 10 Rauwolfia alkaloids

95

Kieselgel G (Silica Gel)

0.05

Separation of 10 Rauwolfia

95

BuOH, 60:30:10

16.

Heptane:Me-CO-Et: Pyridine, 70:15:15

17. Heptane:Me-CO-Et: Pyridine, 70:15:15

Solvent System

18. Heptane:Me-CO-Et: Pyridine, 7 0 : 1 5 : 1 5

0

Aluminum oxide G

Rf -

Appliaation and C m e n t s

Referenoe

0.90

Separation of 10 Rauwolfia alkaloids

95

Whatman SG 4 1 (Silica 0.64 Gel)

Separation of 10 Rauwolfia alkaloids

95

20. n-BuOH:glacial HOAc:H20, 4 : l : l

Kieselfel G (Silica Gel)

0.75

Separation of 10 Rauwolfia alkaloids

95

21.

n-Bu0H:glacial H0Ac:H 0, 4 : l : l 2

Aluminum oxide G

0.97

Separation of 10 Rauwolfia alkaloids

95

22.

1sooctane:Et 0: xylene:EtOAc2, 45:40:15:5

Silica Gel G

0.00

9 6 , 97 Separation of Rauwolfia alkaloids for quantitative analysis

23. Ethylene dichloride: EtOAc:n-BuOH, 6 0 : 3 0 : 10

Silica Gel G

0.58

Separation of Rauwolfia 9 6 , 97 alkaloids for quantitative analysis

19.

P

Sorbent

Q\

n-Bu0H:glacial HOAc:H20, 4 : l : l

24.

Me C0:Pet. Ether: C C ~:isooctane, 3 5 : $0:20:15

Silica Gel G

0.32

9 6 , 97 Separation of Rauwolfia alkaloids for quantitative ana1ysis

25.

1sooctane:Me C0:nBuOH, 58:33.8: 8.4

Silica Gel G

0.40

Separation of Rauwolfia alkaloids for quantitative analysis

96

Solvent System

0

4

Sorbent

Rf -

Application and Comments

Reference

26. Me C0:Pet. Ether: Silica Gel G 2 glacial HOAc, 45:45:10

0.22

Separation of Rauwolfia 96, 97 alkaloids for quantitative analysis

27. Me C0:MeOH:glacial H d c , 70:25:5

Silica Gel G

0.80

Separation of Rauwolfia 96, 97 alkaloids for quantitative analysis

28.

Pet. Ether:Me CO: Et2NH, 70:2O:fO

Silica Gel G

0.60

Separation of Rauwolfia alkaloids for quantitative analysis

96

29. Me2CO:MeOH:Et2NH, 70:20: 10

Silica Gel G

0.96

Separation of Rauwolfia alkaloids for quantitative analysis

96

30. Me CO:CC14:Pet. Etzer , 45:45:30

Silica Gel G

0.43

Separation of Rauwolfia alkaloids for quantitative analysis

97

31.

H O:EtOH:CHC13, 58:42:2

Cellulose

1.00

Detection of cholinesterase inhibitors at low levels

98

32.

C6H6:EtOH, 9:l

Aluminum oxide

33.

n-Bu0H:Me-CO-Et : H20, 65:25:25

Silica Gel G

Quantitative analysis 0.95

Separation of reserpine from its degradation products

100 34

Solvent System 34.

P

CHCl3:Me2C0, 70:30

Sorbent Silica Gel G

Rf 0.35

Application and Comments Separation of reserpine from other drug substances

Reference 101

Table 8 Visualization of Reserpine on Thin Layer Plates

No.

Result

Reference

1

Iodine Vapor

2

Dragendorff Reagent

3

Acetylchloride, then heat

4

1% Ceric Sulfate in 10% H2S04

greenish brown, turning brown after 5 minutes at 105°C

95

5

Frohde's Reagent (Sulphomolybdic acid)

yellow-brown

95

6

5% Ferric Chloride in 50% HN03

yellow-green turning greenbrown after 5 minutes at 105°C

95

7

Iodoplatinate

pink

95

8

0.5% Phosphomolybdic acid in 50%

yellow-green

96

yellow-green

96

P 0 \o

Treatment

87

brown spot

88 89

m03 9

1% Ammonium Vanadute in 50% HN03

No.

Treatment

Result

10

Spray with source of cholinesterase (human blood plasma suitable), followed by 1 part 0.6% bromthymol blue in 0.1N NaOH and 15 parts aqueous 1% acetylcholine chloride

blue spots on yellow background

98, 99

11

500 mg p-dimethylaminobenzaldehyde in 50 ml conc. H SO

greenish-black spot

101

2 4

Reference

Table 9 Paper Chromatography Systems for Reserpine

No. Solvent Systems

Immobile Phase

Rf -

Application and C m e n t s

Reference

1.

C6H6

50% ethanolic formamide .96

Distinguish reserpine from 112, 116 synthetic precursors and related compounds. Detection lhit, 1 mcg.

2.

C6H6

50% ethanolic fonnami.de .75 containing 5% ammonium formate

Distinguish reserpine from 112 synthetic precursors and related compounds. Detection limit, 1 mcg.

3.

C6H6:C6H12, 1:l

50% ethanolic formamide .61

Distinguish reserpine from 112 synthetic precursors and related compounds. Detection limit, 1 mcg.

4.

C6H6:C6H12, 1:l

50% ethanolic formamide .20 containing 5% amnonium formate

Distinguish reserpine from 112 synthetic precursors and related compounds. Detection limit, 1 mcg.

50% ethanolic formarni.de .OO containing 5% aunnonium formate

Distinguish reserpine from 112 synthetic precursors and related compounds. Detection limit, 1 mcg.

2 c

5 * ‘gH12

No. Solvent Systems

Immobile Phase

6.

Shake isooctane: Me CO: formamide, C H :Formamide, 106:30 6 180:50:5 together, discard lower layer, add 2 parts C H and filter. eatirate chamber with N 3 vapor

7.

C6H6:C6H12, 1:1

Rf

Application and Comments

Reference

.56

Identification of reserpine 113, 114 and related compounds in drug formulations

Formamide:MeOH, 70:30

Estimation of reserpine in 115 Rauwolfia root

8. C6H6:CeHa2, .i:l satura e wi h propylene glycol

Propylene glycol: MeOH;HOAc, 50:50:1

Quantitative analysis of raw materials

9.

Et-CO-Me:Me CO: HCOOH:H20 , $0:2:1:6

Whatman No. 1

.86

117

10. Et-CO-Me:Me2NH:H 0, 2 921:2: 77

Whatman No. 1

.95

117

i-Bu-CO-Me:HCOOH: Whatman No. 1 H20, 10 parts ketone saturated with 1 part 4% formic acid

.44

117

11.

116

e

1J

No. -

Solvent S y s t e m

I m m o b i l e Phase

Rf

12.

CHC13:MeOH:HCOOH: H 0 , 10 p a r t s CHC13 2 saturated w i t h a m i x t u r e of 1 p a r t MeOH and 1 p a r t 4% HCOOH

Whatman No. 1

.91

117

13.

C H :Et-CO-Me:HCOOH: 6 6 H 0 , 9 parts C H6 2 and 1 p a r t Et-6O-Me saturated w i t h 1 p a r t 2% HCOOH

Whatman No. 1

.12

117

14.

C H :HCOOH:H20, 10 pP&s c H satu-

W h a t m a n No. 1

.05

117

A p p l i c a t i o n and C o m m e n t s

Reference

rated w f t f : 1 p a r t 2% f o r m i c acid 15.

A c e t i c A c i d : 5% aqueous s o d i u m acetate, 10: 90

a.

Shake w i t h nWhatman No. 5 4 2 BuOH, added i n paper small portions, u n t i l saturation of t h e aqueous phase i s j u s t achieved

.34

D e t e c t less than 1 m c g

118

No.

Solvent System b.

Immobile Phase

Same as a except Whatman No. 542 replace n-BuOH paper with i-pantanol

16. n-Bu0H:C H6: equal parts 1.5~ NH OH and 1.5N (NH4f2C03, 80:5:15

Whatman No. 1

Rf -

Application and Comments

Reference

.34

Detect less than 1 mcg

118

.90

Radio-assay for reserpine in biological samples

48

Table 10 Visualization of Reserpine on Paper Chromatograms

P VI

Reference

Treatment

Result

1.

ObsCmre under low or high pressure Hg lamp

Strong green fluorescence

112

2.

Observe under low pressure Hg lamp after spraying with .0025% solution of fluorescein in 0.5M annnonia

Strong green

112

3.

Observe under W lamp after spraying with 3% solution of sodium nitroprusside in 50% trichloroacetic acid

Strong green

112

No. -

c

4.

5.

Brown spot, reaction weak Spray with mixture containing 5 ml HC1, 95 ml ethanol, and 1 g pdimethylaminobenzyldehyde, heat briefly at lOO"C, respray with nitroprusside solution (No. 3 above), and heat again at 100°C for 3-5 minutes Dragendorff Reagent (2% potassium bismuth tetraiodide in 0.01N HC1)

112

117

ROGER E . SCHIRMER

7.3 System 1,

Column Chromatography Description

Reference

column: Solka-Floc and Celite 545, 1:l

128

Eluting Solvent: 5N acetic acid nation

Application: Quantitative determipharmaceutical preparations.

-

2. Column: A 200 x 22 m id column packed in 129-131 four layers is used. 1) bottom layer 1 g celite +0.5 ml of fresh 2% NaHC03, 2 ) 1 g celite +0.5 ml of fresh 0.5% citric acid solution, 3) 0.5 g celite +0.5 ml H 0, and 4) 2 1 m g equivalent of reserpine sample and 1 m l dimethylsulfoxide and 2 g celite.

-

Eluting Solvent: Chloroform Application: Quantitative determination of reserpine in formulations. 3. Column: 1) Bottom layer consisting of 113, 114 1.5 g celite 545 and 0.4 ml ethanol and 1 ml 2% NaHCO , and 2) top layer of 10 g celite 545 and 30 ml of a solution prepared by dissolving 1.05 g citric acid in water, diluting to 50 ml with water, and adding 20 ml ethanol. Eluting Solvent: Shake together 100 ml CHC13, 200 ml isooctane, 100 ml water and 40 ml ethanol. Discard aqueous layer and filter. Application: Quantitative determination of reserpine in formulations.

4.

Column: Celite 450 and fonnamide layer 132, 133 resulting from shaking together 15 ml heptane, 110 ml CHC13, 1 ml morpholine, and 25 ml formamide.

Eluting Solvent: CHCl layer obtained as described under "column." 416

RESERPINE

System

Description

Reference

Application: Separation and determination of reserpine, deserpidine, and rescinnamine. 5.

Colnmn: Acid activated aluminum oxide

134

Eluting Solvent: Methanol Application: Separation and quantitation of reserpine in Rauwolfia extracts.

6.

Column: Dowex 50-X-2 cation exchanger

135

Eluting Solvent: Methanol/ammonia,

411

Application: Quantitation of reserpine in formulations.

7. Column: Aluminum oxide equilibrated with CHC13.

136

Eluting Solvent: Ethanol/CHC13 , 1/99 Application: Separation of reserpine from weakly basic alkaloids in R. Serpentina extract. Column: Alumina

8.

137

Eluting Solvent: Gradient elution with benzene containing 0-40% ethanol. Electrophoresis Reserpine has been separated from rescinnamine, reserpic acid and serpentine by electro49fy33s on paper with 5N acetic acid as the electrolyte. Under the conditions employed (8 volts/cm, 1.2 mA, 5 hours) rescinnamine was not completely separated from reserpine. Electrophoretic separations of reserpine from other Rauwolfia alkaloids were also reported in references 6, 121 and 136. 7.4

417

ROGER E. SCHIRMER

7.5

Countercurrent D i s t r i b u t i o n The t h r e e solvent systems described i n Table 7 have been used t o s e p a r a t e r e s e r p i n e from o t h e r Rauwolfia a l k a l y j g s by countercurrent d i s t r i b u t i o n . ‘ Kidd and Scott a l s o reported several solvent systems, but found t h e u s e of d i e t h y l e t h e r : chloroform ( 3 : l ) as t h e mobile phase and a pH 3.1 b u f f e r (16.3 g c i t r i c a c i d and 16.1 g Na HPO *12H 0 i n 1 l i t e r water) a s t h e s t a t i o n a r y phase 2 gave tke be& separation of t h e a l k a l o i d s of R. Serpentina and R. Vomitoria. Other countercurrent systems may be found i n references 140-142. 8.

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

Reserpine has been determined by t i t r a t i o n i n C H C l 3 s o l u t i o n using 0.1N H C l O i n dioxane as t i t r a n t and de4 t e c t i o n of t h e endpy&tl~&th methyl r e d , methyl yellow, o r potentiometrically. ’ T i t r a t i o n s with anionic surf ac t an , y g h - as sodium l a u r y l s u l f a t e , have a l s o been reported Reserpine has been determined t i t r i m e t r i c f&+y i n formul a t i o n s a f t e r an appropriate e x t r a c t i o n . Sun extracted with 0.3N c i t r i c a c i d , alkalyzing with NH OH, back extracting i n t o CHCl drying, taking up i n t o .01N H SO4, 3’ and f i n a l l y back-tifEgting with 0.01N NaOH using met?iyl red i n d i c a t o r . Sakurai e x t r a c t e d r e s e r p i n e with CHCl from i n j e c t i o n s o l u t i o n s a l k a l i n i z e d with NH OH and then 3 4 t i t r a t e d with 0.002N p-toluenesulfonic a c i d dissolved i n ethylene glycol: 2-propanol ( 1 : l ) . Reserpine has a l s o been determined by p r e c i p i t a t i o n as i t s tetraphenylborate s a l t , d i s s o l u t i o n of t h e p r e c i p i t a t e i n acetone,lttd t i t r a t i o n with AgNO using silver electrodes; j q ~ dby micro-Zeisel J e t e m i n a t i o n of i t s s i x methoxy groups.

its

.

9.

Electrochemical Analysis

I n a c i d i c media, a l k a l o i d s containing t h e 6-methoxyindole nucleus undergo a one e l e c t r o n oxidation which probably involves i n s e r t i o n of a hydroxyl group i n t o t h e aromatic portion of t h e molecule. I f t h e molecule contains a v a i l a b l e n i t r o g e n with an unshared p a i r of e l e c t r o n s , a two e l f S i r o n oxidation occurs with formation of a n Noxide. 15$Leserpine does not undergo polarographic reduct ion. 418

RESERPINE

Reserpine has been analyzed coulometrically by reaction with chlorine generated electrochemically from HC1. l'Qe electrochemical coefficient was 0.0007885 mgfma-sec. 10. Spectrophotometric Analysis 10.1

Infrared The infrared absorption of reserpine in the 5.06.5 micron region has beef5tsed for quantitation of this compound in formulations. The band intensity was measured following extraction of the reserpine into chloroform. 10.2

Ultraviolet The ultravio€p6,f~f"~~fP~20f85eserpinecan be but the possiused for quantitation, bility of interference from related alkaloids, excipients, and degradation products requires that the reserpine be isolated from these other substances prior to measurement. The separation from roots and crude pfsga$g5ions has been ; from formy16 accomplished by extensive extraction lations by paper chromatography (System No. 8, Table 9) ; and from crude extracts, raw materials, and formulations by electrophoref$3,~3~paper using 5N acetic acid as the The absorbancels{ the final sample is electrolyte generally measured at 268 nm. Page has reported a semiautomated procedure for reserpine in tablets using a Technicon Auto-Analyze@ system with both ultraviolet and colorimetric detection. Ultraviolet spectrophotometry has also been evaluated for the deteytgation of foreign alkaloids in reserpine preparations.

.

10.3

Colorimetric The most commonly used colorimetric procedures for reserpine involve oxidation of the compound to 3,4didehydroreserpine with nitrite and measurement of the absorbance of the oxidation product at about 390 nm. The reaction is carried out in solutions containing 5-15 pgfml or ethanol acidified with reserpine in sulfuric aci This procedure has been appli 96,160,165 , cry& Rauwolfia preparationsf2lF0 f8??$bt ,ions An automated version of this and animal feeds. assay for determining reserpine in tablets has been

efkbef6.ethanol -

419

ROGER E . SCHIRMER

183 reported by Page. An a l t e r n a t e procedure employs oxidation of reserpine with n i t r i t e inlgrjef& acid followed by ext r a c t i o n i n t o chloroform. The absorbance of t h e chloroform l a y e r i s measured a t 465 nm. This procedure is s u i t a b l e f o r 50-300 pg of r e s e r p i n e , and has t h e advant t h a t hydrolysis products of r e s e r p i n e do not i n t e r f e r e . 887 A v a r i a t i o n of t h i s procedure i n which t h e sample i s t r e a t e d with amyl n i t r i t e r a t h e r than with aceySfj a c i d sodium n i t r i t e reagent has a l s o been reported. Reserpine has a l s o been analyzed c o l o r i r n e t r i c a l l y by reactiof7yiflj2vanillin (absorbafyg of 0.1 a t 532 nm w i t h

-

-

17 pg/ml) , ’ amifqgy~+yidine, plggylisocyanate, xanthydrpS6 (measur iodine,977 50-500 pg a t 500 nm), and sodium glyoxalate FeC13.

-

10.4

Ion-Pair Extraction with Spectrophotometric Detecy {p Booth reported a procedure f o r t h e a n a l y s i s of r e s e r p i n e i n formulations by e x t r a c t i n g t h e r e s e r p i n e i n t o chloroform from pH 4.0 phosphate b u f f e r as an ion-pair w i t h bromcresol purple. A f i n a l s o l u t i o n concentration of 2.8 pg/ml gave an absorbance of 0.188 a t 402 nm. Pro181 cedures employing bromcresol green and methyl orange i n place of bromcresol purple have a l s o been reported. 10.5

Fluorescence Since 3,4-didehydroreserpine i s s t r o n g l y f l u o rescent (Section 2.2.3), t h e s e n s i t i v i t y of t h e c o l o r i metric methods employing oxidation of r e s e r p i n e t o t h i s product (Section 10.3) can be increased by using f l u o r i metric detection. N i t r i t e oxidation and f l u o r i m e t r i f 84 determination have been used f o r a n a l y s i s of t a b h?f-9188 f o r feeds containing r e s e r p i n e a t t h e ppm level. automated s i n g l e - t a P & p l g a s a y based on t h i s procedure has a l s o been reported. Reagents o t h e r than n i t r i t e have been us @i8f?90fsyelop f luorescencf93fp$tuding hydrogen peroxide, sg+zpious a c i d , pgpiygnesulfonic acid i n a c e t i c and vanadium penta c i d , 2 0 0 ~ y ~ ~ v a n a adcii cd , oxide. The vanadium pentoxide p r o c ~ $ ~ ~ ~ Obeen ljas automated f o r use i n s i n g l e t a b l e t a s s a y s , The selenious acid procedure has beey9ysyd4to determine r e s e r p i n e i n b i o l o g i c a l samples.

’

420

RESERPINE

Kollistrat~h s a~s ~r e~p o r t e d t h a t ThCl g r e a t l y enhances t h e f l u o r e s c e n c e of r e s e r p i n e i n e t h a n o t , methanol, a c e t o n e , and dioxane, b u t d i d not determine t h e mechanism r e s p o n s i b l e f o r t h e enhancement.

11. Analysis of Reserpine i n B i o l o g i c a l Systems Reserpine levels hayGgbeen deteyg&ned f l u o r h e t r i c a l l y a d j u s t e d t h e pH of i n t i s s u e samples. Poet and Hess samples t o 8.5 w i t h b o r a t e b u f f e r , e x t r a c t e d w i t h heptane o r petroleum e t h e r , back-extracted i n t o d i l u t e s u l f u r i c a c i d and f i n a l l y developed f l u o r e s s 5 n c e u s i n g t h g 4 s e l e n i o u s a c i d o x i d a t i o n procedure, Glaszko and Maronde adjusted t h e pH o f plasma and u r i n e samples t o 3.7-4.0 w i t h c i t r a t e b u f f e r , e x t r a c t e d w i t h e t h y l e n e d i c h l o r i d e , and developed f l u o r e s c e n c e w i t h odium n i t r o p r u s s i d e followed by hydrogen peroxide. Zsoter” analyzed p l f A a and u r i n e samples u s i n g a m o d i f i c a t i o n of J a k o v l j e v i c ‘ s f l u o r i m e t r i c procedure. The most widely used methods f o r detygminins r e s e r p i n e i n t i s s u e s a r e t h o s e employing C o r H labeled r e s e r p i n e , S p e c i f i c i t y i s obtained he r a d i o a c t i v e 53’65 t h i n l a y e r chromad e t ermina , ~ i r , ~ t j t g s , g 9 5 t ;o~r ~paper ~ ~ ~ chromatogra~2 togre”Xg’54965’66 * PhY Other procedures may b e found i n t h e r e f e r e n c e s c i t e d i n S e c t i o n 4. The b i o a v a i l a b i l i t y of r e s e r p i n e a d m i n i s t e r e d o r a l l y as a c o p r e c i p i t a t e w i t h p o l y v i n y l p y r r o l i d o n e o r w i t h b i l e a c i d s has been determined using t h e b l e p h a r o p t o t 3655% P i t y of t h e p r e p a r a t i o n a s a measure of a v a i l a b i l i t y . 212 P t o s i s w a s r a t e d using t h e s c a l e devised by Rubin g . &. These s t u d i e s included examination of t h e d i s s o l u t i o n rates of t h e c o p r e c i p i t a t e s and t h e r e l a t i o n s h i p between d i s s o l u t i o n rate and b l e p h a r o p t o t i c a c t i v i t y .

%a!:i!

42 1

ROGER E. SCHIRMER

References 1. E. Schlittler, Rauwolfia Alkaloids with Special Reference to the Chemistry of Reserpine, In "The Alkaloids" (R.H.F. Manske, ed.), Vol. VIII, Academic Press, New York, 1965. 2. R.B. Woodward, F.E. Bader, H. Bickel, A.J. Frey, and R.W. Kierstead, J. Am. Chem. SOC. 78, 2023, 2657 (1956). 3. R.B. Woodward, F.E. Bader, H. Bickel, A.J. Frey, and R.W. Kierstead, Tetrahedron 2, 1 (1958). 4. L. Velluz, B. Muller, R. Joly, G. Nomine, A. Allais, J. Warnant, R. Bucourt, and J. Jolly, Bull. SOC. Chim. France p. 145 (1958) 5. L. Velluz, G. Muller, R. Joly, G. Nomine, J. Mathieu, A. Allais, J. Warnant, J. Valls, R. Bucourt, and J. Jolly, Bull. SOC. Chim. France p. 673 (1958). 6. F.A. Hochstein, K. Murai, and W. H. Boegemann, J. Am. Chem. SOC. 77, 3551 (1955). 7. "The Merck Index," 8th Edition, Merck & Co., Inc., Rahway, New Jersey (1968). 8. W.E. Rosen and J.N. Shoolery, J. Am. Chem. SOC. 83, 4816 (1961). 9. H.B. MacPhillamy, C.F. Huebner, E. Schlittler, A.F. St. Andre, and P.R. Ulshafer, J. Am. Chem. SOC. 77, 4335 (1955). 10. M.M. Robison, R.A. Lucas, H.B. MacPhillamy, R.L. Dziemian, I. Hsu, R.J. Kiesel, and M.J. Morris, Abstr. Papers Am. Chem. SOC. 139th Meeting, St. Louis, Missouri, 1961, p . 3N. 11. W.E. Rosen and-J: M. O'Connor, J. Org. Chem. 26, 3051 (1961). 12. J.R. Platt, J. Chem. Phys. l7, 484 (1949). 13. J.R. Platt, J. Chem. Phys. l9, 101 (1951). 14. "Physical Data of Indole and Dihydroindole Alkaloids," Eli Lilly and Co., Indianapolis, Indiana (1964). 15. G. Barth, R.E. Linder, E. Bunnenberg, and C . Djerassi, Helv. Chim. Acta 55, 2168 (1972). 16. W.H. McMullen, H.J. Pazdera, S.R. Missan, L.L. Ciaccio, and T.C. Grenfell, J. Am. Pharm. Assoc. 44, 446 (1955). 17. S. Udenfriend, D. Duggan, B. Vasta, and B. Brodie, J . Pharmacol. Exptl. Therap. 120, 26 (1957). 18. G.G. Guilbault, "Practical Fluorescence," p. 325, Marcel Dekker, Inc., New York (1973). 19. R.P. Haycock, P.B. Sheth, and W.J. Mader, J. Am. Pharm. Assoc. 4.8, 479 (1959). 20. R.D. Rauh, T.R. Evans, and P.A. Leermakers, J. Am. 422

RESERPINE

Chem. SOC. 90, 6897 (1968). 21. R.D. Rauh, T.R. Evans, and P.A. Leermakers, J. Am. Chem. SOC. 9 l , 1868 (1969). 22. D.L. Dexter, J. Chem. Phys. 21, 836 (1953). 23. T. Forster, Disc. Faraday SOC. 271, 1 (1959). 24. C. Djerassi, M. Gorman, A.L. Nussbaum, and J. Reynoso, J. Am. Chem. SOC. 75, 5446 (1953). J.M. 25. A. Furlenmeier, R. Lucas, H.B:MacPhillamy, Mueller, and E. Schlittler, Experentia 2, 331 (1953). 26. L. Dorfman, A. Furlenmeier, C.F. Huebner, R. Lucas, H.B. MacPhillamy, J.M. Mueller, E. Schlittler, R. Schwyer, and A.F. St. Andre, Helv. Chim. Acta 37, 59 (1954). 27. N. Neuss, H.E. Boaz, and J.W. Forbes, J. Am. Chem. SOC. 76, 2463 (1954). 28. W.E. Rosen, Tet. Letters p. 481 (1961). 29. P. Baudet, C1. Otten, and E. Cherbuliez, Helv. Chim. Acta 47, 2430 (1964). 30. E. Wenkert and D.K. Roychaudhuri, J. Am. Chem. SOC. 78, 6417 (1956). 31. H.A. Rose, Anal. Chem. 26, 1245 (1954). 32. W.C. Hamilton, Sc. 169, 135 (1970). 33. M. Langejan and H.F.L. Liefferink, Pharm. Weekblad 91, 847 (1956). 34. Th.J. Hakkesteegt, Pharm. Weekblad 105,829 (1970). 35. M. Kaern and M. Tbnnesen, Farm. Revy. 57, 553 (1958). 36. E. Wenkert and L.H. Liu, Experentia ll, 302 (1955). 37. H.B. MacPhillamy, L. Dorfman, C.F. Huebner, E. Schlittler, and A.F. St. Andre, J. Am. Chem. SOC. 77, 1071 (1955). 38. Th.J. Hakkesteegt, Pharm. Weekblad 105,801 (1970. 39. A.J. Gaskell and J.A. Joule, Tetrahedron 23, 4053 (1967). 40. J. Bayer, Pharmazie 3, 468 (1958). 41. F. Ljungberg, Farm. Tidskr., 62, 693 (1958). 42. D. Banes, J. Wolff, H.O. Fallscheer, and J. Carol, J. Am. Pharm. Assoc. 45, 710 (1956). 43. K.G. Krebs .and N. Futscher, Arzneimittel-Forsch. l0, 75 (1960). 44. Th.J. Hakkesteegt, Pharm. Weekblad 103,297 (1968). 45. E. Wright and T.Y. Yang, J. Pharm. Sc. 6 l , 299 (1972). 46. H. Potter and R. Voigt, Pharmazie 22, 436 (1967). 47. 0. Weis-Fogh, Pharm. Acta Helv. 35, 442 (1960). 48. P. Numerof, M. Gordon, and J. M. Kelley, J. Pharmacol. Exptl. Therap. 115, 427 (1955). 49. H. Sheppard and W.H. Tsien, Proc. SOC. Exptl. Biol. Med. 90, 437 (1955). 423

ROGER E. SCHIRMER

50. R.E. Stitzel, L.A. Wagner, and R.J. Stawarz, J. Pharmacol. Exptl. Therap. 182, 500 (1972) .3 51. L.A. Wagner, Subcellular distribution of [ HIreserpine, Diss. Abstr. Int. B 2870 (1970). 52. A.J. Glazko, W.A. Dill, and L.M. Wolf, J. Pharmacol. Exptl. Therap. 118,377 (1956). 53. H. Sheppard, R.C. Lucas, and W.H. Tsien, Arch. internat. pharmacodyn. 103, 256 (1955). 54. H. Sheppard, W.H. Tsien, E.B. Sigg, R.A. Lucas, and A.J. P l m e r , Arch. internat. pharmacodyn. 113,160 (1957). 55. R.J. Stawarz, Factors influencing the microsomal metabo lism of tritium-labelled reserpine in vitro. Diss. Abstr. Int. B 33, 5990 (1973). 56. L. Manara, P. Carminati, and T. Mennini, Eru. J. Pharmacol. 20, 109 (1972). 57. S.J. Enna and P.A. Shore, Biochem. Pharmacol. 20, 2910 (1971). 58. L. Manara and S . Garattini, Eru. J. Pharmacol. 2, 139 (1967). 59. L. Manara, T. Mennini, and P. Carminati, Eur. J. Pharmacol. l7, 183 (1972). 60. L.A. Wagner and R.E. Stitzel, J. Pharm. Pharmacol. 24, 396 (1972). 61. E.A. DeFelice, Experentia 13,373 (1957). 62. S.M. Hess, P.A. Shore, and B.A. Brodie, J. Pharmacol. Exptl. Therap. 118,84 (1956). 63. H. Sheppard, W.H. Tsien, A.J. Plunrmer, E.A. Peets, B.J. Giletti, and R.A. Schulert, Proc. SOC. Exptl. Biol. Med. 9 7 , 717 (1958). 64. R.F. Maronde, J. Haywood, D. Feinstein, and C. Sobel, J. Am. Med. Assoc. 184,7 (1963). 65. P. Numerof, A.J. Virgona, E.H. Cranswick, T. Cunningham, and N . S . Kline, Psychiat. Res. Rep., Wash. 9, 139 (1958). 66. A.R. Maass, B. Jenkins, Y. Shen, and P. Tannenbaum, . l, 366 (1969). Clin. Pharmacol. Ther. 0 67. T.T. Zsoter, G.E. Johnson, G.A. Deveber, and H. Paul, Clin. Pharmacol. Ther. 14, 325 (1973). 68. T.K. Adler and M.E. Latham, Proc. SOC. Exptl. Biol. Med. 73, 401 (1950). 69. L.A. Wagner and R.E. Stitzel, J. Pharm. Pharmacol. 21, 875 (1969). 70. G.F. Placidi, G. Ciccone, A. Moschino, E. Gliozzi, and G.B. Cassano, J. Nucl. Biol. Med. 16,32 (1972). 71. M.M. Dhar, J.D. Kohli, and S.K. Srivastava, J. Sc. Ind. Research (India) 179 (1955).

=,

x,

424

RESE RPlNE

72. M.M. Dhar, J.D. Kohli, and S.K. Srivastava, Indian J. Pharm. l8, 293 (1956). 73. J.D. Kohli, M.M. Dhar, and S.K. Srivastava, J. Sci. 167 (1956). Ind. Research (India) 74. S.K. Srivistava, M.M. Dhar, and J.D. Kohli, J. Sci. Ind. Research (India) 73 (1957). 75. M. Mirolli, Sc. 149, 1503 (1965). 76. The United States Pharmacopoeia XVIII. 77. British Pharmacopoeia 1973. 78. The Pharmacopoeia of Japan, 7th Edition, Part 1 (1961). 79. C. Korczak-Fabierkiewicz and G.H.W. Lucas, Proc. Can. SOC. Forensic Sci. 4, 233 (1965). 80. M. Brandstaetter-Kuhnert, A. Kofler, R. Hoffman, and H. C. Rhi, Sci. Phann. 33, 205 (1965). 81. H. Kaneko, Chem. Phann. Bull. (Tokyo) 5, 318 (1958). 82. A. Hofmann, Helv. Chim. Acta 37, 314, 849 (1954). 83. R. Cortesi and H. Laubie, Bull. SOC. pharm. Bordeaux 93, 116 (1955). 84. R. San-Martin-Casamada,Rev. Real Acad. Farm. Barcelona, No. 12, 1 (1968). 85. R. Bonino, Rev. assoc. bioquim. Argentina 20, 229 (1955). 86. S . Cheng, J. Chinese Chem. SOC. (Taiwan) Ser I1 4, 112 (1957). 87. T. Fuwa, T. Kido, and H. Tanaka, Yakuzaigaku 25, 138 (1965). 88. M. Ikram, G.A. Miana, and M. Islam, J. Chromatog. 11, 260 (1963). 89. A. Kaess and C. Mathis, Ann. Pharm. Franc 23, 739 (1965). 90. E. Stahl, "Thin-Layer Chromatography: A Laboratory Handbook," p . 448. Springer-Verlag, New York (1969). 91. D. Waldi, K. Schnackerz, and F. Munter, J. Chromatog. 6, 61 (1961). 92. L. Manara, Eur. J. Pharmacol. 2, 136 (1967). 93. K. Teichert, E. Mutschler, and H. Rochelmeyer, Z. anal. Chem. 181,325 (1961). 94. M. Ikram and M.K. Bakhsh, Anal. Chem. 36, 111 (1964). 95. W.E. Court, Can. J. Pharm. Sc. 1, 76 (1966). 96. W.E. Court and M.S. Habib, J. Chromatog. 80, 101 (1973). 97. M.S. Habib and W.E. Court, Can. J. Pharm. Sc. 8 , 81 (1973). 98. J.J. Menn and J.B. Bain, Nature 209, 1351 (1966). 99. M.E. Getz and S.F. Friedman, J. Assoc. Offic. Agr. Chem. 4 6 , 707 (1963). 100. N.A. Mirzazade, Azerb. Med. Zh. 47, 26 (1970): Chem.

my my

425

ROGER E. SCHIRMER

Abstr. 73, 38588t (1970). 101. W.J. Weaver, J. Phann. Sc. 55, 1111 (1966). 102. M.F. Bartlett, B.F. Lambert, H.M. Werblood, and W.I. Taylor, Jr., J. Am. Chem. SOC. 85,475 (1963). 103, J. Baumler, and S. Rippstein, Pharm. Acta Helv. 36, 382 (1961). 104. Seyson, Mottet, Crispen, Hennau, Gloesener, Dvred, Vanhaelen, Louvet, Toth, Stainier, J. Phann. Belg. 26, 292 (1971): Chem. Abstr. 75, 10133OM (1971). 105. R.R. Paris, R. Rousselet, M. Paris, and J. Fries, Ann. Pharm. Franc 23, 473 (1965). 106. D. Giacopello, J. Chromatog. l9, 172 (1965). 107. M.S. Habib and W.E. Court, J. Pharm. Pharmacol. 23, 230s (1971) 108. M. Sahli, Arzneimittel-Forsch. l2, 55 (1962). 109. M. Sahli, Arzneimittel-Forsch. l2, 155 (1962). 110. F. Schlemmer and E. Link, Phann. Ztg. 104,1349 (1959). 111. E. Ullman and H. Kassalitzky, Arch. Pharm. 295, 37 (1962). 112. K. Macek and S. Vanecek, Coll. Czechoslov. Chem. Commun. 26, 2705 (1961) 113. D. Banes, J. Carol, and J. Wolff, J. Am. Pharm. Assoc. 44, 640 (1955). 114. J. Carol, D. Banes, J. Wolff, and H.O. Fallscheer, J. Am. Pharm. Assoc. 45, 200 (1956). 115. B.P. Korzun, A.F. St. Andre, and P.R. Ulshafer, J. Am. Pharm. Assoc. Sc. Ed. 46, 720 (1957). 116. H.J. Pazdera, W.H. McMullen, L.L. Ciaccio, S.R. Missan, and T.C. Grenfell, Anal. Chem. 29, 1649 (1957). 117. L. Reio, J. Chromatog. 3, 458 (1960). 118, R.J. Boscott and A.B. Kar, Nature 176,1077 (1955). 119. J. Bayer, Ceskoslov. farm. 9 , 396 (1960): Chem. Abstr. 55, 9791f (1961). 120, F. Kaiser and A . Popelak, Ber. 92, 278 (1959). 121. H. Kaneko, Yakugaku Zusshi 2,512 (1958): Chem. Abstr. 52, 13190e (1958). 122. E B . Kar and R.J. Boscott, Indian J. Pharm. 18,204 (1956). 123. K. Macek, J. Hacoperkova, and B. Kakac, Phannazie ll, 533 (1956). 124. F. Machovicova, Ceskoslov. farm. 5, 310 (1957). 125. N.Y. Tsarenko, V.P. Georgievskii, and M.S. Shraiber, Aptechn. Delo, 14, 49 (1965): Chem. Abstr. 64, 5257d (1966). 126. E. Vidic and J. Schuette, Arch. Pharm. 295, 342 (1962). 127. A. Becker, Z. anal. Chem. 174,161 (1960). 128. W.F. Bartelt and E.E. Hamlow, J. Am. Pharm. Assoc. 44, 426

RESERPINE

129. 130.

131. 132. 133. 134. 135. 136.

137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153.

660 (1955). S. Barkan, J. A s s . O f f i c . A n a l . Chem. 5 2 , 113 (1969). F.M. Kunze and S. Barkan, J. Assoc. O f f i c . Anal. Chem. 51, 1324 (1968). E L . Dow and R.C. Grant, J. Assoc. O f f i c . A n a l . Chem. 53, 1106 (1970). E L . Hayden, L.A. Ford, and A.E.H. Houk, J. Am. Pharm. ASSOC. 47, 157 (1958). D. Banes, A.E.H. Houk, and J. Wolff, J. Am. Pharm. Assoc. 47, 625 (1958). R. K l a u s a n d G. Weber, J . Chromatog. 48, 446 (1970). E . H a b e r l i and E. Beguin, Pharm. Acta Helv. 34, 65 (1959). K. Yamaguchi, H. S h o j i , and M. I t o , Eisei Shikenjo J (1958): Chem A b s t r . 53, 17418e Hokoku No. 76, 8 (1974). E.E. van Tamelen and C.W. T a y l o r , J. Am. Chem. SOC. 2, 5256 (1957). M. S a h l i , Pharm. Acta Helv. 33, 1 (1958). D.A.A. Kidd and P.G.W. S c o t t , Pharm. Pharmacol. 2, 176 (1957). Banerjee and Hausler, B u l l , C a l c u t t a School Trop. Med. -3 , 111 (1955). C. Djerassi, M. Gonnan, A.L. Nussbaum, and I. Reynoso, J. Am. Chem. SOC. 76, 4463 (1954). M.W. Klohs, M.D. Draper, F. Keller, and W. Malesh, Chem. & Ind. 1264 (1954). R. V a s i l i e v , V. S c i n t e e , and M. Mangu, F a r a c i a Bucharest) 2, 239 (1961). R. Vasiliev, V. S c i n t e i e , and M. Mangu, Rev. Chim. (Bucharest) l l , 180 (1960). I.M. Roushdi and R.M. S h a f i k , J. Phann. SC. U.A.R. 2, 65 (1968). F. P e l l e r i n , 3.A. G a u t i e r , and D. Demay, Ann. Pharm. Franc 22, 495 (1964). S. Cheng, Chemistry (Taiwan), 213 (1957). H. S a k u r a i , Y. Yoneda, and H. Machida, Takamine Kenkyusho Nempo l0, 154 (1958). C.W.R. Phaf, Pharm. Weekblad 2, 517 (1960). E. Kahane and M. Kahane, Ann. pharm. f r a n c . 16,726 (1958). M.J. A l l e n and V.J. Powell, J. Electrochm. SOC. 105, 541 (1958). L. Hruban, A. Nemeckova, and F. Santavy, Acta Univ. Palackianae Olomucensis 1 6 , 5 (1958): Chem. A b s t r . 54, 12490f (1960). A.E. Kalinowska and J. Bartnik-Kurzawinska, Acta Polon.

427

ROGER E . SCHIRMER

Pharm. l9, 45 (1962). 154. W.R. Maynard, Jr., J. Assoc. Offic. Anal. Chem. 41, 676 (1958). 155. E.H. Sakal and E.J. Merrill, J. Am. Phann. Assoc. 43, 709 (1954). 156. D. Banes and J. Carol, J. Assoc. Offic. Agr. Chem. 38, 866 (1955). 157. B.C. Bose and R. Vijay vargirja, J. Pharm. Pharmacol. 11, 456 (1959). 158. K A . Mannell and M.G. Allmark, Drug Standards 24, 5 (1956). 159. D. Banes, J. Assoc. Offic. Agr. Chem. 41, 488 (1958). 160. C.R. Szalkowski and W.J. Madar, J. Am. Pharm. Assoc. 45, 613 (1956). 161. KYa. Tsarenko and M.S. Shraiber, Farmatsevi. Zh. (Kiev) l9, 34 (1964): Chem. Abstr. 63, 6861c (1964). 162. Analyst 85, 755 (1960). 163. R.P. Haycock and W.J. Mader, J. Am. Pharm. Assoc. 46, 744 (1957). 164. R.P. Haycock, P.B. Sheth, R.J. Connolly and W.J. Mader, J. Assoc. Offic. Agr. Chemists 42, 613 (1959). 165. D. Banes, J. Assoc. Offic. Agr. Chemists 40, 798 (1957). 166. D. Banes, J. Wolff, H.O. Fallscheer and J. Carol, J. Am. Pharm. Assoc. 45, 708 (1956). 167. A.W,M. Indemans, I.M. Jakovljevic, and J.J.A.M. v.d. Langerijt, Pharm. Weekblad 94, 1 (1959). 168. Z. Nawakowska, I. Wilczynska and W. Zyzynski, Acta Polon. Pharm 21, 161 (1964). 169. A.W.M. Indemans, Pharm. Weekblad 108, 641 (1973). 170. V. Scarselli, Boll, SOC. ital. biol. sper. 34, 1132 (1958). 171. D. Banes, J. Am. Pharm. Assoc. 44, 408 (1955). 172. D. Banes, J. Assoc. Offic. Agr. Chemists 39, 620 (1956). 173. H. Wachsmuth and L. van Koeckhoven, J. Pharm. Belg. 18, 378 (1963). 174. Z. Jung, Ceskoslov. farm. 5, 299 (1957). 175. N. Nakamura and T. Yoshida, Takeda Kenkyusho Nempo l8, 25 (1959). 176. J. Bartos, Ann. Pharm. Franc. l.9, 610 (1961). 177. J.E. Gardner and S.J. Dean, Drug Standards 28, 50 (1960). 178. H.P. Rieder and M. Bohmer, Helv. Chim. Acta 42, 1793 (1959). 179. R.E. Booth, J. Am. Pharm. Assoc. 44, 568 (1955). 180. H.-S. Chou, C.-Y. Ku, C.-L. Chin, C.-Y. Ch'en, C.-L. 42 8

RESERPINE

181. 182. 183. 184. 185. 186. 187. 188. 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208.

Hu, S.-L. Lo, and H.-S. Chao, Yao Hsueh Hsueh Pao l3, 280 (1966): Chem. A b s t r . 65, 8673a (1966). E.A. DeFelice, Experentia 14, 159 (1958). D. Banes, J. Assoc. O f f i c . Agr. Chemists 40, 796 (1957). D.P. Page, J. Assoc. O f f i c . Anal. Chemists 53, 815 (1970). D. Banes, J . Am. Pharm. Assoc. 4 6 , 601 (1957). F. T i s h l e r , P.B. Sheth, and M.B. Giaimo, J . Assoc. Offic. Agr. Chemists 46, 448 (1963). W . J . Mader, R.P. Haycock, P.B. Sheth, and R.J. Connolly, J. Assoc. O f f i c . Agr. Chemists 43, 291 (1960). W.J. Mader, R.P. Haycock, P.B. Sheth, R.J. Connolly, and P.M. Shapoe, J. Assoc. O f f i c . Agr. Chemists 44, 13 (1961). W . J . Mader, G . J . P a p a r i e l l o , and P.B. Sheth, J. Assoc. O f f i c . Agr. Chemists 45, 589 (1962). B.N. Kabadi, A.T. Warren, and C.H. Newman, J . Pharm. Sc. 58, 1127 (1969). B.N.Kabadi, J. Pharm. Sc. 60, 1862 (1971). E.B. Dechene, J. Am. Pharm. Assoc. 44, 657 (1955). E.B. Dechene, J. Am. Pharm. Assoc. 47, 757 (1958). R.B. Poet and J.M. K e l l y , Abstr. 126th Meeting Amer. Chem. SOC., p. 83c (1954). S.M. Hess, P.A. Shore, and B.B. Brodie, J. Pharmacol. Exptl. Therap. 118, 84 (1956). I . M . J a k o v l j e v i c , J.M. Fose, and N.R. Kuzel, Anal. Chem. 34, 410 (1962). R. S t a i n i e r , Fannaco 24, 167 (1968). Pharmacopoeia Belgique I V . R. S t a i n i e r , J. Pharm. Belg. 28, 115 (1973). R. S t a i n i e r , H.P. Husson, and C.L. Lapiere, J . Pharm. Belg. 28, 307 (1973). T. Urbanyi and A. O'Connell, A n a l . Chem. 44, 565 (1972). S. Barkan, J . Assoc. O f f i c . Anal, Chemists 55, 149 (1972). T . Urbanyi and H. S t o b e r , J. Assoc. O f f i c . Anal. Chemists 55, 180 (1972). T. Urbanyi and H. S t o b e r , J. Pharm. Sc. 2,1824 (1970). G. K a l l i s t r a t o s , J. Less-Common Metals 29, 226 (1973). M.H. Malone, H . I . Hochman, and K.A. N i e f o r t h , J. Pharm. Sc. 55, 972 (1966). M. G i b a l d i , S. Feldman, and T.R. Bates, J . Pharm. Sc. 57, 708 (1968). YR. Bates, 3. Pharm. Pharmacol 2,710 (1969). R.G. S t o l l , T.R. Bates, K.A. N i e f o r t h , and J.

429

ROGER E . SCHIRMER

209. 210. 211. 212.

Swarbrick, J . Pharm. S c . 58, 1457 (1969). L. Decato, J r . , M.H. Malone, R . G . S t o l l , and K . A . N i e f o r t h , J . Pharm. S c . 58, 273 (1969). L. Decato, J r . , R.G. Stall, T . R . Bates and M.H. Malone, J. Pharm. S c . 60, 140 (1971). E.I. Stupak and T . R . B a t e s , J. Pharm. S c . 61, 400 (1972). B . Rubin, M.H. Malone, M . H . Waugh, and J.C. Burke, J. Pharmacol. Exptl. Therap. 120, 125 (1957).

430

SPIRONOLACTONE

John L . Sutter and Edward P. K. Lau

JOHN L. SUTTER AND EDWARD P. K . LAU

Contents 1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor 2. Physical Properties

2.1 Infrared Spectm 2.2 Nuclear Magnetic Resonance Spectm 2 . 3 Ultraviolet Spectm 2.4 Mass Spectm 2.5 Optical Rotation 2.6 Melting Range 2 . 7 Differential Scanning Calorimetry 2.8 Solubility 3. Synthesis

4. Stability and Degradation 5. Drug Metabolic Products and Pharmacokinetics 6. Methods of Analysis 6.1 6.2 6.3 6.4 6.5 6.6

Partition Coefficient Phase Solubility Spectrophotometric Analysis Colorimetric Analysis Fluorometric Analysis Chromatographic Analysis 6.61 High Pressure Liquid Chromatography 6.62 Thin Layer Chromatography

7. Acknow1edgements

8. References

432

SPI RONOLACTONE

1. Description 1.1 Name, Formula, Molecular Weight Spironolactone is 17-hydroxy-7a-mercapto-3-oxo17a-prep-4-ene21-carboxylic acid y- lactone 7acetate,

C24H3204S

Molecular Weight: 416.59

1.2 Appearance, Color, Odor Spironolactone is a yellowish-white crystalline powder, with a faint mercaptan odor. 2. Phvsical Prouerties

2.1 Infrared Spectrum The infrared absorption spectrum of a spirono-

lactone reference standard compressed in a KBr disc is shown in Figure 1. Essentially the same infrared spectrum is observed in chloroform solution. The following assignments have been made for absorption bands in Figure 1: Assignment

Wavenumber, an1775 1670 - 1690

5-membered lactone carbonyl 3-ketone, 7-thioester carbony1 4,5 - double bond

1620 433

P W A

Figl. I n f r a r e d Spectrum of S p i r o n o l a c t o n e

SPIRONOLACTONE

2.2

Nuclear Mametic Resonance Smctrwn The NMR spectrum of a spironolactone reference standard i n deuterated chloroform is shown i n Figure 2. Spectral assignments are as follows:

Chemical Shift (PPm)

2.3

Type

Assignment

5.7 4.0 2.4

Broad quartet Broad s i n g l e t Singlet

1.2 1.0

Singlet Singlet

4 - H 7 - H 7-thioacetate protons 19-methyl protons 18-methyl protons

Ultraviolet Spectrum The u l t r a v i o l e t absorption spectrum of a spirono lactone reference standard i n methanol is shown i n Figure 3. The molar absorptivity of spironolactone i n methanol i s 19.6 X l o 3 a t the absorbance maximum a t about 238 nm.

2.4

Mass S D e c t m The low resolution m a s s spectrum of spironolactone shown i n Figure 4 was obtained with an AEI Model Ms-30 mass spectrometer. A molecular ion was observed a t m/e 416. The base peak i n the spectrum was a t m/e 341, corresponding t o loss of .SCOCH3. Structure assignments are as follows :

m/e

Assignment

43 26 7

CH3CO+ 340 - Lactone ring 340 - methyl W - HSCOCH3 M? - .SCOCH3 IvP - (CH2CO+CH3)

325 340 341 359

435

% Relative Intensity

89 26 12 43 100 20

P

w

Q\

Fig. 2

N u c l e a r Magnetic Resonance Spectrum o f S p i r o n o l a c t o n e

Fig. 3 Ultraviolet Spectrum of Spironolactone

Fig. 4 Mass Spectrum of Spironolactone

SPI RONOLACTONE

M-f - (CH co) M-f - (H26 + CH3) M-f

374 383 416 2.5

36 1 0.4

Optical Rotation The following s p e c i f i c rotation values i n chloroform' have been reported f o r spironolactone : [a]&

=

-403.8'

CCt1;g6

=

-110.00

c.15';~

=

-

45.8O

- 38.8' [~ti;;~ =

C ~L Ji . -1 ~ -~ 36.3' ~ 2.6

Melting Range The USP l a melting range of spironolactone is 1980 - 2070C.* A reference standard l o t of the compound was found t o melt from 205.40 t o 206.90C. Melting and r e s o l i d i f i c a t i o n a t lower temperatures is sometimes noted.

2.7

Differential Scanning Calorimetry The DSC thermogram of a spironolactone reference standard c r y s t a l l i z e d from ethanol and water shown i n Figure 5 was obtained on a Perkin-Elmer DSC-1B d i f f e r e n t i a l scanning calorimeter a t a heating rate of 2OoC/minute i n an atmosphere of nitrogen. A s l i g h t exothermic peak was observed beginning a t about 160OC, and an endothermic melting peak was found a t about 210°C.3 Spironolactone chemical c r y s t a l l i z e d from d i f f e r e n t solvents can y i e l d d i f f e r e n t thermograms. Samples c r y s t a l l i z e d from methanol have 439

I

P P

0

I

I

140

150

160

170

1

1

180 190 TETPERATURE, O C

Fig. 5 DSC Thermogram of Spironolactone

I

I

I

200

210

220

SPI RONOLACTONE

been shown to give thennograms with two transition peaks: an endotherm at about 120°C and an exothenn at about 14OOC. These transition geaks can be removed by heating the sample at 105 C or refluxing in water for four hours. A sample crystallized from n-propanol, however, gave only one endothermic peak at 2100C. Thermograms of spironolactone crystallized from ethyl acetate have shown two transition peaks: one endotherm at about llOOC and an another at about 207OC. This behavior suggests that spironolactone can exist in a number of polymorphic f o m . 2.8

Solubility Solubilities of spironolactone in various solvents at 25OC are given in the following table:4 Solvent

Solubility, mg./ml.

Water

2 . 8 x 10-2

Methanol

6.9

Ethanol (USP)

27.9

Chloroform

50 2.4 x 10-1

Heptane 3. Synthesis

The synthetic route to spironolactone shown in Figure 6 has been reported in papers by Cella, Brown and Burtner', and Cella and Tweit. Carbonation of the Grignard reagent of l7a-ethynyl-5androstene-38,178-diol (I) yielded an acetylenic acid (11). Selective reduction of the acetylenic bond was accomplished by catalytic hydrogenation over palladium on calcium carbonate, using dioxane and pyridine as solvents. Treatment of the product with mineral acid yielded the unsaturated lactone, 3- (38,178-dihydroxy5-androstene-17a-yl) - propenoic acid lactone (111) , 441

JOHN L . SUTTER AND EDWARD P. K. LAU

Figure 6.

Spironolactone Synthesis

442

SPI RONOLACTONE

which was easily reduced to the saturated lactone (IV) by hydrogen over palladium on charcoal. oppenauer oxidation of the product afforded 3-(3-0x0-178hydroxy-4-androsten-17a-yl) propionic acid lactone (V). maturation at c6 was then introduced by treatment with chloranil. Finally, the resulting 17-hydroxy-3-0x0-17a-pregna-4,6-diene-21-carboxylic acid-y-lactone (VI) w a s reacted with thioacetic acid, yielding 17-hydroxy-7a-mercapto-3-0x0-17a-prep-4ene-21-carboxylicacid y-lactone 7-acetate, spironolactone (VII)

.

4. Stability and Degradation Spironolactone has been found to decompose to the dienone, canrenone. The reaction is not a facile one in either the pure chemical or its dosage forms, since canrenone forms only to the extent of 1% or less over a period of five years at 400C.' 5.

Drug Metabolic Products and Phannacokinetics The structures of spironolactone metabolites in man identified so far are summarized in Figure 7. Gochman and Gantt, using the fluorimetric method described in Section 6.5, demonstrated that spironolactone MI)is readily converted to the dethioacetylated metabolite, canrenone (VI) , in humans. ' Subsequent studies by Zicha et a19 and by Wagner et all showed the presence of at least six metabolites in the urine of humans after ingestion of spironolactone. Tentative structures for the unknown metabolites were proposed by these investigators;9, "however, rigorous identification of the metabo l ites was not attempted.

"

Karim and Brown established that elimination of the thioacetate group of spironolactone in vivo is not quantitative. Besides canrenone, nese mvestigators identified a major sulfur-containingmetabolite, 4-andros3-(3-0x0-7a-methylsulfinyl-6~,17~-dihydroxyten-17a-yl) propionic acid y-lactone (Metabolite C, (VIII), Figure 7), in the urine of humans. Tentative

-

443

Figure 7.

Spironolactone Metabolism

444

SPI RONOLACTONE

structures were also proposed for three other minor sulfur-containing metabolites, (IX) , (X) , and (XI). Under the gas chromatographic conditions reported by Chamberlain for the determination of canrenone, it was found that Metabolite C (VIII) was converted to A4-3,6-dioxo steroid (XII). Further, under these conditions, spimnolactone (VII) as well as labile metabolites (IX), (X), and (XI), were converted to canrenone (VI)

.

Further unpublished observations have shown that metabolite C (VIII) had the same retention time as an unknown urinary polar metabolite observed by Chamberlain. The pharmacokinetics of spironolactone and canreno?: were investigated by Sadee, Dagcioglu and Schroder Determination of plasma levels following equivalent oral administration of spironolactone or canrenone led to the conclusion that 79% of spironolactone is dethioacetylated to canrenone. The terminal loglinear phase half-life of canrenone in plasma following oral administration of spironolactone ranged from 17 to 22 hours. Fluorescence assay revealed that from 14 to 24% of oral doses of spironolactone were excreted in the urine as fluorogenic metabolites within five days.

.

6. Methods of Analysis 6.1 Partition Coefficient Spironolactone is preferentially extracted into heptane from water. The partition coefficient is 3.5 at 25%. 6.2 Phase Solubility Phase solubility analysis of spironolactone can be carried out by equilibration in methanol at 25%. Figure 8 shows the phase solubility diagram of a spironolactone reference standard.

445

JOHN

L.SUTTER AND EDWARD P. K. LAU

~

Sample: Solvent: Equilibration: Extrapolated Solubility:

Spironolactone Methanol 24 h r . @ 25OC 8.7 mg./g.

_ - -

I

I

I

I

1

SYSTEM CCBPOSITION

Figure 8.

I

I

I

I

(mg./g. solvent)

Phase Solubility Diagram of Spironolactone

446

SPI RONOLACTONE

6.3

Spectrophotometric Analysis The u l t r a v i o l e t absorption spectrum of spironolactone is the basis f o r t h e USP XVIII assay f o r the compound. The absorbance maximum a t abo? 238 nm. in methanol i s used f o r quantitation.

6.4

Colorimetric Analysis Reaction of spironolactone with methanolic hydroxylamine hydrochloride and ferric perchlor a t e yields a red ferric hydroxamate complex having an absorbance maximum a t about 515 nm.' The absorbance a t this wavelength is linear with concentration over a range of 5 mg./ml. t o 29 mg./ml. The colored complex is s t a h l e f o r up t o 2 hours. The method is used f o r analysis of t h e compound and a variety of i t s dosage forms. Canrenone, the principal degradation product of spironolactone, does not interfere. Isonicotinic acid hydrazide may be reacted with spironolactone i n methanolic solution, yielding a soluble yellow product whose absorbance rises t o a plateau beginning a t about 375 m. "The molar absorptivity i s 2.2 X 103 a t about 380 nm. The method has not yet been adopted f o r quantit a t ive analysis.

6.5

Fluorometric Analvsis Spironolactone may be dethioacetylated under mild acid o r alkaline conditions, yielding the corresponding 4,6 -dienone, canrenone, which, i n 62% s u l f u r i c acid, i s converted t o a fluorThis compound has an excitescent trienone. a t i o n maximum a t 483 nm. and an emission maximum a t 525 m. The fluorescence is useful f o r quantitation of canrenone in plasma over the range 40-160 ng./ml.' The procedure may be used t o determine spironolactone in the presence of i t s major dethioacetylated metabolites, by measuring fluorescence in 62%H2SOq,$oth with and without p r i o r dethioacetylation.

''

441

JOHN L. SUTTER A N D E D W A R D P. K . L A U

6.6

Chromatographic Analysis 6.61 High Pressure Liquid Chromatography Spironolactone may be separated from canrenone on an octadecyl silane column using methanol - water mobile phase, and detected with the aid of a continuously variable wavelength W detector. Spironolactone, which is eluted first, may be detected at about 238 nm. and canrenone may be observed at \$s absorption maxinnun at about 283 nm. 6.62 Thin Layer Chromatography

Thin-layer chromatographic systems and corresponding R values for spironolactone are summarized in the following table: Solvent System

Adsorbent

Ethyl acetate, 100%

Silica Gel GF (Woelm)

Benzene: Ethyl acetate: Methanol 73: 25: 2

Silica Gel G

Detection 1,293

4

R 0.53

0.67

4

Detection: 1. Observe under short wave W. 2.

Spray with 50% H SO , heat at 80 C for 10 minutes, observe under long wave W.

3. spray with Phosphamolybdic Acid.

4. Spray with Phosphamolybdic Acid, heat at 80 C. for 10 minutes.

448

SPI RONOLACTONE

7.

Acknowledgements The authors wish t o express t h e i r appreciation t o Mr. E. Brown, Dr. H. Dryden and Mr. R. Tweit f o r t h e i r helpful discussions on synthesis; t o Dr. R. Bible, Mr. A. Damascus, Dr. J. Hribar and Miss Lydia Swenton for t h e i r assistance i n the preparation of the sections on I R , NMR and Mass Spectra; t o Dr. A. Karin f o r sharing with them his knowledge of spironolactone metabolism and phaxmacokinetics ; and t o Mrs. Lorraine Wearley f o r searching the l i t e r a t u r e and assembling various sections of this manuscript. The expert s e c r e t a r i a l assistance of Mrs. Corey Leone is also gratefully acknowledged.

449

JOHN L. SUTTER AND EDWARD P. K. L A U

8.

References 1. Anthony, G., Searle Laboratories, personal c o m i c a tion. 2.

"The United States Pharmacopeia" XVIII,

p. 681.

3. Marshall, S . , Searle Laboratories, personal comunication.

4. Aranda, E., Searle Laboratories, personal comication. 5. Cella, J. A., Brown, E. A. and Burtner, R. R., J. Org. Chem. 24, 1109 (1959). 6. Cells, J. A. and Weit, R. C., J. Org. Chem. 24, 1109 (1959). -

7. Baier, M. E., Searle Laboratories, personal comication. 8.

Gochman, N. and Gantt, C.L., J. Pharmacol. Exp. Ther. 135, 312 (1962).

-

9. Zicha, L., Weist, F., Scheiffarth, F. and Schmid, E., Arzneimittel-Forsch. 14, 699 (1964). 10.

Wagner, I]., Weist, F. and Zicha, L., ArzneimittelForsch. 17, 415 (1967).

-

11. Karim, A. and Brown, E. A., Steroids 20, 41

(1972).

12.

Chamberlain, J., J. Chromatog. 55, 249 (1971). L

13. Karim, A,, Searle Laboratories, personal commication.

450

SPI RONOLACTONE

14.

Sadee, W., Dagcioglu, M. and Schroder, R., J. Pharmacol. Exp. "her. 185, 686 (1973).

15.

Seul, J., Searle Laboratories, personal connnunication.

-

16. Wearley, L. , Searle Laboratories, personal connnunication. 17

Sadee, W., Dagcioglu, M., and Riegelman, S., J. Phan. Sci. 61, 1126 (1972).

18.

Sadee, W., Riegelman, S. and Johnson, L. F., Steroids 1 7 , 595 (1971).

19.

Runser, D. J. and Fortier, C., Searle Laboratories, personal c o m i c a t i o n .

45 1

TESTOSTERONE ENANTHATE

Klaus Florey

TESTOSTERONE ENANTHATE

CONTENTS

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 Rotation 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Solubility 2.9 Crystal Properties

3.

Synthesis

4.

Stability

5.

Drug Metabolic Products

6.

Methods of Analysis 6 . 1 Elemental Analysis 6.2 Spectrophotometric Analysis 6.3 Colorimetric Analysis 6.4 Chromatographic Analysis 6.41 Paper 6.42 Thin-Layer 6.43 Column

7.

References

-

Degradation

453

KLAUS FLOREY

1.

Description 1.1 Name, F o r m u l a , M o l e c u l a r Weiqht Testosterone enanthate (heptanoate) is 17-~~(1-oxoheptyl)oxy~-4-androsten-3-one. OCO ( C H 2 ) 5CH3 I

Mol. W t .

c2 6H4Oo 3 1.2

400.61

A p p e a r a n c e , C o l o r , Odor White, c r y s t a l l i n e , o d o r l e s s powder,

Physical Properties 2 . 1 I n f r a r e d Spectrum T h e i n f r a r e d s p e c t r u m (KBr p e l l e t of t e s t o s t e r o n e e n a n t h a t e i s p r e s e n t e d i n F i g u r e 11 2.

.

Nu clear M a g n e t i c Resonance S p e c t r u m The NMR s p e c t r u m i s p r e s e n t e d i n Figure 2. I t w a s o b t a i n e d on a 60 MHz s p e c t r o meter i n deuterochloroform 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 an i n t e r n a l r e f e r e n c e . The f o l l o w i n g p r o t o n a s s i g n m e n t s were made2 : Protons a t Chemical S h i f t Coupling Constants T ( i n H2) C-4H 4.20 s i n g l e t C-17aH 5.38 t r i p l e t 16H, 17H = 8 . 5 C-18H 9.17 s i n g l e t C-19H 8.82 s i n g l e t ester methyl 9.13 m u l t i p l e t 2.2

-

454

Figure 1.

Infrared Spectrum of Testosterone Enanthate(KBr Pellet). Instrument: Perkin-Elmer 621.

F i g u r e 2.

N M R S p e c t r u m of T e s t e r o n e E n a n t h a t e in D e u t e r a t e d C h l o r o f o r m . I n s t r u m e n t : P e r k i n - E l m e r R12B.

21378 5139815

782L15

MASS/CHARGE

Figure 3.

Low R e s o l u t i o n Mass S p e c t r u m of T e s t o s t e r o n e E n a n t h a t e . I n s t r u m e n t : AEI-902.

KLAUS FLOREY

2.3

Ultraviolet Spectrum Squibb House Standard (Lot 78245) gave a single band, due to the 3-keto-4 ene system3:

A A

max 240 nm E E m

42g3

max 240 nm El% 1cm

4107

2.4

Mass Spectrum The low-resolution mass spectrum,shown in Figure 3, demonstrates the expected M+ of m/e 400. There is progressive loss of carbons from the alkyl portion of the acylate which gives rise to the ions at m/e 315, 316 from the hydrocarbon portion of the chain and the m/e 288 ion with the additional l o s s of the acylate carbonyl. The loss of the entire C-17 group yields the m/e 270 and 271 ions. The ions at m/e 85 and 113 represent the hydrocarbons, CgH13, and C7H130, respectively. Fragment ions occur through a progressive l o s s of D-ring, C-ring and B-ring carbons. The m/e 147 and 124 ions are diagnostic for A4-3-ketones. The assignment of some of the diagnostic ions is depicted below2 (-1H)288

.

458

'

TESTOSTERONE ENANTHATE

2.5

Rotation The following rotation were reported: +78O (dioxan)4 +78.0° (Squibb House Standard Lot 7

78245)

+7 go +7 5-7608

Melting Ranqe The following melting range temperature temperatures (OC) were reported: 2.6

36-37. !j4 3 37-38 (Squibb House Standard Lot 78245) 35 (from alcohol-ether) 366 36-377 36-378 2.7

Differential Thermal Analysis Endotherm at 37OC.

Solubility Insoluble in water; very soluble in ether, soluble in vegetable o i l s 2 3 . 2.8

Crystal Properties The powder X-ray diffraction pattern is presented in Table 19. It is not too well resolved, since due to the low melting point of testosterone enanthate, the sample partially liquified during analysis. 2.9

459

KLAUS FLOREY

Table I Powder X-ray Diffraction Pattern of Testosterone Enanthate’

0.39 0.15 0.12 0.15 0.25 0.25 0.25 0.25 0.30 0.28 0.33 0.77

14.5 9.20 8.00 7.30 6.40 6.30 6.18 6.00 5.80 5.46 5.27 5.10

4.80 4.69 4.60 4.49 4.20 4.11 4.03 3.72 3.52

*d = interplanar distance

A= 1.539

0

1.00 0.25 0.30 0.27 0.45 0.27 0.27 0.22 0.20

n k 2 sine

A;

Radiation:Kal and Ka2 Copper

**

Relative intensity based on highest intensity of 1.00

460

3.

Synthesis T e s t o s t e r o n e e n a n t h a t e i s p r e p a r e d by a c y l a t i o n o f t e s t o s t e r o n e w i t h enanthic anhydride i n pyridine4.

E n a n t h i c a c i d and an i o n exchanger8 and e n a n t h i c a c i d c h l o r i d e 6 j 7 I t can a l s o be have a l s o been used i n s t e a d o f e n a n t h i c a n h y d r i d e . p r e p a r e d by t r a n s e s t e r i f i c a t i o n o f t e s t o s t e r o n e p r o p i o n a t e and methyl enan t h a te5. S t a b i l i t y , Degradation T e s t o s t e r o n e e n a n t h a t e i s s t a b l e as a s o l i d . I n solution photolytic d e g r a d a t i o n of t h e A-ring i s p o s s i b l e when exposed t o u l t r a v i o l e t l i g h t o r o r d i n a r y f l u o r e s c e n t l a b o r a t o r l i g h t n i n g ( c f . 1 6 ) . The k i n e t i c s o f s a p o n i f i c a t i o n have been s t u d i e d ' ' 9 18. Mucor f u n g i p o s s e s s e s t e r a s e s a b l e t o s a p o n i f y t e s t o s t e r o n e e n a n t h a t e among o t h e r s t e r o i d e s t e r s l g . 4.

KLAUS FLOREY

5.

Drug Metabolic Products Generally, the metabolic fate of testosterone enan thate is deacylation to testosterone It then follows testosterone metabolism, mostly to 17-keto steroids such as 4-androsten-3,20-dione and estrogenic substances. Data have been reported for human urine after intramuscular injectionlO,ll, rats12, their prostates and livers13. The fate in maternal peripheral blood and cord venous blood has also been studiesl4. Tissue distribution and excretion of labeled testosterone enanthate was studied in steers15.

.

6.

Methods of Analysis 6.1 Elemental Analysis

Calc.%

C 77.95

H 10.07

0 11.98

6.2

Spectrophotometric Analysis The U . V . maximum at 240 nm can be used for quantitation determination in pharmaceutical preparations20,21,22, 6.3 Colorimetric Analysis Reaction with isoniazid and determination of the resulting hydrazone at 380 nm is the basis of the compendia1 assay23. 6.4 Chromatographic Analysis 6.41 Paper The quantitative determination and separation from testosterone by paper chromatography in oily vehicles has been described by Roberts and Florey26. After spotting, the paper strips were impregnated with 30% diethylene glycol monoethyl ether (Carbitol) in chloroform and developed for 2-1/2 hours with methyl cyclohexane saturated with diethylene glycol 462

monoethyl e t h e r . After locating of the s t e r o i d spots with a fluorescent p a d d l e , t h e s p o t s a r e e l u t e d and r e a c t e d w i t h i s o n i c o t i n i c a c i d h y d r a z i d e i n methanol c o n t a i n i n g a l s o h y d r o c h l o r i c a c i d . The a b s o r b a n c e of t h e y e l l o w h y d r a z o n e i s r e a d a t 415 nm. A s i m i l a r procedure,

% w

using impregnation with propylene glycol, phenylglycol and p r o p y l e n e g l y c o l , p h e n y l g l y c o l e t h y l e t h y l e t h e r and m e t h a n o l ( 1 : 1 : 2 ) e t h e r and h e p t a n e (1:1:200) a s d e v e l o p i n g s o l v e n t and 2 , 4 - d i n i t r o p h e n y l hydrazone a s s p r a y r e a g e n t h a s a l s o been r e p o r t e d 2 7 . 6 . 4 2 Thin-Layer T h i n - l a y e r chromatographic systems have been compiled i n T a b l e 11. T a b l e I1 Absorbent S o l v e n t System Rf. Ref. Silica gel G 31 24 50% A c e t i c A c i d impreg. w i t h m i n e r a l o i l 26 24 50% E t h a n o l -25 Benzene-Acetone ( 4 :1) Silica gel -25 Silica gel Benzene-Methanol(9: 1) Silica gel P e t - e t h e r , Benzene , A c e t i c A c i d , W a t e r (67:33:85: 35) Alumina Benzene-Acetone (4: 1) Silica gel Methanol-Water (9: 1) impreg.with corn o i l D e t e c t i o n systems: U.V.

light,

sulfuric acid

-

alcohol.

KLAUS FLOREY

Column Column c h r o m a t o g r a p h y o n s i l a n i z e d c h r o m a t o g r a p h i c s i l i c e o u s e a r t h u s i n g 95% e t h a n o l - h e p t a n e s o l v e n t system f o l l o w e d b y t h e i s o n i a z i d r e a c t i o n i s t h e b a s i s o f t h e compendia1 a s s a y of t e s t o s t e r o n e e n a n t h a t e i n o i l p r e p a r a t i o n s 23 6.43

.

7. 1.

References B.

T o e p l i t z , The S q u i b b I n s t i t u t e , P e r s o n a l

Communication. 2.

3. 4.

5. 6.

7. 8.

9. 10. 11. 12.

13.

I. Cohen, The S q u i b b I n s t i t u t e , P e r s o n a l Communication. G. A. B r e w e r , J r . , The S q u i b b I n s t i t u t e , P e r s o n a l Communication. K. Junkmann, J. K a t h o l , H. R i c h t e r , U. S. P a t e n t 2 , 8 4 0 , 5 0 8 (1958) : K. Junkmann, Arch. Expt. P a t h o l . Pharmacol. 2 1 5 , 8 5 ( 1 9 5 2 ) .

A.

S. A . A l t e r , S p a n i s h P a t e n t 241,206 ( 1 9 5 8 ) ; C.A. 54,3532a ( 1 9 6 0 ) . T. Kasugai, J a p a n . P a t e n t 1 4 , 0 8 9 ( 1 9 6 2 ) : C.A. 59,10182a ( 1 9 6 3 ) . H. C i e s l i k , H. Koprowska, A. Walczak, Pol. P a t e n t 56,358(1968)C.A.71,39286~(1969). M. U l r i c h , A. Novacek a n d F. S t e j s k a l , Czech. P a t e n t 95,825(1960)C.A. 55,15551d .( 1 9 6 1 ) . Q. Ochs, The S q u i b b I n s t i t u t e , P e r s o n a l Communication. J. A . S c h n e i d e r and A. S c h u c h t e r , A r z t l . Wochschr. 9 , 3 9 2 ( 1 9 5 4 ) G. D o e r n e r , F. S t a h l and R. Z a b e l , Endokrinologie 45,121(1963). M. Wenzel, L . P i t z e 1 a n d P. E. S c h u l z e , Angew.Chem. I n t . E d . E n g 1 . 1 , 2 1 1 ( 1 9 6 8 ) . J. S h i m a z a k i , H. K u r i h a r a , Y. I t o , K.Shida a n d K. Kirao, Gunma J. Med. S c i . 1 4 , 3 1 3 464

TESTOSTERONE ENANTHATE

14. 15.

(1965) : C . A . 66,263296 (1967) i b i d . 14,326 (1965) ; F A . 66,622631 ( 1 9 6 7 ) . H. H. Simmer, M. V r F r a n k l a n d and M. G r e i p e l , S t e r o i d s 2 , 2 2 9 ( 1 9 7 2 ) . F . X . G a s s n e r , R. P. M a r t i n , W. Shimoda and J. W. Algeo, F e r t i l i t y and S t e r i l i t y 11,49(1960). D.R. B a r t o n and W. C. T a y l o r , J.Am.Chem. SOC. 8 0 , 2 4 4 (1958) : J. Chem. SOC.1958,2500. M. Schenk and K. Junkmann, Arch.Exper. P a t h . and Pharmacol, 227,210 ( 1 9 5 5 ) . 2. V e s e l y , J. P o s p i c e k and J . T r o j a n e k , C o l l e c t . Czech. Chem. Commun. 3 4 , 6 8 5 ( 1 9 6 9 ) . K.A. Kohcheenko, G. K. S k r y a b i n , B. K. E r o s h i n , L. M. Kogan and J.V.Torgor, 181 (1965) : P r i k l . Biokhim. i . M i c r o b i o 1 . C.A.63,9018~(1965). N. H. Coy, The S q u i b b I n s t i t u t e , P e r s o n a l Communication. A . R e g i n a t o , A n a l e s f a c . quim. y. f a r m . , Univ. C h i l e lO,48(1958)C.A.54,6028f ( 1 9 6 0 ) . S. Kanna, M. Takuma and S. Watanabe, Yakuzaigaku 25,145(1965)C.A. 64,15676d (1966). U. S. P. 1 8 t h e d i t i o n . D. S o n a n i n i and L. Anker, Pharm. Acta Helv. 4 2 , 5 4 ( 1 9 6 7 ) . S. Hara and K. Mibe, Chem. Pharm. B u l l . 15,1036 ( 1 9 6 7 ) . H. R. R o b e r t s and K. F l o r e y , J . P h a r m . S c i . 51,794 ( 1 9 6 2 ) . A. N i l u f e r , Turk. I j i y e n T e c r u b i . B i y o l . D e r g i s i 2 l , l 4 9 ( 1 9 6 1 ) C . A . 59,6200f ( 1 9 6 3 ) .

-

16. 17.

18. 19.

L,

20. 21. 22.

23. 24. 25. 26.

27.

-

-

L i t e r a t u r e s u r v e y e d t h r o u g h December 1972. The h e l p of H. Gonda and A. Mohr 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 i s g r a t e f u l l y acknowledged.

465

THEOPHY LLINE

Jordan L . Cohen

THEOPHYLLINE

Table of Contents Page 1.

2. 2.1

2.2 2.3 2.4 2.5 2.6 3.

4.

5. 6. 6.1 6.2

6.3 7. 8.

Description 1.1 Name: Theophylline 1.2 Formula and Molecular Weight 1.3 Hydrates 1.4 Salts 1.5 Appearance, Color, Odor and Taste Physical Properties Spectra 2.11 Infrared Spectrum 2.12 Nuclear Magnetic Resonance Spectrum 2.13 Mass Spectrum 2.14 Ultraviolet Absorption Spectrum Optical Rotation Me 1t ing Range Solubility Dissociation Constant Dipole Moment Synthesis Isolation and Purification Theophylline Stability and Compatibility Methods of Analysis Identification Tests Ouantitative Analytical Methods 6.21 Ultraviolet Spectrophotometry 6.22 Visible Spectrophotometry 6.23 Gas-Liquid Chromatography 6.24 High Pressure Liquid Chromatography 6.25 Thin Layer Chromatography Paper Chromatography 6.26 6.27 Column Chromatography Polarography 6.28 Titrimetry 6.29 6.291 Complexometric Titration 6.292 Photometric Titration 6.293 Non-Aqueous Titration 6.294 Miscellaneous Titrations Bioassay Methods Pharmacokinetics References

3

3 3 3 3

4 4 4 4

6 6 6 10 10 10 12 12 12

14

14 15 15 16 16 17 17 17 18 19 19 19 19 20 20 20 20 20 20 23

467

JORDAN L. COHEN

1.

Description 1.1

Name: Theophylline Theophylline1,2,3 is designated by Chemical Abstracts as 1, 3-dimethylxanthine. It is also known as theocin. 1.2

Formula and Molecular Weight

180.17

1.3

Hydrates Theophylline has been reported to exist in both anhydrous and monohydrate forms. The anhydrous form is obtained by drying finely powdered drug at 15OoC for three hours4

.

1.4

Salts Sodium and potassium salts as well as a large number of less basic salts and/or complexes have been prepared to increase the water solubility of theophylline for parenteral administration. The ethylenediame salt (aminoph~lline)~,~ is the most widely utilized form of this drug. Other theophylline salts which have been prepared inc ude sodium and alumi urn glycinates7, h xamethylene iamine , monoethan lamine8, trietha olamine , glucamine methylglucamine8 ethylglucamine , 2-diethylaminoethanol10, choliy5" and sodium acetatel2. Theophylline aminoisobutanol and 2-carbamoylphenoxyacetic acid, sodium salt14 have been prepared and studied clinically as has a niacinamide-theophylline complex 15,16. Theophylline has also been shown to form stable, complexes with saccharinl7, phenobarbita118, papaverine19, caffeinel5, sulfosalicylic

8

F

468

8

4

THEOPHYLLINE

acid15, benzyl alcohol15, and d -, and tate15.

is- naphthalene ace-

1.5

Appearance, Color, Odor and Taste Theophylline occurs as a white, odorless, crystalline powder with a bitter taste. 2.

Physical Properties

2.1

Spectra 2.11

Infrared Spectrum The IR spectrum of anhydrous theophylline is shown in Figure 1, This was recorded on a KBr pellet with a Perkin-Elmer model 21 spectrophotometer20. This spectrum is consistent with the literature interpretation which is presented in Table I21, Table I Infrared Spectrum of Theophylline

IR Absorption Band h)

Interpretation

1

N-H (stretch) C=O (stretch) C=C (stretch) C-N (stretch) C-H (bending) C-N, C-0 (vibration)

2.90 5.86, 5.98 6.20 6.40

6.92

7 . 7 , 8.0

The spectrum is generally consistent with those of other purine bases although the placement of the methyl groups prevents enolization and is reflected in differences in the 2.5-3.5Pregion of the spectra. The region from 5.8-8.2 is nearly identical for the three methylated xanthines; i.e. theophylline, theobromine and caffeine, however, the fingerprint region between 8.5 and 13.0kis quite dissimilar and could be used for different iation.

469

Figure 1.

Infrared Spectrum of Theophylline Reference Standard.

THEOPHYLLINE

2.12

Nuclear Magnetic Resonance Spectrum The 60 MHZ proton magnetic resonance spectrum run in deuterodimethylsulfoxide o a Perkin-Elmer R-12 B of theoThe spectrometer is shown in Figure 2$0 phylline has been reported in the leteratuT2 and the assignments are given in Table 11.

.

Table I1

NMR Spectral Assignments for Theophylline Proton Assignment

No.

N-CH (position 3) N-CH (position 1) N-H C=C-H Impurity

3 3 1 1

-

Chemical Shift (h 6.83 ( 8 ) 6.63 (s) -3.4 (b) 2.01 ( 8 ) 7.55

s= singlet, b= broad

2.13

Mass Spectrum The low resolution, electron-impact mass spectrum of theophylline us&g a 70 e.v. ionizing energy is illustrated in Figure 3. The resultant fragmentation represented by this spectrum is not readily explainable and is only in Frtial agreement with a literature massspectral report 3. The peak at 180 m/e corresponds to the base peak, which is also the molecular ion and the peak at m/e 123, which represents loss of CONCH3, are c o m n to both spectra. other peaks observable in Figure 3 occur at m/e 95, 68, 53 and 41 while the literature report indicates major peaks at 137, 109, 82, 67 and 55 m/e. Another, more recent, literature report24 suggests that fragmentation from m/e 1 2 3 9 9 5 is due to loss of CO and that m/e 95-68 is due to loss of HCN from metastable ion calculations. Apparently ring opening occurs which results in a further complex fragmentation pattern. 2.14

Ultraviolet Absorption Spectrum The ultraviolet spectrum of theophylline, K and K Laboratories, recrystallized from water, recorded on a Perkin-Elmer Hitachi, Model 124 Recording Spectrophoto47 1

Figure 2.

NMR Spectrum of Theophylline Reference Standard in DMSO-dfj

THEOPHYLLINE

“v-m

I

Figure 3.

Low Resolution Mass Spectrum of Theophylline

473

P

4 P

WAVELENGTH ( nm) Figure 4.

Ultraviolet Spectrum of Theophylline in 0.1 N NaOH.

THEOPHY L L l N E

meter in 0.1 N NaOH is illustrated in Figure 4. In addition the spectra was also recorded in 0.1 N HC1, pH 6.3 phosphate buffer and CHC13 with all of the results given in Table 111. Table I11 Ultraviolet Absorption of Theophylline Amax. (nm)

271 271 274 271

1oLymax. 1.02 1.04 1.28 1.04

Solvent 0.1 N HC1 pH 6.3 buffer 0.1 N NaOH CHC13

All of these spectra exhibited a single maximum in the spectral region between 230-300 nm. The nature of the spectra, as well as the calculated molar absorptivities are in good agreement with published liter1 ~ ~ a n 6 at 271 nm. of ature datz. Turner and 0 ~ 0 reported 1.02 x 10 in pH 6.3 phosphate buffer, while the value of 6 at 274 nm. in 0.1 N NaOH calculated from the data of Schach and Waxler26 compares closely with the observed value above.

475

JORDAN L. COHEN

2.2

Optical Rotation Theophylline exhibits no optical activity.

2.3

Melting Range The original synthetic literature27 reported a melting point 0 ; 264O for theophylline subsequently the melting range has been reported to be between 271° and 274O C.28

c

2.4

Solubility The reported solubility of theophylline is 8.3 mg/ml in water; 12.5 mg/ml in ethanol; 11.f,Tg/ml in , Several chloroform; and sparingly soluble in ether basic salts have been prepared which enhance the water solubility of theophylline and have been utilized therapeutically intended for oral, rectal and parenteral administration. (See Section 1.4) In addition, there are numerous literature reports of altered theophylline solubility due to specific interactions wi a variety of chemical substances, Leuallen and 0 ~ 0 1 ~ ’ have demonstrated marked increases in theophylline solubility in the presence of a wide variety of primary, secondary andd tertiary aliphatic amines. They attributed this increased solubility to salt formation as well as hydrophobic interactions. Paruta and Irani3O reported a three-fold increase in theophylline solubility in alcohol-water and dioxane-water mixtures with maximal solubility occurring at a dielectric constan of 40 in these mixed solvent systems. A British patent” reports a sixfold increase in theophylline water solubilit in the presence of guiacol. Several other have reported specific interactions of theophylline with a multitude of planar organic molecules in aqueous solution usually resulting in an increase in the solubility of of both interacting species, For a thorough discussion of the nature of these specific molecular interactions and the magnitude of this solubility effect see Cohen and Connors. 37 Not surprisingly there has been some NMR evidence suggesting that theophylline also forms 1:1 complexes with itself (dimerizes). 38,39 Theophylline also demonstrates altered solubility in the presence of several inorganic salts as illustrated in Table IV.

paper^^^'^^

476

THEOPHYLLINE

Table IV Effect of Inorganic Salts on Theophylline Solubility Salt -

Effect

NaI, KI, NaCNS, KCNS

Increased theophylline solubility

NaC1, KC1, Na2SO4

Decreased theophylline solubility

NaBr, KBr

No effect on theophylline solubility

Dissociation Constant Theophylline is a weakly acidic compound with the proton on the nitrogen in position 7 being dissociable. The pKa of 8.6 determined both potentiometrically and spectrophotometri all is in good agreement with other literature datah5y4y,42 reporting a pKa-8.6 in aqueous solution, The0 hylline is very weakly basic and pKb's of 13.555.43 and 11.5 have been reported in the literature from aqueous potentiometric mea surements 2.5

"'$8

.

2.6

Dipole Moment The dipole moment of theophylline has been deter mined in three different solvents with the following values being reported (all in D ye units):& = 3.94 in an@,= 3.94 in bendio~ane~= ~ ;4.6 P in 90% phenol"; zene47. Synthesis Although present in coffee, tea and cocoa as well as other natural sources, theophylline is made available commercially by total synthesis. Theophylline was first syntliesized by Traube27 as 'an intermediate in the total synthesis of caffeine starting from urea, This classic procedure, which is still utilized, is illustrated in Figure 5. Several subsequent syntheses which are modifications of this procedure and are claimed to either increase the reaction y Id or simplify the process, have also been reportedfig, 49. 3.

411

JORDAN L. COHEN

o=O

Ie o

CH+THp,,

-I

C=O

CH3-N

CH3-NH

I

C=O

NCCH7COOH

CH2

I

I

I

CN

CH3-NH

CH3-NH

J

alkalai

0

cH3-k3N0 (HNOr, c H 3 k ( )

NH

0

NH

0 I

I

m3

j. C

H

3

k

0 3 NH2

0

C H 3 ~ ~ " ' NH3 * "

NH2

0

dH3 H

0

I

k:ki

cH3"jii 0

I

CH3

Figure 5.

Chemical Synthesis of Theophylline 478

THEOPHYLLINE

Uric acid has been used as the starting c o m p ~ u n d . ~ ~ , ~ ~ In addition various intermediates have been prepared, including 1,3-dimethyl-4-amino-5-formylaminouraci152 1,3dimethyl-4,5-diamino-2,6-dih dro~yformylpyrimidine~3,1,3dimethyl-5,6-diaminourscily5~ and 1,3-dimethyl-4-imino-2.6dio~yhexahydropyrimidine~5, from which theophylline has been synthesized.

4.

Isolation and Purification Because of its significantly increased solubility in hot water2, theophylline can be purified by recrystallization from water. The anhydrous form can be obtained by finely powderin the resulting solid and drying at 150° C. Adjusting the pH of an aqueous solution for three hours to 4-6 allows optimal extraction with chloroform while pH 5-7 is required using 1,2-dichloroethane.56 Saturation of the aqueous phase with sodium or amonium sulfate and addition of 5-15% isopropyl alcohol to the organic phase has been shown to markedly increase the extraction efficiency26,57,58.

8.

Sephadex G-10 adsorption chromatography has been used to isolate theophylline from biological samples also containing several closely-related xanthines59. Thin layer chromatographic separation of theophylline, caffeine and theobromine on silica gel-G using an ethylacetate, acetone, butanol, 10% NH,!+OH(5:4: 3: 1) mixture has also been reported60. Cohen and Garrettson have demonstrated base line separation of these same three compounds using reverse-phase high performance liquid ~hromatography~8. 5.

Theophylline Stability and Compatibility Theophylline solutions are generally quite stable over the entire pH range. Strongly alkaline solutions (pH) 12) show decom osition and apparent ring opening Solutions of theophylline have after several weeks been shown to be susceptible to oxidation at position 8 forming 1,3-dimethyluric acid in the presence of methylene blue which acts as a photosensitizing dye61. Because of its low solubility and relatively high pKa theophylline will precipitate from aqueous solutions if the pH drops below 9 unless present in concentrations less than the

e.

419

JORDAN

L. COHEN

water solubility. All acidic salts, therfore, are potentially incompatible with theophylline in solution, As discussed earlier theophylline forms generally soluble complexes with a variety of other compounds. In the solid state however, these complexes may lead to formation of eutechtic mixtures as in the cases of theophylline, phenobarbital and papaverine hydrochloride62 and theophylline riboflavin interactions63 which have been reparted. Theophylline ethylenediame (aminophylline), which is commonly employed in alkaline parenteral solutions in concentrations considerably greater than the solubility of theophylline, has many incompatibilities associated with it. Since the solutions are alkaline, but essentially unbuffered, all acidic substances produce precipitation of the free theophylline when mixed together with aminophylline solutions. Limited exposure to air results in carbon dioxide absorption and also leads eventually to precipitation of the free drug. For a more complete discussion and listing of the incom atibilities of aminophylline Remmington3, MartindalePl or a series of papers may be consulted. in the hospital pharmacy

6.

Methods of Analysis 6.1

Identification Tests Theoph lline has been qualitatively identified by infrared21, "MR 2 and mass spectroscopy23, thin-layer60 and paper chromatography69 using color localization reactions as well as by a multitude of specific color formation tests designed to distinguish it from caffeine, theobromine and other xanthines and purines, Brown needles are produced from the aqueous reaction of theophylline and ammoniacal AgN03 70 while reaction with ammoniacal silver and subsequent treatment with thallium acetate yields light brown ball shaped masses71. A combination of 2,6dichloroquinone chlorimide and theophylline in sodium borate solution gives a blue color and subsequently forms a red-violet precipitate72. Sanchez73 has reported theophylline to produce a semi-quantitative red color when reacted with diazo-p-nitroaniline. More recently Feigl, et. a1.74, have shown that microgram quantities of theo-

!!

480

THEOPHYLLINE

phylline can be detected in the presence of other xanthines by heating at 15Oo-18O0 C. with excess Hg(CN)2. 6.2

Quantitative

Analytical Methods

6.21

Ultraviolet Spectrophotometry Shack and Waxler described a remarkable ultraviolet spectrophotometric method for the analysis of theophylline in aqueous solutions as well as biological fluids in 1949 26. This procedure involves extraction from the sample adjusted to pH 7.4 using 5% isopropyl alcoholchloroform followed by back-extraction of the drug into 0.1 N NaOH and measurement of absorbance at 274 nm. They found the method to be sensitive to about 1 mcg/ml and demonstrated its applicability to theophylline analysis in water, plasma, urine and even tissues. Although it lacked specificity, it was rapid and sensitive and is still widely utilized for routine blood level determinations of theophylline. Gupta and L ~ n d b e r greported ~~ a modified procedure using differential spectrophotometry and estimating the theophylline concentration from absorbance readings at 285 nm and a standard curve. Although their method was somewhat less sensitive, 10 mcg/ml in plasma, it was specific for theophylline in the presence of phenobarbital; a drug frequently taken with theophylline and a serious interference in the Shack and Waxler procedure. A similar ultraviolet procedure was developed for the analysis of theophylline in dosage forms which avoided the back-extraction into NaOH. The absorbance of the organic extract was measured at 271 nm and the theophylline concentration determined from a beers 1 w plot of standards which was linear from 2-15 m~g/ml.~' A method utilizing extraction and combination of two successive volumes of CHClg-isopropyl alcohol (20:l) from an aqueous sample to which sodium chloride had been added reports improved sensitivity of 0.5 mcg/ml from aqueous sarnple~'~, A method involving oxidation of theophylline with K2Cr2q in acid and steam distillation of the product which is measured at 257 nm has also been reported applicable to plasma samples78. It is, however, not specific for theophylline and marginally sensitive for therapeutic

JORDAN L. COHEN

plasma levels of 10-20 mcg/ml. 6.22 Visible Spectrophotometry Several methods involving derivitization of theophylline and subsequent colorimetric analysis have been reported, Coupling reactions have been described with p-nitroaniline79, and p-aminobenzenesulfonic acidso exhibiting colorimetric absorption maxima at 510 nm and 482 nm respectively, While the sensitivity was less than ultraviolet methods, acceptable standard curves enabled quantitation of 50-5,000 mg. Chemical reaction of theophylline with murexide and thiophene enabled quanitation of 300-2,500 mg. amounts by measuring absorbance at 6601m.~~ 6.23 Gas-Liquid Chromatography A gas chromatographic method for theophylline in dosage forms using a 6 f-ot, 1.5%, SE-30 chromosorb W column has been reported88 Helium was used as the carrier gas and temperature program was required to separate the individual xanthines. Elefant, et a183 reported a GLC procedure for tablets containing ephedrine, phenobarbital and thophylline. Using a 6', o.d., 3% HI-EFF 8 BP on 100/120 mesh Gas Chrom Q, theophylline eluted as a moderately sharp peak after 14 minutes at a column temperature of 250° C. Recently, Shah and R i e g e l m a ~reported ~~~ a GLC procedure applicable to therapeutic plasma and saliva determinations of theophylline with a sensitivity of 1 mcg/ml. The dipropylated analog was formed by derivitization using tetrapropylamnonium hydroxide and chromatoThe retention graphed on a 3% OV-17 column at 190'. time was 3.6 minutes with a sharp peak resulting.

.

6.24 High Pressure Liquid Chromatography The author has developed a high performance liquid chromatographic method for the analysis of theophylline in biological fluids58. A reverse-phase 2', Phenyl Corasil column with a 5% CH CN in pH 8.5 phosphate buffer mobile phase permits determ nation of as little as 0.5 mcg/ml using an ultraviolet detector. Baseline separation of theophylline, theobromine and caffeine is also achieved,

2

482

THEOPHYLLINE

6.25 Thin Layer Chromatography Several thin layer chromatographic systems for the separation of theophylline, theobromine and caffeine and subsequent quantitation have been developed and are summarized in Table V. Table Va4 Summary of Thin Layer Chromatographic Literature Data Solid Silica Gel G 11

11

11

Aluminum Oxide 11

II

II

11

11

Solvent

Rf Theophyl- Caffeine Theobromine .35

.20

.40

.50

.23

,26

.54

-46

.31

.50

.49

.44

CHC13-BuOH .54 (98:2) CHC13-C2H50H 1.00 (99:1) CgHg'C2H OH .15 (93: 2) C ~ H ~ - C Z H ~ O H .47 (95:5) C6Hg-C H OH .58 f98: 10) .75 CgHg'C2H OH (88:20)

.30

.15

.10

.10

.06

.02

.08

.04

.11

.06

.27

.19

CHCl Ether 3'(85: 15) CHC13-CH30H (95:5) CC14-CHC1 -CH30H (8: 1) CHClg-acetoneCH30H (1: 1:1)

3:

483

JORDAN L. COHEN

wantitation was most often achieved by densitometry at 254nm. Fluorescence inhibition scanning at 254 nm. enabled quantitation of 50 mcg/ml of theophylline on a sili a gel plate and a 95:5 CHC13-C2H50H developing solventg5. Two papers report the use of Draggendorfs Reagent for the quantitative estimation of theophylline in amounts as low as 0.5 mcg. Schunack, et alg6 utilized two different solvent systems, benzene-acetone (30:70), Rf= 0.06 and chloroform-ethanol-formic acid (88:10: 2), Rf= 0.45 while Senanayake, et alg7 utilized an 8:5:1 mixture of chloroform, carbontetrachloride and methanol and found an Rf for theophylline of 0.31. 6.26 Paper Chromatography There'areseveral references in the literature to paper chromatographic syst or the separation and quantitation of theophylline. illustrated by Berger and Hedrickg9 who developed two different percentage mixtures of acetonitrile and aqueous buffer to achieve separations of theophylline from theobromine and caffeine. Quantitation by diazotization and fluorescence quenching densitometry following a paper chrgvatographic separation has been described by Kala, et al. 6.27

Column Chromatography Theophylline has been separated and quanti tively determined by column adsorption chromatography. A silicic acid column with 5% butanol in chloroform was utilized and the eluent analyzed by ultraviolet spectrophotometry

H-

.

Polarograph Dusinsky an: Cavanakg2 report on a non-specific polarographic determination of theophylline involving the bromine oxidation to methyl parabanic acid. The halfwave potential was reported to be -0.72 v. and accuracy for dosage form analysis was of the order of 3%. 6.28

6.29 Titrimetry There are numerous literature reports describing titrimetric quantitative analytical methods for theophylline due to its acidic properties and reactivity with a variety of compounds. 484

THEOPHY LLlNE

6.291 Complexometric Titration Theophylline has been determined by reaction with excess copper (11) ions, usually as amine salts and then back-titration with omplexon I11 (Murexide) to a visual indicator endpoint.939 st Theophylline also forms molecular complexes with silver ions and has been determined b both direct titration with si ver nitrate using visualJ5 or potentiometric detection" and by backtitration after the addition of excess anrmoniacal silver nitrate using potassium thiocyanate9 7. 6.292

Photometric Titration Fleischery6 has reported a photometric titration determination of theophylline by coupling with Fast Blue. Sensitivity from aqueous solutions is claimed to be 2 mcg/ml. 6.293 Non-Aqueous Titration Theophylline can be determined by titration in a nonaqueous solvent using perchloric acid in acetic acid as a titrant.99 Endpoint determination can be done either wi h a visual indicator such as Nile BlueYID or Crystal Violet or potent iometricallylo2. Theophylline has also been titrated as an acid in dimethylformamide using sodium methoxide and thymol blue as an indicator.103 6.294 Miscellaneous Titrations Other titrimetric procedures re orted for theophylline determination include iodometric114,105, bromometriclo6 amperometric1°7, coulometriclo8 and radiometric109'

.

6.3

Bioassay Methods The diuretic activity of theophyllig$_was de'l a in termined on a relative scale by Lipshitz, et " dogs by plotting the log dose versus the log of the diuretic effect. With urea normalized to 1.0, thophylline was found to be 115 timesas effective. Pharmacokinetics Because of the availability of methodology for the analysis of theophylline in plasma as early as 194926 there are numerous literature reports detailing the dispo7.

485

JORDAN L. COHEN

sition of this drug in humans following administration by several routes. Although theophylline is generally well absorbed following oral administration, considerable variation does occur depending upon dosage form, formulation and which salt form of theophylline being administered. Hydroalcoholic solutions produce peak plasma levels in 60 minutes which decline below acceptable thera ti levels four hours after single dose administration.pfy, lE2 Tablets demonstrate peak levels from 2-4 hours with availability dependent upon formulation, while sustained release tablets provide peak levels at 6 hours and provide therapeutic levels up to 10 hours‘13. A non-alcoholic liquid suspension administered as a soft gelatin capsule showed absorption characteristics between those for a hydroalcoholic solution and a sustained release tablet114. This same study suggests that there is significantly slower absorption of theophylline ethylenediamine (aminophylline) than plain theophylline in comparable oral doses in the same subjects on a cross-over study desi n. This contradicts earlier findings by Waxler and and additional studies are being performed. Theophylline is conrmonly administered rectally, particularly in asthmatic children,but has been shown to be poorly and erratically absorbed from normal suppository base preparations showing peak plasma levels considerable lower than from comparable oral doses,and occurring 1-3 hours after administration116, Theophylline retention enemas, however, have been shown to produce plasma consideration--time profiles similar to those following an intravenous dosel14. The drug appears to be fully bioavailable from this route if allowance is made for a normal 30-60 minute absorption dela and subsequent effect of partial first-pass metabolism. Correlation of plasma levels and clinical response is readily achievable and this route has been proposed as the o timal method of theophylline administration in children118

.

Theophylline disposition following intravenous, intramuscular and oral administration can be described by okinetic mode1119 with an an open two-compartment -phase dissappearance of average plasma half-life

486

THEOPHY LLlNE

4 . 4 hours, Wide patient-to-patient variation with halflives ranging from 2.5-9.5 hours presents possible serious management roblems for asthmatics on chronic theophylline therapyP20 The drug is relatively widely and rapidly distributed into all tissues with high rates of blood flow and has a volume of distribution (Vd) of 0.3v kg. and a half-life for distribution (o(-phase) of 0.12 hours, Erythrocytes apparently also take up significant amounts of theophylline rapidly. While there was found to be no statistically significant differences in the pharmacokinetic parameters between rmal volunteers and asthmatic subjects, Maselli, et allf' showed that asthmatic children evidenced significantly shorter -phase half-lives than adults with a mean of 2.65 hours compared to the 4;4 hour half-life for the adult population.

.

#

A recent study122 has shown theophylline to be bound about 60% to plasma proteins at therapeutic plasma concentrations of 12-15 mcg/ml This same study, as well as that of Shah and RiegelmanS$ indicates that saliva levels of theophylline are approximately 50% of those in plasma and the disappearance of theophylline from saliva parallels that from plasma over the normal therapeutic concentration range. This may lead to a simplified method for monitoring theophylline concentrations in clinical pharmacokinetic studies for patients undergoing chronic therapy. Theophylline has been shown to be extensively metabolized in vivo with 13% of the administered dose appearing in the urine as 3-methylxanthine) 35% as 1,3dimethyl-uric acid and 19% as l-methyluric acid123. Although considerably more work needs to be performed in this area, the variations in plasma half-life in adults and the marked decrease in plasma half-life in children is probably related to differences in metabolic activity in individual subjects.

487

JORDAN L.COHEN

References 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

The United States Pharmacopoiea, XVIII, p.724 (1970) Merck Index, 8th Ed,, p. 1034 (1968). Remmington Pharmaceutical Sciences, 14th Ed., p. 1157, (1970). J. L. Cohen, Ph.D. Dissertation, University of wis3-consin (1969). :erman Patent 223,695 (Oct. 18, 1970). U . S . Patent 919,161 (April 20, 1909). Brit. Patent 624,696 (June 15, 1949). U.S. Patent 1,867,332 (July 12, 1932). U.S. Patent 2,161,114 (June 6, 1932). German Patent 739,334 (Aug. 12, 1943). Martindale, The Extra Pharmacopoiea, 26th Ed. , p. 357, (1972). Ibid, p. 362, 1972. F. Steinberg and J. Jensen, J. Lab, Clin. Med., 30, 769 (1945). German Patent 1,106,923 (May 18, 1961). R. Reiss, Arzneimittel-Forsch, 11, 669 (1961); C.A. 55 25976 b (1961). B. M. Cohen, J. of Asthma Res. , 4, 75 (1966). Brit. Patent 1,109,964 (April 18, 1968). U.S. Patent 2,017,279 (Oct. 15, 1935). A. Mossini, Boll. Chim. Farm., 78, 261 (1939). This spectrum was kindly provided by Dr. K. Florey, Squibb, Medical Research, New Brunswick, N. J. E. R. Blout and M. Fields, J. Am. Chem. SOC., 72, 479, (1950). R. Otinger, G. Boulin, J. Reisse and G. Chiurdogiu, Tetrahedron, 2l, 3435 (1965). G. Spiteller and M. FriedmaM, Monatsh, 93, 632 (1962) C.A. 58, 1018 g (1964). G. S. Rao, K. L. Khanna and H. H. Cornish, J. Pharm. g. , 6l, 1822 (1972). A . Turner and A. Osol, J. Am. Pharm. Assoc. , Sci. Ed., 38, 158 (1949). J. A. Schack and S. H. Waxler, J. Pharmacol. Exp. -*, Ther 97, 283 (1949). W. Traube, g . 33, 3052 (1900). H. Doser, Arch. Pharm. , 281, 251 (1943). E. E. Leuallen and A. Osol, J. Amer. Pharm. ASSOC., Sci. Ed. , 38, 92 (1949).

-

-2

16. 17. 18, 19. 20. 21, 22. 23. 24. 25. 26. 27. 28. 29.

-

-

48 8

THEOPHYLLINE

30. A. N, Paruta and S. A. Irani, J, Pharm. Sci., 55,1055 (1966). 31. British Patent 932,874 (July 31, 1963). 32. J. L, Cohen and K, A. Connors, Amer. J. Pharm. Ed., 31, 476 (1967). 33. T. Higuchi, J. Pharm. Sci., 53, 644 (1964). 34. R. Huettenrauch, W, Suess and U. Schmeiss, Pharmazie, 24, 646 (1969), 35. British Patent 1,109,964 (April 18, 1968). 36. T. Higuchi and K. A. Connors, Advances in Anal, Chem. and Instrumentation, Vol. k, Interscience, New York, 1965, p.117-212. 37. J. Li Cohen and K. A. Connors, J. Pharm. Sci., 59, 1271 (1970). 38. A. L. Thakkar, L. G. Tensmeyer and W. L. Wilham, J. Pharm. Sci;, 60, 1267 (1971). 39. D. Guttman and T. Higuchi, J. Pharm. Sci., 60, 1269 (1971)40. V. P, Gusyakov, Aptechnoe Delo., S, 30 (1959); C.A. 54: 3033 f (1960). 41. H. V. Maulding and M. A. Zoglio, J, Pharm. Sci., 60, 309 (1971). 42. H. Miyamoto, Sci. Rep. Niipata Univ., E, 23 (1969); C.A. 72: 83560 t (1969). 43. K. Lizk and C. Peciai, Chem. Zvesti., l.6, 692 (1962); C.A. 58: 8409 h (1963). 44. K. J. Evstratova and A, I. Ivanova, Farmatsiya, 17, 41 (1968); C.A. 69: 46128 a (1968). 45. H. Weiler-Feichenfeld and E. Bergmann, Israel J. Chem., 6, 833 (1968); C.A. 70: 77174 n (1969). 46. H. Miyazaki, Osaka Dalgaku Igaku Zasshi, 2, 4306 (1959); C.A. 54: 14339 a (1961). 47. H. Weiler-Feilchenfeld and 2. Neiman, J. Chem. SOC., -By 596 (1970). 48. B. Gepner and L. Kreps, J. Gen. Chem. (U.S.S.R.); C.A. 41: 9bd (1947). 49. E. I. Abromova and V. I.Khmelevskii, Med. Prom. S.S.S.R., 18, 35 (1964); C.A. 61: 1865 c (1964). 50. V. I. Khmelevskii, Med. Prom. S . S . S . R . 12, ll(1958); C.A. 53: 11761 g (1958). 51. E. I. Abramova, V. I. Khmelevskii and Y. L. Shneidermann, Med. Prom. S.S.S.R., l.5, 31 (1961); C.A. 56: 6097 g (1962). 52. German Patent (East) 64722 (November 20, 1968); C.A.

-

-

489

JORDAN L. COHEN

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

71: 22136 8 (1969). F. L. Grinberg, J: Appl. Chem. U.S.S.R., 2,1461 (1940); C.A. 35: 3974' (1941). German Patent 2,058,912 (June 16, 1971); C.A. 75: 63836 w (1971). B. Bobranski and Z. Synowiedski, J. Amer. Pharm. Assoc., Sci. Ed., 37, 62 (1948). A. I. Shkadova, l9, 56 (1970); C.A. 72: 136462 w (1970). V, P.. Shah and S. Riegelman, J, Pharm. Sci., 63, 1283 (1974). J. L. Cohen and L. K. Garrettson, Acad. of Pharm. Sci. , Annual Meeting, Nov, 1974 (abstract). L. Sweetman and W. L. Nyhan, J. Chromatog., 32, 662 (1968). V. M. Pechenikov, A. Z. Knizhnik and P. L. Senov, Farm. Zh. , 23, 41 (1968) ; C.A. 70: 22932 c (1969). P. A. Friedman, Biochem. Biophys. Acta, 166, 1 (1968) E. G. Glass, Pharm. Praxis Beil Pharmazie, 8, 197 (1964); C.A. 61: 14474 a (1964). A. Koshiro, Yakuzaigaku, $9 (1967); C.A. 69: 99331 t (1968). R. Misgen, Amer. J. Hosp. Pharm., 22, 92 (1965). J. A. Pate1 and G. L. Phillips, h e r . J. Hosp. Pharm., 23, 409 (1966). g e r . J. Hosp. Pharm., 24, 440 (1967). h e r . J. Hosp. Pharm., 27, 67 (1970). J. Hosp. Pharm., 28, 228 (1970). H. K. Hegi, Pharm. Acta Helv., 34, 105 ( 1959); C.A. 53: 16466 e (1959). C. K. Glycart, J. Assoc. Off. Agr. Chem. ' -919 512 (1936). L. Rosenthaler, Pharm. Acta Helv., 14, 218 (1939); C.A. 34: 7782l (1940). H. W. Raybin, J. h e r . Chem. SOC. , 67, 1621 (1945). J. A. Sanchez Boll. SOC. Quim., Peru, 9, 197 (1943); C.A. 2:228g3 (1944). F. Feigl, D. Goldatein, E.K. Libergott, Anal. Chim. 49, 573 (1970). R. C. Gupta and G, D. Lundberg, Anal. Chem., 45, 2403 (1973). L. 0. Kircheuko, F. Kagan and F. Yu, Farm. Zh., 25, 42 (1970); C.A. 73: 28951 8 (1970)

-

i7,

-

a,

490

THEOPHYLLINE

77. B. K. Kim, Rachelle Laboratories, Personal Connnunication, Jan. 1973. 78. A. Villa and A. Pistis, Farmaco. Ed, Pract., 25, 717 (1970); C.A. 74: 30565 h (1971). 79. H. Raber, Sci. Pharm. , 32, 122 (1964); C.A. 61: 11853 e (1964). 80. M. P. Castro and R. R. Mendoza, Inform, Quim. Anal,, 17, 158 (1963); C.A. 61: 537 d (1964). 81. H, Wachsmuth and L. Van Koechoven, J. Pharm, Belg., 18, 366 (1963); C.A. 61: 8131 b (1964). 82, J. Reisch and H. Walker, Pharmazie, 21, 476 (1966); C.A. 66: 22234 u (1967). 83. M. Elefant, L. Chafetz and J. M. Talmadge, J. Phanu Sci -’ 56 1181 (1967). 84. L. J. Kraus and E, Dumont, J. of Chromatog., 48, 96 (1970). 85. V. Massa, F. Gal, P, Suspulgas and G. Maestre, Trav. SOC. Montpellier, 2,167 (1971); C.A. 75: 83949 P (1971). 86. W. Schinack, E. Mutschler and H. Rochelmeyer, Deut. Apotheker-Ztg., 105, 1551 (1965); C.A. 65: 16791 f (1966). 87. U. M. Senanayake and R. 0. B. Wijesekrea, J. Chromatog. , 32, 75 (1968). 88. E. G. C. Clarke and S. Kalayci, Nature, 198, 783 (1963). 89. B. A..Berger and C. E. Hedrick, Anal. Biochem., ’ ,16 260 (1966). 90. H. Kala, H. Moldenhauer and K. Wolff, Pharmazie, 14, 519 (1959); C.A. 54: 10239 i (1960). 91. A, J. Shingler and J. F. Carlton, Anal, Chem., 3 l , 1679 (1959). 92. C. Dusinsky and T. Cavanak, Ceskosiov. Farm., 1, 511 (1958); C.A. 53: 8539 g (1959). 93. A, G. Mindermann, K. Leupin and R. Baum, Pharm. Acta Helv., 39, 390 (1964); C . A . 61: 4154 d (1959) 94. G. H. Morait, M. Coada, V. Turailet and G. H. Clogolea, Farmacia, 2,521 (1964); C.A. 62: 403 d (1965). 95. M. E. Shub and E. N. Tsitrin, Med. Prom. S.S.S.R., 11, 43 (1957); C.A. 52: 10500 a (1958).

- 0 )

-

-

49 1

JORDAN L. COHEN

96. F. Pellerin and G. Leroux-Mamo, Ann. Pharm. Fr., 29, 153 (1971); C.A. 75: 52853 y (1971). N. Stevens and D. J. Wilson, J. her. Pharm. 97. Assoc., 2,314 (1937). 98. G. Fleischer, Pharmazie, 11, 208 (1956); C.A. 51: 8365 a (1957). 99. M. B. Devani, C. J. Shishoo, and D. J. Bhut, J. Pharm. Sci., 57, 1051 (1968). 100. H. Ellert, T, Jasinski and F. Pawelczak,& A Polon. Pharm., 16,235 (1959); C.A. 54: 1182 d (1960). 101. R. Reiss, Z. Anal, Chem,, 167, 16 (1959); C.A. 53: 18729 e (1959). 895 102. c. Peciar and K. Linek, Chern. Zvesti., (1961); C.A. 58: 3271 g (1963). 103. M. A. McEniry, J. Assoc. Off. Agr. Chem,, 40, 926 (1957); C.A. 52: 5750 e (1958). 104, C. Runti, Farmaco Ed. Prat., 11,218 (1956); C.A. 53: 6537 f (1959). 105. P. Plumer, J. Pharmacol. Exp. Ther., 93, 142 (1948). 106. H. Raber, Sci. Pharm., 34, 202 (1966); C.A. 66: 22249 c (1967). 107. G. Bozsai and L. Mosonyi, Pharm. Zentrahalle, 103, 250 (1964); C.A. 61: 8131 e (1964). 108, 2. Kalinowska, Acta Polon, Pharm,, 20, 193 (1963); C.A. 62: 1516 e (1965). 109. J. Kormicki and E. Mikolajek, Dissertations Pharm., 16, 565 (1964); C.A. 62: 11628 c (1965). L. Lipshitz, Z, Hadidian and A. Kerpcsar, J, 110. Pharmacol., 2, 97 (1943); C.A. 38: 1615 (1944). 111. A. R. Flora, Curr. Ther. Res. Clin. Exp., 2, 611 (1970). 112. C. Bruda, R. B. Miller, S. T. Leslie, E. G. Nicol and I. Thomson, J. Clin. Pharmacol., l4, 385 (1973). 113. E, B. Truitt, V. A. McKusick and J. C. Krantz, J. Pharmacol. Exp. Ther. , l.0, 309 (1950). 114. R. H. Jackson, R, Garrido, H. I. Silverman and H. Saleno, Ann. Allerpy, 3 l , 413 (1973). 115. S. H. Waxler and H. B. Moy, J. her. Pharm. Assoc., Sci. Ed., 49, 619 (1960). 116. A. S. Ridalfo and K. G, Kohlstaedt, Am. J. Med. Scj,

-

is,

w.

492

THEOPHYLLINE

116. 237, 585 (1959). 117. T S . Segal and E. B. Weiss, Annals of Allergy, 29, 136 (1971). 118. J. W. Yunginger, M. Shigeta, I. Smith, M. Gwen and H. Keitel, Annals of Allergy, 24, 469 (1966). 119. P. A. Mitenko and R. I. Ogilvie, Clin, Pharmacol. Ther. , 2,509 (1973). 120. J. W. Jenne, E. Wyze, F. S. Roodand and F. M. MacDonald, Clin. Pharmacol, Ther., 2,349 (1972), 121. R. Maselli, G . L. Casal and E. F. Ellis, J. Pediatr. 76, 777 (1970). 122. R. Koyoosko, E, F. Ellis and G. Levy, Clin. Pharmacol. Ther.,l5, 454 (1974). 123. H. A . Cornish and A, A. Christman, J, Biol. Chem., 228, 315 (1957).

-

-

493

TYBAM ATE

Philip Reisberg, John Kress, and Jerome I. Bodin

TYBAMATE

CONTENTS 1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor,Mode of Occurrence 2 . Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Mass Spectrum 2.4 Thermal Characteristics 2.41 Melting Range 2.42 Boiling Range 2.43 Differential Scanning Calorimetry 2.5 Solubility Characteristics 2.51 Solubility in Several Solvents 2.52 Partition Coefficients 2.6 Optical Rotation 3 . Synthesis 3.1 Purification and Crystallization 4. Stability 5. Drug Metabolic Products 6. Methods of Identification and Analysis 6.1 Elemental Analysis 6.2 Identification ‘rests 6.21 Derivative Formation 6.22 Colorimetric Identification Test 6.3 Cobalt Cyanate Colorimetric Assay 6.4 Hypochlorite Colorimetric Assay 6.5 Volumetric Analysis 6.6 Spectrophotometric Analysis 6.61 Infrared Identification 6.62 Nuclear Magnetic Resonance 6.7 Gas Chromatographic Methods 6.71 Tybamate in Dosage Forms 6.72 Tybamate in Biological Fluids 6.8 Thin-Layer Chromatographic Methods 6.81 Identity and Purity of Commercial Tybamate 6.82 Identification in Presence of Other Drugs 7. Pharmacokinetics 8. References 9. Acknowledgements

495

PHILIP REISBERG e t a /

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

1.1 Name, Formula, Molecular Weight Tybamate i s 2 - (hydroxymethyl) -2-methylIt is also p e n t y l b u t y l c a r b a m a t e 'carbamate. known a s 2-methyl-2-propyltrimethylene b u t y l c a r bamate carbamate, and as N-butyl-Z-methyl-2propyl-1,3-propanediol dicarbamate. 0

CH3

0

I II H2N-C-O-CH2-C-CH2-O-C-NH-CH2-CH2-CH-3 II

I

C13H26N204, M o l e c u l a r Weight:

274.36

1 . 2 Appearance;Color, Odor, Mode of Occurrence Tybamate o c c u r s as a w h i t e , c r y s t a l l i n e powder or as a c l e a r , c o l o r l e s s , v i s c o u s l i q u i d , which may c o n g e a l t o a s o l i d form on s t a n d i n g . I t h a s a m i l d c h a r a c t e r i s t i c o d o r and a b i t t e r

taste

.

2. Physical Properties

2 . 1 I n f r a r e d Spectrum The i n f r a r e d s p e c t r u m o f N. F. Tybamate Reference S t a n d a r d (1) i s shown i n F i g u r e 1. The spectrum was o b t a i n e d on a 0.5% d i s p e r s i o n o f tybamate, p r e v i o u s l y d r i e d u n d e r vacuum a t 30° t o 35OC for 4 h o u r s , i n a p o t a s s i u m bromide d i s c . The f o l l o w i n g band a s s i g n m e n t s have been made ( 2 ) : C h a r a c t e r i s t i c of Wave Number, cm. -1

NH s t r e t c h i n g

3350 1720 1540 1255 620

Amide I band Amide I1 band Amide I11 band Amide IV band

496

P

W

4

Figure 1

-

IR spectrum of N.F. Tybamate Reference Standard in KBr dispersion.

PHILIP RElSBERGetaL

2 . 1 I n f r a r e d Spectrum Cont'd. A mjneral o i l (Nujol) mull of c r y s t a l l i n e tybamate can be p r e p a r e d much more e a s i l y t h a n a s a t i s f a c t o r y K B r d i s c , mainly because o f t h e low m e l t i n g r a n g e o f tybamate. The i n f r a r e d s p e c t r u m o f a 1%m i n e r a l o i l mull of tybamate i s shown i n F i g u r e 2 .

2.2 N u c l e a r Magnetic Resonance Spectrum The NMR s p e c t r u m as p r e s e n t e d i n F i g u r e 3 was o b t a i n e d by k i n g a 12.5% s o l u t i o n o f tybama t e i n carbon t e t r a c h l o r i d e c o n t a i n i n g t e t r a m e t h y l s i l a n e as t h e i n t e r n a l s t a n d a r d . The f o l l o w i n g peak a s s i g n m e n t s have been made: Chemical S h i f t ,ppm. P r o t o n

No. of P r o t o n s

9

(a)

0.9

CH3-C

(b)

1.3

C-CH2 -CH2- C

a

(c)

3.05

N-CH2

2

(d)

3.8

C-CH2-0

4

N -H N-H2

3

(e) 5.2

-

5.7

26

The peak a s s i g n m e n t s i n d i c a t e d i n F i g u r e 3 are shown on t h e f o l l o w i n g s t r u c t u r a l r e p r e s e n t a t i o n o f tybamate:

498

Figure 2

-

IR spectrum of N.F. Tybamate Reference Standard in Nujol mull.

VI

0 0

Figure 3

-

NMR spectrum of N.F.

Tybamate Reference Standard.

TYBAMATE

2.3 Mass Spectrum The low r e s o l u t i o n mass spectrum o f tybama t e ( 3 ) , shown i n F i g u r e 4 , e x h i b i t s t h e e x p e c t e d M+ o f m/e 2 7 4 . Long c h a i n a l k y l g r o u p s , p a r t i c u l a r l y t h o s e a t t a c h e d t o n i t r o g e n , fragment on t h e carbon-carbon bond p t o t h e n i t r o g e n f u n c t i o n w i t h r e t e n t i o n o f t h e c h a r g e on t h e a l k y l moiety. Cleavage of t h e N-butyl group r e s u l t s i n an m/e 43 i o n . The m/e 4 1 i o n , C3H5, arises i n t h e c l e a v a g e s of t h e branched hydrocarbon p o r t i o n o f t h e molecule. L i k e w i s e , an m/e 43 i o n arises i n t h e f r a g m e n t a t i o n o f t h e C3H7 moiety o f t h e branched hydrocarbon. The m/e 55-57 i o n s have t h e composition C 4 H 7 t o C Hg. The fragment i o n s r e s u l t i n g from d i r e c t bon8 c l e a v a g e are: m/e 2 9 , 43, 4 4 , 7 2 , 7 4 , 158, 2 0 0 , 2 1 4 , 231, and 245. The d i a g n o s t i c peak f o r c a r b a m a t e s , m/e 62, was p r e s e n t and was shown by C o u t t s ( 4 ) t o be NH2COOH2+. Through r e a r r a n g e m e n t , HOCONH2 i s l o s t from t h e M+ t o form t h e m/e 213 i o n . The same loss can o c c u r from t h e m/e 213 i o n t o form, t h e m/e 1 7 0 i o n . I n a p a r a l l e l f r a g m e n t a t i o n mode, t h e e l e m e n t s of OCONHz are l o s t from t h e m/e 231 i o n t o y i e l d t h e m/e 1 7 1 i o n . The v e r y i n t e n s e h i g h mass m/e 158 i o n can be formed from e i t h e r t h e m/e 200 i o n through l o s s o f CH3-CHmCHz ( 4 2 amu) o r from t h e M+ by t h e l o s s o f OCONHC4Hg. F i n a l l y , t h e m/e 184 i o n can a r i s e through t h e loss o f C 2 H g from t h e m / e 213 i o n . The mass spectrum i s i n agreement w i t h t h e s t r u c t u r e proposed f o r tybamate i n 1.1.

2 . 4 Thermal C h a r a c t e r i s t i c s 2 . 4 1 Melting Range C r y s t a l l i n e tybamate i n f i n e powder form, d r i e d under vacuum f o r 4 h o u r s a t 3Oo-35'C, melts w i t h i n a range o f 2' between 49' and 54'C.

2.42 Boilin

Ran e

s a t a p p r o x i m a t e l y 1500152'C a t a p r e s s u r e o f 0.06 mm. o f mercury. m

501

t t

t n

0 N

MASS/CHARGE

Figure 4

-

Mass Spectrum of N.F. Tybamate Reference Standard.

TYBAMATE

2 . 4 Thermal C h a r a c t e r i s t i c s C o n t ' d .

2.43 D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y T f e i r e 5 was o b t a i n e d w i t h a P e r k i n I E l m e r DSC-lb i n s t r u ment by f i r s t c o o l i n g t h e e n c a p s u l a t e d sample t o O°C w i t h a m i x t u r e o f d r y i c e and methanol. The sample was t h e n h e a t e d a t t h e r a t e o f 10°C p e r minute u s i n g a r e c o r d e r c h a r t s p e e d o f 1 i n c h p e r m i n u t e , and an o r d i n a t e s e n s i t i v i t y o f 8 m i l l i c a l o r i e s p e r s e c o n d . A s h a r p m e l t i n g endotherm o c c u r s a t a b o u t 46OC. P u r i t y c a n b e e s t i mated by comparison w i t h a r e f e r e n c e s t a n d a r d . 2.5 S o l u b i l i t y C h a r a c t e r i s t i c s

2.51 S o l u b i l i t y i n S e v e r a l S o l v e n t s S o l u b i l i t y of tybamate i n s e v e r a l s o l v e n t s was d e t e r m i n e d a t room t e m p e r a t u r e . Approximate s o l u b i l i t y d a t a i s shown i n T a b l e I . 2.52 P a r t i t i o n C o e f f i c i e n t s The p a r t i t i o n c o e f r i c i k n t s f o r tybama t e were d e t e r m i n e d f o r s e v e r a l s o l v e n t s y s t e m s a t room t e m p e r a t u r e . These a r e shown i n T a b l e 11. 2.6 O p t i c a l R o t a t i o n A 5 % a l c o h o l i c s o l u t i o n o f tybamate i s o p t i c a l l y i n a c t i v e when t e s t e d by t h e USP p r o cedure.

3. S y n t h e s i s Tybamate h a s been s y n t h e s i z e d ( 5 , 6 ) by t h e s e q u e n c e o f r e a c t i o n s shown i n F i g u r e 6 . The f i r s t s t e p i s the p r e p a r a t i o n of 2-methyl-2p r o p y l - 1 , 3 - p r o p a n e d i o l by r e a c t i n g 2 - m e t h y l p e n t a n a l and formaldehyde i n t h e p r e s e n c e o f p o t a s s i u m h y d r o x i d e . The d i o l is t h e n r e a c t e d w i t h d i e t h y l c a r b o n a t e i n t h e p r e s e n c e o f sodium m e t h y l a t e t o form 5-methyl-5-propyl-2-m-dioxanone. T h i s i s d i s t i l l e d and r e a c t e d w i t K 28% aqueous ammonia s o l u t i o n forming 2 - m e t h y l - 2 503

PHILIP REISBERG eta/.

Figure 5

-

DSC thermogram o f N . F . Tybamate Reference Standard

.

504

TYBAMATE

TABLE I

Approximate S o l u b i l i t y o f Tybamate

m l s . S o l v e n t Required per g . o f Tybamate

Solvent Water

2000

Ethyl Alcohol

0.6

Chloroform

0.5

Ethyl Ether

1

Propylene Glycol

2

TABLE I 1

P a r t i t i o n C o e f f i c i e n t s f o r Tybamate Part i t i on C oe f f i ci en t

S o l v e n t System Cottonseed Oil/Water

28

>loo

Chloroform/Water Carbon Tetrachloride/Water

10

Benzene/Water

43

0.2

Heptane/Water

505

CH3 I CH3-CH2-CH2-C-CH0 I

H

-

“””\ ICH2OH C

CH 2O KOH

CH3 - CH2 - CH2

( 2 -methy 1pent an a1 )

CH3\

m 0

(2-methyl-2-propyl1,3-propanediol)

0 I /CHZ-O-C-NH2

NH40H

CH3-CH2-CH2

CH3-CHZ-CH2

CH3 3

(C2Hs0) 2C=0

2

FIG. 6:

1/

CH2 -0- C -NH2

SYNTHESIS OF TYBAMATE

J.

TYBAMATE

3. S n t h e s i s C o n t ' d . ProPY k r o x y p r o p y l carbamate. This i n t u r n i s t r e a t e d w i t h b u t y l i s o c y a n a t e t o form N - b u t y l 2-methyl-2-propyl-l,3-propanediol d i c a r b a m a t e (tybamate)

.

3.1 P u r i f i c a t i o n and C r y s t a l l i z a t i o n

T h e m p u r e s y n t h e s i z e d tybamate p r o d u c t i s c r y s t a l l i z e d from h e x a n e - b u t a n o l , o r x y l e n e naptha solvent mixtures. S u c c e s s i v e c r y s t a l l i z a t i o n s o f tybamate from t r i c h l o r e t h y l e n e , SDA-3A a l c o h o l , and from h o t w a t e r by d i l u t i o n w i t h c o l d water w i l l r e move most i m p u r i t i e s . The l i q u i d form o f tybamate may b e conv e r t e d t o t h e c r y s t a l l i n e form by d i s s o l v i n g t h e f o r m e r i n a m i x t u r e o f one p a r t o f t r i c h l o r e t h y l e n e and two p a r t s o f h e x a n e , and c o o l i n g . 4. S t a b i l i t

d i s v e r y s t a b l e c h e m i c a l l y ( 7 ) as a s o l i d o r as a l i q u i d . I f d e g r a d a t i o n s h o u l d o c c u r , i t c o u l d be v i a t h e r m a l o r h y d r o l y t i c c l e a v a g e o f t h e carbamate g r o u p . As N - s u b s t i t u t i o n i n c r e a s e s , s t a b i l i t y i n c r e a s e s and t h u s tybamate would be e x p e c t e d t o b e more s t a b l e t h a n meprobamate which h a s no N - s u b s t i t u t i o n . It has been shown t h a t e v e n c a r b a m a t e s o f t h e meprobamate t y p e do n o t d e g r a d e r e a d i l y u n d e r t h e above c o n d i t i o n s . ( 7 ) I t i s s t a b l e i n d i l u t e a c i d o r i n d i l u t e a l k a l i , and i s n o t b r o k e n down a t 37OC i n g a s t r i c o r i n t e s t i n a l f l u i d . Hot a l k a l i o r s t r o n g a c i d h y d r o l y s e tybamate t o y i e l d t h e c o r r e s p o n d i n g d i o l , ammonia, b u t y l a m i n e and c a r b o n d i o x i d e . Dosage forms o f tybamate ( t a b l e t s , soft g e l a t i n c a p s u l e s ) have shown no loss o f p o t e n c y a f t e r a 5 y e a r p e r i o d a t room t e m p e r a t u r e .

507

PHILIP REISBERG eta/.

5, Drug Metabolic Products Douglas, Ludwig, Schlosser, and Edelson (8) have identified 4 compounds in the urine of both dogs and cats after administration of tybamate. These are: the unchanged drug, meprobamate (I), hydroxymeprobamate (11), and the major metabolite, The metabolites are hydroxytybamate (111). shown in Figure 7. 6. Methods of Identification and Analysis 6.1 Elemental Analysis A typical result obtained on medicinal grade tybamate using the F & M Model 185 CH & N Analyser (9) is shown below: % Theoretical

E lemen t Carbon Hydrogen Nitrogen

56.90 9.55 10.21

0 Found

56.51 9.45 10.30

6.2 Identification Tests 6.21 Derivative Formation A crystalline derivative of tybamate with xanthydrol in glacial acetic acid can readily be prepared. This reaction is characteristic of carbamates.(lO) H

‘

0

OH

H O \ II N-C-R

H

/

R-8-N-H2

0

+

H20

R = balance of Tybamate molecule

508

TY BAMATE

(I) Meprobamate 0

(11) Hydroxymeprobamate 0

II

CH2-0-C-NH2

/ -I / \ O HcH3\

CH3 C- CH2

CHz-O-C-NHz

I H

II

0

(111) Hydroxytybamate

0

II

FIG. 7: METABOLIC PRODUCTS OF TYBAMATE 509

PHILIP REISBERG e t a / .

6 . 2 2 C o l o r i m e t r i c I d e n t i f i c a t i o n Test A red c o l o r is formed when tybamate

i s h e a t e d w i t h p-dimethylaminobenzaldehyde, a n t i mony t r i c h l o r i d e , and a c e t i c a n h y d r i d e . (11) 6 . 3 Cobalt Cyanate C o l o r i m e t r i c Assay

Tybamate i s h y d r o l y s e d i n a l k a l i n e a l c o h o l i c medium t o y i e l d c y a n a t e i o n which forms a b l u e complex w i t h c o b a l t i o n . The a b s o r p t i o n i s determined a t 590 nm. (12) An automated method b a s e d on t h e c o b a l t complex h a s been d e v e l o p e d f o r N - u n s u b s t i t u t e d carbarnates and c a n be a p p l i e d t o tybamate. (13) 6 . 4 H y p o c h l o r i t e C o l o r i m e t r i c Assay

A t pH 10.5 tybamate reacts w i t h hypochl o r i t e t o form an " a c t i v e " c h l o r i n e d e r i v a t i v e . The e x c e s s h y p o c h l o r i t e i s decomposed w i t h p h e n o l i n d i l u t e a c i d . The c h l o r i n a t e d compound i s r e a c t e d w i t h e x c e s s p o t a s s i u m i o d i d e and t h e l i b e r a t e d i o d i n e i s measured c o l o r i m e t r i c a l l y a t 357

nm. (14)

6 . 5 Volumetric A n a l y s i s s-

upon t h e s o l v o l y s i s o f t h e u n s u b s t i t u t e d carbamate group by sodium methoxide i n a nonaqueous medium. The e x c e s s sodium methoxide i s t i t r a t e d w i t h 0.1N hydrochl o r i c a c i d u s i n g p h e n o l p h t h a l e i n T.S. as t h e i n d i c a t o r . (11) C e r r i , e t a1 (15) have shown t h a t t h i s method i s s p e c i f i c for t h e non-N-subs t i t u t e d c a r b a m a t e s . They have p o s t u l a t e d t h e f o l l o w i n g mechanism f o r t h e r e a c t i o n : (1)

0

OH

I

II

R-0-C-NH2 (2)

R-0-C=NH

OH I R-0-b-NH

ONa

I

+

CH3ONa+R-O-b=NH

510

+ CH30H

TYBAMATE

6.5 Volumetric Analysis Cont'd. (3)

ONa

I

R-O-CmNH-R-OH

+

NaO-CEN

I n anhydrous p y r i d i n e , t h e e q u i l i b r i u m shown i n the f i r s t equation is strongly s h i f t e d t o the r i g h t . The e n o l i c s p e c i e s r e a c t s w i t h t h e sodium methoxide. I n aqueous medium, t h e k e t o form i s f a v o r e d and t h e r e a c t i o n w i t h a l k a l i i s no l o n g e r quantitative. 6.6 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 6.61 I n f r a r e d I d e n t i f i c a t i o n The i n f r a r e d a b s o r p t i o n s p e c t r u m o f a 1 0 % s o l u t i o n o f tybamate i n c h l o r o f o r m i s comp a r e d w i t h t h e s p e c t r u m o b t a i n e d w i t h N.F. Tybama t e R e f e r e n c e S t a n d a r d . No e x t r a n e o u s bands s h o u l d be found. M i n e r a l o i l ( N u j o l ) m u l l s have a l s o been u s e d f o r o b t a i n i n g tybamate I R s p e c t r a . 6.62 N u c l e a r Magnetic Resonance An NMR method was d e v e l o p e d f o r t h e a n a l y s i s o f meprobamate t a b l e t s u s i n g m a l o n i c a c i d as an i n t e r n a l s t a n d a r d . (16) A s i m p l e a d a p t i o n o f t h i s method c a n be u s e d f o r tybamate. (17) P o s s i b l e d e g r a d a t i o n o f tybamate t o N-butyl-2-methyl-2-propyl-3-hydroxypropylcarbama t e can be d e t e c t e d by o b s e r v i n g t h e NMR s i g n a l s o f t h e p r o t o n s o f t h e methylene g r o u p s a t t a c h e d t o oxygen, I n t y b a m a t e , t h e r e a r e 2 s u c h g r o u p s and t h e y a p p e a r as a s i n g l e t . I f h y d r o l y s i s o c c u r s , t h e s i n g l e t w i l l d e c r e a s e and a new s i g n a l w i l l s t a r t a p p e a r i n g down f i e l d from t h e methylene s i n g l e t . 6.7 Gas Chromatographic Methods 6 . 7 1 Tybamate i n Dosage Forms A method f o r t h e d e t e r m i n a t i o n o f tybamate i n dosage forms i s made a v a i l a b l e by a d a p t a t i o n o f t h e procedure o f Rabinowitz, e t 511

PHILIP RElSBERGetaL

6 . 7 1 Tybamate i n Dosage Forms Cont'd. a1.(18) T h i s i n v o l v e s a simple e x t r a c t i o n p r o cedure, followed by chromatography on a 3.8% OV1 7 column w i t h meprobamate a s t h e i n t e r n a l s t a n d a r d . Other methods u s i n g d i f f e r e n t i n t e r n a l s t a n d a r d s have been r e p o r t e d . (19,ZO)

6.72 Tybamate i n B i o l o g i c a l F l u i d s A method for t h e d e t e r m i n a t i o n o f tybamate i n b i o l o g i c a l f l u i d s has been r e p o r t e d by Douglas, e t a 1 , ( 2 1 ) which employs g l a s s c o l umns packed w i t h 3.89 UC-W98 methyl s i l i c o n e on 80-100 mesh D i a t a p o r t S. A l i n e a r r e l a t i o n s h i p has been e s t a b l i s h e d u s i n g d i b u t y l phthal a t e a s t h e i n t e r n a l s t a n d a r d . Other carbarnates a r e determined s i m i l a r l y . 6.8 Thin-Layer Chromatographic Methods 6.81 I d e n t i t y and P u r i t y o f Commercial

a 1 0 % s o l u t i o n of tybamate f -F i n chloroform i s s p o t t e d on a s i l i c a g e l p l a t e . The chromatogram i s developed w i t h a mixture of chloroform and acetone ( 4 : 1 ) , t h e p l a t e i s a i r d r i e d f o r s e v e r a l minutes, sprayed w i t h a s a t u r a t e d s o l u t i o n of antimony t r i c h l o r i d e i n c h l o r o form and then w i t h a 31 s o l u t i o n o f r e d i s t i l l e d f u r f u r a l i n chloroform. A f t e r 10 t o 1 5 minutes dark s p o t s a r e v i s i b l e on a w h i t e t o grey background which slowly darkens. Pure tybamate shows o n l y one s p o t , Commercial m a t e r i a l may have t r a c e q u a n t i t i e s of meprobamate and 2-methyl-2propyl-3-hydroxypropyl carbamate w i t h Rf v a l u e s o f 0.36 and 0 . 5 1 r e l a t i v e t o tybamate. (22) 6.82 I d e n t i f i c a t i o n i n Presence o f Other &%a1 o t h e r TLC methods a r e a v a i l 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 tybamate i n t h e presence of o t h e r p s y c h o t r o p i c drugs i n blood or u r i n e . P o s i t i v e i d e n t i f i c a t i o n has been o b t a i n e d by t h e u s e of 5 d i f f e r e n t chromatographic systems, (19,ZO) 512

TYEAMATE

7 . Pharmacokinetics

Peak serum c o n c e n t r a t i o n s o f tybamate were obt a i n e d a b o u t 1 hour a f t e r o r a l a d m i n i s t r a t i o n . The major m e t a b o l i t e found i n t h e u r i n e was h y d r o xytybamate. Serum l e v e l s o f tybamate were d e t e r mined i n male mongrel dogs by t h e c o l o r i m e t r i c method o f Hoffman and Ludwig (25) a f t e r o r a l a d m i n i s t r a t i o n of 1 5 mg./Kg. Metabolic s t u d i e s were c a r r i e d o u t a f t e r t y b a m a t e , 100 mg./Kg., was g i v e n d a i l y f o r 5 d a y s . The d i s t r i b u t i o n of tybamate i n r a t t i s s u e was o b s e r v e d a f t e r o r a l a d m i n i s t r a t i o n o f 3 t o 7 mg. o f c a r b o n - 1 4 l a b e l e d d r u g . Tybamate was r a p i d l y a b s o r b e d ; a maximum b l o o d serum l e v e l i n dogs o f a b o u t 10 mcg./ml. occurred within 1 hour a f t e r administration. Serum h a l f - l i f e was a b o u t 3 h o u r s and no d r u g was d e t e c t a b l e a f t e r 24 h o u r s , N e i t h e r tybamate n o r i t s m e t a b o l i t e s were r e t a i n e d by r a t t i s s u e , b u t were e x c r e t e d a l m o s t e n t i r e l y i n t h e u r i n e d u r i n g a 24 h o u r p e r i o d . (5)

513

PHI LIP RE ISBE R G e t a / .

8 . References

1. A. Z . Hayden, W. L. Brannen, and C . A. Yaciuw, J . Ass. O f f i c . Agr. Chem., 49, 1109 (1966). 2 . K J . Bellamy, "The I n f r a r e d S p e c t r a o f Complex Molecules", 2nd e d . , J o h n Wiley and S o n s , I n c . , New York, N . Y . , 1 9 6 4 , chap. 1 2 . 3. K . F l o r e y and P. T. Funke, S q u i b b I n s t i t u t e f o r M e d i c a l R e s e a r c h , p e r s o n a l communication. 4 . R. T. C o u t t s , J . Pharm. S c i . , 6 2 , 7 6 9 ( 1 9 7 3 ) . 5 . F. M. Berger and B. J , L u d w -i g. , T . S . P a t e n t 2,724,720-(1955). 6. B. J. Ludwig and E. C. P i e c h , J . Amer. Chem. SOC., 73, 5779 (1951). ams and F. A. Baron, Chem. Revs., 65, 7. !67*:1965). 8. J. F. Douglas, B. J . Ludwig, A. S c h l o s s e r , and J . E d e l s o n , B i o c h e m i c a l Pharmacology, 1 5 , 2087, ( 1 9 6 6 ) . A. Brenner, Carter-Wallace, I n c . , un9. published data. 1 0 . E . F. S a l i m , J . I . Bodin, H. B. Zimmerman, and P. R e i s b e r g , J. Pharm. S c i . , 5 5 , 1439 (1966). 11. "The N a t i o n a l Formulary", 1 3 t h e d . , Mack P u b l i s h i n g Co., E a s t o n , P a . , 1 9 7 0 , p.749. 1 2 . G . Devaux, P. Mesnard, and J . Cren, P r o d . Pharm., 1 8 , 2 2 1 (1963). C u n e n . L. J . Heckman. a n d G. J . 13. P a p a r i e l l o , J . Pharm. S c i . , 58, 1537 (1969) 1 4 . G. H. E l l i s and C. A. Hetzel. Anal. Chem.. 3 1 , 1090 ( 1 9 5 9 ) . 1 5 . K C e r r i , A. S i a l t i n i , and U. G a l l o , Pharm. Acta Heyv., 34, 1 3 (1959) 16. 3. W. T u r c z a n a n d T. C . K r a m , J . Pharm.Sci. 56, 1 6 4 3 ( 1 9 6 7 ) . 1 7 . TT A. B r e n n e r a n d P. R e i s b e r g , CarterWallace, I n c . , u n p u b l i s h e d d a t a . 1 8 . M. P. R a b i n o w i t z , P. R e i s b e r g , a n d J. I . Bodin, J . Pharm. S c i . , 6 1 , 1974 ( 1 9 7 2 ) . 1 9 . R. C . G r a n t , F.D.A. B y - L i n e s , 2 ( 1 ) : 1 0 - 1 4 , J u l y (1971). ~

514

.

TY BAMATE

20. C . C a r d i n i , V. Q u e r c i a , a n d A. C a l o , J-. Chromato r 37, 190 ( 1 9 6 8 ) . 21. OUI!" . .sa B. S m i t h . and J . A . S t o c k a g e , J . Pharm. S c i . , 5 8 , 145 ( 1 9 6 9 ) . 2 2 . A . E. Martin, A. H. R o b b i n s C o . , p e r s o n a l communication. 23. I . Z i n g a l e s , J. Chromatogr., 31, 405 ( 1 9 6 7 ) . 29, 283 2 4 . T. W. McConnell, J. C h r o m a t o g r , (1967). 25. A. J . Hoffman and B. J . Ludwig, J. A m e r . Pharm. A s s . , S c i Ed., 4 8 , 740 ( l g r

Y77PT'

General References

1. B . J . Ludwig, L.S. P o w e l l , and F.M. B e r g e r , J . Med. Chem., 1 2 , 462 (1969). 2 . B . J . Ludwig a n d 7 . R . P o t t e r f i e l d , Adv. 9, Pharmacology and Chemotherapy, 1971). 9 . Acknowledgements The a u t h o r s wish t o e x p r e s s t h a n k s t o D r . K. F l o r e y and D r . P.T. Funke o f t h e Squibb I n s t i t u t e f o r Medical Research f o r p r o v i d i n g t h e mass s p e c t r a l d a t a , t o Urs. B . J . Ludwig and D . R e i s n e r o f Wallace L a b o r a t o r i e s f o r s u g g e s t i n g improvements i n t h e m a n u s c r i p t , and t o Mr. I . A . Brenner, Miss R. Rader, and Mrs. M. B e l a s o n f o r a s s i s t a n c e i n its preparation.

515

ADDENDA

ADDENDA

Ampicillin 6.21

Spectrophotometric determination of ampicillin sodium in the presence of its degradation and polymerization products. H. Bundgood, J. Pharm. Pharmac. 26, 385 (1974).

Chloramphenicol 3.

Biosynthesis of Chloramphenicol. Origin and Degradation of the Aromatic Ring. W. P. O'Neill, R. F. Nystrom, K. L. Rinehart Jr. and D. Gottlieb, Biochemistry l2, 4775 (1973).

Chlordiazepoxide Hydrochloride 6.6

Determination of Chlordiazepoxide Hydrochloride and its Major Metabolites in Plasma by Differential Pulse Polarography. M. R. Hackman, M. A. Brooks, J. A. F. de Silva and T. S. Ma, Anal. Chem. 46,1075 (1974).

Diazepam 6.33

Quantitative Determination of Medazepam, Diazepam and Nitiazepam in Whole Blood by Flame-Ionization Gas-Liquid Chromatography. M. S. Graeves, Clin. Chem. 2 0 , 141 (1974),

518

ADDENDA

Dexamethasone 5.

Radioimmunoassay for Dexamethasone in Plasma. M. Hichens and A. F. Hogans, Clin. Chem. 20,266 (1974).

Fluphenazine Hydrochloride 5.

Metabolism in rats; in vitro and urinary metabolites. H. J. Gaertner, U. Breyer and G. Liomin, Biochem.Pharmac. 23, 303 (1974). Formation of identical metabolites from piperazine and dimethylaminosubstituted phenothiazine drugs in man, rat and dog. U. Breyer, H. J. Gaertner and A. Prox, Biochem. Pharmac. 23,313 (1974)

.

6.72

Thinlayer chromatography of phenothiazine derivative and analogues. A. deleenheer, J. Chromatog. 75, 79 (1973).

6.74

Gas-liquid chromatographic analysis of fluphenazine and fluphenazine sulfoxide in the urine of chronic schizophrenic pa tien ts M. I. Kelsey, A. Keskiner, E. A. Moscatelli, J. Chromatog. 75,294 (1973).

.

519

ADDENDA

Meprobamate 6.63

GLC Determination of Meprobamate in Water, Plasma and Urine. L. Mortis and R. H. Levy, J. Pharm. Sci. 63,834 (1974).

Methadone Hydrochloride 5.4

Biliary excretion of methadone by the rat; identification of a para-hydroxylated major metabolite. R. C. Baselt and M. H. Bickel, Biochem. Pharmac. 22,3117 (1973).

Methaaualone 5.

Identification of Free and Conjugated Metabolites of Methaqualone by Gas ChromatographyMass Spectrometry R. Bonnichsem, Y. Marde and R. Ryhage, Clin. Chem. 20,230 (1974). Qualitative and Quantitative Determination of Methaqualone in Serum by Gas Chromatography. M. A . Evenson and G. L. Lensmeyer, Clin. Chem. 20,249 (1974).

Propoxyphene Hydrochloride

4.

Fluorometric Determination of Propoxyphene. J. C. Valentour, J. R. Monforte and I. Sunshine, Clin. Chem. 20, 275 (1974).

520

ADDENDA

Sulfamethoxazole 5.

Modification of an Automated Method for Measurement of S ulfamethoxazole and its Major Metabolite in Biological Fluids. A. Bye and A. F. J. Fox, Clin.Chem. 20,288 (1974).

Triamcinolone 2.4

Mass spectrometry of some corticosteroids and related compounds of pharmaceutical interest. P. Taft, B. A . Lodge and M. B. Simard, Can. J. Pharm.Sci. -7,53 (1972).

Triamcinolone Acetonide

5.

6@-Hydroxylation of trimcinolone acetonide by a hepatic enzyme system. D. Kupfer and D. Partridge, Arch. Biochem. Biophys., 140, 23 (1970).

Triflupromazine Hydrochloride 5.

Formation of identical metabolites from piperazine and dimethylaminosubstitu ted phenothaizine drugs in man, rat and dog. U. Breyer, H. J. Gaertner and A. Prox, Biochem. Pharmac. 23,313 (1974).

52 1

ADDENDA

6.72

Thinlayer Chromatography of phenothiazine derivatives and analogues. A. deleenheer, J. Chromatog. 75 (1973).

522

ERRATA

ERRATA

Fluphenazine Enanthate 6.2

Vol. I1 p. 258 Neutralization equivalent: calc. 275. not 285.

Triamcinolone 1.1

Vol.1 p.369 Molecular weight: 394.45 not 434.49

524

CUMULATIVE INDEX Italic numerals refer to Volume numbers. Acetaminophen, 3, 1 Acetohexamide, 1, 1;2, 573 Alpha-Tocopheryl Acetate, 3, 1 1 1 Amitriptyline Hydrochloride, 3, 127 Ampicillin, 2. 1 ;4, 5 17 Cefazolin, 4, 1 Cephalexin, 4, 2 1 Cephalothin Sodium, I , 319 Chloral Hydrate, 2, 85 Chloramphenicol, 4, 4 7 , 5 17 Chlordiazepoxide, 1, 15 Chlordiazepoxide Hydrochloride, I , 39; 4, 517 Chlorprothixene, 2, 6 3 Clidinium Bromide, 2, 145 Clorazepate Dipotassium, 4, 9 1 Cloxacillin Sodium, 4 , 1 1 3 Cycloserine, I , 5 3 Cyclothiazide, I , 66 Dexamethazone, 2, 163; 4, 5 18 Diatrizoic Acid, 4, 137 Diazepam, 1, 79; 4 , 5 17 Digitoxin, 3, 149 Dioctyl Sodium Sulfosuccinate, 2, 199 Diphenhydramine Hydrochloride, 3, 173 Disulfiram, 4, 168 Echothiophate Iodide, 3, 233 Erythromycin Estolate, I , 101;2, 573 Estradiol Valerate, 4, 192 Ethynodiol Diacetate, 3, 253 Fludrocortisone Acetate, 3, 28 1 Fluorouracil, 2, 22 I

525

Fluphenazine Enanthate, 2, 245; 4, 523 Fluphenazine Hydrochloride, 2, 263; 4, 5 I8 Flurazepam Hydrochloride, 3, 307 Halothane, I , 119;2, 573 Hydroxyprogesterone Caproate, 4 , 209 lodipamide, 3, 333 Isocarboxazid, 2, 295 Isopropamide,2, 315 Isosorbide Dinitrate; 4, 225 Levartereno1 Bitartrate, 1, 49; 2, 573 Levallorphan Tartra?e, 2, 339 Meperidine Hydrochloride, I , 175 Meprobamate, I , 209; 4, 519 Methadone Hydrochloride, 3, 365;4, 519 Methaqualone, 4 , 2 4 5 , 5 19 Methyprylon, 2, 363 Norethindrone, 4, 268 Norgestrel, 4, 294 Nortriptyline Hydrochloride, I , 233; 2, 573 Oxazepam, 3 , 4 4 1 Phenazopyridine Hydrochloride, 3 , 4 6 5 Phenelzine Sulfate, 2, 383 Phenformin Hydrochloride, 4, 3 19 Phenoxymethyl Penicillin Potassium, I , 249 Phenylephrine Hydrochloride, 3,48 3 Primidone, 2,409 Procainamide Hydrochloride, 4, 333 Propiomazine Hydrochloride, 2,439 Propoxyphene Hydrochloride, I , 301 ;4, 519

CUMULATIVE INDEX

Reserpine, 4, 384 Secobarbital Sodium, 1, 343 Spironolactone, 4, 431 Sulfamethoxazole, 2, 467; 4, 520 Sulfisoxazole, 2 , 4 8 7 Testosterone Enanthate, 4, 452 Theophylline, 4, 466 Tolbutamide, 3, 5 13 Triamcinolone, I, 367;2, 571;4, 520,523 Triamcinolone Acetonide, 1,397;2, 57 1 ; 4,520

Triamcinolone Diacetate, I, 423 Triclobisonium Chloride, 2, 507 Triflupromazine Hydrochloride, 2,523; 4, 520 Trimethaphan Camsylate, 3, 545 Trimethobenzamide Hydrochloride, 2, 55 1 Tropicamide, 3, 565 Tybamate, 4 , 4 9 4 Vinblastine Sulfate, 1, 443 Vincristine Sulfate, I, 463

A 5 8 6 c 7 D € F G H

526

8 9 O 1 2

1 3 1 4