Analytical Profiles of Drug Substances

Volume 3
Edited by

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

Contributing Editors

Norman W. Atwater Salvatore A. Fusari Olenn A. Brewer, Jr. Erik H. Jensen Lester Chafetz Boen T. Kho Jack P. Comer Gerald J. Papariello Bernard 2. Senkowski

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

Academic Press New York and London 1974

A Subsidiary of Harcourt Brace Jovanovich, Publishers

EDITORIAL BOARD

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

David E. Guttmaa Erik H. Jensen Boen T. Xho Arthur F. Michaelis Oerald J. Papariello Bernard Z. Senkowski Frederick Tishler

AMERICAN PHARMACEUTlCAL ASSOCIATION COPYRIGHT 1974, BY THE ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD.

Library of Congress Cataloging in Publication Data Main entry under title:

Analytical profiles of drug substances. Compiled under the auspices of the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences. Includes bibliographical references. 1. Drug-Collected works. 2. Chemistry, Medical and pharmaceutical-Collected works, I, Florey, Klaus, 11. Brewer, Glenn A. 111. Academy of Pharmaed. ceutical Sciences. Pharmaceutical Analysis and Control 1. Drugs-Analysis-Yearbooks. Section. [DNLM: QV740 AA1 ASS] RM300 .AS 6 615'.1 7 0-187 259 ISBN 0-12-260803-5 (v. 3)
PRINTED IN THE UNITED STATES OF

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AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

N. W. Atwater, Searle and Company, Chicago, Illinois
W. F. Beyer, The Upjohn Company, Kalamazoo, Michigan

K. W. Blessel, Hoffmann-LaRoche, Inc., Nutley, New Jersey

R . H. Bishara, Eli Lilly and Company, Indianapolis, Indiana C. A . Brewer Jr., The Squibb Institute for Medical Research, New Brunswick, New Jersy L . Chafetz, Warner-Lambert Research Institute, Morris Plains, New Jersey E. M . Cohen, Merck, Sharp and Dohme, West Point, Pennsylvania J. P. Comer, Eli Lilly and Company, Indianapolis, Indiana

R . D. Daley, Ayerst Laboratories, Rouses Point, New York

J. E. Fairbrother, The Squibb Institute for Medical Research, Moreton, Wirral, England K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey R . I. Fryer, Hoffmann-LaRoche, Inc., Nutley, New Jersey

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

C. A . Caglia, Jr., Warner-Lambert Research Institute, Morris Plains, New Jersey

vii

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

D. E. Guttman, School of Pharmacy, University of Kentucky, Lexington, Kentucky I. J. Holcombe, Parke, Davis and Company, Detroit, Michigan
I. M. Jakovljeric, Eli Lilly and Company, Indianapolis, Indiana

E. H. Jensen, The Upjohn Company, Kalamazoo, Michigan B. T. Kho, Ayerst Laboratories, Rouses Point, New York E. P. K. Lau, Searle and Company,Chicago, Illinois H. H. Lerner, The Squibb Institute for Medical Research, New Brunswick, New Jersey
A . F. Michaelis, Sandoz Pharmaceuticals, East Hanover, New Jersey G. J. Papariello, Wyeth Laboratories, Philadelphia, Pennsylvania

C. R. Pilla, Wyeth Laboratories, Philadelphia, Pennsylvania E. L. Pratt, The Sterling-Winthrop Research Institute, Rensselaer, New York B. C. Rudy, Hoffman-LaRoche, Inc., Nutley, New Jersey B. 2. Senkowski, Hoffmann-LaRoche, Inc., Nutley, New Jersey
C. M . Shearer, Wyeth Laboratories, Philadelphia, Pennsylvania

viii

PREFACE
Although the official compendia list tests and limits for drug substances related t o identity, purity, and strength, they normally do not provide other physical or chemical data, nor d o they list methods of synthesis or pathways of physical o r biological degradation and metabolism. For drug substances important enough to be accorded monographs in the official compendia such supplemental information should also be made readily available. To this end the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences, has undertaken a cooperative venture t o compile and publish Analytical Profiles of Drug Substances in a series of volumes of which this is the third. Reviews and comments received so far have reinforced our belief that the series fills a need and they have strengthened our determination to continue. The enthusiasm and cooperative spirit of our contributors have made these profiles possible. All those who have found the profiles useful are earnestly requested t o contribute a monograph of their own. The editors stand ready to receive such contributions. Beginning with Volume 2 a cumulative index has been added, t o facilitate the correction of errors and t o encourage the addition or relevant new information. 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 t o provide physico-chemical and analytical information on 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 compendial status. Klaus Florey

i x

ACETAMINOPHEN

John E. Fairbrother

JOHN E. FAIRBROTHER

CONTENTS
1.
2.

3.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color Physical Properties 2.1 Spectra 2.11 Infra-red Spectrum 2.12 Ultra-violet Spectrum 2.13 Fluorescence Spectrum 2.14 N.M.R. Spectrum 2.15 Mass Spectrum 2.2 Physical Properties of the Solid 2 . 2 1 Melting Characteristics 2.22 Density 2.23 Vapor Pressure and T.G.A. 2.24 D.T.A. and D.S.C. 2.25 Crystal Characteristics 2.26 X-ray Diffraction 2.3 Powder Characteristics 2 . 3 1 Static Charge 2.32 Flow Properties 2.33 Compression Characteristics 2.34 Surface Area and Porosity 2.4 Solubility 2.41 Solubility in Aqueous Solvents 2.42 Solubility in Water Miscible Solvents 2.43 Solubility in Solvents Immiscible with Water 2.44 Rate of Dissolution 2.5 Physical Properties of Solutions 2 . 5 1 Cryoscopy 2.52 Ionisation and pH 2.53 Dipole Moment 2.54 Refractive Index 2.55 Adsorption from Solution 2.56 Partition Coefficients Molecular Complexes

ACETAMINOPHEN

4.

5.

6.

Synthesis and Purification 4 . 1 Chemical Synthesis 4.11 Synthetic Routes 4.12 Purification 4.13 Impurity Profile 4.14 Reference Standards 4.2 Biosynthesis 4.21 Metabolism of Phenacetin and Acetani1ide 4.22 Prodrugs 4.23 Microbial Biosynthesis Stabi1ity 5.1 Stability to Light 5.2 Stability of Solid Acetaminophen to Heat Stability of Solutions of Acetamino5.3 phen 5.4 Stability to Oxidation 5 . 5 Compatibility with Excipient Materials 5.6 Compatibility with Aspirin 5.7 Physical Incompatibilities Analytical Chemistry 6.1 Identity Tests 6.2 Methods of Analysis 6.20 Gravimetric Procedures T itrimet ric Procedures 6.21 Polarographic Procedures 6.22 6.23 U.V. Spectrophotometric Procedures 6.24 Photocolorimetric Procedures 6.25 Ion-Exchange Chromatrographic Procedures 6.26 Partition Chromatographic Procedures 6.27 Paper and Thin-Layer Chromatographic Procedures 6.28 Vapor' Phase Chromatographic Procedures High - Pressure Liquid Chrom6.29 atographic and Gel Filtration Procedures 6.3 Automated Procedures 6.4 Radiochemical Procedures

JOHN E. FAIRBROTHER

Determination of Trace Impurities and Degradation Products 7 of Acetami nophen and 6.6 Luids and Tissues 6.61 Determination in Urine 6.62 Determination in Serum, Plasma and Whole Blood 6.63 Determination in Tissues and Orsans 7. Metabolic Transzormations 7.1 Metabolism in Man 7.11 Adults 7.12 Newborn Infants 7.2 Metabolism in Animals 8. Drug Availability 8.1 Pharmacokinetics 8.2 Protein Binding 8.3 Interactions with Other D,rugSubstances 8.4 Biopharmaceutics 9. Toxicity References
6.5

ACETAMINOPHEN

1.

Description

1.1

Name, Formula, Molecular Weight Generic names - Acetaminophen1, Paracetamol and Acetophenum2. Chemical names - 4 - Hydroxyacetanilide; p-hydroxyacetanilide; pacetamidophenol; p-acetaminophenol; p-acetylaminophenol; N-acetyl-paminophenol.
1

gHgN02 1.2

Mol. wt.

151.16

Appearance, Color, Odor, Taste

White, odorless, crystalline powder, possessing a bitter taste. 2. Physical Properties

2.1

Spectra 2.11 Infrared Spectrum

Infrared spectra of solid dis ersions of acetaminophen in potassium bromide3 t 7 and in 6 Nujo16, have been recorded. In the solid state the carbonyl stretching band appears at 1659 c m - I (1650 cm-1; ref. 3 ) , the N-H stretching band at 3326 cm-1 and a broad 0-H stretching band at 3162 cm-1. In solution the C=O,N-H and 0-H stretching bands occur at higher frequencies.

JOHN

E. FAIRBROTHER

Solvent Chloroform Dichloromethane 1,4-Dioxan

Stretching Band 1686cm-1 ( 9 ) 1690cm 1700cm-1 (8) 1692cm-1 (8)

c =o

N-H

0-H

Stretching Stretching Band Band 3435cm (6) 3588cm-1(8

Several other authors10,11,12,15,16 report infrared spectra of acetaminophen. The infrared spectra of acetaminophenl4 in KBr and in a mineral oil mull are presented in figures 1 and 213. 2.12 Ultraviolet Spectrum

The U.V. spectrum of acetaminophen has been recorded in a number of solvents, showing two bands in each. The long wavelength band corresponds to the A l g + B2u transition while the short wavelength band corresponds to the IT^ + T co transition1. *

Fig.

1.

I n f r a r e d spectrum of acetaminophen (KBr p e l l e t )

Fig. 2.

Infrared spectrum of acetaminophem (Mineral Oil Mull)

ACETAMINOPHEN

TABLE 1 Absorption maxima of acetaminophen in neutral solvents Solvent

K band
248-249mp. 249-250mu. 250mv. 250mp. 244-245mi.l. 278mp. 264mi.l. 247mp. 242.5243.5mp.

B band

Referenca

Me thano1 Ethanol (abs.) n-Butanol iso-Propanol Cyclohexane Cyclohexane Ether Ether (dry) Water

3.18 about 290mi.l. 4, 8,19 20 19 19 8 19 about 283mi.l. 8 about 283mp. 8,19,23

The addition of acid to aqueous and alcoholic solutions does not give any observable change in the sition of the maximum of the main band 7 I 16r~~,19r21,22* In 10-1M caustic alkali acetaminophen ionises to give the pacetamidophenolate ion and the maximum of the main band is shifted bathochromically, in aqueous solution from 243 mu. to about 258 mu. 19,20,22,23 anpSin methanolic solution from 248 mp. to 262 mp.

JOHN E. FAIRBROTHER

TABLE 2 Molar absorptivities ( c ) of acetaminophen in different solvents Solvent Ethanol Wavelength 249 mu. 288 mu. Me thano1 Ethanol/O.lN Hydrochloric Acid Water (pH 2 to 3) Water ( p H 7.2) (Clark and Lubs Buffer) 0.1N Sodium Hydroxide 0.01N Sodium Hydroxide Water (pH 10 to 11 1 249 mu. 249 mu. 242 mp. 242.5mp.
E -.

References 4,8,19,24 19,24 3 26 25

13,090 to 14,000 2,000 to 2,120 13 I 600 13 ,750

0 ca .11 ,0 0

10,037 10,820 10,830 ca.10,500

8
21 24 25

257 mp.
258 mu. 258 mu.

The ultraviolet spectra of acetaminophen in ethanol (95%) and in 0.01N Sodium hydroxide (aqueous) are presented24 in figures 3a and 3b.

10

FIGURE 3a.

Ultraviolet Spectrum of Acetaminophen ( e t h a n o l 95%)

11

FIGURE 3b.

Ultraviolet Spectrum of Acetaminophen (0.01N sodium hydroxide)

12

ACETAMINOPHEN

2.13

Fluorescence Spectrum

Acetaminophen has been reported25 ,27 to exhibit fluorescence in neutral and acidic solution (excitation at 330 mp. and emission peak at 400 mp.) Nang et a1.25 also observed fluorescence in aqueous alkaline conditions (excitation at 315 mp. and emission peak at 430 mu.). However, recent attempts 24,28 to confirm these findings have been unsuccessful and it has been suggested28 that the earlier ob'~ s e r v a t i o n ~27~could have resulted from the Raman emission of water in conjunction with poorly aligned monochromator systems. Acetanilide is not fluorescent436 and it is unlikely that the introduction of a para-hydroxylgroup into the molecule would change this characteristic.

2.14

N.M.R.

Spectrum

Puar and Funke201 recorded the N M. R. spectrum of acetaminophen in dimethylsulphoxide - d6 (see figure 4) and assigned the observed chemical shifts in the following manner. 69.07

HO

5
13

69.58

62.00
0

\ & NH

II - C-CH

66.70d ( 9.0) 67.35d(9.0)

Theriault and Longfield181 used the spectrum as determined in deuterated acetone to identify acetaminophen, formed by Amanita muscaria as a conversion product of acetanilide.
N.M.R.

ACETAMINOPHEN

2.15

Mass Spectrum

The effects of substituents on the mass spectral fragmentation of para-substituted acetanilides has been studied in detail29 ,30,446 but unfortunately examination of the p-hydroxy com ound was omitted in each case. Burtis et al.y1 give the main peaks of the mass spectrum of acetaminophen as m/e 151, 135, 121, 109, 95, 81 and 55. The molecular ion undergoes a mass l o s s of 42 to give the base peak of m/e 109. This results from the re-arrangement of a proton of the acetyl group to the phenyl ring, followed by cleavage of the amide bond with the loss of CH CO (m/e 42). This metastable transition gi6es a strong diffuse peak. Fales, Milne and Law444 recorded the mass spectrum of acetaminophen, reporting the most abundant peaks as m/e 109, 151, 43, 80 and 81. The relative abundancies for m/e 40 to 152 are tabulated444. Puar and Funke201 have also recorded the high-resolution mass spectrum of acetaminophen (see figure 5 ) and suggest the following scheme of fragmentation:-

15

JOHN E. FAIRBROTHER

The occurrence of all three alternative modes of fragmentation of m/e 109 is supported by observation of corresponding metastable ions and high resolution data. Milne, Fales and Axenrod 445 have recorded the isobutane chemical ionisation mass spectrum of acetaminophen indicating the peaks found to be m/e 152, 153 and 151.

16

ACETAMINOPHEN

1595 A C E T A M I N O P H E N

'Ih)

LJ -:
35

38

2
Z
J 2

25 0
28

0
t t

1
d

8
3

MASS/CHARCE TOTAL UNSEALED INTENSITY. 692QN B A S E P E A K IS '13.93 PERCENT OF T O T A L

Fig.

5.

Mass spectrum of acetaminophen

17

JOHN E . FAIRBROTHER

2.2

P h y s i c a l P r o p e r t i e s of t h e S o l i d
2.21

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

31 The m e l t i n g p o i n t f i r s t quoted f o r acetaminophen ( 1 7 9 O C ) a p p e a r s t o be e r r o n eous. Subsequent d e t e r m i n a t i o n s gave m e l t i n g p o i n t s o f 165 t o 168OC f o r r e l a t i v e l y u n p u r i f i e d m a t e r i a l 32 t o 39 and a m e l t i n g r a n e of 1 6 8 t o 1690C f o r p u r i f i e d m a t e r i a l 40 to 49. More r e c e n t l y improved p u r i f i c a t i o n p r o c e d u r e s have been developed g i v i n g m a e r i a which m e l t s i n t h e range 1 6 9 t o 1 7 1 0 C . 4i to i8. It is t h i s m e l t i n g r a n g e t h e r e f o r e which i s quoted i n t h e c u r r e n t r e f e r e n c e books3 t 4 and o f f i c i a l compendia 14,21. Kuhnert-Brandsttitter 4 9 , 5 0 has recorded t h e melting p o i n t using a Kofler h o t s t a g e a s 1 6 7 t o 1 6 9 O C and c a r e f u l l y d e s c r i b e s the melting process. From 14OoC t o t h e m e l t i n g p o i n t g r a i n s , hexagonal prisms and rhomboids sublime. R e s i d u a l c r y s t a l s i n t h i s tempe r a t u r e range grow i n t o hexagonal t o p o l y h e d r a l g r a i n s and prisms. The m e l t s o l i d i f i e s t o a g l a s s and g i v e s u n s t a b l e columnar a g g r e g a t e s a t l l O C on which r e c t a n g u l a r prisms of t h e s t a b l e m o d i f i c a t i o n a r e induced from a b o u t 1 4 0 O C . T h i s u n s t a b l e m o d i f i c a t i o n (11) melts i n t h e r a n g e 154 t o 156OC.
2.22

Density

Fels4' reported the s p e c i f i c g r a v i t y o f acetaminophen a t 2 1 O C a s 1.293.

2.23
(T.G.A.)

Vapor P r e s s u r e and T . G . A .

Thermogravirnetric A n a l y s i s f a i l e d t o d e t e c t any l o s s of v o l a t i l e s from a sample of acetaminophen N F 30.

..

J a e c k e l and P e ~ e r l e ~ ~ ~ measured t h e dependence of t h e c o n d e n s a t i o n c o e f f i c i e n t

18

ACETAMINOPHEN

on the partial pressure over an evaporating crystal face of acetaminophen. Measurements of the vapor pressures on single crystal faces as functions of the partial pressure were made using a torsion balance. 2.24 D.T.A. and D.S.C.

Differential Thermal Analysis D.T.A.) of a sample of acetaminophen N.F. gave436 a sharp Examinati0x-1~~~ of melting endotherm at 171OC. a sample of B.P. grade material by Differential Scanning Calorimetry (D.S.C.) similarly gave an endotherm at 171OC. On cooling the sample and rescanning a different pattern was obtained showing the sample melting at 157OC and also an From the D.S.C. 431 exotherm occurred at 67OC. data a value of 6.8 K cals./mol. was obtained for the Latent Heat of Fusion. 2.25 Crystal Characteristics

Morse31 in a paper describing the first reported synthesis of acetaminophen, recorded that it crystallised in the form of white monoclinic prisms. Kuhnert-Brandstatter49r50 has described visual changes which take place in crystalline acetaminophen durin the melting process F e l obtained two app~ ~ ~ (see Section 2.21). arently isomorphous crystalline forms of acetaminophen on recrystallisation from ethanol. From his optical examination of these crystals, Fels4O assigns them to the monoclinic system with symmetry 2/m; C2h a: b: c
6
=

1.3688
115O

1.5103 40

49.5'

For the two isomorphous forms he makes the following face assignments:-

19

JOHN E. FAIRBROTHER

Form 1

and

Form 2

m =

c =
f =
p 3

The observed a n g l e s between t h e s e f a c e s a r e g i v e n and i n some c a s e s compared w i t h calculated values. Form l i s r e p o r t e d t o be gapa b l e of undergoing t r a n s f o r m a t i o n ( t o 001/010/ 100) b u t Form 2 does n o t . T h i s d a t a h a s been s y s t e m a t i s e d i n t h e Barker Index of C r y s t a l s 4 3 4 . D i s p e r s i o n of t h e o p t i c a l a x e s i s v e r y s t r o n g i n acetaminophen, r < v . A v e r y s t r o n g negative birefringence i s exhibited. F a i r b r ~ t h e r ~found t h a t c r y s t a l l i s a ~ t i o n of acetaminophen from a wide r a n g e of s o l v e n t s gave e s s e n t i a l l y two t y p e s of c r y s t a l h a b i t . Hexagonal prisms (by c r y s t a l l i s a t i o n from a l c o h o l s , e s t e r s , k e t o n e s , water, d i o x a n and a c e t o n i t r i l e ) and s l e n d e r rhombohedra1 n e e d l e s (by c r y s t a l l i s a t i o n from benzene, t o l u e n e , d i c h l o r o e t h a n e and s e v e r a l o t h e r c h l o r i n ated solvents). Examination of t h e s e two c r y s t a l t y p e s by D . S . C . , i . r . and x-ray d i f f r a c t i o n (powder) f a i l e d t o show any e v i d e n c e of polymorphism.
2.26

x-ray D i f f r a c t i o n

Coy and Ochs433 have r e c o r d e d t h e x-ray powder d i f f r a c t i o n p a t t e r n f o r a sample of acetaminophen N.F. (see F i g . 6 and Table 3 ) .

20

ACETAMINOPHEN

TABLE 3 X-ray Powder Diffraction Pattern of Acetaminophen (7032-LKR-242) Interplanar Distances Relative Intensities

d (8)
7.36 6.42 5.78 5.30 4.90 4.70 4.38 3.81 3.68 3.37 3.29 3.21 3.08 2.75 2.48 2.44 2.34 2.3 Powder Characteristics 2.31 Static Charge

i/i1
0.26 0.20 10 .0 0.13 0.66 0.19 0.34 0.65 0.90 0.74 0.11 0.06
0.09

0.20 0.07 0.11 0.07

Acetaminophen particles flowing through a hopper acquire a negative static charge51. This charge is reduced by the addition of tablet lubricants and by small quantities (0.5%) of water. 2.32 Flow Properties

The bulk density of acetaminophen granules falls with increasing water content. This represents a rise in internal cohesion and causes a deterioration in flow properties52r 53. Spher0nization~~9 granules for tablet compression of
21

Fig. 6.

X-ray powder-diffraction pattern of acetaminophen

ACETAM INOPH EN

improves granulation flow rate. The presence of water in acetaminophen granules increases the angle of repose52. 2.33 Compression Characteristics

Uniaxial compression of crystalline acetaminophen ives a pressure cycle typical of a Mohr's body5zr59 producing capping and laminating compacts. The effects moisture content and granulation have on the com ression characteristics have been studied 541 5,58,59,60,456.

2.34

Surface Area and Porosity

The surface area of acetaminophen powder compacts has been studied by the BrunauerEmmett-Teller 1B.E.T.) low-temperature nitrogen The change of this adsorption p r ~ c e d u r e ~ ~ . surface area with changes in moisture content and/or compression pressure of the compacts has been studied57158. 2.4 Solubility 2.41 Solubility in Aqueous Solvents

The solubility of acetaminophen in distilled water has been described by several authors. Temperature 2O0C 25OC 37OC
l0O0C

Solubility (mg./ml.) about 11.3 about 14.5 11.66 13.85 about 19 about 20 about 52

References 61 3,21I 63 8,64 65 66 67 3,61,62,63

In pH 6.0 buffer solution at 37OC its solubility has been recorded68 as 23.8 mg./ml.
23

JOHN E. FAIRBROTHER

P a r u t a and showed t h e solubi l i t y p r o f i l e of acetaminophen i n dioxan-water mixtures t o correlate i n v e r s e l y w i t h t h e polari t y ( d i e l e c t r i c c o n s t a n t ) p r o f i l e of t h e s o l v e n t f o r m i x t u r e s c o n t a i n i n g more t h a n 30% w a t e r .
A s i m i l a r study65 conducted w i t h sucr o s e s o l u t i o n s ( a s s o l v e n t s ) gave t h e o p p o s i t e e f f e c t , t h e s o l u b i l i t y of acetaminophen decr e a s i n g w i t h d e c r e a s i n g d i e l e c t r i c c o n s t a n t of t h e s o l v e n t ( i . e . w i t h i n c r e a s i n g s u c r o s e concentration)

Goldberg e t a1.66 examined t h e solubi l i t y of acetaminophen i n aqueous u r e a s o l u t i o n s and found a l i n e a r i n c r e a s e i n acetaminophen solu b i l i t y with i n c r e a s i n g u r e a c o n c e n t r a t i o n . T h i s i n c r e a s e d t h e s o l u b i l i t y a t 37OC from about 1 9 mg./ml. ( i n water) t o about 31 m g . / m l . ( i n 3.0 Molar u r e a s o l u t i o n ) . T h e authors66 a t t r i b u t e t h e s o l u b i l i z i n g e f f e c t t o an i n t e r a c t i o n o c c u r r ing i n s o l u t i o n between urea and t h e acetaminoThe s o l u b i l i t y of acetaminophen i n water phen. i s r e a t l y i n c r e a s e d i n t h e p r e s e n c e of phenazone 7 O f 7 1 by a p r o c e s s thought t o i n v o l v e hydro en A s i m i l a r s i t u a t i o n is observed 435 i n bonding. t h e presence of c a f f e i n e b u t t h e o p h y l l i n e h a s been shown435 t o reduce t h e s o l u b i l i t y of a c e t a minophen.
2.42

S o l u b i l i t y i n Water M i s c i b l e Solvents

Solvent Ethanol Ethanol ( 9 5 % ) Ethanol Methanol Acetone A c et o n e Propylene Glycol 1 in 1 in 1 in 1 in 1 in 1 in 1 in

10 7 8 10

63 21 61

13 20
9

61,63 21,63 ( c o n t ' d)

61 21

24

ACETAMINOPHEN

Solvent Propylene Glycol Propylene Glycol Glycerol Glycerol

1 in 1 in 1 in 1 in
10

Reference
63 61 21,63 61

50 40 50

The f o l l o w i n g s o l u b i l i t i e s have been determined under c o n t r o l l e d c o n d i t i o n s .

Solvent Water c o n t a i n i n g 2% e t h a n o l Propylene Glycol Dioxan

2.43

Temperature
(OC)

Solubility (mg / m l . )

Ref.
72 68 69

26.5 37 25

23.9 156 90

S o l u b i l i t y i n S o l v e n t s Immiscible w i t h Water Reference

1 i n 50 Insol. Insol. Insol. Insol.

Solvent Chloroform Benzene

Ether

Petroleum E t h e r Pentane

3,61,63 4,61,63 3,61,63 4 4

The following s o l u b i l i t i e s have been determined under c o n t r o l l e d c o n d i t i o n s : Solvent Cyclohexane Theobroma O i l

S o l u b i l i t y a t 37OC

(mg /ml 1

Reference
67 68

0.0015

2.16

2.44 Rate of D i s s o l u t i o n

D i s s o l u t i o n r a t e s t u d i e s conducted by Goldberg e t a 1 . 6 6 examined t h e d i s s o l u t i o n of

25

JOHN E. FAIRBROTHER

monoparticulate layers73 of acetaminophen, alone, and in fused and physical mixtures with urea. The dissolution of samples of pure acetaminophen followed pseudo-zero order kinetics over a 5 min. period. Coarse particles (50-60 mesh) gave data in reasonable agreement with the "cube root law" 7 4 thus representing a system requiring correction for the decrease in surface area during dissolution. Finer material (100-120 mesh) gave data more in agreement with a planar surface dissolution model. The eutectic and physical mixtures with urea gave biphasic dissolution curves, the rate constant of the first part being approximately twice that for pure acetaminophen of similar particle size, while the second rate constant closely resembled that for pure acetaminophen. This suggests the urea is leached out leaving a matrix of effective surface area comparable with that of the pure acetaminophen. Mattok, McGilveray and Mainville75 studied the dissolution of eight different lots of formulated acetaminophen tablets using the USP XVIII - NF XI;i,jrotating basket) method and None of the methods two other methods gave complete correlation with the blood and urine profiles obtained with the same samples. All of the recently manufactured samples required less than 15 mins. for 50% dissolution but stored samples showed greatly diminished dissolution rates in some cases.

Chow and studied the dissolution rate of acetaminophen and its physical mixtures and complexes with caffeine and theophylline. The anhydrous form and the monohydrate of the 1:l acetaminophen-caffeine complex showed more than two and a half times the However, dissolution rate of acetaminophen. the hexahydrate of the acetaminophen-caffeine complex and the 1:l acetaminophen-theophylline complex both showed a reduction in acetaminophen dissolution rate relative to pure acetaminophen.

26

ACETAMINOPHEN

2.5

Physical Properties of Solutions 2.51 Cryoscopy

Several eutectics of acetaminophen have been described in the literature:Eutectic with Eutectic Eutectic Reference Temperature Composition ( % Acetaminophen) (OC)
115 136 115

Phenacetin Benzanilide Urea Acetylsalicylic Acid Phenazone

118.2 83 104

37 28.5 59.5

85 6 6

The cryoscopic properties of acetaminophen in naphthalene have been reported by Auwers 86. 2.52 Ionisation and DH

Acetaminophen is a weak acid its saturated aqueous solution having a pHi4 of 5.3 to 6.5 at 25OC. pKa values for acetaminophen have been quoted betwe 9.078 and 9.580 and also recently as 10.15995. Two papers describe the determination of the pKa value of acetaminophen b spectrophotometric procedures. Talukdar et al.15 obtained a value of 9.35 + 0.05 (uncorrected for ionic strength) at 2: 5 % procedure described by Roth and Bunnett Dob68 et al.79 a value of 9.55 + 0.03 (at 25OC) using the procedure described by Albert and Serjeant82. 2.53 Dipole Moment

The dipole moment of acetaminophen has been determined8 in 1,4-dioxan solution using a Dipolmeter DMOl (heterodyne beat apparatus) and
21

JOHN E. FAIRBROTHER

the molecular dipole moment ca ulated using the method described by Hedestrandk5

This result is in good agreement with the value of 3.96D reported by Lutskii et al.84. Tomlinson's8 value yields a Molar Orientation Polarisation (P2-) of 325.4658 cm3. Lutskii et a1.84 quote a value for 1 . 1 ~ ~ LI calc.) of - 0.53 D (i.e. 1.1 obs.

2.54

Refractive Index

Microscopic studies with the Kofler hot stage49tSO showed melts of acetaminophen to have a refractive index of 1.5403 at 174oC (for red light) and at 181 - 182OC (for sodium light). Using an Abbe refractometer solutions of acetaminophen in 1,4-dioxan and in methyl alcohol show linear increase of refractive index with concentration up to 3.6% w/w and 10.8% w/w respectively94. From the equation:

=n n (observed) (acetaminophen) + "(methanol) ( 1-4 (x = weight fraction of acetaminophen in solutim)

a refractive index for acetaminophen of 1.608 (21C, white light) was calculated94 (methyl alcohol solution). Measurement of the nD in ethanol has been used87 to quantitatively determine the concentration of acetaminophen in two component mixtures.

28

ACETAMINOPHEN

2.55

Adsorption from Solution

The quantitative adsorption of acetaminophen from 2% ethanol solution was investigated for the solid adsorbents, nylon, cellulose triacetate and cellulose by Ward and Upchurch72. The influence of temperature, time, solubility and solvent were examined. Cellulose did not adsorb acetaminophen while nylon adsorbed almost twice as much as cellulose triacetate. Desorption studies indicated that adsorption occurred through hydrogen bond formation, the preferred mechanism being through the amido hydrogen of the acetaminophen and the carbonyl oxygen of the adsorbent. Brook and Munday89 have examined the adsorption of acetaminophen on a dextran gel (methylated Sephadex (LH-20)) and suggest a similar mechanism of hydrogen bond formation. 2.56 Partition Coefficients

Acetaminophen is preferentially extrac ted into ether from acid and weakly alkaline a ueous solutionsg0 g1 I 92. Brodie and Axelrod 9Yi92 examined the effect of pH on the partition of acetaminophen between ether and aqueous solution saturated with NaC1. PH Volume ratio (ether/water)
5 5
S

4.0 7.0 9.0 10.0 11.0 13.0

Fraction Extracted in ether phase (ref. 9 2 ) (ref. 91) 0.91 0.85 0.61 0.57
0 0 .

5 5 5

0.88 0.88 0.89 0.79 0.62

0.0

Partition coefficients for acetaminophen between other organic phases and water have been described.
29

JOHN

E. FAIRBROTHER

Organic Phase

Partition Coefficient
(PI

Log P Hansch Hydrophobic Substituent Constant

(TI)

Ref.

67 93 88

Cyclohexane 0.000075 Chloroform/ Ethanol about 0.44 l-Octanol l-Octanol (part ion it with pH 7.2 buffer) 6.237 +
2 .o%-

n.a. n.a. 0.55

n. a. n.a. -0.61

0.795

-0.36

Similar information may also be derived from the R values obtained in specially designed reversed phase silica gel thin layer chromatographic systems. Tomlinson8 employed two systems of this kind. (see Section 6.27)
3.

Molecular Complexes

Acetaminophen has been reported to interact with chloral95 and with sorbitolg6tg7r 98. Possibly these interactions may result in molecular complexes but insufficient data is available for any interpretation of this kind. A molecular complex of acetaminophen with pyramidon (1:l) has been made99 and acetaminophen is known to hydrogen bond onto the surfaces of nylon72 and rayon72.

30

ACETAMINOPHEN

Lach and CohenlOO demonstrated the solubilisation of acetaminophen with alpha - and beta cyclodextrins (Schardinger dextrins) The cyclodextrins exist in the form of cyclic chains having a relatively large open space within each molecule ( 6 a for alpha - and 82 for beta - cyclodextrin) The interaction of acetaminophen with the cyclodextrins produces non stoichiometric inclusion complexes of the clathrate typelol. Beta - cyclodextrin solubilises acetaminophen to a greater extent than alpha-cyclodextrin, the respective slopes of the interaction isotherms being 1.100 and 0.395.

When mixed with henazone (antipyrine), acetaminophen was reportedyo2 to give a syrupy mass. Ridgway and Johnson70 independently found that phenazone solubilised acetaminophen in water and that an equimolar molecular complex crystallised from solution. The complex was also obtained from alcoholic or acetone solution It showed a congruent and from melts7l. melting point (109.5 to 110.50C)71 and a hydrggen bonded structure has been proposed by Dearden for the complex. Chow and prepared (1:l) complexes of acetaminopheq with caffeine and with theophylline by a process of crystallisation from aqueous solution. These complexes were shown to exist in several hydrated forms:Heat of M.pt. K1:l Acetaminophen Degree of (OC) (l.mole) Solutim (1 1) Complex Hydration : at 25OC with (K cal./ mole) 6.5 anhydrous ca.145 59.4 Caffeine monohydrate 75-80 10.1 Caffeine hexahydrate 4 2 - 5 0 Caffeine 192-195 16.1 Theophylline anhydrous Theobromine does not form a complex with acetaminophen in aqueous solution435.

31

JOHN E. FAIRBROTHER

4.

Synthesis and Purification 4.1 Chemical Synthesis 4.11 Synthetic Routes

Acetaminophen was first synthesised by Morse31 in 1878 by reduction of p-nitrophenol with tin in glacial acetic acid. The p-aminophenol produced by the reducing action of the tin was not isolated, being acetylated in situ by the acetic acid. Tingle and W i l l i a m s 3 3 w e d the Morse synthesis but found it necessary to increase the acetic acid concentration to 100% by the addition of acetic anhydride. Vignololo3 simplified the synthesis by employing p-aminophenol a6 his starting material which he acetylated with acetic acid. Friedlander32 modified this process slightly by acetylating the p-aminophenol (from p-nitrophenol) with acetic anhydride in place of acetic acid. Many preparative methods have since been described employing the acetylation of p-amino henol with acetic acid and/or acetic anh dride33 I s 4 I 36 I 37 I 39 I 41 I 42 I 43I 44 I 48I104 I 105I106 I187 I108; in some cases anh drous sodium acetate also has been added34 I 3% I 37 I 41I 42. The p-aminophenol has been produced in numerous ways includin the electrolytic reduction of nitrobenzene4%I the alytic hydrogenation of p-nitrophedirect o no143 I 10s I gG , the sulphide reduction of p-nitrosophenolLu'.
A typical43 reaction sequence is :

32

0 0 0. 0
ACETAMINOPHEN

N02

NH,HCI

zr1{5"to
Pd/C
NHCOCH,

p.s.i.g.) 3ob

OH /0 4H Neutralise to

OH

NH2

Acetic

Anhydride

OH

OH

In some processes the p-aminophenol is not isolated but is acetylated in situ as it is formed45,47,109,110. An alternative to the acetylation procedure uses the action of ketene 111 in p-aminophenol. Other synthetic pathwa s involve the such as saponification of esters36 I 112I lY3 4acetamidophenylacetate,the hydroxylation of anilides b chemica1114 I electrolytic115 or enzymatic116 processes, and the decomposition of diazo compounds such as p-AcNHC H N BFq117. Acetaminophen has also been synt gide8 from p-hydroxyacetophenone hydrazone

2%.

4.12

Purification

Crude acetaminophen is in most cases furified by recrystallisation from hot water39 I 5 t 48t110. Coloured impurities are removed during the recrystallisation by treatment with
33

JOHN E. FAIRBROTHER

activated charcoal39'48 and oxidation is suppressed the addi on of small quantities of NaHS031f8, Na2S204" or h y d r o ~ u l p h i t e ~ ~ . One process48 controls the pH to 6 . 5 during the recrystallisation by the addition of ammonia. A number of processes seek to purify the p-aminophenol intermediate in a similar manner with activated carbon and Na2S204 43,44, 437,438 and in one case44 also by extraction of the aqueous p-aminophenol with an organic solvent such as benzene, toluene, hexane etc., to remove impurities such as azoxybenzene and azobenzene. A patent by Hahn and Q ~ i n n ~ ~ deals specifically with the purification of acetaminophen and related compounds made from crude discoloured intermediates (p - aminophenol). The discoloured acetaminophen is dissolved in hot water, acidified (pH 1 to 5 ) with a non-oxidising mineral acid and kept in a non-oxidising atmosphere (H2,C020r S02). The solution is agitated with activated charcoal, filtered and allowed to crystallise in the presence of an alkaline reducing sulphite, bisulphite, or hydrosulphite.
4.13

Impurity Profile

The following impurities have been detected in acetaminophen:Substance Origin Amount Reference in commercial (pharmaceutical) grade material
119

p-Nitrophenol Synthetic precursor t 0.025% p-Aminophenol Synthetic intermediate p-Ch loro Impurity +lo p.p.m. acetanilide
34

7,14(first suppl) ,21 7,14 cont ' d

ACETAMINOPHEN

Substance

Origin

Amount Reference in commercial (pharmaceutical) grade material

119 120 121

0-Acetyl para- Impurity from over- none detected cetamol ( DAPAP ) acetylationl- to 1.3% 1 of paracetamo1
Azobenzene Azoxybenzene By-products of reduction of nitrobenzene (precursor)

44 44

Guinone Quinonimine meri-Quinonimine Inorganic Chloride Inorg l ic al Su1$ate Inorganic Sulphide Water 4.14

Oxiuation Give a bluish of p-amin- or greyish color to acetaminophen ophenol (synthetic intermediate)

46 46 46

0.02%

7,14 7,14
7 I 14,21

Not detected

0.5%

Reference Standards

A National Formular Reference Standard exists f o r acetaminophenY 4

4.2

Biosynthesis 4.21 Metabolism of Phenacetin and Acetanilide

35

JOHN E. FAIRBROTHER

Acetaminophen is the main metabolite of both acetanilide and phenacetin (acetophenetidin) in man and in animals. Acetanilide was introduced by Cahn and Hepp12 as an analgesic and antipyretic in 1887. Investigating the metabolism of acetanilide, Mdrner (1889) 122 isolated potassium p-acetamidophenyl sulphate as a double salt with potassium ethyl oxalate from human urine. He also isolated a glucuronide tentatively identified as a conjugated p-acetamidophenol. Confirmation of this work was provided by Greenberg and Lester 123,124 and shortly after by Smith and Williams 12 Who demonstrated that in the rabbit 70% of the administered dose was excreted in the urine as the glucuronide conjugate of acetaminophen and 12% as acetaminophen sulphate. The metabolic fate o aypanilide has since been studied in detail12 r 1 In the case of phenacetin MBrner128 similarly isolated acetaminophen sulphate and the conjugated glucuronide from human urine. Smith and Williams130 showed that 54% of the dose administered to rabbits was recovered in the urine as conjugated acetaminophen, (47% glucuronide and 7% sulphate). In man, Brodie and Axelrodgl found up to 82% of the administered dose in the urine as conjugated acetaminophen More reand about 3 % as free acetaminophen. cent papers231,132 give an essentially similar picture. A comparative study133 of the availability of acetaminophen administered orally as such and as pnenacetin gave availability ratios (acetarninophen/phenacetin) in two studies as 1.04 and 1.06.

4.22

Prodrugs

Prodrugs are defined134 as having physico-chemical properties different from the parent drag but retaining qualitatively identical pharmacologic effects and reverting to the parent drug in the body.
36

ACETAMINOPHEN

c t ophen forms numerous ester proKotenko and MokhortlEj5 desdrugs67 ,13' cribe an ethoxyphenylmethylacrylamide homopolymer and its copolymer with o-carboxyphenylmethacrylamide which as analogs of phenacetin may be considered as prodrugs of acetaminophen. Extensive studv has been made of the release of acetamino-

4.23

Microbial Biosynthesis

Theriault and Longfield1' studied the 8 microbial conversion of acetanilide to acetaminophen. An unidentified Streptomyces species RJTS539 gave a peak yield of 405mg./litre of acetamirr ophen from 1000mg./litre of acetanilide. Amanita muscoria F-6 gave a mixed yield of acetaminophen and 2'-hydroxyacetanilide. 5. Stability 5.1 Stability to Light

Acetaminophen is slightly light sensitive in solution63 and may degrade by a mechanism involving pre-dissociation of the N-C bond as in the case of acetanilide1711172. 5.2 Stability of Solid Acetaminophen toHeat

Dry, pure, acetaminophen is very stable at temperatures up to at least 45OC. Should it however, be contaminated with traces of p-aminophenol or be exposed to humid conditions such that hydrolysis to p-aminophenol takes place, then further oxidative degradation of the paminophenol occurs121 characterised by a gradual color change through pink to brown and eventually to black. This involves the breakdown of the paminophenol to quinonimine and related compounds 46.

37

JOHN E. FAIRBROTHER

5.3

Stability of Solutions of Acetaminophen

The degradation of acetaminophen in aqueous solution appears to be both an acid catalysed and a base catalysed reaction173 I 174. It is first order with respect to the concentration of acetaminophen and first order with respect to the hydrogen and hydroxyl ion concentrationl73. Koshy and proposed reaction mechanisms for the acid and base catalysed hydrolysis of acetaminophen and determined the specific reaction constants (k') over the pH range 2 to 9.

pH

k' (hours-' x
35OC 7OoC - 9ooc 168.3 31.03 10.76 8.37 6.98 13.16 25.37 66.62 (K

Ea
16.69 17.99

t\ at 25OC (years) 0.78 5.83 15.39 19.78 21.80 12.59 7.13 2.28

2 3 4 5 6 7 8 9

2.52

29.13 7.40

2.56

17.42 17.99 17.42

6.58 19.02

The above studies17 were carried out isothermally. Zoglio et a1.175 repeated part of the study (pH 2 buffer) but used a nonisothermal linear temperature programmed technique. The results obtained were in good agreement with those of Koshy and Lach yielding a value for Ea of 17.0 K cal./mole and a value for k' (at 35OC) of 1.95 x 10-4 hr.-1. Zoglio et al. calculated the activation energy by comparing analytical data with Arrhenius model degradation curves using a digital computer. This approach was further improved by Kay and Simon176 who recalculated the data of Zoglio et al. using an analog computer system.

38

ACETAMINOPHEN

5.4

Stability to Oxidation

Acetaminophen is relatively stable to aerial oxidation unlike its hydrolysis product p-aminophenol. Acetaminophen has been used as an antioxidant for carotene in mineral o i l solution177, a heat stabilizer for p ~ l y a m i d e s l ~ ~ and as an antioxidant, stabilizer and short-stopping agent for synthetic rubber latexesl79. 5.5 Compatibility with Excipient Materials

The compatibility of acetaminophen with a wide ran e of excipient materials has been reported156 20 170.
5.6

Compatibility with Aspirin

Acetaminophen has been formulated in numerous commercial tablet preparations with aspirin. In some cases a third active drug substance such as caffeine, codeine phosphate or salicylamide is also present. Acetaminophen is known85 to form a eutectic product with aspirin (m.p. 118.2OC) and there is also some evidence to suggest that the two substances interact chemically to produce salicylic acid and diacetyl-p-aminophenol (pacetoxyacetani1ide)

Koshy et al.180 found up to 4mg./tablet of diacetyl-p-aminophenol (DAPAP) in commercial products and studied the formation of DAPAP in laboratory prepared mixtures of acetaminophen and aspirin after storage for up to 1 month at 5OoC. They also noted that magnesium stearate appeared to accelerate the formation of DAPAP. Boggiano, Drew and Hancock12'in a later study confirmed the formation of DAPAP in formulations containing acetaminophen and aspirin (compressed tablets and uncompressed mixtures) on storage at elevated temperatures ( 6 0 O C ) . They
39

JOHN E. FAIRBROTHER

also suggested that codeine phosphate and magnesium stearate both accelerate the formation of DAPAP

Kalatzis1' refutes the findings of 2 both the previous authors180,120, and shows DAPAP to be present as a synthetic impurity in commercial grades of acetaminophen and consequently is present in conunercial products containing acetaminophen. In stability evaluation experiments with mixtures of acetaminophen and aspirin stored at 45OC for up to 2 months no DAPAP was formed. Samples of the acetaminophen/ aspirin mixtures spiked with DAPAP in fact showed a gradual decline in DAPAP content if stored under humid conditions at elevated temperature.
5.7

Physical Incompatibilities

Acetaminophen shows physical incompatibility with antipyrine, Irgapyrin, Irgaphen, 2phenylquinoline-4-carboxylic acid and diphenhydramine hydrochloridelo2, mixtures with these substances becoming sticky on mixing. Rheological examination68 of acetaminophen in microcrystalline cellulose-carboxymethylcellulose gels shows some evidence of an interaction between the acetaminophen and MCC - CMC. Under humid conditions and at elevated temperatures acetaminophen discolours in the presence of codeine phosphate or caffeinelzl.
6.

Analytical Chemistry
6.1

Identity Tests

Acetaminophen may be identified by its melting point50 (see Section 2.21) and its eutectic temperatures with phenacetin50, benzanilide50 or urea66. It may be identified by measurement of hysical parameters such as infrared spectrum14rs1 or G.L.C. retention time182.
40

ACETAMINOPHEN

Acetaminophen yields numerous derivatives many of which have clearly defined melting points:Reagent Benzoyl chloride KOH 4-Nitrobenzoy1 chloride-pyridine Derivative
0Benzoy1 M.p. (OC1

Ref.
62,183 21 135 137 106

-acetaminophen 0-(4-Nitrobenzoyl)-acetamin'ophen Succinyl chloride bis (p-acetam-pyridine inopheny1) succinate Phthaloyl chloride 0-Phthaloyl -pyridine -acetaminophen Et2S04- alkali Phenacetin Ally1 bromide

171 210 225227 235237 134136 93

42 0-Allylacetaminophen 1-Fluoro-2,4p- (2,4-dinitro 197- 184 .198 dinitrobenzene phenoxy ) -acetanilide) 174 41 2 ,6-dibromoBr /C HC1 acetaminophen 158 185,186 conc.HN03/conc. 2-nitroH2S04 (-5oC) acetaminophen Diazotised aniline m-acetamino226 34,36 HC1 o-hydroxyazobenzene l-Nitroso-210-acetamido337.5 190 naphthol-HN03 SH-benzo- [ ] a phenoxazonium nitrate

Acetaminophen gives a characteristic violet-blue color reaction with a ferric chloride test solution3,14' 21 and may be distinguished from phenacetin by the color formed with T h i s involves oxiLiebermann's reagent3,Zl. dation of the acetaminophen with acid dichromate to slowly give a violet coloration in contrast to phenacetin which gives a red coloration.
41

JOHN E. FAIRBROTHER

Feig1187 describes a spot test for acetaminophen claimed to have a sensitivity of lpg. The test uses a procedure involving the nitrosylation of the amine group followed by its hydrolysis to a diazonium group which is subsequently coupled with 1-naphthol to give a red precipitate. Le Perdriel et al. 186 found that in the case of acetaminophen the initial nitrosylation reaction proposed by Feigl did not occur but 2-nitro-4-acetaminophen01 is being formed instead. Acidification of the final Feigl test solution (containing 1-naphthol) (as applied to acetaminophen) produces a yellow-orange coloured solution whereas in the contrasting cases of acetanilide, phenacetin and p-aminophenol; red, violet and black precipitates are formed. Paper and thin-layer chromatography have been used extensively to separate acetaminophen from other substances and the combination of Rf value and chromogenic response to spray Of reagents may be used as an identity test. particular note are the papers by Gumprecht and Schwartzenburg1B8 (paper chromatography of isomeric monosubstituted phenols) and by Goenechea 189 (thin-layer chromatography of analgesics related to acetanilide).
6.2

Methods of Analysis 6.20 Gravimetric Procedures

Poethke and Kdhne184 describe the quantitative precipitation of acetaminophen with l-fluoro-2,4-dinitrobenzene in a sodium bicarbonate-dimethylformamide medium to give p-(2,4dinitrophenoxy) acetanilide. The precipitation is carried out over a 4 hour period and is claimed to give a precision of + 0 . 3 % . Caffeine, phenazone, 4-aminophenazone, phenacetin and codeine phosphate do not interfere.
42

ACETAMINOPHEN

6.21

Titrimetric Procedures

Acetaminophen may be determined by titration with sodium nitrite after prior acid hydrolysis of the acetaminophen to p-amin p Both visual511911440and potentiometric1 9 s ,P J S O l Ce4+ quantitatively end-points have been used. oxidises acetaminophen thus rendering it possible to titrate acetaminophen with 0.1N Ce(S04)~ in an ethanolic HC1 medium1g3 I lg4. Chatten and Orbecklg5 attempted to titrate acetaminophen with perchloric acid in various acetic anhydride based solvents but were unable to obtain an end-point. Acetaminophen may be successfully titrated in a dimethylformamide medium with 0.1N The sodium methoxide (in benzene-methanol) end-point may be determined visually using anlg6 azo violet indicator1g2 or potentiometrically

Laurentlg7 also using dimethylformamide solution tit-ratedacetaminophen visually to a thymol blue end-point employing 0.1N Me4NOH (in benzene-methanol) as titrant. Fogg et al. 2o employed a similar system with 0.1M Bu4NOH as titrant, a N2 atmosphere and potentiometric endpoint detection using a calomel reference electrode filled with EtqNBr saturated dimethylformamide. 6.22 Polarographic Procedures

The anode polarographic behaviour of acetaminophen has been studiedl98 at the waximpregnated graphite electrodelgg. This system employs a solution of acetaminophen in aqueous ethanol/phosphate buffer (l:l), pH 7.1 and gives a value for ES vs. S.C.E. of 333mV. Brockelt 2oo describes a cathode polarographic procedure for the determination of acetaminophen after nitration with 5N HNO3. The solution containing nitrated acetaminophen is treated with potassium hydroxide and phosphoric acid to give a
43

JOHN E. FAIRBROTHER

solution pH of 5 . 8 and examined polarographically (E4 versus S . C . E . - 0.38V). Shearer et al. 441 found that with the use of a glassy carbon electrode, acetaminophen could be determined polarographically with a peak potential of about + 0.5V versus S . C . E . This procedure is capable of selectively determining acetaminophen in the presence of paminophenol ( E # versus S . C . E . + 0.2V) and thus may be used as a stability-indicating assay. The water content of the acetate-acetic acidmethanol supporting electrolyte significantly alters the measured peak current for a given concentration of acetaminophen and thus has to be limited. 6.23 U.V. Spectrophotometric Procedures

The British Pharmacopoeia 1963223 and National Formulary XI62 both adopted U.V. spectrophotometric procedures for the determination of acetaminophen in acetaminophen tablets. In both procedures the tablets are extracted with an anhydrous alcoholic solvent (B.P.ethanol and N.F.-methanol), the extract acidified with a small amount of dilute hydrochloric acid and then further diluted with the alcohol. The acetaminophen concentration is determined by spectrophotometric measurement of the extinction of the solutions(249 mu.) and its content calculated against, a standard E (1 percent 1 cm.) ( B . P . method) or, the extinction given by a sample of the N.F. Reference Standard.
U.S.

Brown and Gwilt26 challenged the official B.P.223 method on the grounds of cost of the solvent and the use of a standard extinction coefficient. They proposed the adoption of an alternative procedure employing 0.01N NaOH as both extractant and spectrophotometric solvent (extinction measured at 257 mu). This procedure was subsequently adopted for use in
44

ACETAMINOPHEN

the Addendum 1964224 to the B.P. 1963 and has been continued in the B.P. 196821. Rogers202 examined the effects of slitwidth on the precision of the B.P.224t21 assay and calculated that for an extinction error of 0 . 2 % a maximum half-intensity slit width of 1.7mp. may be used (calculated for Hilger and Watts, Uvispek H.700 and Unicam SP. 700 spectrophotometers) 203 The U . S . National Formulary (XI1 and XIII14 editions) retains the use of the acidified methanol solvent but employs a revised extraction procedure in which the acetaminophen is extracted from an aqueous suspension of the ground tablet with a mixture of chloroform and ethanol (3:l). Ivashkiv93 has studied the parameters of this14 procedure as applied to the assay of Squibb Acetaminophen Tablets and reports the (75:25) ratio of chloroform to ethanol to be critical. Also examined were the partition coefficient of acetaminophen between the solvent phases (see Section 2.56) and the effect of the grinding procedure used, on the extraction of the acetaminophen. The results obtained indicate micro-milling should not be employed in the sample preparation.

Acetaminophen has been determined spectrophotometrically ( 2 5 0 mp.) after partition into n-butan0120 from sodium bicarbonate solution. This facilitates the determination of acetaminophen in the presence of aspirin20. In a similar manner acetaminophen may be determined in formulated tablets of the acetaminophen-phenazone complex204 (see Section 3) by selective retention of the acetaminophen in 0.1N sodium hydroxide solution after partitioning with chloroform. The alkaline solution containing the acetaminophen is acidified with hydrochloric acid and the acetaminophen determined spectrophotometrically at 245 mp.

45

JOHN E. FAIRBROTHER

Differential spectrophotometry has been used to determine acetaminophen in mixtures with Hdberli and Bgguin205 other drug substances. determined acetaminophen in mixtures with salicylamide by differential spectrophotometric measurement at two wavelengths (255 and 301 mu). Shane and X~wblansky~ used differential spectrophotometry to determine acetaminophen in the presence of aspirin, salicylamide and caffeine in analgesic tablets. Their procedure is based on the observation that the subtraction spectrum obtained by measuring the U.V. absorption of the p-acetamidophenolate ion (pH 10) against p-acetamidophenol (pH6) yields an absorption maximum near to the isobestic point of zero absorbance for salicylamide (263.5 mu.). Under the same subtraction conditions caffeine and aspirin (which is converted to sodium salicylate) do not exhibit any absorbance from 255 to 340 mu. Routh et al. 206 employ a similar procedure for the determination of acetaminophen in the presence of aspirin and salicylic acid. Acetaminophen has been determined spectrophotometrically in mixtures with other drug substances by several procedures involving preliminary ion-exchange (see Section 6.25) or partition chromatographic (see Section 6.26) separation of the acetaminophen.
6.24

Photocolorimetric Procedures

The majority of the published colorimetric methods for the determination of acetaminophen are based on one of three systems. These are nitration, oxidation or hydrolysis to paminophenol followed by diazotisation and phenolic coupling. G i ~ z a r d lnitrated acetaminophen with ~~ a mixture of nitric and sulphuric acids at -5OC
46

ACETAMINOPHEN

to yield 2-nitro-4-acetamidophenol. Horn225 described the nitration of phenacetin with nitric acid and Brockelt20 applied this procedure to the nitration of acetaminophen. This involved nitration of an aqueous solution of acetaminophen with 5N nitric acid at room temperature for 20 minutes. Brockelt found no absorption maximum for the nitrated acetaminophen in the range 380 to 750 my. and decided to use the color formed in an assay procedure measuring the light absor(mid-point of the straight ption at 428 mp. part of the light absorption curve). and also Rosenthaler226 reFeig1 ported that amides such as acetaminophen undergo nitrosylation with nitrous acid to yield an Nnitroso compound which may be saponified to give a diazonium compound suitable for phenolic Koen5 noted that in acidic medium coupling. with sodium nitrite, acetaminophen gives a yellow color which changes to an orange color on rendering alkaline and uses this color to quantitatively determine acetaminophen. Le Perdriel et a1.186 found that acetaminophen did not form a nitroso compound on reaction with sodium nitrite and dilute hydrochloric acid but the 2-nitro-4-acetamidophenol described by Girard. They found that this reaction could be used as the basis of a colorimetric assay procedure. The solution containing nitrated acetaminophen is made alkaline with sodium carbonate solution and the light absorption measured at the absorption maximum occurring between 440 and 445 mp. Inamdar and Kaji227 employ a similar procedure but do not make the final solution alkaline. This yields a solution of nitrated acetaminophen giving absorption maxima at 375 and 395 mp., the latter wavelength being used for quantitative measurement. Hanegraaff and Chastagner continued the work of Le Perdriell86 studying the mechanism of the nitration of acetaminophen and modified the spectrophotometric assay procedure.
47

JOHN

E. FAIRBROTHER

They greatly increased the concentration of acid employing a very strong mixture of nitric and sulphuric acids in addition to the use of sodium nitrite and measured the extinction at 375 mu. The method of Le Perdriel ha thoroughly evaluated by Chafetz et al. have attempted to optimize the reaction parameters and claim the procedure has an excellent precision and is well suited to automated techniques442. This procedure229 is claimed to be specific for the determination of acetaminophen in the presence of a range of excipient materials and drug substances commonly found in formulations containing acetaminophen. The p-nitrobenzoyl esters of acetaminophen were prepared by Tin le and Williams35 and More reby Reverdin and Cuisinier 83 in 1906. cently the absorption spectra of the 4-nitrobenzoic acid230 and the 2,4- and 3,5-dinitrobenzoic231 acid esters of acetaminophen (as well as the trans-4-nitrocinnamic acid ester232) have been thoroughly studied and may represent useful colorimetric reagents for the determination of acetaminophen.

Oxidative reactions have been used in the determination of acetaminophen. Basu250 hydrolysed acetaminophen with hydrochloric acid to give p-aminophenol which was then oxidised with 0.1N potassium dichromate to give a violet coloured oxidation product. This dye was quantitatively extracted into isobutyl alcohol and its absorbance measured spectrophotometrically at 550 mu. Brodie and Axelrodg2 found that paminophenol from the acid hydrolysis of acetaminophen could be oxidised with sodium hypobromite and the oxidation product coupled with phenol to form an indophenol dye (absorption maximum 620 to 630 mp.) This procedure has been used to determine acetaminophen in biological material
48

ACETAMINOPHEN

92r251 and has also been used to determine the level of free p-aminophenol in acetaminophen621 203 ,252. The Brodie-Axelrod procedureg2 requires the neutralisation of an acid solution of p-aminophenol with alkali and should the neutralisation point be passed to give an excess of alkali, degradation of the p-aminophenol results. This roblem has been overcome by Murfin and Wragg253, 554 who have been able to remove the need for preliminary hydrolysis of the acetaminophen to p-aminophenol. In their manual procedure254 the acetaminophen solution is added to a hydrochloric acid-sodium hypochlorite mixture (pH 3.4; ca. 0.25% available chlorine) and the excess hypochlorite is removed with sodium arsenite. The quinone chlorimide thus formed is then coupled with phenol in the presence of a borate buffer (pH 9.9) to give a stable blue The procedure indophenol dye (A max. 625 mp.). yields results with good precision, the mean relative standard deviation obtained by the authors being 0.36%. N i n ~ m i y a ~oxidised acetaminophen ~ directly with potassium ferricyanide in sodium hydroxide solution at OOC and then formed an indophenol dye ( A max. 635 mu.) by coupling with phenol. The coloured product was tentatively identified by Ninomiya as N-(p-hydroxypheny1)p-benzoquinone imine. Sakurai and Umeda2560xidised acetaminophen with chloramine T in the presence of 2,4dinitrophenyl hydrazine to give a coloured pbenzoquinone imine 2,4-dinitrophenylhydrazone. The color produced can probably be used in a similar manner to that described in a further paper by Umeda257 which describes the oxidation of acetaminophen with ceric ammonium sulphate in acidified ethanolic solution and is subsequently reacted with 3-methyl-2-benzothiazoline hydrazone. This reaction mixture on neutralisation with tri49

JOHN E . FAIRBROTHER

ethanolamine yields a blue violet color (A max. 5 8 0 mp.) which facilitates quantitative spectrophotometric measurement. Routh et al. 206 employed a stable free radical, diphenylpicrylhydrazyl, to abstract a hydrogen atom from acetaminophen (in ethylene dichloride solution) thereby promoting a process of radical coupling. This results in a reduction of the violet color of the diphenylpicrylhydrazyl ( A max. 527 mp. ) with the formation of yellow diphenylpicrylhydrazine. The decrease in the intensity of the violet color is used to measure the concentration of acetaminophen. Dedicoat and Symonds443 found that in a pH 8.0 borate buffer acetaminophen reduces Folin and Ciocalteau's reagent to give a stable This was best blue color (A max. 700 mp.) produced by heating the reaction mixture at 100C for 10 minutes and could be used to determine acetaminophen in the presence of several other analgesic drugs.

A number of procedures are based on the acid hydrolysis of acetaminophen to paminophenol which is then coupled with a suitA much used procedure employs the able agent. - naphthol reaction of the p-aminophenol with in alkaline solution, extraction of the coloured reaction product into n-butanol followed by This measurement of the extinction at 6 3 5 mp. procedy55,i54based on the work of Greenberg and Lester and the later papers of Kosh and LachZ09 and Gwilt, Robertson and McChesney 3 3 , the latter authors employing an 0: - naphthol reagent containing potassium dichromate.

Brodie and Axelrod 92 modified the procedure and diazotised the p-aminophenol - naphthol to before reacting it with alkaline give a dye exhibiting an h max. at 5 1 0 mp. This procedure was also employed by Carlo et a1.234 after slight modification.
50

ACETAMINOPHEN

Kos 235 reacted the p-aminophenol (obtained by acid hydrolysis of acetaminophen) with alkaline B - naphthol (without diazotisation) to give a green color having an absorption maximum at 420 mu.

Mouton and Mason236 used trichloroacetic acid to hydrolyse acetaminophen to paminophenol which they then diazotised and coupled with N1-diethyl-N-1-naphthyl-propylenediamine. The dye formed was extracted into amyl alcohol and the extinction determined at 590 mp. This procedure was modified slightly by Heirwegh and Fevery237 who substituted N- (1-naphthyl) ethylenediamine as coupling agent ( A max. 596mp.). I v a ~ h k i v ~ ~also examined this procedure has ~ critically, finding at least 4 hours incubation at room temperature is required for complete color development. Other procedures have been described where the p-aminophenol obtained has been cou led with alkaline anisaldehyde239, acid vanillin2go and other reagents241,247. It is possible to couple diazotised reagents directly with acetaminophen but they react on1 s l 0 w l y 7 9 ~ ~ ~ Dobsg, Stgrba and ~. VebeFLa79, 42 have studied the kinetics of these reactions and propose a reaction mechanism.

Acetaminophen has also been shown to couple with the diazotised reagents shown in Table 4 thus presenting the opportunity for possible spectrophotometric measurement. Kondo et al. 246 have examined the absorption spectra (and bathochromic shift) produced by the reaction of acetaminophen in alkaline methanolic solution with an p-nitrobenzene diazonium fluoroborate reagent. Acetaminophen has been shown to produce colors of potential spectrophotometric use by interaction with 1nitroso-2-naphthol, 2-nitroso-1-naphthol and
51

JOHN E. FAIRBROTHER

disodium-3-h dr sulphonate 2x8

-4-nitros0-2~7-naphthalenedi5%.

TABLE 4 Diazotised Reasents CaDable of Coupling wit; Acetaminophen Reagent Color of Ref, Product Diazotised aniline (acid) 7% Yellow Diazotised 1,4-Napthylamino-sulphonic acid (alkali) Red 243 Diazotised 2-NaphthylYellow243 amino-8-sulphonic acid(alka1i) brown Diazotised lf4-Amido-acetoRed 243 naphthalide-6-sulphonic acid (alkali) Diazotised 4-Nitro-6-chloroOlive 244 2-aminophenol brown Diazotised 4,6-Dinitro245 2-aminophenol

6.25

Ion-Exchange Chromatographic Procedures

Ion-exchange column chromatography has been used to separate acetaminophen from its decomposition product, p-aminophenol, from mixtures containing other medicinal agents and from dosage forms (see Table 5)

Street and Niyogi211f213 separated acetaminophen from phenacetin (acetophenetidin) phenobarbitone and salicylic acid by two dimensional chromatographic development on diethylaminoethylcellulose ion-exchange paper (Whatman DE 20). Initial separation was by simple development in a 0.2N ammonium hydroxide solvent. This was followed by ionophoretic development at right angles in the same solvent (5mA; 250V.).

52

TABLE 5 Ion-Exchange Chromatographic Separation of Acetaminophen Separation from Ion-Exchange medium

200- 400 mesh

Elutrient

Quantitation

Reference

VI

20% glacial Titrimetry in DMF acetic acid solution with sodium (hydroxide form) in ethanol methoxide 7 0 % methanolU .V. SpectroPhenobarbitone Dowex 1 - X1 photometry (249mu. ) 0.1N HC1 in Differential U.V. 7 0 % ethanol Spectrophotometry p-Aminophenol Amber 1ite Water U.V. Spectrophotometry ( 2 4 4 mv.) p-Aminopheno1 U.V. SpectrophotoChlorobenzoxazolin:yt ffie IR-120 Water metry ( 2 4 4 mu.) Sulphadimethoxine Caffeine Theophorin Tartrat

Elixir formulation Dowex 1

X8,

196
207 205 208 209

Phosphate Cation Exchange Water Phenylephrine HC1 Resin, Alginic Pyrilamine Maleate acid, Mesh 40-100 (B.D.H.)

U.V. Spectrophotornetry (249 mu.)

210

JOHN E. FAIRBROTHER

6.26

Partition Chromatographic Procedures

Koshy213 separated acetaminophen from admixture with caffeine and aspirin by partition chromatography employing consecutive sulphuric acid and sodium bicarbonate impregnated Celite columns. Caffeine was retained on the acid column and aspirin on the alkaline column. Acetaminophen which is not ionised under either set of conditions passed through the columns in the diethyl ether solvent. Levine and Hohmann214 noted that the above system was unable to separate acetaminophen from neutral or weakly acidic compounds. To overcome this weakness they replaced the sulphuric acid by hydrochloric acid and the sodium bicarbonate by a sodium carbonate-sodium bicarbonate buffer (pH 10.1). The sample containing acetaminophen is incorporated into the acid impregnated Celite before it is packed into the first column which is placed directly above the second alkaline column. The two columns are washed with chloroform and then the acetaminophen is eluted with ether. The acetaminophen is determined spectrophotometrically in acid methanol solution (249mp.) following evaporation of the ether. This procedure will successfully allow the determination of acetaminophen in the presence of many coadministered drug substances. This rocedure214 was slightly modified by HohmannSlS who contained the two packing materials in a single column as separated segments. The modified procedure shown to be satisfactory for the determination of acetaminophen in liquid preparations was adopted by the NFXIII for the determination of acetaminophen in Acetaminophen Elixir. Both procedures
54

215 have been

ACETAMINOPHEN

s u c c e s s f u l l y a p p l i e d 2 1 6 to 219 t o t h e d e t e r m i n a t i o n of a c e t a m i n o p h e n i n o t h e r f o r m u l a t e d p r o ducts. F u r t h e r evaluation220 of t h e s i n g l e column p r o c e d u r e i n d i c a t e d t h a t optimum r e s u l t s were o b t a i n e d if 1N h y d r o c h l o r i c a c i d and 0 . 5 % e t h a n o l i n chloroform a r e used i n p l a c e of t h e c o n c e n t r a t e d h y d r o c h l o r i c a c i d and c h l o r o f o r m .
H a m i 1 o n 221 h a s d e s c r i b e d a f u r t h e r t m o d i f i c a t i o n t o t h e two-column p r o c e d u r e i n which t h e columns a r e s e p a r a t e d a f t e r t h e c h l o r o form wash and a c e t a m i n o p h e n i s e l u t e d f r o m t h e a c i d column w i t h water-washed e t h y l a c e t a t e .

have used a t h r e e Koshy e t a l . column s y s t e m ( b a s e d on t h e Levine222 s y s t e m ) t o s e p a r a t e d i a c e t y l - p - a m i n o p h e n o l from t a b l e t f o r m u l a t i o n s c o n t a i n i n g acetaminophen, a s p i r i n and c a f f e i n e .

6.27

P a p e r and T h i n Layer Chromatographic Procedures

A number of t h i n l a y e r and p a p e r chroma t o g r a p h i c methods have b e e n found s u i t a b l e f o r t h e 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 a c e t a m i n o phen. T h e q u a l i t a t i v e a s p e c t s of t h e s e methods a r e summarised i n T a b l e s 6 t o 11.

Reversed phase chromatography w a s used by Tomlinson8 i n a s t u d y d e s i g n e d t o c o r r e l a t e Rf c h a r a c t e r i s t i c s w i t h c h e m i c a l s t r u c t u r e f o r a series of s u b s t i t u t e d a c e t a n i l i d e s including acetaminophen. I n t h i s study8 t w o separate s t a t i o n a r y p h a s e s were u s e d on s i l i c a g e l G plates.

55

JOHN E. FAIRBROTHER

Stationary Phase l-octanol liquid paraffin (B.P.)

Mob i1e Phase acetone/water (1:9) acetone/water (2:8)

Rf at 20 + 0.5OC (av. of lo-results) 0.758 (0.740 to 0.779) 0.713 (0.710 to 0.716)

Semi-quantitative procedures relying on the visual comparison of sample spot size and intensity with standards have been described by Klutch and Bordunl31 and by Shand267. Bkh et a1.270 described a quantitative procedure in which the acetaminophen is acid hydrolysed to p-aminophenol which is then separated (lO-lOOug./ spot) by thin layer chromatography on a Silica Gel G layer. The p-aminophenol is eluted with 0.5N sodium hydroxide solution and determined A further spectrophotometrically at2zj0 mu. paper by the same authors employs chromatographic separation of the acetaminophen (without prior hydrolysis) on a layer of Silica Gel GF followed by elution with methanol and spectrophotometric determination at 245 mu. The procedure may be used to determine acetaminophen (60-3OOpg. /spot) in serum with a precision of + 5%. Cummings, King and Martin265 describe a similar quantitative procedure that employs elution of the acetaminophen from the silica gel with water rather than with methanol. 6.28 Vapor Phase Chromatographic Procedures (G.L.C., V.P .C.)

Acetaminophen presents difficulties for quantitative G.L.C. determination as a result of the pronounced elution peak tailing caused by its polar hydroxyl group. Koibuchi et al. 278 overcame this problem by acetylating the hydroxyl group to give N,O-diacetyl-p-aminophenol (DAPAP) which gave a good symmetrical peak after elution from a 1%
56

TABLF: 6 Paper Chrmtographic Systems for Acetaminophen

paper
W h a m 3M M

Direction
Two dimensional

Solvent System a) Isopropanol/aq. mnia/water (8:l:l) b) Benzene/propionic : acid/water (loo0

F+

Value
0 ; 83

Use
Characterisation in human urine

Reference 481

development, ascending

0.24

700:41)

Whatman No. 1 ascending w amt a No.1 ht amn

II

l n
4

Matman No. 1

11

w amt a No. 1 ascending ht amn impregnated with t r b u t y r i n Wa hNo. 1 ascending

Water Mineral s p i r i t s saturated with water Toluene saturated w i t h water Phosphate buffer (pH 7.4) Benzene/gl acetic acid/water/n-butanol (38:38: 17 :7) n-Butanol/lO% aq. m n i a (2:l) Benzene/acet i c acid/water (4:4:2)

0.83
0.00
0.03

Chromatographic study of isomeric phenols

It

188
II

II

II

0.80

Identity test Isolation from microbial culcultures Separation from other analgesics

3 181
2

n. a.
0.61

Grade FN1 ascending (VEB Spezialpapierf abrik) Grade FN1 ascending impregnated w i t l i 4% sodium bicarbonate (pH9)

0.04

other analgesics

Separation from

Thin Layer Chromatographic Systems for Acetaminophen (Neutral Systems)

TABLE

Absorbent Silica Gel GF Silica Gel G Silica G e l GF Silica Gel H F Silica Gel GE' Silica G e l
G

solvent system
&k?thanol

Rf Value
0.50

Use -

Reference
259 260 259

Methyl ethyl ketone

&k?thylethyl ketone

0.50

0.70

Butanone Chlorofom/diethyl ether

(85:15)

0.44
0.00

Chloroform/acetone ( 90 :1 ) 0.09 0 Chlorofom/methanol (80: 20) 0.75 BenZene/acetone (20 :1 ) 0 0.33 Chlorofobzene/ acetone ( 65 :10:25) Acetone/n-butanol/ water (50:40:10)
0.33 0.92

Silica G e l G Silica G e l G Silica G e l G Silica G e l GE' Silica Gel G

Separation from phenazone Separation from chlorpromzine Separation f c n phenara zone Identity test Separation from phenazone Separation from butcbarbitone Identity test Separation from other analgesic metabolites as abwe Determination of acetaminophen and mtabolites in serum Determination of acetaminophen and metabolites in urine

2 68 259
191
261 262 189 2 63

Two dimnsional develop-ent a) Chloroform/acetone (90: n a . 10) b) Chloroform/benzene/ na .. acetone (65 :5 :30)

264

TABLE

Thin Layer Chromtographic Systems for Acetaminophen (Alkaline Systems) Adsorbent S i l i c a Gel GF S i l i c a G e l GF S i l i c a Gel GF Solvent system

Rf Value
0.47
0.80

Use -

Reference 181 263

Chlorofom/95% rraethanol/ammnia (85: 15: 1) chlorofom/iso-propanol/ 35% aq. m n i a (45:45:10)

Silica G e l G S i l i c a Gel c;F

Chloroform/iso-propanol/ 33% aq. ammnia (80:15:5) lower phase/methanol ( 90 :5 ) Butylacetate/acetone/nbutanol/lO% aq. m n i a ( 5 0 :40: 30: 10: ) Cyclohexane/chloroform/ pyridine (20:60:5)

0.79
0.67 0.05
0.06

Isolation from microb i a l cultures Determination of acetaminophen and metabolites i n serum

as above

263

I d e n t i t y test Separation from phenazone Identity test

269 259 268

Thin Layer Chromtographic Systems for Acetaminophen (Acidic

Adsorbent Silica Gel G
Silica Gel G
S i l i c a G e l GF

m L E 9 l

Systems)

solvent system Chloroform/ethanol/acetic acid (88:10:2) Chloroform/acetone/acetic acid (80:18:2) &nzene/nethanol/acetic acid (45:8:4)

Rf Value Chrmtogratn diagram as above

Use Reference SeparationTFom WAP 1 2 1 and other analgesics as above 121,180

0.58

Determination of acetaminophen and

263

S i l i c a Gel GF
m

S i l i c a G e l (F

Al oxide

B r inlaMn
GF

Al oxide GF
B r inlaMn A1 oxide GI?

Brinlrman

Mhy1 acetate/nethanol/ 0.82 water/acetic acid ( 60 :30: 9 :1) Double developxent Chrmtogratn a) Benzene/diethyl ether/ acetic acid/rraethanol illustrated (120: m: 18 :1) b) E t h y l acetate/diethyl ether (80:20) 0.10 Toluene/benzene/water/ acetic acid (2:2:1:2) (upper phase) Chloroform/mthanol/water/ 0.35 acetic acid (20:10:20:1) (lmer phase) Cyclohexane/n-propano 1/ 0.84 water/acetic acid (20: 20:20:1) (upper P h - 4

metablites in serum Determination of 265,266 acetaminophen and metabclit=s in urine

Separation from W A P 120 and other analgesics

Separation from other 154 roetabolites

as above

154

Separation of 131 phenacetin metabolites cont'd

......

?IABLE 9 (cont'd) Thin Layer Chramatographic Systems for Acetaminophen (Acidic Systems)

Adsorbent A1 oxide GF
minlanan Al oxide GF Silica Gel G
S i l i c a Gel W
Brinlcman

Solvent system

Rf Value

Use

Reference

Ethylene dichloride/methanol/ 0.40 water/acetic acid (20:10:20:1) (1 phase) Ethylene dichloride/nrethanol/ 0.16 water/acetic acid (30:5:10:3) Butyl acetate/chloroform/ 0.46 85% formic acid (60:40:20) Dichloroethane/ethyl acetate/ 0.65 98% formic acid (60:20:20)

Separation of 131 phenacetin metabolites

as above

131

Identity test as above

269
263

TAEu;E 10

Reagents for Papa Chrm-aphic Reagent Color

Visualisation of Acetaminophen Sensitivity (pg acetaminophen)

1 U.V. .

2. U.V.

3. 4. 5.
O,
h ,

6.

Blu-rey Fluorescence a f t e r Yellow spraying w i t h 0.5% ethanolic oxine Diazotised p-nitroaniline Violet 5%ethanolic f e r r i c chloride Violet 15%aq. f e r r i c chloride/ 1% potassium ferricyanide (1:l)Deep blue aq. Ferric chloride/potassium Violet ferricyanide/Millon's reagent

- Fluorescence
-

Reference
2 2 2,258 2,181

20 1

< 1
20

2 2 2 3 188

(2: 2: 2) 7. Amnoniacal silver n i t r a t e (0.W) 8. Rromine/starch/potassium iodide 9. C e r i c m n i u m n i t r a t e

Black

Blue

purple

Reagents for Thin Layer Chranatographic Visualisation of Acetaminophen

Reagent Color Dark spot Black Black Dark blue Grey blue
Blue
( ug

TABLE

1 1

1 U.V. .
2.

3. 4.

5.
6.
o \

7.
8. 9. 10. 1. 1

(254mp.) - fluorescence quenclling 5%aq. s i l v e r n i t r a t e 10% aq. s i l v e r n i t r a t e 10% e r r i c chloride and 0.5% f potassium ferricyanide in w a t e r 5% aq. f e r r i c chloride Folin and Ciocalteu reagent pDimethylaminabenzaldehyde/ hydrochloric acid Iodine vapor Pauly reagent Diazotised o-dianisidine Diazotised sulphanilic acid

Sensitivity acetaminophen) 0.5

Reference 267 259, 262,267 189, 264 181, 267 189 263 189

0.25-0.5 0.2 0.1 < 5 0.5

Yellcw n.a. n.a.

n.a.
n.a.

n.a. n. a. n. a. n.a.

180 131 131 120

JOHN E. FAIRBROTHER

DEGS column. The acetaminophen was acetylated with a pyridine-acetic anhydride reagent employing strictly controlled reaction conditions to suppress the formation of N,N,O-triacetyl-paminophenol (TAPAP), a secondary product of the reaction. Quantitation was effected by peak height ratio measurement using an internal standard and almost theoretical recoveries were obtained from laboratory prepared mixtures (standard deviation 0.4 to 0 . 5 % ) . Prescott has more recently employed a similar procedure for the determination of acetaminophen in plasma (standard deviation about 3 . 5 % )

PreScott2 used a N- tr imethy 1sily1imidazo1e (TMSI) reagent to selectively silylate the hydroxyl group. In a separate procedure he280 used a N,O-bis (trimethylsilyl) acetamide (BSA) reagent which produces a di-TMS derivative by silylating both the hydroxyl group and the amide nitrogen. He reports280 near theoretical recoveries (TMSI procedure) for acetaminophen from plasma and urine with standard deviations of 1.8 and 2 . 8 % respectively (calculation from peak height ratio with an internal standard).

286 to 29Q to form derivatives suitable for quantitative G.L.C. determination2791280,2811292.

cetaminophen may be readily silylated

The direct G.L.C. determination of acetaminophen has been described27212741275 but the accuracy seems generally to be inferior to that of the indirect procedures and the working range for sample size is narrower as a result of poor peak symmetry. Table 12 gives details of the various G.L.C. systems described for the separation, identification and quantitative determination of acetaminophen.

64

G.L.C. Colunm

(V.P.C.)

TAJ3LE 12 Determination of A c e t a m i n o p h e n

Support

Column Stationary Phase

Column Temp.

R e t e n t i o n Detector Time System

Internal T y p of
Standard Deter-

Reference

mination
ca.1 min.
A m i t r i p t y - Analgesic line hydro-Preparations chloride 3 min. E l e c t r o n External Metabolic Capture Standard (Sr-90) ca.3 min. E l e c t r o n QualitativeClinical Capture
F.I.D.

C h r m s o r b W 10% E-W-982 195OC (AWDE.r=S) 80/100 (mesh) Anakr0111 AS 1% SE-30 plus 20O0C 80/90 1% carbowax

272 154 258

20M
o \

6% QF-1-0065 60/70

16OoC

VI

(Tritium
Foil) F.I.D.

C h r o m s o r b W 2% SE-30 plus 180C (Awl 0.1%TristeGas Chror~i0

3.5&.

Toxicological 273

arin

100/120

3% OV-17
2% FFAP

165OC

Aeropak 30 70/80 (in silanised

2 4OoC

ca.5 min. KCl-T.I.D.AmbarbitalPhannaceutica1 2 7 4 Preparations 6.5 min. F.I.D. D i p h e n y l bktabolic 275 phthalate
10 mins. E l e c t r o n Capture (Sr-90)

Column)
Ana?-xm AS m/90

0.5% SE-30 190C plus 0.5% Carbcwax 2 0 M

External
Standard

Metabolic

131

G.L.C.

(V.P.C.)

TABLE: 12 (cont'd) Determination of Acetamhaphen

Column SUPpo*
Gas Chrom Q

Column
Stationary Phase

Column Retention Detector

Temp.
Time

system

standard
External Standard

Internal

Type of
Determination

Reference

1% HI-EFF-8BP 22OoC ~a.17min. F.I.D. plus 10% SE-52 Chrcdrosorb W 10% Apiezon L 2lOoC 2.4 relF.I.D. (AWHQS) ative t o barbitme 190C n.a. n.a. Cklrmsorb W 5% SE-30 or 6o/m 3%Neopentyl or (silanised) glycol s u c c h a t e 2003 poljjester 225 C n.a. F.I.D. Chmrmsorb G 5%Carhowax 70/8O 2oM Gas C h r o m Q 3% HI-EFF-8BP 22OoC 3 . m . F.I.D. 100/120 (as 0-acetyl acetaminophen) 8O/l00
Chrmsorb W 1% Diethylene- 180C ca. 8 m i n . F. I.D. 60/80 glycol succin(as 0-acetyl (AW-silanised)a t e polyester acetaminophen) as cilrom Q 5%Apiezon L MOOC 9 min. (as F.I.D. 100/120 T.M.S. ether of

Pharmaceutical 182 Preparations Qualitative 3 Pharmaceutical 2 7 6 Preparations

n.a.

External Standard

Analgesic mixtures N-butyryl- Metabolic p-aminophen01 (as 0acetyl deriv.) N,@diiso- Antipyretic butyryl-p- Preparations aminupherd
n-Hexade-

180

277

278

cane

Pharmaceutical 279 Preparations

TAE3LE 12 (contd)

G.L.C. Column

(V.P.C.)

Determination of Acetaminophen Retention Time

Column
Stationary Phase

Column
T~IT~.

Detector
System

Internal
Standard

Type of
Determination

Ref.

support
Gas Chrom Q 80/100
Gas Worn Q

280

5% ov-1

l55OC

ca.15.6 m i n . (as F.I.D. B.S.A.* deriv.)

10% OV-17

200OC

8/0 010

ca.17.6 min. (as F.I.D.

T.M.S. I. **)

p-BramMetabolic acetanilide (as J3.S.A. deriv ) p-chloroMetabolic acetanilide

280

(as T.M.S.I.
5%OV-101
Program- 10.9 m i n . (as med from B.S.T.F.A.*** 100 t o deriv.) 3oooc a t 10 deg./ min. 160~ n.a.(as B.S.T. F.A. *** deriv. )

**

deriv.)

Chrmsorb W (W 8 / m 01

F.I.D.

Qualitative
Pathological

1 1

Gas Ckrom Q

3%OV-1

F.I.D.

Gas

Chrom Q

OV-17

1woc

3 . 3 min. (as H .M. s D

deriv. )

..

F.1.D-

Metabolic acetanil ide (as E.S.T.F. A.*** deriv.) ~ooosane (as bktabolic H.M.D.S. -k deriv. )

p-Bram-

281

447

JOHN E. FAIRBROTHER

* ** ***
-t

B.S.A. T.M.S.I. B.S.T.F.A.

L.M.D.S.
6.29

N,O-bis (trimethylsilyl) acetamide N-trimethylsilylimidazole bis (trimethylsilyl) trifluoroacetamide (Regisil) hexamethyl disilazane/trichloro methyl silane

High-pressure Liquid Chromatographic and Gel Filtration Procedures

.,-

Burtis, Butts and R i e ' anyl first described the use of high-pressure liquid chromatography in the determination of acetaminophen and its glucuronide metabolite in urine. Their procedure employed a high-pressure anion exchange chromatographic system293 with a U . V . detector and gave very long retention times in excess of 16 hours. Anders and Latorre295 have more recently developed an improved procedure capable of resolving acetaminophen and its glucuronide and sulphate conjugates present in urine within a total elution time of 40 min. Henry and S ~ h m i t ~ described a rapid '~ high-pressure anion exchange chromatographic procedure for the determination of acetaminophen in analgesic tablets using a peak area ratio measurement with an internal standard. A plot of peak area versus concentration was linear for both acetaminophen and the salicylamide3interna1 standard over a dynamic range of 5 x 10 This gave a possible range for acetaminophen determination of 3 mg. to about 50 pg. per sample injection.

have improved Stevenson and on this procedure and describe a rapid high-pressure liquid chromatographic assay for acetaminophen in a wide range of analgesic tablets claiming a precision giving a relative % standard
68

bur ti^^'^

ACETAMINOPHEN

deviation of 0.79 (using an external standard). Details of the procedures are given in Table 13. Jagenburg, Nagy and Re)djer297 separated the conjugated metabolites of acetaminophen by a gel filtration technique using Sephadex G-10 (Pharmacia) but no mention is made of the elution of acetaminophen. Brook and Munday8 studied the interaction of a series of compounds including acetaminophen with Sephadex G-10 and Sephadex LH-20 eluting with 0 . 1 N sodium hydroxide solution. 6.3 Automated Procedures Ederma et a1.282 automated the Brodie and Axelrodg2 procedure for the determination of acetaminophen in serum. The ether extraction of acetaminophen from the serum and its backwashing into dilute caustic soda remained as manual procedures but the conversion of the acetaminophen to p-aminophenol and the colorimetric determination were automated using an Auto Analyser system, A later paper283 describes the application of basically the same system to the determination of acetaminophen in whole blood. Shane and X ~ w b l a n s k yautomated their ~~ differential U.V. spectrophotometric procedure (see Section 6.23) for the determination of acetaminophen in the presence of aspirin, salicylamide and caffeine. Alber and O ~ e r t o n ~also deter~ mining acetaminophen in the presence of salicylamide and caffeine used a G.L.C. system with automated peak height measurement and calculation. This system employed an amplified KC1 thermionic detector system with a direct feed into the analog-to-digital converter of a PDP 1 2 A L I N C System computer. It is suggested that the sample preparation could also be automated. Daley, Moran and Chafetz442 automated

69

TAFLE 13 High-Pressure L i q u i d Chrmtographic Determination of Acetaminophen

Insizxumnt
"W-ANALYSER" (Oak Ridge National Lab-

Column S i z e
and Packinq

C o l m Temp.

Flw

Elution

oratory) w i t h Pnotmter Detector (260 and 29Qnl.l.)

C
. I

0.45x2OQ~1. 25uC i n m w b g t o 6WC after Dowex 1-X8 1 6 h r . (5 t o 1 0 ~ ) 1-2000 p.s.i.g.

and Pressure

Rate
(ml.
/ii.

Retention Tk2

Ref.

3 0

1 ~AmnxliumChlo- Acetaminophen ride-Acet i c 16.5 hr. Acid Buffer pH 4.4 Acetaminophen 0 . 0 1 5 M (38Qnl. ) glucwonide 1.OM (37cknl.) 22.4 hr. 4.0M (36cknl.) 6.0M (525m1.)

1, 1 293

Du Pant We1

0.2UooOCm. T e n m a t u r e 820 L i q u i d n. a. Chrmatograph Zipax coated 1200 p.s.i.g. w i t h Model 410 w i t h strong Photare!ter anion exDetector change resin
( 2 5 4 ~1 .

90

Buffer (Fisher ca. 2 mins. Gram-Pac) pH 9.2 containing 0.005M m n i u m nitrate

294

(cont'd..

...)

TABLE 13 (cont'd) High-pressure Liquid ChrOIMtographic Determination of Acetaminophen

Instrurrent

Column Size and Packing

Column Temp.

and Pressure

Flow

Elution

Retention

Ref.

V a r i a n WS-1030 O.lOx25Ocm. 80C with Photometer Detector LSF pellicular -loo0 (254 nu.) anion exp.s. i.g. change resin

X,

4
r

1 O . W formic Acetaminophen acid (pH3) con- 3.6 min. taining 1.OM Acetaminophen potassium c h l o r - l g l m m i d e ide (gradient 2.7 min. system also Acetaminophen given) sulphate 9.5 min.

295

with Photorreter LSF pellicular 925-1030

Detector (254 nip.)
anion exchange p.s.i.9. resin

varian ~ ~ - 1 0 001 x c . 0 0 mm .

6O 0 c

8.6

1.OM Tris Buffer (pH 9.0)

Acetaminophen 649 secs. (+ 1.08%) -

296

JOHN E . FAIRBROTHER

the colorimetric procedure of Chafetz et al.229 (see Section 6.24). Murf in253 has automated a colorimetric procedure based on the chromogenic reaction of acetaminophen with an acid hypochlorite - alkaline phenol reagent system. The procedure which may be used for single tablet assay of acetaminophen, alone or in combination with aspirin and codeine phosphate is based on a Technicon 25-channel system preceded by a sampling unit and a Technicon continuous filter. The complete procedure from commencement of sampling to the recording of the maximum color takes only 11 mins. and yields results with a coefficient of variation of about 0.4%. The sampling time takes 2 min. 15 secs. followed by a wash time of 45 secs. thus permitting the examination of up to 20 samples per hour on a continuous basis. 6.4 Radiochemical Procedures

Davison et al. 284 described the preparation of N-(114C-Acetyl)-p-aminophenol from sodium hydrosulphite washed, p-aminophenol and (acetyl 14C) acetic anhydride giving a product with an activity of 0.88pC/mg. Koss et a1.285 prepared quantities of acetaminophen labelled on the nucleus or acetyl side chain. N-Acetyl-2,6-14C-p-aminophenol was prepared by the reaction of sodium nitromalondialdeh de monohydrate with 1,3-14C-acetone to give 2,61XC-p-nitrophenol. This was then reduced and concurrently acetylated to give the required product. N- ( l-14C-Acetyl)-p-aminophenol was produced by reacting p-aminophenol in peroxide free tetrahydrofuran with l-14C sodium acetate. The determination of radioactivity in the or ans of Wistar-Rats dosed with either of the lagelled compounds was carried out employing
72

ACETAMINOPHEN

a Packard Scintillation Counter. Samples were dissolved in a benzalkonium chloride solution, decolorised with hydrogen peroxide and a scintillator solution added which contained naphthalene, PPO and POPOP. Radiolabelled metabolites were estimated after thin layer chromatographic separation using a Berthold T.L.C. Radioactivity Scanner. 6.5 Determination of Trace Impurities and Deqradation Products

The impurity profile of acetaminophen has already been discussed (see Section 4.13). The early compendia1 procedures for the determination of p-arninophenol in acetaminophen relied on colorimetric limit tests employing either a sodium nitro-prusside reagent21 or the phenol-hypobromite reaction62. The latter procedure was shown252 to be capable of quantitative use having a precision of about + 5% for a p-arninophenol level in acetaminophen-of 0.012%. More recently the NF XI11l 4 I 7 has adopted a thin layer ch;omatographic procedure for the determination of traces ( 4 0.025%) of paminophenol in acetaminophen. The procedure uses silica gel (HR grade) plates and a methyl ethyl ketone/acetic acid (9:l) solvent system. Visualisation is achieved with an acid p-dimethylaminocinnamaldehyde spray reagent and the size and intensity of the sample spot is compared with a standard spot. p-Chloroacetanilide levels in acetaminophen were determined by Savidge and Wraggllg using a thin layer chromatographic separation which employed a solvent mixture of cyclohexane/ acetone/diisobutylketone/methanol/water (100:80: 30:S:l). This procedure was designed for a p0.03%. The chloroac tanilide limit test of limits the level of p-chloroacetan1JF XIIIlg
73

JOHN E . FAIRBROTHER

ilide to 10 p.p.m. and describes a thin layer chromatographic procedure (solvent-chloroform/ benzene/acetone (65:10:25)) capable of this sensitivity

Savidge and Wragg showed their T.L.C. procedure to be capable of separating O-acetylacetaminophen (DAPAP) from acetaminophen and using this procedure found levels of up to 0.09% DAPAP in commercial samples of acetaminophen. Several other T.L.C. procedures have been described12 ,121,180 for the determination of DAPAP in acetaminophen and quantitative determinations have also been made using a G.L.C. systemlb0. (see Section 5.6). The limitation of the content of quinoniraine type oxidation products has been achieved mainly by close control of the white color of acetaminophen. 6.6 Determination of Acetaminophen and its Metabolites in Body Fluids and Tissues
6.61

Determination in Urine

Tne majority of the published work centres on the determination of free and conjugated acetaminophen in human and animal urine. Lester and G ~ e e n b e r g determined the ~~ metabolites of acetanilide by colorimetric reaction with a - naphthol of the p - aminophenol produced after acid hydrolysis. Acetaminophen was selectively determined by the same colorimetric procedure after extraction into ethylene dichloriae from urine, salted out with dibasic potassium phosphate. Smith and Williams125 examining rabbit urine containing acetanilide metabolites, hydrolysed etrier extracted urine by heating with acid thus lherating p-aminophenol from the acetam14

ACETAMINOPHEN

inophen conjugates. The p-aminophenol was determined gravimetrically after isolation. Brodie and Axelrodg2 determined free acetaminophen in urine by a procedure involving the extraction of the acetaminophen from sodium chloride saturated urine into a solvent mixture of isoamyl alcohol and diethyl-ether. The extracted acetaminophen was then acid hydrolysed to p-aminophenol and determined colorimetrically Conafter diazo coupling with a - naphthol. jugated acetaminophen was calculated by difference from total acetaminophen determined as total p-aminophenol obtained by direct acid hydrolysis of the urine. In this case the p-aminophenol was determined by the phenol/hypobromite colorimetric procedure. and Lach208 modified the Lester and GreenEz$y24 procedure separating the paminophenol from acid hydrolysed urine on an ionexchange column prior to colorimetric determination. Several authors91,126,251,264,298,299 have used slight modifications of the two procedures described by Brodie and Axelrodg2 for the determination of free and conjugated acetaminophen in both human and rabbit urine. Levy and Yamada3O0 used the Brodie and Axelrod92 procedure but deconjugated the metabolites by incubation with an enzyme mixture rather than re1 ing on acid hydrolysis. Heirwegh and Fevery 2y7 retained the acid hydrolysis procedure for deconjugation of the tabolites but substituted the Bratt~n-Marshall~~' diazotisation procedure for the diazo coupling with = - naphthol. Lower, Murphy and Bryan302 employed both enzyniic hydrolysis and the Bratton-Marshall colorimetric procedure for the assay of acetaminophen glucuronide in urine. In this case the sample preparation involved a preliminary fractionation step on a cation-exchange resin.
A third colorimetric procedure303 has also been described involving diazo coupling of
75

JOHN E. FAIRBROTHER

p-aminophenol (from acid hydrolysis of acetaminophen and metabolites) with o-cresol. Welch et al. 304 examining the metabolism of acetaminophen in animals determined the conjugated metabolites in urine (after B - glucuronidase and sulphatase hydrolysis) by a colorimetric procedure involving the formation of ionpairs of acetaminophen with methyl orange. Acetaminophen and its conjugated metabolites have been determined in urine after thinlayer chromato raphic se aration by U.V. spectrophotometryl31 I 363 I 265 I 267 I 270 and by measurement of r a d i o a ~ t i v i t y ~ ~ ~ . Vapor phase chromatography has been used extensively to measure acetaminophen and its metabolites in urine. Grove275 determined free acetaminophen in urine by a direct G.L.C. procedure employing an ether extract of treated urine. Klutch and B0rdun1~~r154 also determined conjugated acetaminophen using a preliminary enzymic (6-qlucuronidase) hydrolysis step. Prescott, Steel and Ferrier292 describes a procedure for the determination of both free and conjugated acetaminophen in urine. Their procedure requires the formation of the trimethylsilyl derivative of acetaminophen which is then chromatographed. Conjugated metabolites are enzymically hydrolysed to give free acetaminophen suitable for trimethylsilylation. Improve me t pproach have also been described 277,38$f2k!??f4$. High pressure liquid chromatography has been used to determine acetaminophen, acetaminophen glucuronide and acetaminophen sulphate direct without hydrolysis or derivative formation l1thq5. Similarly gel filtration procedures297 may be used but the chromatographic separation is tedious.

76

ACETAMINOPHEN

S- (1-acetamido-4-hydroxyphenyl) cysteine and 1-acetamido-4-hydroxyphenylmercapturic acid, minor metabolites of acetaminophen have been determined in urine by a gel filtration procedure 297. The cysteine compound has also been determined305 in urine following ion-exchange chromatographic separation by a ninhydrin colorimetric procedure.

6.62. Determination in Serum, Plasma and Whole Blood The procedures for the determination of acetaminophen and its metabolites in blood are essentially similar to those described above for its determination in urine. Lester and Greenberg124 treated both blood and plasma samples with tungstic acid to precipitate proteins and determined the acetaminophen derivatives using the same procedure as described for urine. Gwilt, Robertson and Mc Chesney306 described a procedure for the determination of free and total acetaminophen in plasma and in whole blood which is essential1 a modification of the Lester and Greenberg134 procedure. In this procedure whole blood is triturated with anhydrous sodium sulphate to give a dry friable mass from which free acetaminophen is extracted with 1.5% isopentanol in diethylether. Acetaminophen is back washed with alkali, hydrolysed with acid to give p-aminophenol which is coupled with alkaline a-naphthol as in the Lester and Greenberg procedure. However, the green solution so produced is then saturated with potassium chloride and the chromophore extracted into butanol for spectrophotometric measurement. This is claimed to increase the sensitivity of the procedure 2% times. The Brodie and Axelrod procedures91,92, 126'307 for plasma and serum are essentially as described for urine after suitable sample preparation. These procedures have also been automated282 I 283 for the determination of acetamin77

JOHN

E. FAIRBROTHER

ophen in blood. The Heirwegh and F e ~ e r y ~ ~ ~ procedure which employs the Bratton-Marshall colorimetric system has been used for determinations in serum as described for determinations in urine. This procedure has also been used by Ivashkiv308 who critically evaluated the reaction parameters. Routh et al. 206 employed two procedures for the determination of acetaminophen in serum or plasma, one employing differential U.V. absorption spectrophotometry and the other the decolorisation of diphenylpicrylhydrazyl dye. Bdch, Pfleger and R ~ d i g e r ~ ~ ~ determined acetaminophen in serum by a quantitative thinlayer chromatographic procedure, the acetaminopkn eluted from the sample spot being quantified by a U . V . spectrophotometric procedure. Koss et al. 2 a 5 used quantitative thin layer chromatography to determine radiolabelled acetaminophen and its metabolites in human serum, measurement being made with a radio-autography scanner. Free acetaminophen has been determined in plasma by vapor phase chromatography by several authors275~277,280,2a1~2g2. The chromatographic procedures in each case are those described for the determination in urine. The samplz7Yreparation however, differs slightly. Grove extracts the acetaminophen into ether from plasma saturated with solid ammonium sulphate. Thomas and Coldwell281 also extract the acetaminophen into ether but buffer the plasma to pH 7.4 with phosphate buffer and then saturate the solution with sodium chloride. In all the papers by Prescott and cow0rkers27~ 280 1 292 the plasma is buffered to pH I 7.4 with phosphate buffer and the acetaminophen extracted into ethyl acetate. Amsel and Davison 447 also use extraction into ethyl acetate.

78

ACETAMINOPHEN

6.63

Determination in Tissue and Organs

Brodie and Axelrodg2 determined acetaminophen and total conjugated p-aminophenol in homogenised tissue (emulsified in acid1309 essentially using the same procedures they described for similar determinations in urine. Gwilt, Robertson and McChesney306 used a very similar procedure to Brodie and Axelrod homogenising the tissue in 0 . 1 N hydrochloric acid, neutralising and buffering to pH 6.6 before extracting the free acetaminophen. Davison et a1284 and Koss et al. 285 describe the radioassay of total acetaminophen in tissue and organ homogenates using radiolabelled acetaminophen and also describe the separation of free acetaminophen and the individual conjugates in bile by a radioautographic procedure.
7.

Metabolic Transformations 7.1 Metabolism in Man

7.11

Adults

Lester and Greenberg124 and Brodie and Axelrod911921126 established that acetaminophen is the main metabolite of acetanilide and acetophenetidin (phenacetin). Thus the main metabolites excreted in the urine after administration of acetanilide, acetophenetidin, bucetin310 or acetaminophen are the glucuronide and ether sulphate conjugates of acetaminophen124I 125 I 1 2 6 . Acetaminophen sul hate had already been isolated in 1889 by MBrnerls2 from the urine of patients who had received acetanilide. Smith and Williams1251130 isolated crude acetaminophen lucuronide from rabbit urine and Shibaski et al. 3 6 6 purified this isolated material and also produced it synthetically. Minor metabolites have been identified
79

JOHN E. FAIRBROTHER

by Jagenburg and Toczko305 and by Jagenburg, Nagy and Rddjer297. These are the cysteine and mercapturic acid conjugates of acetaminophen. The recent findings of Nery311 of four new metabolites of acetophenetidin suggest that the list of acetaminophen metabolites may not et be complete 329. Focella, Heslin and Teitel4 8 identified a metabolite of acetophenetidin isolated from dog urine as 4-hydroxy-3-methylthioacetanilide. This substance may also be a metabolite of acetaminophen. In the same ~ t u d y ~ 4 ~S - ( 1 the acetamido-4-hydroxyphenyl) cysteine found by Jagenburg and Toczko305 was tentatively identified more correctly as 3-[(5-acetamido-2-hydroxyphepyl)thio] alanine.

5;

Burtis et al.ll described the formation of 3-methoxy-acetaminophen (by a girl with a neuroblastoma) after treatment with acetophenetidin. They11 ascribe the formation of this metabolite and its excretion as the glucuronide to an induced activity of the hydroxylase and catechol 0-methyl transferase enzyme systems caused by the high level of acetaminophen (see also refs. 331 and 332). Fig. 7 . The metabolic routes are summarised in

The relative amounts of free acetaminophen and its sulphate and glucuronide conjugates excreted in the urine var with the individual. Typical results265,29Y,300,312 for a dose of 1 to 2 gm. acetaminophen show 75 to 90% of the dose is excreted in the urine with the acetaminophen and its metabolites distributed (approximately) in the following manner:Free acetaminophen 2 to 5% (of total excreted) Acetaminophen glucuronide 55 to 75% (but some results are much lower) Acetaminophen sulphate 20 to 40% Acetaminophen 3-cysteine 0.5 to 7 % (only 3 resula Acetaminophen 3-mercapturic acid 5 to 7% (only 3 results).
80

ACETAMINOPHEN

HO acetaminophen glucuronide
acyinophen

OH

NHCOCH

NHCOCH3

$SC12y

HC O2H

@scH2c
3

I HC02H

HNCOCH

OH

OH H2 3-[(5-acetamido-2-hydroxyphenyl) thiolalanine

l-acetamido-4hydroxyphenyl-mercapturic acid
FIGURE 7

Metabolic Pathways of Acetaminophen

81

JOHN E. FAIRBROTHER

Patients suffering with chronic hepatitis and with liver cirrhosis show a decrease in the blood serum and urine levels of acetaminophen glucuronide and increased levels of free acetaminophen. This results from a corresponding decrease in the activity of lucuron ltransferase in the pathologic l i v e r s 2 3 q , 3 1 3 1 3 1 ~ , 3 1 5 . 3 1 6 , 3 1 7 . Renal insufficiency does not effect the ratio of free to conjugated acetaminophen in the plasma but through a decrease in glomerular filtration it may increase the plasma level of total acetThe metabolaminophen by as much as 4-fold318. ism of acetaminophen to its sulphate can be blocked by salicylamide which competes with the This effect acetaminophen for sulphate3O0 I 319. can be counteracted by L-c steine, a well absorbed source of sulphate350. This may be due to sulphate availability being the capacitylimiting factor3001320. Salicylamide also decreases the excretion of acetaminophen glucuronide possibly by a similar mechanism. Salicylic acid has no significant effect on the formation of acetaminophen sulphate and g l u ~ u r o n i d e ~ ~ ~ . 7.12 Newborn Infants

Vest and co-workers 307r323 found that in newborn infants acetaminophen (produced by the administration of acetanilide) is much more slowly conjugated to the glucuronide than in older children and adults. Similar results have been obtained after the administration of acetamino hen324,325,3261327 and it has been suggested32g1327 that the urinary excretion and blood levels of acetaminophen conjugates depend on the maturity of the glucuronide-forming enzyme system (glucuronic acid transferase and uridine diphosphate glucuronic acid) and the development of renal tubular function. 7.2 Metabolism in Animals

Clark328 demonstrated that the metabolic pathways of acetaminophen in man and dog were

82

ACETAMINOPHEN

similar. It has been shown in rats285 that about30% of the dose was secreted with the bile in 4 hr. The acetaminophen in the rat bile was shown to be almost completely conjugated to the glucuronide (83%) and the sulphate (14%) and only about 2.5% free acetaminophen was still available.

Cats dosed with acetaminophen metabolise the drug in a different manner from man, dog, rabbit and rat, in that less than 6% of the dose was excggied in the urine as acetaminophen glucuronide and less than 2 % as acetaminophen sulphate304. It has been reported3331334 that the cat has an impaired ability to form glucuronides, and this defect has been attributed to the lack Q35the glucuronyl transferase enzyme in the liver. The cat, however, does excrete acetaminophen in the urine, in a conjugated form (capable of enzymic hydrolysis with 6-glucuronidase) and it has been suggested by Welch et a1.304 that it may be conjugated with cysteine.

These 13% of the dose the urine as an does not appear

same authors304 found that 10 to administered to cats appears in aromatic primary amine but this to be p-aminophenol.

Acetaminophen is metabolised in the rat and rabbit in a similar manner to that in ma with different ratios of the metabolites125 ,Y29Yt 263,330, 8. Drug Availability 8.1 Pharmacokinetics

Many authors have described various aspects of the harmacokinetics of acetaminophen 68 80 91 124 127,148,234,285,292,298,299 300,306 , , 325,336 t o 347
83

JOHN E. FAIRBROTHER

G w i l t e t a l . 336 examined t h e a b s o r p t i o n o f a c e t amino hen i n man 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 . They336 f o u n d t h e h i g h e s t a v e r a g e b l o o d l e v e l o f t o t a l a c e t a m i n o p h e n was r e a c h e d a f t e r between 30 and 9 0 min. d e p e n d i n g on t h e i n d i v i d u a l . The e f f e c t s of f o o d and s l e e p on t h e a b s o r p t i o n and e x c r e t i o n of a c e t a m i n o en h a v e b e e n examined by Koss e t a l . 2 8 5 folMcGilveray and Mattok 4 s 2 lowed t h e a d m i n i s t r a t i o n (100 mg./kg.) of l a b e l l e d a c e t a m i n o p h e n i n r a t s , showing t h a t r a p i d a b s o r p t i o n occurs i n t h e f i r s t h a l f h o u r , o n l y a b o u t 4 0 % of t h e d o s e r e a c h i n g t h e s m a l l i n t e s t ine. This gradually reaches t h e l a r g e i n t e s t i n e where t h e c o n t i n u e d a b s o r p t i o n a p p e a r s t o b e compensated f o r by b i l i a r y s e c r e t i o n of acetaminophen ( a b o u t 30% of t h e d o s e ) .

The p l a s m a h a l f l i v e s r e p o r t e d v a r y a s shown i n T a b l e 1 5 .
TABLE 1 5 Acetaminophen Plasma Half

L i f e i n Man

Author B r o d i e and A x e l r o d
Carlo e t a l . G w i l t et al. Prescott e t a l .

Plasma h a l f - l i f e ( h o u r s1

(tf)

Ref.
1 26

Heald and Evans Prescott e t al. McGilveray e t al. Careddu e t a l .

1.5 2.4 2.3 2.7 2.03 (mean of 8 s u b j e c t s ) 2.94 (mean of 10 s u b j e c t s ) 2 . 0 + 0.1 (17 scbjects) 3.02 + 0 . 3 2.23 - 0 . 5

234 336 34 7 322 317 339

315

The e l i m i n a t i o n r a t e c o n s t a n t s f o r f r e e (unchanged) a c e t a m i n o p h e n and i t s c o n j u g a t e d m e t a b o l i t e s f o r man h a v e been d e t e r m i n e d by s e v eral authors265,312,319,339,348,454.

84

ACETAMl NOPHEN

8.2

Protein Binding

The bindin of acetaminophen to n lon72 cellulose triacetateq2 and to dextran gels84; has been described in Section 2.56. Hansch and Helmer88 related this work to the octanol-water partition coefficient and ultimately to the binding of acetaminophen to natural polymers such as proteins. Dearden and T o m l i n ~ o n ~ ~ examined have the binding of acetaminophen to bovine serum albumin (BSA) using a dynamic dialysis method finding the association constant to be sufficiently low to give a fairly high free drug concentration in the bloodstream over a relatively long time period.

Koss et al. 285 measured the binding of radiolabelled acetaminophen onto serum protein using a Sephadex filtration technique and found that about 18% of the acetaminophen is bound to the serum albumin over a wide acetaminophen concentration range. Hartshorn 349 quotes the amount of acetaminophen bound to the plasma proteins as about 25%.
8.3 Interactions with Other Drug Substances

The analgesic activity of acetaminophen has been claimed to be enhanced by the co-administration of a number of other analgesics and harmacologically active drug substances2t285I 300, 521,322,350-356, Levy and Yamada300 examined the effects of salicylamide on the pharmacokinetics of acetaminophen, showing that salicylamide retards the excretion rate of acetaminophen conjugates. This was shown to be accompanied by a competitive inhibition of the formation of acetaminophen and salicylamide conjugates in the blood implying increased therapeutic availability of free acetaminophen.
85

JOHN E. FAIRBROTHER

Niwa and N a k a ~ a m a ~ ~ l found that acetaminophen and ant ipyrine (phenazone) mutually inhibit the metabolism of each other in the rat and rabbit and showed that penetration of acetaminophen and antipyrine through excised intestine is mutually inhibted by the other drug substance. Heald and Evans322 determined the effect of antipyrine (as acetaminophen-antipyrine complex) on the plasma level of free acetaminophen in man (10 subjects). Their results suggest that antipyrine prolongs the peak plasma level of free acetaminophen. Acetaminophen has been re orted to be anta onistic to a number of drugs3251349 350,353 3551361. It has also been reported to show synergism of anti-inflammator c iv wi h other anti-inflammatory drugs3 5 4 t o st8 an$ 362 to 367.
8.4

Biopharmaceutics

Assessment of the bioavailability of acetaminophen has been made using both in vitro measurement of dissolution rate and in vivo pharmacokinetic methods. Goldberg, Gibaldi and Kanig66 used dissolution rate measurement to evaluate the potential increase in the bioavailability of acetaminophen after fusing it with urea to form eutectic mixtures. Lach and CohenlOO carried out similar studies employing alpha and beta cyclodextrins to increase the dissolution rate of acetaminophen (see also Section 3). Many authors have used the measurement of acetaminophen plasma levels and/or urinary levels to demonstrate ts bioavailability. Mattok 339 and c o - ~ o r k e r s ~ ~ rI 345 have used both in vivo and in vitro procedures and attempted to correlate the results. Levy133 used the areas under the acetaminophen plasma concentration vs. time curvesI to estimate the comparative systemic
86

ACETAMINOPHEN

availabilities of acetaminophen when administered orally as such and when administered as acetophenetidin (phenacetin). Mattok and co-workers75,339,345 used these techniques to compare the bioavailability of acetaminophen in eight commercial tablet formulations, a formulated elixir and a simple laboratory prepared solution and showed no significant differences between them. Gwilt et a1.336 examined the plasma levels given by seven acetaminophen formulations and later also examined an eighth formulation containing acetaminophen and sorbitol. The plasma levels of free acetaminophen.given by this acetaminophen-sorbitol formulation were significantly higher than those given by the seven other formulations. Sorbitol was claimed336 to potentiate the absorption of acetaminophen and thus is claimed to enhance the antipyretic and analgesic effects of the drug96~97198. Walters 385 has critically examined these claims using in vitro methods and concludes that sorbitol does not form an absorbable complex with acetaminophen and that the enhanced activity of acetaminophen in tablets containing sorbitol may result solely from the improved dissolution rate. Bloor and Morrison455 examined the effects of solubilization of acetaminophen by Tween4' (a polyoxyethylene sorbitan monopalmitate) on its rate of diffusion. Carlo et al. 234 examined the effect on bioavailability obtained by formulating acetam-. inophen in an effervescent tablet form. The effervescent formulation gave higher and more rapidly attained plasma concentrations of acetaminophen than an ordinary non-effervescent formulation but did not maintain plasma levels as efficiently as the latter. Bru452 makes similar
87

JOHN

E. FAIRBROTHER

claims. The effect of vehicle composition on the rectal absorption of acetaminophen from su pository formulations has been examined6812981 455. Incorporation of enzymes having hyaluronidase and chrondrosulphatase activity into acetaminophen formulations has been claimed to enhance acetaminophen absorption from and rectally administered dosage forms3sb!?3?6;1 ly 371~372~3731374. These claims were later refuted in a study by Brustier et a1.375. The administration of acetaminophen by percutaneous absorption from s lution in dialkyl Modification sulphoxides has been reported376 of drug availability can be effected by formulation as a sustained release (timed-release) dosage form. Several such formulations have been described for acetaminophen377I 3781379 1380 381,382. Timed release may also be effected by formulation of the drug in a microencapsulated form. This method of presentation has also been used to mask the taste of acetaminophen383138~.

9.

Toxicity

The acute and chronic oral toxicity of acetaminophen in man and animals has been well to Overdosin a e reported3~8r67~386 398. amino hen can cause hepatic necrosis317,996,s92I 3961 go 4 0 5 and also in a few cases of heav overrenal insufficiency3181347386,3961x06 to I dosin 414 421 91I 124,304,%b:s9k:?f!$? fff5e%s4%ve been discussed

88

ACETAMINOPHEN

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181. Theriault R.J. and mngfield T.H., A w l . Microbiol. 15 - (6), 1431-1436 (1967) 182. Rader B.R. and Aranda E.S., J. Pharm. Sci. 57 (5) 847-851 (1968) , 183. bverdin F. and Cuisinier L., Chem. E3er. 2, 3793-3797 184. Pcethke W and Kdhne H., Pharm. Zentralhalle Ml., . - (9-lo), 63*635 (1965) 104 185. G i r a r d A., wlll. SOC. C M . 3 ( 4 ) , 772-779 (1924) 5 186. Le P e r d r i e l F , Hanegraaff C z C h a s t a g n e r N. and . De Mntety E. , Ann. Pha.rm. Fr. - (3) 227-237 26 , (1968) . 187. Feigl F , Anal. Chem. 27, 1315-1318 (1955) 188. G m p r e c h t D.I. and SchEtzenburg Jr., F. , J. ChrcaMtog. 23 (l), 134-141 (1966) J. Chromatog 38 (11, 145-147 (1968) 189. Goenechea 190. Masuo lhneda, Y a k u g a k u Zasshc 84 (9), 839-845 (1963) PrivaFE ccmnmication 191. Fairbrother, J.E. ,SU (,based on Falex 0; ,A u s t r a l a s . J. Pharm., 37, 7 (1956) 1 192. Aftalion H., K e i m N. and Sterescu M., Rev. Chim. 1 ( B u c h a r e s t ) 1 , 49 (1960) 193. Kalinmska Z x . and Hasztar H., Farm. Pol. 23 (5-6), 447-450 (1967) 21, 194. Kalhowska Z.E. and H as ztar H., Farm. Pol. (15-161,570-573 (1965) 53 195. Chatten L.G. and Orbeck C.K., J. Pharm. Sci. (11) 1306-1308 (1964) ,

1121 (1967)

S z

97

JOHN E . FAIRBROTHER

196. H u n t J. , Fthcdes H.J. and B l a k e M.I., Can. J. Phann. Sci. - (11, 20-21 (1971) 6 197. Lament 0. , J. P h m . Belg. 25, 157-159 (1970) 198. N a s h R.A., Skauen D.M. and G d y W.C., J. Am. Pharm. Assoc. 47, 433-435 (1958) 199. G a y l o r V.F. , C o n r a d A.L. and Lander1 J.H., Anal. Chem. 29, 224 (1957) 200. EirockeE G. , Pharmazie 20 (3), 136-140 (1965) 201. puar M.S. and m e P.T= sq-uibb private C c m m i c a t ion 202. Wers A.R., J. Pharm. Phannacol. 17, 325 (1965) d. 203. N a t i o n a l F o m l a r y , 12th E. , AmerEan Pharmaceutical A s s o c i a t i o n , Washhgton, D.C., 1965, pp. 1e12. 204. Hoyland D. , Squibb Private C a m n u n i c a t i o n l . i n . h. ACta HelV. 34. 205. m i E and w E 1 P65-78 (1959) 206. Fauth J.I., Shane N.A., Arrendondo E.G. and Paul W.D., C l h . C m 14 ( 9 ) , 882-889 (1968) h . 298 207. DFbbem H.W. and S z o l z G., Arch. Pharm. -1 175-184 (1965) 208. Koshy K.T. and Lach J.L., Drug Standards, 28, 53-56 (1960) 209. K o s h y K.T. and Lach J.L., Drug Standards, 2, 85-87 (1960) 210. De Fabrizio F., J. Phann. Sci. 57 (4), 644-645 (1968) 211. S t r e e t H.V. and N i y o g i S.K., N a t u r e , 190, 537, 718 and 1199 (1961) .. 212. Street H.V. and N i y c g i S K , Analyst, 86,671-673 (1961) 1) 213. Koshy K.T. , J. Phann. Sci. 53 ( 0 , 1280-1282 (1964) 214. Levhe J. and Hohruann J.R. , J. Assoc. O f f i c . Anal. C h e m i s t s 49 (3), 533-536 (1966) 215. Hohmnn JX., J. A s s o c . O f f i c . Anal. C h e m i s t s 53 (3), 591-594 (1970) 216. V a l a t i n P., Squibb Private Comunication 217. V a l a t i n P., Squibb Private C a m n u n i c a t i o n 218. Ivashkiv E., and V a l a t i n P., Squibb Private Comroylicatian 219. Ivashkiv E. , Squibb Private C o m n u n i c a t i o n 220. Ivashkiv E., Squibb Private C o m n u n i c a t i o n

98

ACETAMINOPHEN

221. 222. 223. 224. 225. 226. 227. 228. 229. 230. 231. 232. 233. 234. 235. 236. 237. 238. 239. 240. 241. 242. 243. 244. 245.

J. A s s o c . Offic. Anal. Chemists 54 ( 4 ) , 895-899 (1971) Levine J., J. Am. Pharm. ASSOC., Sci. - 687 46, (1957) B r i t i s h Pharmacopoeia, 1963, Pharrn. Press, London, p. 558. B r i t i s h Pharmacopoeia, 1963, Addendum 1964, Pharm. Press, London, p. 55-56. Horn D., Pharm. Zentralhalle, 90, 296 (1951) Rosenthaler L., Pharm. A c t a Helv. 25, 365-367 (1950) I n a d a r M.C. and K a j i N . N . , Indian J. Pharm. 31 (3) I 79-81 (1969) Haneg-raaff C. and Chastagner N . , Ann. P h m . Fr. - ( U ) , 663-672 (1969) 27 Chafetz L., Daly R.E., Schriftman H. and Lcanner J.J., J. Pharm. Sci. 60 (3) , 463-466 (1971) Belotsvetov A.V. , G l u z d c o v V.A. and Larina M.K., Zh. Obshch. Khh. 36 ( 7 ) , 1198-1201 (1966) L a r i n a M.K., BelotGetov A.V. , Zh. Obshch. K h h . - (12), 2664-2699 (1967) 37 Belotsvetov A.V., Ehina T.F. and Larina M.K., Zh. Obshch. K h h . 36 (7) , 1202-1204 (1966) G w i l t J . R . , Robertson A. and PkChesney E.W., J. Pharm. Pharmacol. 15, 440-444 (1963) C a r 1 0 P.E., CambosG N.M., Feeney G.C. and Smith P.K., J. Am. P h m . AsSOC., S c i . Ed., - (7) 44 396-399 (1955) Kos J. , Farm. Glasnik 22 ( 2 ) , 51-53 (1966) Mouton M.M. and M s n K, Ann Pharm. Fr. ao ( 0I 1) 759-762 (1960) Heirwegh K.P.M. and Fevery J . , C l i n . Chem. (31, 215-219 (1967) Ivashkiv E . , Squibb P r i v a t e Cormmication D'Sowa A.A. and Shenoy K.G., Can. J. Pharm. Sci. 3 (4), 90-92 (1968) vaughan J . B . , J. Pharm. Sci. 58 (41, 469-470 (1969) H. Pharmazie 22 (1) 2 7 - 2 9 (1967) , Dobas I., & b &a V. m-VerEefa M. , Chem. Ind. (London) 1968 (51), 1814 Deut. Reich. Patent 146,265 (Nov. 9, 1903) t o Dahl and Co. in Barmen P a t e n t , Swiss 286,500 (Feb. 16, 1953) t o C i b a Ltd. Patent, G e r . (East) 12,865 (Mar. 5, 1957) t o VEB Fabenfabrik Wolfen

Hamiltm J.L.,

a.

1 8 2

Mhllin ,

99

JOHN E. FAIRBROTHER

246. Tatsuo Kondo and Iwao Kawashiro,. Shokuhin Eiseigaku Zasshi, 6 (51, 433-436 (1965) 247. Inoue Tekio, Tatsuzawa Masayoshi, Hojo Mash and Okawara Akira, E i s e i Kagaku, 1 (11, 24-27 6 (1970) 248. Masuo umda, Yakugaku Zasshi, 84 (9), 836-838 (1964) au 249. M s o Umda, Yakugaku Zasshi, 84 (9), 839-845 (1964) 250. Easu A.P., Indian J. Pharm., g ( 1 2 ) , 280 (1968) 1 251. Welch R.M. and C m e y A.H. , C l i n . Chm. 1 (12), 1064-1067 (1965) 252. Hoyland D., Squibb Private Ccarmunication 253. Murfin J.W., Analyst 2, 663-669 (1972) 254. Mwfh J.W. and Wragg J.S., Analyst 97, 670-675 (1972) Ninaniya, yakugaku Zasshi 85 (51, 394-399 255. ~ e r u o (1965) au 256. Hiroshi Sakurai and M s o m a , Yakugaku Zasshi 82, 1282-1286 (1962) 257. Gsuo Umeda, Yakugaku Zasshi 83, 951-956 (1963) 258. Shinsaku Imashuku and La Brosz E.H., C l i n . Chem. 17 (2), 122-124 (1971) 259. Fairbrother J.E. and Johnson B.A., Squibb Private

260. Datta D.D. and Ghosh D., Indian J. P h m . - (51, 28 133-134 (1966) 261. C i e r i U.R., J. P h m . Sci. 58 (12), 1532-1535 (1969) J. P h m . Sci. 262. Shun-Ichi Naito and Kazuo F&i, - (lo), 1217-1220 (1969) 58 263. Blich H., Pfleger K. and Rildiger W. , 2. Klin. Chm. - (3), 110-114 (1967) 5 264. Goenechea S., 2. Klin. Chm. 7 (4), 346-349 (1969) 265. Cumnings A. J. , King M.L. , and-hartin B.K. , Brit. J. Pharmacol. Chemther. 29 (21, 150-157 (1967) 266. Juichim Shibasaki, Etsuko Sadakane, Ryoji Konishi and Tarmtsu Koizuni, Chm. Pharm. Bull. 2 (U), 2340-2343 (1970) . 267. Shand S , Squibb Private Camnulication 268. Stahl E., "Thin Layer Chranatography" Springer, Berlin-Gdttingm-Heidelberg, 1965. 269. Zarnack J. and Pfeifer S., Pharmazie 19, 216 (1964) 270. glich H., Hauser H., Pfleger K. and Rmger W., 2. Klin. Chgn. 4 (6), 288-290 (1966) 271. Lindberg N.OT, Aktiebolaget Draco, Lund, Sweden,
Private Comnmication 100

Ccmrmnication

ACETAMINOPHEN

272. 273. 274. 275. 276. 277. 278. 279. 280. 281. 282. 283. 284. 285. 286. 287. 288. 289. 290. 291.

Nieminen E , Bull. Narcot. 23 (l), 23-28 (1971) . McMartin C. and street H.V.,J. Chromatog. - (21, 22
274-285 (1966) Alber L.L. and Overton M.W., J. A s s . Offic. Anal. Chm. 54 (3) 620-624 (1971) Grove T I J. Chranatog. 59 (2), 289-295 (1971) Ryabtseva I.M., Kuleshom-M.I., Rudenko B.A. and Kucherov V.F., Izv. Akad. Nauk SSSR, S e r . Khim. (12) I 2676-2680 (1970) Prescott L.F., J. Pharm. Pharmacol 23, 807-808 (1971) Masanobu Koibuchi , Toshio Shibazaki, Tsutanori

Minamikawa and Yukio Nishimra, Yakugaku Zasshi

- (7), 88
(1971)

Kipping S.A.B., The Boots C m y Ltd., Nottingham, Private Cammication 23 Presoott L.F., J. Pharm. Pharmacol. - (21, 111-115
243 (1972) Ederma H.M., Skerpac J., Cotty V.F. and Sterbenz F.,

877-881 (1968)

24,

Thanas B.H. and Coldwell B.B., J. Pharm. Pharmacol.

Automation i Analytical Chemistry, Technicon n Symposium, 1966, p. 288-231. Ederrna H M , Cotty V.F. and M e m K J , Advan. .. .. AutcaMt. Anal. , Technicon Int. Congr. (Chicago)
1969 (pub. 1970), 2, 179-181

Davison C. , Guy J.E., Levitt M. and Smith P.K., J. Phamacol. Eqtl. Therap. 134, 176-183 (1961) Koss F.W., Mayer D. , Haindn. and Kabbara T., Arzneh.-Forsch. - (9) , 1218-1222 (1970) 20 Reikhsfel'd V.O., Tr. Konf. po Probl. Primeneniya Korrelyatsion. Uravnenii v Organ. Khim., Tartusk. Gos. Univ., Tartu (1962) (11, 214-249 Reikhsfel'd V.O. , Prokhorova V.A. and Pmia V.A., Kinetika i Kataliz 6 (11, 171-176 (1965) Reikhsfel'd V.O. a d Korol'ko V.V. , Reaksionnaya Sposobnost. Organ. Scedin., Tartusk. Gos. Univ.

2,

Reikhsfel'd V.O. and Prokhorova V.A.,

K h h . 35 (10) 1826-1829 I

( 2 ) , 77-87

(1965)

Zh. Obshch.

ReikhsGl'd V.O. , Intern. Symp. Organosilicon Chen. Sci. Comnun., Suppl., Prague (1965) 34-40 Reikhsfel'd V.O., Khim. Prakt. Primen. Krerrmiiorg. Win., Tr. Sovesch. (19661, 7-23
101

(1965)

JOHN E. FAIRBROTHER

Prescott L.F., S t e e l R.F. and F e r r i e r W.R., C l i n . Pharmac01. Ther. 11 (4)I 496-504 (1970) (4) , 293. Burtis C.A. and Wzren K.S., C l i n . Chem. 2 9 e 3 0 1 (1968) 3, 294. H e n r y R.A. and Schnit J.A., C h r c m a t c g r a p h i a - 1161 2 0 (1970) 55, 295. Anders M.W. and Latorre J.P., J. C h r o m t o g . 409-413 (1971) 296. Stevenson R.L. and Burtis C.A., J. ChrcaMtog. - 253-261 (1971) 61, 297. Jagenburg R., Nagy A. and RcMjer S., Scand. J. C l i n . Lab. Invest. 22 (11, 11-16 (1968) 298. Hobel M. and TalebiK M. , Arzneimittel - Forsch. 10, 653-656 (1960) 299. Juichiro Shibasaki, R y o j i Konishi, Yoshinori T a k e d a and T m t s u Koizumi, Chem. P h m . B u l l . (Tokyo) 1 9 (9), 1800-1808 (1971) 60 300. Levy G. and Y m d a H. , J. Pharm. Sci. - (2) , 215-221 (1971) 301. B r a t t o n A.C., Marshall E.K., Babbitt D. and Hendrickson A.R., J. B i o l . Chem. 128, 537 (1939) 302. Lower G.M. , Murphy S.B. and B r y a n T T . , C l i n . Chim. A c t a . 29 (31, 421-427 (1970) 6, 303. Tonpsez S.L. , Ann. C l i n . Biochem. - (41, 81-82 (1969) 304. Welch R.M., C o n n e y A.H. and Bums J.J., Biochem. Pharrnacol. 15 (5), 521-531 (1966) 305. Jagenbmg O x . and Toczko K. , Biochem. J., 92, 639643 (1964) 306. W i l t J . R . , R o b e r t s o n A. and McChesney E.W., J. Pharm. Pharmacol. 15, 440-444 (1963) 307. V e s t M.F. , S t r e i f f y . R . , A.M.A. J. D i s e a s e s of C h i l d r e n , 98, 688-693 (1959) 308. Ivashkiv E T Squibb Private C c m m n i c a t i o n 309. Brodie B.B., Udenfriend S. and Baer J . C . , J. B i o l . Chem. 299 (1947) 310. Shibasaki J., K o i z u n i T . , Tanaka T. and N a k a t a n i M., Chem. B u l l (Tokyo) 16, 1726 (1968) 311. N e r y R., Biochem. JY 122 (3) , 317-326 (1971) 312. Levy G. and -&dh C.- G., J. P h m . S c i . 9, (4) 608-611 (1971) 313. Hart'umr C.H., and P r e l l w i t z W., K l i n . W o c h e n s c h r . - (17) I 1010-1014 (1966) 44
292.

1 4

18 6,

102

ACETAMINOPHEN

14, 314. V e s t M.F. and F r i t z E., J. C l i n . Pathol. - 482 (1961) 315. Careddu P. , Mereu T. and Apolloni T. , Minerva Pedi& 13, 1619-1622 (1961) 316. Every J. and D e G r o o t e J. , Acta Hepato-Splenol. - (1), 11-18 (1969) 16 317. Frescott L.F., Roscoe P., Wright N. and Brown S.S., Lancet (1971) 519-522 , 318. Dubach U C I K l h . Wochenschr. - (5) I 261-264 .. 46 (1968) 319. Levy G. and Yamada H., J. Pharm. Pharmacol. 22, 964-965 (1970) 320. Buech H., R m l W., Pfleger K., Eschrich C. and
T e x t e r N. , Naunyn-Schmiedebergs Arch. Pharmakol. Exp. Pathol. 259 (31, 276-289 (1968) 321. N i w a H. and N Z y m T. , Yakugaku Zasshi, - (5) , 88

542-548 (1968) 322. Heald A.F. and Evans R. (1959)

, Squibb Private

Ccarmunic-

ation 323. V e s t M.F.,

Schweiz.med. hbchenschr. - 102-105 89,

324. Careddu P., Mereu T. and Apollonio T., Boll. SOC. i t a l . biol. sper. 37, 359-363 (1961) 325. Careddu P. , Pacenizereni L. , Apollonio T. and Mereu T. , Minerva Pediat. 14 (40), 1047-1049 (1962) 326. Mereu T. , Apollonio T. , P a G i - S e r e n i L. and Careddu P., Lancet (1962 - I) 1300 327. V e s t M.F. and Rossier R . , Ann. N.Y. Acad. Sci. - (1), 183-197 (1963) 11 1 328. Clark B.B., Symposium on N-acetyl-p-minophenol. p. 23-34, I n s t i t u t e f o r t h e Study of Analgesic and Sedative D r u g s , Elkhart, 1951 329. Klutch A., Harfenist M. and Conney A.H., J. Med. Chem. 9 , 63 (1966) 330. Bray HTG. , Thorpe W.V. and White K. , Biochem. J. , - 423-430 (1952) 52, 331. Inscoe J.K., Daly J. and Axelrod J., Biochem. Ph-col. - (8)I 1257-1263 (1965) 14 332. Axelrod J., Science 140 (3566) 499-500 (1963) , 333. Wbinson D. and W i l l 5 R.T. , Biochem. J., 68, 23P (1958) 334. Borrell S., Biochem. J . , 70, 727 (1958) 335. Dutton G. J. and Greig C . G Y Biochem. J. , 66, 52P (1957)
103

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336. G w i l t J.R.,

Robertson A., GoL. and Blanchard A.W., J. Phann. Pharmacol. 15, 445-53 (1963) Barry H. J. Pharm. 337. J a f f e J.M., Colaizzi J.L. Sci. 60, (ll), 1646-1650 (1971) 338. WeikerJ.H. and Lish P.M., Arch. I n t . P h a n r a d y n .

"her. 119, 398-408 (1959) I.M., Mattok G.L., Fooks J.R., Jordan 339. McGilv=y N. and Cook D., Can. J. P h m . S c i . 5 (2), 38-42 (1971) 340. S t r i c k e r H., Pharm. Ind. 31 (111, 794-799 (1969) J. Pharm. Pharmacol. 341. Dearden J.C. and Tcsnlinsm-E., - Suppl., 68s-72s (1971) 23, 342. Dearden J.C. and Tcanlinson E., J. Phann. Pharmacol. - Suppl., 73s-76s (1971) 23, 343. Salzn?ann K., Therapiemche 12, 1034 (1962) 344. D i t t e r t L.W. and Adams H.J., J. Phann. Sci. 2, 1269 (1968) 345. Mattok G.L., W i l v e r a y I.M. and Cook D., Can. J. Pharm. Sci. - (21, 35-38 (1971) 6 346. W i k e l J . H . , J. Am. Pharm. Assoc., Sci. Ed. 2, 477-479 (1958) 347. Prescott L.F., Sansur M., Levin W., Conney A.H., C l i n . Pharmacol. Therap. 9, 605-614 (1968) . 348. N e l s o n E and Morioka T., J. Pharm. Sci. 52. (91, 864-868 (1963) 349. H a r t s h o r n E.A., Drug I n t e l l i g e n c e and C l i n i c a l Pharmacy 6, 50-54 (1972) 350. Grotto M., D f i s t e h S. and Sulman F.G., Arch. Int. Pharmacody~. 155 (2), 365-372 (1965) 351. Takagi K., Tajiii-pagi I., Kayaoka S., Okabe S . , Shigenobu K., Fukao T., K a w a s h h K. and Taga F., Yakugaku Zasshi 88 (61, 779-783 (1968) 352. Grosto M., H a r e f a 73 (31, 90-93 (1967) F., A c t a Physiol. Scand. 353. Boreus L.O. and S&g - 266-271 (1953) 28, 354. Botha D., Mueller F.O., Krueger F.G.M., Melnitzky H., V e m a k L. and b u w L., Em. J. Pharmacol. 6 (3), 312-321 (1969) 355. Grotto M., Harokeach H a i v r i 11, 152/172-157/167 (1965) .. 356. Patent, U S , 3,439,094 (15 Apr. 1969) to W l e J F (Warner-Lambert Pharmaceutical Co ) 357. Patent, U.S. 3,482,021 (2 D e c . 1969) t o Gosling R.H. (Geigy Chemical Corp.)

..

104

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358. Patent, Ger. Offen. 2,058,893 (9 Jun. 1971) t o Wilhelmi G. (CIBA-GeigyA<) 359. Lechat.P., Delaeu D. and Bunot O., Therapie - (41, 20 867-877 (1965) 360. Lechat P., Delaeu D., Fontagne J. and m o t O., Therapie, 21 (31, 565-570 (1966) 361. B u s h H., Gerhards W., Pfleger K., Rueaiger W. and Ihnmrel W., Biochan. Pharmacol. - (81, 1585-1599 16 (1967) 362. Patent, B r i t . 901,674 (25 J u l y , 1962) t o Johnson W.J. (F.W. Homer Ltd.) 363. Patent, U.S. 3,053,737 (Sept. 11, 1962) t o Johnson W. J. 364. Corte G. and Johnson W . J . , Proc. Soc. Exptl. B i o l . W.97 751-755 (1958) 365. MacK&ie D.H.H. and smythe H.A., Can. Conf. Res. Rhemat. Diseases, 2nd., Toronto - 148-149 1960, (Pub. 1961). 366. Levillain R., Cluzan R., David G. and Cayeux Ph., Therapie 2 (21, 539-548 (1965) 367. Weichselbaum T.E. and Margraf H.W., Proc. Soc. Exptl. B i o l . M d 107, 128-131 (1961) e. Yakugaku Zasshi 88 (71, 368. Niwa H. and Nakaym-T., 843-846 (1968) 369. Patent, Belg. 668,418 (Dec. 16, 1965) t o Riviere J.

370. Patent. F r . M 4,196 (July 4, 1966) t o Riviere J. (Laboratoire Solac) 371. Patent, Fr. M 4,825 (Mar. 20,1967) to Riviere J. 372. Patent, Fr. M 4,519 (Nov. 21,1966) t o Riviere J. 373. Patent, F'r. M 5,575 (Jan. 2,1968) to C e n t r e d'Etudes pour 1 Industrie Pharmaceutique ' 374. Patent, Fr. M 5,022 (May 29, 1967) t o C e n t r e d ' Etudes pur 1'Industrie Pharmaceutique 375. Brustier V., Aubert D., Amselem A., Gazz. I n t . Med. Chir. 74 (41, 357-367 (1969) r i c 376. Patent, B 1,043,104 (Sept. 21, 1966) t o Senior N. and Madinaveitia J.L. (Imperial Chemical 377. Patent, U.S. 3,133,863 (May 19, 1964) t o Tansey R.P. 378. Patent, B r i t . 1,019,146 (Feb. 2, 1966) t o Hermelin V.M.
(Strong Cobb A m e r Inc. ) Industries Ltd. )

(Laboratoire Solac)

105

JOHN E . FAIRBROTHER

379. Patent, B r i t . 1,021,924 (Mar. 6, 1966) t o W t h , K l i n e and French Laboratories 380. Patent, N e t h . Appl. 6,512,130 (Mar. 21, 1966) t o 381. Patent, U.S. 3,279,998 (Oct. 18, 1966) to Raff A.M. and Svedres E.V. (Smith, Klhe and French
Laboratories)

Gaunt W.E.

382. Patent, Brit. 1,125,882 (Sept. 5, 1968) t o Shepard M. (Key Pharmaceuticals Inc.) 383. Patent, Ger. Offen. 1,917,930 (Nov. 6, 1969) t o P-11 T.C. and Anderson J.L. (National Cash
Register Co.) and Sloan F.D., D r u g and Cosmetic Industry, March 1972, 34-38, 902, 9OD, 117-121 385. Walters V., J. Pharm. Pharmacol. - Suppl. 228s20,

384. Bakan J.A.

231s (1968) 386. Ebyer T. and muff S., J. Am. Med. A s s o c . 218, 440-441 (1971) 387. Boxill G.C., Nash C.B. and Wheeler A.G., J. Am. Pharm. ASSOC., Sci. Ed. 47, 479-487 (1958) 388. Renault H., Rohrbach P. and D u g n i o l l e J . , Therapie 1 , 3 e 3 0 6 (1956) 1 389. z t c h f i e l d J.T. and Wilcoxon F., J. Phannacol. Exptl. Therap. 96, 99 (1949) 390. Burn J.H. I Finn= D.J. and Goodwin L.G., "Biological Standardisation", 1950, Oxford Press, London, p.114. 391. S t m r G.A., McLean S. and Thorns J., 'Ibxicol. A w l . P h m c o l . 1 (l), 20-28 (1971) 9 e. 11 392. Manolov P., Suwemenna M d 14 ( 0 ,33-37 (1963) 393. Miller M.M., Squibb Private C&munication 394. Boyd E.M. and Bereczky G.M., Brit. J. Pharmacol. Chamtherapy - (31, 606-614 (1966) 26 395. Swinyard E.A., Brown W.C. and Goo&nan L.S., J. Pharmacol. Exptl. Therap. 106,319 (1952) J. Physiol. Pharmacol. 396. Boyd E.M. and H o g a n S.E., 46 (2), 239-245 (1968) , 397. &d E.M., J. C l i n . P h m c o l . 10 (4), 222-227 (1970) - l 4 (2), 205-213 (1971) 398. Boys E.M. I C l h . Pm. J. 399. Dimn M.F., N i ~ m and Prezcott L.F., J. Pathol. 103, 225-229 (1971) 400. E i d s m D.G.D. and Eastham W.N., B r i t . M e d . J. (19661,g , 497 401. Thomson J.S. and Prescott L.F., B r i t . Med. J. (1966),g, 506

can.

106

ACETAMINOPHEN

402. 403. 404. 405. 406. 407. 408. 409. 410. 411. 412. 413. 414. 415. 416. 417. 418. 419. 420. 421. 422. 423. 424.

Pimstone B.L. and Uys C.J., S. Afr. M e d . J., 42, 259 (1968) Maclean D. , P e t e r s T.J., Brown R.A.G. , m a t h i e M., mines G.F. , Robertson P.G.C., Lancet (19681, 849 R s P.G. , Brit. Med. J. , (19691, i,381 oe Toghill P.J., W i l l i a m s R. , Stepheng J.D. and C a r r o l l J . D . , Gastroenterology 56, 773 (1969) P r e s c o t t L.F., J. P h a n n ~ P h a r m c o l .- 331-353 18, (1966) Prescott L.F., Lancet (19651, ii, 91-95 C a l d e r I C , Funder C.C. , G r e e n C.R., Ham K.N. .. and Tange J.C. , Brit. Med. J. (1971) -, 518-521 4 Smith P.K., Davison C. and Scdd M.A. , (1956) , Proceedings of the International P h y s i o l o g i c a l Congress, B r u s s e l s , p. 836. E i s a l o A. and T a l a n t i S., A c t a Med. Scand. 169, 655-660 (1961) Angervall L. , Lehmnn L. and L i n c o l n K. , A c t a P a t h o l , Microbiol. Scand. 154, 274-282 (1962) Angervall L. , LehsMnn L. a n T L i n c o l n K., A c t a P a t h o l . Microbiol. Scand. 154, 283-286 (1962) Angervall L., LehTMnn L. a n ? L i n c o l n K. , A c t a P a t h o l . Microbiol. Scand. 154, Suppl. 61-63 (1962) Angervall L. , LemL. and Bengtsson U. , A c t a Med. S c a d . 175 ( 2 ) , 155-160 (1964) K i e s e M. and-zel H. , Arch. Exptl. P a t h o l . Pharmakol. 242, 551-554 (1962) Schwerd W., Med. W l t (1962) 2348-2349 Heubne.r W., Arch. Ekptl. P a t h o l . Pharmakol. 72 239-247 (1913) S c h n i t z e r B. and Smith E.B., Arch. P a t h o l . 81 ( 3 ) , 264-267 (1966) J3aader H., G i r g i s S., Kiese M., Menzel H. and S k r o h t L., Arch. E x p t l . P a t h o l . Pharmakol. 214, 317-334 (1961) L e s t e r D., J. P h a m c o l . E x p t l . Therap. 77, 154-159 (1943) D o l l G. and Hackenthal E., Arzneimittel-Forsch. 13, 68-71 (1963) Bums J.J. and Conney A.H., Proc. Eur. Soc. Study D r u g T o x i c i t y , 6, 76-82 (1965) Dern R.J. , B e u t i e r E. and A l v i n g A.S. , J. Lab. C l i n . Med. 45, 30-39 (1955) C l a u s e n E. and Larsen O.A., A c t a Pharmacol. T o x i c o l . - ( 2 ) , 135-140 (1965) 22

107

JOHN

E. FAIRBROTHER

425. 426. 427. 428. 429. 430. 431. 432. 433. 434. 435. 436. 437. 438. 439. 440. 441. 442. 443. 444. 445. 446.

b y d M. and Sheppard E.P., B r i t . J. Pharmacol. C h e m t h e r a p y - (21, 497-505 (1966) 27 D i k s t e i n S., G r o t t o M., Zor U., T m r i M. and S u h F.G., J. Endccrinol. 36 ( 3 ) , 257-262 (1966) Thanas J.M., Nakaue H.S. and Reid B.L., Poultry Sci.

- (S), 46

D i k s t e i n S., Zor U., Ruah D. and S u b a n F.G., poultry Sci. 45 ( 4 ) , 744-746 (1966) Lloyd T.W., G c e t (1961), i, 114 Jacobson H., Squibb Private Comnunication C l a r k e G., Squibb Private C m i c a t i o n Jaeckel R. and Peperle W., 2. N a t u r f o r s c h .

1216-1219

(1967)

171-172 (1960) Coy N.H. and Ochs Q., Squibb Private C c g n n u n i c a t i o n Porter M.W. and Spiller R.C., The Barker Index of C r y s t a l s , V o l . 2, P a r t 2 , 1956, H e f f e r and Sons L t d . , C a m b r i d g e (M. 1769A and M. 1769B). Chaw Y.P. and Repta A.J., J. Pharm. S c i . 61 ( 9 ) , 1454-1458 (1972) W i l l i a m s R.T. and Bridges J.W., J. C l i n . Pathol. 17, 371-394 (1964) Patent, G e r . ( E a s t ) 81,119 (12 Apr. 1971) t o

s,

Patent, Ger. O f f e n . 2,121,164 ( 1Nov. 1971) t o 1 Schulman H.L. , Baron F.A. and Minberg A.E. ( H a l l , Howard and Co.) Jalal I.M., M a l i n w s k i H.J. and Smith W.E., J. P h m . Sci. 61 ( 9 ) , 1466-1468 (1972) Elste U. and Duda H., B u t . Apth.-Ztg. 112 ( 1 9 ) , 711-715 (1972) Shearer C.M., C h r i s t e n s o n K., Mukherii A. and Paperiello G i J . , J. P h m . S c i . 6 1 (iO), 1627-1630 (1972) D a l y R.E., Mran C. and C h a f e t z L., J. Pharm. Sci. 6 1 ( 6 ) , 927-929 (1972) Dedicoat H. and Symnds D.C., J. Assoc. Publ. Anal. - 14-17 (1972) 10, Fales H.M., Milne G.W.A. and Law N.C., Arch. Mass Spectral Data 2 ( 4 ) , 6-1 (1971) Mihe G.w.A., Fales H.M. and m n o T., -. e rd 1 C h m . 43 (13), 1815-1820 (1971) Lageoty., L i m u z i n Y. and Maire J.C., J. Organmtal. Chem. 38, 23-27 (1972)

H i n s d o r f G.K.

108

ACETAMINOPHEN

447. 448. 449. 450. 451. 452. 453. 454. 455. 456.

and Davison C., J. Pharm. Sci. 6 1 ( 9 ) , (1972) Fccella A . , Heslin P. and Teitel S. , Can. J. Chem. 50, 2025-2030 (1972) Fairbrother J . E . , Squibb Private C o m i c a t i o n Patent, G e r . 1,493,727 (31Aug. 1972) t o Koenig C., Schmitz P. and P e l s t e r H. (Bayer A.-G.) Peters G., Baechtold-Fowler N. , Bonjour J., e t al., Arch. ToxFkol. 28 ( 4 ) , 225-269 (1972) Patent, Fr. m-de 2,092, 893 (3 Mar. 1972) t o Bru J. R.P. Scherer Ltd., Capsule N e w s lo ( 2 ) , 3 (1972) McGilveray I.J. and Mattok G.L., J. Pharm. Pharmacol 24, 615-619 (1972) Blmr J.R. and Mrrison J.C., J. Phann. Pharmacol. 24, 927-933 (1972) Patent, B r i t . 1,287,431 (31 Aug. 1972) t o Lakenorris W. and Slater J.E. (Aspro-Nicholas Ltd. )
Amsel L.P.

1474-1475

This profile a t t q t s t o cover the published l i t e r a t u r e on acetaminophen up t o Chemical Abstracts, V o l m 77, Issue 21

109

dl-ALPHA-TOCOPHERYL

ACETATE

Bruce C. Rudy and Bernard 2. Senkowski

BRUCE C. RUDY AND BERNARD 2 . SENKOWSKI

INDEX

Analytical P r o f i l e

dl-Alpha-Tocopheryl Acetate

1.

Description 1.1 Name, Formula, M o l e c u l a r Weight 1 . 2 Appearance, C o l o r , Odor 1 . 3 I s o m e r i c Forms 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 Mass Spectrum 2.5 O p t i c a l R o t a t i o n 2.6 M e l t i n g Range 2.7 D i f f e r e n t i a l Scanning C a l o r i m e t r y 2 . 8 Thermal G r a v i m e t r i c A n a l y s i s Synthesis S t a b i l i t y Degradation Drug M e t a b o l i c P r o d u c t s Methods of A n a l y s i s 6.1 Elemental Analysis 6.2 Thin Layer Chromatographic A n a l y s i s 6.3 Gas-Liquid Chromatographic A n a l y s i s 6.4 Direct S p e c t r o p h o t o m e t r i c A n a l y s i s 6.5 C o l o r i m e t r i c A n a l y s i s 6.6 S p e c t r o f l u o r o m e t r i c Analysis 6.7 T i t r i m e t r i c A n a l y s i s Acknowledgements References

2.

3.
4.

5.
6.

7.
8.

112

dl-ALPHA-TOCOPHERYL

ACETATE

1. Description

1.1 Name, Formula, Molecular Weight dl-Alpha-tocopheryl acetate is a racemic mixture of 2,5,7,8-tetramethy1-2-(4',8',12'-trimethy1tridecy1)-6chromarol acetate.

'2 3' 13 ' 5 1.2

Molecular Weight:

472.76

Appearance, Color, Odor dl-Alpha-tocopheryl acetate occurs as a yellow, nearly odorless, clear, viscous oil. Isomeric Forms There are four possible enantiomeric pairs of diastereoisomers which result from the three asymmetric centers present in the alpha-tocopheryl acetate molecule (the asymmetric centers are marked with a small circle in the above structural formula). dl-Alpha-tocopheryl acetate contains an equimolar mixture of the eight isomers. 2. Physical Properties 1.3

2 - 1 Infrared Spectrum The infrared spectrum of dl-alpha-tocopheryl acetate is presented in Figure 1 ( ) 1 . The spectrum was measured with a Perkin-Elmer 621 Spectrophotometer on a capillary layer of the liquid between KBr discs. The assignments for the characteristic bands in the infrared spectrum are listed in Table I ( ) 1. Table I Infrared Assignments for dl-Alpha-Tocopheryl Acetate Frequency (cm-1) 2943 and 2861 Characteristic of CH3 stretching vibrations
113

PI

rd
L)

OD

0 -

- -

L)

Ll

M
CJY

a
al

w
H

Ll

h rd

33NVlllWSNWl K

114

dl-ALPHA-TOCOPHERY

L ACETATE

2920 a n d 2850 1752 1456 1363 1206 2.2

CH2 s t r e t c h i n g v i b r a t i o n s C=O s t r e t c h i n g v i b r a t i o n s CH2 and CH3 d e f o r m a t i o n s CH3 symmetric d e f a m a t i o n s C-0-C s t r e t c h i n g v i b r a t i o n s

N u c l e a r Magnetic Resonance Spectrum (NMR) The s p e c t r u m shown i n F i g u r e 2 w a s o b t a i n e d on a J e o l c o 60 MHz NMR by d i s s o l v i n g 92.4 mg of d l - a l p h a tocopheryl acetate i n 0 . 5 m l of CDC13 c o n t a i n i n g tetram e t h y l s i l a n e a s a n i n t e r n a l r e f e r e n c e ( 2 ) . The s p e c t r a l a s s i g n m e n t s a r e g i v e n i n T a b l e I1 ( 2 ) . T a b l e I1

NMR Assignments o r dl-Alpha-Tocopheryl '

Acetate

Proton

Number o f Protons 12 3 21 2 9 3 2

Chemical S h i f t (ppm) 0.85 1.21 1.0-1.6 1.75 1.96,2.00,2.07 2.29 2.57

Mu1 t i p l i c i t y Doublet (Ja-d = 5 HZ) Singlet Complex M u l t i p l e t T r i p l e t (Je-h = 6 . 8 Hz) T h r e e O v e r l a p p i n g singlets Singlet T r i p l e t (Jh-e = 6 . 8 Hz)

a b c&d e f g h
2.3

U l t r a v i o l e t Spectrum (UV) When t h e UV s p e c t r u m of d l - a l p h a - t o c o p h e r y l a c e t a t e w a s s c a n n e d from 350 t o 220 n m , two maxima and two minima were o b s e r v e d . One maximum is l o c a t e d a t 285 n m ( E = 2.24 x l o 3 ) w i t h a s h o u l d e r a t 287 nm ( E = 2 . 2 1 x l o 3 ) and t h e o t h e r maximum o c c u r s a t 279 n m ( E = 1 . 9 8 x l o 3 ) . The minima a r e l o c a t e d a t 281 nm and 253 nm. The s p e c t r u m shown i n F i g u r e 3 w a s o b t a i n e d from a s o l u t i o n of 1 2 . 4 1 1 mg of d l - a l p h a - t o c o p h e r y l a c e t a t e p e r 100 m l o f cyclohexane(3).

115

BRUCE C. RUDY AND BERNARD 2 . SENKOWSKI

Figure 2

NMR Spectrum of dl-Alpha-Tocopheryl

l ~ ~ [ ' ~ ~ ~ l

Acetate
~ ~ ~ ~ l l

240

180

I20

116

dl-ALPHA-TOCOPHERYL

ACETATE

Figure 3 Ultraviolet Spectrum of dl-Alpha-Tocopheryl Acetate

.8 -

.7

.6

; a
m

.5-

% m
a

4-

.3-

.2

.I

0-

1
117

BRUCE C. R U D Y A N D B E R N A R D 2. SENKOWSKI

Mass Spectrum The mass s p e c t r u m of d l - a l p h a - t o c o p h e r y l a c e t a t e was o b t a i n e d u s i n g a CEC 21-110 mass s p e c t r o m e t e r w i t h a n i o n i z i n g e n e r g y o f 70 e V . The o u t p u t from t h e mass s p e c t r o m e t e r w a s a n a l y z e d and p r e s e n t e d i n t h e form of a b a r g r a p h , shown i n F i g u r e 4 , by a V a r i a n 100 MS d e d i c a t e d computer s y s t e m ( 4 ) . The mass spectrum of dl-alphat o c o p h e r y l a c e t a t e i s c h a r a c t e r i z e d by t h e a b s e n c e of numerous f r a g m e n t a t i o n p r o c e s s e s . The m o l e c u l a r i o n peak o c c u r s a t m / e 472. The b a s e peak a t m / e 430 o c c u r s from t h e l o s s of k e t e n e from t h e m o l e c u l a r i o n . F r a g m e n t a t i o n of t h e non-aromatic r i n g o c c u r s , y i e l d i n g a peak a t m / e 207 ( w i t h t h e a c e t a t e group p r e s e n t ) and a peak a t m / e 165 ( a f t e r l o s s of t h e k e t e n e g r o u p ) . An i n - d e p t h a n a l y s i s o f t h e mass s p e c t r a of t o c o p h e r o l s h a s r e c e n t l y been p u b l i s h e d by Scheppele e t a l . (5). Optical Rotation dl-Alpha-tocopheryl a c e t a t e i s a n equimolar m i x t u r e of t h e o p t i c a l i s o m e r s of a l p h a - t o c o p h e r y l a c e t a t e 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 . M e l t i n g Range dl-Alpha-tocopheryl a c e t a t e is an o i l a t room temperature. I t s o l i d i f i e s a t a t e m p e r a t u r e of -27.5OC 2.7 2.6

2.4

2.5

(6).

D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) A DSC s c a n w a s r u n by c o o l i n g t h e head of t h e DSC which h e l d t h e sample pan c o n t a i n i n g ;he dl-alpha-tocopheryl A f t e r h o l d i n g t h e t e m p e r a t u r e a t -9OOC a c e t a t e t o -9OOC. f o r 30 m i n u t e s , t h e t e m p e r a t u r e w a s i n c r e a s e d a t a r a t e o f 10/minute. Only a n e x t r e m e l y weak e n d o t h e r m i c t r a n s i t i o n w a s observed s t a r t i n g a t a b o u t -44OC. It a p p e a r s t h a t when d l - a l p h a - t o c o p h e r y l a c e t a t e s o l i d i f i e s i t forms a g l a s s i n s t e a d of a c r y s t a l l i n e m a t e r i a l ( 7 ) . Thermogravimetric A n a l y s i s (TGA) The TGA of d l - a l p h a - t o c o p h e r y l a c e t a t e i n a n i t r o g e n atmosphere showed no w e i g h t l o s s from ambient t o 210OC. A s i n g l e w e i g h t l o s s c o r r e s p o n d i n g t o 100% of t h e sample o c c u r r e d between 210' and 37OoC ( 7 ) .

2.8

118

u (d u
a,
4
rl
U

a,

F :
0
U

h k a,

. 3 RI
a m h F : 1 a
l
*rl

I rl

a
w
0

BRUCE C. RUDY A N D BERNARD Z . SENKOWSKI

Synthesis dl-Alpha-tocopheryl a c e t a t e i s p r e p a r e d by t h e r e a c t i o n scheme shown i n F i g u r e 5. Trimethylhydroquinone is condensed w i t h racemic i s o p h y t o l t o y i e l d d l - a l p h a - t o c o p h e r o l which is t h e n a c e t y l a t e d ( 8 ) . S t a b i l i t y Degradation dl-Alpha-tocopheryl a c e t a t e i s p r a c t i c a l l y u n a f f e c t e d by t h e o x i d i z i n g i n f l u e n c e o f a i r and u l t r a v i o l e t l i g h t ( 6 ) . When i t is r e f l u x e d i n a c i d i c and and b a s i c s o l u t i o n s i n t h e a b s e n c e o f oxygen, t h e m o l e c u l e is h y d r o l y z e d t o t h e f r e e dl-alpha-tocopherol. I f oxygen is p r e s e n t , t h e d l a l p h a - t o c o p h e r o l , once formed, w i l l o x i d i z e r a p i d l y t o t h e quinone. The r a t e of o x i d a t i o n i s much f a s t e r i n t h e b a s i c solution. Drug M e t a b o l i c P r o d u c t s dl-Alpha-tocopheryl a c e t a t e i s m e t a b o l i z e d as o u t l i n e d i n Figur e 6 (9,lO). The e s t e r i s r e a d i l y c o n v e r t e d i n t h e animal t o f r e e a l p h a - t o c o p h e r o l which i s t h e n f u r t h e r m e t a b o l i z e d t o a l p h a - t o c o p h e r o l quinone and a n a l p h a t o c o p h e r o l dimer. The a l p h a - t o c o p h e r o l q u i n o n e may b e reduced t o t h e c o r r e s p o n d i n g hydroquinone o r f u r t h e r o x i d i z e d t o a l p h a t o c o p h e r o n i c a c i d (9,10,11). 6. Methods of A n a l y s i s 6.1 5. 4.

3.

Elemental A n a l y s i s The r e s u l t s from t h e e l e m e n t a l a n a l y s i s of d l a l p h a - t o c o p h e r y l a c e t a t e a r e l i s t e d below (12).

E l emen t
C H

% Theory

% Found
78.55 11.05

78.76 11.09

Thin Layer Chromatographic A n a l y s i s (TLC) A TLC system which c a n b e used t o s e p a r a t e d l a l p h a - t o c o p h e r y l a c e t a t e from i t s major m e t a b o l i t e s i s as f o l l o w s . The s a m p l e is a p p l i e d t o a S i l i c a G e l G p l a t e and s u b j e c t e d t o a s c e n d i n g chromatography u s i n g cyc1ohexane:die t h y l e t h e r ( 4 : l ) as t h e d e v e l o p i n g s o l v e n t . A f t e r t h e s o l v e n t h a s ascended 10 t o 1 5 c m , t h e p l a t e is a i r d r i e d , sprayed w i t h c o n c e n t r a t e d s u l f u r i c a c i d , and warmed i n an oven a t 105OC f o r 5 m i n u t e s . The a p p r o x i m a t e Rf v a l u e s 6.2
120

Figure 5 Synthesis of dl-Alpha-Tocopheryl Acetate

a 3

TRIMETHYLHYDROQUINONE

RACEMIC

ISOPHYTOL

Acetic Anhydride

C"3
dl - ALPHA-TOCOPHEROL

dl- ALPHA-TOCOPHERYL ACETATE

Figure 6

Metabolic Products of dl-Alpha-Tocopheryl Acetate

ALPHA-TOCOPHERYL ACETATE

deestwificatian

w w

/
O +

ALPHA- TOCOPHEROL (major)

dl-ALPHA-TOCOPHERY

L ACETATE

are l i s t e d below (13).

a l p h a tocopherol a l p h a t o c o p h e r y l acetate a l p h a t o c o p h e r o l quinone

0.5 0.7 0.9

Gas-Liquid Chromatographic A n a l y s i s (GLC) A r e c e n t c o l l a b o r a t i v e s t u d y has shown dl-alphat o c o p h e r y l a c e t a t e may r e a d i l y b e s e p a r a t e d and a s s a y e d by GLC ( 1 4 ) . The p e r t i n e n t experimental c o n d i t i o n s as w e l l a s t h e r e t e n t i o n t i m e s a r e given i n T a b l e 1 1 1 . Table I11
GLC Method f o r dl-Alpha-Tocopheryl

6.3

Acetate

Column:

Support: S t a t i o n a r y Phase: Detector: Temperature (OC) Injection Port: 300 Column : 280 Detector: 300 C a r r i e r Gas Flow Rate: $40 ml/min. of n i t r o g e n Quantities Injected: c10 mcg i n n-hexane R e t e n t i o n Time (minutes) 22 alpha-tocopherol alpha-tocopheryl acid s u cc i n a t e 23 alpha-tocopheryl acetate 26 dotriacontane ( i n t e r n a l s t a n d a r d ) 30

4 - 8 f e e t , 2-3 mm i . d . , Pyrex o r s t a i n l e s s steel S i l a n i z e d Diatomaceous E a r t h 5-10% SE-30 Hydrogen flame i o n i z a t i o n

Direct S p e c t r o p h o t o m e t r i c Analysis Direct spectrophotometry may b e c a r r i e d o u t on dl-alpha-tocopheryl a c e t a t e provided t h e r e a r e no i n t e r f e r e n c e s p r e s e n t . The maxima f o r dl-alpha-tocopheryl a c e t a t e a r e dependent on t h e c h o i c e of s o l v e n t used. I f m cyclohexane is used as t h e s o l v e n t , t h e maximum a t 285 n may b e used f o r q u a n t i t a t i o n .

6.4

I23

BRUCE C. RUDY A N D BERNARD 2 . SENKOWSKI

Colorimetric Analysis A i n d i r e c t method f o r t h e a n a l y s i s of dl-alphan t o c o p h e r y l a c e t a t e u t i l i z e s t h e Emmerie-Engle c o l o r i m e t r i c p r o c e d u r e ( 1 5 ) . The d l - a l p h a - t o c o p h e r y l a c e t a t e is b a s e hydrolyzed i n anhydrous e t h a n o l t o t h e f r e e t o c o p h e r o l . The s o l u t i o n i s a c i d i f i e d t o p r e v e n t a i r o x i d a t i o n , water i s added, and t h e d l - a l p h a - t o c o p h e r o l i s e x t r a c t e d i n t o die t h y l e t h e r . The e t h e r i s e v a p o r a t e d under n i t r o g e n and t h e r e s i d u e i s immediately d i s s o l v e d i n anhydrous e t h a n o l . F e r r i c c h l o r i d e i s added a l o n g w i t h 2 , 2 ' - b i p y r i d i n e , b o t h i n anhydrous e t h a n o l . The m i x t u r e i s s h a k e n v i g o r o u s l y and timed f o r 10 m i n u t e s . The a b s o r b a n c e of t h e r e d s o l u t i o n i s measured a t 520 n ( 1 6 , 1 7 ) . A review of t h e m Emmerie-Engle and c e r r i c s u l f a t e t i t r i m e t r i c methods f o r Vitamin E was p u b l i s h e d by Lehman (18). Spectrofluorometric Analysis The i n t e n s e u l t r a v i o l e t f l u o r e s c e n c e e x h i b i t e d by a l p h a - t o c o p h e r o l h a s p r o v i d e d t h e b a s i s f o r a s i m p l e and e x t r e m e l y s e n s i t i v e method f o r t h e d e t e r m i n a t i o n of f r e e a l p h a - t o c o p h e r o l and a l p h a - t o c o p h e r y l a c e t a t e i n plasma ( 1 9 , 2 0 ) . Two m l of plasma are d i l u t e d w i t h 2 ml of w a t e r and 4 ml of e t h a n o l and t h e n e x t r a c t e d w i t h 8 ml of hexane. The f r e e a l p h a - t o c o p h e r o l i s d e t e r m i n e d by d i l u t i n g a 1 ml a l i q u o t of t h e hexane p h a s e w i t h 3 ml of e t h a n o l and meas u r i n g t h e i n t e n s i t y of t h e f l u o r e s c e n c e produced a t 340 nm by e x c i t i n g t h e sample a t 295 nm. The a l p h a t o c o p h e r y l a c e t a t e i s determined by d i f f e r e n c e a f t e r h y d r o l y z i n g a 4 ml a l i q u o t of t h e hexane p h a s e w i t h LiAlH4 t o c o n v e r t any acetate t o a l p h a - t o c o p h e r o l and measuring t h e i n t e n s i t y of t h e f l u o r e s c e n c e as above. A p l o t of f l u o r e s c e n c e v e r s u s c o n c e n t r a t i o n of a l p h a t o c o p h e r o l was l i n e a r o v e r t h e r a n g e o f 0 . 6 ug/ml through 40 pg/ml ( 2 0 ) . The l i m i t of d e t e c t i o n i s about 0.01 pg/ml ( 1 9 ) . T i t r i m e t r i c Analysis dl-Alpha-tocopheryl a c e t a t e ( a b o u t 250 mg) i s d i s s o l v e d i n 25 m l of anhydrous e t h a n o l , 20 m l of 5 N e t h a n o l i c s u l f u r i c a c i d is added and t h e s o l u t i o n r e f l u x e d f o r 3 h o u r s t o e f f e c t complete h y d r o l y s i s t o t h e f r e e d l - a l p h a tocopherol. After t h e s o l u t i o n i s c o o l e d , i t is t r a n s f e r r e d t o a 200-ml v o l u m e t r i c f l a s k and d i l u t e d t o volume w i t h 50 m l of 0.5N e t h a n o l i c s u l f u r i c a c i d and 20 m l of water. Two d r o p s of diphenylamine T . S . a r e added and t h e 6.7 6.6

6.5

124

dl-ALPHA-TOCOPHERY

L ACETATE

s o l u t i o n is t i t r a t e d w i t h 0.01N c e r i c s u l f a t e u n t i l a b l u e end p o i n t i s reached which p e r s i s t s f o r 1 0 seconds. A b l a n k is r u n and any n e c e s s a r y volume c o r r e c t i o n made. Each ml of 0.01N c e r i c s u l f a t e i s e q u i v a l e n t t o 2 . 3 6 3 8 mg of d l - a l p h a - t o c o p h e r y l a c e t a t e ( 1 7 ) . Acknowledgments The a u t h o r s wish t o acknowledge Mrs. A. M. Ormsby f o r t y p i n g many of t h e A n a l y t i c a l P r o f i l e s and M r s . L. B. Rubia f o r drawing and l e t t e r i n g many of t h e f i g u r e s . The h e l p a f f o r d e d by t h e S c i e n t i f i c L i t e r a t u r e Department and t h e Research Records O f f i c e of Hoffmann-La Roche I n c . i n t h e l i t e r a t u r e s e a r c h e s also is g r a t e f u l l y acknowledged.

7.

125

BRUCE C. RUDY AND BERNARD 2.SENKOWSKI

8.

References
1.

2.
3, 4. 5.
6.

7. 8. 9.
10.

11.

12.

13. 14. 15. 16. 17. 18. 19.

20.

Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. Johnson, J. , Hoffmann-La Roche Inc., Personal Communication. Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc. , Personal Communication. Scheppele, S . E., Mitchum, R. K., Rudolph, C. J., Kinneberg, K. F. , and Odell, G. V. , L i p i d s , 1, 297 (1972). Merck Index, Eight Edition, p. 1115, 1968. Moros, S., Hoffmann-La Roche Inc., Personal Communication. Surmatis, J., and Weber, J., U . S . Patent 2,728,278 (1955). Gallo-Torres, H E., Miller, 0 N., Hamilton, J. G., . . and Tratnyek, C., L i p i d s , 5, 318 (1971). Draper, H. H., and Csallany, A . S . , "Metabolism o f Vitamin E", DeLuca, H. F., and Suttie, J. W. (editors) , flae Fat-SoZubZe Vitamins, The ZTniversity of Wisconsin Press, pp. 347-353, 1970. Simon, E. J., Eisengart, A . , Sundheim, L., and Milhorat, A . T., J . B i o l . &em.. 221, 807 (1956). Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. Bolliger, H., and KGnig, A . , Hoffmann-La Roche Inc., Unpublished Data. Rudy, B. C., Mahn, F P., Senkowski, B. Z., . Sheppard, A. J. , and Hubbard, W. D. , J . A . 0. A. C., November, 1972 (In press). Emmerie, A., and Engel, C., Nature, 142, 873 (1938). United S t a t e s Pharmacopeia XVIII, p. 914, 1970. National Formulary X I I , p. 760, 1970. Lehman, R. W., J . Pham. S c i . , 53, 201 (1964). Duggan, D. E., Arch. Biochem. Biophys., 9, 116 (1959). Hansen, L. G., and Warwick, W. J., Am. J . Clin. Path., 46, 133 (1966).

126

AMITRIPTYLINE HYDROCHLORIDE

Kenneth W. Blessel, Bruce C. Rudy, and Bernard Z. Senkowski

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2.SENKOWSKI

INDEX Analytical Profile

1. Description

Amitriptyline Hydrochloride

1.1 1.2

Name, Formula, Molecular Weight Appearance, Color, Odor

2.

Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 Differential Scanning Calorimetry 2.9 Thermogravimetric Analysis 2 . 1 0 Solubility 2 . 1 1 X-ray Crystal Properties 2 . 1 2 Dissociation Constant Synthesis Stability Degradation Drug Metabolic Products Methods o f Analysis 6.1 Elemental Analysis 6.2 Phase Solubility Analysis 6.3 Thin Layer Chromatographic Analysis 6.4 Gas-Liquid Chromatographic Analysis 6.5 Colorimetric Analysis 6.6 Fluorescence Analysis 6.7 Non-Aqueous Titration Acknowledgements References

3.

4. 5. 6.

7.
8.

128

AMlTRlPTYLlNE HYDROCHLORIDE

1. D e s c r i p t i o n
Name, Formula, Molecular Weight A m i t r i p t y l i n e h y d r o c h l o r i d e i s lO,ll-dihydro-N,Ndimethyl-5H-dibenzo [ a , d ] cycloheptene-A5, '-propylamine hydrochloride.

1.1

C20H23N*HC1

Molecular Weight:

313.87

Appearance, Color, Odor A r n i t r i p t y l i n e h y d r o c h l o r i d e i s an o d o r l e s s , o f f w h i t e c r y s t a l l i n e powder.

1.2

2.

Physical Properties

I n f r a r e d Spectrum The i n f r a r e d spectrum of a m i t r i p t y l i n e hydroc h l o r i d e is shown i n F i g u r e 1 (1). The sample w a s d i s p e r s e d i n f l u o r o l u b e f o r t h e r e g i o n of 4000-1350 cm-I and i n m i n e r a l o i l t o r e c o r d t h e spectrum i n t h e r e g i o n of 1350-600 cm-1. The f o l l o w i n g assignments of bands i n t h e spectrum have been made (1).

2.1

Band
3057 cm-l 2949 and 2825 c m - l 2921 and 2852 cm-l 2545-2428

Assignment Aromatic CH s t r e t c h Asymmetric and symmetric CH3 stretch Asymmetric and symmetric CH2 stretch C h a r a c t e r i s t i c of H C 1 s a l t of t e r t i a r y amine Four a d j a c e n t hydrogens on benzene r i n g

cm-l

767 and 757 cm-l

129

Figure 1 Infrared Spectrum of A m i t r i p t y l i n e Hydrochloride

WAVELENGTH
25
10 0
3

(MICRONS)
7
I

4
I

5
I

6
I

8 I

9 1 0 1 2 1 5 I I I I I I I I I

20
10 0

80
w
c

- 80
-60

k $20-

a a

-40

-20
I 3OOO
I 2500

I OL 4OOO 3500

I
ZOO0

I
1700

I
1400

I
1100

I
800

500

FREQUENCY

(CM-')

AMlTRlPTYLlNE HYDROCHLORIDE

Nuclear Magnetic Resonance Spectrum (NMR) The NMR spectrum of amitriptyline hydrochloride is shown in Figure 2 ( 2 ) . The spectrum was recorded using a solution of 54.7 mg/0.5 ml in CDC13. The spectral assignments are shown in Table I ( 2 ) .
2.2

Table I
NMR Spectral Assignments for Amitriptyline Hydrochloride

Proton Identification N,N-dimethyl Protons on 7 membered ring and methylene protons Methine pro tons Aromatic protons Proton of Hydrochloride
2.3

Total No. Chemical Shift of Each (ppm) Multiplicity

6
2.65

Singlet Multiplet (unresolved) Triplet Mu1tiplet Singlet (broad)

2.75-3.34
5.8

1
8

7.2
12.5

Ultraviolet Spectrum The ultraviolet spectrum of amitriptyline hydrochloride is shown in Figure 3 ( 3 ) . The spectrum was recorded on a solution which contained 1.00 mg in 100 m l of 0.1N HC1. A maximum was observed at about 239 nm ( E = 1.4 x 104) and a minimum at 228-231 nm. Fluorescence Spectrum The excitation and emission spectra of amitrip4 . The sample tyline hydrochloride are shown in Figure 4 ( ) was dissolved in methanol at a concentration of 1 mg/ml and the spectra were recorded using a Farrand MK-1 recording spectrofluorometer. Excitation at 302 nm produced emission with a maximum at 357 nm.
2.4

Mass Spectrum The low resolution mass spectrum of amitriptyline is shown in Figure 5 ( 5 ) . The spectrum was recorded on a CEC 21-110 mass spectrometer using an ionizing energy of 70 eV, which was interfaced with a Varian data system
2.5
131

132

Figure 3 Ultraviolet Spectrum

of

Amitriptyline Hydrochloride

I .(

O.!

0.f

0. ;

0.c

z a
m
Iz:

in m U

0.:

0.4

0.3

0.2

0. I

0.0
210

250

300
NANOMETERS

3 50

133

z U z

AlISN31NI

134

. 7
I
I

l a s e

8
AlISN31NI 3A11Vl3Y

135

KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD Z . SENKOWSKI

100 MS. The d a t a system accepted t h e o u t p u t of t h e s p e c t r o m e t e r , c a l c u l a t e d t h e masses, compared t h e i r i n t e n s i t i e s t o t h e b a s e peak and p l o t t e d t h i s d a t a as a series of l i n e s whose h e i g h t s were p r o p o r t i o n a l t o t h e i n t e n s i t i e s . The molecular i o n w a s measured a t m / e 277 ( f r e e b a s e ) . The c h a r a c t e r i s t i c f e a t u r e of t h i s compound is i t s s t r o n g tendency t o l o s e N i n o r d e r t o a t t a i n a r o m a t i c i t y and/or r i n g c l o s u r e . The main fragments are t h e loss of N(CH3)2 and CH2N(CH3)2 from t h e s i d e c h a i n t o form m / e 233 and 219 r e s p e c t i v e l y . Each o f t h e s e s p e c i e s then l o s e s hydrogens, g i v i n g rise t o m / e 231, 217, and 215. Complete l o s s of t h e s i d e c h a i n g i v e s rise t o m / e 192 and m / e 85. The b a s e peak o c c u r s a t m / e 58 and i s due t o CH2N(CH3)2 ( 5 ) . A h i g h r e s o l u t i o n spectrum w a s found t o b e f u l l y comp a t i b l e w i t h t h e low r e s o l u t i o n scan. Optical Rotation A m i t r i p t y l i n e h y d r o c h l o r i d e does n o t e x h i b i t optical activity.
M 1t i n g Range e The m e l t i n g range r e p o r t e d i n USP X V I I I i s 195-198OC when a Class I procedure i s used ( 6 ) .

2.6

2.7

D i f f e r e n t i a l Scanning Calorimetry (DSC) The DSC curve f o r a m i t r i p t y l i n e h y d r o c h l o r i d e a t a s c a n r a t e of 10C/min. i s shown i n F i g u r e 6 ( 7 ) . The curve was recorded w i t h a P e r k i n E l m e r DSC-1B under a n atmosphere of n i t r o g e n . A s i n g l e endotherm was observed, t h e e x t r a p o l a t e d o n s e t of m e l t i n g o c c u r r i n g a t 193.0 5 0.2OC and t h e peak a t 197.5 5 0.2OC. A l l temperatures a r e c o r r e c t e d . The v a l u e c a l c u l a t e d f o r AHf was 6.7 kcal/mole f o r t h e m e 1 t i n g endo therm. Thermogravimetric Analysis (TGA) The TGA curve f o r r e f e r e n c e s t a n d a r d a m i t r i p t y l i n e h y d r o c h l o r i d e e x h i b i t e d no l o s s of weight from A s i n g l e weight l o s s o c c u r r e d i n t h e temperature 30-195OC. range of 195-317OC which accounted f o r 100% of t h e sample weight ( 7 ) . Solubility The s o l u b i l i t y d a t a o b t a i n e d f o r r e f e r e n c e s t a n d a r d a m i t r i p t y l i n e h y d r o c h l o r i d e is l i s t e d i n Table I1 ( 8 ) . 2.10 2.9

2.8

136

AM1TR IPTY LINE H Y DROCH LOR IDE

Figure 6

DSC Curve of A m i t r i p t y l i n e H y d r o c h l o r i d e

A Hf =6.7kcal / m o l e

t
4

Endothermic

Exothermic

I
I70

I
I80

I
190
OC

I
200

I
210

137

KENNETH w. BLESSEL, BRUCE c. RUDY, AND BERNARD z . SENKOWSKI

Table I1 S o l u b i l i t y of A m i t r i p t y l i n e Hydrochloride Solvent petroleum e t h e r (30-60C) diethyl ether water 2 -propano1 3A a l c o h o l chloroform 95% e t h a n o l benzene methanol S o l u b i l i t y (mg/ml)

0.30 0.50 >SO0 53 * 313

5.0 >500

>500. >SO0 -

Crystal Properties The x-ray powder d i f f r a c t i o n d a t a f o r a sample of r e f e r e n c e s t a n d a r d a m i t r i p t y l i n e h y d r o c h l o r i d e i s g i v e n i n Table I11 (9). The o p e r a t i n g parameters of t h e hstrument are given below. I n s t r u m e n t a l Conditions General E l e c t r i c Model XRD-6 Spectrogoniometer Generator: Tube t a r g e t : Radiation : optics :

2.11

Goniometer: Detector:

50 KV, 12-112 MA Copper CU Ka = 1.542 8 01 D e t e c t o r s l i t .' M.R. S o l l e r s l i t 3' Beam s l i t 0.0007" N i f i l t e r 4O t a k e o f f o a n g l e Scan a t 0.2 2 0 p e r minute Amplifier g a i n - 1 6 c o u r s e , 8.7 f i n e Sealed p r o p o r t i o n a l c o u n t e r tube and DC v o l t a g e a t plateau P u l s e h e i g h t s e l e c t i o n EL 5 volts Eu - o u t Rate meter T.C. 4 2000 CIS f u l l s c a l e

138

AMlTRl PTYLINE HY DROCHLORlDE

Recorder: Samples:

Chart speed 1 inch per 5 minutes Prepared by grinding at room temperature

Table I11 Interplanar Spacings in Amitriptyline Hydrochloride from Powder Diffraction Data 28 11-50 12.78 13.46 14.86 15.66 16.06 16.34 16.72 17.72 18.58 18.90 19.26 20.24 20.94 21.34 21.87 22.94 23.42 24.32 24.88 25.78 26.62 27.30 27.58 28.16 29.00 d) &* 7.69 6.93 6.58 5.96 5.66 5.52 5.42 5.30 5.01 4.78 4.70 4.61 4.39 4.24 4.16 4.06 3.88 3.80 3.66 3.58 3.46 3.35 3.27 3.23 3.17 3.08 28 IITo% -

21 10
16 9 4 15 18 1 74 13 36 9 100 10 4 60 7 9 3 19 16 9 3 9 4

29.36 29.52 30.10 30.96 31.28 31.74 31.98 32.46 32.76 33.80 34.12 34.98 35.30 36.00 38.26 39.02 39.66 40.04 40.54 41.22 41.88 42.06 43.24 43.88 44.22 44.70 45.30

3.04 3.03 2.97 2.89 2.86 2.82 2.80 2.76 2.73 2.65 2.63 2.57 2.54 2.49 2.35 2.31 2.27 2.25 2.23 2.19 2.16 2.15 2.09 2.06 2.05 2.03 2.00

6 3 5 7 6 3 2 3 3 3 6 2 1 1 2 1 2 3 2 2 2 2

4 4 7

3 2

*d = (interplanar distance) - nh 2 Sin e

**I/

I0

= relative intensity (based on highest intensity of 100)

139

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2.SENKOWSKI

D i s s o c i a t i o n Constant The d i s s o c i a t i o n c o n s t a n t f o r a m i t r i p t y l i n e hydrochloride was determined u s i n g a g r a p h i c a l meihod i n volving t h e pH dependence of t h e water s o l u b i l i t y . The v a l u e f o r t h e pKa determined by t h i s method was 9.4 (10). Synthesis Two s y n t h e t i c r o u t e s t o a m i t r i p t y l i n e are shown i n Figure 7. The f i r s t , r e a c t i o n sequence I (11,12), i n v o l v e s t h e a d d i t i o n of a Grignard r e a g e n t followed by d e h y d r a t i o n w i t h HC1 o r a c e t y l c h l o r i d e , w h i l e I1 (13) u s e s a cyclopropyl Grignard r e a g e n t followed by r i n g opening w i t h dimethylamine t o form t h e d e s i r e d compound. A number of a l t e r n a t i v e syntheses have been d e s c r i b e d i n t h e l i t e r a t u r e 3.

2.12

(14-17).
S t a b i l i t y Degradation The s t a b i l i t y of a m i t r i p t y l i n e h y d r o c h l o r i d e , i n t h e bulk form, was s t u d i e d under c o n d i t i o n s of e l e v a t e d tempera t u r e o r exposure t o l i g h t (18). It was found t o b e s t a b l e a t room temperature and a t 45OC f o r a p e r i o d i n excess of two months. It showed some decomposition a f t e r two months a t 100C, and when exposed t o l i g h t , a s evidenced by t h e formation of a brownish d i s c o l o r a t i o n of t h e powder. A 1% aqueous s o l u t i o n was found t o be s t a b l e a t OC, room temperature, and 45OC, a s w e l l as f o r a p e r i o d of 60 hours a t IOOOC. Drug Metabolic Products Hucker and P o r t e r (19) demonstrated t h a t very l i t t l e of a dose of a m i t r i p t y l i n e i s e x c r e t e d unchanged i n humans. Other i n v e s t i g a t o r s (20-23) have demonstrated t h e p r e s e n c e of t h e major m e t a b o l i c products shown i n F i g u r e 8. Facino and Corona (20) have demonstrated m e t a b o l i t e s I, 111, IV, t h e two isomers o f V, and VI i n t h e organs of r a b b i t s . Eschenoff and Rieder (21) demonstrated m e t a b o l i t e s I, 11, and V and i n a d d i t i o n r e p o r t e d t h e e x i s t e n c e of t h e N-oxide m e t a b o l i t e ( V I I ) i n s t u d i e s on rats and humans. They r e p o r t e d t h a t t h e metabolism o f a m i t r i p t y l i n e i n t h e s e two species i s n e a r l y i d e n t i c a l ( 2 4 ) . I n a d d i t i o n , Facino and Corona (25) l a t e r r e p o r t e d t h e e x i s t e n c e of an a c i d i c m e t a b o l i t e t o which they a s c r i b e d t h e s t r u c t u r e of t h e c a r b o x y l i c a c i d which would b e formed by o x i d a t i o n deaminat i o n of t h e drug.

4.

5.

140

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD Z . SENKOWSKI

Figure 8 Metabolic Products of Amitriptyline Hydrochloride

CH(CH1)zN(CHJ2 AMlTRlPTYLlNE HYDROCHLORIDE

f 0

CI

IV

142

AMlTRl PTY LINE HYDROCHLORIDE

6.

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

Elemental Analysis The r e s u l t s o f an e l e m e n t a l a n a l y s i s of a sample of r e f e r e n c e s t a n d a r d a m i t r i p t y l i n e h y d r o c h l o r i d e i s p r e s e n t e d i n T a b l e I V below (32). Element

C

Theory % 76.53 7.70 4.47 11.30

Found % 76.44 7.79 4.50 11.26

H
N

c1
6.2

Phase S o l u b f l i t y Analysis A phase s o l u b i l i t y a n a l y s i s f o r a m i t r i p t y l i n e h y d r o c h l o r i d e is shown i n F i g u r e 9 . The s o l v e n t u s e d w a s a c e t o n e and t h e e x t r a p o l a t e d s o l u b i l i t y w a s 17.81 mg/g (8). Thin Layer Chromatographic A n a l y s i s A number of t h i n l a y e r chromatographic s y s t e m s f o r a m i t r i p t y l i n e h y d r o c h l o r i d e h a v e been d e s c r i b e d i n t h e l i t e r a t u r e . A r e p r e s e n t a t i v e number o f t h e s e s y s t e m s are shown i n T a b l e V. Methods of d e t e c t i o n were n o t i n c l u d e d s i n c e t h e s e are many times d e t e r m i n e d by t h e i n d i v i d u a l p r e f e r e n c e s of t h e a n a l y s t o r t h e p a r t i c u l a r s e p a r a t i o n d e s i r e d (26). Table V
TLC Systems f o r A m i t r i p t y l i n e H y d r o c h l o r i d e

6.3

Adsorbent
Silica G e l

S o l v e n t System benzene:dioxane:NH3 (60 :35 :5)

&
0.74

Reference

27
27 27 28

Silica G e l

e t h a n o 1 : a c e t i c a c i d : 0.65 H20 (50 :30 :20) b methanol : u t a n o l (60 :40) 0.37

S i l i c a Gel
Silica G e l

+ 0.1M

NaOH cyc1ohexane:benzene: 0.72 d i ethylamine (75 :1 5 :10)

143

Figure 9
I -

z
W

>
J
0
\

25

0
v)

W I -

20

0
v)

LL

0 w
c

15

PHASE SOLU B ILI TY AN ALY S IS Sample: Amitriptyline Solvent = Acetone / Slope : 0.04 o o Equilibration : 20 hrs at 25OC Extrapolated Solubility * 17.81 mg/g of Acetone

P P

2 t
v)

10

0
0.

0
V

z 0
I J

0
v)

0 0

25
SYSTEM COMPOSITION
:

50

75

100

mq OF SAMPLE PER g SOLVENT

AMITR IPTY LINE HYDROCHLORIDE

Silica G e l S i l i c a Gel Silica G e l S i l i c a Gel

+ 0.1M + 0.1M + 0.1M + 0.1M

NaOH NaOH

methanol acetone methanol

0.50
0.34 0.41

28 28

KHSO4
KHS04

28 28

95% e t h a n o l

0.28

Gas-Liquid Chromatographic Analysis (GLC) A GLC method f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n of a m i t r i p t y l i n e has been d e s c r i b e d (29). The method w a s developed f o r measuring t h e a m i t r i p t y l i n e c o n t e n t of plasma. The lower l i m i t of d e t e c t i o n is 20 ng/ml. The chromatographic c o n d i t i o n s a r e given below. Column

6.4

5 f t . x 114 i n . s i l a n i z e d
glass Chromosorb W (80-100 mesh) 1 polyvinyl pyrrolidinone % and 3% Versamid 900 Flame i o n i z a t i o n 205OC

Support Liquid Phase

Detection Oven Temperature Detector Temperature Injection Port Temperature C a r r i e r Gas -

240C
Maximum N i t r o g e n , 50 ml/min.

C o l o r i m e t r i c Analysis A m i t r i p t y l i n e h y d r o c h l o r i d e can b e determined c o l o r i m e t r i c a l l y u s i n g t h e methyl orange r e a c t i o n . The method i n v o l v e s b u f f e r i n g t h e s o l u t i o n c o n t a i n i n g t h e compound a t a pH v a l u e of 4 . 3 , adding the methyl o r a n g e and e x t r a c t i n g t h e r e s u l t i n g complex i n t o e t h y l e n e d i c h l o r i d e . The absorbance of t h e e t h y l e n e d i c h l o r i d e e x t r a c t i s meas u r e d a t about 430 n and t h e amount of a m i t r i p t y l i n e calm c u l a t e d by comparison w i t h a c a l i b r a t i o n curve prepared from pure a m i t r i p t y l i n e . This method can b e used t o determine a m i t r i p t y l i n e i n t h e p r e s e n c e of i t s N-demethylated m e t a b o l i t e s by t h e a d d i t i o n of acetic anhydride b e f o r e ext r a c t i o n s i n c e primary and secondary amines do n o t r e a c t w i t h methyl orange i n t h e p r e s e n c e o f a c e t i c anhydride (30). Fluorescence A n a l y s i s A s e n s i t i v e f l u o r i m e t r i c a s s a y h a s been developed f o r a m i t r i p t y l i n e h y d r o c h l o r i d e i n b i o l o g i c a l samples (31).

6.5

6.6

KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD 2 . SENKOWSKI

The b i o l o g i c a l material t o b e analyzed is homogenized and a n e q u a l volume of methanol added. Then 0 . 2 g of borax, 1 5 m l of heptane and 1 m l of d i s t i l l e d w a t e r i s added t o a 1 m l a l i q u o t of t h e methanolic sample. The heptane l a y e r a f t e r s e p a r a t i o n , is t h e n e x t r a c t e d w i t h p e r c h l o r i c a c i d . The a c i d e x t r a c t is t h e n heated i n a b o i l i n g water b a t h f o r 10 min. and cooled. The f l u o r e s c e n c e of t h e carbonium i o n generated by t h e h e a t i n g p r o c e s s i s t h e n measured a t 555 nm u s i n g a n a c t i v a t i o n wavelength of 305 nm. The f l u o r e s c e n c e i n t e n s i t y was found t o b e l i n e a r w i t h a m i t r i p t y l i n e conc e n t r a t i o n i n t h e range of 0.05-5.0 mcg/ml.

T i t r i m e t r i c Analysis A non-aqueous t i t r a t i o n w i t h D e r c h l o r i c a c i d i n acetic a c i d i s t h e p r e f e r r e d method f o r ' t h e a n a l y s i s of bulk a m i t r i p t y l i n e hydrochloride. The sample is d i s s o l v e d i n g l a c i a l a c e t i c a c i d . Then mercuric a c e t a t e T.S. and c r y s t a l v i o l e t T.S. are added. The s o l u t i o n i s t i t r a t e d with 0.1N p e r c h l o r i c a c i d t o a green end-point. Each m l of 0.1N p e r c h l o r i c a c i d i s e q u i v a l e n t t o 31.39 m of a m i t r i p g t y l i n e hydrochloride ( 6 ) .
Acknowledgments The a u t h o r s wish t o acknowledge t h e a s s i s t a n c e o f t h e Research Records O f f i c e and t h e S c i e n t i f i c L i t e r a t u r e Department of Hof fmann-La Roche I n c .
7.

6.7

146

AMlTRlPTYLlNE HYDROCHLORIDE

8. References
1. Hawrylyshyn, M,, Hoffmann-La Roche Inc., Personal Communication. 2 . Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. 3. Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. 4. Boatman, J., Hoffmann-La Roche Inc., Personal Communication. 5. Benz, W., Hoffmann-La Roche Inc., Personal Communication. 6. The United S t a t e s Pharmacopeia XVIII, pp. 38-40 (1970). 7. Moros, S., Hoffmann-La Roche Inc., Personal Communication. 8. MacMullan, E., Hoffmann-La Roche Inc., Personal Communication. 9. Hagel, R., Hoffmann-La Roche Inc., Personal Communication. 10. Green, A. L., J . Pharm. Pharmac.,E, 10 (1967). 11. Hoffmann-La Roche, F. and Co., A . G., Belgian Patent 577,057 (1959). 12. Merck and Co. Inc., Belgian Patent 584,061 (1960). 13. Hoffsommer, R. D., Taub, D., and Wendler, N. L., J. Org. Chem., 2, 1829 (1962). 14. Protiva, M., et al., J . Med. Pharm. Chern.,&, 411 (91. 16) 15. Villani, F. J., Ellis, C. A . , Teichman, C., and Bigos, C . , J . Med. Pharm. Chem.,?, 373 ( 9 1 . 16) 16. Winthrop, S . , et al., J . Org. Chern.,Z, 230 (1962). 17. Merck and Co, Inc., United States Patent 3,205,264 (1965) 18. Schmidli, B., Hoffmann-La Roche Inc., Unpublished Data. 19. Hucker, H. B. and Porter, C. C., Federation Proc., - 172 (1961). 20, 20. Facino, R. M. and Corona, G. L., J . Pharm. Sci., 58, 764 (1969). 21. Eschenhof, E. and Rieder, J., ArzneimitteZ-Forsch , - 957 (1969). 19, 22. Hucker, H. B., PharmaeoZogis$ k, 171 (1962). 23. Diamond, S., Current Therap. Res., 7, 170 (1962).

147

KENNETH W. BLESSEL. BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

24. Eschenhof , E. and Rieder, J., Deut. Apotheker-Ztg., - 1202 (1968). 108, 25. Facino, R., Santagostino, G. and Corona, G., Biochem. PharmacoZ., 2 , 1503 (1970). 26. Comer, J. P. and Comer, I., J . Pham. S c i . , 56, 413 (1967) 27. Cochin, J. and Daly, J. W., J. PhamacoZ. ExptZ. Therap., 139, 160 (1963). 28. Fike, W. W., AnaZ. Chem.,E, 1697 (1966). 29. Braithwaite, R. A . and Widdop, B., CZin. Chim. Acta , - 461 (1971). 35, 30. Silverstein, R. M., AnaZ. Chem.,g, 154 (1963). 31. Eschenhof, E. and Rieder, J. , Hoffmann-La Roche Inc. , Unpublished Data. 32. Scheidl, F., Hoffmann-La Roche Inc., Personal Communication.

148

DIGITOXIN

Ivan M . Jakovljevic

IVAN M. JAKOVLJEVIC

CONTENTS
1.

2.

3. 4. 5.

6.

DESCRIPTION 1.1 R e g i s t e r e d Names 1.2 Chemical Name 1 . 3 Formula, S t r u c t u r e , M o l e c u l a r Weight 1 . 4 Appearance PHYSICAL PROPERTIES 2.1 I n f r a r e d Spectrum 2.2 N u c l e a r M a g n e t i c Resonance S p e c t r u m 2.3 U l t r a v i o l e t Spectrum 2.4 Mass S p e c t r u m 2.5 Optical Rotation 2.6 O p t i c a l R o t a t o r y D i s p e r s i o n (ORD) a n d C i r c u l a r D i c h r o i s m (CD) 2.7 M e l t i n g Range 2.8 X-Ray D i f f r a c t i o n P a t t e r n 2.9 Polarography 2.10 S o l u b i l i t y SYNTHESIS STABILITY METABOLISM, P R O T E I N BINDING A N D C L I N I C A L ASSAYS 5.1 Metabolism 5.2 P r o t e i n Binding 5 . 3 D e t e r m i n a t i o n i n Blood 5.3.1 Chemical Methods 5.3.2 P h y s i c a l Methods 5.3.3 Radioimmunoassay ( D e u t e r i u m and T r i t i u m Labeled D i g i t o x i n ) METHODS O F ANALYSIS 6.1 I d e n t i f i c a t i o n Tests 6.2 Elemental Analysis 6 . 3 Chromatography 6.3.1 Column Chromatography 6.3.2 Thin L a y e r Chromatography 6.3.3 P a p e r Chromatography 6.3.4 Gas Chromatography 6.3.5 High S p e e d Liquid Chromatography

150

DIG ITOX IN

Colorimetric Analysis Fluorometric Analysis Electrophoresis Automated Assay 7. CLEAVAGE OF CARDIAC GLYCOSIDES 8. BIOLOGICAL ACTIVITY 8.1 Characteristic Structural Features 8.2 Bioassay 9 , ACKNOWLEDGMENT
6.4 6.5 6.6 6.7
10. REFERENCES

15 1

IVAN

M. JAKOVLJEVIC

DESCRIPTION Digitoxin is a cardiotonic glycoside obtained from D i g i t a l i s p u r p u r e a LinnQ, D i g i t a l i s l a n a t a Ehrhart , and other suitable species o f D i g i t a l i s leaves, Registered Names 1.1 Digitoxin is designated by the following names: C A R D I G I N (Nat ,Drugs) , C R Y S T O D I G I N (Li 1 ly) , D I G I C O R Y L (Roussel) , D I G I L O N G (Roehringer) , D I G I JYERCK (Merck) , D I G I P A N , D I G I S I D I N (Winthrop) , D I G I T A L I N E N A T I V E L L E (Varick), D I G I T O R A (Upjohn), D I G I T O X I N (Sandoz) , D I G I T O X O S I D E (W.H.0,) , D I G I T R I N (Astra) , L A N A T O X I N (Beiersdorf) , P U R O D I G I N (Wyeth) , P U R P U R E N , P U R P U R I D (Promonta), 1.2 Chemical Name
1.

3B-(D-Digitoxosyl-D-digitoxosyl-D-digitoxosyl-oxy)-14~-hydroxy-5~-card-2O(2Z)-enolide, 1.3 Formula, Structure, Molecular Weight

c 4 1H6 4 0 1 3

Mol.Wt.

764.94

C18H3109

C23H3404

(tridigitoxose)

digitoxigenin

THE CONFORMATIONAL ARRANGEMENT

Digitoxin belongs t o the cardenolide series, which consists of a steroid nucleus with a 5-membered unsaturated lactone ring at C-17. As in most other glycosides, the sugar i s present in the six-membered ring form (pyranoid
152

DIGITOXIN

form) w i t h t h e c h a i r c o n f o r m a t i o n . The s t e r o i d framework i s c o n s i d e r a b l y b e n t a t e i t h e r e n d o f t h e m o l e c u l e , which i s a n import a n t s t e r i c requirement f o r c a r d i o t o n i c a c t i v i t y . S a t u r a t i o n of t h e l a c t o n e r i n g g r e a t l y r e d u c e s t h e c a r d i o t o n i c a c t i v i t y . The u n s a t u r a t e d l a c t o n e r i n g must be a t t a c h e d i n t h e B - c o n f i g u r a t i o n . E p i m e r i z a t i o n r e d u c e s p h a r m a c o l o g i c a l a c t i v i t y by a t l e a s t 400 times. O p e n i n g t h e l a c t o n e r i n g by a l k a l i n e h y drolysis also results i n loss of activity',
1.4

Appearance

Very s m a l l e l o n g a t e d , r e c t a n g u l a r p l a t e s from d i l u t e d e t h a n o l , o r m i c r o c r y s t a l l i n e powder, w h i t e o r p a l e b u f f , o d o r l e s s , v e r y 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 ( I R ) s p e c t r u m o f d i g i t o x i n , USP r e f e r e n c e s t a n d a r d , i s g i v e n i n F i g . 1 . The I R s p e c t r u m was t a k e n i n a K B r p e l l e t on a Beckman IR-12 s p e c t r o m e t e r . IR s p e c t r a l a s s i g n m e n t s o f d i g i t o x i n a r e as follows2: W a v e l e n g t h of V i bra ti on Mode 8

A b s o r p t i o n (CM-')
t

3575 3440 2960, 2935 1740 1630 1448, 1403, 1 3 7 9 , 1 3 6 7 , 1348

-OH

s t r e t c h Inon-hydrogen bonded) -OH s t r e t c h ( h y d r o g e n bonded) CH s t r e t c h , C H 3 , CH2 stretch C E O s t r e t c h (a,B u n s a t u rated lactone] CH s t r e t c h [ c o n j u g a t e d d o u b l e bond)
C H d e f o r m a t i o n (CH3, C H 2 , C-CHJ, CH)

153

I V A N M. JAKOVLJEVIC

1162 1125 1075 1060 1040

3O-OH d e f o r m a t i o n ZO-OH deformation C-0- s t r e t c h ( g l y c o s i d i c ether) C-0- s t r e t c h ( c y c l i c e t h e r oxygens ) 1-OH d e f o r m a t i o n

The IR s p e c t r a o f 36 g l y c o s i d e s a n d t h e i r a g l y c o n e s w e r e s t u d i e d . G l y c o s i d e s were c h a r a c t e r i z e d by a d o u b l e t i n t h e r e g i o n 1099-1031 and 1 0 6 6 - 1 0 1 3 cm-'.' Examination of t h e I R s p e c t r a of d i g i t o x i n r e v e i l e d t h e p r e s e n c e o f two p o l y m o r p h s . One o f t h e p o l y m o r p h i c c r y s t a l s was o b t a i n e d b y r e c r y s t a l l i z a t i o n from 85% e t h a n o l and t h e o t h e r from t h e c o l d ethanol evaporation1 , 2.2 Nuclear Magnetic Resonance Spectrum The N M R s p e c t r u m o f d i g i t o x i n i s c o m p l e x e v e n a t 2 2 0 MHz, h o w e v e r 1 7 p r o t o n s i g n a l s may b e s e e n t o low f i e l d o f 2.5 ppm b o t h i n C D C 1 3 nnd CD3SOCD3 a f t e r e x c h a n g e w i t h D,O t o remove t h e s i g n a l s f o r O H , They may b e a s s i g n e d w i t h r e a s o n a b l e c e r t a i n t y from chemical s h i f t and c o u p l i n g c o n s t a n t s , The 6 v a l u e s a r e l i s t e d a s f o l l o w s : I n C D C 1 , : 2 . 7 9 ppm ( b r o a d e n e d t r i p l e t ) , p r o t o n a t 1 7 ; 3 . 2 0 , 3.23, 3 . 2 7 ( o y e r l a p p i n g ) , J a a = 1 0 . 0 Hz, = 2.8, p r o t o n s 4 l o ' , 16' Jae ( n u m b e r i n g f r o m t h e a n o m e r i c p r o t o n away f r o m t h e oxygen i n e a c h s u g a r r i n g s e q u e n t i a l l y s t a r t i n g w i t h p o s i t i o n 3 on t h e s t e r o i d ) ; 3 . 7 7 u n r e s o l v e d p r o t o n s 5 ' , l l ' , 1 7 ' ; 4 . 0 1 , p r o t o n 3 ; 4.10, p r o t o n 1 5 ' ; 4 . 2 2 , u n r e s o l v e d p r o t o n s 3' and 9 ' ; 4.78, 4 . 9 7 , J g e m - - 1 8 . 0 Hz, J z 1 - 2 2 = 1 . 8 , C H 2 a t 2 1 ;

4.84,

4.88,

4.91,

( o v e r l a p p i n g ) Jaa- 10, p r o t o n s

1 3 ' ; 5.88, p r o t o n a t 22.5 See F i g . 2 , P o s i t i o n s o f t h e a c e t y l groups i n p a r t i a l l y a c e t y l a t e d c a r d e n o l i d e s were e s t a b l i s h e d b y t h e a n a l y s i s o f NMR s p e c t r a . Chemical s h i f t s o f p r o t o n s belonging t o s p e c i f i c a c e t y l groups d i f f e r s u f f i c i e n t l y t o serve as d i a g n o s t i c e h a r a c t e r i s t i c s . l', 7',
154

DIGITOXIN

3000 2500

2000
I

1600
I

1400

FREOUENCY 1200 1000

1

900
0

850

800
1

750

700
I

65
I

9 10 WAVELENGTH

11

12

13

14

15

F i g . 1 I R S p e c t r u m of D i g i t o x i n , U S P R e f e r e n c e S t a n d a r d . K B r P e l l e t . I n s t r u m e n t : Beckman I R - 1 2 Spectrophotometer.

500
I

400
I

300
I

200

100
I

80

70

60

50

40

30

20

10

PPM

( 6 )

Fig. 2 NHR S p e c t r u m of D i g i t o x i n , U S P R e f e r e n c e S t a n d a r d . I n s t r u m e n t : V a r i a n T-60A.

155

I V A N M. JAKOVLJEVIC

The s i g n a l s o f t h e e q u a t o r i a l p r o t o n s o f d i g i t o x o se molecules a r e a l s o o f d i a g n o s t i c value a s t h e i r p o s i t i o n c h a n g e s when t h e a d j a c e n t a x i a l g r o u p i s acetylated6. 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 c u r v e o f a s o l u t i o n i n m e t h a n o l shows a p e a k a t 218 nm, E 17.4 x 2.4 Mass S p e c t r u m Mass s p e c t r o m e t r i c d a t a were o b t a i n e d u s i n g e l e c t r o n i m p a c t a n d low r e s o l u t i o n on a C.E.C. 110 mass s p e c t r o m e t e r . The f r a g m e n t a t i o n pattern i s t y p i c a l of t h e s t e r o i d portion only. The h i g h e s t s i g n i f i c a n t mass i s a t m/e 357 w h i c h represents t h e digitoxigenin fragment. 2.5 Optical Rotation I n v e s t i g a t o r s have determined t h e o p t i c a l r o t a t i o n under d i f f e r e n t conditions:

[a];'=
[a];'=
20=

+4.8'

(c31.2 i n d i o x a n e ) ' (cp2.5 in chlor~form)~

a b o u t +18' a b o u t +21' a b o u t +13'

(cxl.0 i n c h l o r ~ f o r m ) ~ (cl.1.0 i n methanol)'

[a];'=

2.6

O p t i c a l Rotatory Dispersion (ORD) C i r c u l a r Dichroism (CD)

and

The c i r c u l a r d i c h r o i c (CD) s p e c t r a were r e c o r d e d on a C a r y 60 s p e c t r o p o l a r i m e t e r , e q u i p p e d The O R D c u r v e shows a w i t h Model 6 0 0 2 C D u n i t . p o s i t i v e [ a ] of 1 3 . 1 a n d a p o s i t i v e C o t t o n e f f e c t o f [ a ] 1 2 . 3 x l o - ' a t 254 nm a s d o many s t e r o i d s . S e e F i g . 3. The C D c u r v e shows a p o s i t i v e e f f e c t o f [ a ] 11.1 x 1 O - j a t 238 nm. These s t u d i e s were done i n m e t h a n o l . 7
156

DIGITOXIN

S t e r e o c h e m i c a l e f f e c t s a r e found i n card e n o l i d e s w i t h a - k e t o l groups i n t h e 11,12 p o s i tion.

Fig. 3 O R D a n d C D S p e c t r a of D i g i t o x i n , U S P Reference Standard. Instrument: Cary 60 Spectrop o l a r i m e t e r , e q u i p p e d w i t h Model 6 0 0 2 C D U n i t .
.?.

200

250

nm

300

350

2.7

M e l t i n g Range

The m e l t i n g p o i n t o f d i g i t o x i n is 256 257C.(anhydrous).e Digitoxigenin has a m e l t i n g p o i n t 25OoC. The e f f e c t o f p r o t e c t i v e e n v i r o m e n t s s u c h a s a ) immersing t h e s u b s t a n c e under s i l i c o n e o i l , b ) u n d e r an a t m o s p h e r e o f n i t r o g e n a n d c ) i n a n e v a c u a t e d , s e a l e d c a p i l l a r y t u b e were s t u d i e d on a m i c r o s c o p e h o t s t a g e , The m e l t i n g p o i n t s w h i c h a r e obtained under such conditions a r e o f t e n higher12. 2.8

The X-ray d i f r a c t i o n p a t t e r n o f d i g i t o x i n conforms t o t h e f o l l o w i n g p a t t e r n . dA

1/11

7
20 30 30

X-Ray D i f f r a c t i o n P a t t e r n

dA
6.26

I/I

dA

I/I

15 9.07
8.01 7.2b

5.95 5.40

7.00

5.63 5.32

40 I00 50 40
157

4 . bO
4.62

4.32
4.10

40 40 70
30 30

13

3.93

I V A N M. JAKOVLJEVIC

dA 3.75 3.62 3.52 3.39 3.30 3.23

2.9

1/1 '
30 7 7 7 20 7

dA
3.06 2.89 2.75 2.60 2.52 2.43

I/I'
7 7 7 3 3 3

dA
2.37 2.20 2.10 2.05 2.01 1.95

1/11
3 3 3 3 3 3

Polarography

A study of the polarographic characteristics of digitoxin in 50% ethanolic solution containing tetraethylammonium hydroxide as electrolyte, showed an average half-wave potential o f -1,965 volts. The diffusion current wave height versus concentration graph indicated that quantities as low as 2 mcg could be determined. The method has been successfully applied to the tincture o f d i g i t a l i ~ ' ~ 1 5 . )

2.10

Solubility

Digitoxin is practically insoluble in water (1 g dissolves in about 100 liters at 20'C). One gram dissolves in about 40 m l chloroform, 6 0 m l ethanol, and 400 m l ethyl acetate. It is also soluble in ether, petroleum ether, benzene and vegetable oils. SYNTHESIS Digitoxigenin, a typical member o f t h e cardenolide family has been synthesized using as the starting material m e t h y l - 3 B - a c e t o x y - 1 4 B - h y droxy-Sf3-etinate by a seven-step sequence16. In the last step a solution ofa,B-unsaturated ester was treated with SeO2 by boiling under reflux for 10 hours. The filtrate was poured into water and the product isolated with ether. Acid hydrolysis o f digitoxigenin acetate yielded digitoxigenin (M.p. 246-249'C and [ a ] +19' in ethanol).
3.

158

DIGITOX IN

4.

STABILITY

The s t a b i l i t y o f two l i q u i d e x t r a c t s f r o m t h e l e a v e s o f D i g i t a l i s p u r p u r e a was e x a m i n e d . B o t h p r o d u c t s c o n t a i n e d d i g i t o x i n a n d g i t o x i n . The a c t i v i t y o f e a c h d r u g was d e c r e a s e d b y more t h a n 1 0 % o f t h e i n i t i a l v a l u e i n less t h a n t h r e e months a t 20". The r a t e o f d e c o m p o s i t i o n was g i t o x i n > d i gitoxin". S t o r a g e of d i g i t o x i n p r e p a r a t i o n s for o n e y e a r did not s i g n i f i c a n t l y decrease t h e i r potency s t o r e d a t t e m p e r a t u r e s u p t o 3OoC. The p o t e n c y was c h e c k e d by B a l j e t c o l o r i m e t r i c a s s a y a n d by t h e b i o l o g i c a l method a c c o r d i n g t o t h e S w e d i s h P h a r m a c o p e i a XI No b r e a k d o w n o f d i g i t o x i n i n t a b l e t s , i n j e c t i o n s o r s o l u t i o n s was f o u n d when s t o r e d f o r 5 y e a r s i n t h e d a r k up t o 3OoC. l 9 5. METABOLISM, P R O T E I N B I N D I N G A N D CLINICAL ASSAY 5.1 Metabolism

Digitoxin i s completely absorbed followi n g o r a l i n g e s t i o n and i t s f u l l e f f e c t a p p e a r s a s r a p i d l y a s by i n t r a v e n o u s i n j e c t i o n * ' . The l i v e r i s t h e main s i t e o f d e t o x i f i c a t i o n o f d i g i t o x i n , I t m e t a b o l i z e s v e r y r a p i d l y . One m e t a b o l i t e h a s been i d e n t i f i e d : digoxigenin-di-digitoxoside, p r o d u c e d by h y d r o x y l a t i o n a n d t h e l o s s o f o n e m o l e cule of sugar. Ten d a y s a f t e r an i n j e c t i o n , h a l f o f t h e d o s e i s s t i l l p r e s e n t i n t h e b o d y , a n d some remains a f t e r 2 0 days. Following t h e a d m i n i s t r a t i o n o f m a i n t e n a n c e d o s e s o f 0 . 1 t o 0 . 3 mg d a i l y , 1 0 % o f t h e dose i s e x c r e t e d unchanged. An e x p e r i m e n t a l m e t h o d h a s b e e n d e v e l o p e d i n order t o study t h e metabolic degradation of dig i t o x i n a n d d i g o x i n and c h a n g e s i n l i p i d s o l u b i l i t y o f t h e r a d i o a c t i v e m a t e r i a l i n p l a s m a 2 ' , The changes occur a f t e r t h e a d m i n i s t r a t i o n o f r a d i o a c t i v e l y l a b e l l e d g l y c o s i d e s i n t o an i s o l a t e d p e r fused l i v e r system (guinea p i g ) o r t o i n t a c t r a b b i t s , The m e t a b o l i c d e g r a d a t i o n o f d i g i t o x i n a n d digoxin i n t h e l i v e r r e s u l t s i n t h e formation of
159

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t h e mono- a n d b i s - d i g i t o x o s i d e s o f d i g o x i g e n i n a n d o f c o n j u g a t e d p r o d u c t s , T h e s e m e t a b o l i t e s a r e more p o l a r t h a n o r i g i n a l g l y c o s i d e s . An i n c r e a s e d p l a s ma-chloroform c o e f f i c j e n t i n d i c a t e s a change i n t h e r a t i o o f p o l a r / n o n p o l a r s u b s t a n c e s i n t h e ext r a c t e d medium. 5.2 Protein Binding The p r o t e i n b i n d i n g o f d i g i t o x i n i s thought t o account f o r t h e higher plasma l e v e l s . The r e s u l t s were o b t a i n e d b y t h e R b E 6 u p t a k e i n hibition technique, suggesting its probable value as a c l i n i c a l l y a p p l i c a b l e q u a n t i t a t i v e m e t h o d f o r t h e d e t e c t i o n o f commonly u s e d d i g i t a l i s g l y c o s i d e s i n p l a s m a . Com a r i s o n r e s u l t s b y t w o l a b o r a t o r i e s were p r e s e n t e d 2 ! The p r o t e i n b i n d i n g c a p a c i t y o f poorly s o l u b l e c a r d e n o l i d e s i n water is d e t e r m i n e d from the saturation concentration of these substances b o t h i n p r o t e i n s o l u t i o n and i n t h e i r u l t r a f i l t r a t e i n microscale. Figures about t h e binding o f d i g i t o x i n , d i g o x i n e t c . t o human s e r u m p r o t e i n , and t h e b i n d i n g o f d i g i t o x i n t o serum p r o t e i n s o f d i f f e r e n t s p e c i e s , a r e p r e s e n t e d . The c a r d e n o l i d e serum p r o t e i n b i n d i n g i s a f f e c t e d by c a l c i u m i o n s 2 3 .

5.3

D e t e r m i n a t i o n i n Blood 5.3.1

Chemical Methods Digitoxin concentration i n the b l o o d o f o r a l l y d i g i t a l i z e d p a t i e n t s was q u a n t i t a t i v e l y determined employing a combination o f TLC and a f l u o r o m e t r i c method: 30 m l v e i n b l l d was d i l u t e d t o 300 m l w i t h w a t e r , a n d t h e h a e m o l y s a t was e x t r a c t e d w i t h c h l o r o f o r m , T h e c h l o r o f o r m e x t r a c t was e v a p o r a t e d u n d e r m i l d c o n d i t i o n (temp. n o t e x c e e d i n g 40OC.). T h e r e s i d u e was d i s s o l v e d i n 5 0 % a q u e o u s methanol and t h e n e x t r a c t e d w i t h p e t r o l e u m e t h e r . R e m a i n i n g m e t h a n o l was e x t r a c t e d w i t h c h l o r o f o r m , The r e s i d u e a f t e r c h l o r o f o r m e v a p o r a t i o n ( r e d i s s o l v e d i n a n e x a c t a m o u n t o f c h l o r o f o r m ) was a p p l i e d t o K i e s e l g e l G p l a t e s . Mobil p h a s e : m e t h y l e n c h l o ride/isopropanol/formamide, 8 0 : 1 9 : 1 . S p r a y r e a g e n t :

160

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a m i x t u r e o f chloramine and t r i c h l o r o a c e t i c a c i d , A f t e r s p r a y i n g t h e p l a t e s were h e a t e d a t 115OC. f o r 1 0 min. O p t i m a l f l u o r e s c e n c e i n U V l i g h t was a t 365 nm24. D i g i t o x i n i n b l o o d p l a s m a was d e t e r m i n e d b y enzyme p - e s t e r h y d r o l a s e a n d ATP-ase i n h i b i t i o n technique2'. P h y s i c a l Methods Cardiac g l y c o s i d e s can b e d e t e r mined i n b i o l o g i c a l f l u i d s f r o m c o n c e n t r a t i o n s o f 1 n g / m l b y t h e i r i n h i b i t i o n o f t h e u p t a k e o f Rb by r e d b l o o d c e l l s . The g l y c o s i d e e x t r a c t was i n cubated a t 37'C. f o r 2 hours w i t h dimethyl s u l f o x i d e , r e d b l o o d c e l l s , a n d a s o l u t i o n o f RbC1. The Rb r e m a i n i n g i n t h e s u p e r n a t a n t i s m e a s u r e d by atomic absorption spectrometry26, Radioimmunoassay ( D e u t e r i u m and Tritium Labeled Digitoxin) A l l t h r e e prot o n s i n t h e u n s a t u r a t e d b u t e n o l i d e r i n g can b e exchanged i n a b a s e - c a t a l y z e d p r o c e s s . The e x change takes p l a c e even under v e r y mild c o n d i t i o n s and t h e r i n g d o e s n o t o p e n . D i g i t o x i n was t r e a t e d w i t h t r i e t h y l a m i n e and d e u t e r i u m o x i d e , and U V , IR a n d N N R d a t a showed t h a t t h e compound f o r m e d c o r r e s p o n d s t o t h e 21,21,22-trideuterodigitoxin. Similar r e a c t i o n t a k e s p l a c e w i t h t r i t i u m o x i d e . The e x c h a n g e i s l i m i t e d t o t h e t h r e e p r o tons i n the unsaturated butenolide ring2'. C l i n i c a l l y a p p l i c a b l e radioimmunoa s s a y t e c h n i q u e s f o r measurement o f serum d i g i t o x i n have been used t o determine l e v e l s o f t h i s drug from 250 p a t i e n t s . U n l a b e l e d d r u g i n t h e p a t i e n t s 8 serum d i s p l a c e s t r i t i a t e d d i g i t o x i n (added i n v i t r o ) f r o m s p e c i f i c a n t i b o d y b i n d i n g s i t e s . The p r o cedure r e q u i r e s one hour2'. The p h a r m a c o d y n a m i c s o f d i g i t o x i n i n man h a v e b e e n s t u d i e d u t i l i z i n g a s e n s i t i v e (0.2 n g / m l ) s p e c i f i c r a d i o i m m u n o a s s a y . P a t i e n t s r e c e i v i n g 0 . 1 mg o f d i g i t o x i n d a i l y h a d a mean s e r u m d i g i t o x i n l e v e l o f 25 n g j m l , a n d 4 4 n g / m l w a s d e t e c t e d i n p a t i e n t s r e c e i v i n g 0.2 mg d a i l y 2 ' . 5.3.3 5.3.2

161

IVAN M. JAKOVLJEVIC

In another study unlabeled d i g i t o x i n i n t h e unknown s a m p l e c o m p e t e s w i t h a t r i t i a t e d d i g i t o x i n tracer f o r b i n d i n g s i t e s of h i g h a f f i n i t y r a b b i t a n t i b o d i e s t o a human s e r u m a l b u m e n - d i g o x i n c o n j u g a t e . Free l a b e l e d d i g i t o x i n was s e p a r a t e d from t h e antibody-bound f r a c t i o n by ads o r p t i o n t o d e x t r a n - c o a t e d c h a r c o a l . The m e t h o d i s s e n s i t i v e t o 2 ng/ml o r l e s s . 6. METHODS O F ANALYSIS 6.1 Identification Tests

D i s s o l v e about 1 m g of d i g i t o x i n i n 2 m l o f a s o l u t i o n p r e p a r e d by mixing 0.3 m l o f a 9% aqueous f e r r i c c h l o r i d e s o l u t i o n and 50 m l o f g l a c i a l a c e t i c a c i d , and u n d e r l a y w i t h 2 m l of s u l f u r i c a c i d : a t t h e zone o f c o n t a c t o f t h e two l i q u i d s a brown c o l o r i s p r o d u c e d , and i t g r a d u a l l y changes t o l i g h t g r e e n , t h e n t o b l u e , and f i n a l l y t h e entire acetic layer acquires a blue c o l o r 3l , D i s s o l v e a b o u t 0 . 2 mg o f d i g i t o x i n i n 2 m l o f a f r e s h l y p r e p a r e d 1 i n 100 s o l u t i o n o f m-dinitrobenzene i n e t h a n o l , and a l l o w t o s t a n d f o r 1 0 min, w i t h f r e q u e n t s h a k i n g . Add 2 m l o f a m i x t u r e o f 1 volume o f a 10% tetramethylammonium h y d r o x i d e a n d 2 0 0 volumes o f e t h a n o l , a n d m i x : a r e d - v i o l e t c o l o r develops s l o w l y and t h e n f a d e s .
6.2
4

Elemental Analysis
3

l H 6 41

Elemental a n a l y s i s o f d i g i t o x i n as : C 64.4% H 8.4% 0 27.2%

6.3

Chromatography 6.3.1

Column C h r o m a t o g r a p h y Aluminum o x i d e , s i l i c e o u s e a r t h , a n d S e p h a d e x a r e a d s o r b e n t s commonly u s e d f o r t h e s e p a r a t i o n o f c a r d e n o l i d e g l y c o s i d e s and t h e i r metabolites,

162

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The USP X V I I I e m p l o y s a column o f s i l i c e o u s e a r t h previously cleaned with hydrochlor i c a c i d , a n d t h e n a c t i v a t e d a t 500Cc. Formamide i s added as t h e s t a t i o n a r y phase. D i g i t o x i n i s e l u t e d with a mixture of benzene/chloroform, 3:l. Aluminum o x i d e d e a c t i v a t e d w i t h 3% w a t e r and p a c k e d i n a column o f 1.5 cm d i a m e t e r t o a height o f 10 cm h a s b e e n u s e d s u c c e s s f u l l y f o r t h e s e p a r a t i o n o f d i g i t o x i n from i t s m e t a b o l i t e s . E l u t i o n i s a c h i e v e d w i t h 100 m l o f c h l o r o f o r m f o l lowed b y 35 m l o f 2 % e t h a n o l i n c h l o r o f o r m , a n d f i n a l l y w i t h 250 m l o f 10% e t h a n o l i n c h l o r o f o r m . D i g i t o x i n and i t s m e t a b o l i t e s a r e f o u n d i n t h a t p o r t i o n o f e l u a t e b e t w e e n 275 and 425 m l ( t h i s i n c l u d e s t h e chloroform prewash)12. S e p h a d e x G-200, s w e l l e d w i t h a m i x t u r e o f w a t e r / m e t h a n o l , 7 : 3 , was e m p l o y e d f o r t h e s e p a r a t i o n o f d i g i t o x i n and i t s m e t a b o l i t e s . S e p h a d e x was p a c k e d i n a column o f 1 c d i a m e t e r m t o a h e i g h t o f 1 5 cm. The s a m p l e was e l u t e d u s i n g t h e a b o v e m i x t u r e . C a r d e n o l i d e s were i n t h e f r a c t i o n between 1 0 and 2 5 m 1 3 . Thin Layer Chromatography A r a p i d s e p a r a t i o n of d i g i t o x i n from d i g o x i n and a c e t y l d i g i t o x i n c a n b e a c h i e v e d a p p l y i n g 2p1 o f a 0 . 0 1 % s a m p l e s o l u t i o n i n c h l o roform/methanol, 1:l t o K i e s e l g e l G p l a t e s . A s t h e e l u e n t a c h l o r o f o r m / m e t h a n o l , 9 : l m i x t u r e was u s e d , D e t e c t i n g a g e n t : h y d r o c h l o r i c a c i d . The s p o t s o f d i g i t o x i n ( R f 0 . 3 3 ) , d i g o x i n (Rf 0 . 2 4 ) a n d a c e t y l d i g i t o x i n (Rf 0 . 4 8 ) w e r e d a r k brown a f t e r 5 min. D r y i n g a t l l O C . f o r 5 min a n d exami n a t i o n u n d e r u l t r a v i o l e t l i g h t ( 3 6 5 nm) showed brown s p o t s f o r d i g i t o x i n a n d a c e t y l d i g i t o x i n , w h i l e d i g o x i n s p o t was b l u e . The l i m i t o f d e t e c t i o n i s 0.05 mcg3. D i g i t o x i n was s e p a r a t e d f r o m d i g o x i n on S i l i c a Gel G p l a t e s w i t h c h l o r o f o r m / m e t h a n o l , 8 8 : 1 2 d e v e l o p i n g s o l v e n t . The z o n e s were l o c a t e d by s p r a y i n g w i t h 1% i o d i n e i n c h l o r o f o r m , t h e n removed f r o m t h e p l a t e a n d e x t r a c t e d w i t h chloroform/methanol, 1:l mixture. After c e n t r i f u g a t i o n , a 7 m l a l i q u o t o f s u p e r n a t a n t was e v a p 163

6.3.2

I V A N M. J A K O V L J t V l C

o r a t e d t o d r y n e s s . The r e s i d u e was d r i e d a n d t h e n t r e a t e d w i t h d i x a n t h y l u r e a r e a g e n t and t h e chromophor r e a d a t 5 3 5 n m 3 4 ) 3 5 . A method f o r t h e d i r e c t q u a n t i t a t i v e e v a l u a t i o n o f d i g i t o x i n , d i g o x i n and a c e t y l d i g i t o x i n o n T L C u s i n g s p e c t r o f l u o r o m e t r y was i n v e s t i g a t e d . The o n l y r e a g e n t u s e d was h y d r o c h l o r i c a c i d , L i n e a r s t a n d a r d c u r v e s were o b t a i n e d when t h e a r e a u n d e r t h e f l u o r o m e t r i c c u r v e was c o r r e l a t e d w i t h t h e amount o f g l y c o s i d e s a p p l i e d . The o p t i m a l r a n g e f o r t h e s p e c t r o f l u o r o m e t r i c d e t e r m i n a t i o n o f t h e s e t h r e e g l y c o s i d e s was a b o u t 0.25 mcgj6. Separation of t h e cardiac glycos i d e s d i g i t o x i n a n d d i g o x i n from t h e i r 2 0 , 2 2 - d i h y d r o d e r i v a t i v e s c a n b e a c h i e v e d b y m u l t i p l e TLC on c e l l u l o s e f i l m s An u l t r a m i c r o f l u o r e s c e n t s p r a y r e a g e n t f o r d e t e c t i o n and q u a n t i t a t i o n o f d i g i t o x i n a n d o t h e r c a r d i o t o n i c g l y c o s i d e s on T L C was d e s c r i b e d . The s p r a y r e a g e n t c o n s i s t s o f a s c o r b i c a c i d , methanol, h y d r o c h l o r i c a c i d and hydrogen p e r o x i d e . The l i m i t s o f d e t e c t i o n were 0 . 0 1 m c g 3 , A p p l i c a t i o n of d i f f u s i o n a n d f l u o r e s c e n c e t o t h e d i r e c t d e t e r m i n a t i o n of d i g i t o x i n was a c h i e v e d by c o n v e r t i n g d i g i t o x i n i n t o a f l u o r e s c e n t d e r i v a t i v e b y means o f a r e a g e n t c o n taining p-toluenesulfonic acid, hydrochloric acid, a s c o r b i c a c i d and h y d r o g e n p e r o x i d e . S e n s i t i v i t y : 0 . 3 - 1 rncg3. A T L C s y s t e m on s i l i c a g e l G p l a t e s h a s been d e v e l o p e d u s i n g as t h e mobile s o l v e n t a m i x t u r e of methylene chloride/methanol/ formamide, 80:19:1, and s p r a y i n g t h e p l a t e s w i t h a c i d - f e r r i c c h l o r i d e j l . The same t e c h n i q u e c a n b e used f o r t h e i d e n t i f i c a t i o n o f d i g i t o x i n , digoxin and a c e t y l d i g i t o x i n , a n d f o r t h e d e t e r m i n a t i o n o f any g i t o x i n p r e s e n t i n t h e i r drug f o r m u l a t i o n s , Digoxin i s n o t a c t i v a t e d t o v i s i b l e f l u o r e s c e n c e a t room t e m p e r a t u r e b y a c i d - f e r r i c c h l o r i d e r e a g e n t . T h e r e f o r e a n y f l u o r e s c e n c e p r e s e n t immedia t e l y a f t e r s p r a y i n g i s due t o g i t o x i n a l o n e , H e a t i n g t h e p l a t e a t 100C. d e s t r o y s t h e g i t o x i n f l u o r e s c e n c e and c o n v e r t s t h e d i g o x i n t o a f l u o -

164

DIGITOXIN

r e s c e n t a n h y d r o d e r i v a t i v e w h i c h may b e s e e n u n d e r b o t h UV and v i s i b l e l i g h t " ' . Paper Chromatography After s e p a r a t i o n by p a p e r chromat o g r a p h y i n formamide s a t u r a t e d m e t h y l e t h y l k e t o n e / x y l e n e , 1 : l m i x t u r e , d i g i t o x i n a n d d i g o x i n were d e t e r m i n e d w i t h x a n t h y d r o l ( 1 0 - f o l d excess o f rea g e n t i n a c e t i c a c i d / h y d r o c h l o r i c a c i d , 9 9 : l mixt u r e ) . I t was n e c e s s a r y t o h e a t t h e r e a c t i o n m i x t u r e f o r 2 0 m i n . a t 60'C. A 1 : l c o m p l e x ( A m a x 535 n m ) , s t a b l e f o r 3 h o u r s was f o r m e d . Beer's law was obeyed o v e r t h e r a n g e 1-20 m ~ g / m l " ~ * " * . A b u t e n o l i d e r i n g s p e c i f i c method of q u a n t i t a t i v e paper chromatographic analysis of d i g i t o x i n u s i n g 2,4,2',4'-tetranitrodiphenyl is d e s c r i b e d . P a p e r : S c h l e i c h e r a n d S c h U l l 2043b i m p r e g n a t e d w i t h formamide. Developing s o l v e n t : methylethylketone/xylene, 1 : l s a t u r a t e d w i t h f o r mamide. T h e c h r o m a t o g r a m was d r i e d f o r 1 5 min. a t 60'C. 6.3.3

"'

6.3.4

Gas C h r o m a t o g r a p h y

Trimethylsilyl ether derivatives o f d i g i t o x i n , d i g o x i n and g i t o x i n h a v e b e e n shown t o b e r e s o l v a b l e on a g a s C h r o m a t o g r a p h i c column p a c k i n g c o n t a i n i n g as a l i q u i d p h a s e 2.5% O V - 1 o r OV-17 on C h r o m o s o r b W. Gas c h r o m a t o g r a p h y was p e r formed on a Barber-Colman 5000 s e r i e s i n s t r u m e n t equipped with hydrogen flame d e t e c t o r . During i s o t h e r m a l o p e r a t i o n i n j e c t i o n p o r t and column b a t h t e m p e r a t u r e s were i d e n t i c a l , D e t e c t o r t e m p e r a t u r e was m a i n t a i n e d a t 34OoC. I n t h e c a s e o f t e m p e r a t u r e p r o g r a m m i n g , i n j e c t i o n p o r t t e m p e r a t u r e was i d e n t i c a l t o t h e s t a r t i n g t e m p e r a t u r e : 240'C."" An i m p r o v e d m e t h o d f o r t h e g a s chromatographic i d e n t i f i c a t i o n o f d i g i t a l i s card e n o l i d e s as t h e i r a n h y d r o d e r i v a t i v e s h a s b e e n developed, r e s u l t i n g i n g r e a t l y reduced r e t e n t i o n times and e n h a n c e d r e s o l u t i o n . R e t e n t i o n d a t a o f 18 c a r d e n o l i d e s on t h r e e l i q u i d p h a s e s a r e r e p o r t ed. S p e c t r a l e v i d e n c e i s p r e s e n t e d showing t h a t t h e t e r t i a r y 14B-OH g r o u p i s n e i t h e r a f f e c t e d b y esterification nor e t h e r i f i ~ a t i o n ~ ~ .
165

IVAN M. JAKOVLJEVIC

High Speed Liquid Chromatography High speed liquid chromatography has been used to examine steroids and steroid conjugates such as digitoxin or digoxin. In these studies reverse phase liquid partition chromatography was employed. The columns consisted of a cyanoethylsilicone polymer (Dupont ZipaxR ANH) and the mobile phase was a mixture containing 2.5% methanol and 97.5% water, The compounds were detected by means of a 254 nm photometerk6.
6.3.5

6.4

Colorimetric Analysis

The methods for the determination of cardiac glycosides can be divided into three general groups based on: 1- the sugar moiety, 2 - the butenolide moiety, and 3 - the steroid part of the molecule. 1- A s far back as 1885, a colorimetric methodk7 was published using equal amounts of sulfuric acid and ethanol with the addition of ferric chloride. Others4 used a solution of ferric sulfate in concentrated sulfuric acid, or added ferric chloride to a solution of glycoside in acetic acid and then underlaid the Kiliani reagent. Many methods employ xanthydrol as the reagent for digitoxoseSo. 2 - The reagent employing picric acid in alkaline ethanol is the most frequently used5. There are many modifications and applications of this reactions2#5 3 ) 5 4 ? 5 5 ? The official U S P XVIII method depends on a chromatographic separation on siliceous earth in the presence of formamide, and a reaction in the butenolide side chain by picric acid in alkaline solution. The application of m-dinitrobenzene for the quantitative determination of cardiac glycosides is very successfuls6, as well as 1,3,5-trinitrobenzene in alkaline medium5. Some authors use 2,4-dinitrodiphenylsulfone in alkaline ethanol. The other rea ents used are 2-naphthoquinone -4 -sul fonat e , and 2 ,2 ,4.4 t et ran i t rodiphenyl

166

DIG ITOX IN

The r e a g e n t s b a s e d upon t h e r e a c t i o n i n b u t e n o l i d e r i n g have a wide a p p l i c a t i o n i n c a r d i a c glycosides metabolism s t u d i e s , These r e a g e n t s r e a c t with any glycoside o r i t s metabolite that s t i l l contains the intact butenolide ring. 3- Methods b a s e d upon t h e r e a c t i o n i n t h e s t e r o i d m o i e t y a r e f o r t h e most p a r t f l u o r o metric, 6.5 Fluorometric Analysis M e t h o d s b a s e d upon t h e r e a c t i o n i n t h e s t e r o i d moiety.'are m a i n l y d e h y d r a t i o n t y p e of rea c t i o n s such as t h a t u s i n g syrupy phosphoric a c i d 6 2 , o r e q u a l amounts o f h y d r o c h l o r i c a c i d and g l y c e r o l a s t h e d e h y d r a t i n g a g e n t s 6 3 . Hydro e n , or p e r o x i d e , h y d r o c h l o r i c a c i d and methanol64' a mixture of s u l f u r i c and phosphoric a c i d s w i t h t h e a d d i t i o n o f f e r r i c c h l o r i d e 6 6 are a l s o used. The f l u o r o p h o r obtained with a mixture o f acetic anhydride, a c e t y l c h l o r i d e and t r i f l u o r o a c e t i c a c i d , s u p p o r t s t h e t h e o r y , b a s e d on N M R , I R and f l u o r e s c e n c e a c t i v a t i o n s p e c t r a l d a t a , t h a t a low y i e l d o f a h i g h l y c o n j u g a t e d f l u o r o p h o r O f s u b s t i t u t e d 3.4-benzpyrene i s o b t a i n e d 6 7 . 6.6 ElectroDhoresis

8,

~~

~~

D i g i t o x i n may b e d e t e c t e d and e s t i m a t e d i n human a u t o p s y t i s s u e s b y p a p e r e l e c t r o p h o r e s i s a c c o r d i n g t o a n a u t h o r 6 * who u s e d a m i x t u r e o f o x a l i c a c i d , b o r i c a c i d and e t h a n o l t o d e v e l o p d i f f e r e n t c o l o r s d e p e n d i n g upon t h e compound. The l i m i t f o r i d e n t i f i c a t i o n was a b o u t 1 5 mcg f o r d i g i t o x i n , a n d 1 0 mcg f o r d i g i t o x i g e n i n . The d i g i t o x i n was t o t a l l y d e g r a d e d i n t h e t i s s u e s f o l l o w i n g p u t r e f a c t i o n f o r t h r e e months6', 6 . 7 Automated Assay An a u t o m a t e d p r o c e d u r e u s i n g a s t a n d a r d T e c h n i c o n a u t o m a t i c a n a l y z e r s y s t e m is d e s c r i b e d f o r t h e u n i t dose a n a l y s i s o f d i g i t o x i n and d i g o x i n i n t a b l e t s 7 ' . The t e c h n i q u e i s b a s e d on t h e f l u o r o m e t r i c measurement o f t h e d e h y d r a t i o n produ c t s of t h e c a r d i o t o n i c s t e r o i d s r e s u l t i n g from t h e i r r e a c t i o n w i t h hydrogen p e r o x i d e and hydro167

I V A N M. JAKOVLJEVIC

c h l o r i c a c i d , The automated system as d e s c r i b e d i s capable of analyzing 1 2 t a b l e t s p e r hour,

C L E A V A G E O F C A R D I A C GLYCOSIDES The i s o l a t i o n o f c a r d i a c a g l y c o n e s f r o m p l a n t m a t e r i a l i s made d i f f i c u l t by t h e l a c k o f r e l i a b l e methods t h a t w i l l m a i n t a i n t h e g e n i n s i n t a c t a f t e r hydrolysis of t h e i r glycosides. A procedure i s described f o r c l e a v i n g t h e suga r bond o f d i g i t o x i n i n o r g a n i c m e d i a a n d u n d e r m i l d a c i d c o n d i t i o n s . Maximum y i e l d s were o b t a i n e d i n 30 min. o f r e a c t i o n t i m e a t 5OoC: i n a medium c o n s i s t i n g o f a n h y d r o u s t e t r a h y d r o f u r a n made 0 . 0 0 2 N with r e s p e c t t o p e r c h l o r i c acid. Digitoxigenin i s s t a b l e u n d e r t h e s e condition^^^.
7.

8.

B I O L O G I C A L ACTIVITY 8.1 C h a r a c t e r i s t i c S t r u c t u r a l F e a t u r e s

From t h e s t u d i e s o f b i o l o g i c a l a c t i v i t y 7 2 four characteristic structural features of the g e n i n s c a n be e a s i l y i d e n t i f i e d as e s s e n t i a l f o r cardiac activity: 1- The L a c t o n e R i n g : The d o u b l e bond o f the lactone ring is apparently necessary f o r card i a c a c t i o n . The r u p t u r e o f t h e l a c t o n e r i n g r e s u l t s i n a loss o f c a r d i a c a c t i o n . 2 - The Hydroxy Group on C - 1 4 Atom: T h i s hydroxy group i s i m p o r t a n t and i t s m o d i f i c a t i o n results i n a significant loss of activity. I f t h i s g r o u p i s removed, t h e i m p o r t a n t s t e r e o c h e m i c a l rel a t i o n s h i p i s d e s t r o y e d so t h a t l o s s o f a c t i v i t y c o u l d b e d u e e i t h e r t o t h e loss o f t h e 1 4 - h y d r o x y l group, o r t o t h e a l t e r a t i o n o f t h e c i s C / D r i n g arrangement 3 , 3- S u g a r s a t C-3 Atom: I n t h e c a s e o f t h e a g l y c o n e , t h e a t t a c h m e n t o f o n e o r more s u g a r s a t C-3 u s u a l l y r e s u l t s i n i n c r e a s e d a c t i v i t y . 4- Stereochemical Arrangements: A llcislt f u s i o n o f t h e C and D r i n g s i s n e c e s s a r y f o r a c t i v i t y . Other types of n a t u r a l s t e r o i d s have t h e " t r a n s " c o n f i g u r a t i o n . The p r e s e n c e o f two h y d r o x y l g r o u p s a t C - 1 2 a n d C-16 i n t h e m o l e c u l e o f d i -

DIGITOXIN

gitoxigenin diminishes the activity hy two-thirds. 1 4 ~ ~ , 1 5 u - e p o x y - 1 4 - a n h y d r o d i g i t o x i g e n i nis practically inactive. 8.2 Bioassay Experiments were made t o examine the suktability of young chicks for the assay of digitalis glycosides, The jugular vein was cannulated and a volume of the test solution was infused. The dose was repeated at 5 min. intervals until cardiac arrest was noticed. The order of susceptibility of tested animals was as follows: pigeon > chick> rat74. ACKNOWLEDGMENT The author acknowledges the invaluable help of Miss Adele Hoskin for her assistance in t h e 1ite rat u r e s e a r c h .
9.

lo.

REFERENCES
Chen,K.K.,Ann.Rev.Physiol. 7,677 (1945). Kossoy,A.D. and Underbrink,F.D.,Eli Lilly and Company, personal communication, Khalique,A. et al., Sci.Res.Pakistan 3,177 (1966). Takuma,O. et al., Bunseki Kagaku, 17(1),53 (1968). Boaz,H.E.,Eli Lilly and Company, personal communi cat i on. Voigtlaender,H.W. et al., Arch.Pharm. 3 0 1 , 2 0 8
(1968).

1. 2.
3.

4. 5.
6.

7.
8.

10,
11,

9.

12.

E l i Lilly and Company, personal communication. The Merck Index, VIII Edition, 1968. British Pharmacopoeia, 1968. Djerassi,C. et al., Helv.Chim.Acta 41,250 (1958). Fieser and Fieser, Steroids, Reinhold Publish. Corp., 1959. (, 2 0 ) Mueller,L. , Microchem.J., z 1 (1968).

Beasly,F.W.,

I V A N M. JAKOVLJEVIC

13. 14. 15.

16. 17. 18. 19.

20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
31.

32. 33. 34. 35. 36. 37. 38. 39.

Parson et al., Henry Ford Hosp.Med.Bull., 6,365 (1958). Hilton,J.G,, Science, 110,526 (1949). Hilton,J.G., J.Pharmacol,Exptl.Therap. 100,258(1950). Danieli,N. et al., Tetrahedron 22,3189(1966). Lutomski,J. , Herba Pol. 12,243 (1966). Samuelsson,G., J.Mond,Pharm. 4,112 (1961). Samuelsson,G., Acta Pharm.Suecica 1(6),227 (1964). Clarke,E.G.C., Isolation and Identification of Drugs, London, 1969. Glover,A., Klin.Wochenschr. 49,1062 (1971). Shapiro,W., Circulation 4O0supp1.3,l84(1969). Pfordte,K. et al., Acta Biol.Med.Germ. 25, 19 (1970). Seipe1,H. et al., Klin.Wochenschr. 46,1257 (1968). Bentley,J.D., Circulation 41(1),67 (1970). Bourdon,R. et al., Ann.BiorClin.(Paris) 27 1) 6 (10-12) ,651 (99. Haberland,G. et al. , Naturwissensch, 56(10) , 516 1196n. Smith,T.W. et al., Circulation 4O(Suppl.3), 1 8 8 n969). Morrison,J. et al., Clin.Res. 18(4),668(1970). Smith,T.W., J.Pharrnacol.Exptl.merap.l75(2), 352 (1970). The United States Pharmacopeia, XVIII. Pfordte,K. et al., Z.Med.Labortechn. 11,272 (1970). Hoeke,M. et al., Pharm.Weekhlad 104,877 (1969). Rican-Fister et al., J-Chromatogr. 41(1),91

Johnston,E. J. et a1 Frijns,J.M.G,J.,

. , J. Pharm.Sci.

Pharm.Weekblad 105,209 (1970). Rabitzsch,G., J.Chromatogr, 35,122 (1968) * Jelliffe,R.W. et a l . , J.Chromatagr. 27,172 (1967). Eide-Juergensen et al., Plants Med.98 (Suppl.5) ,112 (1971).

55 ) ,531 -( 5(1966) .

(1969).

DIG ITOX IN

40.

41.

(1971). Pharm.Zh. (Kiev) 26(3) ,42 f 1971). 42. Dzyuba,N.P. et al,, Khim.Farm.Zh. S(llj,Sl (1971). 43. Rabitzsch,G. et al., J.Chromatogr. 4l,96 (1969). 44. Wilson,W.E. et al., Analyt.Chem. 41(6),810 (1969). 45. Tan,L., J.Chromatogr. 45,68 (1968). 46. Henry,R.A. et al., J.Chromatogr.Sci. 9,513 (1971). Lafon,P., Compt.Rend. 100,1463 (1885). 47. 48. Kiliani,H., Arch.Pharm. 234,273 (1896). 5,275(1895). 49. Keller,C., Ber.Pharm.Ges. Pesez,M., Ann.Pharm.Franc.-ED104 (1952). 50. s , 7 1 , 8 4 (1918). 51. Baljet,H., Schweiz.Apoth.Ztg. 52. Bel1,F.K. et al., J.Am.Pharm.Assoc.,Sci.Ed. 37,297 (1948). 53. Bel1,F.K. et al., J.PharmaZl.Exptl.Therap. 88,14 (1946). Quart.J.Pharm. 5,172 (1932). 54. Dyer,F.J., Kennedy,E.E., J.Am,Pharm.Assoc.,Sci.Ed. 55. 39,25 (1950). 5 ( 2 ) ,949(1935). 56. Morel,A., Bull.Soc.Chim.FrGce Kimura,M., J. Pharm.Soc. Japan 2 7 9 9 1 (1951). 57. Pharm,Weekblad 93,245 (1958). 58. Tattje,D.H.E., 59. Warren,A. T. et al. , J.Am. P h a r m x s s o c . ,Sci .Ed. 37,186 (1948). Doelker E. et al., Pharm.ActaHelv. 44,647 60. (1969). 61. Rabitzsch,G. et al. , Pharmatie a ( 5 ) ,262 (1969). 62. Petit,A. et al., B u l l . S o c . C h i m . F r a n c e - ~ , 2 8 8 (1950). 63. Jensen,K.B., Acta Pharmacol.Toxico1. 8,101 T1952). 64. Jensen,K.B., Ibid. 9,66 (1953). 65. Wells,D. et al., J.Fharm.Pharmaco1. 13,389 (1961). 66. Tattje,D.H.E., J.Pharm.Pharmaco1, 6,476(1954). 67. Jakovljevic,I.M., Anal.Chem. 35(10),1513 (1963). Dzyuba,N.P. et al.,

Dow,M.L.

et al.,

J.Pharm.Sci.

%(2),298

171

I V A N M. JAKOVLJEVIC

68. 69, 70. 71. 72. 73. 74.

Dyes,H.P., Pharm.Weekblad 95,682 (1960). Bors,G. et al., Farmacia (Bucharest) 15(5), 269 (97. 16) Cullin, L.F. et al. , J.Pharm.Sci. 59(5),697 (1970). Frey,M.J. et al., Anal.Biochem. 36,78 (1970). Henderson,F.G., Digitalis, G r u n e E Stratton Publ.,New York-London 1969. Wright,S.E., The Metabolism of Cardiac Glycosides, Charles C. Thomas Publ. 1960. 89(6), Fukuda,H. et al,, J.Pharm.Soc.Japan 866 (1969).

***********
During the preparation of these analytical profiles, the following, most recent, papers on different topics of digitoxin have been found: 1- Gisvold,O. Acetyldigitoxin and acetyldigoxin from Digitalis lanata. J.Pnarm.Sci. - 1320 (1972). 61, 2- suss, w. Extraction of digitalis leaves with the aid of ultrasonics. Pharmazie - 615 (1972). 27, 3- Watson, E. et al. Identification of submicrogram amounts of digoxin, diIsolation by chromatography., gitoxin J. Chromatogr. - 157 (1972). 69, 4- Potter, H. et al. TLC analysis of digitalis glycosides. Pharmazie - 315 (1972). 27, 5- Butler, V.P.,Jr. Assay of digitalis in the blood. 14, Prog.Cardiovasc.Dis. - 571 (1972). 6- Bodem, G. et al. Determination of digoxin and digitoxin in the blood.. Klin.Wochenschr. - 57 (1973). 51, THE L l T E R A T U R E SEARCff WAS CONDUCTEV UP TO MAY 1 9 7 3 .

.....

..

172

DIPHENHYDRAMINE HYDROCHLORIDE

Ira J. Holcomb and Salvatore A . Fusari

IRA J. HOLCOMB AND SALVATORE A. FUSARI

1.

Description
1.1
1.2

Name, Formula, Molecular Weight Appearance, color, Odor

2.

physical properties
2.1 2.2 2 -3 2 -4 2 -5 2 .6 2 -7 2 .8 2.9

Infrared Spectrum Nuclear Magnetic Resonance ultraviolet Spectrum Mass Spectrum optical Rotation Me Iting Range Differential Thermal Analysis Solubility crystal Properties 2.91 optical crystal Properties 2.92 X-Ray Diffraction Distribution Coefficients Aggregation: Micelle Formation PK; values Metal Complex Formation and Binding

2.10 2.11 2.12 2.13 3.

Synthesis

4. Stability
5.

Degradation

Drug Metabolic Products Identification:

Pharmacokinetics

6.
7.

Microchemical Tests

Methods of Analysis 7.1

7.2

Elemental Analysis Spectrophotometric Analysis

174

DIPHENHYDRAMINE HYDROCHLORIDE

7.21 7.22 7.23

7.24

Elemental Analysis Separation Methods Prior to Spectrophotometric Assay Methods Based on Conversion to Benzophenone Prior to Spectrophotometric Assay Method Based on Conversion to Chloranilic Acid Prior to Spectrophotometric Assay

7.3

Colorimetric Analysis 7.31 Ion-Pair Extraction Methods 7.32 Ammonium Reineckate Methods 7.33 Picric Acid Method 7.34 Method Based on Molle Reaction 7.35 Miscellaneous Colorimetric Methods Titrimetric Analysis 7 . 4 1 Direct Methods of Titration 7.42 Separation Prior to Titration 7.421 Reineckate salt Formation 7.422 Complexometric Method 7.423 Slurry Method 7.424 Ion Exchange Method 7.425 Extraction Method 7.43 Miscellaneous Titrimetric Methods Fluorometric Analysis Automated Analysis Biological Assay Gravimetric Analysis Chromatography 7.91 Paper Chromatography 7.92 Thin Layer chromatography 7.93 Gas Chromatography

7.4

7.5 7.6 7.7 7.8 7.9

175

I R A J. HOLCOMB A N D SALVATORE A . FUSARI

7.94 7.95
8.0

D i r e c t Methods on N e u t r a l columns 7.932 D i r e c t Methods on B a s i c columns 7.933 o x i d a t i o n to Benzophenone P r i o r t o G a s Chromatography Column c h r o m a t o g r a p h y Electrophoresis

7.931

References

176

DIPHENHYDRAMINE HYDROCHLORIDE

Description 1.1 N a m e , Formula, Molecular Weiqht Diphenhydramine h y d r o c h l o r i d e i s 2(dipheny1methoxy)-N,N-dimethylethylamine hydroc h l o r i d e -1 S i x a d d i t i o n a l chemical names are l i s t e d i n The Merck Index2, along w i t h twelve t r a d e names. One a d d i t i o n a l name i s Dimedrol. The e m p i r i c a l formula i s C17H21NO'HCl w i t h a molecular weight of 291.82.

1.

. HC1
The CAS R e g i s t r y Number is 58-73-1 for 2- (diphenylmethoxy) -N, N-dimethylethylamine and f o r t h e h y d r o c h l o r i d e , 147-24-0.

1.2 2.

Appearance , c o l o r , odor White, o d o r l e s s , c r y s t a l l i n e powder. 1

Physical Properties 2.1 I n f r a r e d Spectrum The i n f r a r e d spectrum of diphenhydramine h y d r o c h l o r i d e i s p r e s e n t e d i n F i g u r e 1. The S a d t l e r Reference Number i s 9382. The spectrum is used f o r c o n t r o l pur o s e s . 1 i 3 S p e c t r a a r e p r e s e n t e d by de ~ o o'I4 and W a l l a c e . 5 s The i n f r a r e d band assignments a r e g i v e n i n Table I .

177

.c

C 0

F r e q u e n c y (cm-1)

Fig. 1.

I n f r a r e d Spectrum o f Diphenhydramine H y d r o c h l o r i d e , U.S.P. Parke-Davis 8 Co. Lot No. 5 6 3 4 6 3 . Instrument: Perkin-Elmer 621, Phase:

KBr, 1:300.

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179

IRA J. HOLCOMB AND SALVATORE A. FUSARI

Nuclear Maqnetic Resonance In Figure 2 the nuclear magnetic resonance spectrum of diphenhydramine hydrochloride is presented. The spectral peak assignments7 are presented in Table 11. The Sadtler NMR Reference Number is 14360. ultraviolet Spectrum The ultraviolet spectrum of diphenhydramine hydrochloride is presented in Figure 3. The absorptivities at 258 nm. listed in Table 111 gompare well with the literature values of 15.4 in methanol and 16.59 in an aqueous system. Mass Spectrum, LOW Resolution11 A plot of the relative intensities vs. mass/charge ratio is presented in Figure 4 and summarized in Table IV. The ionization potential is 70 electron volts. 2.4 Optical Rotation Diphenhydramine hydrochloride is not optically active. Melting Range Diphenhydramine hydrochloride melts in the range 167O to 172C. The actual range in which the compound melts is usually less than 2OC. The melting point is affected by the rate of heating1* as shown in Table v. Data was obtained from melting point scans using the Mettler FP-1. 2.6 2.5 2.3

2.2

180

c W c

Fig. 2.

Nuclear Magnetic Resonance Spectrum of Diphenhydramine Hydrochloride, U.S.P., Parka-Davis & Co. l o t No. 5 6 3 4 6 3 . Instrument: V a r i a n A-60. Solvent.

D20. Sweep Offset: 0 cps.

x7 167 YY 0 & i u I
0

0
d

I n

?
m

?c!

182

Fig. 3. Ultraviolet Spectrum of Diphenhydramine H y d r o c h l o r i d e , U.S.P., Parke-Davis & Co.

Lot N O . 5 6 3 4 6 3 . Instrument: Cory 14.

IRA J. HOLCOMB A N D SALVATORE A . FUSARI

Table 111.

Absorptivities'O
&

of

D iphenhydr amine Hydrochloride ,

Parke, Davis

Co., Lot N o . 563463

Aqueous Medium (pH 3): wavelength 267 nm(s) 263 nm(s) 257.5 nm(s) 252 nm Methanol, absolute: 268 nm 264 nm 258 nm 252 nm 7.95 12.1 15.5 12.8 232 353 452 374 a (l%, 1 cm.)
9.3

e 270 370 476

12.7
16.3

13.95

406

184

BC

K
0
Y

70

-u C a

60

* 5 0 >

:
0

.+

4c

30

m
lc

Fig. 4.

Mass Spoctrum of Diphenhydramine Hydrochloride, U.S.P., Parke-Davis & CO. Lot NO. 563463. lnstru men t:

Lr/lA
I

XI0

Xloo

200

250

F i n n ig a n Q u a dr u pol e Mass Spectr o mete r, M ode I 1015.

IRA J. HOLCOMB AND SALVATORE A. FUSARI

Table Iv Low R e s o l u t i o n Mass Spectrum Assignments f o r Diphenhydramine Hydrochloride Measured Mass Relative I n t e n s ity

S t r u c t u r a l Assignments

256

10.67

H C3 -CH2-N , H CH3
4-

183

10 - 0

( O \ H

4-

(oyc 1 67

C13H110

+
C13Hll

30 .O

165

52.67

a
186

+
C13H9

152

22.00pJ-$J1+

C12H8

DIPHENHYDRAMINE HYDROCHLORIDE

Table Iv( cont .)

Measured Mass

Relative Intensity

S t r u c t u r a l Assiqnments

58

100

C3H8N

45

12

2H7

187

Table V.

M e l t i n g P o i n t and R a n g e 1 2 of D i p h e n h y d r a m i n e
Parke, D a v i s
&

Hydrochloride,

co .,

Lot No.

563463
Mid-Point

S t a r t Temp. ( O c .)

Heating Rate

Ranqe

163 158 O

1 OC/min.

167 -7-168-6(0-9) 167 -7-168 (1- 0 ) -7

168 -7-169.3 ( 0 -6) 168 -7-170-0(1- 3 )

168.1 168.2 169 -0 169 -4

3 OC/min.

DIPHENHYDRAMINE HYDROCHLORIDE

D i f f e r e n t i a l Thermal A n a l y s i s The DTA curve o b t a i n e d u s i n g a M e t t l e r TA 2000 is shown i n F i g u r e 5 . The p e r c e n t p u r i t y found f o r t h e sample, Diphenhydramine Hydroc h l o r i d e , USP, Parke, Davis & c o . , Lot N o . 593125, i s 99 .62%.13 Solubility The s o l u b i l i t y of diphenhydramine i n w a t e r i s 0.7 rng./m1.lo S o l u b i l i t i e s of diphenhydramine h y d r o c h l o r i d e have been determined14 and a r e p r e s e n t e d i n Table VI. c r y s t a l Properties The o p t i c a l c r y s t a l l o r a p h i c c o n s t a n t s Diphenhydramine have been r e p o r t e d by Keenan h y d r o c h l o r i d e i s d e s c r i b e d as c o l o r l e s s , m o s t l y s i x - s i d e d p l a t e s w i t h l e n g t h w i s e c l e a v a g e . The n20 v a l u e s are: a , 1 . 6 0 2 ; p , 1 . 6 2 5 : and y , 1.630; a f l 5 0.002. I n p a r a l l e l p o l a r i z e d l i g h t , ext i n c t i o n is p a r a l l e l and t h e s i g n of e l o n g a t i o n i s n e g a t i v e . S h e l l 1 6 a l s o r e p o r t e d on o p t i c a l c r y s t a l l o g r a p h i c p r o p e r t i e s and gave t h e d e n s i t y a s 1.189.
2.9

2.7

2.8

.'?

x-Ray D i f f r a c t i o n The X-Ray d i f f r a c t i o n d a t a f o r diphenh dramine h y d r o c h l o r i d e were r e p o r t e d by The compound h a s been run by Krcl* Gadret''. and t h e d i f f r a c t i o n p a t t e r n i s p r e s e n t e d i n Figure 6 . The c a l c u l a t e d I'd spacings18 f o r t h e d i f f r a c t i o n p a t t e r n a r e g i v e n i n Table VII. The 28 a n g l e s were c o r r e c t e d on t h e d i f f r a c t i o n p a t t e r n u s i n g known v a l u e s f o r c a l c i t e added t o a sample.
I'

2.92

Fig. 5 Diphenhydramine H y d r o c h l o r i d e , D.T.A Curve, Parke-Davis & CO.

Lot NO. 593125

Instrument: M e t t l e r T A 2 0 0 0 AHz7.495 kcal. M e l t i n g p o i n t : 1 6 8 . 3 O C .

DIPHENHYDRAMINE HYDROCHLORIDE

Table V I

S o l u b i l i t y 1 4 of Diphenhydramine

Hydrochloride i n Various S o l v e n t s Solubility, mq ./ml.

Solvent
Water

858

Methanol
A l c o h o l , 95% Chloroform

599
408

3 94
35

Isopropyl Alcohol
Acetone

16

191

z 2
z
0
c

E
c u a C

DlPH EN H Y DRAMIN E HYDROCHLORIDE

Table VII

Diphenhydramine Hydrochloride :

calculated 'Id" Spacings and I/I, Values

Radiation: Filter: d (Ao)

8.56 7.97 7.19 6.74 5.70 5.42 5.30 4.78 4.59 4.37 4.05 3.85 3.59 3.40 3.35 3 -33 2.98 2.91
Ni

C u G , h 1.5418

( 1
10 0 2

( 1
5

( 1
13 5 20 24 2 12 7 d

2.86 2.78 2 -75 2.70 2.60 2.58 2 -53 2.46 2.42 2.29 2.19 1.98 7.19 4.05 24 4.37 20

4 1

( 1
1 2

( 1 ( 1 ( 1 ( 1
1 1 1
8.56 7

l1 11 / 1 100
6 4 3

193

IRA J. HOLCOME AND SALVATORE A . FUSARI

Distribution Coefficients Doyle h a s determined t h e d i s t r i b u t i o n b e h a v i o r o f - diphenhydramine h y d r o c h l o r i d e i n ch1oroform:water and e t h e r :water. A l o g a r i t h m i c d i s t r i b u t i o n diagram i s p r e s e n t e d i n t h e s t u d y f o r s e l e c t i o n of p a r t i t i o n chromatographic systems . I 9 The d i s t r i b u t i o n of diphenhydramine h y d r o c h l o r i d e between ch1oroform:water as a f u n c t i o n of aqueous p - t o l u e n e s u l f o n i c a c i d w a s a l s o s t u d i e d by Doyle .20 The s o l v e n t composition e f f e c t on t h e p a r t i t i o n of amines, i n g e n e r a l , h a s been s t u d i e d . 2 1 Konyushko22 examined t h e e f f e c t of pH on t h e d i s t r i b u t i o n of diphenhydramine between water and chloroform. The e x t r a c t i o n of diphenhydramine w i t h chloroform i n p r e s e n c e of W F , KF, K C 1 , KBr, K I and KSCN a s s a l t i n o u t a g e n t s a t 20 and p 3 h a s been r e p o r t e d . 29 H Aggregation - M i c e l l e Formation Diphenhydramin h y d r o c h l o r i d e forms Attwood2 h a s deteraggregates i n s o l u t i o n . mined t h e c r i t i c a l m i c e l l e c o n c e n t r a t i o n u s i n g s c a t t e r i n g a t a n a n g l e of 9 0 t o t h e i n c i d e n t beam and determining t h e i n f l e c t i o n p o i n t s i n t h e p l o t v e r s u s t h e molal c o n c e n t r a t i o n .
2.11

2.10

PKA v a l u e s AndrewsLo determined t h e i o n i z a t i o n c o n s t a n t of diphenhydramine h y d r o c h l o r i d e a t 0 O , p d = 9.67, and 25 O , p A = 9.12 i n w a t e r . These K v a l u e s compare w e l l w i t h t h o s e o b t a i n e d by L ~ r d o f ~p ~ = 9.00 i n water. deRoos28 h a s i K A determined t h e p& a t 2 0 t o b e 9.06 i n w a t e r .
2.12

194

DIPHENHYDRAMINE HYDROCHLORIDE

The pK4 of Diphenhydramine H d r o c h l o r i d e , USP, Lot 563463, h a s been determined2S; i n a water:methanol (1: 1) system t o be 8 . 4 . Since t h e p& v a r i e s s l i g h t l y w i t h t h e a l c o h o l c o n t e n t , the value obtained i s acceptable. 2.13 Metal complex Formation and Binding Diphenhydramine h y d r o c h l o r i d e forms complexes26 w i t h v a r i o u s m e t a l i o n s such as Cu++, co++, and N i + + . Evidence f o r i n t e r a c t i o n of diphenhydramine h y d r o c h l o r i d e w i t h s t rene-maleic30 and sodium carboxymethylcelluloseYl h a s been r e p o r t e d . Synthesis The f i r s t method p a t e n t e d f o r t h e 32 s y n t h e s i s of diphenhydramine w a s by R i e v e s c h l i n 1947, a s s i g n e d t o Parke, Davis & C o . The g e n e r a l method i n v o l v e s t h e r e a c t i o n of bromodiphenylmethane w i t h t h e a p p r o p r i a t e d i a l k y l a m i n o a l c o h o l i n t h e p r e s e n c e of anhydrous sodium c a r b o n a t e . The d i a l k y l a m i n o a l c o h o l used i s dimethylamino e t h a n o l (see F i g u r e 7 ) . The diphenhydramine b a s e t h a t i s formed i s t h e n converted t o t h e H C 1 s a l t .
A v a r i e t y o f s y n t h e t i c methods have appeared i n t h e l i t e r a t u r e . 3 3 , 34, 35 I n t h e m a j o r i t y o f methods, t h e b a s e , diphenhydramine i s formed f i r s t and t h e n c o n v e r t e d t o t h e hydrochloride. I n some i n s t a n c e s , diphenhydramine h y d r o c h l o r i d e may be formed d i r e c t l y by rearrangement of a q u a t e r n a r y ammonium s a l t 3 6 , 37 ( s e e F i g u r e 8 ) . The f r e e b a s e can a l s o be formed as t h e r e s u l t of a d e c a r b o x y l a t i o n reaction38 (Figure 9 ) .

3.

195

IRA J. HOLCOMB AND SALVATORE A. FUSARI

Q
I
I

CHZ + Brz

hv

HCI
7

H C - O C H ~ C H ~-

H C - 0 -cH~-cH~-N', CH3

(b2 150-165')

Fig. 7.

Synthesis of Diphenhydramine H y d r o c h l o r i d e .

196

\ /

u N

I u

I97

Fig. 8. Synthesis of D i p h e n h y d r a m i n e Hydrochloride: R e a r r a n g e m e n t R e a c t i o n

CHzC~-O-C-COzNa
/

230 -300 c

HC-0-CHZCHZ-N /

AH3 CY

HCI

Fig. 9 . Synthesis of Diphenhydramine Hydrochloride : Decarboxylation Reaction

DIPHENHY DRAMINE HYDROCHLORIDE

4. stability - Deqradation The earliest published detailed work on the stability - decomposition o diphenhydramine hydrochloride is that of Nogami59 in 1961. The kinetics of the decomposition was examined in an acidic and alkaline medium. In an acidic medium, diphenhydramine undergoes fairly rapid decomposition, whereas the compound is fairly stable in an alkaline solution. The decomposition in an acid medium is due to hydrolysis of the ether linkage. The rate determining step is first order and catalyzed by hydrogen ion. The principle degradation products are benzhydrol and 2- (dimethylamino) ethanol. Earlier observations on the decomposition of diphenhydramine hydrochloride were in relation to the effect of h drogen peroxide4 0 t 41and ultraviolet light43 on the compound. The decomposition products with hydrogen peroxide are toluene, benzophenone, benzyl alcohol, benzoic acid and phenolic substances in addition to dimethylaminoethanol. The benzhydrol under the conditions used undergoes further reactions. Under ultraviolet irradiation, the principle decomposition products are benzhydrol and dimethylaminoethanol . The work by Nogami3 on of diphenhydramine hydrochloride in part by d e R ~ o s ~ 1963 in a in ~ stability of the ether bond in a benzhydryl ethers. the stability was confirmed study on the series of

199

I R A J. HOLCOME A N D SALVATORE A. FUSARI

Drug M e t a b o l i c P r o d u c t s - Pharmacokinetics I n t h e i n i t i a l work by Glazko and coworkers 44, 45 on t h e m e t a b o l i c f a t e o f diphenhydramine h y d r o c h l o r i d e , r a t s and g u i n e a p i g s w e r e examined a t d e f i n i t e t i m e s a f t e r subcutaneous i n j e c t i o n s . The h i g h e s t c o n c e n t r a t i o n s of diphenhydramine w e r e found i n t h e l u n g s , w i t h lower c o n c e n t r a t i o n s i n t h e s p l e e n , l i v e r and muscles. Peak c o n c e n t r a t i o n s occurred i n about one hour w i t h a f a i r l y r a p i d drop o v e r a s i x hour p e r i o d . Diphenhydramine w a s demonstrated i n human u r i n e i n s m a l l amounts by e x t r a c t i o n and u l t r a v i o l e t a b s o r p t i o n . 5. The r e s u l t s o b t a i n e d u s i n g r a d i o a c t i v e carbon i n c o r p o r a t e d i n t o t h e ci p o s i t i o n of t h e benzhydryl group of diphenhydramine a g r e e w i t h t h e chemical a n a l y s i s . 4 5 I n r a t s t h e maximum r a t e of e x c r e t i o n occurred i n t h e f i r s t seven h o u r s . Radioautographs p r e p a r e d from u r i n e samples showed a t l e a s t s i x d i f f e r e n t r a d i o a c t i v e compounds p r e s e n t , one of which was d iphe nh yd r a m i n e

K i k k a ~ a d e~ t i f i e d benzhydrol and dimethyli~ n aminoethanol a s m e t a b o l i c p r o d u c t s i n v i t r o and i n v i v o . An a c i d i c compound w a s a l s o d e t e c t e d , b u t not i d e n t i f i e d

D r a ~ h 48 ~u,s i n g t r i t i u m l a b e l e d ~ diphenhydramine i n r h e s u s monkey plasma found t h e major m e t a b o l i t e t o be a deaminated c a r b o x y l i c a c i d d e r i v a t i v e of diphenhydramine, (diphenylmethoxy) a c e t i c a c i d . The a c i d , t h e mono- and d i - d e a l y k y l a t e d d e r i v a t i v e s of diphenhydramine and t h e N-oxide d e r i v a t i v e w e r e i d e n t i f i e d c h r o m a t o g r a p h i c a l l y . The diphenyl-

200

DIPHENHY DRAMINE HY DROCHLORI DE

methoxyacetic a c i d i s e x c r e t e d a s t h e g l u t a m i n e conjugate. Kinke14 examined plasma l e v e l s of diphenhydramine a f t e r s i n g l e dose o r a l administ r a t i o n of diphenhydramine h y d r o c h l o r i d e c a p s u l e s a t d i f f e r e n t l e v e l s i n human v o l u n t e e r s . Peak plasma l e v e l s were o b t a i n e d 2 t o 3 h o u r s followi n g t h e dose. I n a m u l t i p l e dose s t u d y , a r e l a t i v e l y c o n s t a n t plasma l e v e l i s o b t a i n e d a f t e r t h r e e days. 50 A d d i t i o n a l work h a s been done on t h e m e t a b o l i t e s i n r e l a t i o n t o d i f f e r e n c e s observed between s p e c i e s . The major m e t a b o l i t e i n a l l s p e c i e s s t u d i e d , e x c e p t t h e r a t , w a s (diphenylmethoxy) a c e t i c a c i d . T h i s m e t a b o l i t e i s conj u g a t e d w i t h glutamine i n t h e monkey and g l y c i n e i n t h e dog. I d e n t i f i c a t i o n : Microchemical T e s t s The f i r s t c o l l e c t i o n o f microchemical t e s t s f o r d e t e c t i o n and i d e n t i f i c a t i o n of diphenhydramine w a s d e s c r i b e d by ~ a l e y l i n 1948. The r e a c t i o n s and r e s u l t s are summarized i n
6.

Table V I I I .

~ o l l d e s c r ~b e d an a d d i t i o n a l c o l o r e ~ i r e a c t i o n i n 1950 i n which t h e r e a g e n t , H2SO4, 90%, and HNO3, lo%, r e a c t s t o g i v e a r e d - v i o l e t c o l o r changing slowly t o y e l l o w . The r e s u l t i n g mixture i s d i l u t e d w i t h w a t e r t o g i v e an orangeyellow c o l o r and t h e n t h e t u r b i d m i x t u r e becomes a violet-rose. chloroform i s added and mixed w e l l : the s e p a r a t e d chloroform layer i s v i o l e t and t h e aqueous l a y e r becomes c o l o r l e s s . The c o l o r r e a c t i o n i s due t o b e n z h y d r o l .

201

Table V I I I .

Identification of Diphenhydramine Microchemical Tests 51 Observation Definite crystalline form

Hydrochloride:

Mandelin reagent Marquis reagent

h)

Red oily globules Canary yellow, reddish-orange then chocolate brown canary yellow then reddish-orange Canary yellow to orange-red orange Yellow Orange then reddish-orange and wine color on dilution Orange brown and yellow-green on shaking

0
h)

Mecke reagent Frohde reagent H2SO4, conc. K2Cr207- H2SO4 Resorcinal

H2SO4

% Furfural, 1 , over H2S04

Table VIII

(cont -) Reagent

observation orange-red
Cherr y-r e d

chromic a c i d , 5%
Foucery r e a g e n t
Name Reagent Compositions:

precipitate

Mandelin r e a g e n t
W

N 0

Ammonium v a n a d a t e , 1 g . i n LOO m l . concentrated s u l f u r i c aci d s u l f u r i c acid-formaldehyde. Two m l . of a 40% s o l u t i o n of formaldehyde mixed w i t h 45 m l . of w a t e r and 5 5 ml. of c o n c e n t r a t e d sulfuric acid Selenous a c i d , 0 . 5 g , i n s u l f u r i c a c i d , 100 ml Ammonium molybdate, 0 .l% i n c o n c e n t r a t e d s u l f u r i c acid Quinone, 1 g

Marquis r e a g e n t

Mecke r e a g e n t Frohde r e a g e n t

Foucery r e a g e n t

. in

acetic a c i d : a l c o h o l ( S :100)

IRA J. HOLCOMB A N D SALVATORE A. FUSARI

A d d i t i o n a l microchemical tests have been d e s c r i b e d b y A ~ t e r h o f f ~ ~~ 0 and ~N ~ ~ h o f *f ~ ~ , 0 1 e Clarke56 i n 1957 gave, i n a d d i t i o n t o observat i o n s , t h e s e n s i t i v i t y of t h e t e s t s used (Table 1x1. Clarke57 h a s l d e s c r i b e d a method f o r t h e r a p i d d e t e c t i o n of b a s i c drugs i n u r i n e t h a t u t i l i z e s many o f t h e r e a c t i o n s d e s c r i b e d i n procedures t h a t require a m i n i m u m of equipment. A d d i t i o n a l r e a g e n t s which form c r y s t a l l i n e p r e c i p i t a t e s w i t h diphenhydramine are f l a v i a n i c acid58, 4 , 4 I -dibromodibenzenesulf onamide59, and 8 h droxy-7-iodoquinoline-5s u l p h o n i c a c i d-68 .

7.

Methods of A n a l y s i s 7.1

Elemental A n a l y s i s The e l e m e n t a l a n a l y s i s of Diphenhydramine Hydrochloride, USP, Lot 563463, i s p r e s e n t e d below : Element % calculated ReportedG1 69.97 C 69.99 7.60 H 7.56 N 4.80 4.84 c1 12.15 12.09 7.2 S p e c t r o p h o t o m e t r i c Assay

D i r e c t Methods Methods i n which t h e sample i s d i l u t e d t o the proper c o n c e n t r a t i o n for absorbance r e a d i n g i n t h e u l t r a v i o l e t r e g i o n have been r e p o r t e d by S e t n i k e r 6 2 and D e m i r 6 3 Corrections using orthogonal functions f o r i r r e v e l a n t
~

7.21

204

T a b l e IX.

Microchemical Identification Tests and Sensitivity 5 6 Observations Needles, some curved plates yellow color ye 1l o w ye 1low yellow

Reagent Gold bromide/HCl Potassium triiodide Formaldehyde-sulfuric acid (Marquis) Ammonium vanadate Ammonium molybdate Selenium dioxide

Sensitivity 0.1 pg 0.1 p g

0.1 pLJ
0.1

0 1 pg .

0.1 pg

I R A J. HOLCOMB AND SALVATORE A. FUSARI

absorption in tablets64 has been applied with a .% recovery of 99.2 2 1 8 . Separation Methods Prior to Spectrophotometric Assa column chromatography 0 alumina65 : thin layer chromatography with an alumina layer66, direct extraction from a basic solution67, ion exchange chromatography68, and extraction from a 5% hydrochloric acid ~ o l u t i o n ~7orhave been ~ used prior to the spectrophotometric assay. Methods Based on Conversion to ~enzophenonePrior to Spectrophotometric As say Diphenhydramine hydrochloride is oxidized to benzophenone b either dichromate in a sulfuric acid medium, y2 or permanganate in a basic medium73. The benzophenone is either steam distilled or separated by extraction into hexane or heptane and determined spectrophotometrically. Conversion to Chloranilic Acid Prior to Spectrophotometric Assay Diphenhydramine has been determined in drugs by conversion to chloranil with FeC13 in hydrochloric acid and hydrogen peroxide74 The chloranil formed is extracted and hydrolyzed to chloranilic acid (2,5-dichloro-3,6-dihydroxyp-benzoquinone) with potassium hydroxide. The absorbance measured at 331 nm. The conversion 0% to chloranilic acid is constant, but not 1 0 . 7.24 7.23 7.22

206

DIPHENHY DRAMlNE HYDROCHLORIDE

Colorimetric Assay 7.31 lon-Pair Extraction Methods The method commonly used for routine control procedures is based on the extraction of diphenhydramine with methyl orange into chloroform. The procedure was initially for studied by Dill and G l a z k ~ ~ ~use in the determination of diphenhydramine in body tissues. A recent modification involves the addition of methanol after the complex is completely extracted into chloroform to prevent adsorption of the methyl orange-diphenhydramine ion-pair onto the walls of the f l a ~ k 7 ~ . Other dyes that have been used for the colorimetric assay are bromocresol green77 bromocresol p ~ r p l e 7 ~ , bromothymol blue78, er iochrome blue SE79 I tetrabromophenolphthalein ethyl ester*O and eosin811 8 2 .
I

7.3

Ammonium Reineckate Methods Bandelin83 separated diphenhydramine as the reineckate salt followed by solution of the salt in acetone and colorimetric estimation at 525 nm. A very comprehensive paper on the identification and determination of nitrogenous bases with ammonium reineckate was presented by K U ~ n - T a t tin which the mole com~~ position of diphenhydramine reineckate is given as ~ 2 1 ~ 2 8 c r ~ 7 O S q . salt decomposes at 178 The 180
O

7.32

Picric Acid Method Picric acid has been used for the colorimetric determination of diphenhydramine in the urine of rabbits and man85.

7.33

207

IRA J. HOLCOMB AND SALVATORE A. FUSARI

Method based on Molle R e a c t i o n Horn86 examined s e v e r a l d i f f e r e n t procedures f o r t h e d e t e r m i n a t i o n of diphenhydramine h y d r o c h l o r i d e , one o f which i s based on t h e M O l h r e a c t i o n d e s c r i b e d e a r l i e r . The compound i s r e a c t e d w i t h a mixture o f s u l f u r i c a c i d and n i t r i c a c i d ( 9 : 1 ) , d i l u t e d w i t h w a t e r and t h e c o l o r e d compound formed e x t r a c t e d w i t h chloroform. The absorbance of t h e chloroform l a y e r w a s t h e n determined w i t h a f i l t e r t y p e instrument. Miscellaneous C o l o r i m e t r i c Methods D iphe nh ydr a m i n e h a s been d e t e r mined by e x t r a c t i o n of a chloroform s o l u b l e complex w i t h c o b a l t t h i o c y a n a t e 8 7 , 88, *9. Diphenhydramine reacts i n a 2 : l mole r a t i o and i n chloroform i s measured i n t h e r e g i o n 590 t o 625 nm. 7.35

7.34

Diphenhydramine can be e x t r a c t e d from an acetate b u f f e r , p H 5 , c o n t a i n i n g i o d i d e w i t h a 0.5% I 2 s o l u t i o n i n e t h y l e n e d i c h l o r i d e g O . c o n d i t i o n s f o r t h e complex format i o n of diphenhydramine w i t h H ( T 1 Br4) have been examined w i t h subsequent d i s p l a c e m e n t b y b r i l l i a n t green. The c o l o r r e a c t i o n w i t h d i e t h y l o x a l a t e g 2 and t h i o b a r b i t u r i c g 2 acid w a s used t o determine diphenhydramine i n p i l l s g 3 . T i t r i m e t r i c Analysis 7.41 D i r e c t Methods of T i t r a t i o n The o f f i c i a l methodl f o r t h e a s s a y of diphenhydramine h y d r o c h l o r i d e i s by
7.4

208

DIPHENHYDRAM INE HYDROCHLORIDE

nonaqueous t i t r a t i o n w i t h 0.1N p e r c h l o r i c a c i d i n t h e p r e s e n c e of mercury (11) a c e t a t e u s i n g c r y s t a l violet as indicator. Work on t h e nona ueous t i t r a t i o n and a c e t o n i t r i l e method w a s r e p o r t e d by Ekablad" w a s examined as a s o l v e n t by Mainvi1leg5. S e v e r a l a c i d s a l t s of diphenhydramine were t i t r a t e d w i t h p e r c h l o r i c acid i n g l a c i a l a c e t i c a c i d using t h e glass-glass retarded potentiom e t r i c method f o r d e t e c t i o n o f t h e e n d p o i n t g 6 . The endpoint h a s been d e t e c t e d conductimetr i c a l l y g 7 . Diphenhydramine and a c i d s a l t s can be t i t r a t e d d i r e c t l y i n anhydrous p r o p i o n i c acid with p e r c h l o r i c a c i d using glass-calomel electrodes98. Diphenhydramine can a l s o be d e t e r mined u s i n g a 0.004M s o l u t i o n of sodium l a u r y l s u l f a t e o r sodium d i o c t y l s u l f o s u c c i n a t e a s t h e t i t r a n t g g , 100. The r a t i o of t h e a n i o n i c s u r f a c e a c t i v e agent t o t h e base i s not i n t e g r a l , but approximate and c o n s t a n t . separations prior t o Titration 7.421 Reineckate S a l t Formation The r e i n e c k a t e of diphenhydramine may be decomposed by h e a t i n g i n an a l k a l i n e medium and a Volhard t i t r a t i o n performed t o determine t h e t h i o c y a n a t e c o n t e n t l o 2 . The s a l t formed may a l s o be determined b r o m a t ~ m e t r i c a l l y ~ ~ ~ , r e s u l t s are about 5% low. The chromium (111) c o n t e n t c a n b e determined w i t h v e r y ood a c c u r a c y f 0.5% u s i n g a c h e l a t o m e t r i c method 184
#'' '

7.42

209

IRA J. HOLCOMB A N D SALVATORE A. FUSARI

Complexometric Method Diphenhydramine forms an i n s o l u b l e s a l t w i t h bismuth which, from t h e r e a g e n t r e p a r e d , releases an e q u i v a l e n t amount of EDTA1g5 The l i b e r a t e d EDTA i s t i t r a t e d w i t h 0.lM ZnSO4 a t p 9.1 u s i n g eriochrome b l a c k T H as i n d i c a t o r .

7.422

S l u r r y Method and c h a t t e n l O 7 p r e s e n t e d s l u r r y methods f o r t h e s e p a r a t i o n of a n t i h i s t a m i n e s f r o m t a b l e t o r c a p s u l e material. The sample i s simply s l u r r i e d w i t h c h l o r o f o r m and f i l t e r e d . The f i l t r a t e i s t i t r a t e d w i t h acetous perchloric acid a f t e r g l a c i a l a c e t i c a c i d i s added. Tuckermanlo8 used a m i x t u r e of magnesium o x i d e and s i l i c e o u s e a r t h for pret r e a t m e n t of a n aqueous i n j e c t i o n followed by washing w i t h warm chloroform i n t o g l a c i a l a c e t i c a c i d . T h e base is then t i t r a t e d w i t h 0.1N perc h l o r i c a c i d u s i n g p-naphtholbenzein as i n d i c a t o r .

7.423

c 1a ir l u b

7.424

Ion Exchanqe Method Ion exchange columnsl09, 110

have been used i n t h e d e t e r m i n a t i o n o f a n t i h i s t a m i n e s w i t h subsequent t i t r a t i o n of t h e effluent

7.425

E x t r a c t i o n Method
A c o l l a b o r a t i v e s t u d y on

t h e e x t r a c t i o n method was r e p o r t e d by H e i m l l l . The f r e e base i s e x t r a c t e d w i t h e t h e r and d e t e r mined by t i t r a t i o n . R e c o v e r i e s w e r e 99-101%. Miscellaneous T i t r i m e t i c Methods p-Toluenesulfonic a c i d i n c h l o r o form112 and m e t h a n e s u l f o n i c a c i d i n g l a c i a l 7.43

210

DIPHENHYDRAMINE HYDROCHLORIDE

a c e t i c a c i d l l 3 have been used as t i t r a n t s f o r In diphenhydramine w i t h v i s u a l i n d i c a t o r s aqueous s o l u t i o n , s i l i c o t u n g s t i c a c i d l i 4 w i t h m e t a n i l yellow or con o r e d as i n d i c a t o r w a s recommended by ~ramml and t h e compound h a s a l s o been t i t r a t e d w i t h 0 . W n i t r a n i l i c a c i d u s i n g a p o l a r o raph f o r d e t e c t i o n of t h e endpoint i n 0.01~K C 11 % 1

Fluorometric Analysis Weak f l u o r e s c e n t i n t e n s i t y w a s observed f o r diphenhydramine when t r e a t e d w i t h 3% H202 1 1 7 , b u t no a n a l y t i c a l u s e w a s made of t h i s observat i o n . Martinll8 treated a residue containing diphenhydramine w i t h c o n c e n t r a t e d s u l f u r i c a c i d and perchloric acid t o o b t a i n f l u o r e s c e n c e a t 525 nm. w i t h e x c i t a t i o n a t 3 7 5 nm. L i m i t of d e t e c t i o n observed w a s 0.02 w./ml. Glazko119 used f l u o r e s c e n t dyes t o e x t r a c t diphenhydramine as an ion-pair and i n c r e a s e d the s e n s i t i v i t y o f d i r e c t e x t r a c t i o n methods s e v e r a l hundred f o l d over t h e use of methyl orange and c o l o r i m e t r y . Automated A n a l y s i s Robertson120 has p r e s e n t e d an automated method of a n a l y s i s f o r amine drugs based on acid-dye methods. B r O m O C r e S O l p u r p l e i s used f o r diphenhydramine. The automated and manual method a g r e e q u i t e w e l l w i t h a 0.4% l a b e l c l a i m d i f f e r e n c e . F u s a r i h a s p r e s e n t e d an u l t r a v i o l e t method f o r c o n t e n t u n i f o r m i t y of diphenhydramine samplesl21.
7.6

7.5

21 1

I R A J. HOLCOMB A N D SALVATORE A . FUSARI

Biological Assay Chen122 used isolated guinea pig ileum for the assay of histamine and diphenhydramine in vitro. A linear relationship is observed between dose and effect. Gravimetric Analysis U ~ e n used ~ ~ picrate method to o ~ the determine diphenhydramine gravimetrically. The picrate is filtered, washed with water and ether, dried and weighed. Chromatography 7.91 paper chromatography The results of chromatography on paper are summarized in Table X for diphenhydramine hydrochloride. Thin Layer Chromatography An excellent review of the thin layer chromatography methods for diphenhydramine was presented by Comer130 in 1967. Additional mobile phases used on silica gel prior to 1967 are presented by Kampl31, (1) CC14:BUOH:MeOH:25% W O H (40:30:30:1), and (2) Petroleum ether:ether:EtzNH (20:80:1) and : by F ~ w a l ~ ~ , :MeOH:NqOH (98 1:1) DiphenCHC13 hydramine has also been chromatographed on thin layers of alumina using @H:EtOH (9:1) or (9:1.5)l33; 6H:EtOH :HOAc ( 3 :1.2 : - 5 ) 133; 0 CHC13:BuOH (98:2)134; CHC13:Me2CO and 6H:EtOH (9:1)134. 7.92 7.9 7.8

7.7

212

Table

x.

Paper Chromatoqraphy of

Diphenhydramine Hydrochloride Mobile Phase n-BuOH:HOAc:H20 n-BUOH:HCl, 0.5N

( 0 1 :50) 4:0
L .

Rf

Pretreatment none none none Impregnated with s o h . of petroleum (180-215O C ) and petroleum ether Impregnated with s o h . of petroleum (180-215Oc) and petroleum ether none none Treated with p H 3 buffer

Ref crence
124

0.85
0-68

(90:30)

124 124

h ,

+
w

I sopropyl alc. :HCl, 0.5N (90:30) EtOH:H2O:QOH (55:43 :2)

1.00
0-31

125

EtOH:H20:NH40H

(95:3:2)

0-76

125

n-BUOH s t . with 1N HC1 a. C6H6:HOAC:H20 (::) 441

0-60
0 -70

126 126
127

n-BuOH sat. with pH 3 buffer 0.63

Table

x cont .) (

Mobile Phase
n-BUOH

Rf
0.67

Pretreatment Treated w i t h pH 5 b u f f e r

Reference

s a t . with p 5 b u f f e r 0.63 H

127

n-BuOH s a t . with p 6 . 5 H buffer nB O - U H s a t . with p 7 - 5 H buffer

n-BUOH:H20 (50:50) w i t h 1 g . c i t r i c a c i d (use upper l a y e r )
n-BUOH s a t . with 1 N HC1

Treated with p 6 . 5 b u f f e r 127 H Treated w i t h p 7.5 b u f f e r 127 H

0.91

0 -96

Dipped i n 5% sodium dihydrogen c i t r a t e Whatman No. 4

128

129

DIPHENHYDRAMINE HYDROCHLORIDE

Additonal systems for the thin layer chromatographic examination and detection of diphenhydramine are presented in Table XI. The reverse phase paper chromatographic s stem developed by Vecerkova has been modifiedlT2 to run on thin layers of silica gel in about 3 hours and will separate diphenhydramine from bromodiphenhydramine. Thin layer chromatography has been used prior to assays by the spot-area method143 and by the acid-dye reaction after elution from the plate.144 Gas chromatography 7.931 Direct Methods on Neutral columns The majority of published gas chromatographic systems for diphenhydramine hydrochloride involve injection of the free base on columns differing in polarity. 7.93

4 MacDonald 1 used a 6 ft column of 1% SE-30 on 100-120 mesh Gas Chrom P. Retention time for diphenhydramine was 6.3 min. : column temperature 173 O ; injection port, 256 Oc; c argon flow rate 60 ml./min. using an argon @-ray ionization detector.

146 Kazpk presented % additional data on 1 SE-30 columns at different temperatures. 147 MacDonald compared four .8 columns in 1964 and found 0 0 % PDEAS on a 120/ 170 glass bead column to be most successful for

215

T a b l e XI. Thin Layer chromatography

of Diphenhydramine Hydrochloride Mobile Phase Sorbent Rf

CHC13 :Me2CO (9: 1) MeOH CHC13:EtOH
c

Reference

Kieselgel G w/ fluorescent indicator

I1

00 .8

El Gendi135

0.22 0.31 0.33

0.90
II

(9:1)
(8:2)

I1

CHC13:EtOH

I1

I1

EtOAc:MeOH:QOH(85:10:5) CHC13 :MeOH (9: 1)

1 sopropyl ether :EtOH (8: 2) MeOH : H40H ( 100 : -5) N 1

Silica Gel G Silica Gel G

Davidow 136 Bastos 137

I1

0.76 0 -11
0.77

II

ISOprOpyl ether:Me2CO(l:l)

Kieselgel G or GF

0.55

Eiden

138

0.48 f

Table XI. (cont .) Mobile Phase Isopropanol Benzene :dioxane :HOAc (50:40 : 10) Sorbent Kieselgel c7 or GF Rf 0.50 s 0.50 f

Reference Eiden
II I1

0.07 s 0.51

Cyc10hexane:EtOAc:Et~NH (65:30 :5) Et0Ac:cyclohexane:dioxane: Gelman silica g e l glass microMeOH : 0 H2 : OH (50:50:10 :lo: 1.5 :O .5) fiber s h e e t s Et0Ac:cyclohexane: NH40H :MeOH : H20 (70:15:2:8:0.5) EtOAc : cyclohexane :MeOH: NH4OH (70:15: 10: 5) Gelman silica g e l glass microfiber sheets

0.71

Kaistha 139

0 -86

Kaistha

13 9

0.91

I1

c,
m

k 0

3.r

al

i ?
Y

X 0

5:
00

9'

4Jm

w-

** N x0** 0 3

dn
218

c s E

DIPHENHY DRAMINE HYDROCHLORIDE

the antihistamines. Diphenhydramine has a retention time of 2.5 min. on a 6' column at 175OC. with an argon flow rate of 60 ml./min.

% Jain148 used 1 Hi-Eff-8B on 100/120 mesh silanized Gas Chrom P. Retention times of diphenhydramine given are relative to .6 methapyrilene; 0.43 at 160Oc. and 0 4 at 190". Gas chromatography was used for the determination of diphenhydramine in blood after an extraction with acetone-ether

A mixture of Hi-Eff-8BP, 1%, and 10% SE-52 on Gas Chrom Q was used by ~ a d e r l ~ application to single and multiple in ~ component drugs. The column temperature was 22OOc. Relative retention time was 0.83 to pentobarbital. presented a general article on gas chromatography in which diphenhydramine was chromatographed on 3 Phenyl % Methyl Silicone (OV 17) on Gas Chrom Q at 175O (6 ft., 4 mm. I.D.). A rapid, direct analysis of antihistamines was reported by Reiss151 in which the sample is dispersed in water, diluted to volume, filtered and injected. Relative retention times to chlorpheniramine maleate are reported on two columns both 4 ft x 0.25 in. 0.d. glass: 2% SE-30 and 2% Carbowax 20M on 8 / 0 mesh DlatOpOrt S, 0.52; and 1 % silicone 010 0 oil DC-200 on 60/80 mesh Diatoport S, 0.63. column temperature was 210 OC.

219

I R A J. HOLCOMB AND SALVATORE A. FUSARI

D i r e c t Methods on B a s i c Columns S t e e l e reported152 on t h e use of a column w i t h 5% Apiezon L and 4.5% potassium hydroxide. column t e m p e r a t u r e w a s 138O f o r t h e f i r s t 6 min. a f t e r i n j e c t i o n and t h e n r a i s e d a t 6Oc./min. t o 275C. R e t e n t i o n t i m e f o r diphenhydramine w a s 3.64 min.

7.932

Oxidation t o Benzophenone P r i o r t o G a s Chromatoqraphy Oxidation w i t h 0.033M Cr2O3 f o r 60 min. c o n v e r t s diphenhydramine t o benzophenone which can b e determined i n nanogram amounts w i t h a p r e c i s i o n of 1.5% u s i n g e l e c t r o n capture gas c h r ~ m a t o g r a p h y l ~ ~ . Column chromatography ~ e v i n e l b 4r e p o r t e d on t h e part i t i o n i n g of diphenhydramine between 2 N H C 1 and chloroform on a C e l i t e column. The diphenhydramine i s e l u t e d w i t h a mixture of 90 m l . chloroform c o n t a i n i n g 1 m l . of a c e t i c a c i d a f t e r a prewash o f t h e column w i t h d i e t h y l e t h e r . Doyle h a s examined d i s t r i b u t i o n diagrams and s e l e c t e d C e l i t e p a r t i t i o n chromatog r a p h i c systems f o r v a r i o u s s e p a r a t i o n s on t h e b a s i s o f t h e diagrams. The e f f e c t s of s o l v e n t composition on t h e column p a r t i t i o n chromatographr5gf a m i n e s h a s a l s o been examined by Doyle and some i n f o r m a t i o n on diphenhydramine was p r e s e n t e d . Electrophoresis The e l e c t r o p h o r e s i s of diphenhydramine h a s been carried o u t by B a r u f f i n i 124 7.95 7.94

7.933

220

DIPHENHY DRAMINE HYDROCHLORIDE

i n d i f f e r e n t pH b u f f e r s a t 7 volts/cm. f o r 3 h o u r s . M i g r a t i o n i s o p t i m a l a t low pH w i t h 8 2 mm. d i s p l a c e m e n t toward t h e cathode a t pH 2 .l. Der likowski 156 examined t h e e l e c t r o p h o r e s i s of diphenhydramine i n 1 9 6 5 . zones w e r e detected w i t h a Dragendorff r e a g e n t .
The l i t e r a t u r e h a s been reviewed through
1972.

Acknowledgment The most c a p a b l e a s s i s t a n c e of M r s . L u c i l l e Kelly, I n f o r m a t i o n S p e c i a l i s t , parke, Davis & C o , i s g r a t e f u l l y acknowledged. The a u t h o r s a l s o wish t o thank M i s s Beverly Jozwiak f o r h e r p a t i e n c e i n t h e p r e p a r a t i o n and c o r r e c t i o n of t h i s manuscript.

22 1

IRA J. HOLCOMB AND SALVATORE A. FUSARI

8. References

1. USP XVIXI, p . 207 (1970) 2. The Merck Index, p . 386, Eighth Edition, Merck and Co., Inc., Rahway, N . J . (1968) 3 . deRoos, A. M., pharm. Weekbl., 102, 1071-7 ( 9 7 . C A. 68, 6240d (1968) 16) 4. deRoos, A. M., ReCl.TraV. Chim. Pays-Bas - 1368-71 (1968). C. A. 70, 72303 u 87, (1969) 5. Wallace, J. E., BiggS, J. D., Dahl, E.V., Anal. Chem. - 831-4 (1966) 38, 6. Schoeb, E. J., parke, Davis & co., Personal Communication 7. Scott, R. B., Parke, Davis & co., Personal Communication 8. Kracmar, J., Kracmarova, J . , Cesk. Farm. - 16-24 (1966) c. A. 64, 19323a (1966) 15, 9. Friedlaender, A. S , Friedlaender, S , Vandenbelt, J. M., J . Allergy - 229 20, (1949) 10. Vandenbelt, J. M., Parke, Davis & Co., personal Communication 11. Okerholm, R. A., Scott, R. B., Parke, Davis & Co., Personal Communication 12. Luers, R. B., Parke, Davis & co., Personal Communication 13. Quan, J. H., parke, Davis & Co., Personal Communication 14. Terhalle, M., parke, D a v i s & Co., personal Communication 15. Keenan, G. L., J. Am. Pharm. ASSOC., S c i . Ed. 36, 281-2 (1947) 16. Shell, J. W., Witt, It. F., Poe, c. F. Mikrochim. Acta, 1960, 31-7. c . A. 58, 2649g (1963)

222

DIPHENHYDRAMINE HYDROCHLORIDE

17. 18. 19. 20.

21.
22.

23.

24.

25.
26. 27.
28.

29. 30.

31.

G a d r e t , M., Bregeve, C B u l l . SOC Pharm. Bordeaux 103, 153-8 (1964) Krc, J., p a r k e , Davis & C o . , Personal Comunicat i o n Doyle, T . D . , Levine J., J. Assoc. O f f i c . Anal. chem., 51, 191-199 (1968) Doyle, T . D . , Anal. Chem., 39, 1282-87 (1967) Doyle, T. D . , P r o c t o r , J . B . , J . A ~ S O C . O f f i c . A n a l . chem., 55, 328-34 (1972) Konyushko, U. S . , Guverich, T . Sh., K a r t a s h e r a , L . Kh. S b . Nauchn. Tr. Vitebskogo Med I n s t . 1964, 189-91. C . A . 64, 7974e (1966) P e t r a s h k e v i c h , S F , S t a r o b i n e t s , G L. , V e s t i Akad. Navuk B e l a r u s , SSR, S e r . IChim, Navuk ( 5 ) , 35-8 ( 1 9 7 0 ) . C. A. 74, 1 1 5 8 2 6 ~(1971) F a r h a d i e h , B . , H a l l , N . A . , Hammarlund, E . R., J . Pharm. S c i . , 56, 18-23 (1967) Attwood, D . , J . Pharm. pharmac. 24, 7 5 1 (1972) Andrews, A . c., Lyons, T . D . , ~ ' B r i e n , T . D . , J . Chem. SOC., 1962, 1776-80 Lordi, N. G . , C h r i s t i a n , J. E., ~ m . Pharm. ASSOC., S c i . E d . , 45, 300-5 (1956) deRoos, A . M., Rekker, R . F . , Nauta, W . T . , Arzneim. F o r s c h . 20, 1763-5 ( 1 9 7 0 ) . C . A . 74, 41739u (1971) S p U r l O x , C . H . , Parke, Davis & c o . , P e r s o n a l Communication Shima, K . , M i z o j i r i , K . , I c h i h a s h i , S . , Yamamoto, R . , J . Pharm. SOC. Jap. 90, 774-7 (1970) Kennon, L., Higuchi, T., J . Am. Pharm. A S S O C . , 45, 157-60 ( 1 9 5 6 ) . C . A . 50, 8139a ( 1 z 6 )

. .

J.

223

IRA J. HOLCOMB AND SALVATORE A. FUSARI

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32.

.,

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DIPHENHYDRAMINE HYDROCHLORIDE

47. 48. 49. 50.

51. 52. 53. 54. 55.

56. 57. 58. 59.

60.

61. 62.

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232

ECHOTHIOPHATE IODIDE

Raymond D. Daley

RAYMOND D. DALEY

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 U l t r a v i o l e t Spectra 2 . 4 Mass Spectra 2.5 D i f f e r e n t i a l Thermal Analysis 2.6 S o l u b i l i t y 2.7 C r y s t a l Properties 2.8 Melting Point Synthesis Stability

3.
4.

-- Degradation

5.
6.

Drug Metabolic Products Methods of Analysis 6 . 1 Elemental Analysis 6 . 2 U l t r a v i o l e t Spectrophotome t r i c Analysis 6 . 2 1 Direct U l t r a v i o l e t Absorption Measurement 6.22 I n d i r e c t U l t r a v i o l e t Absorption Method 6.3 Titrimetric Assay Method 6 . 4 Thin Layer Chromatography 6 . 5 Other Tests

234

ECH OTH I OPHATE I OD I DE

1 .

Description
11 .

Name, Formula, Molecular Weight

The Chemical Abstracts name for echothiophate iodide is 2 [ (diethoxyphosphiny1)thiol -N,N,N-trimethyl 1. ethanaminium iodide, starting with Volume 76 ( ) Previously the Chemical Abstracts name was (2-mercaptoethy1)trimethylauunonium iodide S-ester with 0,O-diethyl phosphorothioate. The CAS Registry No. is [ S U - l O - O ] . The British Pharmaceutical Codex lists the compound as ecothiophate iodide (2), and The Merck Index lists several other names (3).

C 9H23IN03PS 12 .

Mol. Wt.:

383.23

Appearance, Color, Odor

White crystalline pwder, with a slight mercaptan-like odor. 2 . Physical Properties 21 . Infrared Spectra

Figure 1 is an infrared spectrum of one of the crystalline forms of echothiophate iodide. This form will be designated as Form I in Section 2.7, Crystal Properties. The spectrum was run in several sections: (a) from 200 to 540 cm as a mineral oil mull on polyethylene; (b) from 470 to 1360 cm as a mineral oil mull between potassium bromide plates, with the 900 to 1055 cm and 1220 to 1275 cml regions run in two thicknesses; (c) from 1360 to 4000 cm as a perfluorinated oil mull between potassium bromide plates. The spectrum was obtained with a Beckman IR-12 spectrophotometer. follows ( 4 ) :
Some of the absorption bands can be assigned as

235

Figure 1.

Infrared spectrum of echothiophate iodide, perfluorinated and mineral o i l mull.

ECH OTH I OPH ATE I 0 0 I DE

3000 c ' m 1240 Cm" 1157 c ' m 975 c m ' 2.2

C-H Stretching P=O Stretching

P-0-Ethyl Vibration P-0-C (Alkyl) Vibration

Nuclear Magnetic Resonance Spectra

' b o proton magnetic resonance spectra of echothiophate iodide are shown in Figures 2 and 3 . These were run in D 0 and in CDCl , on a Varian A-60A 60 MHz NMR spectraneger, with a te2ramethylsilane reference ( 5 ) .

The NMR spectra of organic phosphorus compounds are complicated by coupling of the proton signals with that of phosphorus. This coupling causes readily observable splitting of the lines from methylene protons in the groups P-0-CH and P-S-CH2, with JpH coupling constants of 9 Hz ( 6 f . The proton NMR spectral assignments are given in Table I (5). A phosphorus NMR scan indicates a chemical shift of about -28 ppm for the phosphorus in echothiophate iodide in aqueous solution, relative to a phosphoric acid reference ( 7 ) . This is consistent with literature values for this structure ( 8 ) .
2.3

Ultraviolet Spectra

Figure 4 shows the ultraviolet absorption spectrum of echothiophate iodide, run on a Cary Model 14 spectrophotometer. The sample was dissolved in water. The maximum at 226 nm has an absorptivity of 1.34 x 104 liters per mole cm. This absorption is essentially that of the iodide ion (the ultraviolet spectrum of a potassium iodide solution exhibits a imum at 226 nm with an absorptivity of about 1.35 x liters per mole cm)

2.4

Mass Spectra

Attempts to obtain the mass spectrum of echothiophate iodide were unsuccessful; the compound apparently
237

00

Figure 2.

Proton NMR spectrum of echothiophate iodide, D20 solution.

bJ

Figure 3 .

Proton NMR spectrum of echothiophate iodide, CDC13 solution.

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Figure 4 .

Ultraviolet spectrum of echothiophate iodide i n aqueous solution vs water, 1 a c e l l s ; 25.0 mcg/ml, 250 mcg/ml, 2.50 mg/ml. n

RAYMOND D. DALEY

decomposed i n the heated i n l e t of the mass spectrometer (5). 2.5 D i f f e r e n t i a l Thermal Analysis

Figure 5 shows the d i f f e r e n t i a l thermal a n a l y s i s curve of echothiophate iodide, run a t 10C per minute on a Dupont Model 900 instrument. The only thermal event below 2OO0C is the melting point, which occurs a t 122OC on t h i s scan. When the m a t e r i a l was run a t 2OoC p e r minute, the melting endotherm was observed a t 125.5OC ( 9 ) . 2.6 Solubility

The s o l u b i l i t y of echothiophate iodide a t room temperature is as follows: Approximate Solvent Solubility, wI m l Water Methanol Ethanol (952) 2-Propanol Ace t oni t r i l e Chlorof orm Acetone Die thy1 Ether Petroleum Ether Benzene Ethyl Acetate 2.7 Crystal Properties

> 500 > 250 > 120

4 25 250 8 1 1 1 1

< < < <

Two c r y s t a l forms of echothiophate iodide have been observed. The x-ray powder d i f f r a c t i o n patterns a r e given i n Table 11. These were obtained with a Norelco diffractometer, using n i c k e l - f i l t e r e d copper Ir radiation. U
2.8 Melting Point The following melting points have been reported: 124-4.5OC 138
(10) (11)

242

N P w

Figure 5.

Differential thermal analysis scan of echothiophate iodide.

RAYMOND D. DALEY

TABLE I1

X-Ray Puwder Diffraction Data for Echothiophate Iodide

Form I

d , A'

1/11 23 9 99 66 22 41 10 100 52 10 25 56 66 8 46 13 60 21 2 70 92 22 33 1 1 6 22 4 15 9 3 8 13 15 12 31
244

d, A '
10.00 6.79 6.68 5.40 4.99 4.64 4.45 4.37 4.21 4.06 4.00 3.95 3.81 3.73 3.60 3.41 3.34 3.28 3.22 3.14 3.07 2.96 2.92 2.86 2.81 2.77 2.71 2.67 2.43 2.34

Form I1 1/11
64 24 15 100 40 47 56 56 22 28 17 92 24 20 30 35 30 4 24 13 12 24 13 7 24 6 21 7 13 1 1

10.54 8.00 6.14 5.90 5.49 5.30 5.01 4.86 4.75 4.49 4.33 4.21 4.16 4.10 4.01 3.94 3.82 3.69 3.61 3.54 3.49 3.45 3.40 3.28 3.24 3.20 3.16 3.12 3.06 3.01 2.97 2.95 2.92 2.89 2.82

ECHOTHIOPHATE IODIDE

TABLE I1 (Cont'd.)
d , A' 1/11 d , A' -

1/11 -

2.74 27 .0 2.67 2.58 2.53 25 .0 24 .8 2.44 24 .0 2.37 2.31 2.27

3.
Synthesis

10 16 25 13 4 6

4 14 3 4 4 7

Two methods f o r preparing echothiophate iodide have been published. The r e a c t i o n s a r e shown i n Figure 6.
I n the f i r s t method ( l o ) , the sodium s a l t of dimethylaminoethyl mercaptan is prepared by t r e a t i n g dimethylaminoethyl mercaptan hydrochloride with sodium. The product is treated with diethylchlorophosphate t o y i e l d 0,O-diethyl-S-$ -dimethylaminoethyl thiophosphate. This m a t e r i a l is t r e a t e d with methyl iodide t o make echothiophate iodide. I n the second method ( l l ) , a mixture of diethylchlorophosphate, dimethylaminoethyl mercaptan, and triethylamine i n e t h e r is refluxed. The mixture is f i l t e r e d t o remove the insoluble triethylammonium chloride and d i s t i l l e d t o obtain the 0,O-diethyl-S-6 -dime thylaminoe thy1 thiophosphate. This material is t r e a t e d with methyl iodide t o make echothiophate iodide.

4.

Stability

-- Degradation

Hussain e t a 1 (12) have shown t h a t echothiophate iodide decomposes by a t l e a s t two mechanisms. I n the pH range 2 4 t o 5, the major r e a c t i o n i s hydrolysis of one . of the C-0 bonds t o form ethanol and the monoethyl e s t e r .
245

1 .

(a)

(CH3l2NH -CH2CH2-SH

+ 2Na *(CH3)2N-CH2CH2S- + 2Na+ + H2

0
0

(b)

(CH3)2N-CH2CH2S-

+ + Cl-P(OC2H5)20 f 0

(CH3)2N-CH2CH2-S-P(OC2H5)2
0

+ C1-

(c)

( c H ~ > ~ N - c H ~ c H ~ - s - P ( o cC H ~ I ~ + ~H )
(CH3)2N-CH2CH2-SH

I: (cH~)~N+cH~cH~-s-P(oc~H~)~-J I-

2 .
N P

(a)

t + C1-P(OC2H5)2 +

(C2H5)3N
0 9

rn

( c H ~ ) ~ N - c H ~ c H ~ - s - P ( o c ~[ ( ) ~ H ~ ) ~ N + ~ c ~ + H~c ~
C H ~ ) ~ N - C H ~ C H ~ - S - + ( - + ~ HH ~ I C C ~)~

(b

I: ( c H ~ )+CNH ~ C H ~ -tS - P C O C ~ HI- ) ~ J ~ ~

Figure 6.

Synthetic Methods for Echothiophate Iodide.

Ol h

+
X
I

Om
n

r-l hl

x l h

+
W

O+pr
U

3
a . d .. a
W
0) Y 0)

0
0

X
ON X

hl
I

+
h

+ :
hl

ON X

m
n hl
n LA
v U

+
X u

m
hl n
W

-X&

u
W

LA

x N

0
v

0
rr)

O f $

rn
hl X

+ :
X

uhl

T
r(

0th
QI

2- .
N

2
a

rn
X

+ :
m
n

3
m

X -

v
U

X u

v U
n
W

n
W

241

RAYMOND D. DALEY

. In the pH range 9 5 to 12, the major reaction is hydrolysis of the S-P bond to yield (2-mercaptoethyl) trimethylammonium iodide and diethylphosphoric acid. . These reactions are shown in Figure 7 At intermediate pH, both react ions occur. 5 . Drug Metabolic Products No metabolic products have been reported. 6. Methods of Analysis 61 . Elemental Analysis

The elemental composition of echothiophate iodide is as followst Element - 'K Theory Carbon Hydrogen Iodine Nitrogen Oxygen Phosphorus Sulfur 62 . 28.21 6.05 33.11 3.65 12.52 80 .8 8.37

Ultraviolet Spectrophotometric Analysis 6.21 Direct Ultraviolet Absorption Measurement

Although the ultraviolet absorption at 226 nm has been used in hydrolysis studies (12), it was useful only because it increased as the echothiophate iodide hydrolyzed. The iodide ion is the principal absorbing species at this wavelength in echothiophate iodide solutions (see Section 2 3 , so that this maximum .) can be used only indirectly to measure the echothiophate cation concentration. 6.22 Indirect Ultraviolet Absorption Method

An ultraviolet assay for echothiophate cation is possible, using hydrolysis to thiocholine, followed by reaction with 4,4'-dithiopyridine to form an
248

ECHOTHIOPHATE lODl DE

ultraviolet absorbing material (13). The method is based on that of Grassetti and Murray ( 4 . The ultraviolet 1) quantitation is essentially an alternative to the titration described in Section 6.3, but requires less sample. The echothiophate iodide is hydrolyzed quantitatively to thiocholine in 20 minutes in pH 12.0 phosphate buffer. The hydrolyzed sample is then treated . with a solution of 4,4'-dithiopyridine in pH 23 phosphate buffer. The final solution has a pH of 6.2, and the 4.4I-dithiopyridine reacts with thiocholine to form 4-thiopyridone. 4-Thiopyridone has an absorption m maximum at 323 n . A blank is prepared by mixing a portion of the original echothiophate iodide solution, before hydrolysis, with a solution of 4,4'-dithiopyridine. Echothiophate iodide assayed by the titration procedure is used as a standard (13).
63 .

Titrimetric Assay Method

The USP method for assay of raw material and dosage forms is iodimetric titration of the thiocholine formed by hydrolysis of the echothiophate iodide. In the USP XVIII procedure, the sample is hydrolyzed with sodium hydroxide ( 5 . It has been shown recently that 1) greater specificity is obtained when hydrolysis is conducted with a pH 12 buffer (16). It is necessary for the pH to be as high as 12 in order that the hydrolysis be completed in 20 minutes. On the other hand, too high a pH increases interference from possible impurities (16).

6 4 Thin Layer Chromatography .

The following systems have been found useful for separating echothiophate iodide from possible degradation products: (a) Silica Gel G (E. Merck) with methanolwater-concentrated ammonium hydroxide (2: 2: 1) developing solvent and iodine vapor detection (17); (b) Cellulose F (E. Merck) with butanol-acetic acid-water (4:1:5) developing solvent and iodine vapor detection ( 8 . 1)

249

RAYMOND D. DALEY

65 .

Other Tests

A microscopic identity test for echothiophate iodide has been reported. Echothiophate iodide in aqueous solution forms a crystalline precipitate with amnonium reineckate ( 9 . 1)
7.

Acknowledgments

The writer wishes to thank Dr. B T Kho for his . . review of the manuscript, Dr. G . Schilling of Ayerst . . Research Laboratories and Dr. W E Krueger of the State University of New York at Plattsburgh for their NMR data and interpretation, the library staff for their literature search, the numerous other contributors who provided information for this profile, and Mrs. Kay Mannan for typing the profile.

250

ECHOTHIOPHATE IODIDE

REFERENCES C A. 76, 2946 (1972), Chemical Abstracts Index Guide. . British Pharmaceutical Codex 1968, Supplement 1971, The Pharmaceutical Press, London, 1971. 3. The Merck Index, 8th Ed., Merck and Co., Inc., Rahway, N J , 1968. . . 4 L J. Bellamy, The Infra-red Spectra of Complex . . Molecules, 2nd E . John Wiley and Sons, Inc., New d, York, 1958. . 5 G Schilling, Ayerst Research Laboratories, personal . communi ca tion. . . . 6 . H Babad, W. Herbert, and M C Goldberg, Anal. Chim. Acta 41, 259-68 (1968). 7 W E.Krueger, State University of New York at . . Plattsburgh, personal communication. . . . . 80 V. Mark, C H Dungan, M M Crutchfield, and J. R. Van Wazer, Top. Phosphorus Chem. 2, 227-457 (1967). . 9 F. Q Gemmill, Ayerst Laboratories, Inc., personal . communica ti on. . . . . . . 10 H M Fitch, U S Patent 2,911,430; C A 54, 4386h. 11. L-E. Tammelin, Acta Chem. Scand. ll, 1340-9 (1957). . . . 12 A Hussain, P Schuman, V. Peter, and G Milosovich, J. Pharm. Sci. 57, 411-8 (1968). . . . 13. N G Nash and F DiBernardo, Ayerst Laboratories, Inc., personal communication. . 14. D R Grassetti and J. F Murray, Jr., Arch. Biochem. . . Biophys. 119, 41-9 (1967). 15. Echothiophate iodide monographs, Pharmacopeia of the United States of America, 18th Revision, Mack Printing C . Easton, Pa., 1970, pp. 220-1. o, . . . 16. C. Warner, F DiBernardo, A B y l w , A Hussain, and B T Kho, J. Pham. Sci. 60, 1548-9 (1971). . . . 17. A Bylow, Ayerst Laboratories, Inc., personal communication. . 18. G. R Boyden, Ayerst Laboratories, Inc., personal communication. . . . . . . 19. L G Chatten, A C Napper, and P J. Barry, J Pharm. Sci. 56, 834-8 (1967). 1 . 2 .

The above references cover the literature through 1972.

25 1

ETHYNODIOL DIACETATE

Edward P. K. Lau and John L . Sutter

EDWARD P. K. LAU AND JOHN L. S U T E R

Contents
1 Description . 1.1 Name, Formula, Molecular Weight. 1.2 Appearance, Color, Odor.
2.

Physical Properties
2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum M s Spectrum as Optical Rotation Melting Range Differential Scanning Calorimetry Thermogravimetric Analysis Solubility

3.

Synthesis Stability and Degradation Drug Metabolic Products and Phannacokinetics Methods of Analysis Phase Solubility Spectrophotometric Analysis Colorimetric Analysis 6.4 Fluorometric Analysis 6.5 Titrimetric Analysis 6 6 Chromatographic Analysis . 6.61 Column Chromatography 6.62 High Pressure Liquid Chromatography 6.63 Thin Layer Chromatography
6.1 6.2 6.3

4.
5. 6.

7.
8.

Acknowledgments References

254

ETHYNODIOL DIACETATE

1 Description .

1 1 Name, Formula, Molecular Weight . Ethynodiol Diacetate is 19-Nor-17a-pregn-4-en20yne-3B, 17-diol Diacetate.

Weight: 348.52 1.2 Appearance, Color, Odor Ethynodiol diacetate is a white to off-white, essentially odorless powder. 2 Physical Properties . 2.1 Infrared Spectrum n The infrared absorption spectrum of a ethynodiol n B diacetate reference standard compressed i a K r pellet is shown in Figure 1 The compound exhibits essentially . the same infrared spectrum in chloroform solution. Tne following assignments have been made for absorption bands in Figure 1

,'

Qn.

-1

Assignment
C r . ( 3 I : Acetylenic C-H
C=O

3315 1740
1670

stretching stretching

: Acetate Carbonyl

C=C

: Ethylenic stretching

255

ETHYNODIOL DIACETATE

n
1275, 1028

CH - C - 0 - : Acetate C-0 3 stretching

I1

2.2 Nuclear Magnetic Resonance Spectrum

The NMR spectrum of ethynodiol diacetate in deuterated chloroform is shown i Figure 2. Spectral assignments n are as follows:2 Chemical Shift (PPm) 5.00-5.41 2.58

Type
Broad Singlet Singlet

Ass ignment

Protons at C-3 and c-4

CGCH : 0

Ethynyl proton

2.03
0.90

Sing1et Sing1et

-0-C-CH : Acetyl methgl protons

-a

: C-18 Aethyl protons

2.3 Ultraviolet Spectrum Ethynodiol diacetate does not absorb between 420 nm and 210 nm. A peak is observed at 204 nm which is not convenient for quantitative determination.
of the compound in methanolic 0.7 N HC1, for 10 minutes on a steam bath. The resulting soTution o f diene exhibits the absorption spectrum sham in Figure 3, with max-

The USP XVIII assay procedure involves acid hydrolysis

ima at about 229 nm, 236 nm and 244 nm. The peak at 236 nm is used for quantitative determination.3 2.4 Mass Spectrum

The low resolution mass spectrum of ethynodiol diacetate shown in Figure 4 was obtained with an AEI Ihdel M5-30
251

FIG. 3: ULTRAVIOLET SPECXRLIM OF ETHYNODIOL DIACETATE

259
h)

UY

0 10

0 4 PJ

In

CI)

co

Wavelength (mp)

'LNI ' 1 3 M I

ETHYNODIOL DIACETATE

mass spectrometer. A molecular ion was observed at m/e 384. The base peak i the spectrum was at m/e 43, COTn responding to (31 a+. Structure assignments are summarized below: '
m/e
384 369 342 324
30 9

Assignment
M'f

% Relative Intensity

1.0 1.4 0.2

M- CH3'
M- m2C0

M- C H 3 0 I
M- (QI + M3COOH) 3 M- (CHzCO + M C O ) 3OH M- (2 CH3cooH) M- ( 2 MC C + M,') 3OH

8.5
1.6

282

2.2 4.0

2 64
249 43 2.5

1.4
100.0

c)13co+
Optical Rotation

n The following specipc rotation values i chloroform have been reported.

261

EDWARD P. K. LAU AND JOHN L. SU7TER

2.6

Melting Range

"lie melting range given i n the U P XVIII is 126'to S 132OC.

2.7

Differential Scanning Calorimetry

The D C tliermogram of ethynodiol d i a c e t a t e obtained a t S a heating rate of SC/minute is shown in Figure 5 . Tne endothermic change observed a t about 126OC corresponds t o the melting of the drug. T e decomposition h temperature is 228OC.

'

2.8

Thennogravimetric Analysis

"lie TGA spectrum of ethynodiol diacetate i n Figure 6 was produced under a nitrogen sweep a t a heating rate of 10C/minute. A rapid weight loss was observed from about 21OoC t o 26OOC. Another Fapicl weight loss was seen s t a r t i n g a t about 400 C.
2.9

Solubility

S o l u b i l i t i e s i n various solvents a t 25OC are given in the following table: Solvent Solubility, mg ./d.
0.0014

Water
Methanol Ethanol Chloroform Heptane

>so
>50
)SO

18

262

ETHYNODIOL DIACETATE

80

100

120

140

160

TEMPERATURE oc

263

EDWARD P. K. LAU AND JOHN L. SUTTER

TGA SPECIRJM OF FI1NNODlC)L DIAEI'XTE

FIG, 6:

100

80

.-. s
9
0 "

60

40

20

100

200

300

400

500

TEGW?ATJRE OC

264

ETHYNODIOL DIACETATE

3.

Synthesis Ethynodiol diacetate has been synthesized by routes utilizing both estradiol 3-methyl ether ( ) and 38I hydroxyandrost-5-en-17-one (11) as starting materials. In the former method, 9 9 l o , l'outlined in Figure 7 , estradiol 3-methyl ether (I) is reduced by the WillsNelson modification of the Birch procedure12,togive the 1,4-dihydro derivative (111). Oppenauer oxidation of (111)13 yields the 17-ketone (IV), which is then ethynylated,14 giving the enol ether intermediate, ( ) V. Reaction of (V) with dilute acetic acid produces norethynodrell (VI) Treatment of either (V) or (VI) with aqueous mineral acid gives norethindrone (VII), which is then converted to ethynodiol (VIII) by reduction with sodium borohydridelO, 16. The diol is then diacetylated with acetic anhydride and pyridine, yielding ethynodiol diacetate (IX)

Alternatively, as shown in Figure 8, peracid treatment of 3fi-hydroxyandrost-5-en-17-one (11) yields the 5,6 a-epoxide (X) Perchloric acid cleavage of (X) results in the 5,6-diol (XI); acetylation then gives the 3, 5,6-triacetate (XII) , which reacts selectively with bicarbonate to give the 3fi,68-diol-5a-acetate (XIII) , Selective acetylation at C-3 followed by lead tetraacetate and iodine functionalization of C-19 then yields the 68, 19-oxide (XIV). Bicarbonate hydrolysis of (XIV) followed by chromic acid oxidation of the resulting alcohol affords X) the key intermediate ( V , which, when treated with zinc and zinc chloride in methanol gives 19-hydroxyandrostenedione (XVI). Treatment of (XVI) with chromic acid affords the acid (XVII), which on heating in pyridine is decarboxylated to give the 5 (10)-dehydro derivative (XVIII). Selective ketalization of (XVIII) at C-3 is accomplished by treatment with weak acid in methanol, yielding (XIX). Ethynylation at C-17 then gives the 3-dimethyl ketal of norethynodrel (XX), Weak acid cleavage of (XX) gives norethynodrel (VI) , while more vigorous acid treatment gives norethindrone (VII) , Conversion of (VII) to ethynodiol diacetate (IX) is accomplished as previously described,

265

EDWARD P. K. LAU AND JOHN

L. SUTTER

FIG. 7:

SYNTHESIS OF E O IL " D0

DIACETAE

a3

&&
I

013

I11

d
m c - &3 ; & = Q13
IV

I/

a; c&
VI

a - & ; : &

@' d

VI I

HO
VIII

266

ETHYNODIOL DIACETATE

FIG. 8:

SyN?HESIS OF ETHYNODIOL DIACETATE

I1

XI

I(

XI I

H
Ac6

Aco OH

XI11

XIV

267

EDWARD P. K . LAU A N D JOHN L. SUTTER

FIG. 8: (CONT.)

XVI I

WIII

268

ETHYNODIOL DIACETATE

4.

S t a b i l i t v and Demadation Ethynodiol diacetate appears to be very s t a b l e as a solid. The degradation of ethynodiol diacetate in both a c i d i c and basic alcoholic solutions is s h m i n Figure 9. I n the acidic alcohol solution, t h e primary degradation product was found to be the diene ( I ) . In basic alcohol solution, the primary degradation product was found t o be the d i o l
(11). l 9

5.

Drug hletabolic Products and Pharmacokinetics

The major metabolites of ethynodiol diacetate in urine a r e shown i n Figure 10. These metabolites were ;$enThe t i f i e d by Kishimoto, Kraychy, Ranney and G a n t t , metabolism of ethynodiol diacetate by rat and human l i v e r was reported by Freudenthal, Cook, Forth, Rosenf e l d and Wall, *' They found t h a t the biotransformation reactions involved i n the in v i t r o metabolism include deacetylation, saturation 3 r i n g A, aromatization of ring A, formation of 3-ketone and an6-bond formation. A method of analysis of very low levels of t h e metabolite norethindrone has been developed by Freudenthal , Cook and Wall. 2 2 The principle of t h i s method is t o convert the cold noreth' rone by enzyme reduction i n the prest o t r i t i a t e d 17-a-ethynylestraneence of NADF'H-4 3,17- B-diol

The pharmacokinetic p r o f i l e of the t o t a l tritium label and metabolic composition i n the plasma a t e r an o r a l administration of ethynodiol diacetate-6,7- I t o a human subject was studied by Karim, Ranney, Cook and B r e s ~ l e r . ~ 3 The ab orption rate constant (k) of the t o t a l label was 0.79% per hour, the peak plasma level being attained a f t e r 3 hours. The elimi a t i o n r a t e constant (K) of the per hour ( h a l f - l i f e 25 hours). t o t a l label was 0.0276% The volume o f d i s t r i b u t i o n (V) was found t o be 33L and n the metabolic clearance rate (MCR) 21.9L per day. O chloroform extraction of the pooled plasma, 20% of the radioactivity was obtained as a f r e e f r a c t i o n which on TLC analysis gave two major spots tentatively identified as saturated dihydroxy metabolites and norethindrone. Eighty percent of the pooled plasma radioactivity was pres

269

FIG. 9: CEGRADATION OF ETHYNODIOL DIACETATE IN ACIDIC F7 BASIC SOLUTION

ETHYNODIOL DIACETATE

FIG. 10: MAJOR METABOLITES OF ETHYNODIOL DIACETATE IN URINE

27 1

EDWARD P.

K. LAU AND JOHN L. SUTTER

ent as water-soluble conjugates which on acidic hydrolysis furnished two major aglycones having chromatographic mob i l i t i e s similar t o the two major spots. In a plasma samp l e taken one hour after administration of the labeled drug, 58% of the radioactivity was associated with the conjugated metabolites, 12.5% with the spot identified as saturated dihydroxy metabolites and 19.7% with the spot identified as norethindrone.
6 . Methods of Analysis

6.1

Phase Solubility
Phase s o l u b i l i t y analysis can be carried out by equilibrating the drug substance i n hexane a t 25%. Figure 1 shows the phase s o l u b i l i t y diagram of a 1 reference standard run.

6.2

Spectrophotometric Analysis Ethynodiol diacetate does not have a useful spectrum f o r d i r e c t U.V. analysis. The solution of diene res u l t i n g from acid treatment has an absorbance maximum a t about 236 nm. The USP XVIII assay is based on t h i s reaction.

6.3

Colorimetric Analysis
A variety of colorimetric methods have been developed t o detect and t o determine ethynodiol diacetate.
6.31

Reaction of ethynodiol diacetate with antimony t r i c h l o r i d e in dry chloroform containacetic anhydride produces a v i o l e t ing 1% color. The absorbance of the solution a t 565 nm. is l i n e a r w i t h ethynodiol concent r a t i o n over a range of 5-60 mcg./5 ml. Tne method has been adapted f o r the analysis of ethynodiol dosage forms.25 None of the other steroids comonly found in o r a l estrogen-progestin combination dosage forms int e r f e r e . A chloroform solution of a n t h n y

212

ETHYNODIOL DIACETATE

FIG. 11:

PHASE SOLUBILITY

& = Q s

WIPE: ETHYNODIOL DIACETAE

EXTRAPOLATED S O ~ I L I T Y :27.9 mg./g. solvent

SLOPE: 0.0% EQUILIBRATICN: 24 hrs. at 2SC

SOLW:

H E M

20

40

00

llu

100

120

27 3

EDWARD P. K. LAU AND JOHN L. S U l T E R

trichloride has also been proposed as a n spray reagent for ethynodiol diacetate i quantitative thin layer chromatography.
6.32

Reaction of ethynodiol diacetate with 52% sulfuric acid for 5 minutes at room temperature yields a solution having an absorbance maximwn at 484 nm. This method may be used for quantitative determination of ethynodiol diacetate, provided that a preliminary separation from other steroids is made, Reaction of ethynodiol diacetate with hydroxylamine hydrochloride and ferric chloride produces a deep red color which serves to distinguish the compound from steroids having no ester group, The color is not sufficiently stable for use in a quantitative determination. *

6.33

6.4

Flwrometric Analysis Ethynodiol diacetate can be quantitatively determined by flwrometry in 65%sulfuric acid solution, with an activation wavelength of about 458 nm. and measuring fluorescence at about 520 nm. Separation from other steroids is necessary due to their interference. The limit of sensitivity of the method is 4 mcg./lOO d.

6.5

Titrimetric Analysis
6.51

Ethynyl Titration - ethynodiol diacetate reacts stoichiometrically with silver nitrate in tetrahydrofuran. The nitric acid produced can be titrated with sodium hydroxide, either potentiometrically o r using phenolphthalein as indicator. One equivalent of the compound is titrated. Ester Saponification - ethynodiol diacetate may be saponified with a h o r n amount of standardized alcoholic potassium hydroxide.
274

6.52

ETHYNODIOL DIACETATE

The excess base is then titrated with hydrochloric acid, either potentiometrically or using phenolphthalein as indicator.2 9 One equivalent of the compound is saponified.
6.6

Chromatographic Analysis
6.61

Column Chromatography - the quantitative separation of ethynodiol diacetate and mestranol in dosage f o m on Sephadex LH-20 has been reported.

6.62 H i g h Pressure Liquid Chromatography

ethynodiol diacetate can be separated from its possible degradation products and quantitatively determined by reversephase high pressure liquid chromatography, using a W o n t ODs column and methanol-water eluants. 3 1 Thin Layer firomatography - TLC systems and corresponding Rf values of ethynodiol diacetate are summarized in the following table:

6.63

Thin Layer Chromatography of Ethynodiol Diacetate Solvent System Adsorbent cyclohexane: isopropanol (97:3)
(95:s)

Detection
1, 2

3
0.40

Reference
32

SG

benzene :methanol SG benzene:acetone

(80:20)

1, 2
3, 4

0.77

33

SG

0.68

33 33

chloroform: methanol
(90: 10)

SG

3, 4

0.76

27 5

EDWARD P. K. LAU A N D JOHN L. SUlTER

methylene chlor- SG ide:methanol:water (150:9:0.5)

3, 4

0.84

33

SG

Silica gel.
1. Spray with 50%H2S04, heat at 8OoC for 10 minutes.
2.
3.

Detection:

Spray with phosphomolybdic acid.

H SO4; heat at 100C for 38 mmutes.

Spray with concentrated

4.
7.

Observe under short wave U.V.

Acknowledgments The authors wish t o express their appreciation to Dr. N. W Atwater, Dr. R. Bible, Dr. F. Colton, M. . r A. J. Damascus and Dr. J. Hribar for their help in preparing sections of the manuscript. The expert secretarial assistance of Miss Mia Mulder is also gratefully acknowledged, as is Mrs. Lorraine Wearley's aid in preparing the figures,

276

ETHY NOD1OL D IACETATE

8.

References
1. Damascus, A. J., Searle Laboratories, personal communication.

2.
3. 4. 5. 6.

Bible, R., Searle Laboratories, personal cmunication. Searle Laboratories Method of Analysis No, RS24-817. Hribar, J., Searle Laboratories, personal communication.

Damascus, A. J., Searle Laboratories, personal communication.

"The United States Phannacopeia" XVIII, p. 259 (1970). Carey, S . and Anthony, G . , Searle Laboratories, personal c o m i c a t i o n .

7.
8. 9.
10.

Chow, A. and Marshall, S., Searle Laboratories, personal c o m i c a t i o n .

Colton, F. B., U.S. Pats, 2,691,028 (1954); 2,725,389 (1955). Colton, F. B., U.S. P a t , 2,843,609 (1958). Steroids 10

11. Klimstra, P. D. and Colton, F. B., 4 1 1 (1967).

12.
13. 14. 15.

Birch, A. J. and Smith, H., J. Chem. SOC. -' 1951 1882. Oppenauer, R. V., Org. Syn. 21, 18 (1941), Stavely, H. E., J. Am. Chem. SOC. - 79 (1939). 61, Colton, F . B., U.S. Pats, 2,655,518 (1952); 2,691,028 (1954); 2,725,378 (1955).

277

EDWARD P. K. LAU AND JOHN L. S U l T E R

16.
17.

Sondheimer, F, and Klibansky, Y., 1 5 (1959)

Tetrahedron

5,

Pappo, R. and Nysted, L . , U.S. Pat. 3,176,014 (1965). Hagiwara, H., Noguchi, S., and Nishikawa, M., Pharm. Bull. (Tokyo), - 84 (1960). 8,

18.

&em.

19. Bollweg, M. and Baier, M., Searle Laboratories, personal comunication.

20. 21.

Kishimoto, Y., Kraychy, S., Ranney, R. E. and Gantt, C. L., Xenobiotica - 237-52 (1972) 2, Freudenthal, R. I., Cook, C. E., Forth, J., Rosenfeld, R. and Wall, M. E , , J. Phannacol. Exp. Ther. - 468-73 (1971). 177, Freudenthal, R. I . , Cook, C. E. and Wall, M, E., "Progress Report No. 2", RTI Project No. CN-385, Research Triangle I n s t i t u t e , Research Triangle Park, N. C., 1971.

22.

23.

Karin, A,, Ranney, R. E., Cook, C. E. and Bressler, R., "Pharmacokinetics and Plasma Metaboli t e s of SC-11800 (Ethynodiol Diacetate) in a Human Subject", Searle Laboratories Progress Report.
Root, A. and Chow, R., Searle Laboratories, personal c o m i c a t i o n . Pasini, R. and Gavazzi, G., J. Pharm. Sci. -' 58 872-4 (1969). 93, Keay, G. R., Analyst - 28 (1968).

24.

25.

26.

27- Seul, C. J., Searle Laboratories, personal cornmunication.

28.

Jack, M., Searle Laboratories, personal comunicat ion. Brown, V., Searle Laboratories, personal communication.
278

29.

ETHYNODl OL DIACETATE

30.

Fernandez, A. L., and Noceda, V. T., J. Pharm. Sci. -9 58 740 (1969).

31. Wood, N . , Searle Laboratories, personal comunication.

32.
33.

Smith, B., Searle Laboratories, personal comunicat ion.

Simard, M. B. and Lodge, B. A., J. Chromatog. 51, 517 (1970).

279

FLUDROCORTISONE ACETATE

Klaus Florey

KLAUSFLOREY

CONTENTS

1 .
2.

3. 4 . 5.

6.

7.

a.
9.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, color, Odor Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Solubility, Dissolution, Partition Coefficient 2.9 Crystal Properties Synthesis Stability, Degradation Drug Metabolism 5.1 Pharmacokinetic 5.2 Metabolic Products 5.3 Microbiological Transformations Methods of Analysis 6.1 Elemental Analysis 6.2 Direct Spectrophotometric Analysis 6.3 Colorimetric Analysis 6.4 Polarographic Analysis 6.5 Chromatographic Analysis 6.51 Paper 6.52 Thin Layer 6.6 Bioassay 6.7 Other Determination in Body Fluids and Tissues. Determination in Pharmaceutical Preparations References

282

F LUDROCORTISON E ACETATE

Description 1.1 Name, Formula, Molecular Weight Fludrocortisone Acetate is 9a-fluorollf3, 17a,21-trihydroxy-4-pregnane-3,2O-dione, 21acetate: also 9a-fluorohydrocortisone acetate: 9a-fluoro-17-hydroxycortisone 21-acetate;gafluorocortisol 21-acetate; fluodrocortisone 21-acetate; fluohydrisone, 21-acetate, fluohydrocortisone 21-acetate, SQ 9321.
1.

21 HzOCOCH3 L O

2 3H3 lF06 '

M.W. 422.48

1.2 Appearance, Color, Odor Fludrocortisone Acetate is a white crystalline, odorless substance. Physical Properties 2.1 Infrared Spectra Mesley' has reported four polymorphic forms and their infrared spectra. Form A - as received from the British Pharmacopoeia. Form B - evaporation of chloroform solution at room temperature followed by heating at looo for 15 minutes. Form C - (amorphous) usually obtained by evaporation of chloroform or acetone solution
~

2.

KLAUS FLOREY

a t room t e m p e r a t u r e . Form D - may be o b t a i n e d by s p o n t a n e o u s c r y s t a l l i z a t i o n from f o r m C. T h e s e forms g i v e t h e f o l l o w i n g c h a r a c t e r i st i c a b s o r p t i o n peaks ( c m - l ) Form A: 1412 1339 1274 1239 ( s h ) 1022 958 940 819 777 680 Form B: 1418 1339 1272 1226 1195 1020 958 945 868 782 678 Form C: 1418 ( s h ) 1267 1236 1198 1023 959 936 870 782 680 Form D: 1406 1344 1267 1232 1198 1020 955 942 869 678

The i n f r a r e d f r e q u e n c i e s of m o d i f i c a t i o n s a n d s o l v a t e described by Kuhnert-Brandstaetter and G a s s e r 2 (see a l s o S e c t i o n 2.9) a r e p r e s e n t e d i n T a b l e 1. Table 1 9a-Fluorohydrocortisone Acetate Frequencies ( c m - l ) CIO and C l C 1738,1716,1651,1629 (w)

Form Modification I

OH

3440 3350 I1 3500 (sh) 3460 V 3510 3370 3300 ( s h ) VI 3525 3500 (w) 3350 Methyl a c e t a t e 3510 solvate 3320 3230 ( s h ) 3515 Ethyl a c e t a t e solvate 3360

1756,1720,1650,1617 1760,1745,1730,1721 1650,1611 ( w ) 1761,1750,1730,1647

1761,1748,1738,1722 1643 1759,1738,1725,1652

284

FLUDROCORTISONE ACETATE

Benzene s o l v a t e

3500 3320

1761,1749,1736,1721, 1644

D i m ethylformamide s o l v a t e

3360 1740,1721,1660,1624 3320 ( s h )

The i n f r a r e d spectrum of S q u i b b House S t a n d a r d ( b a t c h #48004-001) i n f i g u r e 1 r e p r e s e n t s m o d i f i c a t i o n I. (Form A ) 3 N u c l e a r Magnetic Resonance Spectrum The NMR spectrum of f l u o d r o c o r t i s o n e a c e t a t e is presented i n f i g u r e 2. The s t r u c t u r a l d a t a p r e s e n t e d in T a b l e 2 a g r e e w i t h t h e a s s i g n e d s t r u c t u r e 4 . The two h y d r o x y l p r o t o n s a t C 1 1 and C 1 7 exchange w i t h d e u t e r i u m .
2.2

Table 2
NMR S p e c t r a l Assignments of SQ 9321a

Proton a t c4

C h e m ic a 1 S h i f t , 6 (ppm)

c-11
C- 18

c-19 c-21 c-21

C-21 Acetate Ox(Exchangeab1e)

5.64 4.10 0.77 1.49 4*78 5.06

s
b

s
s

ABq;J=17.0 H z

2.10 5.00 b, 5 . 4 3 s b= b r o a d

a = DMs0-d~ s= s i n g l e t ABq = AB q u a r t e t

285

Figure 1. Infra Red Spectrum of Fludrocortisone Acetate (Squibb House Standard batch 48004-001) from KBr/Chloroorm. 1nstrument:Perkin Elmer 21.

Figure 2. NMR Spectrum of Fludrocortisone acetate (batch 76682) in deuterated DMSO (1nstrument:Varian XL-100).

KLAUS FLOREY

U l t r a v i o l e t Spectrum max 238 nm; F r i e d a n d Sabo5 r e p o r t e d c C = 16,800 i n e t h a n o l . 2 . 4 Mass Spectrum The l o w - r e s o l u t i o n mass s p e c t r u m o f SQ 9 , 3 2 1 (see f i g u r e 3 ) shows t h e e x p e c t e d M+ a t m/e 422. C o r t i c o s t e r o i d s g e n e r a l l y show fragment a t i o n p a t t e r n s r e s u l t i n g from t h e loss o f D-ring s u b s t i t u e n t s (cf A n a l y t i c a l P r o f i l e s , Triamcinolone, Triamcinolone Acetonide, Triamcinolone 16, 17-diacetate) . I n a d d i t i o n , f l u o r i n a t e d steroids a l s o h a v e f r a g m e n t a t i o n pathways i n v o l v i n g the loss o f HF. Thus, t h e f r a g m e n t a t i o n pathways shown below d e p i c t t h e losses o f t h e s e g r o u p s .

2.3

m / e 422 M+

m/e

363

-C0CH20Ac

m/e

362 34 2

m/e

292

m/e
m/e
301

1-HF

303

H2 m / e 344

m/e 283
The base p e a k of m/e

42 i s from t h e a c e t y l p o r t i o n of t h e 21-acetate. Although n o t a s i n t e n s e a s t h o s e from A-ring d i e n o n e s , t h e m / e 121-123 (CgHg-110)and t h e m / e 135-137 (C Hll-130) i o n s s u p p o r t t h e p r e s e n c e o f t h e A - r i n g enone group. The mass s p e c t r u m i s c o n s i s t e n t w i t h t h e proposed s t r u c t u r e 6 .

288

Figure 3. Low r e s o l u t i o n mass s p e c t r u m of F l u d o r c o r t i s o n e Acetate. ( S q u i b b s t a n d a r d batch 48004-001) I n s t r u m e n t : AEI-MS-902.

KLAUS FLOREY

2.5

Optical Rotation Solvent +143O Chloroform +127O Acetone +149O Dioxane +145- 15O0Dioxane

Ldi~

Ref 5 5 7 8

2.6

M e l t i n q Range Ref 5 7 8

233-234' 230 ( decomp. ) 220-233O ( decomp. )

I t was n o t e d 5 t h a t o c c a s i o n a l samples s t a r t e d t o m e l t a t 2 0 5 - 2 0 8 , r e s o l i d i f i e d and e v e n t u a l l y m e l t e d a t 226-228 (See s e c t i o n 2 . 1 0 ) . The m e l t i n g b e h a v i o r by h e K o f l e r method h a s been described a s follows :

A t 210 d r o p l e t s s t a r t t o form.

The r e s i d u a l c r y s t a l s grow t o g r a i n s , s q u a r e s , and hexagons t h a t f i n a l l y a g g r e g a t e t o a mosaic. Three o r f o u r d i f f e r e n t forms a r e produced a t 1600 i n t h e glassy solidified m e l t . The b u l k c o n s i s t s of long s t a l k e d s p h e r u l i t e s of Form I1 t h a t m e l t a t 208-212O and l e a f y , p a r t i a l l y f a n l i k e r a d i a t e s p h e r u l i t e s o f Form I11 t h a t m e l t a t 205-208O and e x h i b i t low-order i n t e r f e r e n c e c o l o r s . Form I V appears only r a r e l y a s fibrous-twisted s p h e r u l i t e s . The m e l t becomes brown i n c o l o r . The e u t e c t i c t e m p e r a t u r e w i t h p h e n o l p h t h a l e i n i s 202O. (For t h e m e l t i n g b e h a v i o r o f p o l y m o r p h i c forms see a l s o s e c t i o n 2.9. ) D i f f e r e n t i a l Thermal A n a l y s i s Squibb S t a n d a r d ( b a t c h 48004-001) e x h i b i t s a s h a r p endotherm a t 23OoC1O. 2.7

290

FLUDROCORTISONE ACETATE

2.8

Solubility''

I n water: 0 . 0 4 mg/ml; i n acetone 5 6 mg/ml; i n chloroform 20 mg/ml; i n ether 4 mg/ml. The d i s s o l u t i o n b e h a v i o r of c r y s t a l l i n e f l u d r o c o r t i n s o n e a c e t a t e and i t s p e n t a n o l and e t h y l a c e t a t e l g o l v a t e s were s t u d i e d by Shef t e r and The i n i t i a l d i s s o l u t i o n rates of t h e Higuchi s o l v a t e w e r e s i g n i f i c a n t l y h i g h e r t h a n t h e nons o l v a t e d form. Flynn determined t h e p a r t i t i o n c o e f f i c i e n t between e t h e r and w a t e r a s 45.7.i3

C r y s t a l Properties The o p t i c a l c r y s t a l l o g r a p h i c p r o p e r t i e s of f l u d r o c o r t i s o n e a c e t a t e (probably m o d i f i c a t i o n A) and f l u d r o c o r t i n one i t s e l f have been p r e s e n t e d as f o l l o w s by B i l e s T4

2.9

System Fludrocortisone Fludrocortisone acetate Fludrocortisone Orthorhombic Tetraqonal optic Orientation xxll c ~ a l l 22 I b 1

Crystal Habit Columnar

Optic
S iqn

Axial

00 Columnar Refractive Indexes a (w) B (5) Y 1.575 1.588 1.646

1.604

Fludrocortisone acetate

Photomicrographs of the two crystals also were presentedl4.

w t

II a
IIC

1.538

--

29 1

FLAUS FLOREY

The existance of several polymorphs has already been reported in section 2.1. A s many as six may exist, according to Kuhnert-Brandstaetter and Gasser15 but classification of the polymorphic conditions proved very difficult since except for modification I (m.p. 225-233OC), all other modifications show only very slight differences in their melting temperatures which are in the 205-215O range. Modification I11 and IV were not obtained in pure form. A further complication is the formation of solvates from a variety of solvents (see section 2.8). The powder X-ray diffraction pattern of fludrocortisone acetate (Form A ) is presented in table 3l6: Table 3 Re lative Relative .dd Intensity Intensity** 12.40 0.07 3.77 0.23 0.16 3.70 0.16 9.10 8.70 0.10 3.56 0.16 0.15 3.53 0.13 7.40 0.28 3.44 0.12 6.80 6.50 0.59 3.29 0.32 6.30 0.65 3.20 0.13 6.20 0.18 0.98 3.10 0.40 3.03 0.18 5.78 5.62 1.00 2.89 0.13 5.49 0.17 2.81 0.18 5.15 0.35 2.69 0.13 0.15 0.35 2.55 4.80 0.35 2.45 0.12 4.62 0.63 2.39 0.09 4.50 0.15 2.32 0.20 4.33 4.20 0.18 *d= (interplanar distance)ni'L 4.12 0.29 Asin 0 4.02 **based on highest intensit 3.94 0.12 of 1.00 Radiation: k a l an 3.83 0.17 Ka, Copper 1nstrument:Phillips

292

F LUDROCORTISON E ACETATE

Synthesis F l u d r o c o r t i s o n e A c e t a t e (Fig. 4 ) was f i r s t s y n t h e s i z e d by F r i e d and Sabo by t r e a t m e n t of 1 t h e epoxide 1 1 w i t h hydrogen f l u o r i d e . Compound VII h y d r o c o r t i s o n e a c e t a t e ) was found a s a byproduct of t h e r e a c t i o n 5 , 7 9 4 2 . Other approaches r e p o r t e d a r e i n t r o d u c t i o n of t h e 4-double bond v i a bromination ( I V and V ) , a l b e i t i n low y i e l d 1 7 , and osmium t e t r o x i d e o x i d a t i o n of t h e A17(20) p r e c u r s o r ( V I ) 1 8 . I t can be p u r i f i e d from V I I v i a t h e benzene adduct19. A method f o r t h e p r o d u c t i o n of dense c r y s t a l h a s been p a t e n t e d z 0 . I t can be d e a c e t y l a t e d t o 5. I t can s e r v e a s f l u o r o h y d r o c o r t i s o n e (11) s t a r t i n g m a t e r i a l f o r 9a-fluoro-prednisolone (cf. r e f . 2 1 ) . For m i c r o b i o l o g i c a l c o n v e r s i o n t o t r i a m c i n o l o n e see s e c t i o n 5 . 2 .
3.

4.

Stability-Degradation F l u d r o c o r t i s o n e a c e t a t e i s very s t a b l e a s a s o i i d . I n aqueous and a l c o h o l i c s o l u t i o n s t h e a - k e t o l s i d e c h a i n , a s i n a l l such c o r t i c o s t e r o i d q i s prone t o o x i d a t i v e rearrangement and degradat i o n a t a l k a l i n e pH*s.

I t h a s been r e p o r t e d 2 2 t h a t h y d r o c o r t i s o n e and p r e d n i s o l o n e , when exposed t o u l t r a v i o l e t l i g h t or ordinary fluorescent laboratory lighting i n a l c o h o l i c s o l u t i o n s , undergo p h o t o l y t i c d e g r a d a t i o n of t h e A-ring. Since fludrocortisone a c e t a t e h a s t h e same A-ring a s h y d r o c o r t i s o n e i t p r o b a b l y a l s o i s l a b i l e under t h e s e c o n d i t i o n s .

Drug Metabolism 5 . 1 Pharmocokinetics The d i s t r i b u t i o n i n r a t t i s s u e s and o r g a n s was s t u d i e d w i t h t r i t i u m l a b e l e d f l u d r o cortisone23. The k i n e t i c s of metabolism were determined i n man, dog, r a t , monkey, and g u i n e a
293

5.

CH2OCOCH3 H20COCH3 H20COCH3

c=o

CH2OCOCH3 CH20COCH3 I

c=o

CH20COCH3

I11 I11
w
\D

VI VI
C H OCOCH3
L O

\D

C H OCOCH3
L O

Br Br

Br

I R=COCH3

Br

I1 R=H V

I R=COCH3 VI I I1 R=H

VI I

Figure 4 Figure 4

FLUDROCORTISONE ACETATE

pig after I . V . and intraduodenal administration. Depending on species,50% or more of the steroid remained unchanged 30 minutes after adminis tration24. Fludrocortisone and it' s acetate had the same pharmocokinetic profile in dogs. The blood level reached a peak between 4 and 8 hours25. Silber26 found that introduction of fluorine at position 9 prolonged the plasma half-life and depressed urinary excretion after oral and I . V . administration to dogs as compared to hydrocortisone. Disappearance of fludrocortisone acetate after incubation with rat liver slices27-31 or perfusion of rat liver32 was a l s o studied. Metabolic Products After incubation of fludrocortisone W ith rat liver slices S ~ h r i e f e r s ~ ~ identified 9a-f luoro-5@-pregnan-l1@, 17a, 21-trihydroxy3,20-dione and 9a-fluoro-5@-pregnan-3@, ll@, 17a, 21-tetrahydroxy-20-one. There was no evidence for 5a-or 20-hydroxy metabolites. Bush and Mahesh3' identified the following metabolites in human urine: 9a-Fluoro-3a,lip, 17a,20,21 pentahydroxy-5@pregnane 9a-Fluoro-3a,ll~,17a,20,21 pentahydroxy-58pregnane 9a-Fluorotetrahydrocortisol 9a-Fluoroallotetrahydrocortisol 9a-Fluoro-20,20-dihydrocortisol 9a-F 1uorocortiso1 9a-Fluoro-ll@-hydroxyetiocholanolone
9a-Fluoro-11B-hydroxyandrostanone

5.2

Bush and M a h e ~ h ~ ~ the far greater noted proportion of 5a-(H) steroids than found with the halogen-free parent steroid. The expected 11-ketone steroids were completely absent.
295

KLAUS FLOREY

Microbiological Transformation The f o l l o w i n g m i c r o b i o l o g i c a l t r a n s f o r m a t i o n s o f f l u d r o c o r t i s o n e and i t ' s a c e t a t e have been r e o r t e d : 1 - h y d r o x y l a t i on35, l-dehydrogenation 9 6 -hydroxy 1 t i o n 38, 16-hydroxya l a t i ~ n ~ ~a l s o under T r i a m c i n o l o n e , (see A n a l y t i c a l P r o f i l e s o f Drug S u b s t a n c e s , V o l . l ) , For and 20-carbonyl r e d u c t i o n t o 20-hydroxy140. t r a n s f o r m a t i o n by mixed c u l t u r e s see r e f . 41.

5.3

',

6.

Methods of A n a l y s i s 6 . 1 Elemental A n a l y s i s Element % Theory 65.39 C 7.39 H 4.52 F

6.2

Repor t e d 3 65.32 7.26 4.50

Direct Spectrophotometric Assay The u l t r a v i o l e t a b s o r p t i o n band a t 238 nm ( s e e 2 . 3 ) i s due t o t h e a , @ u n s a t u r a t e d The a b s o r b a n c e i s u s e f u l k e t o n e of t h e A-ring. a s a measure of p u r i t y from e x t r a n e o u s m a t e r i a l s and h a s been s o used8, a l b e i t a t 242 nm. 6.3 C o l o r i m e t r i c Methods
A number of c o l o r i m e t r i c methods f o r

i d e n t i f i c a t i o n , d i f f e r e n t a t i o n from o t h e r s t e r o i d s and q u a n t i t a t i o n have been a p p l i e d t o fludrocortisone acetate. Based on r e a c t i o n w i t h t h e A-ring a r e t h e i ~ o n i a z i d k ~ ( ~ max 382 nm i n e t h a n o l ) and 2,4-dinitrophenylhydra~ine~~methods. Based on r e d u c t i o n of t h e d i h y d r o x a c e t o n e sidechain a r e the blue tetrazoliumYO, PorterA S i l b e r 3 O , and Nessler' s r e a g e n t 4 4 methods. b l u e chromogen ( k m a x 625 nm) i s produced b y reacting fludrocortisone acetate with 2,6-di-tert-butyl-p-cresol i n a l k a l i n e s o l u t i o n45

R e a c t i o n s w i t h a p h e n o l , hydroquinone,phosphorics u l f u r i c a c i d m i x t u r e (amber c o l o r ) 4 6 , p - n i t r o so296

FLUDROCORTISONE ACETATE

d i m e t h y l a n i l i n e ( 2max 650 nm) and a s u l f u r i c acid, f r u c t o s e , c y s t e i n e m i x t u r e ( 2 max 548 nm) have been d e s c r i b e d . The l a s t r e a c t i o n h a s a l s o been u s e d t o d e t e r m i n e s g s i d u a l f l u d r o c o r t i n s o n e i n fermentation broths. Chromo ens are a l s o formed i n c o n c e n t r a t e d s u l f u r i c 53 and p h o s p h o r i c acids. 5 1 Polarpgraphic Analysis CohenJL s u b j e c t e d f l u d r o c o r t i s o n e t o f p o l a r a g r a p h i c r e d u c t i o n i n dime t h y 1 ormamide and found two r e d u c i n g waves:
6.4

Wave 1
E 1/2

Wave 2
2.10

(Volts vs. mercury p o o l anode) Id (Diffusion current constant) n (Apparant number of e l e c t r o n s t r a n s f e r r e d

1.66
1.4
0.0 3

1.8
0.69

6.5

Chromatographic A n a l y s i s 6.51

P a p e r Chromatographic A n a l y s i s For p a p e r chromatographic s y s t e m s , see Table 4 .

297

Table 4 System #
1 2 3

Solvent System

4 5
6

7 8

10

11
12

Formamid/Chloroform Methanol/Water/Benzene 1:1:2 Methanol/Water/Ethylacetate/ Benzene 25:25:2.5:47.5 Propylene glycol/Toluene Benzene/Formamide Toluene/Heptane, Methanol/Water 5:5:7:3 Benzene/Methanol/Water 2:l:l Petroleum ether (b.p. 100-120) Toluene/Methanol/Water 67:33 :85: 15 Benzene/Ethanol/Water 2:1:2 Toluene/Petr. ether (b.p. 30-60), Methanol/Water 12:8:13:7 Benzene/Petr. ether (b.p 90-looo), . Methanol/Water 5 5 7 3 ::: Methyl isobutyl ketone/Formamide 20:l

Developing Time Rf Values (hrs) 18 4 4 96

Ref.

33 33 33 29 53 53 53 53 54 54 54 55

--

---5 2- 1/2

0.27 0.9

0.9

0.35

2-1/2
2-1/2

0.18
0.87

FLUDROCORTISONE ACETATE

The following detection systems have been reported: Detection Systems Ref.

u.v.

Tetrazolium Phosphoric acid fluorescence 2,4 Dinitrophenylhydrazine Tollen's Reagent Isonicotinic acid hydrazide

33 33,34 33 53 53

55

System #12 can be used to separate fludrocortisone acetate (Rf 0 . 8 7 ) from fludrocortisone (Rf 0.68) and 16a-hydroxyfludrocortisone (Rf 0 . 3 0 ) . It can be used for the quantitative determination of fludrocortisone acetates5 by dissolving the ground tablets in dimethylformamide, spotting approx 100 mcg. on filter paper impregnated with formamide-methanol 20:80, developing with methyl isobutylketone-formamide 20:1, elution, reaction with isonicotinic acid hydrazide and determination of the absorbance at 4 1 5 nm against a standard, usinz6the genera1 procedure of Roberts and Florey

Thin Layer Chromatographic Analysis Experience with the thin layer chromatography of fludrocortisone acetate is summarized in Table 5.

6.52

299

Table 5 Rf or "running distance" values (for explanation of individual values, see below)
Sys tem

1
0.87
I

a
0.48

b 0.42

Fludrocortisone Acetate Fludrocortisone S y s tem Fludrocortisone Acetate Fludrocortisone

w 0

1.031.14 0.24 0.33 I1

0.46

1.16 2.6 0.62 0.63 0.70 0.01 I11 IV

--

--

0.31

--

VI
0.90 0.76

-0.49

--

-0.29

-0.71

0.55

--

System 157: Kieselguhr G plate; Dichloroethane/methylacetate/water 2: 1: 1 : Spray reagent:Alkaline 2,5-diphenyl-3(4-styrylphenyl)tetrazolium solution; ''Running distances" values related to cortisone acetate = 1 . 0 0 ; Systems A-E58:Kieselguhr GF 254 plates; Spray reagent:Tetrazolium blue: "Running distance" values: A,B,C,E related to hydrocortisone acetate=1.00 D related to hydrocortisone = 1.00 Solvent systems: A- 1,2-Dichloroethane:methanol:water 95:5:0.2 B- 1,2-Dichloroethane:2-methoxyethyl acetate:water 80:20:1 C- Cyc1ohexane:ethylacetate:water 25:75:1 D- Stationary phase: 20% v/v formamide in acetone Mobile phase:Chloroform:ether:water 80:20:0.5 E- Stationary phase: 25% v/v formamide in acetone Mobile phase:Cyclohexane:tetrachloroethane:water 50:50:0.1

FLUDROCORTISONE ACETATE

S y s t e m s a-c59: K i e s e l g u h r G p l a t e s : S p r a y r e a g e n t : T e t r a z o l i u m b l u e . V a l u e s g i v e n a r e Rf values. S o l v e n t systems: a methylene c h 1 o r i d e : t o l u e n e 60:40 b - methylene c h l o r i d e : t o l u e n e 50:50 c - c h l o r o f o r m : t o l u e n e 25: 75

Systems I 1760: S i l i c a g e l p l a t e s , Spray reagent: Vanillinp e r c h l o r i c a c i d sprayed over tetrazolium reagent. V a l u e s g i v e n a r e Rf v a l u e s . S o l v e n t system I - E t h y l a c e t a t e I1 Methylene ch1oride:dioxan: water I11 - C h l o r o f o r m - e t h e r - w a t e r (80:25:0.5) on formamide plate I V - A m y l a c e t a t e - a c e t o n e 1:1 V - Ether

System ~ 1 ~ 5 , S i l i c a g e l GF P l a t e , U.V. d e t e c t i o n o r e l u t i o n and r e a c t i o n w i t h N y d r a z i d . S o l v e n t : Ether-dimethylformamide, a c e t o n e , I n t h i s system A8~14-hydrom e t h a n o l 88:8:2:2. c o r t i s o n e a c e t a t e h a s an R v a l u e of 0 . 8 3 i n relation t o fludrocortisone acetate. 6.6 Bioassay
A s e n s i t i v e b i o a s s a y i s b a s e d on t h e

u r i n a r y Na+/K+ r a t i o , e x p r e s s e d a s p e r c e n t o f t h e c o n t r o l v a l u e a f t e r i n j e c t i o n of f l u d r o c o r t i s o n e a c e t a t e i n t o adrenalectomized rats61.

30

FLAUS FLOREY

6.7 Other Bismuth oxidation to the corresponding 17-ketosteroid has a l s o been used as the basis for an analytical method62.
Determination in Body Fluids and Tissues References mentioned earlier, can be summarized as follows: Thin Layer Chromatography Paper Chromatography Colorimetric Bioassay
8.

7.

References : 2 29,33,34 25,30,31,49 61

Determination in Pharmaceutical Preparations The following references specifically mention analysis in pharmaceuticals. Paper Chromatography Colorimetric References : 55 8,45,47

302

FLUDROCORTISONE ACETATE

9.
1 .

References R. J. M e s l e y , S p e c t r o c h i m i c a A c t a 2 2 , 8 8 9 (1966). M. K u h n e r t - B r a n d s t a e t t e r a n d P. G a s s e r , Microchem. J. 1 6 , 577 ( 1 9 7 1 ) . B. T o e p l i t z , T G 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. M. S . P u a r , The S q u i b b I n s t i t u t e f o r Medical Research, P e r s o n a l Communication. J. F r i e d a n d E . F. S a b o , J. Am. Chem. SOC. 76, 1 4 5 5 ( 1 9 5 4 ) a n d i b i d . 79, 1130 ( 1 9 5 7 ) . A . I . Cohen, The 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. R. F. Hirschmann,R. M i l l e r , J. Wood and R. E . J o n e s , J. Am. Chem. SOC.7 8 , 4 9 5 6 ( 1 9 5 6 ) . James B. Kottemann, Drug Standards 26, 38 (1958). M. K u h n e r t - B r a n d s t a e t t e r , E. J u n g e r a n d A . K o f l e r , Microchem. J. 53, 1 0 5 ( 1 9 6 5 ) . H. J a c o b s o n , The 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. The Merck I n d e x , 8 t h E d i t i o n 1968. E . S h e f t e r a n d T. H i g u c h i , J. Pharm. S c i . 52, 7 8 1 ( 1 9 6 3 ) . G. L . F l y n n , J. Pharm. Sci. 60, 3 4 5 ( 1 9 7 1 ) . J. A . B i l e s , J. Pharm. S c i . 50, 4 6 4 ( 1 9 6 1 ) . M. K u h n e r t - B r a n d s t a e t t e r a n d P. G a s s e r , Microchem J. l , 577 ( 1 9 7 1 ) . . 6 Q. Ochs, The 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. J. E l k s , G. H. P h i l l i p s a n d W. F. W a l l , J. Chem. SOC. 1 9 5 8 , 4001. J. A . Hogg a n d F. H. L i n c o l n J r . , U.S. P a t e n t 2,875,200 (1959) K.G. F l o r e y a n d J. F r i e d , U . S . P a t e n t 2,809,977 (1957). R. P. G r a b e r a n d C. S . S n o d d y , u . S. P a t e n t 2,957,013 (1960).

2. 3.
4.

5. 6. 7. 8. 9.

10.

11.
12.

13. 14. 15. 16. 17.

18.

19. 20.

303

KLAUS FLOREY

21.

22.

23. 24. 25. 26. 27. 28. 29. 30.

31.
32, 33, 34. 35.

36. 37. 38.

F r i e d , K. F l o r e y , E . F. Sabo, J. H. Herz, R. Restivo,A. Borman and F. M. S i n g e r , J. Am. Chem. SOC. 77, 4181 ( 1 9 5 5 ) . W. E . Hamlin, T. C h u l s k i , R. H. Johnson and J. G. Wagner, J.Arn. Pharm.Assoc., SCi. Ed, 49 253(1963) and D. R. B a r t o n and W. C. T a y l o r , J . A m . Chem.Soc. 80, 2 4 4 ( 1 9 5 8 ) , J . Chem. SOC. 1958 2500. H. Wenzl, A . Garbe, H, Nowak, Arzneim.Forschung 1971, 1123. H. Wenzl, Arzneim. -Forsch. 1971,1127. H. Wenzl, A . Garbe, H. Nowak, Arzneim.Forsch. 1971,1115. R. H. S i l b e r and E . R. Morgan, C l i n . Chem. -3 2 170(1956). G. M. Reaven, E n d o c r i n o l o g y 57, 5 8 0 ( 1 9 5 5 ) . E. M. Glenn, R. 0. S t a f f o r d , S. C . L y s t e r and B. J. Bowman, E n d o c r i n o l o g y 61,128 (1957). H. S c h r i e f e r s , W. Korus, and W. D i r s c h e r l , A c t a E n d o c r i n o l . 26, 331 ( 1 9 5 7 ) . J.H.U.Brown and A . Anason, E n d o c r i n o l o g y 62, 103 ( 1 9 5 8 ) . W. Korus and H. L. Krdskemper, K l i n . Wochschr. 38, 938 (1960). H. S c h r i e f e r s and W. Korus, Z . P h y s i o l . Chem. 318, 2 3 9 ( 1 9 6 0 ) . H. S c h r i e f e r s , J. P h y s i o l . Chem. 324, 188 ( 1 9 6 1 ) . I. E. Bush and V. B. Mahesh, Biochem. J. 93,236 (1964). W. J. McAleer, M. A . Kozlowski, T. H. S t o u d t and J. M. Chemerda, J. Org. Chem. 23, 508 ( 1 9 5 8 ) . C . J. S i h , Biochim. Biophys. Acta 62,541 (1962). G. M. S h u l l , U. S. P a t e n t 2 , 7 7 6 , 9 2 7 ( 1 9 5 7 ) . L. L. Smith, J. J. Goodman, H, Mendelsohn, J. P. Dusza and S . B e r n s t e i n , J. Org. Chem. 26,974 ( 1 9 6 1 ) .
A.

J.

304

F LUDROCORTISONE ACETATE

39.

40. 41. 42.

43. 44. 45.

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

55.

56. 57. 58.

R. W. Thoma, J. F r i e d , S. Bonanno, and P. Grabowich, J. Am. Chem. SOC. 79, 4818 ( 1 9 5 7 ) . L. L. Smith, T . F o e l l and T. J. Goodman, B i o c h e m i s t r y L, 353 ( 1 9 6 2 ) . D. Y. Ryu, B. K. Lee, R. W.Thoma and W. E . Brown, B i o t e c h n o l , Bioeng. 1969,1255. N. L. Wendler, R. P . G r a b e r , C. S . Snoddy Jr and F. W. B o l l i n g e r , J. Am. Chem. SOC. 79,4476 ( 1 9 5 7 ) . L. L. Smith and Th. F o e l l , Anal. Chem. 31. 102 ( 1 9 5 9 ) . C. Monder and A . White, E n d o c r i n o l o g y E , 159 ( 1 9 6 1 ) . E. P. S c h u l z and J. D. N e u s s , Anal. Chem. 29,1662 ( 1 9 5 7 ) . E I v a s h k i v , J. Pharm. S c i . 5 1 , 6 9 8 ( 1 9 6 2 ) . (1960; J. V e r d i e r , Ann. Pharm. Franc.18,795 C . A . 55, 14826g ( 1 9 6 1 ) . C. J. S i h , S. C. Pan and R. E. Bennet, Anal. Chem. 32, 6 6 9 ( 1 9 6 0 ) . E . I v a s h k i v , Anal. Chem. 33,1051 ( 1 9 6 1 ) . L. L. S m i t h and W. H. M u l l e r , J.Org.Chem. - 960 ( 1 9 5 8 ) . 23 W. J . N o w a c z i n s k i and P. R. S t e y e r m a r k , Can. J. Biochem. and P h y s i o l . 3 4 , 5 9 2 ( 1 9 5 6 ) . A . I . Cohen, Anal. Chem. 3 5 , 1 2 8 ( 1 9 6 3 ) . L. M. R e i n e k e , Anal. Chem. 2 8 , 1 8 5 3 ( 1 9 5 6 ) . L. L. Smith, Th. F o e l l , R. deMaio and M. Halwer, J. Am. Pharm. Assoc. S c i . Ed. 48, 528 ( 1 9 5 9 ) . H. R. R o b e r t s , The S q u i b b I n s t i t u t e f o r Medical R e s e a r c h , P e r s o n a l Communication (1967). H. R. Roberts and K. F l o r e y , J. Pharm. S c i . , 51,794(1962). X H a l l , J. Pharm. Pharmacol. S u p p l . 1 , 6 9T (1964) C. J. C l i f f o r d , J. V. W i l k i n s o n and

305

KLAUS FLOREY

16,
59.

60. 61. 62,

J. S. Wragg, J. Pharm. Pharmacol. S u p p l . 11T (1964). D. S o n a n i n i , R. H o f s t e t t e r , L. Anker and H. Mdhlemann, Pharm. Acta. 40, 302 (1965). M. S. Moss and H. J. Rylance, J. Pharm. Pharmacol. l8, 1 3 (1965). J. G. L l a u r a d o , K l i n . Wochschr. 34 669 (1956). H. S c h r i e f e r s and W. Korus, J. P h y s i o l . Chem. 313,66 (1958).

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 July 1972.

306

FLURAZEPAM HYDROCHLORIDE

Bruce C. Rudy and Bernard Z. Senkowski

Chemistry reviewed by R. I. Fryer.

BRUCE C. RUDY A N D BERNARD Z . SENKOWSKI

INDEX Analytical Profile

Flurazepam Hydrochloride

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 2.21 Proton Spectrum 2.22 19F Spectrum 2.3 U1 traviolet Spectrum 2.4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 Differential Scanning Calorimetry 2.9 Thermal Gravimetric Analysis 2.10 Solubility 2.11 X-ray Crystal Properties 2.12 Dissociation Constant Synthesis Stability Degradation Drug Metabolic Products Methods of Analysis 6.1 Elemental Analysis 6.2 Fluorine Analysis 6 . 2 1 Organically Bound Fluorine Analysis 6.22 Free Fluoride Analysis 6.3 Thin Layer Chromatographic Analysis 6.4 Gas-Liquid Chromatographic Analysis 6.5 Polarographic Analysis 6.6 Direct Spectrophotometric Analysis 6.7 Colorimetric Analysis 6.8 Fluorimetric Analysis 6.9 Titrimetric Analysis

3.

4.
5. 6.

7 . Acknowledgement 8. References

308

F LURAZEPAM HYDROCHLORIDE

1. D e s c r i p t i o n
Name, Formula, Molecular Weight Flurazepam h y d r o c h l o r i d e is 7-chloro-1-(2[diethylamino] e t h i l ) -5~(o-fluorophenyl)-1,3-dihydro-2_TI-l,4benzodiazepin-2-one d i h y d r o c h l o r i d e .
1.1

C21H23C1FN30.2HC1 1.2

Molecular Weight:

460.83

Appearance, Color, Odor Flurazepam h y d r o c h l o r i d e o c c u r s a s a n o f f - w h i t e t o yellow, n e a r l y o d o r l e s s , 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 Spectrum The i n f r a r e d spectrum of flurazepam h y d r o c h l o r i d e is p r e s e n t e d i n F i g u r e 1 (1). The spectrum was measured with a Perkin-Elmer 621 Spectrophotometer i n a K r p e l l e t B g g c o n t a i n i n g 1 . 0 m of flurazepam hydrochloride/300 m of KBr. The assignments f o r t h e c h a r a c t e r i s t i c bands i n t h e i n f r a r e d spectrum a r e l i s t e d i n Table I ( 1 ) . Table I I n f r a r e d Assignments f o r Flurazepam Hydrochloride Frequency (cm-l) Characteristic of

3066
2500
1683 1560 and 1483 748

a r o m a t i c CH s t r e t c h i n g vibrations h y d r o c h l o r i d e of t e r t i a r y amine C-0 s t r e t c h i n g v i b r a t i o n s aromatic r i n g 4 a d j a c e n t H ' s on phenyl r i n g

309

&I &I-

smcu-0-

a-aIc--

a-m-t--

?:
310

m-

FLURAZEPAM HYDROCHLORIDE

2.2

Nuclear Magnetic Resonance Spectrum (NMR) 2.21

P r o t o n Spectrum The p r o t o n NMR s p e c t r a shown i n F i g u r e 2 were r u n on a J e o l c o 60 M z NMR u s i n g t e t r a m e t h y l s i l a n e as H an i n t e r n a l r e f e r e n c e ( 2 ) . The flurazepam h y d r o c h l o r i d e spectrum, F i g u r e 2A, was o b t a i n e d by d i s s o l v i n g 59.0 m of g sample i n 0.5 m l of methanol-d4. The s p e c t r a l a s s i g n m e n t s are l i s t e d i n T a b l e I1 ( 2 ) . The s o l v e n t peak f o r methanold4 o c c u r s a b o u t 3.27 ppm and i n t e r f e r e s w i t h t h e a s s i g n ments i n t h a t r e g i o n . T h e r e f o r e , t h e spectrum of f l u r a z e Pam b a s e (54.2 mg/0.5 m l CDC13), shown i n F i g u r e 2B, w a s determined and t h e s p e c t r a l assignments p r e s e n t e d i n T a b l e I1 ( 2 ) . 19F Spectrum The 1yF spectrum shown i n F i g u r e 2C w a s o b t a i n e d w i t h a J e o l c o C-60 HL i n s t r u m e n t w i t h a 19F module c r y s t a l modified t o a f r e q u e n c y of 56.446 MIz. Two hundred m of flurazepam h y d r o c h l o r i d e were d i s s o l v e d i n 0 . 5 m l of g methanol c o n t a i n i n g CC13F as t h e i n t e r n a l r e f e r e n c e ( 2 ) . The spectrum c o n s i s t s of a q u i n t e t a t -108 ppm. The c h o i c e of CC13F as t h e i n t e r n a l r e f e r e n c e a l o n g w i t h t h e a s s i g n ment of -108 ppm i s i n accordance w i t h Bovey ( 3 ) . T a b l e I1

2.22

NMR Assignments f o r Flurazepam and Flurazepam Hydrochloride

No. of

Proton

Protons

Chemical S h i f t (ppm) 1.40 ~3.42

Multiplicity Triplet (JH,-H~ = 7Hz) M 1 i p1 t u t e Triplet

Flurazepam Hydrochloride a 6 b
C

6 2
311

'~4.50

BRUCE C. RUDY AND BERNARD 2. SENKOWSKI

Figure 2
A.
C.

B.

NMR Spectrum of Flurazepam Hydrochloride NMR Spectrum of Flurazepam Base 19F NMR Spectrum of Flurazepam Hydrochloride

312

FLURAZEPAM HYDROCHLORIDE

Pro t o n d e f Flurazepam Base a b

C

No. o f Pro t o n s

Chemical S h i f t (pprn)

Mu1t i p 1 i c i t y Sing1 t e Mu1t i p l et Triplet

(J H ~ - H ~ = 7.5Hz)

2 2* 7

~4.50 4.92 7.30-8.15 0.95 2.50 3.47-4.55 3.76-4.85 6.95-7.80

6 6 2 2

d
f

Quartet ( J H ~ - H ~ =5~2) 7. M 1 i p l et u t Two sets of doublets (J = 11 Hz) Mu1t i p l e t

I9F Spectrum of Flurazepam Hydrochloride -108 Q u i n t e t

*Also
2.3

any H20 p r e s e n t i n methanol-d4

U l t r a v i o l e t Spectrum (UV) When t h e W spectrum of flurazepam h y d r o c h l o r i d e w a s scanned from 450 t o 210 nm, t h r e e maxima and t h r e e minima were observed. The maxima a r e l o c a t e d a t 362 n m (E = 3.7 x lO3), 284 n m (E = 1.2 x 1041, and 239 n (E = m 2.8 x lo4). The m i n i m a o c c u r at 333 nm, 263 nm, and 219 nm. The spectrum shown i n F i g u r e 3 was o b t a i n e d from a s o l u t i o n of 1.006 m of flurazepam h y d r o c h l o r i d e f 1 0 0 m l of a c i d i f i e d g methanol (2.8 m l of c o n c e n t r a t e d H2SO4 d i l u t e d t o 100 m l w i t h anhydrous methanol) (4). F1uorescenc.e Spectrum The e x c i t a t i o n and emission s p e c t r a f o r f l u r a z e Pam h y d r o c h l o r i d e (1 mg/ml of methanol) a r e shown in F i g u r e 4 (5). One maximum appears i n t h e e x c i t a t i o n spectrum a t 378 n and one maximum i n t h e e m i s s i o n spectrum a t 492 nm. m

2.4

Mass Spectrum The mass spectrum of flurazepam h y d r o c h l o r i d e w a s o b t a i n e d u s i n g a CEC 21-110 mass s p e c t r o m e t e r w i t h an i o n i z i n g energy of 7 0 ev. The o u t p u t from t h e mass s p e c t m meter was analyzed and p r e s e n t e d i n t h e form of a b a r

2.5

313

314

tn a
w Iw
0

z 4 z

A 1 S N3lN I 1

315

ar a
.?I

k 0

7
t

si

fl

w
8
0
rw
0

316

F LURAZEPAM HYDROCHLORIDE

g r a p h , shown i n F i g u r e 5 , by a V a r i a n 1 0 0 MS d e d i c a t e d comp u t e r system. Due t o t h e e x t r e m e i n t e n s i t y of t h e b a s e peak a t m / e 86, t h e r e l a t i v e i n t e n s i t y of t h e peaks a t t h e h i g h e r mass u n i t s a r e v e r y weak. T h e r e f o r e , t h e peaks from m / e 1 5 0 up were s u b j e c t e d t o a t e n f o l d s c a l e e x p a n s i o n ( F i g u r e 5 - i n s e r t ) ( 6 ) . The p a r e n t peak (+)a t m / e 387 is due t o t h e free f l u r a z e p a m b a s e . The b a s e peak a t m / e 8 6 i s d u e t o t h e (C2H5)2NCH2 f r a g m e n t . The o t h e r m a j o r p e a k s can b e a t t r i b u t e d t o s t e p w i s e f r a g m e n t a t i o n o f t h e p a r e n t + i o n ; i . e . , M - (C2H5)zN = 315, 315 - CH2N = 287 ( 6 ) . 2.6 activity. M e l t i n g Range Flurazepam h y d r o c h l o r i d e m e l t s w i t h d e c o m p o s i t i o n w i t h i n a 5 O r a n g e between 208' and 218OC when t h e USP X V I I I Class Ia p r o c e d u r e i s u s e d ( 7 ) . D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) The t h e r m a l p r o p e r t i e s of f l u r a z e p a m hydrochloride i n t h e m e l t i n g r e g i o n a r e v e r y dependent on p r e v i o u s t h e r m a l h i s t o r y . Using a t e m p e r a t u r e program of 20C/min., t h e e x t r a p o l a t e d o n s e t of a n e n d o t h e r m i c t r a n s i t i o n o c c u r r e d a t 215 . a 0 C f o l l o w e d immediately by t h e e x o t h e r m i c t r a n s i t i o n d u e t o d e c o m p o s i t i o n a t 229.5OC ( F i g u r e 6 ) . A t a s c a n r a t e o f 10C/min., a small endothermic t r a n s i t i o n o c c u r s a t 203.3OC f o l l o w e d by sample d e c o m p o s i t i o n a t Due t o t h e sample i n s t a b i l i t y i n t h e r e g i o n o f 217.5OC. t h e m e l t , t h e AHf was n o t d e t e r m i n e d ( 8 ) . Thermal G r a v i m e t r i c A n a l y s i s (TGA) A TGA performed a t a s c a n r a t e of 10C/minute showed l i t t l e w e i g h t loss f o r f l u r a z e p a m h y d r o c h l o r i d e from ambient t o 190C. A w e i g h t l o s s amounting t o a b o u t 70% of t h e sample o c c u r r e d between 190 and 345OC (8). Solubility The s o l u b i l i t y d a t a f o r f l u r a z e p a m h y d r o c h l o r i d e o b t a i n e d by e q u i l i b r a t i o n f o r 20 h o u r s a t 25OC are g i v e n i n T a b l e 111 ( 9 ) . 2.lo 2.9 2.8 2.7 Optical Rotation Flurazepam h y d r o c h l o r i d e e x h i b i t s no o p t i c a l

3 17

Figure 6
DSC Scan for Flurazepam Hydrochloride

2 3

d 240 230 220 210 200 190 I80 170 I

TEMPERATURE 'C

318

FLURAZEPAM HYDROCHLORIDE

T a b l e I11 S o l u b i l i t y P r o f i l e f o r Flurazepam H y d r o c h l o r i d e Solvent S o l u b i l i t y (mg/ml)

3A al c o h o l benzene chloroform 95% e t h a n o l diethyl ether m e thano 1 p e t r o l e u m e t h e r (3Oo-6O0) 2-propanol water

28.3 0.4 11.1 2 60 0.2 340 0.2 14.6 >500

X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n of f l u r a z e Pam h y d r o c h l o r i d e i s p r e s e n t e d i n T a b l e I V (10). The i n s t r u m e n t a l c o n d i t i o n s a re g i v e n below. Instrumental Conditions General E l e c t r i c Model XRD-6 S p e c t r o g o n i o m e t e r Generator : Tube t a r g e t : Radiation : optics: 50KV-12-1/2 MA Copper Cu K, = 1 . 5 4 2 0.1' D e t e c t o r s l i t M.R. S o l l e r s l i t 3 Beam slit ' 0.0007 i n c h N i f i l t e r 4 take off angle ' Scan a t O.Zo 28 p e r m i n u t e Amplifier g a i n - 1 6 c o u r s e , 8.7 f i n e Sealed proportional counter t u b e and DC v o l t a g e a t plateau P u l s e h e i g h t s e l e c t i o n EL 5 v o l t s ; EU - o u t Rate meter T . C . 4 2000 C I S f u l l s c a l e C h a r t Speed - 1 i n c h p e r 5 minutes

2.11

Goniometer : Detector:

Recorder:

Samples p r e p a r e d by g r i n d i n g a t room t e m p e r a t u r e .

319

BRUCE C. RUDY AND BERNARD 2. SENKOWSKI

Table I V X-ray Powder D i f f r a c t i o n P a t t e r n of Flurazepam H y d r o c h l o r i d e 20 8.32 9.04 11.62 12.49 12.86 15.50 16.40 16.78 17 38 17.94 18.65 19.16 20.05 20.38 20.75 21.66 23.14 23.61 24.32 24.50 25.18 25.96 26.18 26.40 26.96 27.44 27.94 28.46 28.78 29.96 30.54 31.00
*d

d (8)

I/TE
3 20 37 15 1 1 62 1 1 9 7 5 50 100 26 20 19 12 82 39 40 36 98 12 33 24 12 15 18 5 5 19 4 15

28 31.40 32.36 32.92 33 - 3 0 33.88 34.24 34.87 35.20 35.77 36.18 37.28 37.72 38.17 38.78 39.47 40.18 40.86 41.28 41.86 42.20 42.94 43.58 44.24 44.70 46.06 47.00 47.52 48.32 49.08 50.00 50.26 50.84 nX 2 Sin 0

d (8)

IT / E
1 38 7 8 6 7 1 1 14 5 10 5 1 1 1 1 19
3

10.6 9.78 7.62 7.09 6.88 5.72 5.40 5.28 2.10 4.94 4.76 4.63 4.43 4.36 4.28 4.10 3.84 3.77 3.66 3.63 3.54 3.43 3.40 3.38 3.31 3.25 3.19 3.14 3.10 2.98 2.93 2.88

2.85 2.77 2.72 2.69 2.65 2.62 2.57 2.55 2.51 2.48 2.41 2.38 2.36 2.32 2.28 2.24 2.21 2.19 2.16 2.14 2.11 2.08 2.05 2.03 1.97 1.93 1.91 1.88 1.86 1.82 1.82 1.80

3 5 2 5
8 9 9 4 5 7 4 2 5 3 3 3 4

(interplanar distance)

**I/Io= 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)

320

FLURAZEPAM HYDROCHLORIDE

Dissociation Constant The pKa's for flurazepam were determined spectrophotometrically and found to be 1.90 5 0.05 and 8.16 5 0.05 (11). The apparent pKa2 has also been determined from the titration curve in a 2-propano1:water (1:l) mixture and found to be 7.0 5 0.1 ( 1 . In water, the trialkylamino 1) type compounds are stronger bases, on the average, by 0.9 pK units (11,12). Therefore, the estimated pKa2 in water would be 7.9 which is in good agreement with that found spectrophotometrically. Synthesis Flurazepam hydrochloride may be prepared by the reaction scheme shown in Figure 7. 7-Chloro-5-(o-fluorophenyl)-1,3-dihydro-2H-l,4-benzodiazepin-2-one is reacted with diethylaminoethyl chloride in the presence of sodium methoxide to yield flurazepam which is then converted to flurazepam hydrochloride by the addition of hydrochloric acid (13). A complete review of the chemistry of benzodiazepines by Archer and Sternbach presents several pathways to arrive at the basic benzodiazepine ( 1 4 ) . Stability Degradation When sealed amber ampuls with dilute solutions of flurazepam hydrochloride in 0.1N HC1, water, and 0.1N NaOH: 3A alcohol (1:l) were heated in a boiling water bath for one hour, the degradation products shown in Figure 8 were observed by thin layer chromatography (15). In 0.1N HC1 solution the main hydrolysis product was 5-chloro-2-(2diethylaminoethylamino)-2'-fluorobenzophenone hydrochloride. In aqueous solution the main degradation product was 7chloro-5-(o-fluorophenyl)-ly3-dihydro-2H-l,4-benzodiazepin2-one. Finally, in the 0.1N NaOH:3A alcohol (1:l) solution, the main degradation products were 5-chloro-2-(2diethylaminoethylamino)-2'-fluorobenzophenone ,and 2-chlorol0-(2-diethylaminoethyl)-9-acridone. When a solution of flurazepam hydrochloride in water in irradiated with light from a high pressure U.V. lamp for 3 hours, some hydrolysis to 5-chloro-2-(2-diethylaminoethylamino)-2'-fluorobenzophenone occurs ( 6 . Flurazepam hydrochloride, when stored 1) in well closed, light resistant containers, is quite stable.
4.

2.12

3.

321

Figure 7 S y n t h e s i s f o r Flurazepam Hydrochloride

H CI

CH2CH3
ClCH CH N(

CH2CH3

_____)

Naoc%

PHENYL )-I ,3-DI HYDRO- 2 H-1.4BENZODIAZEPINE- 2-ONE

7-CHLORO-5-(&FLUORO-

DIE THYLAMINOETHYLCHLORIDE

C I

K T=C H "
FLURAZEPAM FLURAZEPAM HYDROCHLORIDE

Figure 8 Major D e g r a d a t i o n P r o d u c t s f o r Flurazepam H y d r o c h l o r i d e i n Acid, Basic, and Aqueous S o l u t i o n s

&\
FLURAZEPAM

HYDROCHLORIDE

nw

$H2CHZ"C2H32
&?.ti2

bf

CI

a6
I

CH2CH2NlC2H&,

CI

fJ?n 8
CH CH N(C2H5)2 1 2 2 ACRIDONE

BENZOPHENONE

7-CHLORO-54 O-FLUOR& PHENYLkt3-DIHYDRO -2 HI.4-BENZWUZEPIN-2-ONE

BENZOPMENONE

BRUCE C. RUDY AND BERNARD 2. SENKOWSKI

5. Drug Metabolic Products The metabolic pathways of flurazepam in dog and man are shown in Figure 9 (17-22). Initially, five metabolites plus the intact drug were found in the urine of dogs. Four of these metabolites were identified as monodesethylflurazepam, didesethylflurazepam, flurazepam-N1-ethanol, and N1-desalkyl-3-hydroxyflurazepam (17,ZO). The fifth metabolite was tentatively identified as a phenolic derivative of N1-desalkyl-flurazepam by mass spectrometry (17). The major metabolite found in the dog urine is flurazepamN1-acetic acid (17,19). In human urine, the flurazepamN1-ethanol was the major metabolite along with smaller quantities of the mono- and didesethyl-flurazepam and N1desalkyl-3-hydroxyflurazepam (17-22).

6. Methods of Analysis
Elemental Analysis The results from the elemental analysis are listed in Table V (23). Table V Elemental Analysis of Flurazepam Hydrochloride Element
X Theory
% Found

6.1

C 54.74 H 5.47 N 9.12 F 4.12 c1 7.69 Cl-(ionic) 15.38

6.2 Fluorine Analysis

54.73 5.46 9.11 4.22 7.86 15.42

6.21 Organically Bound Fluorine Analysis There are several methods available to determine the amount of carbon-bonded fluorine. One of the earlier methods employed the Schoniger Combustion technique followed by thorium nitrate or cerous chloride titration using sodium alizarin sulfonate or murexide as the indicator (24). With the advent of good specific ion
324

Figure 9 Metabolic Products of Flurazepam Hydrochloride

H

NI-DESALKYLFLURUEPAY

FLURAZEPAM Nl-ETHAWL

Cl & o :"
&F

\
c f ga
OH N I -DESALKYL FLURAZEPAM PHENOL

ac=;.I.
N-C-0

CHpCOOH I

8 "

N,-DESALKYL-3HYDROXYFLURAZEPAM

FLURAZEPAY NI-ACETIC ACD

BRUCE

C. RUDY AND BERNARD 2.SENKOWSKI

e l e c t r o d e s , methods were developed t o l i b e r a t e t h e bound f l u o r i n e and d i r e c t l y measure t h e f l u o r i d e c o n c e n t r a t i o n . The r e a g e n t , s o d i u m b i p h e n y l , followed by o x i d a t i o n w i t h hydrogen p e r o x i d e , is used t o l i b e r a t e t h e o r g a n i c a l l y bound f l u o r i n e i n flurazepam h y d r o c h l o r i d e . A f l u o r i d e i o n s p e c i f i c e l e c t r o d e i s used, i n t h e p r e s e n c e of a highi o n i c - s t r e n g t h b u f f e r s o l u t i o n , f o r d i r e c t measurement of t h e l i b e r a t e d f l u o r i d e (25). The l a s t method t o b e p r e s e n t e d f o r t h e a n a l y s i s of t h e carbon-bonded f l u o r i n e i n flurazepam hydroc h l o r i d e i s 19F Nuclear Magnetic Resonance s p e c t r o m e t r y (2). A flurazepam h y d r o c h l o r i d e r e f e r e n c e s t a n d a r d and an i n t e r n a l s t a n d a r d , r e a g e n t g r a d e o-fluorobenzoic a c i d , a r e d i s solved i n methanol and t h e I 9 F spectrum o b t a i n e d and i n t e g r a t e d . From t h i s d a t a an i n t e r n a l s t a n d a r d f l u o r i n e conversion f a c t o r c a n b e c a l c u l a t e d and used t o d e t e r m i n e t h e amount of f l u o r i n e p r e s e n t i n a sample of flurazepam h y d r o c h l o r i d e t h a t i s r u n i n a s i m i l a r manner (26).

6.22 F r e e F l u o r i d e Analysis
The d e t e r m i n a t i o n of any f r e e f l u o r i d e p r e s e n t i n flurazepam h y d r o c h l o r i d e b u l k samples i s c a r r i e d o u t by d i r e c t measurement u s i n g a f l u o r i d e s p e c i f i c i o n e l e c t r o d e . The measurements a r e made i n an a c e t a t e b u f f e r s o l u t i o n (pH 5 . 3 ) . The e l e c t r o d e r e s p o n s e w a s found t o b e l i n e a r throughout t h e working range of 0.08 t o 0.20 m of F-/100 m l of s o l u t i o n ( 2 7 ) . g

Thin Layer Chromatographic Analysis (TLC) S e v e r a l TLC systems f o r t h e s e p a r a t i o n of flurazepam h y d r o c h l o r i d e from its m e t a b o l i t e s and s i m i l a r s t r u c t u r e d compounds are g i v e n i n Table V I . I n each c a s e F t h e sample i s s p o t t e d on a s i l i c a g e l G p l a t e * which is allowed t o develop i n a s a t u r a t e d t a n k u n t i l t h e s o l v e n t f r o n t h a s ascended a b o u t 15 cm. The p l a t e is t h e n removed, a i r d r i e d , and viewed under shortwave and longwave U.V. radiation.

6.3

*I f

t h e sample s o l u t i o n is t o o a c i d i c , an a r t i f a c t a p p e a r s a t t h e p o i n t of a p p l i c a t i o n due t o t h e quenching of t h e phosphor i n t h e s i l i c a g e l G p l a t e by t h e a c i d . F

326

FLURAZEPAM HYDROCHLORIDE

Table VI Rf Values for Flurazepam in Various Developing Solvents Solvent System diethyl ether:diethylamine (75:2) methylene ch1oride:ethyl ether: methano1:conc. ammonium hydroxide (240:360:8:3) ethyl acetate:conc. ammonium hydroxide (200:1) ethyl acetate: ethanol:conc. ammonium hydroxide (100:10:0.3)
benzene:methanol:glacial (9:1:1)

- Value Reference R
0.6
28

0.2

28 20 20
20

0.14
0.38 0.26 0.00, 0.05*

acetic acid 20

chloroform:acetone (17 :3)

*When
6.4

the plate is developed two times in the same system

Gas-Liquid Chromatographic Analysis (GLC) The acid hydrolysis of blood extracts containing flurazepam and its metabolites has been used by deSilva et al. (29) to prepare the respective benzophenones as volatile derivatives for gas chromatography. This method is an adaptation of the method developed for GLC of diazepam and its metabolites (30). When the benzophenones were chromatographed at 21OoC on a 2 feet x 114 inch column containing 2% Carbowax 20M-TPA, they showed an excellent response to detection by electron capture which was linear between 10 and 40 ng. The main disadvantage of hydrolysis to the benzophenone is the lack of specificity for a given benzodiazepine. A method recently published by Sine et al. (31) for chromatographing flurazepam directly, utilizes a 3 feet x 2 mm glass column packed with 3.8% SE-30 on Chromosorb W (AW-DMCS, 80-100 mesh). The GLC is equipped with a hydrogen flame ionization detector and the column temperature is about 23OoC. The patient's serum is adjusted to pH 7.4 and extracted with chloroform. The chloroform is evaporated, the residue is dissolved in acidic methanol (1 ml HCl/liter methanol) and chromatographed.

327

BRUCE C. RUDY AND BERNARD 2. SENKOWSKI

This method w i l l s e p a r a t e flurazepam from diazepam and chlordiazepoxide. P o l a r o g r a p h i c Analysis P o l a r o g r a p h i c a n a l y s i s of flurazepam hydroc h l o r i d e h a s been c a r r i e d o u t i n Britton-Robinson B u f f e r The halfwave p o t e n t i a l o c c u r s a t -0.78 V.versus a t pH 4.4. a s i l v e r / s i l v e r c h l o r i d e r e f e r e n c e e l e c t r o d e and is prop o r t i o n a l t o c o n c e n t r a t i o n . T h i s wave is a t t r i b u t e d t o the r e d u c t i o n of t h e azomethine (,C=N) f u n c t i o n a l group and v a r i e s w i t h pH ( 3 2 ) . Direct Spectrophotometric Analysis Direct s p e c t r o p h o t o m e t r i c a n a l y s i s is used t o determine t h e q u a n t i t y of flurazepam h y d r o c h l o r i d e p r e s e n t i n capsules. A q u a n t i t y of t h e c a p s u l e c o n t e n t s is atc u r a t e l y weighed and t h e flurazepam is e x t r a c t e d i n t o a c i d i f i e d methanol ( s e e s e c t i o n 2 . 3 ) . The methanol s o l u t i o n is f i l t e r e d and a p p r o p r i a t e s u b d i l u t i o n s made t o y i e l d a f i n a l s o l u t i o n c o n t a i n i n g 1 . 0 m of flurazepam hydrog c h l o r i d e p e r 100 m l of a c i d i f i e d methanol. The absorbance of t h i s s o l u t i o n along w i t h a s o l u t i o n of flurazepam hydroc h l o r i d e r e f e r e n c e s t a n d a r d s i m i l a r l y prepared is measured v e r s u s a c i d i f i e d methanol a t t h e 239 NO maximum. From t h i s d a t a t h e c o n c e n t r a t i o n of flurazepam h y d r o c h l o r i d e i n t h e c a p s u l e s is c a l c u l a t e d ( 3 3 ) . C o l o r i m e t r i c Analysis Flurazepam h y d r o c h l o r i d e forms a i o n - p a i r complex w i t h bromocresol green i n a p 5 . 3 b u f f e r . This c o l o r e d H complex is e x t r a c t e d i n t o chloroform and i t s absorbance measured a t t h e 415 nm maximum. A p l o t of c o n c e n t r a t i o n v e r s u s absorbance is l i n e a r from 0 t o 2.5 m of flurazepam g h y d r o c h l o r i c p e r 100 m l of chloroform ( 3 4 ) . 6.8 F l u o r i m e t r i c Analysis
A f l u o r i m e t r i c a n a l y s i s f o r t h e d e t e r m i n a t i o n of flurazepam h y d r o c h l o r i d e and i t s m e t a b o l i t e s i n blood and

6.5

6.6

6.7

u r i n e h a s been d e s c r i b e d by d e S i l v a and S t r o j n y ( 2 0 ) . This a s s a y i n v o l v e s s e l e c t i v e e x t r a c t i o n i n t o d i e t h y l e t h e r from blood b u f f e r e d t o pH 9 o r u r i n e made b a s i c w i t h NaOH, then back-extracted i n t o 4N H C 1 , and hydrolyzed t o t h e r e s p e c t i v e benzophenones. The benzophenones are t h e n c y c l i z e d t o t h e 9-acridone d e r i v a t i v e s i n dimethylformamide

328

FLURAZEPAM HYDROCHLORIDE

i n t h e p r e s e n c e of K2CO3. These d e r i v a t i v e s a r e s e p a r a t e d by TLC, e l u t e d from t h e s i l i c a g e l , and t h e i r f l u o r e s c e n c e determined i n methanol:O.lN HC1 ( 4 : l ) . T h i s method a l l o w s q u a n t i t a t i o n i n t h e r a n g e of 0.003 t o 10.0 mcg of compound/ ml of blood o r u r i n e ( 2 0 ) . T i t r i m e t r i c Analysis Flurazepam h y d r o c h l o r i d e i s assayed by d i s s o l v i n g about 0 . 6 gm of sample i n g l a c i a l a c e t i c a c i d , adding exc e s s mecuric acetate, and t i t r a t i n g w i t h 0.1N p e r c h l o r i c a c i d i n g l a c i a l a c e t i c a c i d . The end-point is determined p o t e n t i o m e t r i c a l l y u s i n g a g l a s s - c a l o m e l e l e c t r o d e system. Each m l of 0.1N p e r c h l o r i c a c i d is e q u i v a l e n t t o 23.04 m g of C21H23C1FN30*2HC1. Acknowledgment The a u t h o r s would l i k e t o thank D r . P. S o r t e r and t h e S c i e n t i f i c L i t e r a t u r e Department as w e l l as t h e Research Records Department of Hoffmann-La Roche I n c . f o r t h e i r h e l p i n t h e l i t e r a t u r e search f o r t h i s Analytical Profile.

6.9

7.

329

BRUCE C. R U D Y A N D BERNARD Z . SENKOWSKI

8.

References

1. Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. 2 . Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. 3. Bovey, F. A . , NucZear Magnetic Resonance Spectroscopy, Academic Press, New York City, pp. 211-214. 4 . Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. 5. Boatman, J . , Hoffmann-La Roche Inc., Personal Communication. 6. Benz, W., Hoffmann-La Roche Inc., Personal Communication. 7. United S t a t e s Pharmacopeia XVIII, 935 (1970). 8 Moros, S . , Hoffmann-La Roche Inc., Personal . Communication. 9. MacMullan, E., Hoffmann-La Roche Inc., Personal Communication. 10. Hagel, R. B., Hoffmann-La Roche Inc., Personal Communication. 11. Toome, V. and Raymond, G., Hoffmann-La Roche Inc., Unpublished Data. 12. Gutbezahl, B. and Grunwald, E., J . Amer. Chem. Soc., - 559 (1953). 75, 13. Sternbach, L. H., Archer, G. A., Earley, J . V., Fryer, R. I., Reeder, E., Wasyliw, N., Randall, L., and Banziger, R., J. Med. Chem., 8_, 815 (1965). 14. Archer, G. A. and Sternbach, L. H., Frem. Review., 68, 747 (1969). 15. Senkowski, B. Z., Hoffmann-La Roche Inc., Unpublished Data. 16. Fryer, R. I., Hoffniann-La Roche Inc., Unpublished Data. 17. Schwartz, M. A . , Vane, F. M., and Postma, E., J . Med. Chem., 1 ,770 (1968). 1 18. Usdin, E., PsychopharmaeoZ. BUZZ., 5, 4 (1970). 19. Schwartz, M. A. and Postma, E., J . .?harm. S c i . , 59, 1800 (1970). 20. deSilva, J . A . F. and Strojny, N., J . Pharm. S e i . , - 1303 (1971). 60, I 21. Randall, L. O., I n t . Symp. Benzodiazepines, Sum., Milan, Italy, 1971:Z.
330

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22.
23. 24.

25. 26.

27.
28. 29.

30.
31. 32. 33. 34.

S c h w a r t z , M. A . , I n t . Symp. Benzodiazepines, Sum., M i l a n , I t a l y , 1971:3. S c h e i d l , F., Hoffmann-La Roche I n c . , P e r s o n a l Communication. Steyermark, A , , Quantitative Organic MieroanaZysis, 2nd Ed., Academic Press, New York, N. Y., 1 9 6 1 , pp. 326-332. J o n e s , B. C . , Heveran, J . E . , and Senkowski, B . Z., J . Pharm. S c i . , 60, 1036 (1971). Rudy, B. C. and Senkowski, B. 2. " A n a l y t i c a l P r o f i l e of F l u o r o u r a c i l " , a c c e p t e d f o r p u b l i c a t i o n i n AnaZyticaZ ProfiZes of Drug Substances, V o l . 2, 1972. J o n e s , B. C . and Heveran, J . E . , Hoffmann-La Roche I n c . , Unpublished Data. Hochhauser, L . , Hoffmann-La Roche I n c . , Unpublished Data. d e S i l v a , J. A . F . , Bader, G . , and Kaplan, J . , Hoffmann-La Roche I n c . , Unpublished Data. d e S i l v a , J. 4. F . , S c h w a r t z , M. A . , S t e f a n o v i c , V., Kaplan, J . , and D'Arconte, L . , AnaZ. Chem., 36, 2099 (1964). S i n e , H. E . , McKenna, M. J . , Law, M. R., and Murray, M. H . , J . Chromatog. S c i . , 1 297 ( 1 9 7 2 ) . 0 L e v i n , M. , Hof fmann-La Roche I n c . , Unpublished Data. G u a s t e l l a , J . , Hoffmann-La Roche I n c . , Unpublished Data. Houghton, R. E . , Hoffmann-La Roche I n c . , Unpublished Data.

331

IODIPAMIDE

Hyam Henry Lerner

HYAM HENRY LERNER

Table of Contents
1.

Description
1.1 1.2

Name, Formula, Molecular Weight Appearance, Color, Odor

2.

Physical Properties

2.1

Spectra
2.11 2.12 2.13 2.14

Infrared Spectra Nuclear (Proton) Magnetic Resonance U l t r a v i o l e t Spectra Mass Spectrometry

2.2

Crystal Properties 2.21 2.22 2.23 2.24

Differential Thermal Analysis Thermal Gravimetric Analysis Melting Range X-Ray Powder Diffraction

2.3

Solution Data 2.31 2.32 2.33 2.34 2.35 2.36 2.37 Solubility Apparent Molecular Weight i n Solution Isotonicity pKa p H Index of Refraction Phys icochemical Data

3.
4.

Synthesis Stability P u r i f i c a t i o n and Analysis f o r Impurities 5.1 5.2 5.3 5.4 5.5 Gel F i l t r a t i o n Complexometric Methods of Separation Countercurrent Distribution Free Iodine and Free Halide Free Amino Compounds
334

5.

IODIPAMIDE

5 .

(Cont'd.) 5.6 5.7

Free Adipic Acid Determination of Water and Conditions f o r Drying

6.

Methods of Analysis 6.1 6.2 6.3 6.4 6.5 6.6 Elemental Analysis I d e n t i f i c a t i o n Tests Direct Spectrophotometric Analysis Organically Bound Iodine Polarograpliy Chromatographic Analysis 6.61 6.62 6.63 6.7 6.8 Paper Chromatography Thin-Layer Chromatography E l e c t r o p h o r e t i c Analysis

X-Ray and B-Particle Dispersion Methods Flame Photometry

7. 8.

Drug Metabolism
References

335

HYAM HENRY LERNER

1 .

Description 11 . Name, Formula, Molecular Weight

Iodipamide is N,N'-adipyl bis (3-amin0-2~4~6 triiodobenzoic acid) Chemical Abstract listings are under the heading benzoic acid, 3,3' (adipyldiimino) bis [2,4,6 triiodol. Other derived chemical names are adipic acid di(3-carboxy-2,4,6 triiodoanilide; N,N'-di- (3-carboxy-2,4,6triiodopheny1)-adipamide and 3,3'- (adipoy1diimino)-bis t2, 4,6-triiodobenzoic acid].

Among the generic and trivial names for this compound are iodipamic acid and adipiodon. Common trade names are Biligrafin and Cholografin. Iodipamide was officially recognized in "National Formulary XI." United States Pharmacopoaeia XVIII continues this name in a monograph for Meglumine Iodipamide Injection.

COOH

YOOH

C20H1416N206 12 . Appearance, Color, Odor

Mol. Wt. 1,139.7

Iodipamide is a white, odorless and tasteless crystalline powder1*11,12. The disodium salt has a sweet, metallic taste followed by a bitter aftertastd2.

336

IOD I PAMI DE

2.

Physical Properties 2.1 Spectra

2.11

Infrared Spectra

The s p e c t r a of iodipamide i n Figures l a and l b were determined on a Perkin-Elmer Model 621 g r a t i n g i n f r a r e d spectrophotometer. Samples of iodipamide were d i s persed i n a potassium bromide p e l l e t or i n mineral The following s p e c t r a l assignments were made by ToeplitzS0 on t h e spectrum obtained from t h e sample d i s persed i n mineral o i l (Figure l b ) :

cm-1
3200 2500, 1900 1690 1610 1530 1280

Assignment
N-H N-H and OH of amido acid C=O o f carboxyl group C=O o f amide secondary amide C-OH of carboxyl group

are The s p e c t r a shown i n Figures l a and dissimilar. A explanation was advanced by Toeplitzi, n who suggested t h a t iodipamide might be r e a c t i n g with potassium bromide.
Herrmannl published an i n f r a r e d spectrum obt a i n e d on a potassium bromide dispersion t h a t agrees qualit a t i v e l y with t h e spectrum i n Figure la. Neudert and RUpke3 published an i n f r a r e d spectrum t h a t does not agree with t h e spectrum i n e i t h e r Figure l a or Figure lb. 2.12

Nuclear (Proton) Magnetic Resonance Spectrum

w a s determined on a Varian XL-100

The MlR spectrum of iodipamide i n Figure 2 NMR spectrometer2 at ambi31O). The sample was dissolved e n t probe temperature i n deuterated dimethylsulfoxide containing tetramethylsilane as an i n t e r n a l reference (Me4Si = 0 ppm). Spectral assignments of t h e peaks are recorded i n Table I.

(z.

337

w m

Figwe la.

Infrared Spectnxn of Iodipamide, Squibb Iot 03122, from KBr pellet. Instrun-ent: PE 1 621 Infrared spectm&oixm=@r

WAVELENGTH

m )

Figure lb, Infrared Spectrum of Iodipamide, Sguibb Lot 03122, from m i n e r d l o i l mull. 1 621 Infrared Sp-olxmeter Instnarent: PE -

zdw
1000

Figure 2.

NMR Spectrum of Iodipamide, Squibb Lot 03122 in lXSO-%. Instrumnt: V a r i a n - m 0 0 NMR Speetrmeter

IODIPAMIDE

In deuterated water and deuterated sodium hydroxide, the peak at 69.86 w a s absent, indicating exchange of the amine proton. The carboxylic acid protons were not located, probably because of hydrogen bonding. High-field methylene resonance indicated the absence of other groups attached t o the methylene groups. Table I
NMR Spectral Assignments

Assignment -cI12-(312-

Chemical S h i f t (Ppm , 6)

No. of Protons 4

17 .8

(s)

-C-CHzaromatic

fi

-NH

-COW

2 3 (m) .6 8 3 (3) .3 9 8 (s) .6 not located

s = singlet; m = nultiplet
2.13 Ultraviolet Spectra

The following u l t r a v i o l e t s p e c t r a l data have been reported f o r iodipamide : Solvent 0.01N NaOH 01 - a H . NN O 0.lT NaOH 0,lR KOH Metbol Methanol 0.15M N a C l 0.lSR Phosphate Bufrer (pH 5 8 .)

Amax, n m
238 236 236 237 238 239 238 238

Reference

70,700 72,000 71,800 72,400 68,000 71,800 73,200 73,200

48 48
1 4 8 4 5 5

Neudert and RtJpke3 reported the E value of n maximum, t o be m 74,700. Ostrow and LevyS reported t h e i r data i n terms of absorbance per micromole of iodine. Sodium iodide, which peaks a t 226 NB, has the same absorbance p e r rnicromole of
the disodium s a l t i n methanol, a t the 239
341

H Y A M HENRY LERNER

iodine as does iodipamide, which suggests t h a t t h e u l t r a v i o l e t absorption of iodipanide r e s u l t s from t h e presence of iodine chromophores

2.14

Mass Spectrometry

No w l e c u l a r ion is observed f o r iodipamide because of i t s low v o l a t i l i t y and because of t h e thermal degragation of t h e compound. Per-trimethyl s i l y l a t i o n by Funke yielded a compound with a molecular ion of m/e 1428, consistent with t h e replacement of four protons by four trimethyl s i l y l groups. The s t r u c t u r e and major fragment a t i o n p a t t e r n are depicted below:
COOH
YOOH

NH-c -(C%),0

C -HN

Other fragments t h a t have been found are due t o l o s s o f I o r ti1 and include:

m/e 1428+m/e m/e 7 4 2 4 m/e m/e 728 3 m/e m/e 728+m/e

2.2

1301 614 601 600

+ I

+ HI
+ I

+ HI

Crystal Properties 2.21 D i f f e r e n t i a l Thermal Analysis

Valenti7 determined t h e DTA of iodipamide on a Du Pont 900 Themnoanalyzer a t a temperature rise of 15O per min. A s i n g l e ondothem a t 308O and a s i n g l e
342

1 0 PAM I DE 01

exotherm a t 314" were detected. . duced i n Figure 3 2.22

The thermogram is repro-

Thermal Gravimetric Analysis

Valenti' determined t h e TGA o f iodipamide on a Du Pont Thennogravimetric Analyzer. When t h e compound was heated a t a rate of 15" per minute under nitrogen sweep, no weight l o s s w a s observed below 250, 2.23 Melting Range

W i l l i reported a melting range f o r i o d i ~ ~ ~ 308", with decomposition, as determined pamide of 306.5 on a Thomas-Hoover Ca i l l a r y Melting Point apparatus. Priewe and Rutkowskilj reported t h e meltin! range t o be 306O 308*, with decomposition. Herrmann reported t h a t t h e compound decomposes above 280O.

Hoevel-Kestermann and Muhlemng determined the melting range on a Kofler Microblock (Reichert) and reported t h e melting range t o be 289 290, with decomposition. This l a t t e r value appears t o be i n e r r o r , when compared with t h e previously c i t e d DTA d a t a (Section 2.21) and measurements made with t h e c a p i l l a r y melting point apparatus.

2.24

X-Ray Powder Diffraction

OchslO obtained t h e X-ray powder d i f f r a c t i o n spectrum of iodipamide on a P h i l l i p s X-Ray Powder D i f fractometer, a t a voltage o f 45 kv and a current of 15 ma. The sample w a s i r r a d i a t e d by a copper source a t 1.54A. Diffraction d a t a f o r Squibb Lot 03122 are recorded i n Table
11.

343

T. OC (CHROMEL: ALUMEL)*

W E IlS,""CIIO*

I" .. */

roll I C l l l COIIIIILIId.

figure 3.

IICA Themxqram of IOaipami.de, Squibb Iat 03122.

Instrumnt:

Du Pant 900 ThermDanalyzer

IODIPAMIDE

Table I1
~

X-Ray Powder Diffraction Pattern o f Iodipamide, Squibb Lot 03122

d (Ao)*
8.80 7.40 5.70 5.5 5.10 4.46 4.40 4.29 4.23 4.16 4.09 3.93 3.80 3.67 3.64 3.47 3.44 3.37 3.30 3.25 3.17 3.14 3.10 2.99 2.96 2.92 2.90 2.84 2.66 2.61 2.56 2.51 2.43 2.32 *d = ( i n t e r p l a n a r d i s t a n c e )

Relative I n t e n s i t y * *
0.25 0.18 0.13 0.19 0.19 0.34 0.60 0.51 0.39 0.34 1.00 0.69 0.15 0.25 0.18 0.16 0.16 0.10 0.15 0.56 0.28 0.39 0.40 0.29 0.43 0.20 0.20 0.14 0.22 0.17 0.25 0.17 0.13 0.13 2 sin 0

nX

** Based on h i g h e s t i n t e n s i t y of 1.00
345

where X = 1.539A

HYAM HENRY LERNER

2.3

Solution Data 2.31 Solubility

The following data were reported f o r t h e s o l u b i l i t y of f r e e acid of iodipamide a t room temperature:

Solubility (mrg /lo0 m l ) Solvent Acetone Ethanol, 95% Ether Chloroform Methanol Water 0.1N sodium hyxroxi de n-hexane Benzene Propylene glycol 0.1N hydrochloric acrd Ethylene glycol Tet rahydro f uran Tet rahydrofurfury1 a1coho1 Ref.12 200

Ref.8 a t 20

100 800 ins o lub 1 e

440 46

Refa7
<20 <20 <20 <20
<20 5,240 <20

insoluble -

8,200

<20
<20

Neudert and R8pke8 a l s o reported t h e solub i l i t y of iodipamide i n acetamide, urethan and phenol, a t the melting point of the solvents, t o be 3 g, 0.5 g, and 0.3 g per 100 g of solvent, respectively.

The s o l u b i l i t y a t 20 of t h disodium and dilithium salts of iodipamide were reported :

346

IODIPAMIDE

S o l u b i l i t y (g/100 ml) Solvent Disodium Salt 350 14 4 4 Dilithium Salt 450

65 3

Water Methanol Tetrahydrofuran Tetrahydro f u r f u r y l alcohol

2.32

Apparent Molecular Weight i n Solution

According t o Neudert and R(lpke8, iodipamide forms micelles and has soap-like properties. Colloidal sol u t i o n s contain molecular aggregates with an apparent molecular weight 16 times that of the empirical formula of iodipamide. 23 .3 Isotonicity

A 14.6% (w/v) s o l u t i o n of t h e disodium s a l t of iodipamide (0.1233M) is i s o t o n i c with physiological s a l t solutionl2*24.

2.34

pKa

The pKa of t h e f r e e a c i d of iodipamide w a s This value may be a composite of r e p ~ r t e d ~or be ~3.5. t ~ pKa1 and pKa2, s i n c e both d i s s o c i a t i o n constants can be expected t o be similar i n value.
2.35

pH

The p of a 1% H suspension of iodipamide w a s Herrmannl proposed limits f o r a satreportedg t o be 3.95. urated s o l u t i o n o f 3.5 3.9.

Iodipamide n e u t r a l i z e d with sodium hydroxide

w a s reported8 t o have a pH of 7.4.

2.36 Index of Refraction

The refractive index of iodipad.de at 21,5O, i n methanol12, is given i n Table 111. Neudert and R1Spke8 reported t h e refractive index of the disodium s a l t , a t a
341

H Y A M HENRY LERNER

concentration of 35 e l l 0 0 i n water, and measured with t h e D-line of sodium, t o be 1.4016.

Table I11

Refractive Index of Iodipamide a t 21.5OC i n Methanol12

$/lo0 m l
"D -

0.0 0.116 0.263 0.348 0.445

2.37

1.3278 1.3283 1.3288 1.3292 1.3294

Physicochemical Data The freezing-point depression (-AT)

of d i s s o c i a t i o n (a), and osmotic pressure (Po) of t h e disodium salt of iodipamide i n aqueous s o l u t i o n were reported8*12, and are recorded i n Table IV.
Table IV Physicochemical Data8,12 f o r t h e Disodium S a l t of Iodipamide :

, degree

Freezing-Point Depression (-AT), Degree of Dissociation (a), and Osmotic Pressure (Po) &/lo0 m l 2.0 5.0 10.0 14.6 21.6
3 .

Molarity 0.0170 0.0422 0.0844 0.1233 0,1825

-AT 0.095 0.226 0.425 0.557

0.720

a -

Po 1.13 2.69 5.04 6.64 8.56

1.00 0.94 0.85 0.72 0.56

Synthesis

Iodipamide is prepared by t h e r e a c t i o n of 2,4,6triiodo-3 aminobenzoic acid with adipic a c i d dichloride13. The former is dissolved i n chlorobenzene and heated t o 110 130%. The a d i p i c acid dicliloride is added dropwise,

348

IODIPAMIDE

r e s u l t i n g i n t h e evolution of hydrochloric acid. When t h e evolution of HC1 has ceased, t h e p r e c i p i t a t e d crude product is f i l t e r e d , with suction, while s t i l l hot. The crude prec i p i t a t e is washed with chlorobenzene, and t h e r e s i d u a l chlorobenzene is extracted by b o i l i n g with methanol. The p r e c i p i t a t e is dissolved i n c a u s t i c methanol, f i l t e r e d through charcoal, and p r e c i p i t a t e d with d i l u t e hydrochloric acid.
A a l t e r n a t e solvent f o r t h e reaction o f 2,4,6-trin iodo-3 aminobenzoic a c i d with a d i p i c acid d i c h l o r i d e is toluenel4.

'elz
0

COOH

C1-C-(CH,),-C-Cl II II

110"-130
C,H,Cl C -HN I

2 HCl

i
COOH COOH

NH

- C -(CH2),-

4.

Stability

Iodipamide is chemically s t a b l e a t room temperature. B extrapolation t o room temperature of t h e d a t a f o r a c t i y vation energy and frequency constant of the reaction between 70 and l l O o , t h e decomposition at t h e N-acyl bond Evolution of iodine w a s calculated t o be 0.1% i n 50 yr.33 a t 90 from very pure iodiparnide i n s o l u t i o n at p 9 is H less than 3% i n 75 hr. The presence of impurities such as under-iodinated compounds , enhances d e c o m p ~ s i t i o n ~ ~ . Formulated neutralized (pli 7.2) solutions containing iodipamide (192 mg/ml) sodium citrate (3.2 mg/ml) a and sodium e d e t a t e (0.28 mg/ml), show l i t t l e o r no l o s s of potency a f t e r storage f o r 5 yr. at room temperaturel5.
5.

P u r i f i c a t i o n and Analysis f o r Impurities

P u r i f i c a t i o n procedures used f o r analysis a r e de.) scribed under Cliromatographic Analysis (Section 66.

349

HYAM HENRY LERNER

5.1

Gel F i l t r a t i o n

Ostrw and Levys attempted t o i s o l a t e and p u r i f y iodipamide on 1.0- x 12.5-cm columns prepared from dextran gel (Sephadex G-10 and G-25, Pharmacia) and polyacrylamide gel (Bio-Gel P-2, Bio-Rad). Samples o f 0.2 m l , containing 0.3 t o 8.6 pmoles o f iodipamide, were e l u t e d with 0.15M sodium phosphate b u f f e r (pi4 5.8). The e l u t e d f r a c t i o n s rere moasured spectrophotometrically at 226, 238, and 255 nm. Results are shown i n Table V. Due t o adsorption of iodipamide on t h e gel, recoveries were not q u a n t i t a t i v e . Contaminants c l o s e l y r e l a t e d t o iodipamide were shown, by paper chromatography o f t h e iodipamide f r a c t i o n s , t o be present even after chromatography on Sephadex Gel G-10.
Table V Gel F i l t r a t i o n of Iodipamide P r o f i l e on Three Different Gels, with p 5.8 Phosphate H Buffer as Elution Solvent3 Component Iodipamide Sephadex Gel G-25 Sephadex Gel G- 10 e l u t e d a t void volume taili n g till 50 m l Bio Gel P-2 8-50 m l (peak a t 16 ml)

(peak a t 20 ml)
10-24 m l (peak a t 15 ml)

8-32 m l

Sodium Iodide

28-50 m l 11-25 m l (peak a t 37 ml) (peak a t 18 ml)

5.2

Complexometric Methods of Separation

Hentrich and P f e i f e r d 4 described methods f o r the p r e c i p i t a t i o n o f ten c o n t r a s t agents as t h e metallic s a l t s o r metallic complex salts. Iodipamic a c i d can be p r e c i p i t a t e d q u a n t i t a t i v e l y by s i l v e r n i t r a t e , cadmium s u l f a t e with thiourea, cadmium s u l f a t e with pyridine, copper s u l fate with thiourea, and copper s u l f a t e with pyridine. Chelatometric methods are a l s o described f o r t h e t i t r a t i o n of excess p r e c i p i t a n t , after separation of t h e p r e c i p i t a t e d salt by f i l t r a t i o n . The formulas of t h e p r e c i p i t a t e d salts and complexes, t h e i r molecular weights, melting ranges, and
350

IODIPAMIDE

t h e equivalent weight o f t h e iodipamide p r e c i p i t a t e d by 1 m l of 0.1M s o l u t i o n of t h e inorganic p r e c i p i t a n t are given i n Table

m.

Table VI S a l t Complexes of Iodipamic Acid34

Equ iv. Weight t o 1 m l of

Precip-

itant

Mol. Formula of Salt

M.W. of Salt

Melting Range

0.1M Preciprtant

CdS04Thiourea ig%ne CsSO4Thiourea

Cd[(NH2)2CSl4*- 1,554.7 C20H1216N206 Cd[(CsH N)4]*C2oH12f6N206 <Cu[(NH2)2CS l 2 ) 'C20H12IgN2'6 1,586.6 1,569.4

237O-238O 290 O 167O

0.1140 g 0.1140 g 0.0570 g

CuSO Pyrijine
5.3

[CU(C~H N)2]'1,359.5 ~20~1256~206

202O-203O

0.1140 g

Countercurrent D i s t r i b u t i o n

S t r i c k l e r e t a120 separated iodipamide and o t h e r c o n t r a s t agents from sera by countercurrent d i s t r i b u t i o n . They used a solvent system composed o f sec-butanol: dilute aqueous ammonia (1:l). Both a 30-tube manual procedure and a 200-tube automatic procedure are described.

--

5.4

Free Iodine and Free Halide

Free i o d i n e can be d e t e c t e d by b o i l i n g iodipamide with water f o r 2 min, f i l t e r i n g , and observing a blue c o l o r a f t e r treatment with s t a r c h . After a c i d i f i c a t i o n o f another p o r t i o n of f i l t r a t e with d i l u t e n i t r i c a c i d and treatment of it with s i l v e r n i t r a t e test s o l u t i o n , t h e presence o f free h a l i d e ions can be detected by t h e

35 1

HYAM HENRY LERNER

occurrence of opalescence o r turbidity1 a2

'.

Hartmann and RUpke33 quantitated free iodide by t i t r a t i n g potentiometrically with 0.001 N s i l v e r nitrate, under a protective cover of nitrogen.

5.5

Free Amino Compounds

Hartmann and RUpke33 determined iodinated impurit i e s having a free amino group, by a k i n e t i c method based on t h e more rapid r e a c t i v i t y of t h e s e impurities with elemental bromine i n acetic acid solution than is shown by iodipamide. Under these conditions, t h e free amino compounds q u a n t i t a t i v e l y s p l i t o f f iodine, which is then oxidized t o iodate by t h e bromine. After destruction of t h e excess bromine, t h e i o d a t e is reduced t o iodine and tit r a t e d with sodium t h i o s u l f a t e , t o a s t a r c h endpoint. Free-iodide compounds a l s o react with bromine and would give p o s i t i v e r e s u l t s by t h i s method. Hoevel-Kestermann and hluhlemann9 described a Bratton-Marshall colorimetric reaction f o r t h e detection of f r e e amino groups. Hartmann and R8pkd3 used the ErattonMarshall reaction t o q u a n t i t a t e f r e e amino compound impurities

56 .

Free Adipic Acid

Hoevel-Kestermann and Wlemanng described a thin-layer chromatographic procedure f o r adipic acid, after its cleavage from i o d i p m i c acid. This procedure is described i n Section 6.2 and can be adapted t o determine free a d i p i c acid.

57 .

Determination of Water and Conditions f o r h y i n g

Herrmannl dried iodipamide at 1 0 5 O f o r 4 hr. Hoevel-Kestermann and Muhlemanng d r i e d iodipamide over phosphorus pentoxide f o r 24 hr. In t h e "National Formulary XI,'@ water is determined by t h e Karl Fischer titrimetric method.

352

lODlPAMlDE

6.

Methods of Analysis 6.1 Elemental Analysis

Element

% Theory

% Reported25

n
N
6.2

21.075 1.238 66.806 2.458 8.422

21-16 1.35 67.28 2.30

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

Infrared (Section 2.11) , paper chromatography (Section 6-61), and thin-layer chromatography (Section 6.62) have been used t o i d e n t i f y iodipamide. S h a m o t i e n k ~i ~ e n t i f i e d iodipamide by b o i l i n g it d~ with t r i c h l o r o a c e t i c a c i d and a 5% aqueous s o l u t i o n of chloramine. Iodipamide gives a cloudy yellow s o l u t i o n with a yellow p r e c i p i t a t e .

The evolution of intense v i o l e t fumes o f l i b e r ated iodine can be observed by heating a sample of iodipamide over an open flame1*9a11.
I d e n t i f i c a t i o n of t h e bound m i n e group can be made by f i r s t a s c e r t a i n i n g t h e absence of free amino groups (Section 5.6) and then cleaving t h e molecule by refluxing i n base and repeating t h e Bratton-Marshall reactiong. Iodipamide may be hydrolyzed by refluxing with hydroiodic acid t o l i b e r a t e a d i p i c acid. The adipic acid can be extracted with e t h e r and chromatographed on s i l i c a gel G plates. The solvent system benzene:dioxane:acetic acid (65:25:29) w a s used. Adipic acid (Rf 0.64) can be det e c t e d v i s u a l l y by spraying t h e developed p l a t e with bromoc w s o l greeng. 6.3
2 2

Direct Spectrophotometric Analysis

The strong u l t r a v i o l e t band of iodipamide a t 238

n i n b a s i c s o l u t i o n and methanol (Section 2.13) can be m

The
353

used f o r d i r e c t spectrophotometric analyses4.

H Y A M HENRY LERNER

absorption band is s a i d t o r e s u l t from t h e i o d i n e chromoDetection and q u a n t i t a t i o n of e l u a t e s from #ores5. chromatographic s e p a r a t i o n s are e a s i l y accomplished by using this band f o r analyses.
6.4

Organically Bound Iodine

Under r e f l w conditions, t h e i o d i n e i n iodipamide can be reduced and replaced by hydrogen, generated by t h e r e a c t i o n of powdered z i n c and sodium hydroxide. The iodide is determined by t i t r a t i o n with standardized s i l v e r n i t r a t e i n a c i d s o l u t i o n i n t h e presence of tetrabromophenolphthalein e t h y l ester i n d i c a t o r solution1

Ates and Ama126 decomposed iodipamide with alka-

l i n e p e n ~ a n g a n a t ,28.~ After decoloration of t h e pennane~

ganate with sodium n i t r i t e and a c i d , they t i t r a t e d t h e l i b o r a t e d i o d i n e with 0.1N sodium t h i o s u l f a t e .

Yakatan and TuckenaanZg reviewed f o u r methods f o r decomposing c o n t r a s t agents t o l i b e r a t e o r g a n i c a l l y bound iodine: A. Parr bomb (fusion with sodium peroxide); B. alk a l i n e permanganate reduction; C. zinc-sodium hydroxide reduction; and D. oxygen f l a s k (Schbniger) combustion. Method D is recommended as a general technique f o r a l l i o d i nated organic compounds because of i t s r e p r o d u c i b i l i t y , s i m p l i c i t y , and r a p i d i t y . For compounds t h a t have a l l t h e iodine atoms ortho o r p a r a t o t h e e l e c t r o n e g a t i v e carboxy l i c acid on t h e aromatic ring, e g iodipamide, Method C (zinc-sodium hydroxide reduction$ is recommended. KrasnovaO recornended a modification of th oxygen-flask combustion method of Yakatan and hickermann 39 Hoevel-Kestermann and hhlemann9 reviewed t h r e e methods f o r l i b e r a t i n g o r g a n i c a l l y bound iodine i n c o n t r a s t agents: A. Parr banb (fusion with sodium peroxide); B. c a t a l y t i c reduction with sodium borohydride; and C. zincsodium hydroxide r e d u c t i o n . They recommend t h e sodium boroiiydride reduction because of i t s s i m p l i c i t y and s h o r t assay time. The sodium borohydride reduction w a s o r i g i n a l l y proposed by EgliS1.

354

IOD1PAM I DE

6.5

Polarography

V a s k e l i s et al.32 determined c o n t r a s t agents potassium chloride containing polarographically gelatin. Iodipamide y i e i d s a s i n g l e waveD with a half-wave p o t e n t i a l of - 1 . 2 ~ vs SCE. Results were q u a n t i t a t e d from prepared c a l i b r a t i o r c u r v e s .

irOm

6.6

Chromatographic Analysis 6.61 Paper Chromatography

Many descending paper chromatographic methods have been found s u i t a b l e f o r t h e i s o l a t i o n and det e c t i o n of iodipamide. These are summarized i n Table VII. Some of t h e references c i t e d give sample preparation techniques and methods of e l u t i n g t h e drug from t h e developed chromatogram t o permit q u a n t i t a t i o n by u l t r a v i o l e t spectrophotometry (Section 2.13) o r o t h e r means. Pileggi e t a1.22 described a method f o r separating 19 organic iodide compounds from blood serum.

--

Table VII Paper Chromatographic Systems f o r Iodipamide Solvent Sys t e m

I I1 I11 IV

Paper Whatman Whatmen Whatman Whatman Whatman Whatman

Method of Detection
1
4 1 3

Reference
35 36 5 26 39 26 39 22

VI

No. No. No. No. No. No.

3 3

A A B B#C B #C D

Solvent Systems

- n-butanol:lc ammonium hydroxide:ethanol (5:2:1) I1 - H20:n-butano1:ethanol (5:4:1) ;upper phase used for
I

development

lower phase t o e q u i l i b r a t e chamber.

355

HYAM HENRY LERNER

Table VII (Contd)

I11

- n-butanol:O.SN- upper phasehydroxide:ethanol:H20 ammonium (20:20:2:1); used f o r development,

- Methanol:2N acetic a c i d ( r a t i o of -sec-butano1:enmronia 4% (3:l)
A.
9.

lower phase + 20 m l o f upper phase used t o e q u i l i b r a t e chamber.

IV

- Ethanol:25% ammonia ( r a t i o o f s o l v e n t s not given)

solvents not given)

V
VI

Methods of Detection Long-wave u l t r a v i o l e t l i g h t . Spray with 10% ceric s u l f a t e and 5% sodium arsen i t e , both prepared i n 1 N s u l f u r i c acid37.

C.

Short-wave u l t r a v i o l e t l i g h t . Spray with mixture o f c e r i c ammonium s u l f a t e and arsenious acid, followed b spraying with 0.5% s o l u t i o n o f methylene bluei 2

D .

6.62

Thin-Layer Chromatography

Thin-layer chromatographic methods found s u i t a b l e f o r t h e separation and detection of iodipamide are summarized i n Table VIII. Some of t h e references c i t e d present sample preparation techniques and methods for e l u t ing t h e drug from t h e developed p l a t e t o p e r m i t q u a n t i t a t i o n by u l t r a v i o l e t spectrophotometry (Section 2.13) o r o t h e r means. Hol lingsworth e t a l .41 separated iodoamino acids and r e l a t e d compounds o n T e m u l o s e p l a t e s , with a solvent system composed o f tert.-butanol:2N ammnia:chloroform (376:70:60). They d i d not report usiiig t h i s technique on iodipamide. However, it seems reasonable t o expect t h a t t h i s system w i l l s e p a r a t e iodinated c o n t r a s t agents. S t a h l and Pfeifle38 reported on s i x systems used t o s e p a r a t e 17 iodinated compounds and Hocvel-Kestermann and Muhlemann separated 8 c o n t r a s t agents with one system.

356

IODIPAMIDE

Table VIII Thin-Layer Systems for Separation of Iodipamide Solvent System I1 I11 Plate
A BDC

Detect ion Syst em a,b

a, C

Rf not given not given 0.27 0.33 03 .3 0.20

00 .9

Reference
9 26,39 26 , 39 38 38 38 40

IV V VI VI I

BBC
A

A A D

a,c a,b a Bb aD b a

Solvent Systems

- Ethyl acet8te:isopropyl alcoho1:ammonia I1 - Methanol:amrPonia, 25% (2:l) I11 - Methanol:2N acetic acid (1O:l) I
IV V

25% (11:7:4)

- Acetone:isopropyl

alcohol:ammonia, 25% (2:2:1) 25% (4:l)

- Isopropyl alcohol:ammonia, - n-butano1:ethanol:lg

A .

VI
VII

- Ethy1acetate:methanol :diethylamine (5:4: 2)

ammonia (5:1:2)
30 g of silica gel HF254, 70 ml of H20 and 0 5 g . of starch.

Plate

B .
C .

Silica Gel G (Merck). Silica Gel H254-366 (blerck). Eastman tThromogramtg #6061 silica gel, plastic plates.

D .

351

HYAM HENRY LERNER

Table VIII (Cont'd) Detection System

A.

Short-wave u l t r a v i o l e t l i g h t (254 m . ) Spray with 50% solution of acetic acid, followed by i r r a d i a t i o n a t 254 MI f o r 10 lain t o give bluev i o l e t spots. Spraying and i r r a d i a t i o n may be repeated t o increase i n t e n s i t y of t h e spots. Spray with 1:l solution of 10% ceric s u l f a t e and 5% sodium arsenite, both i n 1 N s u l f u r i c acid3'.

B .

C .

6.63

Electrophoretic Analysis

Ardoino and P a ~ o n e ~ ~ reported on t h e elect r o p h o r e t i c analysis of c o n t r a s t agents- i n b i o l o g i c a l secret i o n s from t h e l i v e r and kidney. Iodipamic acid migrates at the speed of albumin and with albumin. 6.7 X-Ray and B-Particle Dispersion Methods

Holynska and J a n k i e w i c ~ ~ ~ X-ray fluorescence used and absorption techniques t o determine iodine i n various contrast agents. I n t h e X-ray fluorescence work, e x c i t a t i o n was obtained by i r r a d i a t i o n of t h e sample, from a 2 4 1 h source of 5 mCi a c t i v i t y . Energy of e x c i t a t i o n w a s 60 keV, The fluorescence of t h e K series of iodine (28.5 keV) was measured with a s c i n t i l l a t i o n counter having a 6-mm t h i c k NaI/T1 c r y s t a l . The c h a r a c t e r i s t i c r a d i a t i o n of iodine was separated by means of a single-channel, pulse-amplitude anal y z e r covering t h e t o t a l width of t h e K-peak iodine. t4easurement time was 1 min. A c a l i b r a t i o n curve of t h e whole range of iodine contents investigated w a s made from standard samples of i o d i c acid (HI03). I n t h e absorption method t h e X-ray source w a s 241Am and t h e energy w a s 60 keV. The d e t e c t o r w a s a scint i l l a t i o n counter with a 6-mm thick NaI/T1 c r y s t a l . Absorpt i o n measurements were made by means of a single-channel, pulse-amplitude analyzer i n t h e energy channel of 5 keV covering 60 keV. Calibration curves were again prepared from s u i t a b l e concentrations of i o d i c acid. The authors claim t h e fluorescence technique is t h e method of choice.
358

l OD1PAMI DE

Analyses take less than 5 min, and t h e r e l a t i v e e r r o r is claimed t o be 1-3%, depending on iodine content.

e a1,44 used t h e method of retrograde Mikolajek -tT persion of b e t a p a r t i c l e s t o assay c o n t r a s t media. %l, with about 3 V C i a c t i v i t y deposited on a r i n g was used as t h e source of radiation. A c a l i b r a t i o n curve showing t h e number o f s c a t t e r e d e l e c t r o n s VS. concentration w a s established. Results by t h i s method are i n good agreement with determinations made by conventional metliods.
6.8 Flame Photometry

Shm~otienko~~ determined sodium iodipamide i n pharmaceutical preparations by measuring t h e sodium content by flame photometry. P r i o r t o analysis, t h e iodipamic a c i d w a s p r e c i p i t a t e d by a c i d i f y i n g t h e s o l u t i o n with 2N hydroc h l o r i c acid, and w a s then separated by filtration: Determinations f o r t h e f i l t r a t e were evaluated from a c a l i bration curve of sodium chloride i n t h e range of 4.5 8.5 m 0. g

This procedure lacks s p e c i f i c i t y , s i n c e it is dependent on t h e content of an atom not associated with t h e H a c t i v i t y of iodipamide. Many formulations are a l s o p adj u s t e d with sodium hydroxide and contain o t h e r sodium comsodium c i t r a t e , sodium e d e t a t e , as excipients. In t ose cases, t h i s method would give erroneously high values,

"%'

7.

Drug Metabolism

--

Iodipamide has been demonstrated t o be excreted largeLangecker e t a1.12 rel y i n t h e unchanged form12r16,17. covered 70% of t h e unchanged compound i n t h e K l r o f rabb i t s within 6 h r after dosing. Because of i t s low pKa (3.5)24 and high molecular weight, i o d i amide is not reabStrickler sorbed after its excretion i n t h e b i l e J*19. et al. 2o reported evidence f o r t h e metabolic conversion of iodipamide t o an u n i d e n t i f i e d product. Hydro1 sis of t h e amide linkago was postulated as a p o s s i b i l i t y

2 1.

Deiodination of iodipamide w a s s t u d i e d i n man21 and was found t o be less than 1% t h e given dose. Pileggi of

359

HYAM HENRY LERNER

e t a1.22 described a papor chromatographic screening t e s t F r - & . e m i n a t i o n of iodipamide and i o d i d e i n sera (Section 6.61). A method f o r t h e removal o f iodipamide from blood sera by Craig countercurrent d i s t r i b u t i o n w a s described by S t r i c k l e r e t a1.20 (Section 5 3 . .)

--

M ~ C h e s n o yreviewed t h e literature through February ~~ 1968 i n a chapter e n t i t l e d , l"llie Biotransforrnation of Iodinated Itadiocontrast Agents.

(1)
(2)

C. tlermann, Drug Stand.,

2, 169

(1957).

M. Puar, Squibb I n s t i t u t e , personal communication.

W. Neudert and H. Rbpke

(3)
(4)

(19%).

, s. E., 845 89, , 17,

T. Pomazonska and W. Zyzynski, Diss. Pharm. 319 (1965).

54, -

(5)
(G)

J. D. Ostrow and R. P. Levy, Gastroenterology, 1085 (1968).

P. Funke, Squibb I n s t i t u t e , personal comunication.

(7)

V. Valenti, Squibh I n s t i t u t e , personal comunication.

W. Neudert and H. Hbpke, Chem. - * I Ber

(8)
(9)

(1954).

87 ,

659

kl. tloevel-Kestermann and H. Muhlemann, Pharm. Acta Helv -* 47 394 (1972). --*'
Q. Ochs, Squibb I n s t i t u t e , personal communica-

(10) (11)

tion.
National Formulary X I , p. 176 (1960).

360

IOOIPAMIDE

(12) (13) (14) (15) (16) (17) (18) (19)

E x p o Pathol. Pharmakol.,

H. Langecker, A. Harwart and K, JUnkmann, Arch. 220, 195 (1953).

H. Prime and R. Rutkowski, U. S. Patent 2,776,241 (1957). H. Casselbaum and R. Drux, German (East) Patent 33738 (1964); Chem. -.- 63 11441 b (1965). Abstr

P. Kallos , Squibb Corp.

, personal

communication. (1955).

E. Clerc, Aerztl. Wochenschr.,

10, 1156

N. Y. Acad, E, - - - =., 319 (1963).

H, W.
Fischer, Radiology,

B. H. B i l l i n g , Q. Maggione and M. A. Carter,

&.

84,

483 (1965).

K. H. Kimbel, W. Bbrner and E. Heise, Fortschr. Roentgenprax.,

(20)

H. S t r i c k l e r , E. S a i e r , E. Kelvington, J. Kempic, E, Campbell and R. Graven, 2. Clin. ocrinol. Metab - 15 (1964). 24 -* H. Langecker, A. Hartwart, K. H. Kolb and M. Kramer, Arch. 9, Pathol. Pharmakol., 247, 493 (1964).
V. J. P i l l e g g i , K. J. Henry, M. Segalove and G. C. klami11, C l i n . Chem., 2, 647 (1962).

(21)

(22)

(23)

E. W. McChesney i n t g I n t e n a t i o n a l Encylopedia of Pharmacology and- Therapeutics P. K. Knoefel, Ed., Pergamon Press, New York, 1971, Vol. I , Sect, 76, pp. 147-163.
P. K. Knoefel, Wadiopaque Diagnostic Agents , I Charles C. Thomas, Springfield, Ill., 1961, pp. 41-44.

(24)

(25)

J, Hydro, Squibb I n s t i t u t e , personal communication.

361

HYAM HENRY LERNER

Fak. - Mecm., 11889-1965i). -

0. Ates and H. Amal, Istanbul Univ. E c z a c i l i k .

2, 82 (1966) ; Chem. Abstr.,

--

66,

National Formulary X I , p. 173 (1960). United S t a t e s Pharmacopoaeia XV, p. 356 (1955).

- bi. A.

G. J. Yakatan and M. M. Tuckerman, J. Pham. Sci., 55, 532 (1966).

--

Krasnova, Farmatsi a (Moscow), 18, 57 (1969); Chem. -*-*416. Abstrv 2-9 (

R. E g l i , Z. Anal. Chem.,

---

3,39

(1969).

A. Vaskelis, Y. Gaule and S. Chausovskii, Farmatsiya (Moscow 17 54 (1968); Chem. Abstr., 70, 6 0 8 9 0 ~

- (m -

--

E. Hartmann and H. Rbpke, 2. Anal. Chem., 268 (1967).

---

232,

K. Hentrich and S. P f e i f e r , Pharmazie, 21, 296 (1966); Chem. Abstr., 65, 8672f (1966).

--

T. Soh, Squibb Corp., personal communication. L. Chow, Squibb Corp., personal communication.
S. L i s s i t z k e y , Bull. SOC. Chim. Biol., (1955).

------

37,

89

E. Stahl and J. P f e i f l e , Z. Anal. Chem., 377 (1964).

200,

0. Ates and H. Amal, Istanbul Univ. Eczacilik. Fak. Mecm., 4 , 36 (1968);m=str., 70, co886-9 697.

--

T. Soh, Squibb Corp.

, personal

communication. K. Bondy,

J. Lab. Clin. Med - - - -*-

D.

R. Hollingsworth, M. D i l l a r d and P. 62 346 (1963).

362

IODlPAMlDE

(42) (43)

R. H. Mandl and R J. Block, Arch. Biochem. .

Biophys,
1

81,

25 (1959).

B. Holynska and J. Jankiewicz, Chem. Anal. (Warsaw), 14, 219 (1969); Chem. Abstr., 2, 2=(1969).

--

(44)

E Mikolajek, J. Kormicki and 2. Kawalczyk, .

Pharm. Pharmecol., 6449b-m. -2

2, 523

(1966); Chem. Abstr.,

e.

(45) (46)

Biol. m 9

G. D Shamotienko, Khim. Farm. Zh., . 69 Chem. Abstr - S m y W8)T

-, 0

L, 32

(1968);

. L A. Ardoino and M Pavone, Boll. Soc. Ital. .

(IYOU J

(47) (48) (49)

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G. D. Shamotienko,

(1970); Chem.

S, Willis, Squibb I n s t i t u t e , personal communication.

A. Hoffmann, Squibb I n s t i t u t e , personal communication.
B. Toeplitz, Squibb I n s t i t u t e , personal c o m n i -

cat ion.

3 63

METHADONE HYDROCHLORIDE

Rafik H. Bishara

RAFlK H. BISHARA

CONTENTS

1.

Description 1.1 Nomenclature 1.2 Formula 1.3 Molecular Weight 1.4 Structure 1.5 Appearance, Color, Odor, and T a s t e 1.6 Proprietary Names Physical Properties 2.1 M e l t i n g Range 2.2 Solubility 2.3 Optical Rotation 2.4 pH Range 2.5 D i s s o c i a t i o n C o n s t a n t (pKa) 2.6 Partition Coefficient 2.7 D i f f e r e n t i a l Thermal A n a l y s i s 2.8 Thermogravimetric A n a l y s i s 2.9 O p t i c a l and C r y s t a l l o g r a p h i c P r o p e r t i e s 2 . 1 0 X-Ray Powder D i f f r a c t i o n 2 . 1 1 U l t r a v i o l e t Spectrum 2.12 I n f r a r e d Spectrum 2.13 Nuclear Magnetic Resonance Spectrum 2.14 Mass Spectrum S y n t h e s i s , S t r u c t u r e and R e s o l u t i o n 3.1 S y n t h e s i s and C o n f i r m a t i o n o f S t r u c t u r e 3.2 Resolution R e a c t i v i t y and S t a b i l i t y Drug M e t a b o l i c P r o d u c t s and P h a r m a c o k i n e t i c s 5.1 Absorption 5.2 Distribution 5.3 Metabolism 5.4 Excretion Identification Microchemical R e a c t i o n s

2.

3.

4. 5.

6. 7.

366

METHADONE HYDROCHLORIDE

8.

Methods of A n a l y s i s 8.1 Elemental A n a l y s i s T i t r a t ion 8.2 8.2.1 Non-Aqueous T i t r a t i o n 8 . 2 . 2 Direct T i t r a t i o n 8.3 Chloride Determination 8.4 Ultraviolet Analysis 8.5 Fluorometric Analysis 8.6 Infrared Analysis 8.7 Colorimetric A n a l y s i s 8.8 Polarography 8.9 Bioassay 8.10 S p i n Immunoassay 8.11 R a d i o t r a c e r T e c h n i q u e s 8.12 Column Chromatography 8.13 P a p e r Chromatography 8.14 T h i n L a y e r Chromatography 8.15 Gas Chromatography 8.16 Combined G a s Chromatography-Mass Spectrometry 8 . 1 7 High P r e s s u r e L i q u i d Chromatography

9.
10.

E x t r a c t i o n from B i o l o g i c a l F l u i d s
Determination i n Tissues Bibliography Acknowledgements References

11.
12.
13.

367

RAFlK H. EISHARA

1.

Description

1.1 N o m e n c l a t u r e 6-Dimethylamino-4,4-diphenyl-3heptanone h y d r o c h l o r i d e . 1 , l - D i p h e n y l - l - (2-dimethylaminopropy1)2- b u t a n o n e h y d r o c h l o r i d e 4,4-Diphenyl-6-dimethylamino-3heptanone h y d r o c h l o r i d e . 6-Dimet hylamino-4,4-diphenylheptan-3one h y d r o c h l o r i d e .

1.2

Formula CBlH2,NO*HCl

43.92

1.3
1.4

Molecular Weight
Structure

ApEearance, Color, Odor, a n d T a s t e --WhiFe, e s s e n t i a l l y o d o r l e s s powder. B i t t e r t a s t e f o l l o w e d by s t i n g i n g s e n s a t i o n .

1.5

--

Proprietary Names dl-Form: Adanon h y d r o c h l o r i d e ; A l g i d o n ; A l g o l y s i n ; Amidon h y d r o c h l o r i d e ; AN-148; B u t a l g i n ; D e p r i d o l ; Diadone; Diaminon h y d r o c h l o r i d e ; D o l o p h i n e h y d r o c h l o r i d e ; Eptadone; Fenadone; Heptadon h y d r o c h l o r i d e ; Hoechst 10,820; K e t a l g i n h y d r o c h l o r i d e ; Mecodin; Mephenon; 1.6

368

METHADONE HYDROCHLORIDE

Miadone; Moheptan; Phenadone h y d r o c h l o r i d e ; P h y s e p t o n e h y d r o c h l o r i d e ; Polamidon h y d r o c h l o r i d e ; Symoron; Z e f a l g i n . 1-Form: Levadone; L e v o t h y l . 2. Physical Properties 2.1 M e l t i n Range 8Idk--e- 235.0'C1 232.5 mp 233.0 mp 236.0 mp 1-Form: mp 241.O'Cl mp 2.2 245.0

233.0C2 236.0C3 236.5"C4 246.OoC4

S o l u b i l i t ~ ~ ~ ~ ~ ~ ~ ~ =-metha= h v d r o c h l o r i d e is v e r v s o l u b l e i n water (12 g/100 mi), s o l u b l e i n a l c o h o l ( 8 g/100 ml), i n i s o p r o p a n o l ( 2 . 4 g/ 100 m l ) , and i n c h l o r o f o r m ; p r a c t i c a l l y i n s o l u b l e i n e t h e r and i n g l y c e r i n e . The 1-form o f methadone h y d r o c h l o r i d e h a s s i m i l a r s o l u b i l i t y t o t h e racemic form i n a l c o h o l , i n c h l o r o f o r m and i n e t h e r . O-- i c a l R o t a t i o n - -t -p No o p t i c a l r o t a t i o n is o b s e r v e d w i t h t h e racemic methadone h y d r o c h l o r i d e . F o r t h e 1-form t h e f o l l o w i n g o p t i c a l r o t a t i o n s a r e reported : 1 -145' (C = 2 . 5 ) 2.3 and [a], 2 0 -169" 2.4 (C = 2 . 1 i n a l c o h o l ) 1 9 4

pH Range T h e m o f a 1% s o l u t i o n is between 4 . 5 and 6 . 5 . 1 , 3 , 5 D i s s o c i a t i o n C o n s t---- (pKa) -- v i e t al.8 r e p o r taendt Le t h e pKa of methadone h y d r G h E r i d e , i n water a t 2OoC., t o
369

2.5

--

RAFlK H. BISHARA

be 8.25. O t h e r data on t h e d i s s o c i a t i o n c o n s t a n t of methadone are r e p o r t e d by Marshall' and Beckett.

Partition Coefficient F m o n coef f icGZs of d l - m e t hadone i n heptane/p]C! 7.4 b u f f e r and chloroform/pH 7.4 b u f f e r a t 25 C . a r e 0.84 and 1fb56 r e s p e c t i v e l y . 9 Misra a n d Mule r e p o r t e d 57.3 and 28.3 t o be t h e p a r t i t i o n c o e f f i c i e n t s of t h e 1- and d-isomers, r e s p e c t i v e l y , i n o c t a n o l / pH 7.4 b u f f e r . No e x p e r i m e n t a l d e t a i l s are g i v e n t o e x p l a i n t h i s d i f f e r e n c e between t h e two o p t i c a l isomers. D i f f e r e n t i a l Thermal TZTfferentialhermal of methadone h y d r o c h l o r i d e was performed- u s i n g a DuPont 900 D i f f e r e t t i a l Thermal A n a l y z e r a t a h e a t i n g r a t e of 20 C. p e r min. a n d a n i t r o g e n a t m o s p h e r e . T h e o t h e r m o g r a m ( F i g u r e 1) shows a n endotherm a t 235 C . , w h i c h appears t o be a m e l t , f o l l o w e d immediately by d e c o m p o s i t i o n . TGA , of methadone h y d r o c h l o r i d e w a s performed u s i n g a DuPont 950 T h e r m o g r a v i m e t r i c A n a l y z e r a t a h e a t i n g r a t e of 5 C . p e r min. a n d a n i t r o g e n atmosphere. The thermogram ( F i g u r e 2) s h o w s a w e i g h t loss b e g i n n i n g a t 156.C. and c o n t i n u i n g through decomposition.
2.8 2.7

2.6

A-GagravTmetric-anaysis,

Thermogravimetric Analysis"

h E E of methadone hydrocklEFTaE p r e p a r e d by Huback and 3ones12 f o r o p t i c a l examinat i o n and i d e n t i f i c a t i o n were o b t a i n e d by c o o l i n g a w a r m s a t u r a t e d aqueous s o l u t i o n of t h e compound. The s i n g l e c r y s t a l s were mounted onl a s t a g e g o n i o m e t e r t o measure t h e p r i n c i p a l r e f r a c t i v e i n d i c e s . A rotatory s t a g e w a s used t o measure a l l a n g l e s . Diamond-shaped c r y s t a l s r e s t i n g on a n end face a r e r e p o r t e d . T h e s e c r y s t a l s show s y m m e t r i c a l e x t i n c t i o n , g i v e a n
370

2.9

0 t i c a l and C r y s t a l l o g r a p h i c P r o p e r t i e s

20

40 217

59 236

79 256

98 117 137 276 T, "C (CHROMEL: ALUMEL)

157

177

197

217

Figure 1 lYl?A-thenrogram of methadone hydro&loride taken on a W o n t 900 . D i f f e r e n t i a l Thermal Analyzer

100 90 -

4
I

* 80I-

! 2

7060 -

50

Figure 2.

TGA-themogram of mthadone hydrochloride taken on a DuPont 950 Thenrcgravk&xic Analyzer

METHADONE HYDROCHLORIDE

i n t e r f e r e n c e f i g u r e t h a t shows t h e o b t u s e b i s e c t r i x a t o n e Zdge o f t h e f i e l d , and t h e i r a c u t e a n g l e o f 62 is r e l i a b l e f o r c h a r a c t e r i s t i c d i a g n o s i s . T a b l e 1 summarizes t h e o p t i c a l and c r y s t a l l o g r a p h i c d a t a o f racemic methadone hydrochloride The s i n g l e c r y s t a l d a t a f o r d l methadone h y d r o c h l o r i d e , t a b u l a t e d by B a r n e s and Forsyth,13 i n d i c a t e t h a t t h e space group of t h i s The a u t h o r 9 l i s t e d compound is Cc or C2/c. a = 16.26, b = 9.76, and c = 25.74 9. The m o n o c l i n i c a n g l e was measured as 74 ( i . e . , I3 = The s p a c e g r o u p e x t i n c t i o n o f C2/c is 106'). f a v o r e d by t h e p r e s e n c e of e i g h t m o l e c u l e s p e r c e l l (z = 8 m o l e c u l e s / c e l l ) . A density of 1.178 g./ml. ( a v e r a g e o f 8 measurements) w a s o b s e r v e d f o r c r y s t a l s from d i f f e r e n t p r e p a r a t i o n s ( P c a l c d . = 1 . 1 7 1 g./ml.).

X-Ray Powder D i f f r a c t i o n The x - r a y d i f ? r x t I o n - p o w d e r d a t a o f dl-methadone h y d r o c h l o r i d e o b t a i n e d by B a r n e s and Sheppard14 u s i n g f i l t e r e d CoKa (A = 1 . 7 9 0 A ) r a d i a t i o n a r e i n v e r y good agreement w i t h t h o s e o b t a i n e d by Hubach and J o n e s , 1 2 f o r a s a m p l e from a d i f f e r e n t commericgl s o u r c e , w i t h f i l t e r e d CuKa (A = 1.542 A ) r a d i a t i o n . With c o p p e r r a d i a t i o n , t h e s p a c i n g s of t h e 3 s t r o n g e s t l i n e s , o f t h e p a t t e r n a r e 7 46 A (v.v.s.), 4.55 A (v.v.s.), and 6 . 4 5 (v.s.) w i t h cobalt r a d i a t i o n thg 3 s t r o n g e s t pacings o f t h e p a t t e r n are 4 . 5 7 A (1001, 7.50 (901, and 6 . 4 8 A (701, t h u s merely i n t e r c h a n g i n g t h e " f i r s t " and "second" l i n e s . B a r n e s and Sheppard14 d r e w t h e a t t e n t i o n t o t h e f a c t t h a t while t h e pattern of t h e dl-methadone h y d r o c h l o r i d e is n o t t h e same a s t h a t of t h e d- and t h e 1- isomers, t h e p a t t e r n s of t h e f r e e base i n d-, 1-, and d l - forms a r e i d e n t i c a l . The x-ray powder d i f f r a c t i o n d a t a o f dl-methadone h y d r o c h l o r i d e r e p o r t e d by these a u t h o r s 1 4 are shown below:

2.10

373

TABLE 1 OPTICAL AND CRYSTALLOGRAPHIC PROPERTIES OF RACEMIC METHADONE HYDROCHLORIDE* C r y s t a l system Optic orientation Monoclinic, C l a s s a c u t e a n g l e B = 74

?,

o n l y a p l a n e of symmetry;

t3 v i b r a t i o n d i r e c t i o n is p a r a l l e l t o c r y s t a l l o g r a p h i c axis b. P l a n e o f symmetry a d i r e c t i o n is a c u t e contains axial plane. b i s e c t r i x w h i c h is n e a r l y p e r p e n d i c u l a r t o c r y s t a l l o g r a p h i c axis c.

5
P

Refractive indices, 5893A. ; 25.C. Optic axial angle Observed C a l c d . from s i n V = sin E

a = 1.5713 0.0005, B = 1.6232 f 0.0005, y = 1.6360 0.0005, a = 1.5760 f 0.0005 ' from c r y s t a l s r e s t i n g on a n e n d f a c e

2E = 90 f 1. by c a l i b r a t e d micrometer eyepiece

2V'

= 52.

163 .2

Observed

2V = 52. by r o t a t i n g f r o m o n e o p t i c a x i s t o t h e o t h e r on goniometer (continued )

. ..

0
fi
c,

a
0

P E

2
0
W

cu
Lo
. d

a,

cu
'
d
*rl
v) v)

*
M

II
cd

c,

tr
a,
L)

E a,

a
P
h

0
cd cd * 0
r(

tr

a
cd 0

a
d * a

a, 0 5

0
fi

a
0
IEr:
I *
a,

375

RAFlK H. BISHARA

d (1) 12.4 8.25 7.87 7.50 6.48 6.20 5.92 5.70 4.72 4.57 4.34 4.20 4.14 4; 00 3.87 3.71 3.49 3.33 3.20 3.14 2.11
A scan of

1/11 -

(A) d 3.10 3.03 2.97 2.92 2.84 2.75 2.68

2.60

I/I 1

25 10 5
00

30 3 10 3
5

70 1 20 5 30 100
40

3 20 20 20 5 BB 20 B 5 25 1 B

2.53 2.48 2.30 2.23 2.16 2.11 2.08 2.04 1.99 1.92 1.67

20 15 3 B 20 2
5

1 2 8 3 15 2 3
2

methadone-hydrochloride i n e t h a n o l a t a c o n c e n t r a t i o n o f 0.27 mg./ml., (7.8 x 10'' M) on a Cary 1 5 spectrophotometer, from 400 t o 210 nm. ( F i g u r e 3) shows maxima a t
254, 250, 265, and 203 nm. The corresponding molar a b s o r b t i v i t i e s of t h e s e maxima are 410, 485, 460, and 470 r e s p e c t i v e l y . Hubach and Jones12 r e p o r t e d that a n a l c o h o l i c s o l u t i o n of methadone h y d r o c h l o r i d e exhibited t w o characteristic electronic a b s o r p t i o n bands a t hmax = 294 nm. ( E = 460) and t h e aromatic band a t Amax = 259 nm. (6 = 480). In water s o l u t i o n , t h e long wavel e n g t h maximum is s h i f t e d t o 292 nm. and t h e molar a b s o r p t i v i t y is i n c r e a s e d t o E. = 520. The spectrum of t h e f r e e base, methadone, u s i n g e t h a n o l or hexane as a s o l v e n t , is e s s e n t i a l l y t h e same i n t h e r e g i o n o f t h e 294 nm. band,

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

(e

376

METHADONE HYDROCHLORIDE

20 18 16 14 N

52
5

; 12k

g
4

t
m

10A max 254

(t =

v)

5 0

410) 485) 460) 470)

8-

Amax 2 5 9 ( c

= = =

I
6-

A max 265 ( e
X m a x 293 ( e

0 200

250

300 WAVELENGTH. nm

350

400

F i g u e 3.

Ultraviolet spectrum of mthadone hydrochloride i n ethanol taken on a Cam 15 Spectroeotcmter

377

RAFlK H. BISHARA

However, t h e molar a b s o r p t i v i t i e s a r e E = 7 6 0 i n a l c o h o l a n d E = 830 i n h e x a n e . The u l t r a v i o l e t a b s o r p t i o n s p e c t r a data o f Mule15 o n methadone i n 0 . 1 N h y d r o c h l o r i c a c i d showed Xmax a t 292 nm, ( E = 5 5 4 ) , Xmin a t 2 7 5 nm. ( E = 3 7 2 ) . Data o b t a i n e d i n e t h y l e n e d i c h l o r i d e c o n t a i n i n g 25% i s o b u t a n o l (v/v) are Xmax a t 2 9 5 nm. ( 6 = 4 3 3 ) and Xmin a t 2 8 0 nm. ( E = 3 9 0 ) . Ultraviolet absorption s p e c t r a a t r e d u c e d t e m p e r a t u r e s of methadone n i t r i l e a n d i s o m e t h a d o n e n i t r i l e are p r e s e n t e d by Sinsheimer - al.l6 e t -2.12

Ascanof-methaa&e h v d r o c h l o r i d e i n a

I n f r a r e d Spectrum

p o t a s s i u m bromide p e l l e t on a Beckman IR-12 s p e c t r o p h o t o m e t e r is shown i n F i g u r e 4 . Underbrink" a s s i g n e d t h e f o l l o w i n g b a n d s t o t h e methadone h y d r o c h l o r i d e I R s p e c t r u m :

a.

710-770 c m . ' l

character is t i c f o r aromatic c a r b o n hydrogen o u t of p l a n e bending.

f i n g e r p r i n t region; due t o s k e l e t a l f r e q u e n c i e s and aromatic c a r b o n hydrogen i n p l a n e bending

b.

900-1200 cm."

C.

1300- 1500 c m

.-

characteristic for methylene and methyl bending

d.

1450, 1490, 1580, and 1600 c m . - 1 1 7 0 8 cm."

characteristic for

aromatic r i n g
frequencies

e.

c h a r a c t e r is t i c f o r carbony 1 s t r e t c h i n g

378

100

4000 3800 3600 3400 3200

3000 2800 2600 2400

2200 2000 1900 1800 1700 1600 1500 1400 WAVENUMBEA CM-

1300 1200 1100 10.00

900

800

700

600

500

4nl:

Figure 4.

Infrared spectrum of methadone hydrochloride taken in a K3 pellet on a E r BeduMn IR-12 Spectrqhotorneter

R A F I K H. BISHARA

f.

2400 c m .

-1

characteristic for t e r t i a r y amine hydrochloride characteristic for a l i p h a t i c carbonhydrogen s t r e t c h i n g .

g.

2810-3000 cm."

h.

3000-3080 c m .

-'

characteristic for aromatic c a r b o n hydrogen s t r e t c h i n g .

2.13

N u c l e a r M a g n e t i--R e s o n a n c e S p e c t r u m c -

The NMR s p e c t r u m o f m e t h a d o n e h y d r o c h l o r i d e i n CDC1, c o n t a i n i n g t e t r a m e t h y l s i l a n e as i n t e r n a l s t a n d a r d on a V a r i a n Associates HA-100 is shown i n F i g u r e 5 . The s p e c t r a l a s s i g n m e n t s 1 * are summarized i n T a b l e 2 . The c h e m i c a l s h i f t s a r e m e a s u r e d i n p.p.m. d o w n f i e l d f r o m t e t r a m e t h y l s i l a n e . The m u l t i p l i c i t y of t h e peaks, and t h e a p p r o x i m a t 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 h e r e appropriate. The m e t h y l e n e a n d m e t h i n e p r o t o n s o f Group 3 a n d Group 4, r e s p e c t i v e l y , were i d e n t i f i e d by d e c o u p l i n g . The t w o m e t h y l e n e p r o t o n s of Group 6 are n o n e q u i v a l e n t g r o b a b l y Their because o f c o n f o r m a t i o n a l e f f e c t s . "9 chemical s h i f t s are a s s i g n e d a t approximately 2 . 3 0 a n d 3.15 p.p.m. by t h e p r o c e s s o f e l i m i n a t i o n a n d i n t e g r a t i o n . The p r o t o n s o f t h e N-methyl g r o u p s a r e n o n e q u i v a l e n t d u e t o t h e r e l a t i v e p r o x i m i t y of e a c h methyl g r o u p t o t h e d e s h i e l d i n g cone o f t h e phenyl g r o u p and a p p e a r as a p a i r o f d o u b l e t s (J" 4 Hz) c e n t e r e d a t 2 . 7 5 p.p.m. D i s c u s s i o n o f t h e NMR a n d PMR o f methadone a n d some r e l a t e d s u b s t a n c e s , a n d t h e a p p l i c a t i o n of t h i s t e c h n i q u e f o r s t e r i o c h e m i c a l and o p t i c a l i s o m e r i s m p r o b l e m s a r e f o u n d i n t h e l i t e r a t u r e . 19-23

3 80

381

figure 5.

Nuclear magnetic resomce spectnm of mthadone hydrochlori& i CDCl3 n taken on a varian Associates HA-100 Spectmmter

TABLE 2

NMR SPECTRAL ASSIGNMENTS OF METHADONE HYDROCHLORIDE

Group 1. %-CHMultiplicity Doublet Chemical Shift (p.p.m.1
0.70

J(H=)
6.5

N (CH31 2

2.

CH3-CH2-C-

II

Triplet

0.83

Approx. 2.30
N ( C H1 ~

4.
5.

CHs-CJ-CH2CH3-CH1 N (CH3 2

Mult iplet

Approx. 3.04
2.75

Mu 1t i p let

(continued

. ..

n l

P
d

0
c ,

a , a , C E

0
W

a,

.I4

E
P
(d

u1

0 h

383

R A F l K H. BISHARA

Mass S e c t r u m T h e r e a t i v e mass f r a g m e n t a t i o n p a t t e r n of methadone was o b t a i n e d 2 4 u s i n g a n LKB-9000

2.14

-f--

combined gas chromatograph-mass s p e c t r o m e t e r (GCMS). A f o u r - f o o t s i l i c o n i z e d glass column (2.5 mm. I . D . ) packed w i t h 1% W-98 m e t h y l v i n y l s i l i c o n gum r u b b e r on 80-100 mesh G a s Chrom Q w a s employed a s t h e O K column. The column t e m p e r a t u r e was 1 7 0 C . a n d t h e c a r r i e r gas ( h e l i u m ) flow w a s 4 0 ml./min. An e l e c t r o n e n e r g y of 7 0 e V w a s u s e d f o r i o n i z a t i o n . The c o m p u t e r i z e d mass f r a g m e n t a t i o n p a t t e r n shown i n F i g u r e 6 was o b t a i n e d by u s i n g a H e w l e t t P a c k a r d 2100 c o mp u t e r i n t e r f a c e d d i r e c t l y t o t h e E M S . The a s s i g n m e n t s a n d c o m p o s i t i o n s a r e as follow:
c-

m/e

Assignment -

C o m D o s it i o n
c2 l H 2 7NO

3 09

M+
(M-CH3

294 26 5 223

>+
I
+

c2 O H 2 4NO

(M-N ( C H ~ ) )

(M-CHz-CH-CH,
N(CH3 ) 2

c1g H 2 lo
ClgH150

165 72

C13H9

C 4 H 1 ON

57 44 29

c3 H5O
C2H6N
C2H5

384

12

r
I

294 I
0
E

10

120

110
mle

220

210

Figure 6.

Relative mass fragmntation pattern of mthadone abtained by using an LKB-9000 ccxbined gas chrangogra&-mass s p e c t r c m t e r ((336) mta courtesy of Sullivan, H. R .

RAFlK H. BISHARA

The above data are i n good agreement w i t h t h e h i g h r e s o l u t i o n mass s p e c t r u m 2 5 of methadone h y d r o c h l o r i d e o b t a i n e d by u s i n g a CEC21-11OA mass spectrometer w i t h p h o t o p l a t e r e c o r d i n g . The a s s i g n m e n t s of t h e prominent i o n s are g i v e n i n Table 3. The mass s p e c t r u m of methadone, t h e most a b u n d a n t p e a k s and t h e metastable i o n s are r e p o r t e d by Fales e t a1.26 The i d e n t i f i c a t i o n of methadone by i s s u s n e chemical i o n i z a t i o n mass s p e c t r o m e t r y is p r e s e n t e d by Milne e t --. 2 7 al

3.

S y n t h e s i s , S t r u c t u r e and R e s o l u t i o n
3.1

S n t h e s i s and C o n f i r m a t i o n of S t r u c t u r e b t of t h e U n i t e d States D e p a r t ment of Commerce28 a b o u t Amidone (methadone), t h e new German a n a l g e s i c d r u g no. 10820, i n c l u d e s t h e method g i v e n by t h e German chemists f o r its s y n t h e s i s . I n t h i s method, 1 - c h l o r o - 2 p r o p a n o l is added t o a n aqueous s o l u t i o n of d i e t h y l a m i n e and sodium h y d r o x i d e t o p r e p a r e 1-dimethylamino-2-propanol [l]. To [ 1 3 a s o l u t i o n of t h i o n y l c h l o r i d e i n b e n z e n e is added t o form 1-dimethylamino-2-chloropropane [2 1. Compound [ 2 1 is t h e n dropped on a cool m i x t u r e of sodamide and d i p h e n y l a c e t o n i t r i l e and t h e t e m p e r a t u r e is allowed t o rise. The s o l u t i o n is r e f l u x e d f o r 15 m i n u t e s , cooled, poured on water, and t h e water is removed. The b e n z e n e s o l u t i o n is a c i d i f i e d w i t h h y d r o c h l o r i c a c i d , t h e a q u e o u s acidic l a y e r is made a l k a l i n e w i t h sodium h y d r o x i d e , and t h e p r o d u c t , 1-d imet hy laminopropy 1-2-d ipheny lacet o n i t r i l e [3 1 , is d i s s o l v e d i n x y l e n e and a d d e d t o a s o l u t i o n of ethylmagnesium bromide, h e a t e d , a n d poured o v e r a c i d i f i e d water t o s e p a r a t e t h e hydrobromide of t h e k e t o n e . The l a t t e r compound is d i s s o l v e d i n w a r m water and made a l k a l i n e t o y i e l d a n o i l y base, methadone, which is c r y s t a l l i z e d from methanol. The h y d r o c h l o r i d e of methadone is p r e p a r e d by d i s s o l v i n g t h e base i n alcohol, alcoholic hydrogen c h l o r i d e is added, and upon c o o l i n g , t h e material c r y s t a l l i z e s .
386

--

TABLE 3

HIGH RESOLUTION MASS SPECTRUM ASSIGNMENTS OF METHADONE HYDROCHLORIDE

C a l c u l a t e d Mass 3 09.2061 294.1854 265.1586 236.1433 223.1121 179.0846 178.0779 165.0699 117.0711 115.0543 91.0552 85.0896 72.0814 71.0745 70.0653 T h e o r e t i c a l Mass 309.2093 294.1858 265.1592 236.1439 223.1123 179.0861 178.0782 165.0704 117.0704 115.0548 91.0548 85.0892 72.0813 71.0735 70.0657 Emperical Formula H C 0 - N I

21 20 19 17 16 14 14 13 9 9
7 5

27 24 21 18 15 1 1 10 9 9 7
7

1 1 0 1 0 0 0 0
0

1 1 1 0 1 0
0

0
0

0 0
0

4 4 4

11 10 9
8

1 1 1 1

0
0 0 0

R A F l K H. BISHARA

I t was n o t e d 2 8 , 2 9 t h a t t h e abovem e n t i o n e d s y n t h e s i s would n o t be e x p e c t e d t o l e a d t o methadone [41, b u t t o t h e f o r m a t i o n of t h e isomeric s t r u c t u r e of i s o m e t h a d o n e [5]:

S c h u l t z e t a l . 2 9 e s t a b l i s h e d and p r o v e d s t r u c t u r e [41 f o r T e n a d o n e . In t h e i r s y n t h e s i s , when d i p h e n y l a c e t o n i t r i l e is r e a c t e d w i t h 1-dimethylamino-2-chloropropane [2 1 e i t h e r i n t h e p r e s e n c e of s o d a m i d e 2 8 or p o t a s s i u m t - b u t o x i d e , t h e p r o d u c t is a m i x t u r e of a p p r o x i m a t e l y e u a l amounts of t h e isomeric n i t r i l e s [3] and [ 6 The 2 , 2 - d ipheny l-4-d i m e t h y l a m i n o p e n t a n e n i t r i l e [6] r e a c t s s m o o t h l y w i t h e t h y l m a g n e s i u m bromide t o g i v e methadone [41. T r e a t m e n t o f 2,2diphenyl-3-methyl-4-dimethylaminobutanenitrile [ 3 ] w i t h t h e G r i g n a r d ' s r e a g e n t does n o t g i v e t h e methadone isomer C5l b u t a d i b a s i c p r o d u c t C71 w h i c h a p p e a r s t o be t h e c o r r e s p o n d i n g k e t i m i n e . A l t h o u g h t h e k e t i m i n e s t r u c t u r e is s u p p o r t e d by a n a l y t i c a l d a t a , t h e o r d i n a r y c o n d i t i o n of h y d r o l y s i s f a i l s t o g i v e t h e k e t o n e . 3 0 The s t r u c t u r e of t h e isomeric n i t r i l e s , a n d h e n c e t h e s t r u c t u r e o f methadone, were e s t a b l i s h e d by d e c o m p o s i t i o n of t h e q u a t e r n a r y b a s e s d e r i v e d from t h e m e t h i o d i d e s of t h e n i t r i l e s by t r e a t m e n t w i t h s i l v e r o x i d e . A summary of t h i s s y n t h e s i s is i l l u s t r a t e d i n F i g u r e 7. B r o d e and H i l l 3 1 r e p o r t e d t h e r e a r r a n g e m e n t of t h e isomeric 1 , 2 dimethylaminochloropropanes, d e r i v e d from t h e c h l o r i n a t i o n of 1-dimethy lamino-2-propanol [ 8 ) and 2-dimethylamino-1-propanol [91, t h r o u g h t h e

7.

388

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C H 5CH-CN C6H5/

+ CI-CH-CH2-N

/CH3

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+
CH3

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Figure 7 .

Synthesis of S c f i u l t z et a L

TFadone according to -389

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e t h y l e n e immonium i o n . 3 2 T h e i r d a t a a l s o show t h e c o n v e r s i o n o f [81 t o C91 i n l o w y i e l d s . To a v o i d t h e t e c h n i c a l d i f f i c u l t i e s d u e t o t h e formation of t h e isomeric a m i n o n i t r i l e s , a new s y n t h e s i s w a s d e v e l o p e d by E a s t o n e t a l . 3 3 i n w h i c h d i p h e n y l a c e t o n i t r i l e is c o n d e n s e d a t h p r o p y l e n e o x i d e i n t h e p r e s e n c e o f sodium amide t o y i e l d 3,3-diphenyl-5-methyltetrahydro-2furanoneimine [lo]. When [ l o ] is t r e a t e d w i t h p h o s p h o r u s t r i b r o m i d e , t h e p r o d u c t is 4-bromo2,2-diphenylpentanenitrile [111* On c o n d e n s i n g [111 w i t h d i m e t h y l a m i n e , Compound [6 1 is f o r m e d . Methadone [ 4 1 is p r e p a r e d from [ 6 ] by t h e a c t i o n of e t h y l m a g n e s i u m b r o m i d e a s d i s c u s s e d p r e v i o u s l y . 2 8 The y i e l d s of t h e a m i n o n i t r i l e from t h e h a l o n i t r i l e is below 107;. The major p r o d u c t a l w a y s formed is a n u n s a t u r a t e d n i t r i l e , presumably 2,2-diphenyl-3-pentenen i t r i l e [ 1 2 ] or a m i x t u r e of [ 1 2 ] and 2 , 2 diphenyl-4-pentenenitrile 113 1. The s t r u c t u r e o f methadone e s t a b l i s h e d b y S c h u l t z e t a1.29 w a s c o n f i r m e d by t h e f o l l o w i n g s e r i e s of r e a c t i o n s . Compound [ 6 3 is d e g r a d e d by e x h a u s t i v e m e t h y l a t i o n ( m e t h y l i o d i d e , s i l v e r o x i d e , and h e a t i n g ) t o a n u n s a t u r a t e d n i t r i l e [121, [131 o r a m i x t u r e w h i c h is h y d r o l y z e d w i t h o u t p u r i f i c a t i o n t o y i e l d t h e l a c t o n e of 2,2-diphenyl-4-hydroxyp e n t a n o i c a c i d [14]. The h y d r o l y s i s o f [13] f o r m s t h e same l a c t o n e [141. Long s t a n d i n g o f t h e h y d r o c h l o r i d e o f Compound [ l o ] i n a q u e o u s s o l u t i o n g i v e s t h e l a c t o n e [141. T h e s e f a c t s a r e a c c o u n t e d f o r by t h e s t r a i g h t s t r u c t u r e of t h e a m i n o n i t r i l e [ 6 ] . F i g u r e 8 shows t h e s y n t h e s i s and c o n f i r m a t i o n o f s t r u c t u r e according t o Easton et al.33 -The p r e p a r a t i o n of some isomers, a n a l o g s , 3 5 - 3 9 and r e l a t e d s u b s t a n c e s 4 0 - 4 4 t o methadone is r e p o r t e d i n t h e l i t e r a t u r e . T o l b e r t e t a l . 4 5 s y n t h e s i z e d dl-methadone l a b e l e d w i t h 1 4 C - i n e i t h e r t h e 1 or 2 p o s i t i o n .

--

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C6H5

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Figure 8.

Synthesis and confirmation of mthadone s t r u c t u r e according t o Easton e t - 33 - al,

39 1

R A F l K H. BISHARA

Resolution -_-Methadone h a s o n e a s s y m e t r i c c a r b o n atom and t h e r e f o r e c a n e x i s t a s d e x t r o or l e v 0 f o r m s or as racemic m i x t u r e . The o p t i c a l r e s o l u t i o n of methadone is r e p o r t e d by Brode a n d H i l l , 4 e L a r s e n et and T h o r p e t -. 4 8 t h r o u g h t h e u s e o f - al d - t a r t a r i c a c i d . Howe a n d S l e t ~ i n g e r a n d~ Howe , ~ and T i s h l e r 5 ' r e s o l v e d d l - m e t h a d o n e , o r i t s h y d r o c h l o r i d e , by f o r m i n g t h e e a s i l y p u r i f i e d , w a t e r - i n s o l u b l e d-a-bromocamphor-n-sulfonate o f t h e d- isomer. P u r e d-met hadone is p r e c i p i t a t e d by slow a d d i t i o n o f water. The 1-form is o b t a i n e d , f r o m t h e m o t h e r l i q u o r , by f o r m i n g t h e d - t a r t r a t e s a l t . When t h e 1-methadone is d e s i r e d , t h e d-isomer is removed f r o m a s o l u t i o n i n b u t y l a l c o h o l as t h e p-nitrobenzoyl-lglutamate. The u s e o f a-bromocamphor-n-sulfonic a c i d and p-nitrobenzoyl-L-glutamic a c i d a s t h e resolving agent reduces t h e excessive c r y s t a l l i z a t i o n time and s u b s t a n t i a l l y i n c r e a s e s t h e y i e l d s . ZauggS1 p a t e n t e d a s p e c i a l a p p a r a t u s t o p r o v i d e a new p h y s i c a l method f o r s i m u l t a n e o u s r e s o l u t i o n o f b o t h o p t i c a l isomers o f d l methadone. Zaugg e x p l a i n s t h a t " t h i s i n v e n t i o n is based o n t h e knowledge t h a t a seed c r y s t a l of t h e d e x t r o - r o t a t o r y isomer w i l l a t t r a c t t h e d - i s o m e r i n s a t u r a t e d s o l u t i o n , and when t h e d e g r e e of s a t u r a t i o n of t h e s o l u t e i n t h e s o l u t i o n is i n c r e a s e d , t h e d - i s o m e r w i l l t e n d t o c r y s t a l l i z e o u t o n t h e d - i s o m e r seed c r y s t a l . A t t h e same t i m e , a p o r t i o n o f t h e 1-isomer w i l l t e n d t o c r y s t a l l i z e o u t o n t h e 1 - i s o m e r seed c r y s t a l . T h i s p r o c e s s w i l l c o n t i n u e s o l o n g as t h e s o l u t i o n is s u p e r s a t u r a t e d w i t h t h e composit i o n o r s o l u t e and s e e d e d c r y s t a l s w i l l grow t o s u b s t a n t i a l size. A t t h e conclusion of t h e o p e r a t i o n , i t w i l l be f o u n d t h a t r e l a t i v e l y p u r e c r y s t a l s o f t h e d-isomer and 1-isomer w i l l h a v e b e e n grown o n t h e seed c r y s t a l s . "

3.2

e.,47

R e a c t i v i t y and S t a b i l i t y l o w r e a c t i v i t y of t h e c a r b o n y l g r o u p of methadone [11 is i n d i c a t e d by n o t g i v i n g t h e semicarbazone under t h e u s u a l c o n d i t i o n s and4.

392

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METHADONE HYDROCHLORIDE

r e s i s t i n g r e d u c t i o n w i t h aluminum i s o p r o p o x i d e or s o d i u m amalgam.36 The c o r r e s p o n d i n g c a r b i n o l [ 2 ] is formed w i t h p l a t i n u m o x i d e . A c e t y l a t i o n o f [ 2 ] y i e l d s t h e 0 - a c e t y l d e r i v a t i v e [3-a]. Reaction of [ 2 ] w i t h c h l o r i n a t i n g a g e n t s ( t h i o n y l c h l o r i d e or phosphorus p e n t a c h l o r i d e ) leads t o t h e f o r m a t i o n of a m i x t u r e o f 6-d i m e t hy l a m i n o - 4,4- d ipheny l-2- h e p t e n e [4 1 a n d 3-chloro-6-dimethylamino-4,4-diphenylheptane [51. Alkaline cleavage of t h e e t h y l k e t o group r e s u l t s i n t h e formation of 3-dimethylamino-1,ld i p h e n y l b u t a n e [6 1. Compound [ 6 ] is a l s o formed by r e f l u x i n g 4 - d i m e t h y l a m i n o - 2 , 2 d i p h e n y l p e n t a n e n i t r i l e [7] w i t h potassium hydroxide and t r i e t h y l e n e g l y c o l . Hydrogenation of t h e r e s u l t i n g o l e f i n C91 from t h e Hofmann d e g r a d a t i o n o f [8], t h e m e t h i o d i d e o f [6l (which is a l s o formed by a l k a l i t r e a t m e n t o f t h e m e t h i o d i d e o f [ 111, g i v e s 1 , l - d i p h e n y l b u t a n e [ l o ] . The l a t t e r compound is a l s o p r e p a r e d f r o m e t h y l butyrate [ll] v i a 1,l-diphenyl-1-butanol [12] w h i c h is h y d r o g e n a t e d t o [ l o ] w i t h p a l l a d i u m - c h a r c o a l o r p a l l a d i u m - b a r ium s u l f a t e c a t a l y s t i n t h e p r e s e n c e of a c e t i c a c i d c o n t a i n i n g traces o f p e r c h l o r i c a c i d . A l k a l i t r e a t m e n t o f a,a-diphenylvaleronitrile [131 g i v e s a l o w y i e l d o f [ l o ] a l o n g w i t h a,ad i p h e n y l v a l e r i c a c i d [14]. F i g u r e 9 shows t h e s e r e a c t i o n s . I n a l a t e r r e p o r t by May a n d P e r ~ - i n e h e~ s t r u c t u r e s of [41 a n d [53 were t~ p r o v e n t o be 6-d i m e t hylamino-3,4-d i p h e n y l - 3 h e p t e n e [15 I, a n d 4-chloro-6-dimethylamino-3,4d i p h e n y l h e p t a n e [16], r e s p e c t i v e l y . T h i s is d u e t o t h e Wagner's r e a r r a n g e m e n t of [21 t o g i v e 6-dimethylamino-3,4-diphenyl-4-heptanol [ l 7 ] , [151 and [161 d e p e n d i n g upon t h e r e a c t i o n conditions. I r r a d i a t i o n w i t h gamma r a y s ( c o b a l t - 6 0 1 , u l t r a v i o l e t , or t h e r m a l n e u t r o n s h a r d l y a f f e c t s t h e m e l t i n g p o i n t o f m e t hadone h y d r o c h l o r i d e . 5 3 , 5 4 However, t h e i r r a d i a t i o n p r o d u c e s a brown color, c h a n g e s o f pH i n s o l u t i o n , decreases s p e c i f i c r o t a t i o n , and m o d i f i e s t h e i n f r a r e d spectrum. Additional s p o t s appear
393

394

C3H7C0ZCZH5

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I I PhzCC3H7 -Ph2CC3H7

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Figure 9 .

S o m reactions of nethadone. 36

Reproduced by permission.

METHADONE HYDROCHLORIDE

on t h e t h i n l a y e r chromatogram of t h e i r r a d i a t e d sample. I r r a d i a t i o n is less d e s t r u c t i v e t o t h e s o l i d material t h a n t o t h e a q u e o u s solution. P h o t o l y s i s a n d r a d i o l y s i s of methadone h y d r o c h l o r i d e s o l u t i o n r e s u l t in t h e formation of 3 , 3 - d i p h e n y l - 2 - e t h y l i d e n e - 5 m e t hy 1 e t r a hyd r o f u r a n a n d 3 d i m e t hy l a m i n o - 1,lt d i p h e n y l b u t e n e , r e s p e c t i v e l y . " 9 56 S t o r a g e of $n o r g a n i c s o l u t i o n of methadone free base a t 30 C . shows t h e f o r m a t i o n of methadone-N-oxide b y t h i n l a y e r chrom atography a n d combined gas chromatography-mass s p e c t r o m e t r y a n a l y s e s . 5 7 The r e l a t i v e concent r a t i o n of t h e chemical o x i d a t i o n p r o d u c t , methadone-N-oxide, i n c r e a s e s w i t h t i m e of storage.

5.

Drug M e t-----i c P r o d u c t s a n d----.--- h a r m a c o k i n e t i c s abol -- P 5.1

Absorption A b s o r p t i o n o f methadone is r e l a t i v e l y p r o m p t . E x p e r i m e n t s w i t h "C-methadone show a p p r e c i a b l e c o n c e n t r a t i o n s of 1 4 C i n plasma58 a n d b i l e 5 9 w i t h i n 10 m i n u t e s a f t e r s u b c u t a n e o u s i n j e c t i o n of t h e labeled d r u g . F o l l o w i n g s u b c u t a n e o u s i n j e c t i o n of methadone i n r a t s , 47% of t h e dose r e m a i n s a t t h e i n j e c t i o n s i t e a f t e r 1 h o u r , 6 o 10-15$ a f t e r 2-3 h o u r s , 3% a f t e r 5 h o u r s , a n d v i r t u a l l y n o n e is p r e s e n t a f t e r 24 h o u r s . 6 1 S e v e n t y p e r c e n t of a methadone dose a d m i n i s t e r e d by stomach t u b e t o f a s t e d rats d i s a p p e a r s w i t h i n 2 h o u r s from t h e gastroi n t e s t i n a l t r a c t .'j2 Distribution Methadone m a i n l y l o c a l i z e s i n t h e l i v e r , k i d n e y s , l u n g s , a n d s p l e e n . B l o o d , heart, b r a i n , a n d m u s c l e show o n l y l o w level^.^'-^^ Methadone is a l s o c o n c e n t r a t e d i n t h e a d r e n a l s and Using s e n s i t i v e tracer t h y r o i d . 58,61965 t e c h n i q u e s , 6 5 methadone l e v e l s of 0.6 t o 0.9 p g . / g . of v a r i o u s s e g m e n t s of t h e c e n t r a l n e r v o u s s y s t e m are f o u n d 30 m i n u t e s a f t e r
395

5.2

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s u b c u t a n e o u s a d m i n i s t r a t i o n of 3 mg./kg. This correlates w e l l w i t h t h e i n t e n s i t y and d u r a t i o n of t h e a n a l g e s i c e f f e c t a s d e m o n s t r a t e d by t h e r e s c t i o n t i m e of t h e r a t t a i l t o t h e r m a l s t i m u l u s . E l l i o t t e t a l . 6 1 r e p o r t e d h i g h concen-t r a t i o n of methadone i n t h e g a s t r o i n t e s t i n a l t r a c t a f t e r s u b c u t a n e o u s a d m i n i s t r a t i o n of t h e d r u g . C o n s i d e r a b l e amounts of r a d i o a c t i v i t y a r e found i n t h e p l a c e n t a e and f e t u s e s of t h e p r e g n a n t r a t a f t e r t h e a d m i n i s t r a t i o n of 1 4 C l a b e l e d methadone.61 The methadone c o n c e n t r a t i o n i n t h e b r a i n of t h e f e t u s is 2-3 times t h e c o n c e n t r a t i o n found i n t h e m a t e r n a l b r a i n . " Methadone appears t o be f i r m l y bound t o t i s s u e p r o t e i n . 6 7 However, a c c u m u l a t i o n of t h e d r u g does n o t o c c u r t o a n y great e x t e n t . A large p a r t of t h e methadone p r e s e n t i n t h e whole a n i m a l is f o u n d i n t h e carcass m a i n l y t h e s k e l e t o n , mu s c l e , a n d bone. '9 61' A d i s t r i b u t i o n s t u d y of methadone i n man6' shows t h a t t h e blood c o n c e n t r a t i o n of t h e d r u g is l e s s t h a n b i l e a n d u r i n e c o n c e n t r a t i o n s . The k i d n e y a n d l i v e r c o n c e n t r a t i o n s are a p p r o x i m a t e l y e q u i v a l e n t . B r a i n t i s s u e is t h e p o o r e s t s o u r c e of methadone and l u n g t h e r i c h e s t . B i n d i n g of me t h a d o n g 9to human plasm a a l b u m i n is reported by O l s e n .
5.3

c a r b o n atoms of methadone a r e n o t removed by o x i d a t i o n w a s d e m o n s t r a t e d by E l l i o t t e t a l . ' l No 1 4 C 0 2 is e l i m i n a t e d by r a t s g i v e n m Z h a d o n e labeled w i t h l 4 C i n p o s i t i o n 2 . C o n t r a r y t o these e a r l y data, t h e r e c e n t work by S u l l i v a n 2 4 shows t h a t a b o u t 1s of t h e dose of 14C l a b e l e d methadone i n p o s i t i o n 2 is e l i m i n a t e d as 1 4 C 0 2 . The p r e s e n c e of 4-dimethylamino-2,2d i p h e n y l v a l e r i c a c i d i n u r i n e of humans is a l s o a n i n d i c a t i o n of t h e s i d e c h a i n o x i d a t i o n , 5 7 The l i v e r a p p e a r s t o be t h e o r g a n c h i e f l y r e s p o n s i b l e f o r t h e metabolism of methadone. 9 O- 73

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396

METHADONE HYDROCHLORIDE

The major metabolites of methadone i n humans are shown i n F i g u r e 10.57,74 The primary metabolite o f methadone [11 is formed by N-demethylation t o y i e l d t h e u n s t a b l e t ~ N-desmethylmethadone [2 3 which i s ~ y c l i z e d o ~ 1,5-dimethyl-3 3 - d i p h e n y l - 2 - e t h y l i d e n e p y r r o l i d i n e [3>. F u r t h e r N-demethylation o f [3 1 forms 2 - e t hy 1-5-met hy l-3,3-d ipheny 1-1-p y r r o l i n e [el. Both [3] and [ 4 ] and t h e i r c o r r e s p o n d i n g r i n g hydroxylated analogs, 2-ethylidene-l,5d i m e t hyl-3- (p-hydroxyphenyl - 3 - p h e n y l p y r r o l i d i n e [:5 3 and 2- et hy 1 5-me t hy 1-3- (p- hy d r oxy p heny 1 -3 p h e n y l - 1 - v g r r o l i n e [S 1 are detected i n human , u r i n e .76I n a minor pathway, t h e k e t o group of methadone is e n z y m a t i c a l l y r e d u c e d S B t o form methadol [ 7 ] which is N-demethylated t o y i e l d normethadol [81 which is excreted i n t h e u r i n e . 8 0 I n a r e l a t i v e l y minor pathway t h e s i d e c h a i n o f methadone is o x i d i z e d t o form 4-dimethylamino2 , 2 - d i p h e n y l v a l e r i c a c i d 191 w h i c h s u b s e q u e n t l y N-demethylates, i n p a r t , t o a n o n - i s o l a t e d i n t e r m e d i a t e , 4-met hy lamino-2,2- d ipheny l v a l e r i c acid [lo]. Ring c l o s u r e ( c y c l i z a t i o n ) of t h e i n t e r m e d i a t e [lo] y i e l d s 1,5-dimethyl-3,3d ipheny l-2- p y r r o l idone [ 1 1. 1 In a d d i t i o n t o t h e p r e v i o u s l y mentioned p h e n o l i c metabolites [ 5 3 and c61, r i n g h y d r o x y l a t e d methadone [12] is a l s o found i n t h e u r i n e of s u b j e c t s m a i n t a i n e d o n methadone.57 Methadone N-oxide [13] is found i n u r i n e from s u b j e c t s r e c e i v i n g a s i n g l e dose o f methadone, i n u r i n e from a d d i c t s b e i n g treated w i t h t h e drug, 74 a n d in u r i n e of r a t s . 1 0 However, t h e work o f S u l l i v a n and Due57 i n humans i m p l i e s t h a t t h e r e is some q u e s t i o n a s t o whether t h e methadone N-oxide is a t r u e m e t a b o l i t e o r a n a r t i f a c t caused by o x i d a t i o n ,

Excretion Way and Adler" i n d i c a t e d t h a t less t h a n 10% of methadone is e x c r e t e d unchanged i n t h e u r i n e and i n t h e feces. U r i n a r y e x c r e t i o n s t u d i e s show v a r i o u s c o n c e n t r a t i o n s o f methadone
--I--

5.4

397

RAFlK H. BISHARA

. Figure 10. Major mtabolites of mthadone in h

57,74

398

METHADONE HYDROCHLORIDE

i n u r i n e . Recovery of 4 s t o 35% o f t h e a d m i n i s t e r e d dose is r e p o r t e d . 8 2 - 8 5 Twenty-four h o u r s a f t e r a d m i n i s t r a t i o n of methadone t o r a t s , t h e unchanged d r u g found i n u r i n e and feces is 4-11s and 19-24s r e s p e c t i v e l y from t h e a d m i n i s t e r e d dose.6a However, Way e t g . , 8 2 u t i l i z i n g c o u n t e r - c u r r e n t t e c h n i q u e s , showed t h a t t h e p r e v i o u s v a l u e s are h i g h and . recommended a f a c t o r of 0 8 a n d of 0.25 f o r t h e c o r r e c t i o n of t h e u r i n a r y and f e c a l e x c r e t i o n respectively. B i l a r y e x c r e t i o n is r e p o r t e d t o be a n i m p o r t a n t avenue f o r t h e e l i m i n a t i o n of methadone and its b i o t r a n s f o r m a t i o n p r o d u c t s , 5 9 ~ 6 3 - 65 ~ 8 2 The r e s u l t s of Baselt and Casarett' demonstrated t h a t , i n man, r e n a l e l i m i n a t i o n may become t h e major e x c r e t o r y pathway a f t e r d a i l y d o s e s g r e a t e r t h a n 55 mg. S i x t y p e r c e n t of a 160 m g . dose of methadone p e r day is e x c r e t e d a s unchanged d r u g i n u r i n e . T h e s e r e s u l t s are i n c o n f l i c t w i t h t h o s e o f S u l l i v a n and Due5' who r e p o r t e d t h a t a r e l a t i v e l y small p o r t i o n of a n 8 0 mg. dose of methadone w a s found unchanged i n t h e u r i n e of h e r o i n maintenance s u b j e c t s . U r i n a r y methadone e x c r e t i o n is markedly enhanced by a c i d i f i c a t i o n of t h e u r i n e . ' A sex d i f f e r e n c e i n t h e p a t t e r n o f e x c r e t i o n of methadone and its metabolites is found and is r e l a t e d t o t h e r a t e of b i o t r a n s f o r m a t i o n of t h e drug. Methadone, metabolites C33 and L43 (See F i g u r e 101, are p r e s e n t i n s u f f i c i e n t l y h i g h c o n c e n t r a t i o n i n human sweat t o s u g g e s t t h a t sweat may be a s i g n i f i c a n t r o u t e of e l i m i n a t i o n of t h i s d r u g . 8 6 The mean a p p a r e n t h a l f - l i f e of orallya d m i n i s t e r e d methadone is 15 hours." Following i n t r a m u s c u l a r a d m i n i s t r a t i o n , t h e h a l f - l i f e is 7.3 hours.88 S u b j e c t s of a methadone maintenance program who r e c e i v e a large o r a l dose of 100 or 120 m g . show a mean a p p a r e n t h a l f - l i f e of methadone t o be 2 5 hours.''

399

RAFlK H. EISHARA

A n a l y t i c a l procedures for i s o l a t i o n , d e t e r m i n a t i o n , a n d i d e n t i f i c a t i o n of methadone and its metabolites from b i o l o g i c a l f l u i d s a n d t i s s u e s i n c l u d e colorimetric a n d p h o t o m e t r i c techni ues following i n t e r a c t i o n with indicator 3 6 e 2 , 6 4 , 6 7 9 70, 8 2 - 8 4 p a p e r chromato 72,90,91 column chromato r a hy, 79,8i g a s raphy, 9i2 t h i n l a y e r c h r o ma t o g r a p h y , 9, 6 8 , 7 4 9 7 6 - 7 8 , c h r o m a t o g r a p h y , 9, 9 54, 9 9 7 0 repeated c o u n t e r - c u r r e n t t r a n s f e r , 8 1 radiotracer methods, 4 5 9 5 8 - 6 1 , 659 72, 8 0 i n rare 76 8 1 , 9 2 n u c l e a r m a g n e t i c r e s o n a n c e , 75, 76, g h , 95 and combined gas chromatography-mass s p e c t r o m e t r y . 579 7 4 9 7 7 - 8 0 Identification E m a d o n e h y d F o c h l o r i d e c a n be i d e n t i f i e d by v i r t u e of its c h a r a c t e r i s t i c x - r a y powder d i f f r a c t i o n p a t t e r n , W , I R , a nd NMR s p e c t r a (See 2.10, 2.11, 2.12, a n d 2 . 1 3 ) . The charact e r 5 s t i c m e l t i n g p o i n t of a b o u t 160 C . , or a b o u t 180 C . of t h e c r y s t a l s formed by p i c r o l o n i c a c i d and methadone is a l s o u s e f u l as a n i d e n t i t y t e ~ t . 3 , A d d i t i o n of 2 m l . of m e t h y l o r a n g e ~ test s o l u t i o n 5 t o a 0.5% methadone h y d r o c h l o r i d e s o l u t i o n forms a y e l l o w p r e c i p i t a t e . The m e l t i n g p o i n t of t h e water-washed r e s i d u e , formed by a d d i n g excess s o d i u m h y d r o x i d e s o l u t i o n 3 t o a 5% s o l u t i o n of methadone, is a b o u t 76C. Methadone h y d r o c h l o r i d e reacts p o s i t i v e l y t o t h e chloride characteristic test w i t h s i l v e r nitrate.3~ 5
6.

Microchemical React i o n s The microchemical i d e n t i f i c a t i o n of methadone t h r o u g h t h e f o r m a t i o n of c r s t a l s w i t h c e r t a i n r e a g e n t s h a s b e e n r e p o r t e d . ?12,93-97 Hubach and J o n e s 1 2 l i s t e d s e v e n r e a g e n t s w h i c h produced c h a r a c t e r i s t i c m i c r o s c o p i c c r y s t a l s from d i l u t e a q u e o u s m e t hadone h y d r o c h l o r i d e s o l u t i o n s . E q u a l d r o p s of t h e r e a g e n t and t h e methadone s o l u t i o n a r e mixed on a microscopic s l i d e and a l l o w e d t o s t a n d u n t i l c r y s t a l s d e v e l o p or a l l l i q u i d e v a p o r a t e s . The c r y s t a l s are t h e n
7.
400

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METHADONE HY DROCH LORlDE

o b s e r v e d and examined m i c r o s c o p i c a l l y . The r e a g e n t s u s e d and t h e lowest c o n c e n t r a t i o n o f methadone h y d r o c h l o r i d e from which c r y s t a l s a r e o b t a i n e d are p r e s e n t e d i n T a b l e 4 a l o n g w i t h t h e d e s c r i p t i o n of t h e formed c r y s t a l s . F i v e m o d i f i e d c o b a l t (11) t h i o c y a n a t e s o l u t i o n s g i v e t u r q u o i s e color w i t h m e t h a d o n e . @ 8 A s o l u t i o n c o n s i s t i n g of 0.8% w/v c o b a l t (11) t h i o c y a n a t e i n a ( 2 : 3 ) v/v m i x t u r e of m e t h a n o l o and 1% r t h o p h o s p h o r i c a c i d ( s p . g r . 1.75) g i v e s a c o l o r r e s p o n s e w i t h i n 5 s e c o n d s from t h e a d d i t i o n of t h e r e a g e n t .

8.

Methods of A n a l y s i s
8.1

E l e m e n t a l A n a l y s i s (As C21H2,NO*HC1)
E l e me n t -----

--72.91 8.16 4.05 4.63 10.25

Theory 1

' Determined -$---------- 47,459

73.14 8.03 3.96 72.77, 72.90 7.98, 8.36
3.88

C
H

73.06, 72.95 8.23, 7.99 4.07

N
0

c1
8.2

T i t r a t ion -----8.2.1
_ I -

Non-Aqueous T i t r a t i o n The t e r t i a r y amine g r o u p of methadone h y d r o c h l o r i d e is d i r e c t l y t i t r a t e d i n non-aqueous medium, g l a c i a l a c e t i c a c i d , i n t h e p r e s e n c e of m e r c u r i c a c e t a t e t o t i e up t h e c h l o r i d e i o n . The t e c h n i q u e of t h e non-aqueous t i t r a t i o n of t h e d r u g u s i n g v i s u a l i n d i c a t o r s ,

40 1

R A F l K H. BISHARA

TABLE 4 MICROCHEMICAL CRYSTALLIZATION OF METHADONE HYDROCHLORIDE Concentrat i o n of Methadone HC1 R e a c t i o n

1:1,000 1:500 1:500
1:10,000

Reagent P o t a s s i u m i o d i d e , 5% Potassium ferrocyanide, 5% ( f r e s h s o l u t i o n ) Potassium f e r r i c y a n i d e , 5% ( f r e s h s o l u t i o n )

Colorless c r y s t a 1s
Colorless crystals Yellow crystals

Marme' s r e a g e n t (fresh solution)

Mayer's r e a g e n t Wagner's r e a g e n t

Colorless c r y s t a 1s
Colorless crystals Pale brown crystals

1:20,000
1 : 1,000

Lanthanum n i t r a t e , 20%

1:20

Color less crystals

402

METHADONE HYDROCHLORIDE

c r y s t a l v i o l e t 3 j 5 or m e t h y l v i o l e t j 6 h a s b e e n d e s c r i b e d i n d e t a i l . Each 1 m l . of 0 . 1 N p e r c h l o r i c a c i d , i s e q u i v a l e n t t o 34.59 m g . of C21H2,NO*HC1. P o t e n t i o m e t r i c e n d p o i n t detect i o n has also been used. 9 9

Direct T i t r a t i o n J o h n s o n a n d K i n g l o o developed a r a p i d d i r e c t t i t r i m e t r i c method, u s i n g a n e x t r a c t i v e end p o i n t , f o r d e t e r m i n a t i o n of methadone i n p h a r m a c e u t i c a l p r e p a r a t i o n s . C h l o r o f o r m is a d d e d t o t h e o r g a n i c base d i s s o l v e d i n pH 2 . 8 a c e t a t e b u f f e r s o l u t i o n s o t h a t t h e r a t i o of chloroform t o t h e a q u e o u s p h a s e is a b o u t 3 t o 1. T i t r a t i o n is p e r f o r m e d u s i n g sodium d i c o t y l s u l f o s u c c i n a t e a n d D i m e t h y l Y e l l o w s c r e e n e d w i t h Oracet B l u e as i n d i c a t o r . The c h a n g e of c o l o r of t h e c h l o r o f o r m p h a s e from g r e e n t o p i n k i n d i c a t e s t h e end p o i n t . The a c c u r a c y of t h e method is f 1%.
8.2.2

C h l o r i d e Determination (Mercuric Nitrate T i t r a t i o AsErnxeoPmeZkHaone hydrochloride c o n t a i n i n g a t l e a s t 2 mg. of c h l o r i n e is d i s s o l v e d i n 80 m l . of w a t e r - m e t h a n o l ( 2 0 : 6 0 ) m i x t u r e a n d t h r e e d r o p s of d i p h e n y l c a r b a z o n e s o l u t i o n ( 5 mg./ml. m e t h a n o l ) are a d d e d . The s a m p l e s o l u t i o n is t h e n t i t r a t e d w i t h s t a n d a r d 0.5 N m e r c u r i c n i t r a t e s o l u t i o n t o t h e first s i g n of rose c o l o r , u s i n g a one-ml. microburette. percent chlorine =

8.3

_-----___

________-_

____---

mL m e r c u r i c n i t r a t e x n o r m a l i t y mg. s a m p l e

_--------

35.5

100

p e r c e n t p u r i t y of m e t hadone h y d r o c h l o r i d e = perce hl __-___ n rt c c _---o r ihnleo-rfionuen- dh eXo r100 - t y pe ent c

-. I_ . .

403

R A F l K H. BISHARA

Ultraviolet Analysis Wallace e t a l . 1 0 1 oxidized methadone w i t h b a r i u m p e r o x i d e t o benzophenone w h i c h was e x t r a c t e d i n h e p t a n e a n d measured s p e c t r o p h o t o m e t r i c a l l y a t 247 nm., E = 18,713. T h i s r e p r e s e n t s a n i n c r e a s e i n molar a b s o r b a n c e of a p p r o x i m a t e l y 34 times o v e r t h a t of methadone i n 0.1 N h y d r o c h l o r i c a c i d , g = 554, a t 292 nm. The method is s u c c e s s f u l l y u s e d f o r d e t e r m i n i n g methadone i n b i o l o g i c a l s p e c i m e n s , namely u r i n e , l i v e r , l u n g s , k i d n e y s , stomach, and i n t e s t i n e s .

8.4

--

F--o-o m e t r i c A n a l y s i s u-l-- r The f o r m a t i o n of a f l u o r o p h o r e when methadone is h e a t e d i n a s o l u t i o n of form aldehyde and c o n c e n t r a t e d s u l f u r i c a c i d followed by a d d i t i o n of water is reported by McGonigle. 1 0 2 The f l u o r e s c e n c e is recorded a t a n e x c i t a t i o n w a v e l e n g t h of 270 nm. a n d a n e m i s s i o n w a v e l e n g t h of 450 nm. The a q u e o u s f l u o r o p h o r e is s t a b l e f o r a t l e a s t 1.5 h o u r s a t room t e m p e r a t u r e . T h i s p r o c e d u r e is s u i t a b l e f o r m e a s u r i n g microgram q u a n t i t i e s of methadone. Morphine, heroin, codeine, and c o c a i n e do n o t i n t e r f e r e . However, p r i o r s e p a r a t i o n of amphetamine, m e p e r i d i n e , and q u i n i n e is r e q u i r e d s i n c e t h e y f l u o r e s c e u n d e r t h e c o n d i t i o n s of t h e a s s a y and i n t e r f e r e w i t h t h e d e t e r m i n a t i o n of methadone. The r e l a t i v e s t a n d a r d d e v i a t i o n of t h e methadone a s s a y is 2.2%. A p p l i c a t i o n of f l u o r e s c e n c e and gas c h r o m a t o g r a p h y t o mass d r u g s c r e e n i n g is p r e s e n t e d by S a n t i n g a . l o 3 The smallest d e t e c t a b l e q u f B 4 i t y of methadone is 1 p g . / m l . Lawler et a l . used spectrophotofluorometry for t h e d e t e r m i n a t i o n of methadone i n t i s s u e s .
8.5

8.6

hydrochloride s o l u t i o n is made a l k a l i n e w i t h (1:l) sodium h y d r o x i d e i n water. The p r e c i p i t a t e d base is e x t r a c t e d w i t h chloroform. The e x t r a c t s are d r i e d o v e r anhydrous sodium s u l f a t e a n d t h e chloroform is e v a p o r a t e d u s i n g steam heat a n d
404

Infra a s i s !3 - r e d A nofl ymethadone The sample

METHADONE HYDROCHLORIDE

a c u r r e n t of a i r . The r e s i d u e is d i s s o l v e d i n chloroform a n d t h e a b s o r b a n c e , v e r s u s a chloroform b l a n k , is d e t e r m i n e d a t 5.87 using 0.1 mm. c e l l s a n d a Beckman IR spectrophotometer. The a b s o r b a n c e of t h e s t a n d a r d is d e t e r m i n e d i n t h e same manner a n d t h e m g . methadone h y d r o c h l o r i d e is c a l c u l a t e d from t h e r e l a t i v e r a t i o . U l t r a v i o l e t i r r a d i a t i o n of a m e t h a n o l i c s o l u t i o n of methadone h y d r o c h l o r i d e , f o r 1 . 5 h o u r s , c a u s e s 74% d e t e r i o r a t i o n a s shown by
t h i s method.

Colorimetric A n a l y s i s D r ug s c o n t a i n i n g b a s i c g r o u p s form a colored complex w i t h s u l f o n i c a c i d i n d i c a t o r d y e s s u c h as Methyl Orange a n d Bromcresol G r e e n . lo' The colored complex is t h e n s e p a r a t e d from t h e e x c e s s dye by e x t r a c t i o n i n t o chloroform o r a s u i t a b l e o r g a n i c s o l v e n t a n d t h e q u a n t i t y of t h e complexed d r u g is estimated spectrophotometrically. The methadone-dye complex is formed u s i n g bromthymol b l u e , 83 bromcresol g r e e n , 8 4 bromphenol b l u e , 8 4 b r o m c h l o r o p h e n o l b l u e , 8 4 c h l o r o p h e n o l r e d , 8 4 bromcresol p u r p l e , 8 4 , 1 0 7 a n d m e t h y l o r a n g e . 6 2 9 8 2 With these dyes, a n y basic a m i n e w h i c h c a n form a n o r g a n i c s o l v e n t s o l u b l e d y e complex would react as methadone, h e n c e p r e c a u t i o n s n e e d t o be t a k e n t o e l i m i n a t e i n t e r f e r i n g substances. For the determination of methadone i n t i s s u e s , R i c k a r d s e t al.GO l i b e r a t e d t h e compound by d i s i n t e g r a t i o n of t h e t i s s u e w i t h s t r o n g a l k a l i , ether e x t r a c t i o n , n i t r a t i o n of t h e p h e n y l r a d i c a l s i n methadone, c o l o r d e v e l o p me n t w i t h e t h y l m e t h y l k e t o n e a n d m e a s u r i n g t h e c o l o r a t 565 nm. The r e p r o d u c i b i l i t y a n d s e n s i t i v i t y are f 5% a n d 1 p g . of methadone. However, i t is t o be n o t i c e d t h a t a n y methadone metabolic f r a g m e n t r e t a i n i n g t h e p h e n y l a n d a mi n e g r o u p s would react a s t h e p a r e n t s u b s t a n c e . 8 i N i t r a t i o n of methadone w i t h a m i x t u r e of HN03/H2S04 (1:l) a n d t h e s u b s e q u e n t p h o t o m e t r i c d e t e r m i n a t i o n of t h e
8.7

--

405

RAFlK H . BISHARA

colored d e r i v a t i v e u s i n g f i l t e r S42 (428 nm.) is p r e s e n t e d by S k o r a . l 0 * The method is used f o r d e t e r m i n a t i o n of methadone i n a mpoules, t a b l e t s , and b i o l o g i c a l media (blood, l i v e r a n d u r i n e ) . An a u t o ma t e d method f o r t h e d e t e r m i n a t i o n of methadone h y d r o c h l o r i d e i n d i s s o l u t i o n s a m p l e s w a s d e v e l o p e d by B e c h t e l a n d

B r i c k l e y . l o 9 The d i s s o l u t i o n s a m p l e s a r e s e q u e n t i a l l y s a mp l e d by a n A u t o Analyzer a n d i n j e c t e d i n t o a n a i r - s e g m e n t e d stream of pH 1 . 2 b u f f e r ( U . S . P . ) w h i c h is pumped t h r o u g h a g l a s s mixing c o i l , Bromcresol p u r p l e (BCP) dye s o l u t i o n is t h e n a d d e d t o t h e stream of t h e methadone s a m p l e a n d t h e s o l u t i o n s are mixed i n a T e f l o n c o i l f o r t h e f o r m a t i o n of t h e dyecomplex. The methadone-dye complex is extracted i n t o e t h y l e n e d i c h l o r i d e and t h e color i n t e n s i t y is measured a t 4 2 0 nm. The r e l a t i v e s t a n d a r d d e v i a t i o n (R.S.D.) a n d t h e r e l a t i v e error (R.E.) are f 1.48% and + 0.05% r e s p e c t i v e l y . The s p e c i f i c a s s a y of basic d r u g s i n u r i n e by C M - c e l l u l o s e column c h rom atography u s i n g c o n t i n u o u s d r u g - d y e complex e x t r a c t i o n as a d e t e c t i o n s y s t e m is d e s c r i b e d by McMartin et - a l , lo6 Polarography --- k e t o g r o u p of methadone is n o t The polarographically r e d u c i b l e because t h e double bonds are n o t c o n j u g a t e d . T h e r e f o r e , a n e l e c t r o a c t i v e n i t r o d e r i v a t i v e is prepared. S k o r a l o 8 n i t r a t e d methadone u s i n g a m i x t u r e of n i t r i c a c i d / s u l f u r i c a c i d ( 1 : l ) . N i t r a t i o n is c o m p l e t e d i n 30 m i n u t e s a f t e r h e a t i n g on a b o i l i n g water b a t h . The m i x t u r e is t h e n cooled, d i l u t e d w i t h 5 m l . d i s t i l l e d water and made a l k a l i n e , pH 10, w i t h 5 N NaOH. T h r e e d r o p s of 0.5% g e l a t i n s o l u t i o n are a d d e d a n d t h e s o l u t i o n is p o l a r o g r a p h e d , a f t e r d e o x y g e n a t i o n , i n a p o t e n t i a l r a n g e of - 0 . 4 t o -1.2 V . The h a l f wave p o t e n t i a l of t h e n i t r a t e d methadone is - 0 . 6 4 V r e l a t i v e t o a s a t u r a t e d calomel e l e c t r o d e . A s t r a i g h t l i n e is o b t a i n e d when t h e c o n c e n t r a t i o n of t h e n i t r a t e d methadone
8.8
406

METHADONE HYDROCHLORIDE

d e r i v a t i v e is p l o t t e d v e r s u s t h e d i f f u s i o n c u r r e n t ( h e i g h t of t h e r e d u c t i o n w a v e ) f o r concent r a t i o n s of 2 0 , 30, 40, 50, and 60 p g . / m l . C o n c e n t r a t i o n s lower t h a n 5 Ccg./ml. c a n be d e t e r m i n e d . The r e d u c t i o n wave of methadone n i t r a t e is d i s t i n c t and t h i s method is u s e d f o r a s s a y i n g methadone i n ampoules and t a b l e t s . A p p l i c a t i o n of t h e method t o a s s a y f o r t h e d r u g i n blood, l i v e r and u r i n e is d i s c u s s e d . C a t h o d e r a y p o l a r o g r a p h y of methadone N-oxide i n W a l p o l e ' s a c e t a t e b u f f e r pH 5 g i v e s a r e d u c t i o n wave w h i c h h a s a peak p o t e n t i a l of - 1 . 2 1 V . R e d u c t i o n of t h e same s o l u t i o n w i t h T i C l , / H C l g i v e s methadone. 74 Bioassay S ~ h a u m a n nr ~ D o r t e d a bioassay e~ p r o c e d u r e f o r methadone ;sing i s o l a t e d g u i n e a p i g g u t . The method is s e n s i t i v e t o methadone a t a c o n c e n t r a t i o n as low as lo-* M.
8.9

S p i n Immunoassay F r e e - r a d i c a l t e c h n o l o g y is combined w i t h immunoassay t o y i e l d t h e new method of " s p i n immunoassay" used f o r d e t e c t i o n and a s s a y of small m o l e c u l e s i n b i o l o g i c a l f l u i d s . l l 0 - 1 1 2 An a n t i b o d y is made a g a i n s t t h e h a p t e n e t o be a s s a y e d . The a n t i g e n is first p r e p a r e d by c o u p l i n g t h e h a p t e n e t o b o v i n e serum a l b u m i n and immunizing r a b b i t s or g o a t s . Ammonium s u l f a t e is used t o p r e c i p i t a t e t h e y - g l o b u l i n f r a c t i o n of t h e s e r u m , The h a p t e n e is s p i n l a b e l e d u s i n g a s t a b l e n i t r o x i d e r a d i c a l . The s p i n of t h e u n p a i r e d e l e c t r o n of t h e l a b e l e d compound prod u c e s a m a g n e t i c moment w h i c h is detected and measured i n a n e l e c t r o n s p i n r e s o n a n c e (ESR) spectrometer. Very broad s p e c t r a l p e a k s a r e o b s e r v e d when t h e complex of s p i n - l a b e l e d h a p t e n e w i t h a n t i b o d y i n a c a p i l l a r y t u b e is placed i n t h e c a v i t y of t h e ESR spectrometer. T h i s r e f l e c t s t h e i m m o b i l i z a t i o n of t h e f r e e r a d i c a l a t o r n e a r t h e a n t i b o d y s i t e . Sharp p e a k s r e s u l t from t h e d i s p l a c e m e n t of t h e s p i n l ab eled h a p t e n e by f r e e h a p t e n e (as by methadone
407

8.10

RAFlK H. BISHARA

i n u r i n e or s a l i v a ) . The a m p l i t u d e s of t h e s h a r p p e a k s m e a s u r e q u a n t i t a t i v e l y t h e number of t h e f r e e r a d i c a l molecules tumbling f r e e l y i n s o l u t i o n a n d is a d i r e c t m e a s u r e of t h e h a p t e n e c o n c e n t r a t i o n , A c o n c e n t r a t i o n greater t h a n 5 X M o f methadone h y d r o c h l o r i d e or methadone c y c l i c metabolites is r e q u i r e d t o p r o d u c e s p i n immunoassay r e s p o n s e s e q u i v a l e n t t o 0.5 p g . / m l . (1.8 X M ) o f m o r p h i n e . The advantages, d i s a d v a n t a g e s and comparison o f t h i s t e c h n i q u e t o t h i n l a y e r c h r o m a t o g r a p h y are d i s c u s s e d b y L e u t e e t al.''O

--

Radiotracer Techniques l 4 C and 3H a r e a - = l a b e l methadone. The 1 4 C a c t i v i t y is a s s a y e d a s a t h i n l a y e r of b a r i u m c a r b o n a t e mounted on aluminum d i s c s a n d c o u n t e d u n d e r a b e l l c o u n t e r u s i n g a t h i n mica window5' and a G e i g e r - M u l l e r c o u n t e r . In r e c e n t y e a r s , l i q u i d s c i n t i l l a t i o n c o u n t e r s have b e e n u s e d f o r d e t e r m i n i n g t h e r a d i o a c t i v i t y of l 4 C a n d 3 H l a b e l e d samples.10,66,114-116
8.11

Column Chromatography ----------_A p p l i c a t i o n of C M - c e l l u l o s e column c h r o m a t o g r a p h y f o l l o w e d by c o n t i n u o u s dye complex e x t r a c t i o n is u s e d f o r t h e a s s a y of methadone i n human u r i n e . '06 S a m p l e s c o n t a i n i n g methadone a r e i n t r o d u c e d o n Amberlite XAD-2 r e s i n column a n d e l u t e d w i t h m e t h a n o l , 7 9 9 '17, methanolchloroform- i s o p r o p a n o l ammonia (300: 51, (3:1), ' ' 9 and waterso p r i o r t o f u r t h e r a n a l y s i s . The XAD-2 r e s i n is a s t y r e n e - d i v i n y l b e n z e n e copolymer and h a s t h e c a p a b i l i t y of a d s o r b i n g many w a t e r - s o l u b l e o r g a y & compounds p r i n c i p a l l y by Van d e r Waal forces. The e f f e c t i v e n e s s of r e c e n t l y d e v e l o p e d r e s i n s BRX-SM-1, -2, -4, a n d P o r a p a k t y p e Q a r e compared w i t h XAD-2 r e s i n by Bastos -- e t a1.12* The r e c o v e r y o f 3H-methadone is somewhat s i m i l a r f o r e a c h r e s i n a n d r a n g e s from 4 5 . 4 t o 56.0%.

8.12

''

'"

408

METHADONE HYDROCHLORIDE

Methadone is r e s o l v e d from o p i a t e s a n d d i l u e n t s i n i l l i c i t n a r c o t i c m i x t u r e s by u s i n g a column of SE-Sephadex C-25 i o n - e x c h a n g e r . 12' D i s p o s a b l e c h r o m a t o g r a p h i c columns a r e u s e d t o e x t r a c t methadone from u r i n e . 1 2 2 P a p e r Chromatograph Paperchromatograp y o n b u f f e r e d Whatman N o . 1 is u s e d by A x e l r o d g o t o p r o v e t h e N-demethylat i o n of methadone. T a b l e 5 summarizes t h e p a p e r chromat o g r a p h y s y s t e m s for methadone.
8.13

T h i n L a y e r Chromatography (TLC) A c o m p a r i s o n of t h e t h i n laser chromat o g r a p h y of methadone on s e v e n c o m m e r c i a l l y a v a i l a b l e s i l i c a g e l c o a t e d f i l m s a n d sheets w i t h s i l i c a gel coated glass p l a t e s u s i n g chloroform/n-butanol/ammonium h y d r o x i d e (70:40:5) a n d benzene/dioxane/ethanol/ammonium , h y d r o x i d e ( 5 0 : 5 0 : 5 : 5 ) is p r e s e n t e d by Schweda. l Z 9 The h a n d - c o a t e d s i l i c a g e l l a y e r on t h e g l a s s p l a t e s is t h e most v u l n e r a b l e l a y e r . The f i l m s are s u p e r i o r t o i t . The s i l i c a g e l s h e e t s r e q u i r e c a r e f u l h a n d l i n g . Ho e t a l . 1 3 0 u s e m i n i t h i n l a y e r p l a t e s (3 X 3 c m . ) to detect t h e p r e s e n c e of methadone, 100 p g . / m l . , in an u n h y d r o l y z e d u r i n e s a m p l e . The d e v e l o p i n g t i m e is u s u a l l y 1.5 min. Copenhaver a n d B l o s e l 3 l u s e s l i d e s t o prepare t h i n layer microplates for f a s t d e t e c t i o n of methadone a n d o t h e r d r u g s of abuse i n urine. Guptal32 u s e s d i s p o s a b l e p l a s t i c bags t o r u n t h e t h i n l a y e r chromatography o f methadone, m e t h a d o l , n o r m e t h a d o l , a n d acetylmethadol. I d e n t i c a l r e s u l t s are obtained from p l a t e s d e v e l o p e d i n t h e p l a s t i c bag or i n a g l a s s t a n k . Dole - e t a1.133-135 u s e i o n - e x c h a n g e p a p e r e x t r a c t i o n p r i o r t o TLC. Two-dimensional TLC is u s e d f o r i d e n t i f i c a t i o n of t h e m e t a b o l i t e s of methadone.80 F i s h e r - a l . 1 3 6 c h r o m a t o g r a p h et methadone on p r e c o a t e d , f l e a b l e t h i n l a y e r s h e e t s . The s e n s i t i v i t y of TLC f o r d e t e c t i o n o f methadone is d i s c u s s e d by G o r o d e t z k y . l 3 7

8.14

409

TABU 5
PAPER CHROMATOGRAPHY SYSTEMS FOR METHADONE

- o l v e n t System S
tert. -Amy1 alcohol/ n-butyl ether/water (80: 7: 1 3 1

Paper Schleicher & S c h u l l , #591-C, pH 3.0 pH 4 . 0 pH 5.0 pH 6.0 Schleicher & S c h u l l , #2045b

Detection* IP

( x 100)

Reference

123

35
49 22

56
BCG
78

f
0

water (12:1:7 )

Butanol/formic

acid/

124 91, 124

D i c h l o r o e t hane/ g l a c i a l acetic acid/ water (20:8:2) n-Butanol/c i t r i c a c i d / water (50: 1:50)' Whatman No. 1 d i p p e d i n 5% sod i urn hy d r og e n c i t ra t e D

75

74

1 25

(cont h u e d

...)

TABLE 5 ( c o n c l u d e d )

So -- l v e n t System --

Paper
2

Rf D e t e c t i o -* ( x 1 0 0 ) n IP 86' 95" 86" 86" 95" 95" 86O 95" 86" 93 88 76 75 67 59

Reference 126 126

Acetate b u f f e r ,
pH 1.00 pH 3.30 pH 4 . 5 8 pH 7 . 4 0

Whatman No. 3 impregnated with tributyrin (1% v/v i n acetone)

126

1 2

126 126 128 126 126 128

M/15 P h o s p h a t e b u f f e r , pH 7 . 4 0

'Upper
2

layer was u s e d
127

A c c o r d i n g t o Vogel

*Key - BCG :

bromcresol green, D :

Dragendorf f ,

IP :

iodoplatin a t e

TABLE 6 THIN LAYER CHROMATOGRAPHY SYSTEMS FOR METHADONE Solvent System --Benzene/ethyl acetate/ methanol/ammonium hydroxide ( 0:20:1.2 :0.1) 8 / Et h 1 acetate/me t hano1 y ammonium hydroxide (85:10:5) Rf (X 100) Adsorbent* Detection* - SG SG SG SG SG SG SG SG SG SG SG SG C C SG Reference 10 123 138 117 118 130 133 134,135 136 i37 139 140 15 15 7 6

RS IP
DPNA IP IP IP IP IP

58 68 95 9 6 65 91 9 6 77 82 96.9 99 84 34
59 4 6

IP IP IP BCG
IP

water (25:50 :20:5)

Dioxane/ethanol/pyridine/

Ethanol/glacial acetic acid/ water (60:30:10)

I P D

(continued

..

.)

TABLE 6 (Continued) Solvent System Benzene/dioxane/ethanol/ ammon ium hydroxide ( 50:40:5 :5

Adsorbent * Detect ion* -

(X

Rf 100)

Reference 15 7 6 15 7 6
7 6

C
SG

IP D IP D IP IP IP IP IP IP IP IP

99
75

Benzene/n-butanol/methanol/ water (10 :15 :60 15) :

C SG SG
SG

17 15 17
55

tert .-Amy1 alcohol/n-butyl ether/water (80:7:13

$

n-Butanol/glacial acetic acid/ water (4:1:2) n-Butanol/conc hydrochloric acid, saturated with water ( 0:10) 9 Ethyl acetate/methanol/ ammonium hydroxide (85:10:3 Ethanol/hexane
(8:92)

SG

64
62 57
96

7 6 141

C SG
SG

15 141 142 142 143

SG
SG

31

Ethyl acetate/methanol/ ammonium hydroxide (85:lO:l)

94

(continued

...)

TABLE 6 ( C o n t i n u e d )

Sol ---v e n t System Carbon t e t r a c h l o r i d e Isopropyl e t h e r Benzene Ethylene dichloride

A d s o r b e n t * Detect i o n * ( X 1 0 0 )

IP IP IP IP IP IP IP IP IP IP IP IP

Rf

Reference 144' 144' 144' 144

SG SG SG SG

1 1

27 42 37
58
55

' '

2 P

Met h y l e n e c h l o r i d e
Chloroform Ethyl ether E t h y l acetate n-Butyl a l c o h o l Isopropy1 a l c o h o l Acetone

SG
SG

144' 144

SG SG SG SG

67

144'

73
61 63
79

' 144 '

144 144' 144' 144

SG

M e t hano 1

SG

77
(continued

...

'

Lo

w
lid
r(

Loco

w v
c a w
r(

4
4

r(

w
w
w
rl rl

L o c o
Q,

a,

7
0
Q,

cow
Lo

ma
co
w t coc
0
v

0
Q,

*A

co
0

Q,c,

*
01

ct

B
n

2
2 B

2 2

P
Q,

c,

7
.r(

C
l

c
P

a,

L 1

o
m m

00

m m

u
n

0
rl

0
r(

..
5

rl

c
0 E E
c,
Q,

Lo
n

..
rl
v

;; 2;
003

cd

2;
0

v
C

..

03

cv ..
0
rl

..
0

Q, v

cd

4 ..

5
U

o x

k 0

.r(

c,W

om ..
a, 4-

orl

**

ii u
415

0 w 0 k 0

..

a,

i d

o E

a,

5
cd
0
rl

5
w
k 0
h

0 C cd

c,

rl Ji

0 k 0

TABLE 6 (Continued) Solvent System Ethyl acetate/cyclohexane/ p-dioxane/methanol/water/ ammonium hydroxide (50:50:10:10:1.5 :0.5) Ethyl acetate/cyclohexane/ p-dioxane/methanol/water/ ammonium hydroxide (50:50:10:10:0.5:1.5)

Adsorbent* Detection*
SG

(X

Rf 10 0 ) Reference

SA

83

150

SG

IP

91

150

5 m

Ethyl acetate/cyclohexane/ methanol/water/ammonium hydroxide (70:15:8:0.5:2) Ethyl acetate/cyclohexane/ ammonium hydroxide (50:40:0.1) Benzene/cyclohexane/ diethylamine (15:75:10) Ethyl acetate/dimethylformamide (3:l) tert.-Amy1 alcohol/n-butyl ether/water (14:7:1)

SG

ASA

94

150

SG

BCG

98

150

SG
SG

IP

99 99

123 123

IP

SG

IP

55 123 (continued

...

TABLE 6 (Continued)
Solvent System tert.-Amy1 alcohol/n-butyl ether/wa ter ( 8 0 :7:13
Chloroform/methanol/ammonium hydroxide (85:10:1)

Rf Adsorbent * Detection* ( x 100) SG

Reference 123 151 151 146 152 146

I P I P I P I P IP I P I P IP I P I P I P

8 6
80 67

SG3
SG3
SG

Ethyl acetate/methanol/ 10: ammonium hydroxide (85: 1.5)

z
4

Chloroform/methanol (9:l) Acetone Acetone/ammonium hydroxide (99:l) Methanol Chloroform/methanol (50:50) Chloroform/methanol/ammonium hydroxide (47.5 :47.5 :5) Chloroform/glacial acetic acid/ methanol (47.5:5:47.5)

SG
SG

17 32
20

SG
SG

59
1 6
20

16 4
16 4

SG

146 146

SG
SG

80

54 16 4 (continued

...

TABLE 6 (Continued) Solvent System

Benzene/dioxane/ethanol/ ammonium hydroxide (50:40:5 :5)

Rf Adsorbent* Detection* -x 100) (

SG

Reference 153

IP

98

Methanol/l2 N ammonium hydroxide (100:1.5)

SG SG SG SG
SG

IP I P I P D + I P

42 53 37 97

153 152 128 154 133

E
O0

n-Butanol e thy 1 acetate/ / ethanol/ammonium hydroxide (2:28:14:0.4) Benzene/ether (10:l)

I P

71

A
A

I P I P I P I P I P

20

155 155 156 157

Dichloromet hane/e t her ( 10 :21

Benzene/diethylamine/dioxane/ ethanol (50:5 :40:51

35

SG
SG SG

91 8 6

Dimethylformamide/ethyl acetate (1:3) Ethanol/isopropyl ether (20:80)

11 152 (conth u e d

.. .

TABLE 6 (Continued) Solvent System Acetoacetic ester/chloroform (1:1)

Acetone/chloroform/formic acid (4:16:1) Cyclohexane/diethylamine (9:1)

Adsorbent - -

Detection*
S W

(X

Rf 100)
88

Reference 158 159 159

159

cc
A

+
D D

59 71

SG
SG

\b

Benzene/chloroform/ diethylamine ( 6 :3:1)

Benzene/l,4-dioxane/ethanol/ ammonium hydroxide (100:80:10:11)

D D

8 8

SG

IP

97

154

Acetic acid/chloroform/ methano1 (10:35 :651 Benzene/ethyl acetate/ ammonium hydroxide (35:60:5) Acetone/chloroform/ diethylamine (2:88:10)

SG
SG

S W S W

53

160 1 0 6
92

75

SG

72

(continued

...

TABLE 6 (Continued) Solvent System Benzene/diethylamine/methanol (75:10:15)

Benzene/n-butanol/methanol/ water/ammonium hydroxide (10:15:60:10:5)

Adsorbent* Detection* SG

(X

Rf 100) Reference 7 4
79 92 92

D D D

SG

Ethanol/ethyl acetate/ ammonium hydroxide (50:45:5)

$
0

SG
SG

73 7 3

7 6

Chloroform/dioxane/ethyl acetate/ammonium hydroxide (25:60:10:5 )

I, D, PP

11 6

lIn an ammonium hydroxide atmosphere 2Top layer was used

3

Silica gel GF with 1%

CaSO4.&H2O

*Key

A:

alumina, ASA: ammonical silver nitrate and heat, BCG: bromcresol green, C : cellulose, D: Dragendorff, DPNA : diazotized (continued

. ..

TABLE 6 (Concluded)
p-nitroaniline, I: 1% iodine, IP: iodoplatinate, PP: 0.1% potassium permenganate, RS: radioscanning, SA: 0.5% sulfuric acid, SG: silica gel, S U V : short ultraviolet light

42 1

RAFlK H. BISHARA

T a b l e 6 c o n t a i n s t h e most commonly u s e d t h i n l a y e r chromatography s y s t e m s f o r methadone.

G a s Chromatography --(GC) G a s chromatography s y s t e m s f o r m e t hadone a r e r e p o r t e d - i n - T a b l e 7. A f l a m e i o n i z a t i o n detector is used i n a l l r e f e r e n c e s u n l e s s otherwise i n d i c a t e d .
8.15

----_-

-Combined G a s Chromatography-Mass Spectroscopy ~ G C I ~ E J - - - - ~ The GCMS c o n d i t i o n s and t h e mass f r a g m e n t a t i o n p a t t e r n have been p r e v i o u s l y d i s c u s s e d (See 2 . 1 4 ) . T h i s t e c h n i q u e is u s e d f o r t h e i d e n t i f i c a t i o n o f methadone m e t a b o l i t e ~ ~ 9 , 7 ~ , 7and ~f~ r t h e s c r e e n i n g and 7- o i d e n t i f i c a t i o n of t h e d a n g e r o u s d r u g s of a b u s e . 171, 1 7 2
8.16 High P r e s s u r ----i q u i d Chromatography e L (HPU: 1 High p r e s s u r e l i q u i d chromatography is used by Lorenz1T3 f o r t h e d e t e r m i n a t i o n o f isomethadone i n methadone. A r e v e r s e p h a s e s y s t e m is u s e d , The column, 1 meter l o n g x 2 mm. i . d . is packed w i t h DuPont Permaphase ETH p a c k i n g material. The m o b i l e l i q u i d c o n s i s t s of 1% r e a g e n t ammonium h y d r o x i d e , 15%methanol, and 84% water. The column is o p e r a t e d a t a f l o w r a t e of 5 0 m l . / h r . A 254 nm. d e t e c t o r is u s e d f o r m o n i t o r i n g t h e column e l u e n t . The r e t e n t i o n volumes t o e l u t e isomethadone and methadone a r e 6 m l . and 17 m l . r e s p e c t i v e l y . E x t r a c t i o n from B i o l o g i c a l F l u i d s Most methods u s e d f o r t h e d e t e r m i n a t i o n of d r u g s i n b i o l o g i c a l f l u i d s i n v o l v e three s t e p s , namely s o l v e n t e x t r a c t i o n , c o n c e n t r a t i o n , and d e t e c t i o n o r a s s a y i n g . Each of these s t e p s is time-consuming and d r u g losses due t o a d s o r p t i o n on t o g l a s s w a r e , i n c o m p l e t e t r a n s f e r of s o l v e n t s and e v a p o r a t i o n of v o l a t i l e compounds may lower t h e r e c o v e r y and hence t h e s e n s i t i v i t y . Ramsey and Campbell163 d e s c r i b e d
9.
422

8.17

TABLE 7 GAS CHROMATOGRAPHY SYSTEMS FOR M T A O E EH D N

Column
3 f t . long x 0.125 i n . O.D., 3% OV-17 on 100/

Carrier Gas

N2

Flow Rate ml./min. 30

Colum: Temp. C . 190

7.40 3.32

R e t e n t i o n ReferT i m e , min. ence 68

120 mesh G a s Chrom Q 6 f t . l o n g x 3 mm. I . D . , 2% SE-30 on 80/100 mesh G a s Chrom S

P
W, h

47

215

15

6 f t . l o n g X 2 mm. 3% SE-30 on 8 0 / l O O G a s Chrom Q

I.D., mesh

He
N2

32

2 00

2.80

162

2 M long X 0.125 i n . O.D., 2.5% E - 3 0 1 on 80/100 mesh Chromosorb G 4.75 f t . long x 0.128 i n . d i a m e t e r , SE-30 on 70/80 mesh DMCS 1.22 M l o n g X 5.5 mm. I . D . , 3% OV-17 on 100/ 200 mesh G a s Chrom Q

30

2 00

5.20

163

N2

50

240

14.8

149

7.7 (continued

164

. .. I

TABLE 7 (Continued)

Carrier Column
3% ov-1
3% OV-17

Flow R a t e

-Gas -

N2

3% ov-210

m -- l . / m i n . 30.7

Column Temp. OC. 220 22 0 220 23 0

R e t e n t i o n ReferTime, m i n . ence 2.6 1.7

1.8
4.7

165 165 165 166

5 f t . l o n g X 0.125 O.D., 5% SE-30 on 60/80 mesh C h r omosorb W

P

6 f t . l o n g X 4 mm. I . D . , 1% SE-30 on 100/200 m e s h Anakrom ABS

A A
N2 N2

65 56 70
35 35

180 2 00 210

12.1 4.9 3.3 2 1.2

167l 167 167 153 168

2% OV-225 on G a s Chrom Q 1 . 2 M long X 3 mm. I . D . , 3 . 5 % UCW98 on 80/lOO m e s h C hr omosorb W -A W- D C MS 1 . 2 M l o n g X 4 mm. I . D . , 3 % SE-30 on 8 0 / l O O mesh G a s Chrom Q

190 23 5

He

75

2 10

160

(continued

...

TABLE 7 (Continued)

Column long X 2 . 5 mm. I.D., 1% W-98 on 8 0 / l O O mesh G a s Chrom Q

4 ft.

Carrier Gas
He

Flow R a t e -ml./min. 60

Colum$ Temp. C.
165

Retention Reference Time, min. 4 57,78

2 M long X 0 . 2 5 i n . O.D., 3% OV-17 on 60/80 mesh a c i d washed, D C t r e a t e d MS G a s Chrom Q

N2

65

195

12

92

1 M long X 0.125 i n . O.D., 2% Carbowax on 80/100 mesh a c i d washed, D C MS t r e a t e d Chromosorb G

6 f t . long X 0.25 i n . , OV-1 on 100/120 mesh Chromosorb W 5%

N2

36

180

12

92

Nz

50

23 5

3.3

1.3 M l o n g X 6 . 2 5 mm. O.D., 3.8% UC-W98 on D i a t o p o r t S

N2

16

190 180

6.4 14.2 (continued

77 76

2 M long x 0.25 i n . O.D., 2% SE-30 on SO/lOO mesh Chromosorb G

...

TABLE 7 (Continued)
Column
1 M long x 0.125 i n . O.D., 5% KOH and 2% Carbowax 20 M

Carrier Gas

Flow R a t e

ml./min. 14

Columt Temp. C.
185

175

Retention ReferTime, min. -ence - 7.9 76

Nz

4 f t . long X 3 mm. I . D . , 3.8% W98 on 80/100 mesh Diatoport S

N2

80

5.4

68

fi

6 f t . long x 3 mm. I . D . , 1% y c l o h e x a n e d i m e t h a n o l c s u c c i n a t e on 100/120 mesh D i a t o m i t e CQ

NZ

80

185

4.6

68

2 f t . l o n g x 4 mm. I . D . , 2.5% SE-30 on S O / l O O m e s h Chromosorb G

6 f t . long X 0.25 i n . I.D., 3% OV-1 on S O / l O O

N2

40

2 00

1.7

169

NZ

60

255

1.3

170

mesh Chromosorb W H P

(continued

...

TABLE 7 ( C o n c l u d e d )

Column
6 f t . l o n g X 0.25 i n . O . D . , 3% SE-30 on loo/

G-a s
N2

Carrier

Flow Rate ml./min.

40

Temp.

Columt C. 2 05

R e t e n t i o n ReferTime, min. ence

5.25

145

200 mesh G a s C h r o m Q

'Strontium

90 a r g o n i o n i z a t i o n d e t e c t o r

R A F l K H. BISHARA

a r a p i d method f o r e x t r a c t i o n of methadone i n which t h e c o m p l e t e a n a l y s i s is carried o u t i n one v e s s e l and o m i t t i n g t h e e v a p o r a t i o n s t e p , t o overcome t h e v o l a t i l i t y problems. L y o p h i l l i z a t i o n and l i q u i d - s o l i d e x t r a c t i o n a re used t o detect d r u g s of a b u s e i n u r i n e . l r 2 R e l i a b i l i t y of i d e n t i f i c a t i o n t e c h n i q u e s f o r d r u g s of a b u s e i n a u r i n e s c r e e n i n g program and d r u g e x c r e t i o n d a t a is p r e s e n t e d by K a i s t h a and J a f f e . 1 7 4 The d r u g is a b s o r b e d on c a t i o n exchange r e s i n l o a d e d p a p e r and t h e i o n p a p e r is e x t r a c t e d a t p H 1 w i t h CHC13 and t h e n chromatographed on t h i n l a y e r p l a t e s . The c o n c e n t r a t i o n of methadone i n t h e u r i n e by i o n exchange paper p r o v i d e s a c l e a n e r e x t r a c t t h a n by d i r e c t e x t r a c t i o n of t h e u r i n e . 1 4 7 Dole et -- a l . d e s c r i b e b u f f e r e l u t i o n of t h e i o n exchange paper135 and t h e a p p l i c a t i o n of A m b e r l i t e IR-120 c a t i o n exchange paper p r i o r t o t h i n l a y e r chromatography.l34 Comparison of three u r i n e e x t r a c t i o n t e c h n i q u e s i s r e p o r t e d by K a i s t h a and Jaf fe. 175 "Clean-up" p r o c e d u r e s f o r u r i n e and blood for t h e d e t e r m i n a t i o n of t h e d r u g s of a b u s e are r e v i e w e d by Sohn e t a l . l 7 6 Kaistha - e t a1.177 r e c e n t l y e v a l u a t e d t h e d r u g a b u s e s c r e e n i n g programs, d e t e c t i o n p r o c e d u r e s , development costs, s t r e e t - s a m p l e a n a l y s e s and f i e l d t e s t s .
Determination i n Tissues -_-_-----_---_----__The p r e v i o u s methods emphasize t h e determ i n a t i o n of methadone i n b l o o d s a m p l e s , u r i n e samples and u r i n e s c r e e n s . However, L a w l e r et -- a l . 1 0 4 s u r v e y t h e q u a n t i t a t i v e a s s a y s of methadone i n t i s s u e s . The d r u g i n t h e t i s s u e is examined b y s p e c t r o p h o t o f l u o r o m e t r y f o l l o w i n g formaldehyde t r e a t m e n t and u s i n g e x c i t a t i o n a t 270 nm. and e m i s s i o n a t 4 5 0 nm. Gas liquid chromatography is a l s o used, a f t e r s i l y l a t i o n , on a 3% OV-17 column packed on 100/120 mesh G a s Chrom Q and h e a t e d a t 22OoC. 10.

428

METHADONE HYDROCHLORIDE

Bibliography A comprehensive b i b l i o g r a p h y o f "Methadone, 1929-1971" has been p u b l i s h e d i n t w o parts,l78,l79 L a n g r o d l s o compiled a b i b l i o g r a p h y o f methadone m a i n t e n a n c e t r e a t m e n t o f h e r o i n a d d i c t i o n . The reader is r e f e r r e d t o t h e s e t h r e e lists of r e f e r e n c e s f o r c o m p l e t e l i t e r a t u r e a b o u t methadone. The l i t e r a t u r e s e a r c h f o r p r e p a r i n g t h i s p r o f i l e w a s c o n d u c t e d t h r o u g h A p r i l of 1973.

11.

Theauthorwishes t o t h a n k t h e Merck and Co., Inc., Rahway, N, J., f o r g r a n t i n g t h e p e r m i s s i o n t o u s e some of t h e i n f o r m a t i o n a b o u t methadone h y d r o c h l o r i d e from t h e Merck Index, 8 t h e d i t i o n . Thanks are a l s o due t o Mr. C . E. Hubach and A n a l y t i c a l C h e m i s t r y , and t o D r . E . L. May and T h e m 5 6 T O r g a n i c Chemistry f o r t h e i r permission t o reproduce Table 1 and F i g u r e 9 r e s p e c t i v e l y . The a u t h o r e x p r e s s e s h i s a p p r e c i a t i o n t o D r . A . D. Kossoy, M r . C . D. Underbrink, D r . D. E. Dorman, Mr. H. R . S u l l i v a n , a n d M r . J. L. Occolowitz o f E l i L i l l y and Company f o r a s s i s t a n c e i n p r e p a r a t i o n and d i s c u s s i o n o f t h e i n t e r p r e t a t i o n of v a r i o u s s p e c t r a l d a t a i n t h i s monograph. S p e c i a l t h a n k s go t o Miss Adele Hoskin, Mrs. N e l l V. Ward, and M s JoAnn S p e n c e r f o r r. t h e i r h e l p i n t h e s e a r c h o f t h e voluminous

12.

Acknowledgments

literature.
The a u t h o r is v e r y g r a t e f u l t o Mrs. J a n i s A . Brown f o r h e r i n v a l u a b l e secretarial help i n developing t h e format o f t h i s p r o f i l e and t y p i n g t h e m a n u s c r i p t . The a u t h o r e x t e n d s h i s s i n c e r e a p p r e c i a t i o n t o h i s c o l l e a g u e s a t E l i L i l l y and Company, w i t h a s p e c i a l r e f e r e n c e t o Mr. C . D. Wentling, f o r t h e i r h e l p , c r i t i c i s m , and s u g g e s t i o n s t o improve t h i s a n a l y t i c a l profile.

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88.
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llz,

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METHADONE HYDROCHLORIDE

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.,

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J.,

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439

OXAZEPAM

Charles M . Shearer and Caesar R . Pilla

CHARLES M. SHEARER AND CAESAR R . PILLA

1.

2.

3. 4. 5. 6.

7.

CONTENTS Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, C o l o r , Odor Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance S p e c t r a 2.3 U l t r a v i o l e t S p e c t r a 2.4 Mass S p e c t r a 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 C r y s t a l P r o p e r t i e s 2.9 D i s s o c i a t i o n C o n s t a n t Synthesis Stability Metabolism Methods of A n a l y s i s 6.1 Elemental A n a l y s i s 6.2 G r a v i m e t r i c A n a l y s i s 6.3 D i r e c t 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 6.4 C o l o r i m e t r i c A n a l y s i s 6.5 F l u o r o m e t r i c A n a l y s i s 6.6 T i t r i m e t r i c A n a l y s i s 6.7 P o l a r o g r a p h i c A n a l y s i s 6.8 Chromatographic A n a l y s i s 6.81 Paper Chromatography 6.82 Thin Layer Chromatography 6.83 Gas Chromatography 6.84 Column Chromatography References

442

OXAZEPAM

1.

Description

1.1 Name, Formula, Molecular Weight

The name used by Chemical A b s t r a c t s and t h e N a t i o n a l Formulary XI11 f o r oxazepam i s 7-chloro-1,3dihydro-3-hydroxy-5-phenyl-2&1,4 benzodiazepin-2-one.

H C1N202 15 11 1 . 2 Appearance, C o l o r , Odor

Mol. W t . :

286.72

Oxazepam i s a creamy w h i t e t o p a l e yellow powder having p r a c t i c a l l y no odor.

2.

Physical Properties

2.1

Infrared Spectra

A i n f r a r e d a b s o r p t i o n spectrum of a potassium n bromide d i s p e r s i o n of oxazepam (NF R e f e r e n c e Standard m a t e r i a l ) i s p r e s e n t e d i n F i g u r e 1. This spectrum a g r e e s w i t h p u b l i s h e d s p e c t r a l , 2 * 3 b The s p e c t r a l band a s s i g n ments a r e l i s t e d i n Table I. Table I I n f r a r e d S p e c t r a l Assignments of Oxazepam Wavelength, u V i b r a t i o n Mode Reference OH, NH s t r e t c h 4 3.05 t o 3.20 C=O s t r e t c h 5 5.79 and 5.86 6.19 C=N-s t r e t c h 5 6.35 and 6.72 Aromatic C=C d e f o r m a t i o n s 6 6 12.07 Out of p l a n e CH deformation of 1 , 2 , 4 s u b s t i t u t e d aromatic 13.39 and 14.35 Out of p l a n e CH 6 deformation of mono s u b s t i t u t e d aromatic

443

m M

&

OXAZEPAM

2.2

Nuclear Magnetic Resonance

The 60 M z NMR spectrum, shown i n F i g u r e 2 , was H o b t a i n e d by d i s s o l v i n g oxazepam (NF Reference Standard material) i n deutero dimethylsulfoxide containing t e t r a m e t h y l s i l a n e a s i n t e r n a l r e f e r e n c e . The s p e c t r a l a s s i g n ments a r e l i s t e d i n Table 11. Table 11 NMR S p e c t r a l Assignments of Oxazepam Chemical S h i f t ( & I Protons 4.82 a l i D h a t i c C-H 0-H' 6.35 a r o m a t i c CH 7.52 N-H 10.78

Both

Splitting Doublet Doublet Mu1 t i p 1e t Singlet

t h e hydroxyl and amino p r o t o n s exchanged w i t h D 0. 2 Sadee8 r e p o r t e d a s i n g l e t f o r t h e a l i p h a t i c C-H proton, however, a s i n g l e t was observed i n t h e s e l a b o r a t o r i e s only a f t e r D 0 exchange.

2.3

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

The u l t r a v i o l e t s p e c t r a of oxazepam i n 0.1Nhydroc h l o r i c a c i d , 0.1N sodium hydroxide, and i n pH 7 b u f f e r a r e p r e s e n t e d i n F i g u r e 3. The spectrum of oxazepam i n a l c o h o l i s p r e s e n t e d i n F i g u r e 4. The spectrum and absorpt i v i t i e s of oxazepam i n a l c o h o l a g r e e w i t h p u b l i s h e d v a l u e ~ l , ~ , The a b s o r p t i v i t i e s and maximum wavelengths ~a r e p r e s e n t e d i n Table 111. Table I11 Ultraviolet Spectral Characteristics Absorptivity max (nm) 236 111 41 284 13 362 230 315 236 342 230 318 131 9

Solvent 0.1N HC1

PH 7 0.1N NaOH alcohol

111 11
126 9

445

E 2
I N

446

OXAZEPAM

0.7 1
0.6

0.5
w

0.4

3
a m
Q

0.3
0.2
0.1 0 .o

WAVE LENGTH (nrn)

F i g . 3 - U l t r a v i o l e t S p e c t r a of Oxazepam (NF R e f e r e n c e HCl--pH 7 b u f f e r , 8 I t 0,lNNaOfi. S t a n d a r d ) Solvent-0.1N

WAVE LENGTH (nrn)

Fig. 4

U l t r a v i o l e t Spectrum of Oxazepam (NF R e f e r e n c e Standard) Solvent-alcohol.

447

CHARLES M. SHEARER AND CAESAR R. PlLLA

2.4

Mass S p e c t r a

The mass spectrum of oxazepam (NF Reference Standard m a t e r i a l ) w a s o b t a i n e d by d i r e c t i n s e r t i o n of t h e samp l e i n t o t h e MS-902 double f o c u s i n g , h i g h r e s o l u t i o n mass 6 spectrometer. The sample w a s run a t 200C. and 1.0 x 10t o r r w i t h 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 e.v. The high r e s o l u t i o n d a t a was compiled and t a b u l a t e d w i t h t h e a i d of t h e PDP-8 D i g i t a l computer. A l i n e graph of t h e mass spectrum i s shown a s F i g u r e 5 and t h e major high r e s o l u t i o n d a t a i n Table IV 10 Table IV Mass Spectrum of Oxazepam C a l c u l a t e d Mass Formula C H O N C 1 286.0509 1 5 11 2 2 C15H902NC1 270.0321 269.0481 268.0402 267.0325 259.0399 257.0481 241.0420 239.0376 233.0714 229.0532 205.0765 194.0844
C
C C C

Measured Mass 286.0484 270.0372 269.0474 268.0436 267.0319 259.0427 257.0450 241.0461 239.0379 233.0715 229.0526 205.0738 194.0849

H ON C 1 15 10 2

15 9
H

H ON C 1

15 8
H

H ON C 1

14 10 2 14 10
15H1'' 0

0 NC1

ON C 1

14H8N
' gH90N2 1

C13H10N2C1 C14H9N2 C13H10N2

11 This spectrum i s i n agreement w i t h t h a t p r e s e n t e d by Sadee.

The molecular i o n was a t r n / = 286. The base peak, m/g 257, i s g e n e r a t e d by loss of a formyl r a d i c a l , which may be preceeded by a h y d r i d e m i g r a t i o n from C-3 t o C-5. Addit i o n a l loss of CO g e n e r a t e d an i n d a z o l e a t m / e 229, which then e l i m i n a t e d C 1 g i v e m/e 194. OthFr predominant peaks a r e m/g 269(M - OH) and m/e 259(M -CHN).

50

448

9
E

() I

2
I

l n

FLI

-rl

00

CHARLES M. SHEARER AND CAESAR R. PILLA

2.5 Melting Range The following m e l t i n g p o i n t temperatures have been reported: OC. Reference 5 203 - 204 205 206 12 20 6 13,14

2.6

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

A s t u d y by d i f f e r e n t i a l thermal a n a l y s i s (DTA) r e v e a l s t h a t be m e l t i n g endotherm i s a f u n c t i o n of t h e heating r a t e This i s i l l u s t r a t e d i n Table V. A DTA curve of oxazepam (NF Reference Standard m a t e r i a l ) o b t a i n e d a t a h e a t i n g r a t e of 10/min. i s i n c l u d e d a s F i g u r e 6.

15.

Table V D i f f e r e n t i a l Thermal A n a l y s i s of Oxazepam heating r a t e 5 10 20

min.
OC.

m e l t i n g endotherm

OC.

188 193
201

2.7

Solubility

The following s o l u b i l i t y d a t a were o b t a i n e d a t u n c o n t r o l l e d room temperature: 4.5 mg./ml. i n 95% e t h a n o l 0.03 mg./ml. i n w a t e r 4 mg./ml. i n chloroform These v a l u e s a g r e e w i t h t h e s o l u b i l i t i e s given i n t h e National Formulary XIII.

2.8

Crystal Properties

The X-ray d i f f r a c t i o n p a t t e r n of oxazepam (NF Reference Standard m a t e r i a l ) was o b t a i n e d w i t h a P h i l i p s d i f f r a c t o m e t e r using Cu KG r a d i a t i o n 15. The c a l c u l a t e d d spacings of t h e d i f f r a c t i o n p a t t e r n a r e p r e s e n t e d i n Table

VI.

450

50

100

150 201) 250 300 350 T. OC (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES)

400

450

Fig. 6

DTA Spectrum of Oxazepam (NF Reference S t a n d a r d )

CHARLES M. SHEARER AND CAESAR R . PILLA

*6.6
12.4 13.2 14.8 16.6 17.4 17.7 18.5 19.4 39.8 20.0

28

Table V I X-Ray-Powder, D i f f r a c t i o n P a t t e r n Sample: Oxazepam: NF Reference Standard Source: C u K S 20 d d d 2& 4T3 74 31.0 2T885 13.4 >G5.3 4.210 31.4 2.849 21.1 7.13 21.5 6.70 4.132 32.2 2.780 5.98 3.934 33.3 2.690 22.6 5.34 3.817 34.0 2.636 23.3 5.096 3.662 34.4 2.607 24.3 5.010 3.506 36.8 2.442 25.4 3.363 38.7 2.326 26.5 4.796 4.576 3.279 39.0 2.310 27.2 2.998 29.8 4.482 *Most i n t e n s e peaks 30.4 4.440 2.940

~~

~~

2.9

D i s s o c i a t i o n Constant

The pKa's o f oxazepam i n aqueous s o l u t i o n s were determined s p e c t r o p h o t o m e t r i c a l l y t o be 1.8, a t which a proton i s g a i n e d ; and 11.1, a t which a p r o t o n is l o s t .
3.

Synthesis

One s y n t h e t i c r o u t e f o r t h e p r o d u c t i o n of oxazepam i s given i n F i g u r e 7. The q u i n a z o l i n e 3-oxide can be p r e p a r e d by r e a c t i n g c h l o r a c e t y l c h l o r i d e w i t h 2-amino-5-chlorobenzophenone oximel6. The r i n g expansion s t e w i t h sodium hydroxide was d e s c r i b e d by Bell and coworkersq7. Treatment of t h e r e s u l t i n g benzodiazepin -2-one 4-oxide w i t h a c e t i c anhydride and h y d r o l y s i s of t h e e s t e r w i t h b a s e y i e l d s oxazepam5. Other s y n t h e t i c r o u t e s and t h e chemistry of benzodiazepines i n g e n e r a l a r e d i s c u s s e d i n review articles18,19,20,21. 4. Stability

19 Oxazepam i s s t a b l e a s a s o l i d o r i n a n e u t r a l s o l u t i o n . Acid h y d r o l y s i s produces 2-amino-5-chlorobenzophenone 22. The itnino-carbinol s t r u c t u r e l e a d s t o a number of r e a r r a n ements ( F i g u r e 8 ) upon t r e a t m e n t w i t h a c e t i c a c i d 5 o r b a s s g .

452

OXAZEPAM

'gH!5 2-amino- 5- chlorobenzophenone 2-amino- 5-chlorobenzophenone oxime 6- chloro- 2chloromethyl4-pheny l- quina z o l i n e 3 oxlde

c1

a$
H
II
0

\1

NaOH

c6H5

3-acetoxy-7-chloro-5-phenyl-l,3dihydro- 2E- 1,4-benzodiazepine-3 one

c6H5 7- chloro- 5-phenyl1,3-dihydro-2&-1, 4-benzodiazepine- 2 one 4 oxide

NaOH

6H5 oxa zep am Figure 7

S y n t h e t i c Route o r Oxazepam

453

H H

%
z
0

7 l

'd

8
454

OXAZEPAM

5 .

Metabolism

The major metabolite (greater than 95%) of oxazepam in man has been determined to be oxazepam glucuronide22. 1) Minor metabolites are as follows: 6-chloro-4-phenyl-24 H quinazolinone; 2- amino-5-chlorobenzophenone; 2'-ben~oyl-4~2chloro-2,2-dihydroxyacetanil ide; 2'-benzoyl-4khlorohydroxy-2-ureidoacetanilide; 7-chloro-I 3-dlhydro-Shydr~xy, 5-(phydroxyphenyl)-2&1,4-benzodiazepin-2-one; and 7chloro-l,3-dihydro-3-hydroxy-5-[ 3 (or 4)-hydroxy-4-(or 3) methoxyphenyll-2~-1,4-benzodiazepin-2-one. The first two minor metabolites exist only in the unconjugated state in urine while the othe 8 are present as conjugates as well as in the free form25
6 Methods of Analysis .

61 .

Elemental Analysis

The following data were obtained on NF Reference Standard material7: % Theorv % Found Element C 62.94 62.71 H 3.85 39 .9 N 9.62 9.79 c1 12.41 12.47 6.2 Gravimetric Analysis

Oxazepm,can be precipitated out of a very dilute acidic solution with silicotungstic acid. The precipitate is collected, washed with water, dried at 7OOC. and weighed This method has been applied to dosage forms2. When a solution of Reinecke's salt is added to a dilute solution of oxazepam, a bright rose-violet colored precipitate is formed. The precipitate is then washed, dried, and weighed 2;Z4. 63 . Direct Spectrophotometric Analysis

Salim and coworkers' described an ultraviolet spectrophotometric method of analysis which was applicable to capsules of oxazepam. In this method a sample equiva455

CHARLES M. SHEARER AND CAESAR R. PlLLA

l e n t t o about 50 mg. oxazepam is e x t r a c t e d w i t h a l c o h o l through a s i n t e r e d g l a s s funnel. 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 c o n c e n t r a t i o n of about 4 mcg./ml. i n alcohol. The absorbance i s r e a d on a spectrophotometer a t 229 nm. u s i n g a l c o h o l a s a blank and compared w i t h t h e absorbance of a s t a n d a r d s o l u t i o n of oxazepam. This i s a l s o t h e method s p e c i f i e d i n t h e N a t i o n a l Formulary XIII. A completely automated system c a p a b l e of d i s i n t e g r a t i n g a whole t a b l e t or c a p s u l e , d i s s o l v i n g t h e a c t i v e c o n s t i t u e n t , f i l t e r i n g i t , d i l u t i n g a p o r t i o n of t h e clear f i l t r a t e t o a d e s i r e d volume an o b t a i n i n g a complete UVv i s i b l e scan, h a s been reportedg5. The accuracy of t h e automated method i s comparable t o t h a t of t h e manual spect r o p h o t o m e t r i c method f o r oxazepam. 6.4 Colorimetric Analysis When a s o l u t i o n of R e i n e c k e ' s s a l t i s added t o a d i l u t e s o l u t i o n of oxazepam a b r i g h t r o s e - v i o l e t c o l o r e d p r e c i p i t a t e i s formed. This can be i s o l a t e d and t h e n d i s c e n t r a t i o n determined s p e c t r o solved i n a c e t o n e and i t s p h o t o m e t r i c a l l y a t 525 nm

2yt.

Oxazepam can be hydrolyzed w i t h h y d r o c h l o r i c a c i d t o form g l y c i n e and 2-amino-5- chlorobenzophenone. The aromatic amine can be d i a z o t i z e d w i t h n i t r o u s a c i d and coupled t o naphthylene diamine26, N-alpha naphthy1-NN-(l-naphthyl)ethylenediamine.2 diethy p r ~ p y l e n e d i a m i n e ~ ~ , HC122138, o r a l p h a naphthol29. The c o n c e n t r a t i o n of t h e oxazepam i n t h e r e s u l t i n g c o l o r e d s o l u t i o n can be d e t e r mined s p e c t r o p h o t o m e t r i c a l l y i n t h e v i s i b l e region. 6.5 Fluorometric Analysis Walkens t e i n and coworkers22 a p p l i e d f luorometry t o t h e d e t e r m i n a t i o n of oxazepam i n b i o l o g i c a l f l u i d s . Oxazepam w a s e x t r a c t e d from the body f l u i d s by e t h y l e n e d i c h l o r i d e and t h e f l u o r e s c e n c e measured on a f l u o r o m e t e r equipped w i t h a high p r e s s u r e mercury lamp, a 200-400 m p broad band primary f i l t e r and a B540 (520-660 m p ) broad band secondary f i l t e r . Braun and coworkers30 developed a f l u o r o m e t r i c method f o r determining oxazepam which had a s e n s i t i v i t y of

456

OXAZEPAM

0.005 pg./ml. I n t h i s method an e t h a n o l i c s o l u t i o n of oxazepam i s h e a t e d i n phosphoric a c i d , producing a v e r y i n t e n s e f l u o r e s c e n c e . The f l u o r e s c e n c e i s measured a t an e x c i t a t i o n wavelength of 360 nm. and an emission wavel e n g t h of 475 nm. T h i s method has a l s o been used f o r dosage form a n a l y s i s 3 1 .
S t e i d i n g e r and S ~ h m i d ~ ~ developed two methods f o r determining oxazepam, combining t h i n l a y e r chromatography and fluorometry. I n one method, t h e oxazepam i s s c r a p e d o f f t h e developed TLC p l a t e and e x t r a c t e d w i t h methanol. P e r c h l o r i c a c i d i s added t o t h i s s o l u t i o n and i t is heated. The r e s u l t i n g f l u o r e s c e n c e can then be determined w i t h a f l u o r o m e t e r . The o t h e r method, a l s o r e p o r t e d by L a u f f l e l , 3 i s t o treat the i n t a c t p l a t e with t r i c h l o r o a c e t i c acid, h e a t t h e p l a t e , and determine t h e f l u o r e s c e n c e d i r e c t l y by u s e of a t h i n l a y e r chromatographic p l a t e scanner.
6.6 T i t r i m e t r i c Analysis

Oxazepam can be d i s s o l v e d i n dimethylformamide, and t i t r a t e d w i t h 0.1N tetrabutylammonium hydroxide [ p r e p a r e d i n benzene:methanol (9: 1 ) 1 t o a p o t e n t i o m e t r i c e n d p o i n t u s i n g glass vs. calomel electrodes1. Beyer and Sadee34 determined oxazepam by d i s s o l ving i t i n g l a c i a l a c e t i c a c i d and t i t r a t i n g w i t h 0.05N p e r c h l o r i c acid. The e n d p o i n t w a s determined e i t h e r potent i o m e t r i c a l l y o r v i s u a l l y , u s i n g c r y s t a l v i o l e t a s an i n d i c a t o r . A c e t i c anhydride w a s a l s o used as a s o l v e n t f o r oxazepam, g i v i n g comparable r e s u l t s . 6.7 Polarographic Analysis

The p o l a r o g r a p h i c b e h a v i o r of oxazepam has been d i s c u s s e d i n many papers. F a z z a r i and Riggleman350btained w e l l - d e f i n e d c a t h o d i c waves a t t h e dropping-mercury elect r o d e i n a m i x t u r e of methylene chloride-methyl a l c o h o l w i t h a s u p p o r t i n g e l e c t r o l y t e of 0.1M tetraethylammonium bromide. The halfwave p o t e n t i a l of oxazepam i n t h i s system i s about -1.02V 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 w i t h c o n c e n t r a t i o n . T h i s method w a s a p p l i e d t o c a p s u l e s of oxazepam. O e l s c h l a g e r and coworkers36 a l s o found a l i n e a r 451

CHARLES M. SHEARER AND CAESAR R . PILLA

r e l a t i o n between c o n c e n t r a t i o n and d i f f u s i o n c u r r e n t i n an a c e t a t e b u f f e r system c o n t a i n i n g 20% dimethylformamide. T h i s procedure w a s used t o a s s a y t a b l e t s c o n t a i n i n g oxazepam. They a l s o c o r r e l a t e d half-wave p o t e n t i a l v e r s u s a s a t u r a t e d calomel e l e c t r o d e , w i t h pH as shown below. The pH w a s determined i n a Britton-Robinson b u f f e r c o n t a i n i n g 20% dimethylformamide. 5.1 6.0 7.2 8.2 9.2 P H 2.3 3.4 E % ( V ) -,695 -.825 -.955 -1.005 -1.08 -1.125 -1.185 Oeschlager and coworkers37 f u r t h e r i n v e s t i g a t e d t h e e l e c t r o c h e m i s t r y of oxazepam and found t h a t i n a c i d b u f f e r s i t i s reduced w i t h t h e u p t a k e of 4 e l e c t r o n s t o form 7-

chloro-5-phenyl-1,3,4,5-tetrahydro-2~-1,4-benzodiazepin2-one a t t h e dropping mercury e l e c t r o d e . However, i n a l k a l i n e b u f f e r s t h e u n s t a b l e 4 , 5 dihydro d e r i v a t i v e which i s formed f i r s t by consuming 2 e l e c t r o n , reacts f u r t h e r forming a c y c l i c aldehyde-ammonia adduct which undergoes r e d u c t i o n t o form t h e same p r o d u c t as is formed i n a c i d i c solution.
6.8 Chromatographic A n a l y s i s

Many of t h e more common chromatographic technique6 have been a p p l i e d t o oxazepam. 6.81 Paper Chromatography

Oxazepam h a s been chromatographed on Whatman %1 paper w i t h e i t h e r b u t a n o l , e t h a n o l , w a t e r (17:3:20) upper s h a s e o r b u t a n o l , p y r i d i n e , w a t e r (6:4:3) a s t h e eluant 2 . 6.82 Thin Layer Chromatography

The v a r i o u s e l u a n t systems used f o r t h i n l a y e r chromatography on s i l i c a g e l p l a t e s f o r oxazepam are given i n Table V I I . Table V I I I g i v e s s p r a y r e a g e n t s used f o r t h e d e t e c t i o n of oxazepam on t h i n l a y e r chromatographs. 6.83 Gas Chromatography

Gas chromatography has been used t o analyze oxazepam. The n e c e s s a r y d a t a of t h e v a r i o u s methods are given as Table IX.

458

OXAZEPAM

! k

00 .0 0.03

00 .4 00 .8
00 .8 0.13 0.13
0.14 0.16 0-21 0.23 0.29 0.29 0-35 0.37 04 .6 04 .8 0.50 0.50 0.51 0.52 0.55 0.57 0.58 0.59 06 .0 06 .0 06 .6 06 .8
0.68

Table V I I Thin Layer Chromatography System for Oxazepam Eluant Reference chloroform:e t h a n o m l ) 27 cyc1ohexane:diethylamine:benzene 4 0 (75:20:15) benzene:ethyl acetate (5:l) 41 benzene: ethanol:ammonium hydroxide 4 0 (95:15:5) chloroform:to1uene:methanol (10:9:1) 42 heptane: chloroform:ethanol (10: 1) 10: 43
ch1oroform:cyclohexane:diethylamine
to1uene:nitromethane:methanol (11:8:1) 42 carbon tetrach1oride:methanol (90:10) 44 benzene:methanol:ammonium hydroxide 13 (9O:lO:1) to1uene:diethylamine (80: 20) 44 43 benzene:acet0ne:diethylamine (70:20:10) isopropanol:isopropyl ether (16:84) 45 ch1oroform:methanol (10: 1) 42 ethanol :water ( 96:4 ) 45 methanol:acetone (12:88) 45 45 methanol :methyl acetate (18:82) benzene:ethano1:diethylamine (5:1:0.5) 46 41 ch1oroform:ethanol: acetone (8:l: 1) 17) 44 cyclohexane:diethylamine (85: 44 carbon tetrach1oride:methanol (75:25) 4 0 acetone 40 methanol :ammonium hydroxide (100:1,5) 44 chloroform:acetone:methanol (70:20:10) 47 heptane: ch1oroform:ethanol (5:5:2) 44 ethyl acetate:1,2 dichloroethane (80:20) 43 isopropanol:ammonium hydroxide (20: 1) 44 benzene:acetone:methanol (55:35:10) 4 4 isopropano1:methanol (30:70) 10: ) 44 ch1oroform:methanol: acetic acid (88: 2 44 isopropano1:ammonium hydroxide:water (75:17:18) chloroform:acet0ne:diethylamine (50:40:10) 44 44 ethyl acetate:methanol:acetic acid (80: 10) 20: chloroform:acetic acid:methanol (15:114) 41

(40: 10) 50:

44

0.73

08 .2 08 .4
08 .8

459

CHARLES M. SHEARER AND CAESAR R . PlLLA

Sadee and Van d e r Kleijn38 found t h a t oxazepam r e a r r a n g e d t o t h e q u i n a z o l i n e carboxaldehyde ( F i g u r e 8, S t r u c t u r e IV) during g a s chromatography.
6.84

Column Chromatography

es S c o t t and B ~ m m e rd~ ~c r i b e a h i g h p r e s s u r e l i q u i d chromatography system which w a s used f o r s e v e r a l benzodiazepines, i n c l u d i n g oxazepam. The column was 100 cm. x 1 mm. i.d. s t a i n l e s s s t e e l packed w i t h Waters Assoc i a t e s Durapak rrOPN", 36-75 p m. p a r t i c l e diameter. The e l u a n t w a s a mixture of hexane and isopropanol. An u l t r a v i o l e t d e t e c t o r w a s used. Table V I I I TLC Spray Reagents f o r t h e D e t e c t i o n of Oxazepam Color L i g h t * Reference Reagent Source Bromine water 13 orange vis. I1 Reinecke s o l u t i o n 13 rose 11 Iodine s o l u t i o n 13 brown II 13 Sulfuric acid yellow I1 Dragendorf f 46 yellow It Chlorine-o-toluidine violet 42 II Cerric sulfate-Dragendorff red-or ang e 42 II Diphenylcarbazone 48 l i g h t purple II 48 blue-purple Silver acetate I1 48 lavender Mercuric s u l f a t e uv 40 blue Cinnamaldehyde It 40 Furfural reagent green Zinc (11) c h l o r i d e II hydrochloric acid 44 beige 70% p e r c h l o r i c a c i d 44 yellow-orange v i s . uv 44 Cerium (IV) s u l f a t e yellow Formaldehyde-hydrochloric 11 acid l i g h t blue 44 40% o-phosphoric a c i d 44 1i g h t ye1 low vis. uv Antimony (111) c h l o r i d e 44 l i g h t blue acetic acid 44 via yellow uv 44 blue Vanillin-sulfuric acid 44 yellow vis.

*vis. = v i s i b l e

UV = u l t r a v i o l e t

460

Table I X
G a s Chromatographic System f o r Oxazepam

Column Packing 3% OV 1 on G a s Chrom Q (60180) 3% OV 1 7 on Gas Chrom Q (60180)

Column

Carrier G a s

Column Temperature 245QC. 230OC.

Detector flame i o n i zation electron capture t o t a l ion current flame i o n i zation electron capture

Ref*
49

2 m, x 2 mm. glass

Nitrogen @ 22 ml./min. Argon-me thane ( 90 : 10) @ 100 mlJmin,

H e l i u m @ 20 m l Imin.

4 f t . x 4 mm. glass
3 f t . x 118 i n . glass 6 f t . x 118 i n . stainless steel

50
38 38 38

fi

HP Chrom G

1.5% OV 1 on

200 - 280C. programmed 245C. 245OC.

3% OV 1 7 on Gas Chrom W 3% OV 1 7 on
G a s Chrom W

Nitrogen @ 70 ml./min. Argon-me thane ( 20 : 1 ) @ 70 ml./min.

6 it. x 118 in. s t a i n l e s s steel

CHARLES M. SHEARER AND CAESAR R . PILLA

7.

References 1. E. F. Salim, J. L. Deuble and G. P a p a r i e l l o , J. Pharm. Sci. 2, 311 (1968). 2. J. B a l t a z a r and M. M. Ferreira Brager, Rev. P o r t u g . Farm. 1 7 , 109 (1967)) C - A = 67, 102845e. 3. F. R F a z z a r i , M. F. Sharkey, C. A. Y a c i w and W. 1. Brannon, J. A s s . O f f i c . Anal. Chem., 5l,1154(1968). 4. N. B. Calthup, L. H. Daly and S E. Wiberly, "In. t r o d u c t i o n t o I n f r a r e d and Raman Spectroscopy," Academic Press, New York, 1964, Chapter 13. . . 5. S C. Bell and S J. C h i l d r e s s , J. Org. Chem., 27, 1691 (1962). 6. L. J. B e l l m y , "The I n f r a r e d S p e c t r a of Complex MOlecules," John Wiley 6 Sons, New York, 1954, Chapter 5. 7. B. Hofmann, Wyeth L a b o r a t o r i e s Inc., p e r s o n a l communication. 8. W. Sadee, Arch. Phann., 302, 769 (1969), C.A. 72, 95079f. 9. M. F. Sharkey, C. N. Andres, S W. Snow, A. Major, . T Krm, V. Warner, T. G. Alexander, J. A s s . O f f i S . Anal. Chem., 2, 1124 (1968). 10. T. Chang and C. Kuhlman, Wyeth L a b o r a t o r i e s Inc., p e r s o n a l communi c a t ion. 11. W. Sadee, J. Med. Chem., 13,475 (1970). 12. P o G. S t e c h e r , Ed., "The Merck Index,'' E i g h t h E d i t i o n , Merck & Co., Inc., Rahway, New J e r s e y , (19681, p. 772. 13. L. D. Rodrigues and M. A. P. Alvee, Rev. P o r t . Farm 15, 309 (19651, C.A. 64L 11030f. 14. F. Berte, L. Manzo, M. DeBernardi and G. Benzi, Farmaco ( P a v i a ) Ed. P r a t . , 25, 1 7 7 (1970). 15. N. DeAngelis, Wyeth L a b o r a t o r i e s Inc. , p e r s o n a l communication. . . . 16. L. H. Sternbach, S Kaiser and E Reeder, J Am. Chem. SOC. 82, 475 (1960). . . 1 7 . S C. B e l l , T. S Sulkowski, C. Gochman and s. J. C h i l d r e s s , J. Org. Chem., 2, 562 (1962). . 18. F. D. Popp and A. C Noble, Advan. H e r e r o c y c l i c 8, 2 1 (1967). . 19. S J. C h i l d r e s s and M. I. Gluckman, J. Pharm. S c i . , 53, 577 (1964). L. H. Sternbach, Angew Chem. I n t e r n . Ed., 2, 34 20 (1971).

--

m ,

.-

462

OXAZEPAM

21.

G, A. Archer and L. H.

22.
23. 24. 25. 26.

27.
28. 29. 30.

31.
32.

Sternbach, Chem. Rev., 68, 747 (1968). S. S. Walkenstein, R. Wiser, C. H. Gudmundsen, H. B. K i m m e l and R. A. Corradino, J. Pharm S c i . , 53, 1181 (1964). S. F. Sisenwine, C. 0. Tio, S R. Shrader and H. W. R u e l i u s , Arzneim. Forsch., 22, 682 (1972). B. Grecu and S. Barbu, Farmacie ( B u c h a r e s t ) , l 6 , 199 (1968) C. A. 69, 12,950x. A. J. Khoury and L. J. C a l i , Ann. N. Y - Acad. Sci., 153, 456 (1968). D. H a l o t , Prod. Probl. Pharm., 25, 175 (19701, & & 73, 485672. P. Lafargue, P, Pont and J. Meunier, Ann. Pharm. Franc., 28, 343 (19701, C. A. 73, 1 3 3 9 5 0 ~ . G. Kamm and R. K e l m , Arzneim. Forsch., 2, 1659 (19691, C - A * , 72, 20201f. H. P e l z e r and D. Maass, 1 9 , 1652 (19691, C. A., 7 2 , 2082P. J. Braun, G. C a i l l e and E. A. Martin, Can. J. Pharm. S c i . , 3, 65 (19681, 3, 317184. G, C a i l l e , J. Braun and J. A. Mockle, Can. J . Pharm. S c i . , 5, 78 (19701, C. A., 74, 2 5 0 2 0 ~ .

w.,

Q.,

33.
34. 35. 36. 37. 38. 39. 40. 41.

J. S t e i d i n g e r and E. Schmid, Arzneim. Forsch., 2, 1232 (1970), C. A., 7 3 , 129198g. S. L a u f f e r , E. Schmid and F. Weist, i b i d . , 2,1965 (19691, C. A = , 72, 4 7 4 1 3 ~ . K. H. Beyer and W. Sadee, Arch. Pharm., 2,667 (19671, C. A., 68, 7 2 2 8 4 ~ . F. R. F a z z a r i and 0. H.Riggleman, J. Pharm. S c i . , 58, 1530 (1969). H. O e l s c h l a g e r , J. Volke, G. T. Lim and R. Spang, Arch. Pharm., 302, 946 (19691, C. A., 2, 93359 y. H. Oelschlager, J. Volke, G T L i m and U. Bremer, . . .i b i d 303, 364 (19701, c. A = , 2, 2 0 8 4 4 ~ . W. Sadee, E. Van d e r K l e i j n , J. Pharm. sci., 60, 135 (1971). C. G. S c o t t and P. Bommer, J. Chromatogr. Sci., 8, 446, (1970). I. Z i n g a l e s , J. ChromatoR., 31, 405 (1967). F. J. DiCarlo and J. P. Viau, J. Pharm. Sci., 2, 322 (1970).

463

CHARLES M. SHEARER AND CAESAR R. PILLA

42. 43. 44. 45.

)

- A. M. .

H D. Beckstead and S. J. Smith, Arzneim. Forsch., . 18, 529 ( 1 9 6 8 ) ) C. A * , 69, 54319d.

Schwartq B. A. Koechlin, E. Postma, S-Palmer and G. K r o l , J. Pharmacol. E x p t l . Therap., 149, 423 (1965). 8 F. R. Weist, Arzneim. Forsch., 2, 7 (19681, 68, 8 5 8 8 0 ~ . E. Roder, E. Mutschler and H. Rochelmeyer, z. Anal. Chem 244, 45 (1969). T. S. G l o r i a , Rev. Fac. Farm. Bioquim. Univ. Sao P a u l o , 2, 391 (1966), C. A., 67,8 4 9 1 7 ~ . M. A. Schwartz, P. Bommer, and F. M. Vane, Arch. Biochem. Biophysics, 121,508 (1967). K. K. K a i s t h a a n d J . H. J a f f e , J. Pharm. s c i . , 3, 679 (1972). F. Marcucci, R. F a n e l l i and E. Mussini, J. Chromatog., 37, 318 (1968). J. A. F. d e S i l v a and C. V. P u g l i s i , Anal. Chem., 42, 1725 (1970).
C . 0

46. 47. 48. 49. 50.

464

PHENAZOPYRIDINE HYDROCHLORIDE

Kenneth W. Blessel, Bruce C. Rudy, and Bernard Z. Senkowski

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

INDEX

Analytical Profile

- Phenazopyridine Hydrochloride

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 U1 traviolet Spectrum 2 4 Fluorescence Spectrum . Mass Spectrum 2.5 2.6 Optical Rotation 2.7 Melting Range 2.8 Differential Scanning Calorimetry 2.9 Thermogravimetric Analysis 2 1 Solubilities .0 2 , I1 X-ray Crystal Properties 3.
4.
5.

Synthesis Stability Degradation Drug Metabolic Products

6. Methods of Analysis 6.1 Elemental Analysis 6.2 Phase Solubility Analysis 6.3 Thin Layer Chromatographic Analysis 6.4 Direct Spectrophotometric Analysis 6.5 Coulometric Analysis 6.6 Titrimetric Analysis

7. Acknowledgments
8.

References

466

PHENAZOPYRIDINE HYDROCHLORIDE

1. D e s c r i p t i o n N a m e , Formula, M o l e c u l a r Weight P h e n a z o p y r i d i n e h y d r o c h l o r i d e is 2,6-diamino-3( p h e n y 1 a z o ) p y r i d i n e monohydrochloride.

1.1

Cl1HI1N5

*HC1

M o l e c u l a r Weight:

249.70

Appearance, C o l o r , Odor Phenazopyridine h y d r o c h l o r id e i s a l i g h t t o d ar k r e d , o d o r l e s s c r y s t a l l i n e powder.

1.2

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 a sample of r e f e r e n c e s t a n d a r d p h e n a z o p y r i d i n e h y d r o c h l o r i d e is shown i n F i g u r e 1 (1). The i n s t r u m e n t used w a s a P e r k i n E l m e r Model 621 r e c o r d i n g s p e c t r o p h o t o m e t e r . The sample was d i s p e r s e d i n m i n e r a l o i l i n o r d e r t o r e c o r d t h e s p e c t r u m . The f o l l o w i n g a s s i g n m e n t s have b e e n made f o r t h e bands i n F i g u r e 1 (1). Band 3329 and 3275 3065 2 5 cm-1 1 6 0 1 and 1500 1638 If: 5 cm-1 815 2 5 cm-1 713 682 2.2 Assignment

5 cm-1 cm-l

5 5

25

cm'l 5 cm-l

N-H s t r e t c h a r o m a t i c C-H s t r e t c h aromatic r i n g v i b r a t i o n s NH2 d e f o r m a t i o n s o u t of p l a n e d e f o r m a t i o n of H on pyridine ring o u t o f p l a n e b e n d i n g o f NH2 o u t of p l a n e d e f o r m a t i o n s of H on benzene r i n g

N u l e a r Magnetic Resonance Spectrum (NMR) The NMR spectrum of p h e n a z o p y r i d i n e h y d r o c h l o r i d e is shown i n F i g u r e 2 ( 2 ) . The s o l v e n t used was DMSO-d6, t h e i n t e r n a l r e f e r e n c e w a s t e t r a m e t h y l s i l a n e and t h e

467

a,
7)

.d

4
0 k T I

&

aJ
'd
7)

'rl

(D

8
f

E
W

k u
V a,
v)

a
a,
W
I4

k (D k

33NVlllWSNVUl '10

468

469

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2. SENKOWSKI

solution concentration was 30.9 mg in 0.5 ml of solvent. The spectral assignments are given in Table I (2). Table I
NMR Spectral Data for Phenazopyridine Hydrochloride

Proton Proton on C5

No. of

Each
1

Chemical Coup1ing Shift (ppm) Multiplicity Constant

6.36

doublet

J H ~ - H ~ =HzO ~

Protons meta and para to phenylazo nitrogen 3 Protons ortho to phenylazo nitrogen 2 Proton on C4 Amino protons
2.3

7.25-7.65 7.85-8.02 8.29 -8.7

mu1tip1et mu1 tiplet doublet singlet (broad)

Ultraviolet Spectrum The ultraviolet spectrum of phenazopyridine hydrochloride (0.5 mg/100 ml of acidified 3 A alcohol) in the region of 210-450 nm exhibits two maxima and three minim . The maxima occur at 238-240 nm ( E = 2.2 x lo4) and 390-392 nm (E = 2.4 x lo4), while the minima are at 220 nm, 272 nm and 296 nm respectively (3). The spectrum is shown in Figure 3. Fluorescence Spectrum The excitation and emission spectra o f phenazopyridine hydrochloride (10 pg/ml in methanol) are shown in Figure 4 (4). The instrument used was a Farrand MK-1 recording spectrofluorometer. An excitation wavelength of 341 nm produced an emission spectrum with a maximum at 380 nm.
2.4

Mass Spectrum The low resolution mass spectrum of phenazopyridine is shown in Figure 5 (5). The spectrum was obtained using a CEC 21-110 spectrometer w i t h an ionizing voltage of 70 eV, which was interfaced with a Varian data
2.5

470

Figure 3 U l t r a v i o l e t Spectrum of P h e n a z o p y r i d i n e H y d r o c h l o r i d e

471

z
4

m
( r

m 0

m
4

NANOMETERS

Figure 4
Fluorescence Spectra of Phenazopyridine Hydrochloride

t
W

412

u )

NANOMETERS

L
I

1
I

I
0
0

1
(D

0 *

0 e l

AllSN31NI

3AllVl3LI

473

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2.SENKOWSKI

system 100 MS. The d a t a s y s t e m a c c e p t e d t h e o u t p u t of t h e s p e c t r o m e t e r , c a l c u l a t e d t h e masses, compared t h e i n t e n s i ties t o t h e b a s e peak and p l o t t e d t h i s i n f o r m a t i o n as a series of l i n e s whose h e i g h t s were p r o p o r t i o n a l t o t h e i n tensities. The m o l e c u l a r i o n of t h e f r e e b a s e was measured a t m / e 213. O t h e r c h a r a c t e r i s t i c masses were observed a t m / e 214, c o r r e s p o n d i n g t o (MSH), m / e 1 8 4 , which c o r r e s p o n d s t o t h e l o s s of HN2 from t h e m o l e c u l a r i o n , m / e 1 3 6 , t h e l o s s of a phenyl r i n g from t h e p a r e n t mass, and m / e 108 which is t h e 2,6-diamino-pyridinium m o i e t y ( 5 ) . A h i g h r e s o l u t i o n s c a n confirmed t h e r e s u l t s of t h e low r e s o l u t i o n spectrum. 2.6 activity Optical Rotation P h e n a z o p y r i d i n e h y d r o c h l o r i d e e x h i b i t s no o p t i c a l

M e l t i n g Range P h e n a z o p y r i d i n e h y d r o c h l o r i d e m e l t s w i t h decomp o s i t i o n a t a p p r o x i m a t e l y 235OC when a class I a p r o c e d u r e is used ( 6 ) . D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) The DSC s c a n of p h e n a z o p y r i d i n e h y d r o c h l o r i d e is shown i n F i g u r e 6 ( 7 ) . The c u r v e ' k a s o b t a i n e d w i t h a P e r k i n E l m e r DSC-1B C a l o r i m e t e r . The t e m p e r a t u r e program used w a s 10C/min. i n an atmosphere of n i t r o g e n . Under t h e s e c o n d i t i o n s , t h e exothermic d e c o m p o s i t i o n o f phenazop y r i d i n e h y d r o c h l o r i d e o c c u r s a t a p p r o x i m a t e l y 239OC. Thermogravimetric A n a l y s i s (TGA) The T A s c a n showed no loss of w e i g h t as t h e G t e m p e r a t u r e was r a i s e d from ambient t o 115OC a t a r a t e of 10C/min. ( 7 ) . Solubility The s o l u b i l i t y d a t a o b t a i n e d f o r a sample o f r e f e r e n c e s t a n d a r d p h e n a z o p y r i d i n e h y d r o c h l o r i d e a t 25OC is shown in T a b l e I1 ( 8 ) . The e q u i l i b r a t i o n t i m e w a s 20 h o u r s a t 25OC. 2.10 2.9

2.7

2.8

474

Figure 6

c V

*rl

DSC Curve or Phenazopyridine Hydrochloride u

0 rl

'd

a,

a,

a,

3 1

475

TEMPERATURE "C

KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD 2. SENKOWSKI

Table I1 Phenazopyridine Hydrochloride Solvent

Solubilities

S o l u b i l i t y (rnglml)

3 A alcohol benzene chloroform 95% e t h a n o l diethyl ether 2 propanol m e t hano 1 petroleum e t h e r (30-600) water 1N H C 1 a c i d i f i e d 3A alcohol

2.7 0.3
0.4

3.5 0.2 2.1 2.7 01 . 3.2 0.3

3.2

Crystal Properties The x-ray powder d i f f r a c t i o n d a t a from phenazop y r i d i n e h y d r o c h l o r i d e is p r e s e n t e d i n Table I11 ( 9 ) . The o p e r a t i n g c o n d i t i o n s of t h e i n s t r u m e n t are g i v e n below. Instrument Conditions General Electric Model XRD-6 Spectrogoniometer Generator: Tube t a r g e t : Radiation: optics:

2.11

50 KV, 12-112 MA
Copper Cu Ka = 1.542 0.10 D e t e c t o r s l i t M.R. S o l l e r s l i t 3' Beam s l i t 0.0007" Ni f i l t e r 4 take off angle ' Scan a t O.2O 28 p e r minute Amplifier g a i n - 1 6 c o a r s e , 8.7 f i n e Sealed p r o p o r t i o n a l c o u n t e r t u b e and DC v o l t a g e a t plateau P u l s e h e i g h t s e l e c t i o n EL 5 volts; Eu - Out

Goniometer: Detector:

476

PHENAZOPYRI DINE HYDROCHLORIDE

Recorder: Samples:

Rate meter T.C. 4 2000 C/S f u l l scale C h a r t Speed 1 i n c h p e r 5 minutes P r e p a r e d by g r i n d i n g a t room temperature
T a b l e I11

I n t e r p l a n a r Spacings i n Phenazopyridine Hydrochloride from X-ray Powder D i f f r a c t i o n Data

28
8.68 9.12 10.22 12.88 13.68 14.46 18.10 19.44 20.40 21. 82 22.10 23.06 24.06 25.74 26.38 27.10

d*
10.20 9.70 8.66 6.87 6.47 6.13 4.90 4.57 4.35 4.07 4.02 3.86 3.70 3.46 3.38 3.29

** uIo6 30 84 10 8 100 20 32 19 25 21 10 10 32 85 46

28 -

d* -

UIo3
85 18 23 18 10 6 9 5 6
3 8 2 4 3

27.88 29.28 30.64 31.32 32.84 34.38 36.06 36.58 37.58 40.02 41.14 42.32 43.78 47.00 48.75
n A

3.20 3.05 2.92 2.86 2.73 2.61 2.49 2.46 2.39 2.25 2.19 2.14 2.07 1.93 1.87

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

2 Sin 8

**I/I,=
3.

r e l a t i v e i n t e n s i t y ( b a s e d on h i g h e s t i n t e n s i t y of 100)

Synthesis P h e n a z o p y r i d i n e may b e p r e p a r e d by c o u p l i n g benzene diazonium c h l o r i d e w i t h a,a-diamino p y r i d i n e ( 1 0 ) . S t a b i l i t y Degradation P h e n a z o p y r i d i n e h a s been found t o b e s t a b l e i n d i s t i l l e d water and 0.1N sodium h y d r o x i d e when r e f l u x e d f o r one hour on a steam b a t h . Some d e g r a d a t i o n o c c u r s i n 0.1N
4.

417

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD A. SENKOWSKI

hydrochloric a c i d s o l u t i o n a t r e f l u x temperature. A f t e r one hour a t r e f l u x i t w a s found, by q u a n t i t a t i v e d e n s i t o metry o n a t h i n - l a y e r chromatographic p l a t e , t h a t 20-25% of t h e i n i t i a l amount of m a t e r i a l had degraded. The deg r a d a t i o n p r o d u c t s were y e l l o w b u t no i d e n t i f i c a t i o n w a s attempted. Drug M e t a b o l i c P r o d u c t s The m e t a b o l i c f a t e of p h e n a z o p y r i d i n e h y d r o c h l o r i d e h a s been s t u d i e d i n t h e r a b b i t and i n man (11). When a n o r a l d o s e of 600 m w a s g i v e n t o humans, a b o u t 80%w a s g e l i m i n a t e d i n t h e u r i n e w i t h i n 24 h o u r s . Of t h i s amount, 7.6% appeared as a n i l i n e , 19.9% as N-acetyl-p-aminophenol, 27.1% as p-aminophenol, 45.4% as t h e unchanged d r u g as w e l l as a t r a c e amount of o-aminophenol. Triaminopyridine a l s o w a s d e t e c t e d b u t n o t measured. 6. Methods of A n a l y s i s 6.1

5.

Elemental Analysis The r e s u l t s of a n e l e m e n t a l a n a l y s i s of a sample of r e f e r e n c e s t a n d a r d p h e n a z o p y r i d i n e h y d r o c h l o r i d e a r e p r e s e n t e d i n T a b l e I V (12). Table I V Elemental A n a l y s i s of P h e n a z o p y r i d i n e H y d r o c h l o r i d e Element

C

Theoretical % 52.91 4.84 28.05 14.20

Found % 52.89 4.76 28.00 14.23

H
N

c1

Phase S o l u b i l i t y A n a l y s i s Phase s o l u b i l i t y a n a l y s e s have been c a r r i e d o u t f o r phenazopyridine hydrochloride. A example i s shown i n n F i g u r e 7 ( 8 ) . The s o l v e n t used w a s methanol w i t h a n e q u i l i b r a t i o n t i m e of 20 hours a t 25OC. Thin Layer Chromatographic A n a l y s i s P h e n a z o p y r i d i n e c a n b e d e t e c t e d i n AZO GANTRISIN t a b l e t s u s i n g t h e f o l l o w i n g TLC p r o c e d u r e . The t a b l e t s

6.2

6.3

478

Figure 7

5
I -

2 3
I '

w 4-

"

t o
m m

3'
0

. .
PHASE SOLUBILITY

"
ANALYSIS

s!
0

z g 2o m
k l L

3 0
0

' " E

l -

Sample : Phenazopyridine Hydrochloride Solvent : Methanol Slope: 0.0% Equilibration: 20 hrs at 25OC Extrapolated Solubility : 2.99 m g / g

l l l l l l

KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD 2.SENKOWSKI

are ground t o a powder and a p o r t i o n d i s s o l v e d i n a c e t o n e .

This i s a p p l i e d t o t h e p l a t e ( s i l i c a g e l GF) and developed f o r a t l e a s t 1 0 cm w i t h t h e s o l v e n t m i x t u r e chloroform: heptane:3A a l c o h o l (45:45:10) i n a p a p e r - l i n e d , pres a t u r a t e d tank. Phenazopyridine can be d e t e c t e d v i s u a l l y as a y e l l o w s p o t a t a n Rf of 0 . 4 (13).

Direct S p e c t r o p h o t o m e t r i c A n a l y s i s The s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n o f phenaz o p y r i d i n e h y d r o c h l o r i d e i n 1.ON H C l i s , a c c o r d i n g t o t h e N a t i o n a l Formulary, t h e method of c h o i c e f o r t h e a s s a y of t h e b u l k d r u g (14). The sample i s d i s s o l v e d i n 1 . O N H C 1 and t h e absorbance d e t e r m i n e d a t t h e maximum a t a b o u t 480 nm. The q u a n t i t y of p h e n a z o p y r i d i n e h y d r o c h l o r i d e is c a l c u l a t e d by comparison w i t h a sample o f r e f e r e n c e s t a n dard m a t e r i a l p r e p a r e d and measured i n a s i m i l a r way. The above method is s u b j e c t t o t h e d i s a d v a n t a g e of t h e low s o l u b i l i t y of p h e n a z o p y r i d i n e h y d r o c h l o r i d e i n 1.ON HC1. The s u b s t i t u t i o n of a c i d i f i e d 3A a l c o h o l as t h e s o l v e n t would g i v e a p p r o x i m a t e l y a t e n - f o l d i n c r e a s e i n t h e s o l u b i l i t y and eliminate t h e n e c e s s i t y of h e a t i n g t h e s o l u t i o n i n o r d e r t o d i s s o l v e t h e r e q u i r e d amount of phenaz o p y r i d i n e h y d r o c h l o r i d e . The maximum i n t h i s s o l v e n t o c c u r s a b o u t 390 nm.
Coulometric A n a l y s i s P h e n a z o p y r i d i n e h y d r o c h l o r i d e may b e d e t e r m i n e d coulometrically i n a n a c i d i f i e d water-acetone s o l u t i o n , u t i l i z i n g a mercury p o o l e l e c t r o d e . The sample is reduced a t a p o t e n t i a l of -0.40 v o l t s u n t i l t h e observed c e l l c u r r e n t d r o p s t o 1/1000,of i t s i n i t i a l v a l u e . A b l a n k det e r m i n a t i o n i s c a r r i e d o u t and any c o r r e c t i o n s made. Each coulomb of e l e c t r i c i t y i s e q u i v a l e n t t o 646.9 mcg of phenazopyridine hydrochloride (6). Titrimetric Analysis P h e n a z o p y r i d i n e h y d r o c h l o r i d e may b e a s s a y e d by a p o t e n t i o m e t r i c t i t r a t i o n w i t h HClO4. The sample i s d i s s o l v e d i n water which i s made b a s i c w i t h 10% NaOH and t h e l i b e r a t e d base is extracted i n t o c h l o r o f orm. I t i s t h e n 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 0.01N HClO4, i n dioxane, u s i n g a g l a s s - c a l o m e l ( s l e e v e t y p e ) e l e c t r o d e combination. Each ml of 0.01N HClO4 i s e q u i v a l e n t t o 2.497 mg of p h e n a z o p y r i d i n e h y d r o c h l o r i d e (15).
6.6 6.5

6.4

480

PHENAZOPYRI DI NE HYDROCHLORIDE

Acknowledgments The a u t h o r s wish t o acknowledge t h e S c i e n t i f i c L i t e r a t u r e Department and t h e Research Records Off i c e of Hoffmann-La Roche Inc. f o r a s s i s t a n c e i n t h e l i t e r a t u r e search f o r t h i s Analytical P r o f i l e .

7.

KENNETH W. BLESSEL, BRUCE C. RUDY, A N D B E R N A R D 2. SENKOWSKI

8.

References
1. Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. 2. Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. 3 . Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. 4 . Boatman, J. , Hoffmann-La Roche Inc., Personal Communication. 5 . Benz, W., Hoffmann-La Roche Inc., Personal Communication. 6 . The National Formulary XIII, pp. 538-540 (1970). 7. Moros, S . , Hoffmann-La Roche Inc., Personal Communication. 8. MacMullan, E. , Hoffmann-La Roche Inc., Personal Communication. 9 . Hagel, R. , Hoffmann-La Roche Inc. , Personal Communication. . 10. Chichibabin, Zeide, J Russ. Phys. Chern. SOC., 46,

1216 ( 1 9 1 4 ) . 11. Johnson, J. W. and Burba, J., Federation Proc..

734 ( 1 9 6 6 ) .

g,

12. Scheidl, F. , Hoffmann-La Roche Inc. , Personal Communication. 13. Sokoloff, H., Hoffmann-La Roche Inc., Unpublished Results 1 4 . The National Formulary XIII, First Supplement, p. 24 (1970). 1 5 . Senkowski, B., Hoffmann-La Roche Inc., Unpublished Results.

482

PHENY LEPHRINE HYDROCHLORIDE

Charles A . Gaglia, Jr.

Reviewed by E. L. Pratt and L. Chafetz

CHARLES A. GAGLIA.

JR.

CONTENTS Analytical Profile

Phenylephrine Hydrochloride

1.
2.

3.

4. 5.
6.

7.

1 . 1 Name, F o r m u l a , Molecular W e i g h t 1 . 2 Appearance, C o l o r , Odor, Taste Physical Properties 2 . 0 1 M e l t i n g Range 2.02 S o l u b i l i t y 2 . 0 3 pK 2.04 Optical Rotation 2.05 U l t r a v i o l e t S p e c t r u m 2.06 I n f r a r e d Spectrum 2 . 0 7 N u c l e a r M a g n e t i c Resonance S p e c t r u m 2 . 0 8 Mass S p e c t r u m 2.09 D i f f e r e n t i a1 Thermal A n a l y s i s 2 . 1 0 Thermal G r a v i m e t r i c A n a l y s i s Synthesis S t a b i l i t y - Degradation Drug M e t a b o l i c Products Methods of A n a l y s i s 6 . 1 Direct S p e c t r o p h o t o m e t r i c A n a l y s i s e 6 . 2 Co 1 o r i m t ri c Ana l y s i s 6 . 2 1 I n d o p h e n o l Dye 6.22 Coupling w i t h p - N i t r o a n i l i n e 6.23 Coupling with 4-Aminoantipyrine 6.24 Complexation 6 . 2 5 C o u p l i n g w i t h Nitrous Acid 6.26 I d e n t i f i c a t i o n 6 . 2 7 O t h e r Methods 6 . 3 Chromatographic Methods o f A n a l y s i s 6.31 Paper Chromatography 6.32 T h i n L a y e r Chromatography 6 . 3 3 L i q u i d - L i q u i d Chromatography 6 . 3 4 Gas C h r o m a t o g r a p h y 6 . 3 5 Ion Exchange Chromatography 6 . 4 S p e c t ro f 1 u o r ome t r i c a n d P h o s p h or i met r i c Analysis 6 . 5 Other Methods o f A n a l y s i s References

Description

484

PHENY LEPHRINE HYDROCHLORIDE

1.

Description 1.1

Name, F o r m u l a , M o l e c u l a r Weight P h e n y l e p h r i n e H y d r o c h l o r i d e i s l-rn-Hyd r o xy -a-[ ( m t h y 1 am i n o ) m t h y 1 ] be n zy 1 a 1 co h o 1 h y e e d r o c h l o r i d e ( 1 ) . I t i s a l s o known a s l - a - h y d r o x y - B -me t hyl a m i no- 3 -hyd roxy -1 -e t h y 1 benzene h y d r o c h l o r i d e ; m-methylaminoethanolphenol hydrochlor i d e ; Neo S y n e p h r i n e h y d r o c h l o r i d e ; Meta-Syne p h r i n e h y d r o c h l o r i d e ; A d r i a n o l ; rn-Sympatol ; Meta-Sympatol; Neophryn; I s o p h r i n H y d r o c h l o r i d e ; Oftal frine; Lexatol ( 2 ) .

OH CsHl4ClN02 1.2

Mol. W t .

203.67

Appearance, C o l o r , Odor, T a s t e White o r n e a r l y w h i t e , o d o r l e s s c r y s t a l s having a b i t t e r t a s t e .

2.

Physical Properties 2.01 M e l t i n g Range P he n -y 1 e .p h r i n e H C1

P heny 1 eph r i n e b a s e
*USP Speci f i c a t i

ons

140 139 170 170

145C 143C 177C 171OC

(1)* (3) (1)" (3)

2.02

Solubility F r e e l y s o l u b l e i n water and i n a l c o h o l

2.03 2.04

pK
pK, = 8 . 7 7 ( 4 ) pK2 = 9.84 Optical Rotation [ ] ' = -46.2 t o - 4 7 . 2 " a;
485

(c=l) (2)

Fig. 1 Phenylephrine hydrochloride - IR spectrum o f 1 3 mm. K B r p e l l e t from 1 mg. drug dispersed i n 200 mg. KBr - Instrument: Perkin-Elmer 621

PHENYLEPHRINE HYDROCHLORIDE

2.05

U l t r a v i o l e t Spectrum Solution X max. n m . 0 . 0 5 N HC1

0.05 N NaOH

x 10-3 5.91 1.81 1.65 8.95 3.04 4.28 6.66 1.67

Isosbestic Points 2.06

216 274 279s 2 39 292.5 222.5 260 278.5

I n f r a r e d Spectrum ( F i g u r e 1 ) The s p e c t r u m i n F i g u r e 1 was o b t a i n e d using a Perkin-Elmer 621, I n f r a r e d Spectrophotometer, A 1 3 mm. KBr p e l l e t c o n t a i n i n g 1 mg. B p h e n y l e p h r i n e HC1 a n d 2 0 0 mg. K r w a s u s e d . Chara c t e r i s t i c band a s s i g n m e n t s a r e l i s t e d below. B,and cm 3420, 3450
2 400-2800
A s s i gnmen t

-OH
N H*

1590 1270 1080 900 780 690 2.07

aromatic C-0 s t r e t c h a r o m a t i c C-0 s t r e t c h s e c o n d a r y a l c o h o l a r o m a t i c o u t o f p l a n e bend s i n g l e a r o m a t i c o u t o f p l a n e b e n d meta disubstituted a r o m a t i c o u t o f p l a n e bend meta di substi t u t e d Nuclear Magnetic Resonance Spectrum S o l v e n t : DMSO I n s t r u m e n t : V a r i an A60 C o n c e n t r a t i o n : A p p r o x i m a t e l y 8%

CH

48 I

CHARLES A. GAGLIA. JR.

Phenylephrine Hydrochloride (Figure 2 ) ppm. ( f r o m TMS) Integral I d e n t i f ic a t i on 2.6 ( S ) 2.8 - 3.2 (M)
3

2 -[!I* 7
6.7 9

7.5 (M) 9.8 (B)*

2 1 1 4 3

N-CH 3 CH 2 - N CH
CHOH

aromatic phenolic OH,

NH':

P h e n y l e p h r i n e Base ( F i g u r e 3 ) ppm. ( f r o m TMS) Integral Identi fi cation 2.4 (S) 3 N-CH 3 2.55 ( D ) 2 CH 2 - N 4.5 ( T ) 1 CH 4 . 8 - 5 . 8 (B)* 3 OH ( 2 ) , NH 6.5 7.5 (M) 4 aromatic * D i s a p p e a r on D20 e x c h a n g e

Mass S p e c t r u m The low r e s o l u t i o n mass spectrum was S d e t e r m i n e d on a n M 902 mass s p e c t r o m e t e r . The s a m p l e was i n t r o d u c e d by a h e a t e d d i r e c t i n s e r t i o n p r o b e a t a t e m p e r a t u r e o f 150C f o r t h e h y d r o c h l o r i d e and o f 1 0 0 C f o r the base. Mass No. 167 148 133 121 107 95 77 65 44 38 36 I n t e n s i t y HC1 10 2 1 5 2 4 8 4 100
8

2.08

I n t e n s i t y Base 38 6 4 lo 10 20 15 30 100

---

26

488

Fig. 2 Time averaged NMR spectrum o f phenylephrine hydrochloride, 8 % i n DMSO - Instrument: Varian A-60

F i g . 3 Time a v e r a g e d N M R s p e c t r u m o f p h e n y l e p h r i n e b a s e , a b o u t 8% i n DMSO - I n s t r u m e n t : Varian A - 6 0

n

l-0

v)

PHENYLEPHRINE HYDROCHLORIDE

2.09

D i f f e r e n t i a1 T h e r m a l A n a l y s i s

O n s e t o f Me1 t i n g Peak E n d o t h e r m P h e n y l e p h r i ne HC1 P h e n y l e p h r i ne Base 2.10 142C 172C 144C 174C

Thermal G r a v i m e t r i c A n a l y s i s No s i g n i f i c a n t w e i g h t l o s s u n t i l decomp o s i t i o n a t 230C f o r p h e n y l e p h r i n e HC1.

3.

Synthesis

L e g e r l o t z was t h e f i r s t t o p r e p a r e p h e n y l e p h r i n e h y d r o c h l o r i d e by t h e h y d r o g e n a t i o n o f mhydroxy-w-methylamino-acetophenone i n t h e p r e s e n c e o f c o l l o i d a l p a l l a d i u m ( 5 ) . Bergmann a n d Sul z b a c h e r ( 6 ) s y n t h e s i z e d p h e n y l e p h r i n e by t r e a t i n g 5-(3'-benzyloxyphenyl)-3-methyl-2-oxaz o l i d o n e w i t h 40% h y d r o c h l o r i c a c i d s o l u t i o n . R u s s e l l and C h i l d r e s s ( 7 ) a c h i e v e d t h e same e n d b y r e f 1 u x i n g 3 - b e n z y l o x y - w - m e t h y l mandel ami de w i t h L i A1H4 i n t e t r a h y d r o f u r a n (THF) t o p r o d u c e 3 - b e n z y 1o x y - a-me t h y 1 ami n o-me t h y 1 b e n z y 1 a1 c o h o l h y d r o chloride. T h i s compound was t h e n h y d r o g e n a t e d i n t h e p r e s e n c e o f 5% p a l l a d i u m - C c a t a l y s t u n t i l one e q u i v a l e n t o f H i s consumed. The h y d r o g e n a t i o n o f 3-benzyloxy-a-methylamino-methyl b e n z y l a l c o h o l was a l s o u s e d b y B r i t t e n ( 8 ) a n d , m o s t r e c e n t l y , R i z z i ( 9 ) as t h e l a s t s t e p s o f t h e i r s y n t h e s e s o f phenylephrine.

4.

Stability

Degradation

P h e n y l e p h r i n e h y d r o c h l o r i de i s s t a b l e as a solid. The d e g r a d a t i o n o f aqueous s o l u t i o n s h a s The b e e n s t u d i e d b y E l - S h i b i n i e t aZ. ( 1 0 , l l ) . Above pH 7 , d e g compound i s s t a b l e b e l o w pH 7 . r a d a t i o n o c c u r s and a p p a r e n t l y i n v o l v e s t h e s i d e c h a i n w i t h l o s s o f t h e s e c o n d a r y amine f u n c t i o n . The p h e n o l i c g r o u p r e m a i n s i n t a c t . The decompos i t i o n p r o d u c t s h a v e n o t been i d e n t i f i e d b u t 5 h y d r o x y - N - m e t h y l i n d o x y l has been p r o p o s e d . The p r e s e n c e o f h e a v y m e t a l s , p a r t i c u l a r l y c o p p e r was found t o c a t a l y z e t h e decomposition.

49 1

CHARLES A. GAGLIA, JR

T r o u p a n d Mitchner ( 1 2 ) c h a r a c t e r i z e d t h e a c e t y l a t i o n of p h e n y l e p h r i n e i n t h e p r e s e n c e o f a s p i r i n . The r e a c t i o n i s a p p a r e n t l y a c c e l e r a t e d by t h e p r e s e n c e of p h e n y l e p h r i n e base a n d t h e a v a i l a b i l i t y of a c e t a t e . The amine g r o u p a c e t y l a t e s p r e f e r e n t i a l l y . The hydroxyl g r o u p s become a c e t y l a t e d a f t e r prolonged e x p o s u r e t o a s p i r i n .

Luduena e t aZ. ( 1 3 ) s t u d i e d t h e e f f e c t o f u l t r a v i o l e t i r r a d i a t i o n on p h e n y l e p h r i n e s o l u t i o n s . E p i n e p h r i n e was i d e n t i f i e d a s t h e product. Luduena p o s t u l a t e d t h e e p i n e p h r i n e could f u r t h e r r e a c t t o produce o t h e r compounds. The f i n d i n g s of West a n d W h i t t e t ( 1 4 ) s u p p o r t t h i s p o s t u l a t e . S c h r i f t m a n ( 1 5 ) found from 1 2 t o 28% decompos i t i o n of unbuffered phenylephrine s o l u t i o n s i n one week a t v a r i o u s t e m p e r a t u r e s . He a l s o found u p t o f i v e d e c o m p o s i t i o n p r o d u c t s . The s e c o n d a r y amine f u n c t i o n was a b s e n t in a t l e a s t one of t h e products. Broadly a n d Roberts ( 1 6 ) found a second comp o u n d present i n s t r o n g acid (10 N hydrochloric a c i d ) s o l u t i o n s of p h e n y l e p h r i n e . The compound was n o t i d e n t i f i e d . Misgen ( 1 7 ) d e t e r m i n e d t h e p h y s i c a l compatib i l i t y of p h e n y l e p h r i n e w i t h twenty-seven common i n t r a v e n o u s a d m i x t u r e s . H found on adding a e s o l u t i o n of phenylephrine t o a s o l u t i o n of d i l a n t i n sodium a p r e c i p i t a t e formed w i t h i n two h o u r s .
F a g a r d ( 1 8 ) found p h e n y l e p h r i n e s o l u t i o n s t o be s t a b l e i n brown g l a s s b o t t l e s , g i v e 1 % decomp o s i t i o n a f t e r 11 days i n low d e n s i t y p o l y e t h y l e n e b o t t l e s a n d decompose t o 81% o f i n i t i a l when s t o r e d f o r 130 days i n nylon b o t t l e s .

P e t r a g l i a a n d Dick ( 1 9 ) r e p o r t e d z a t i o n of p h e n y l e p h r i n e s o l u t i o n s t o a d d i n g 0 . 2 % sodium m e t a b i s u l f i t e a n d zine. E l - S h i b i n i e t aZ. ( 1 1 ) i n d i c a t e d

492

the s t a b i l i s u n l i g h t by 0.1% t a r t r a the stabi-

PHENY LEPHRINE HYDROCHLORIDE

l i z i n g e f f e c t o f E D T A on b a s i c s o l u t i o n s o f p h e n y l e p h r i ne. Kisbye and Bols ( 2 0 ) f o u n d t h a t n o r a c e m i z a t i o n o f p h e n y l e p h r i n e o c c u r s a s a f u n c t i o n of pH. P r a t t ( 2 1 ) found phenylephrine o p t i c a l l y s t a b l e i n s o l u t i o n s a t pH 3 . 0 and 6 . 0 when r e f l u x e d f o r 3 hours. Fourneau e t aZ. ( 2 2 ) r e p o r t e d t h e r e a c t i o n o f phenylephrine w i t h aldehydes under "physiological c o n d i t i o n s " t o p r o d u c e a m i x t u r e o f 4 , 6 - and 4 , 8 d i hydroxy-2-methyl-1 y 2, 3 , 4 - t e t r a h y d r o i soquino1ines. 5.
Drug Metabolic Products

R e l a t i v e l y l i t t l e work h a s been r e p o r t e d on t h e m e t a b o l i s m o f p h e n y l e p h r i n e . Bruce ( 2 3 ) r e ported phenylephrine i s excreted i n urine almost e n t i r e l y a s t h e s u l f a t e c o n j u g a t e . Bruce r e ported 82% average t o t a l r e c o v e r y o f phenyle p h r i n e from a t a b l e t f o r m u l a t i o n i n 24 h o u r s . T y p i c a l u r i n e s a m p l e c o n t a i n e d from 86 t o 98% o f t h e recovered p h e n y l e p h r i n e a s t h e s u l f a t e conj ugate

Bogner and Walsh ( 2 4 ) , and C a v a l l i t o e t aZ. ( 2 5 ) showed b l o o d l e v e l and u r i n e e x c r e t i o n p a t t e r n s f o r p h e n y l e p h r i n e h y d r o c h l o r i d e and p h e n y l e p h r i n e t a n n a t e . T h e i r work i n v o l v e d t r i t i u m l a b e l e d d r u g . M e t a b o l i c p r o d u c t s were n o t i d e n t i f ied.
R u b i n and K n o t t ( 2 6 ) r e p o r t e d a f l u o r o m e t r i c procedure f o r determining phenylephrine i n serum.

Dombrowski and P r a t t ( 2 7 ) r e p o r t a p r o c e d u r e f o r determining unmetabolized phenylephrlne i n plasma. 6. Methods o f A n a l y s i s 6.1 D i rect Spectrophotometri c Analysis The u l t r a v i o l e t a b s o r p t i o n band a t 2 7 2
493

CHARLES A . GAGLIA, JR.

nm. i s due t o t h e p h e n o l i c s t r u c t u r e . The a b s o r b a n c e c a L be u s e d t o q u a n t i t a t e p h e n y l e p h r i n e d i r e c t l y (28,29,30) o r a f t e r e x t r a c t i o n ( 3 1 ) . S h i f t i n g t h e maxima t o 290 nm. i n b a s i c s o l u t i o n has a l s o b e e n u s e d a s a d i r e c t a s s a y as w e l l a s a d i f f e r e n t i a l technique t o quantitate phenyle p h r i n e (32,33). O x i d a t i on o f p h e n y l e p h r i n e t o m-hydroxy benzaldehyde and measuring absorbance a t 2 5 7 nm. a n d / g r 315 nm. i n a c i d i c o r n e u t r a l s o l u t i o n s o r a t 2 3 7 , 267 a n d 357 nm. i n b a s i c s o l u t i o n h a s a l s o b e e n c a r r i e d o u t ( 3 4 ) . The oxidation also offers increased s e n s i t i v i t y over d i r e c t U.V. U . V . i s a common d e t e c t i o n t e c h n i q u e f o r t h i n l a y e r , paper and column chromatographic techniques. Col o r i r n e t r i c A n a l y s i s P h e n v l e D h r i n e h a s b e e n id e n t i f i e d a n d q u a n t i t a t e d b y a v a r i e t y o f c o l o r i m e t r i c t e c h niques. I n d o p h e n o l Dye I n d o p h e n o l dye i s f o r m e d b y t h e r e a c t i o n o f p-Me2NC6H4NH2C1 a n d K 3 F e ( C N ) 6 w i t h p a r a u n s u b s t i t u t e d p h e n o l s i n a1 k a l i n e medi a ( 3 5 36,37). The d y e r e s u l t i n g f r o m t h e r e a c t i o n w i t h p h e n y l e p h r i n e has an a b s o r b t i on maximum a b o u t 6 2 0 nm. Coupling with Phenylephrine diazotized p-nitroaniline i n 39). The r e s u l t i n g compound d e t e r m i n e d a t 495 nm. 6.23 6.22 p-Nitroaniline may be c o u p l e d w i t h a c i d s o l u t i o n (38, i s made b a s i c a n d 6.21 6.2

Coup1 i n g w i t h 4-Ami n o a n t ip y r i n e P-aminoantipyrine i s a selective coupling agent f o r phenols w i t h t h e para p o s i t i o n free. The r e a c t i o n i s c a r r i e d o u t i n a l k a l i n e b u f f e r s o l u t i o n pH = 9 i n t h e p r e s e n c e o f K 3 F e (CN),. The r e s u l t i n g a b s o r b t i o n maximum a t 460 nm, i s q u a n t i t a t i v e f o r p h e n y l e p h r i n e (40,41 , 4 2 ) . 6.24 Complexation Phenylephrine forms complexes w i t h
494

PHENY LEPHRINE HYDROCHLORIDE

v a r i o u s s u l f o p h t h a l e i n dyes i n n e u t r a l t o s l i g h t l y b a s i c s o l u t i o n . The r e s u l t i n g complexes a r e then extracted i n t o a polar organic solvent and t h e c o l o r d e t e r m i n e d s p e c t r o p h o t omet ri c a l l y ( 4 3 , 44) C o u p l i n g w i t h N i t r o u s Acid When s o l u t i o n s o f P h e n"v l e.P h r i n e a r e h e a t e d w i t h mercury s a l t s then c o u p l e d w i t h n i t r o u s a c i d , a red c o l o r d e v e l o p s . The peak a t 495 n m . h a s been used t o q u a n t i t a t e p h e n y l e p h r i n e (45,46). Detailed s t u d i e s o f t h e reaction condit i ons have been c o n d u c t e d ( 4 7 , 4 8 ) . Identification P h e n y l e p h r i n e u n d e r g o e s many c o l or r e a c t i o n s . S e v e r a l schemes f o r i d e n t i f y i n g p h e n y l e p h r i n e a l o n e and i n t h e p r e s e n c e o f o t h e r d r u g s have been d e v e l o p e d ( 4 9 , 5 0 , 5 1 , 3 ) . 6.26 6.25

Other Methods Many o t h e r q u a n t i t a t i v e c o l o r rea c t i o n s have been k e p o r t e d ' i n t h e l i t e r a t u r e . The r e a c t i o n p r o d u c t w i t h i o d i c a c i d i s d e t e r m i n e d a t 420 n m . ( 5 0 ) . P h e n y l e p h r i n e r e a c t s w i t h 2 , 6 - d i c h l o r o q u i n o n e i n n e u t r a l s o l u t i o n and i s d e t e r m i n e d a t 625 nm. ( 5 2 ) . The o x i d a t i o n o f p h e n y l e p h r i n e t o an a l d e h y d e f o l l o w e d by r e a c t i o n w i t h t h i o b a r b i t u r i c a c i d (53) o r 3-methylbentoNinthiazolin-2-one (54) i s also quantitative. hydrin r e a c t s w i t h phenylephrine t o produce a p i n k c o l o r w i t h a m a x i m u m a b s o r b a n c e a t 440 n m . (55)
6.27 6.3 C h r o m a t o g r a p h i c Methods o f A n a l y s i s 6.31

P a p e r chromatoqraDh.y has been u s e d t o i s o l a t e phenylephrine from-its decomposition p r o d u c t s ( 1 5 , 5 6 ) and from o t h e r sympathicomi metics (57,58,59,60,37). T a b l e 6.31 -1 summarizes t h e 1i t e r a t u r e f o r p a p e r chromatographic s e p a r a t i o n o f phenylephrine.

P a p e r Chromatography

495

CHARLES A. GAGLIA, JR.

T A B L E 6.31 -1
Sol vent Sys tern
A
B

Method o f V i s u a l i z a t i on
n i nhydri n

R f x 100

Reference
57 15

57 63

d i a z o t i zed psul f a n i l i c dragendorff U.V. deni tometry

n i nhydri n

67

56
60

D
E

n o t a v a i 1a b l e indophenol

-10

37

A = n-butanol /aceti c aci d/water

B = n-butanol /aceti c aci d/water

4:5:1 5:l :3

C = p h e n o l c o n t a i n i n g 15% v / v 0 , I N H C l

D = butanol /to1 uene/aceti c aci d/water 1 0 0 : 1 0 0 : 50:50 E = benzyl a l c o h o l / a c e t i c a c i d / w a t e r

5:2:5

496

PHENYLEPHRINE HYDROCHLORIDE

TABLE 6 . 3 2 - 1

System

R f x 100

Method o f D e t e c t io n s p r a y 1% I 2 i n methanol and/or dragendorff's reagent

I1

Reference 61

I1 I11 IV V VI

21 33 60 45

61 61 61 61 62

II

II

I1

--

p o t a s s i um f e r r i cyanide 0.6% w/v i n 0 . 5 % w / v NaOH quan. U V . densi t o m e t r y n i nhydrin

VII

50

63

S i l i c a Gel P l a t e s Coated w i t h

D e v e l o p i n g Sol v e n t c y c l ohexane/benzene/ diethylamine 75:15:10 ( v / v ) methanol acetone methanol 95% ethanol

I I1 111 IV V

0.1

NaOH

0.1 M NaOH 0 . 1 M NaOH

0 . 1 M KHSOI,

0 . 1 M KHSOI,

497

CHARLES A. GAGLIA. JR.

TABLE 6.32-1 (continued)

S i l i c a Gel P l a t e s Coated with

VI

Developing Sol vent n-butanol/aceti c acid/water 4 : l : 5 v / v organic phase as the developing s o l vent n-butanol l a c e t i c aci d/water 1 2 :1 :7 v / v organic phase as t h e developing solvent

c e l l u l o s e 2 5 0 1-1

VII

s i l i c a gel G

498

PHENYLEPHRINE HYDROCHLORIDE

T h i n L a y e r Chromatography The t h i n l a y e r c h r o m a t o g r a p h i c R f v a l u e s f o r p h e n y l e p h r i n e i n a number o f s o l v e n t s y s t e m s a r e g i v e n i n T a b l e 6.32-1 L i q u i d - L i q u i d Chromatography P h e n y l e p h r i ne l e n d s i t s e l f r e a d i l y t o l i q u i d - l i q u i d chromatography, The d i f f i c u l t y i n e x t r a c t i n g p h e n y l e p h r i n e f r o m aqueous s o l u t i o n s has been u s e d t o a d v a n t a g e t o remove o t h e r compounds f r o m phen l e p h r i n e . L e v i n e and D o y l e ( 6 4 , 6 5 ) and Cox ( 6 6 7 p r e s e n t e d t h e t h e o r e t i c a l aspects o f l i q u i d - l i q u i d p a r t i t i o n systems. T h e i r w o r k i n c l u d e s t h e p a r t i t i on c o e f f i c i e n t s o f many d r u g s and i s p r e s e n t e d s o t h a t o p t i m u m c o n d i t i o n s f o r p a r t i c u l a r s e p a r a t i o n s may be s e l e c t ed. T a b l e 6.33-1 summarizes t h e p r a c t i c a l appl ic a t i ons o f 1 iqu id - 1 iq u i d c h r o m a t o g r a p h i c s e p a r a t i o n s o f phenylephrine. Gas C h r o m a t o g r a p h y Gas c h r o m a t o g r a p h y has been u s e d t o s e p a r a t e , i d e n t i f y and q u a n t i t a t e p h e n y l ephrine. A summary o f t h e gas c h r o m a t o g r a p h i c d a t a i s p r e s e n t e d i n T a b l e 6.34-1. I o n Exchange C h r o m a t o g r a p h y Table 6.35-1 summarizes t h e l i t e r a t u r e on i o n e x c h a n g e s e p a r a t i o n o f p h e n y l e p h r i n e . S p e c t r o f l u o r o m e t r i c and P h o s p h o r i m e t r i c Analysis P h e n y l e p h r i n e has n a t i v e f l u o r e s c e n t p r o p e r t i e s . U d e n f r i e n d et Q Z . ( 8 0 ) r e p o r t e d 270 nm. as t h e w a v e l e n g t h o f e x c i t a t i o n w i t h 305 nm. being the wavelength o f emission. The f l u o r e s c e n c e o c c u r s i n aqueous a c i d s o l u t i o n w i t h a r e p o r t e d s e n s i t i v i t y o f 0.04 p g . / m l . Rubin and K n o t t ( 2 6 ) used a f l u o r o m e t r i c p r o c e d u r e t o d e t e r m i n e p h e n y l e p h r i n e i n serum. Winefordner (81) d e t e r m i n e d p h e n y l e p h r i ne b y i t s p h o s p h o r e s c e n t properties a t l i q u i d nitrogen temperatures i n e t h a n o l ic s o l u t i o n s . The w a v e l e n g t h s o f e x c i t a t i o n a r e 2 9 0 a n d 2 4 0 nm. w i t h p h o s p h o r e s c e n c e
499

6.32

6.33

6.34

6.35

6.4

CHARLES A. GAGLIA, JR.

TABLE 6.32-1

System

R f x 100

Method o f Detect i o n spray 1% I2 i n methanol and/ o r dragendorff reagent

II

Reference 58
I s

I1 I11 IV V VI

21
33
60 45

58 58 58 58 59

I1

II

II

--

p o t a s s ium ferricyanide 0 . 6 % w/v i n 0 . 5 % w/v NaOH quan. U V . d e n s itometry

VII

50

n inh y d r i n

60

S i l i c a Gel P l a t e s Coated w i t h

Developing Sol v e n t Cyc 1 o hex a n e / b e n z e n e / diethylamine 7 5 : 1 5 : 1 0 (v/v) methanol acetone met hano l 95% e t h a n o l

I I1 I11 IV V

0 . 1 M NaOH

0 . 1 M NaOH 0 . 1 M NaOH 0.1 M KHSOk 0 . 1 M KHSOk

500

PHENY LEPHRINE HYDROCHLORIDE

T A B L E 6.32-1

(continued) Developing Solvent n-butanol lacetic a c i d l w a t e r 4 : 1 : 5 v/v organic phase as the developing solvent n-butanollacetic acid/water 12:1:7 v/v organic phase as the developing s o l vent

S i l i c a Gel P l a t e s Coated with

VI

c e l l u l o s e 250

1.1

VII

s i l i c a gel G

501

TABLE 6 . 3 3 - 1

Col umn Support c e l i t e 545

Stationary Phase acetic acid NaCL ( s a t ' d )

Mobile Phase chl oroform wash t h e n ether el u t ion

Other Compounds Present Not Interfering

Reference

codeine 67 d e x t rometh o r p h a n phenyl p r o p a n o l amine HC1 chlorpheniramine p h e n i ramine pyrilamine doxy1 amine s u c c i n a t e t r i p e 1 e n n a m i n e ci t r a t e p h e n y l t o 1 oxamine d i hydrogen c i t r a t e aspirin p h e n y l p r o p a n o l ami ne 68 HC1 d e x t rome t h o r p han H B r glyceryl guai acol a t e acetaminophen chlorpheni ramine p h e n y l t o l o x a m i ne c i t r a t e aspirin phenolphthalein p y r i 1 amine ma1 e a t e g u l f i i;oxgzol e romp en1 r a m i n e ma1 e a t e

c e l i t e 545

pH 5 . 8 b u f f e r pH 5.1 b u f f e r

chloroform wash t h e n e l u t e w i t h 2.4% v/v DEHP i n c h l o r o form

Column Support c e l i t e 545 a c i d washed

T A B L E 6.33-1 ( c o n t i n u e d ) Other ComDounds Stationary P r e s e n t Not Mobile Phase Phase Interfering

Ref e rence
69

sodi u m borate

chloroform wash then a c e t y l a t e and e l u t e a c e t y 1 a t e d phenyle p h r i ne w i t h chloroform saponify same a s above

codeine s u l f a t e m t h a p y r i 1 en e H C1 e p y r i l a m i n e maleate d-amphetamine s u l f a t e

u l
w

c e l i t e 545 aci d washed c e l i t e 545 aci d washed

s o d i um borate various acids a n d bases

codeine phosphate chl orpheni ramine ma1 e a t e chlorpheniramine ma1 e a t e p y r i l a m i n e maleate codeine phosphate phenyl p r o panol ami ne

70

c h 7 o rof orm wash elute w i t h ethanol

71

HC1

Method of Q u a n t i t a t i o n

U.V.

T A B L E 6.34.1
Column Condi t i ons
8 f t . , 3 mm. I . D . , S E 30 1 .15% on gas chromP 100-140 mesh

I ns trumen t a1 Conditions c o l . temp. 135"C, flow r a t e 30 m l . / m i n . i n l e t p r e s . 31 p s i

Derivative base acetone butanone

Reference
72

6 f t . , 3 mm. I.D., QFI-0065 (Dow Corni n g ) 2.8% on chromsorb 6080 mesh

VI

c o l . temp. 135"C, i n l e t a c e t o n e pres. 30 psi B - i o n i z a t i on d e t e c t o r

i n j . 300"C, d e t c . 260C t r i f l u o r o flame i o n i z a t i o n d e t c . acetic acid he1 i urn/hydrogen/ai r flow r a t e 80/80/450 m l J m i n . , resp. program col. 100C t o 200C a t l O " C / min.

72

0 P

2 M g l a s s , 4 mm. I . D . , 0.1% s i l i c o n e o i l ( D C 710) on 60-80 mesh dimethyl-dichlorosilane t r e a t e d g l a s s beads

73 a

6 f t . , 4 mm. I.D., 10% F-60 (methyl p o l y s i l o xane) on gas chrom-P 80-100 mesh

d e t c . 300C f l a m e i o n i - HMDS ( h e x a z a t i on t e m p . program methyldi s i 1 100"C-2OO0C 8 1.5"C/ azane) m i n . n i t r o g e n 12 psi acetone a i r 40 p s i , hydrogen cycl obutanone 20 psi a = assayed t a b l e t s and s y r u p s

74

TABLE 6 . 3 5 - 1 Res in Amberl it e IR-45 Type weakly basic M o b i l e Phase Method o f Quan.
tit r a t i o n

Compounds Present Not Interfering codeine phosphate p o t a s s i um guaiacolsul fonate camphor menthol

Reference

75% e t h a n o l

75

Dowex 50-X-1 Dowex 50-X-2 Dowex 50-X-8 Dowex 50-X-12 Dowex 50-X-16 Dowex 50-W-X-1 Amberl it e I R 120

sulfonic aci d H+ f o r m

w a t e r ash then e ute w i t h 0.5 N

HCI

azo coup1 in g Millon's reagent

APAP t heny 1 d iami ne

HC1

39

caffein

T A B L E 6.35-1

(continued) Method of Quan.

U.Y.

Resin Type A

Type P01Y styrene sulfonic acid N H t form

Mobile Phase g r a d i e n t pH 10-12 c r 0 . 1 5 M t o 0.37 M NHbOH 1 N HC1 i n 50% met ha no1

Compounds P r e s e n t Not Interfering met anep h ri ne p -synephri ne + 13 o t h e r compounds

Re f e ren ce
76

AG 5OW-X-4

strong cation

U.Y.

codeine phosphate 77 chl orpheni rami ne 78 maleate prome t h a z i ne H 1 C methapyrilene H 1 C d e x t romet h o r p h a n H B r p y r i l ami ne ma1 e a t e codeine phosphate acetaminophen 79

A ginic 1 Acid

cation

0.01 N H 1 C

U.Y.

PHENY LEPHRINE HYDROCHLORIDE

o c c u r r i n g a t 390 n m .

Other Methods o f A n a l y s i s A non-aqueous t i t r a t i o n o f p h e n . y l e p h r i n e t o a crystal violet end point u s i n g perchloric a c i d i n d i o x a n e - a c e t i c a c i d medium h a s been r e ported ( 8 2 ) .
6.5

Bromi n a t i on has been used t o d e t e r m i n e phenyle p h r i ne u s i n g bromine w a t e r ( 8 3 ) and coul ometri c a l l y g e n e r a t e d bromine ( 8 4 ) .
T h e i n c r e a s e i n b l o o d pressure o f b o t h r a t s and g u i n e a p i g s i s t h e b a s i s o f a b i o l o g i c a l assay o f phenylephrine ( 8 5 ) . Salicylamide, Na c e t y l -p-aminophenol and c h l o r p h e n i r a m i n e m a l e a t e d o n o t i n t e r f e r e . Sample s i z e s o f 1 . 5 t o 80 u g . have been d e t e r m i n e d . I n t e r f e r e n c e r e f r a c t o m e t r y ( 8 6 ) has been u s e d as a q u a n t i t a t i v e micro method of phenylephrine analysis.

7.

References

1.
2.

3.

4.
5.
6.
7.

8.

The U n i t e d S t a t e s P h a r m a c o p e i a , E i g h t e e n t h R e v i s i o n , 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 . 498. The Merck I n d e x , E i g h t h E d . , Merck & Co. , I n c . , Rahway, N.J. , 1 9 6 8 , p . 817. F . Martin, J . Plzarm. B e Z g . , 6 , 283-93, (1951). S . R i e g e l m a n , L . A . S t r a i t and E . Z . F i s c h e r , J . Pharm. S c i . , 5 2 , 1 2 9 - 1 3 3 , (1962). H . L e g e r l o t z , U.S. 1 , 9 3 2 , 3 4 7 , ( O c t . 2 4 , 1933). E . D. Bergmann a n d M . S u l z b a c h e r , J . O r g . Chem., 1 6 , 8 4 , ( 1 9 5 1 ) . P . B . R u s s e l l and S . C . C h i l d r e s s , J . P h a r m . S c i . , 50, 713, ( 1 9 6 1 ) . A. Z . B r f t t e n , C h e m . I n d . ( L o n d o n ) 2 9 6 8 , ( 2 4 ) , 771 -72 , C A 69 ~ 7 6 7 9 0 ~ .

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. .

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PHENYLEPHRINE HYDROCHLORIDE

69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86.

W . R. C l a r k a n d L. A . R o s e n b e r , J . A s s o c . 0 f i e . A g r . Chem., 4 8 ( 3 7 , 5 7 9 82 (3965f. R. H a t t , J . A s s o c . O f f i c . A g r . Chem., 4 8 ( 3 j , 594-95 ( 19 6 5 ) . D. J . S m i t h , J . A s s o c . Offic. A n a l . Chem. 4 9 ( 3 ) 9 536-41 ( 1 9 6 6 ) . E. Brochmann-Hanssen and A. B . Svendsen, J . Pharm. S c i . , 5 1 ( l o ) , 9 3 8 - 4 1 ( 1 9 6 2 ) . C. H i s h t a a n d R. G . L a u b a c k , J . Pharm. Sci., 58(6) 745-46 ( 1 9 6 9 ) . P . C a p e l l a and E . C . H o r n i n g , AnaZ. Chem. 3 8 ( 2 ) , 31 6 - 2 1 ( 1 9 6 6 ) . M . C . V i n c e n t , E . K r u p s k i a n d L. F i s c h e r , J . A m . Pharm. A s s o c . , S c i . E d . , 46, 85, ( 1957). T . A . Wheaton a n d I . S t e w a r t , A n a l . B i o chem. 12, 5 8 5 - 5 9 2 ( 1 9 6 5 ) . K . 0. M o n t g o m e r y , P . V . J e n n i n g s and M. H . W e i n s w i g , J . Pharm. S c i . , 56(1>, 1 4 1 43 ( 1 9 6 7 ) . Idem. i b i d . , 3 9 3 - 9 5 ( 1 9 6 7 ) . F. De F a b r i z i o , J . Pharm. S c i . , 5 7 ( 4 ) , 644-45 ( 1968). S. U d e n f r i e n d , D. E . Duggan, B. M. V a s t a a n d B . B . B r o d i e , J . Pharmacol. E x p . T h e r . 120, 2 6 - 3 2 ( 1 9 5 7 ) . J . D. W i n e f o r d n e r and M. T i n , A n a l . Chim. A c t a , 31, 2 3 9 - 4 5 ( 1 9 6 4 ) . R. P o h l o u d e k - F a b i n i a n d K. K o n i g , Pharmazie, 13, 7 5 2 - 5 6 ( 1 9 5 9 ) . W . Awe a n d H . S t o h l m a n n , P h a r m a z i e , 12, 647-51 ( 19 5 7 ) . G. P a t r i a r c h e , J . Pharm. B e Z g . , 1 9 ( 7 - 8 1 , 299-317 ( 1 9 6 4 ) , CA 63:16125e. J . S . N a r a v a n e a n d S. S. S a m a r t h , I n d i a n J . Pharm., 2 8 ( 4 ) , 99-102 ( 1 9 6 6 ) . N. H . C h o u l i s , J . Pharm. Sci., 5 6 ( 9 ) , 1206, ( 1 9 6 7 ) . ( L i t e r a t u r e s e a r c h c o v e r s t h r o u g h Decemb e r 1971).

511

CHARLES A. GAGLIA, JR.

Acknowledgment The a u t h o r w i s h e s t o a c k n o w l e d g e t h e a s s i s t a n c e o f Dr. R. C . G r e e n o u g h i n p r e p a r i n g and i n t e r p r e t i n g s p e c t r a l d a t a i n t h i s p r o file.

512

TOLBUTAMIDE

William F. Beyer and Erik H. Jensen

WILLIAM

F. BEYER

AND ERIK H. JENSEN

CONTENTS 1. Description 1.1. Name, Formula, Molecular Weight 1.2. Appearance, Color, Taste, Odor 2. Physical Properties 2.1. Solubility 2.2. Melting Range 2.3. Crystal Properties 2.31. Crystal Morphology 2.311. System and Class 2.312. Axial Ratio 2.313. Interfacial Angles 2.314. Habit 2.32. Optical Properties 2.321. Refractive Indicies 2.322. Molecular Refraction 2.323. Optic Axial Angle 2.324. Dispersion 2.325. Optic Orientation 2.326. Common Crystal Orientation 2.327. Optic Sign 2.33. Fusion Properties 2.34. X-Ray Diffraction 2.4. Infrared Spectrum 2.5. Nuclear Magnetic Resonance Spectrum 2.6. Mass Spectrum 2.7. Ultraviolet Spectrum 2.8. pKa 2.9. Differential Scanning Calorimetry 3. Synthesis 4 . Stability 5. Drug Metabolites 6 . Methods of Analysis 6.1. Elemental Analysis 6.2. Phase Solubility 6.3. Titr imetric 6.4. Ultraviolet Spectrophotometric 6.5. Colorimetric

5 14

TOLBUTAMIOE

7. 8. 9.

6.6. Gas Chromatographic 6.7. Liquid Chromatographic 6.8. Paper Chromatographic 6.9, Thin Layer Chromatographic 6.10. Coulometric Pharmacokinetics and Toxicity Identification References Cited

515

WILLIAM F. BEYER AND ERIK H. JENSEN

1 Description . Name, Formula, Molecular Weight To1butamide is 1-Buty1 3-( to1y 1sul fony1)urea p It is also referred to as: N-(4-methyl-benzenesulphonyl)3N'-n-butyl-urea2, to1 lsulfonylbutylurea~, (p-tolyl-4sulfony1)-1-butylurea , N-(sulfonyl-p-methylbenzene)-N'-nbutylurea3. The most commonly used trade marks are Orinase and Rastinon; 14 additional are listed in the Merck Index 8th Edition3. Tolbutamide is a sulfonamide but it is not a sulfanilamide derivative, 1.1.

- -

' .

0 - - S O 2

- $" - 4 - N C
I

CH3

c4H9

12H 1BN 2' ' 3

Mol. Wt. 270.35

Appearance, Color, Taste, and Odor Tolbutamide is a white, or practically white, crystalline powder, It has a slightly bitter taste and is practically odorless1

1.2.

2.

Physical Properties 2.1.

Solubility Practically insoluble in water but forms watersoluble salts with alkalies. I is soluble in alcohol and t in chloroform1. It is soluble to the extent of 7.8 mg/ml . in toluene and 4 4 mg/ml in ethyl acetate:heptane (1:3)4.

516

TOLBUTAMIDE

M e l t i n g Range The m e l t i n g range of tolbutamide has been r e ported a s 126-132'l and 128.5-129.5'3. 2.3. Crystal Properties 2.31.
C r y s t a l Morphology5

2.2.

2.311. MM2 2.312.

System and C l a s s Orthorhombic, rhombic pyramidal, Axial Ratio a:b:c = 0.4504: 1:0.3864 I n t e r f a c i a l Angles

2,313.

(oil)

= 1380; ( i 2 0 ) A

(iio)

(101) A = 960

(ioi)

= 810; (011) A

Habit Tabular {OlO) w i t h {lOl), {Oll), [i20) Supplementary twinning is common, but u s u a l l y w i t h t h e composition p l a n e and r e e n t r a n t a n g l e s v i s i b l e .

2,314.

2.32.

Optical Properties5 2,321.

1.604;

R e f r a c t i v e I n d i c i e s (5893A) Nx = 1.544; N = 1.550; Nz = y geometric mean = 1.562 2,322. 2,323. Molecular R e f r a c t i o n Observed = 69.4; c a l c u l a t e d = 70.4

O p t i c A x i a l Angle (58938) 2V = 38' c a l c u l a t e d from r e f r a c t i v e i n d i c i e s ; 42' u s i n g M a l l a r d ' s c o n s t a n t . 2.324. Dispersion 1 > v, strong. :

517

WILLIAM

F. BEYER AND E R I K H. JENSEN

2.325. 2.326.

Optic Orientation

a=Y; b = X Common C r y s t a l O r i e n t a t i o n (010) showing c e n t e r e d o b t u s e bisectrix interference figure. 2.327. 2.33. O p t i c Sign Positive

Fusion P r o p e r t i e s When tolbutamide i s melted and slowly cooled, an u n s t a b l e form c r y s t a l l i z e s which, upon r e h e a t i n g , slowly undergoes a s o l i d - s o l i d phase t r a n s f o r m a t i o n t o t h e s t a b l e form5. X-ray D i f f r a c t i o n P r e c e s s i o n and Weissenberg photographs of t h e X-ray d i f f r a c t i o n p a t t e r n of s i n g l e c r y s t a l s of USP Tolbutamide were made i n a study t o update t h e powder d i f f r a c t i o n f i l e 6 . The c r y s t a l s were found t o be orthorhombic, and s y s t e m a t i c absences uniquely determined t h e space group a s P nma. The u n i t c e l l parameters measured were i n good agreement w i t h t h e o r i g i n a l d e t e r m i n a t i o n of Q h e l l 5 . The l a b e l i n g of t h e u n i t c e l l axes given by S h e l l has, howe v e r , been permuted t o a g r e e w i t h t h e I n t e r n a t i o n a l Tables f o r X-ray C r y s t a l l o g r a p h y convention f g r space grgup P nma. The new c e l l dimensions a r e : a = 20.14A; b = 9 . 0 7 ~ ; and
c = 7.781.

2.34.

The DIFMAX computer program was used t o a r r i v e a t t h e indexed r e f l e c t i o n s , 2 8 v a l u e s , and d s p a c i n g s of Table I. I n space group P nma, t h e f o l l m i n g c o n d i t i o n s l i m i t possible reflections:
0,

k, l : k

1 m u s t be even

h , k, o:h m u s t be even

Accordingly, d e l e t i o n s of s s t e m a t i c a l l y a b s e n t r e f l e c t i o n s were made i n Table I.

518

TOLBUTAMIDE

TABLE I
Possible X-ray Diffraction Maxima for Tolbutamide

H
2 1 2 2 0 1 2 3

K
0 0

L
0

2 Theta 8.78 12.19 13.13 14.38 15.00 15.63 17.40 17.44 17.61 19.57 20.02 20.17 21.01 21.48 22.86 23.12 23.21 23.28 24.37 24.53 24.89 25.30 26.33 26.45 26.45 26.48 26.55 26.79 28.28 28.35 28.87 28.96 29.00 30.26 30.60 30.63 519

D
10.0608 7.2507 6.7332 6.1510 5.8998 5.6614 5.0893 5.0780 5.0304 4.5308 4.4299 4.3981 4.2231 4.1312 3.8864 3.8423 3.8278 3.8159 3.6480 3.6253 3.5737 3.5168 3.3807 3.3666 3.3659 3.3627 3.3536 3.3245 3.1526 3.1451 3.0893 3.0792 3.0755 2.9499 2.9187 2.9155

1 0

1 1 1
0 0 2

4
0 3 4 4 2 0

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

1 1 0 2 0 2 1 0 2
0 0

4 0 1
6

1 2 2 1 0 0 1 1 1 2 0 0 2 2 1

1 0 1 1 1 1 1 0 0 1 0 1 0 2 1 1 2 1 2 1 2 1
0 2 2 0

1 2 0 1 1 2 2 2 1

WILLIAM F. BEYER AND ERIK H . JENSEN

TABLE I (Continued)

H
4 2 2 0 5 5 1 2 3 7 6 5 3 4 7

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

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

2 Theta 30.66 30.87 31.57 31.75 31.86 31.98 32.06 33.00 33.14 33.19 33.20 33.51 34.51 34.60 34.68 34.88 35.20 35.23 35.31 35.66 35.75 36.01 36.30 36.53 36.72 37.05 37.14 37.16 37.54 37.77 37.95 38.51 38.76 38.83 38.88 38.93 39.00 39.06 39.75
520

D
2.9123 2.8930 2.8307 2.8154 2.8059 2.7956 2.7883 2.7113 2,7002 2.6960 2.6955 2.6713 2.5960 2.5896 2.5841 2.5697 2.5467 2.5446 2.5390 2.5152 2.5091 2.4911 2.4722 2.4568 2.4448 2.4235 2.4181 2.4169 2.3930 2.3791 2.3683 2.3352 2.3206 2.3169 2.3137 2.3110 2.3069 2.3034 2.2654

6 4 6 8 2 0 1 4 6 8 2 3 8 5
1 3 2 7 8 7 5 4 0

3
0

TOLBUTAMIDE

Figure 1 gives the X-ray powder diffraction pattern for USP Tolbutamide obtained with a General Electric XRD-5 Diffractometer using C u K d 1, 50 KVP, 16 MA, and tray mount6, Infrared Spectrum Tolbutamide can be identified bv means of its infrared spectrum (Figure 2. ) The spectrLm of USP Tolbutamide7 was obtained from a Nujol mull using a Perkin-Elmer Model 421 spectrophotometer. The principal peaks are assigned as follows: 3320, 3190 (urea, NH stretch), 2920, 2850 (alkane, CW stretch including Nujol), 1700, 1660 (urea, C=O stretch), 1600, 1500 (aromatic, C=C stretch), 1, 1555 (urea, amide 1 ) 1460, 1375 (alkane, CH deformation including Nujol), 1335, 1160 (sulfonamide, S=O stretch), and 815 cm-1 (aromatic, CH deformation for para substitution). Other peaks occur at 1320, 1310, 1250, 1220, 1190, 1120, 1095, 900, 740, 725, and 660 cm-l. Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance (NMR) spectrum of tolbutamide was obtained using a Varian instrument Model A-60 D. Figure 3 gives the spectrum8. The tolbutamide was dissolved in deutero chloroform with tetramethylsilam as the internal reference. The NMR proton spectral assignments are given in Table I18. Mass Spectrum Tolbutamide mass-spectral data are given in Table III9. The data were obtained using an Atlas CH4 instrument. The l o s s of 64 mass units from the molecular ion is attributed to the loss of S02, a unique loss of these elements from the middle of the tolbutamide molecule. The molecular ion was observed at mfe 270. At 70 ev the most intense peak was observed at m/e 91 (15.4% of total ionization). This peak can be represented as a C7H7+ion which yields CgHg+(m/e 65; metastable at 46.4) upon expulsion of an acetylene molecule after decomposition. 2.6. 25 .. 2.4.

521

522

0 0

m
0 0

b
a
a ,

i
0

o m a b u , u , a n ~ -

33NWlllHISNWMl %

523

\ I
\Figure 3 . Nuclear magnetic resonance spectrum of tolbutamide

TOLBUTAMIDE

TABLE I1 NMR Spectral Assignments for Tolbutamide Shape Distorted Triplet Broad Multiplet Chemical Shift J (Hz)
0.88

6.5

CH3-CgHqCH3-CH2-CH2-CH2-

Singlet

2.43 3.23 6.57

6.0
6.0

Quartet Triplet

(av.) (av.)

-N_H-CH2H

Apparent Doublet

7.33

8.0

(av.)

CH3 QS

CH3

9
H
S

Apparent Doublet

7.83

8 0 (av.) .

-02S-NK-CO2.7.

Broad Singlet

9.7

Ultraviolet Spectrum The UV spectrum of USP Tolbutamide is shown in Figure 4 . The spectrum was obtained with a Cary 15 spectrophotometer using a 15 mcg/ml solution of tolbutamide in anhydrous ethanol. The spectrum, obtained with a 1-cm cell, shows A max at 228 nm.

525

WILLIAM F. BEYER AND E R I K H. JENSEN

0.3

WAVELENGTH (nm)

Figure 4 .

Ultraviolet spectrum of tolbutamide

526

TOLBUTAMIDE

TABLE 111
Mass Spectral Data for Tolbutamide

ml e 270 255 241 227 215 206 184 171 163 155 139 115 107 99 91 73 72 65 30
2.8.

% total ionization

70 ev 2.2 0.2 0.3 1.1 0.4 6.5 0.2 0.6 0.3 7.1 0.6 2.0 14.0 5.3 3.2 15.4 3.2 2.3 46 . 12.6

19 ev 7.5

35.7 0.5

0.4
1.5 19.4 11.5 3.3 0.1 3.1 0.5 5.7

ioa

The pKa' of tolbutamide by two separate procedures was 5.43 at 25OC and 5.32 at 37.5OC10. Differential Scanning Calorimetry The absolute purity of tolbutamide can be determined using differential scanning calorimetry. The purity of Tolbutamide USP Reference Standard with this technique 12. A Perkin-Elmer Differential Scanning was 99 .51 Calorimeter Model 1 - B was used at a scan speed of 1 2 ' min. at a sensitivity of 2 m callsec f u l l scale. 2.9.

p ~ a

521

WILLIAM F. BEYER AND ERIK H. JENSEN

W F. Beyer and E H Jensen . . .

Synthesis The patent procedure for tolbutamide2 gives the following example for its preparation: 50 gms of n-butyl isocyanate are stirred at RT into a suspension of 96 gms of sodium 4-methyl-benzenesulfamide in 120 ml of dry nitrobenzene, and the mixture is then heated for 7 hours at 100C. After being cooled, the reaction mixture, which is a thick magma, is diluted with methylene chloride or ethyl acetate, and the sodium salt of the sulfonylurea formed is separated by centrifuging. The centrifuged crystalline residue freed from organic solvents is dissolved in 500-600 ml of water heated at 5OoC and decolorized with charcoal. The precipitate obtained by acidification with dilute hydrochloric acid is dissolved in an equivalent quantity of dilute ammonia solution (about 1:20), again treated with charcoal and reprecipitated with dilute hydrochloric acid. In this manner tolbutamide is obtained in analytically pure form in a yield of 70-80 percent of theory. Menzer et all3 list p-tolylsulfonamide and p-tolylsulfonylurea as the primary impurities to be expected in the synthesis of tolbutamide. Stability Because of the absence of p-amino groups, which are common to antibacterial sulfonamides, tolbutamide cannot be acetylated. Its p-methyl group, however, renders tolbutamide susceptible to oxidation, occurring chiefly in biological systems.
4.

3.

Thermal decomposition of tolbutamide has been reported by Menzer et al13, with reformation of p-tolylsulfonamide and synthesis of butylisocyanate. The latter then reacts with unconverted butylamine and ammonia to form N,N-dibutylurea and N-butylurea. The authors also isolated four additional by-products of tolbutamide, one identified as p-tolylsulfonylbiuret. The hydrolysis of tolbutamide in an acid environment was investigated by Vogtl4 and in alkaline solution by Haussler and Hajduls. A quantitative dissociation of tolbutamide to p-toluenesulfonamide and n-butyl isocyanate was reported by Ulrich and Sayighl6 to take place in inert solvent at 160 to 180C. A significant degradation
528

TOLBUTAMIDE

occurred in some o/w creams when the drug wa dissolved in the oil phase of the emulsion at 70 to 80 C 17. No such loss occurred when the sulfonylurea was incorporated in the base at room temperature. The authors concluded that the instability of tolbutamide in the oil phase of the emulsion was due to components containing hydroxyl groups. The investigations were expanded to study the dissociation of tolbutamide at 80OC in twelve primary alcohols and in polyethylene glycol 40018. Tolbutamide was shown to dissociate to give butylamine and p-toluenesulfonyl isocyanate. N-(p-toluenesulfonyl) carbamate, formed by the reaction of the sulfonylisocyanate with the alcohols, was present in the equilibrium mixture. Bottari et all9, in studies investigating the reaction products of tolbutamide and other N-substituted sulfonylureas in alcohols, water, and amines, concluded that dissociation, rather than solvolysis, was the most likely mechanism by which sulfonylureas undergo breakdown at rela ively low temperatures. A report by Chubb and SimmonsZS indicates that tolbutamide reacts with refluxing methanol to form the butylamine salt of methyl p-tolylsulfonylcarbamate. They subscribe to a mechanism of methanolysis for the reaction rather than one of pyrolysis.
5.

Drug Metabolites Oxidation of tolbutamide through its p-methyl group appears to be the principal manner of degradation of tolbutamide in man. The p-methyl group is oxidized to form a carboxyl group, converting tolbutamide into its principal metabolite, 1-butyl-3-p-carboxyphensulfonylurea (carboxytolbutamide)21s22.

The tolbutamide metabolite is highly soluble over the critical acid range of urinary pH values, and its solubility increases with an increase in pH. The measured solubility at pH 5 is 2.8 mg/ml, increasing to 20 mg/ml at pH 5.5; atpH6.0 by extrapolation, the solubility becomes 300 mg/m123. At 37.5OC and at various pH ranges the following carboxyto1butamide:tolbutamide solubility ratios were determined: pH 5.0, 13:l; pH 5.5, 50:l; pH 6.0, 350:11. A pKa1 of 3.54 at 37.5OC has been determined for the metabolitelo.
529

WILLIAM

F. BEYER A N D E R I K H. JENSEN

The amount of m e t a b o l i t e i n u r i n e c a n be d e t e r m i n e d by measuring t h e c o l o r found when amyl a c e t a t e - e x t r a c t e d u r i n e i s added t o 0.1% d i n i t r o f l u o r o b e n z e n e and h e a t e d a t 150'24. 6. Methods o f A n a l y s i s 6.1. E l e m e n t a l A n a l y s i s of t o l b u t a m i d e 4 ,
E 1e men t
% Theory

% Reported 53.43 6.99 10.29 11.88

Carbon Hydrogen Nitrogen Sulfur 6.2.

53.31 6.71 10.36 11.86

Phase S o l u b i l i t y A n a l y s i s Phase s o l u b i l i t y p r o f i l e s o f t o l b u t a m i d e were o b t a i n e d w i t h s o l v e n t s y s t e m s of t o l u e n e and e t h y l a c e t a t e : h e p t a n e ( 1 : 3 ) , g i v i n g s o l u b i l i t i e s of 7.79 k0.15 mg/gm and 4 . 4 1 k0.08 mg/gm r e s p e c t i v e l y 4 . The c a l c u l a t e d p u r i t y of t h e t o l b u t a m i d e sample based on i t s s o l u b i l i t y p r o f i l e i n t o l u e n e was 100.1 k0.70% and i n e t h y l a c e t a t e : h e p t a n e (1:3) i t was 99.6 k0.41%.

Titrimetric The p r o c e d u r e of F r a n c h i 2 5 depends on t i t r a t i o n o f t o l b u t a m i d e w i t h sodium methoxide i n anhydrous p y r i dine-chloroform-methanol.

6.3. I n a r e p o r t e d t i t r i m e t r i c method of a s s a y f o r t o l butamide i n non-aqueous mediaz6, 50 t o 150 mg i s d i s s o l v e d i n 10 m l of anhydrous a c e t o n e o r p y r i d i n e and t i t r a t e d w i t h 0 . 1 1 sodium methoxide t o a p h e n o l p h t h a l e i n e n d p o i n t , I n a m i x t u r e of benzene and methanol ( 2 : 1 ) , thymol b l u e c a n be used a s i n d i c a t o r . A s i m i l a r p r o c e d u r e was r e p o r t e d by Dave and P a t e 1 2 7 . S i m i o n o v i c i and ConuZ8 developed a d i r e c t g t i t r a t i o n method whereby a p p r o x i m a t e l y 300 m o f

530

TOLBUTAMIDE

t o l b u t a m i d e i s d i s s o l v e d i n 25 m l of a c e t o n e p r e v i o u s l y n e u t r a l i z e d t o c r e s o l r e d and t i t r a t e d w i t h 0.1; sodium hydroxide t o a r o s e - v i o l e t c o l o r . I n a p r o c e d u r e depending on h y d r o l y s i s , t h e previous a u t h o r s 2 8 t r e a t e d a p p r o x i m a t e l y 300 m of t o l b u t a m i d e w i t h g 10 m l o f e t h a n e d i o l and 2 m l of conc. h y d r o c h l o r i c a c i d , and a p p l i e d 120-122'C h e a t f o r 30 min. The m i x t u r e was then d i l u t e d with water, t r e a t e d i n a Kjeldahl f l a s k with 1 5 g sodium h y d r o x i d e and t h e amine was d i s t i l l e d o f f i n t o 25 m l o f 0.1N h y d r o c h l o r i c a c i d , t h e e x c e s s d e t e r m i n e d by t i t r a t i o n . Assay v a r i a t i o n o f +1.0% was r e p o r t e d . P a r i k h and Mukherji2' developed a t i t r a t i o n proc e d u r e f o r t o l b u t a r n i d e i n which t o l b u t a m i d e was c o n v e r t e d i n t o i t s sodium s a l t , combining i t q u a n t i t a t i v e l y w i t h s i l v e r n i t r a t e t o form a n i n s o l u b l e s i l v e r s a l t . F e r r i c ammonium s u l p h a t e was used a s t h e i n d i c a t o r and 0.05; ammonium t h i o c y a n a t e a s t h e t i t r a n t . Beyer and Houtman3' d e s c r i b e d automated t i t r i metric procedures f o r the analysis of tolbutamide t a b l e t s u s i n g a modified F i s c h e r T i t r a l y z e r . The r e l a t i v e s t a n d a r d d e v i a t i o n f o r t h e p r o c e d u r e was a p p r o x i m a t e l y 1%. The USP a s s a y p r o c e d u r e 1 f o r t o l b u t a m i d e depends upon t h e t i t r a t i o n o f t h e d r u g i n n e u t r a l i z e d aqueous a l c o h o l w i t h sodium h y d r o x i d e a s t h e t i t r a n t , and phenolp h t h a l e i n as i n d i c a t o r . U l t r a v i o l e t Spectrophotometric S p i n g l e r and K a i s e r J L d e t e r m i n e d t o l b u t a m i d e i n serum a f t e r l y o p h i l i z a t i o n , e x t r a c t i o n w i t h a c i d i f i e d e t h y l a c e t a t e , r e d u c t i o n t o d r y n e s s , and f i n a l l y d i s s o l u t i o n i n methanol. Absorbances a t 228 nm and 280 nm were used i n q u a n t i t a t i n g tolbutamide. F o r i s t e t a132 d e v e l o p e d an a n a l y t i c a l p r o c e d u r e f o r t h e d e t e r m i n a t i o n o f t o l b u t a m i d e i n plasma. The
6.4.

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WILLIAM f. BEYER A N D ERIK H . JENSEN

procedure depends on c h l o r o f o r m e x t r a c t i o n o f weakly a c i d i f i e d plasma, c o n c e n t r a t i o n o f t h e e x t r a c t i o n t o d r y n e s s , d i s s o l u t i o n of t h e d r y r e s i d u e i n e t h a n o l , t r e a t ment o f t h e s o l u t i o n w i t h c h a r c o a l , and measurement of t h e a b s o r b a n c e of a n a l c o h o l i c s o l u t i o n a t 228 nm. Experiments w i t h human and dog plasma gave r e c o v e r i e s o f added t o l b u t a m i d e o f a b o u t 98-99%. R e f i n ments i n t h e method were r e o r t e d by Bladh and Norden3', and D e l a v i l l e and P a l a z z o l i 34

A p r o c e d u r e f o r t h e automated a n a l y s i s of t o l b u t a m i d e t a b l e t s h a s been r e p o r t e d u s i n g Technicon C o r p o r a t i o n ' s AutoAnalyzer equipment35. The a n a l y s i s was c a r r i e d o u t a t a w a v e l e n g t h of 263 nm a t a sampling r a t e of 201hour. A c o e f f i c i e n t o f v a r i a t i o n o f a p p r o x i m a t e l y 1% was o b t a i n e d .
The USP X V I I I l p r o c e d u r e f o r t o l b u t a m i d e t a b l e t s depends upon t h e UV a b s o r b a n c e of e x t r a c t e d t a b l e t s i n c h l o r o f o r m a t a wavelength o f 263 nm. A UV d i s s o l u t i o n r a t e t e s t f o r t o l b u t a m i d e t a b l e t s i s d e s c r i b e d i n t h e 1st supplement t o t h e USP X V I I I u s i n g t r i s ( h y d r o x y m e t h y 1 ) aminomethane b u f f e r a t pH 7.6 and a s t i r r i n g r a t e o f 150 rpm. F i l t e r e d samples a r e r e a d a t a w a v e l e n g t h o f 226 nm. The t e s t s p e c i f i e s t h a t t h e t i m e r e q u i r e d f o r 50% of t h e l a b e l e d amount of t o l b u t a m i d e i n t a b l e t s t o d i s s o l v e i s n o t more t h a n 45 m i n u t e s , Colorimetric McDonald and S a ~ i n s k d e v e l o p e d a c o l o r i i ~ ~ m e t r i c method i n v o l v i n g t h e r e a c t i o n between t o l b u t a m i d e , 2 - n a p h t h o l , sodium n i t r i t e , and c o n c e n t r a t e d s u l f u r i c a c i d forming a red c o l o r . The method i s r e p o r t e d t o be a p p l i c a b l e over t h e c o n c e n t r a t i o n range of approximately 50 mcg t o 1 0 m o f t o l b u t a m i d e p e r m l o f s o l u t i o n . g C h ~ l s k d e~ c r i b e d a method f o r t h e d e t e r i s~ m i n a t i o n o f t o l b u t a m i d e i n serum by e x t r a c t i n g a c i d i f i e d serum w i t h c h l o r o f o r m . A f t e r r e d u c i n g t h e c h l o r o f o r m e x t r a c t t o d r y n e s s , a n a l c h o l i c s o l u t i o n o f p-N-dimethylaminobenzaldehyde i s added and t h e s o l u t i o n reduced t o d r y n e s s . The d r y r e s i d u e i s h e a t e d or 2-112 h o u r s a t 6.5.

532

TOLBUTAMIDE

7 O o C and a l c o h o l i s added. Absorbance o f t h e s o l u t i o n s i s measured a t 395 nm. The a v e r a g e r e c o v e r y o f t o l b u t a m i d e from serum was r e p o r t e d t o be n e a r 100% w i t h a s t a n d a r d d e v i a t i o n o f a p p r o x i m a t e l y 5%.
The c o l o r i m e t r i c t e r m i n a t i o n o f serum t o l b u t amide d e v e l o p e d by S p i n g l e r " depends on t h e r e a c t i o n o f t o l b u t a m i d e and d i n i t r o f l u o r o b e n z e n e a t 15OoC f o l l o w i n g e x t r a c t i o n o f a c i d i f i e d serum w i t h amyl a c e t a t e . The a b s o r b a n c e i s d e t e r m i n e d a t a b o u t 380 nm. A f t e r a s t u d y o f t h e method o f S ~ i n g l e r 3 ~ i g n a r d 3 g s u g g e s t e d improveP, ments t h a t were r e p o r t e d t o i n c r e a s e s p e c i f i c i t y and r a n g e of u s e f u l n e s s . Among t h o s e s u g g e s t e d were p u r i f i c a t i o n of r e a g e n t s and l e n g t h e n i n g t h e h e a t i n g t i m e from 5 t o 30 m i n u t e s a t a t e m p e r a t u r e of 100C i n s t e a d o f 15OoC. D ~ r f m ~ l l rer orted the r e a c t i o n of t o l b u t e p~ ~ amide i n a l k a l i n e media w i t h diacetylmonoxime and N-phenyla n t h r a n i l i c a c i d , f o l l o w e d by a c i d i f i c a t i o n and h e a t and t h e a d d i t i o n o f sodium p e r s u l f a t e and sodium a c e t a t e t o form a b l u e c o l o r . Mesnard and C r o c k e t t 4 1 e x t e n d e d t h e work o f R i c h t e r 4 * , which i n v o l v e d t h e d e t e r m i n a t i o n of a l i p h a t i c amino a c i d s , t o t h e a n a l y s i s of t o l b u t a m i d e i n b i o l o g i c a l f l u i d s . The method i s based on t h e s t r o n g y e l l o w c o l o r a t i o n o f s u b s t i t u t e d ammonium p i c r a t e s i n s e l e c t e d anhyd r o u s s o l v e n t s , i n which p i c r i c a c i d i s e s s e n t i a l l y c o l o r l e s s . Of t h e two a b s o r b a n c e maxima observed (355 nm and 412 nm) t h e maximum a t 412 nm w a s used t o a v o i d a b s o r p t i v e m a t e r i a l s i n blood and u r i n e t h a t c o u l d i n t e r f e r e a t t h e 355 nm wavelength. The a u t h o r s a l s o reviewed o t h e r methods f o r t h e d e t e r m i n a t i o n of t o l b u t a m i d e and o t h e r non-amino hypoglycemic su I f ~ n a m i d e9 s 4 . ~ 4~ Kern45 r e p o r t e d a p r o c e d u r e f o r t h e d e t e r m i n a t i o n of t o l b u t a m i d e i n blood f o l l o w i n g e x t r a c t i o n w i t h e t h y l e n e chloride a t pH 5. A f t e r n i t r a t i o n , d i a z o t i z a t i o n and c o u p l i n g w i t h N(1-naphtyl) e t h y l e n e d i a m i n e , a n a z o dye i s produced t h a t i s measured a t 547 nm.

533

WILLIAM F. BEYER AND ERIK H. JENSEN

A number o f o t h e r i n v e s t i g a t o r s have d e s c r i b e d co l o r ime t r i c p r o c e d u r e s f o r t o 1 u t amid e46-49 b

Gas Chromatographic A g a s - l i q u i d c h r o m a t o g r a p h i c method f o r t h e d e t e r m i n a t i o n of t o l b u t a m i d e i n b l o o d , u r i n e , and t a b l e t s was r e p o r t e d by S a b i h and SabihSO. A method f o r b l o o d and u r i n e i n v o l v e d e x t r a c t i o n o f t o l b u t a m i d e from a c i d i f i e d plasma o r u r i n e and c o n v e r s i o n t o t h e m e t h y l d e r i v a t i v e with dimethylsulfate i n t h e presence of base. A g a s chromatograph (F & M 5755B) w i t h a f l a m e i o n i z a t i o n d e t e c t o r and f i t t e d w i t h a s t a i n l e s s s t e e l column was u s e d . The column was packed w i t h d i a t o m a c e o u s e a r t h (Gas chrom Q) and c o a t e d w i t h 5% DC-200. T e m p e r a t u r e s o f 205-210' f o r t h e column, 320' f o r t h e d e t e c t o r , and 330' f o r t h e i n j e c t i o n p o r t were u s e d . A d d i t i o n a l GLC methods f o r t o l butamide i n blood have b e e n r e p o r t e d by P r e s c o t t and Redman51 and Simmons e t a152 a l s o i n v o l v i n g m e t h y l a t i o n w i t h dimethyl s u l f a t e . L i q u i d Chromatographic A l i q u i d chromatographic a s s a y procedure f o r t o l b u t a m i d e i n t a b l e t s ( a l s o a p p l i c a b l e t o b u l k d r u g ) was d e s c r i b e d by B e ~ e r ~ A~ duPont Model 820 L i q u i d Chromato. g r a p h w i t h a n HCP column ( s t a i n l e s s s t e e l ) 1 M l o n g x 2.1 mm I D and a mobile p h a s e of pH 4.4 monobasic sodium c i t r a t e b u f f e r i n 15% methanol a t a f l o w r a t e of 0 . 3 6 m l / min were used f o r t h e a n a l y s i s . The r e l a t i v e s t a n d a r d d e v i a t i o n w a s less t h a n 2% and r e c o v e r y was q u a n t i t a t i v e , Paper Chromatographic Chakrabarti54 r e p o r t e d a paper chromatographic a n a l y s i s o f t o l b u t a m i d e i n v a r i o u s s o l v e n t s y s t e m s . The developed p a p e r i s r e a c t e d w i t h p h e n y l h y d r a z i n e and s p r a y e d w i t h a s o l u t i o n of ammoniacal Ni2+ g i v i n g p i n k t o v i o l e t s p o t s , d e p e n d i n g on t h e c o n c e n t r a t i o n of t o l b u t amide s o l u t i o n s . Hentrichfj5 d e s c r i b e d a paper chromatographic s e p a r a t i o n o f t o l b u t a m i d e by c h r o m a t o g r a p h i n g t h e b u t y l amine produced when t o l b u t a m i d e i s r e a c t e d w i t h a m o d i f i e d F o l i n r e a g e n t . A f t e r h e a t i n g t h e p a p e r a t 180-200', a brown s p o t i s produced. 534 6.7.

6.6.

6.8.

TOLBUTAMIDE

Abdel-Wahab and E l - A l l a ~ yr~ p o r t e d a procedure e~ u t i l i z i n g paper chromatography f o r r a d i o i s o t o p e s and t h e d e t e r m i n a t i o n of t o l b u t a m i d e . The a u t h o r s employed v a r i o u s d e v e l o p e r s and c o l o r i n g r e a g e n t s such a s n i n h y d r i n . R a d i o - a c t i v a t i o n of d r i e d , developed chromatograms, u s i n g 1131was a p p l i e d t o determine Rf v a l u e s . I n v e s t i g a t i o n s of S35 l a b e l e d t o l b u t a m i d e showed t h a t paper chromatography, accompanied by r a d i o s c a n n i n g o r a u t o r a d i o g r a p h y could be used f o r t h e s e p a r a t i o n , d e t e c t i o n , and d e t e r m i n a t i o n of tolbutamide. A i n f r a r e d i d e n t i f i c a t i o n of tolbutamide i n n human serum employing paper chromatography was r e p o r t e d by K r i v i s and F o r i s t 5 7 . The procedure depends upon ext r a c t i o n of serum w i t h chloroform, e v e n t u a l r e d u c t i o n t o d r y n e s s , d i s s o l u t i o n i n methylene c h l o r i d e , and a p p l i c a t i o n t o prewashed Whatman No. 1 paper. A f t e r development i n a butanol-water-piperidine (81: 17: 2) system by d e s c e n d i n g chromatography, t h e t o l b u t a m i d e zone was e l u t e d w i t h w a t e r . A potassium bromide m i c r o p e l l e t prepared from a methylene c h l o r i d e e x t r a c t i o n of t h e aqueous e l u a t e was t h e n examined by i n f r a r e d spectrophotometry. Thin Layer Chromatographic S t r i c k l a n d b 8 d e s c r i b e d TLC procedures f o r t h e s e p a r a t i o n and d e t e c t i o n o f microgram amounts of t o l b u t amide i n t h e presence of acetohexamide, chlorpropamide, and phenformin HC1. Various s o l v e n t systems and s p r a y r e a g e n t s were used t o determine r e l a t i v e Rf v a l u e s . A s o l v e n t system c o n s i s t i n g of acetone-benzene-water (65:30:5) s e p a r a t e d t o l b u t a m i d e and t h e o t h e r t h r e e a n t i d i a b e t i c a g e n t s . The l i m i t of d e t e c t i o n f o r t o l b u t a m i d e u s i n g UV was about 1 mcg.
A TLC procedure f o r t h e d e t e c t i o n of t o l b u t a m i d e i n blood and u r i n e was r e p o r t e d by Baumler and R i p p s t e i n 5 9 . The d r u g i s e x t r a c t e d w i t h e t h e r and chromatographed u s i n g K i e s e l g e l - c e l l u l o s e ( 1 : l ) a s t h e TLC s u p p o r t and developed w i t h benzene-methanol ( 4 : 1). Tolbutamide a p p e a r s a s a v i o l e t s p o t when t h e developed p l a t e i s sprayed w i t h n i n h y d r i n - s t a n n o u s c h l o r i d e and h e a t e d , t h e n sprayed w i t h a c i d i f i e d n i n h y d r i n and h e a t e d a g a i n .

6.9,

535

WILLIAM

F. BEYER AND E R I K H. JENSEN

Guven e t a160 i d e n t i f i e d t o l b u t a m i d e amongst o t h e r a n t i - d i a b e t i c d r u g s by TLC on s i l i c a g e l u s i n g a s o l v e n t system composed of b u t a n o l - a c e t i c acid-water (10:2:1). A s o l u t i o n of copper s u l f a t e (10%) ammonia (2%) ( 5 : l ) was used t o d e t e c t t h e s p o t s , w i t h t o l b u t a m i d e e x h i b i t i n g a green color. A r e p o r t by Hutzul and Wright61 f o r t h e d e t e c t i o n of s m a l l amounts of i m p u r i t i e s ( f o r example, 0.05% p - t o l u e n e s u l f o n y l u r e a ) i n t o l b u t a m i d e , makes use of a "Moscow" method of TLC developed by Mistryukov62. The p l a t e , 5x8x1/8" f r o s t e d window pane, i s covered w i t h adsorbent and maintained h o r i z o n t a l l y w h i l e t h e d e v e l o p i n g s o l u t i o n i s fed t o one edge by c a p i l l a r y a c t i o n . E-Toluenesulfonylurea is s e p a r a t e d from t o l b u t a m i d e u s i n g Davison s i l i c a g e l a s a d s o r b e n t , benzene-acetonem e t h a n o l - a c e t i c a c i d (70: 20: 9: 1) a s d e v e l o p i n g s o l v e n t , and v i s u a l i z e d u s i n g mists of hypochlorous a c i d , e t h a n o l , and an aqueous s o l u t i o n of a c e t i c a c i d w i t h i o d i d e and s a t u r a t e d w i t h b e n z i d i n e . For t h e d e t e c t i o n of 1toluenesulfonamide, an a d s o r b e n t of aluminum oxide and a developing s o l v e n t of benzene-acetone-methanol (70:20:10) was used. Agents f o r v i s u a l i z a t i o n were t h e same a s t h o s e f o r p t o l u e n e s u l f o n y l u r e a . Menzer e t a l l 3 u s e d a developing system comp r i s e d of c h 1 o r o f o r m : g l a c i a l a c e t i c a c i d (99:l) and s i l i c a g e l H p l a t e s , 0.25 mm t h i c k t o s e p a r a t e t o l b u t amide from i t s by-products. E v a l u a t i o n s were made under UV l i g h t a f t e r spraying with xanthydrol s o l u t i o n . Their work a l s o d i s c l o s e d a new by-product i n t h e s y n t h e s i s of tolbutamide: p t o l y l s u l f o n y l b i u r e t . Table I V summarizes their results.

536

TOLBUTAMIDE

TABLE IV Relative TLC Rf Values for Tolbutamide and By-products Using a Ch1oroform:Glacial Acetic Acid (99:l) Developing Solution Compound Tolbutamide N,N'-Dibutylurea P-Toly lsu 1fonamide P-Toly lsu1fony lurea P-Tolylsulfonylbiuret N-Butylurea
Rf -

0.85 0.56 0.47 0.26 0.13 0.12

Reisch et a163 reported five TLC developing systems using n-butanol in combination with two to three other organic solvents. Spots were detected on silica gel G plates when sprayed with visualizing agents. TLC was in applied by Neidlein et a164 and Glogner et d5 separating tolbutamide from other sulfonamides. 6.10.

A Mercurocoulometric method for the determin-

Coulometric

ation of tolbutamide has been reported by Kalinowski and Korzybski66 and V O ~ C U ~ ~ . Pharmacokinetics and Toxicity The half-life in human subjects for 1 gm of tolbutamide given as a single dose following an overnight fast is reported by McMahon et a168 to be 5.7 hours. The LD50 f o r tolbutamide administered orally to rats is 2,344 mg/ kgm and in mice injected IP is 1,232 mg/kgm68. Inactivation of tolbutamide to carboxytolbutamide occurs in man and is rapidly excreted in urine as the principal metabolite. 7.

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WILLIAM F. BEYER AND ERIK H. JENSEN

8.

Identification I d e n t i f i c a t i o n t e s t s f o r tolbutamide a r e given i n U.S.P. X V I I I 2 . The t e s t s depend upon: a ) t h e i n f r a r e d a d s o r p t i o n spectrum of a m i n e r a l o i l d i s p e r s i o n of t h e drug i n t h e range o f 2 t o 1 2 p; b) f o r m a t i o n of an orange-red c o l o r a f t e r t h e d r u g i s r e f l u x e d w i t h d i l u t e s u l f u r i c a c i d , steam d i s t i l l 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 b e i n g made s t r o n g l y a l k a l i n e , made a l k a l i n e w i t h a c e t a t e and b o r a t e b u f f e r , and r e a c t e d i n an i c e b a t h w i t h p - n i t r o a n i l i n e and sodium hydroxide; and c ) prod u c t i o n of p-toluenesulfonamide ( m e l t i n g between 136' and 141') by r e f l u x i n g i n d i l u t e s u l f u r i c a c i d , c o o l i n g t h e s o l u t i o n , c o l l e c t i n g and p u r i f y i n g t h e c r y s t a l s .

538

TOLBUTAMI DE

9. References

1. 2.

"United States Pharmacopeia," 18th Ed., Mack Printing Co., Easton, Pa. Ruschig, H., W. Aumiiller, G. Korger, H. Wagner, J. Scholz, and A . BPnder, U.S. Patent 2,968,158, January 17, 1961 (assigned to The Upjohn Co.). The Merck Index, 8th Edition, Merck and Co., Inc. Rahway, N.J. (1968).

3.

4 Humphrey, L M., The Upjohn Co., unpublished data. . .

5. Shell, J. W., Anal. Chem., 30, 1577 (1958).

6. Zipple, K. G., C G. Chidester, C. G Waber, and . . D. J. Duchamp, The Upjohn Co., unpublished data.

7. Muelman, P. A. and M. L. Knuth, The Upjohn Co., unpublished data.

8. Slomp, G., The Upjohn Co., unpublished data.

9.

Grostic, M. F., R. J. Wnuk, and F. A. MacKellar, J. Am. Chem. SOC., g,(1966).

10. Forist, A . A , , T. Chulski, Metabolism, 3 , 807 (1956). 11. Bowman, P. B., The Upjohn Co., unpublished data.

12. Perkin-Elmer Trade Publication, Thermal Analysis News Letter No. 5. 13. Menzer, M., J. Presewowski, and U. Haug, Pharmazie - 738 (.i97i). 26, 14. Vogt, H., Pharm. Zentralh, 98, 651 (1959).

15. Haussler, A . and P. Hajdu, Arch, Pharm., (1962).

539

295, 471

WILLIAM F. BEYER A N D ERIK H. JENSEN

16. 17.

U l r i c h , H. and A.A.R. Sayigh, Angew. Chem. I n t . E d . Engl. 2, 724 (1966). B o t t a r i , F., M. F. S a e t t o n e , B. G i a n n a c c i n i , and M. E. LoBrutto, Rass. Dermatol, S i f i l o g r . , 20, 235 (1967). B o t t a r i , F., M. M a n n e l l i , and M. F. S a e t t o n e , J. Pharm. S c i . , 2, 1663 (1970). B o t t a r i , F., B. G i a n n a c c i n i , E. N a n n i p i e r i , and M. F. S a e t t o n e , J. Pharm. S c i . , 61, 602 (1972).

18.
19. 20. 21.

. Chubb, F. L. and D. L Simmons, Can. J. Pharm. S c i . , 1,28 (1972).

L o u i s , L. H . , S. S , F a j a n s , J . W . Conn, W , A. S t r u c k , J. B. Wright, and J. L. Johnson, J. Am. Chem. SOC. 78, 5701 (1956). P h y s i c i a n s Desk R e f e r e n c e , 26 E d . , Medical Economics, I n c . , 1972. Tucker, H. A., "Oral A n t i d i a b e t i c Therapy 1956 1965: w i t h P a r t i c u l a r R e f e r e n c e t o Tolbutamide ( O r i n a s e ) , " C. C. Thomas, P u b l i s h e r , 1965. Nelson, E . , I. O ' R e i l l y , and T. C h u l s k i , C l i n . Chim. Acta, 3 , 774 (1960). Franchi, G., (1956-57).
A t t i . Accad. F i s i o c r i t .

22.

23.

24. 25.

Siena

6,61
1,

26.
27. 28.

Kracmarova, J . , J. Kracmar, C e s k o s l . Farm., 566 (1958). Dave, J. B., and J. L. P a t e l , I n d i a n . J . o f Pharm. Z l , 226 (1959).

S i m i o n o v i c i , R., and I. Conu, Rev. Chim. Bucharest, 107 (1959).

Lo,

540

TOLBUTAM I DE

29. 30. 31. 32.

P a r i k h , P. M., S. P. M ukhe r j i , I n d i a n J. o f Pharrn. , 2l, 110 (1959). Beyer, U. F. and R. L. Houtman, Ann. N.Y:Acad. S c i . , 130, 539 (1965). S p i n g l e r , H. and F. K a i s e r , A r z n e i r n i t t e l - F o r s c h , 6 , 760 (1956).

F o r i s t , A.A., W. L. M i l l e r , J r . , J. Krake, W. A . S t r u c k , Proc. SOC. E x p t l . B i o l . Med., 96, 180 (1957).

33. 34.

14,

Bladh, E . and A. Norden, Ac t a . Pharrnacol. T o x i c o l . 188 (1958).

D e l a v i l l e , G . , and M. P a l a z z o l i , Ann. B i o l . C l i n . ( P a r i s ) , l6, 481 ( 1958) . Beyer, W. F. and R. L. Houtman, Ann. N.Y. S c i . , 130, 535 (1965). Acad.

35.
36. 37. 38. 39. 40. 41. 42.

McDonald, H. and V. Sawins k i , Texas Rep. B i o l . Med., l6, 479 (1958). C h u l s k i , T., ( 1959) .
J. Lab. and C l i n . Med.

53,

490

35, S p i n g l e r , H . , K l i n . Wochschr, - 533 (1957). P i g n a r d , P., Ann. B i o l . C l i n . ( P a r i s ) , l6, 4 7 1 ( 1958) . D or f r nuller , T . , (1957). "Das A r t z l i c h e Laboratorium"

-, 16

Mesnard, P. and R. C r o c k e t t , Rev. Esp. F i s i o l , 163 (1960). R i c h t e r , D., Biochem. J., 32, 2 (1938).

WILLIAM F. BEYER A N D ERIK H . JENSEN

43. Mesnard, P. and R , Crockett., Chim. Anal., 346 (1960).

9,

44

Mesnard, P and R. Crockett, . (1960).

m, 42, 381

45. Kern, W., Anal. Chem. 35, 50 (1963).

46. Wermuth, C. G., and P. Morand, Trav. SOC. Pharm. Montpellier, 20, 234 (1960).
47.

- 170 (1961). 21,

-

Jung, L. M., C G Wermuth, and P Morand, . . .

x,

48. Alessandro, A., R. Emer, and G. Abbondanza, G, Med. Mil., 116,827 (1966). . 49. Kaistha, K. K. and W. N French, J. Pharm. Sci., 57, 459 (1968).
50. Sabih, K. and K. Sabih, J. Pharm. Sci., 59, 782 (1970)

. 51. Simmons, D. L , R. J. Ranz, and P. Picotte, J. Chromatog.71, 421 (1972).

52 53.

. Prescott, L F. and D. R. Redman, J. Pharm. Pharmac., 24, 713 (1972).

Beyer, W. F., Anal. Chem., 44, 1312 (1972).

54. Chakrabarti, J. K., J. Chromatog., 55.

8 , 414 (1962).

Hentrich, K., Pharmazie, l8, 405 (1963).

56. Abdel-Wahab, M. F. and R. M. El-Allawy, Isotopenpraxis, 4, 371 (1968). 57.

5,

Krivis, A. F. and A. A. Forist, Microchem, J. 553 (1961).

542

TOLBUTAMIDE

58. Strickland, R. D., J. Chromatog., 24, 455 (1966).

59. Baumler, J. and S . Rippstein, Dt. ApothZtg. 1647 (1967).
60.

107,

Gcven, K. C., S Gecgil, and 0. Pekin, Eczacilik . BElteni, 2, 158 (1966).

61. Hutzul, M. and G. Wright, Canadian J. Pharm. S t i . , 3 , 4 (1968).

62. Mistryukov, E. A,, Collection Czech. Chem. Commun. 26, 2072 (1961). 63. Reisch, J., H. Bornfleth, and G. L Tittel, . Pharm. Ztz Apotheker Ztg, 109, 74 (1964).

64. Neidlein, R., G. KlGgel, and U. Lebert, Pharm. Ztg. Ver. Apotheker Ztg, 110, 651 (1965).
65. Glogner, P., H. Lange, and R, Pfab, Med. Welt, 52, 2876 (1968).

66. Kalinowski, K. and R. Korzybski, Acta Polon. Pharm. 2, 221 (1963).

67.

Voicu, A., Farrnacia l0, 399 (1962).

68. McMahon, F. G., H. L. Upjohn, 0 S. Carpenter, . J. B. Wright, H. Oster, and W. E. Dulin, Current Therapeutic Research, 4 , 330 (1962).

This monograph is based to a great extent on a preliminary compilation of analytical information on tolbutamide by Dr. Arlington A. Forist. His background data facilitated the preparation of the monograph. Mrs. Betty Breseman deserves special recognition for preparation of the manuscript in its final form. The authors also wish t o acknowledge the valuable secretarial support of Mrs. Marilynn K. Nelson.

543

TRIMETHAPHAN CAMSYLATE

Kenneth W. Blessel, Bruce C. Rudy, and Bernard Z. Senkowski

KENNETH W. BLESSEL, BRUCE C. R U D Y , A N D B E R N A R D 2 . SENKOWSKI

INDEX
1. Description 1.1 1.2 1.3
2.

Name, Formula, Molecular Weight Appearance, Color, Odor Isomeric Forms

Physical Properties 2.1

2.2 2.3 2.4

Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Fluorescence Spectrum Mass Spectrum 2.5 2.6 Optical Rotation Melting Range 2.7 Differential Scanning Calorimetry 2.8 Thermogravimetric Analysis 2.9 2.10 Solubility 2.11 X-ray Crystal Properties Synthesis

3.

4. Stability Degradation
5.

Drug Metabolic Products

6. Methods of Analysis
6.1 6.2 6.3 6.4 6.5 6.6

Elemental Analysis Phase Solubility Analysis Thin Layer Chromatographic Analysis Direct Spectrophotometric Analysis Colorimetric Analysis Non-Aqueous Titration

7. Acknowledgment
8 References .

546

TRIMETHAPHAN CAMSY LATE

1. Description

Name, Formula, Molecular Weight Trimethaphan camsylate is (+)-1.3-dibenzyldecahydro-2-oxoimidazo~4,5-c]thieno [ 1,2-a] -thiolium 2-bxo-10bornane sulfonate.

1.1

C32H40N205S2

Molecular Weight:

596.81.

Appearance, Color, Odor Trimethaphan camsylate is a white crystalline powder which is odorless or has a slight odor. Isomeric Forms Trimethaphan camsylate has four possible isomers, grouped in two pairs of enantiomers.
2.

1.2

1.3

Physical Properties 2.1

Infrared Spectrum The infrared spectrum of a sample of reference standard trimethaphan camsylate is shown in Figure 1 ( ) 1. The spectrum was recorded using a 5% w/v solution in chloroform, utilizing a Perkin Elmer 621 Spectrophotometer equipped with 0.1 mm NaCl liquid cells. The following assignments of some of the bands in the spectrum have been 1. made which are shown in Table I below ( ) Table I Band 1735 c m ' l

Assignment
C=O stretch in the camphorsulfonic acid moiety C=O stretch of the trimethaphan moiety

1700 cm-l

547

8
I n

8
8 3 I

0 0

e;:
0

8:
R W

8'
v)
( u

8 0
8
I n M

8
33NWlllWSNWUl
Yo

548

TRIMETHAPHAN CAMSY LATE

1447 cm-l 701 cm-1

CH2 and CH3 deformations out-of-plane deformations o f t h e monosubs t i t u t e d benzene rings.

Nuclear Magnetic Resonance Spectrum (NMR) The NMR spectrum of a sample of r e f e r e n c e standard trimethaphan camsylate i s shown i n F i g u r e 2 ( 2 ) . The s o l v e n t used was DM!SO-d6 and t h e c o n c e n t r a t i o n of t h e trimethaphan camsylate was 54.8 mg/0.5 m l . Due t o t h e complex n a t u r e of t h e spectrum, a c o n s i d e r a b l e amount of spin-spin decoupling w a s n e c e s s a r y i n o r d e r t o o b t a i n t h e assignments shown i n Table I1 ( 2 ) . A a r b i t r a r y set of n numbers was used on t h e s t r u c t u r a l formula, shown below, f o r ease i n p r e s e n t i n g t h e assignments.
2.2

Table I1

NMR S p e c t r a l Assignments f o r Trimethaphan Camsylate

Proton c18 p r o t o n s
C19

Total No. of Protons

3 3 4 3
2

Chemical S h i f t (ppm)
0.73 1.03

Multiplicity Singlet Singlet Multiplet

Coup1i n g Constant

protons

C14 and C15 pro t o n s

c16 and C20 protons
C12

1.10-1.60
-2.3

Mu1 t i p l e t
Triplet 2 p a i r s of

protons

-3.0
4.0-4.3 4.6-5.0

Cg and Cll protons

J(C.:i)-16

Hz

Doublets

549

550

TRIMETHAPHAN CAMSYLATE

c1,c2 ,c3 ,C4

C6,C7 and C8
p r o tons aromatic protons

1 1

2.0-3.8 4.5-5.9

Two sets of Multiplets

10

7.34

Singlet

U l t r a v i o l e t Spectrum The u l t r a v i o l e t spectrum of r e f e r e n c e s t a n d a r d trimethaphan camsylate (a) is shown i n F i g u r e 3 (3). The c o n c e n t r a t i o n w a s 1.00 mg/ml i n chloroform. The c u r v e shows a maximum a t 258 n (E = 3.9 x lo2) having two m s h o u l d e r s on t h e r i s i n g p o r t i o n of t h e curve. Also shown on t h e same f i g u r e is t h e b a s e l i n e s c a n (b) and t h e u l t r a v i o l e t spectrum of t h e bromocresol g r e e n complex ( c ) w i t h trimethaphan camsylate which is used f o r c o l o r i m e t r i c determination. Fluorescence Spectrum TrimethaPhan camsylate (1 mg/ml i n methanol) has extremely weak e x c i t a t i o n and e m i s s i o n s p e c t r a which are shown i n F i g u r e 4 (4). The i n s t r u m e n t used w a s a Farrand M - r e c o r d i n g 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 a t 290 n K1 m produced an emission spectrum having a maximum a t 416 nm.
Mass Spectrum The low r e s o l u t i o n mass spectrum of a sample of r e f e r e n c e s t a n d a r d trimethaphan camsylate i s shown i n F i g u r e 5 ( 5 ) . The spectrum was o b t a i n e d w i t h t h e a i d of a CEC 21-110 mass s p e c t r o m e t e r a t an i o n i z i n g energy of 7 0 eV, i n t e r f a c e d w i t h a Varian d a t a system 100 MS. The d a t a system a c c e p t e d t h e o u t p u t of t h e s p e c t r o m e t e r , c a l c u l a t e d t h e masses, compared t h e i n t e n s i t i e s t o t h a t o f t h e b a s e peak and p l o t t e d t h e i n t e n s i t i e s as a series of l i n e s whose h e i g h t s were p r o p o r t i o n a l t o t h e i n t e n s i t i e s . Trimethaphan camsylate i s a thiophanium s a l t and a s such h a s v e r y low v o l a t i l i t y , t h e r e f o r e , a h i g h l y c h a r a c t e r i s t i c mass spectrum cannot b e expected. The h i g h e s t mass observed by low r e s o l u t i o n was m / e 488. The b a s e peak was observed a t m / e 9 1 , corresponding t o C H5-CH20. A high r e s o l u t i o n s c a n showed masses up t o m f e 578 which probably arises from t h e l o s s of H20 from t h e molecular i o n . Other masses were observed a t m / e 548, probably due t o t h e l o s s of SO from t h e p a r e n t mass and
551

2.3

2.4

2.5

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD Z.SENKOWSKI

Figure 3 Ultraviolet Spectra (a) (b)

(c)
I.c

Trimethaphan Camsylate Solvent Trimethaphan Camsylate

- Bromocresol

Green Complex

8:
.i
C

!m

I
x)o

350

400 WAVELENGTH,

450 nm.

500

552

0 0
rp

8
I n

v)

W +

o w
4

0
0

rn

0 0

553

--I:

554

TRIMETHAPHAN CAMSY LATE

m / e 545, due t o t h e l o s s of S and H20 from t h e m o l e c u l a r H i o n . These peaks a r e r a t h e r p e c u l i a r i n t h a t t h e y a p p e a r t o a r i s e from fragments c o n t a i n i n g b o t h t h e a c i d and b a s e p o r t i o n s of t h e molecule ( 5 ) .

Optical Rotation The v a l u e r e p o r t e d f o r t h e s p e c i f i c r o t a t i o n of trimethaphan c a m s y l a t e i n t h e United S t a t e s Pharmacopeia XVIII i s "not less t h a n +20 and n o t more t h a n +23' d e t e r mined i n a s o l u t i o n c o n t a i n i n g 400 m i n each 10 m l " ( 6 ) . g A graph of s p e c i f i c r o t a t i o n as a f u n c t i o n of wavelength i s a l s o p r e s e n t e d i n F i g u r e 6 ( 7 ) . The d a t a were o b t a i n e d by c o n v e r t i n g r o t a t i o n v a l u e s from a J a s c o ORD-UV 5 i n s t r u ment t o s p e c i f i c r o t a t i o n . The s p e c i f i c r o t a t i o n w a s z e r o a t 294 n and 225 nm. It can b e s e e n t h a t t h e s p e c i f i c m r o t a t i o n changes r a p i d l y a t wavelengths below 300 nm.

2.6

M e l t i n g Range Trimethaphan c a m s y l a t e melts w i t h decomposition over a range of 230-235'C when a Class I a p r o c e d u r e is used ( 6 ) . D i f f e r e n t i a l Scanning Calorimetry (DSCl The DSC c u r v e f o r a sample of r e f e r e n c e s t a n d a r d trimethaphan c a m s y l a t e is shown i n F i g u r e 7 ( 8 ) . T h i s curve was o b t a i n e d a t a s c a n r a t e of 10C/min. i n a n i t r o gen atmosphere u t i l i z i n g a P e r k i n E l m e r DSC-1B. The ext r a p o l a t e d o n s e t of t h e m e l t i n g endotherm, accompanied by . decomposition, i s 234.9 f 0.loC and t h e peak is a t 238.4 2 0.4OC. A l l t e m p e r a t u r e s have been c o r r e c t e d . Because of t h e decomposition d u r i n g t h e m e l t , t h e AHf cannot b e calc u l a t e d w i t h much r e l i a b i l i t y , however, i t s v a l u e is approximately 1 2 k c a l / m o l e ( 8 ) . Thermogravimetric A n a l y s i s (TGA) The TGA c u r v e f o r r e f e r e n c e s t a n d a r d t r i m e t h a p h a n camsylate showed no weight l o s s from ambient t e m p e r a t u r e t o 265OC a t a h e a t i n g r a t e of 10C/min. A t about 265OC, weight l o s s began which amounted t o approximately 70% o f t h e weight a t 3 4 5 O C ( 8 ) . 2.9 2.8

2.7

555

KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD 2.SENKOWSKI

Figure 6 Specific Rotation vs Wavelength f o r Trlmethaphan Camsylate

+ 400

7 12001600

2000 2400 2800

3200 -

3600
4000 4400

4800

556

TRIMETHAPHAN CAMSYLATE

Figure 7
DSC Curve of Trimethaphan Camsylate

Endothermic

\
I

234.9'C

Exothermic

250

240

230

220

210

"C

557

KENNETH W. BLESSEL. BRUCE C. RUDY, A N D BERNARD 2 . SENKOWSKI

Solubility The s o l u b i l i t y d a t a shown i n T a b l e I11 was obt a i n e d a t 25OC f o r r e f e r e n c e s t a n d a r d t r i m e t h a p h a n cams y l a t e (9). T a b l e I11 S o l u b i l i t y Data f o r Trimethaphan Camsylate Solvent S o l u b i l i t y (rng/ml)

2.10

diethyl ether p e t r o l e u m e t h e r (30-60') 2 propanol 3A a l c o h o l chloroform 95% e t h a n o l benzene methano 1 water

0.01 0.05 20.15 89.06 >500. 175.80 2.59

>500. >500.

Crystal Properties The x-ray powder d i f f r a c t i o n d a t a o r a sample of r e f e r e n c e s t a n d a r d t r i m e t h a p h a n c a m s y l a t e is g i v e n i n Table I V ( 1 0 ) . The o p e r a t i n g p a r a m e t e r s of t h e i n s t r u m e n t are g i v e n below. Instrumental Conditions General E l e c t r i c Model XRD-6 S p e c t r o g o n i o m e t e r Generator: Tube t a r g e t : optics:

2.11

Goniometer : Detect o r :

50 KV, 12-112 MA Cu K = 1 . 5 4 2 8 , 01 D e t e c t o r s l i t .' M.R. S o l l e r s l i t 3' Beam s l i t 0.0007" N i f i l t e r 4 take off angle ' Scan a t 0.2' 28 p e r m i n u t e Amplifier g ain - 1 6 co u r s e, 8.7 f i n e Sealed proportional counter t u b e and DC v o l t a g e a t p 1 te a u a

558

TRIMETHAPHAN CAMSY LATE

Recorder: Samples :

P u l s e h e i g h t s e l e c t i o n EL 5 volts Eu - o u t Rate meter T.C. 4 2000 CIS f u l l scale Chart Speed 1 i n c h p e r 5 mi n u t e s P r e p a r e d by g r i n d i n g a t room t e mp e r a t u r e . Table I V

I n t e r p l a n a r S p a c i n g s i n Trimethaphan Camsylate from Powder D i f f r a c t i o n Data 28 7.04 10.94 12.00 12.26 12.52 12.76 14.14 14.84 16.22 17.54 18. 68 19.24 20.78 21.28 21.94 22.34 22.82 23.28 24.50 24.86 25.20 25.58 25.94
d

(1) *

I/1*** 100

28 26.94 28.06 28.86 29.92 30.42 31.12 31.66 32.54 34.54 35.10 35.86 36.96 37.44 37.84 38.96 39.56 40.40 41.00 41.76 43.00

(1) *
3.31 3.18 3.09 2.99 2.94 2.87 2.83 2.75 2.60 2.56 2.50 2.43 2.40 2.38 2.31 2.28 2 .2 3 2.20 2.16 2.10

ZL1,- **
16 18 14 25 2 4 4 8 10 6 3 3 6 4 4 3 4 1 3 4

12.5 8.09 7.38 7.22 7.07 6.94 6.26 5.97 5.46 5.06 4.75 4.61 4.27 4. 1 8 4.05 3.98 3.90 3.82 3.63 3.58 3.53 3.48 3.43

14 13
19 53 23 52

7
96 81 44 98 57 48 20 21 1 1 10 25 1 1 9 13 4

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

nX 2 Sin 8 of 1 0 0 )

**I/

I0

= r e l a t i v e i n t e n s i t y ( b a s e d on h i g h e s t i n t e n s i t y

559

K E N N E T H W. BLESSEL, BRUCE C. RUDY, A N D B E R N A R D Z . SENKOWSKI

Synthesis Trimethaphan c a m s y l a t e can b e o b t a i n e d as a by-product i n t h e s y n t h e s i s of b i o t i n . The s y n t h e t i c r o u t e s t o b i o t i n have been r e p o r t e d i n s e v e r a l p a t e n t s (11-13).

3.

4.

S t a b i l i t y Degradation A study of t h e s t a b i l i t y of trimethaphan camsylate was c a r r i e d o u t by h e a t i n g t h e material i n t h e d r y form f o r The s o l i d w a s d i s s o l v e d i n d i f f e r e n t p e r i o d s of t i m e . d o u b l e d i s t i l l e d water a t a c o n c e n t r a t i o n of 5% and a n estimate of s t a b i l i t y was made by n o t i n g t h e d e g r e e o f t u r b i d i t y c a u s e d by d e c o m p o s i t i o n p r o d u c t s . It w a s obs e r v e d t h a t on h e a t i n g a t 100C f o r p e r i o d s up t o 2 h o u r s , o n l y a s l i g h t t u r b i d i t y was n o t e d i n t h e s o l u t i o n ( 1 4 ) . The d e c o m p o s i t i o n of t r i m e t h a p h a n c a m s y l a t e u n d e r e x t r e m e c o n d i t i o n s w a s a l s o s t u d i e d . I t was found t h a t r e f l u x i n g a 5% aqueous s o l u t i o n f o r 90 h o u r s c a u s e d a p p r o x i m a t e l y 60% d e c o m p o s i t i o n , c a l c u l a t e d from t h e amount of l i b e r a t e d f r e e acid (15).

Drug M e t a b o l i c P r o d u c t s S c u r r and Wyman ( 1 9 ) r e p o r t e d i n 1954 t h a t t h e metab o l i c p r o d u c t s o f t r i m e t h a p h a n c a m s y l a t e were unknown. A s e a r c h of t h e l i t e r a t u r e from t h a t p o i n t up t o t h e p r e s e n t d i d n o t add t o t h e c u r r e n t knowledge p e r t a i n i n g t o metab o l i s m of t h e d r u g . S i n c e t h e p h y s i o l o g i c a l a c t i o n of t h e d r u g commences and c e a s e s v e r y r a p i d l y , t h e e f f e c t o r n a t u r e of t h e m e t a b o l i t e s i s u n c e r t a i n .

5.

6.

Methods of A n a l y s i s

Elemental Analysis The r e s u l t s of a n e l e m e n t a l a n a l y s i s of a s a m p l e of reference standard trimethaphan camsylate are presented i n Table V ( 1 6 ) . Table V E l e m e n t a l A n a l y s i s of Trimethaphan Camsylate
E l emen t
C

6.1

% Theory

H N S

64.40 6.76 4.69 10.74

560

% Found 64.39 6.81 4.76 10.78

TRIMETHAPHAN CAMSY LATE

Phase S o l u b i l i t y Analysis The r e s u l t s of a phase s o l u b i l i t y a n a l y s i s a s an i n d i c a t i o n of p u r i t y i s shown i n F i g u r e 8 f o r a r e f e r e n c e s t a n d a r d sample o f trimethaphan camsylate ( 9 ) . The s o l v e n t used was a c e t o n e w i t h a n e q u i l i b r a t i o n t i m e of 20 hours a t 25OC. The remainder of t h e e x p e r i m e n t a l c o n d i t i o n s and r e s u l t s a r e shown i n F i g u r e 8. Thin Layer Chromatography A t h i n l a y e r chromatographic system h a s been developed f o r t h e s e p a r a t i o n of h y d r o l y s i s p r o d u c t s of trimethaphan camsylate from t h e p a r e n t s u b s t a n c e (17). The type of p l a t e used w a s s i l i c a g e l G, w h i l e t h e d e v e l o p i n g solvent was methanol: 10% aq. H2SO4 (90 :10) A f t e r t h e sol- t k o n t h a ascended f o r 1 5 cm t h e p l a t e i s a i r d r i e d and sprayed w i t h modified Dragendorff r e a g e n t . The Rf v a l u e of trimethaphan camsylate was 0.5 w h i l e t h a t of t h e major h y d r o l y s i s product was 0 . 7 .

6.2

6.3

Direct Spectrophotometric Analysis Trimethaphan c a m s y l a t e , I n i n j e c t a b l e s o l u t i o n , can b e assayed d i r e c t l y by a UV a b s o r p t i o n procedure. This procedure i n v o l v e s t h e d i l u t i o n of 5 m l of ampul s o l u t i o n (50 mg/cc) t o one l i t e r . The absorbance of t h i s s o l u t i o n i s measured a t wavelengths from 254-259 nm. The maximum a b s o r p t i o n i n t h i s range i s used t o c a l c u l a t e t h e amount of trimethaphan camsylate p r e s e n t i n t h e ampul by comparison w i t h a sample of r e f e r e n c e s t a n d a r d m a t e r i a l prepared and measured i n a similar way (18).

6.4

C o l o r i m e t r i c Analysis Trimethaphan camsylate can be determined c o l o r i m e t r i c a l l y by f o r m a t i o n of t h e bromocresol green i o n p a i r followed by e x t r a c t i o n as d e s c r i b e d i n t h e f o l l o w i n g procedure. A volume of s o l u t i o n e q u i v a l e n t t o a b o u t 100 m g of trimethaphan camsylate is d i l u t e d t o o n e l i t e r . A 10-ml a l i q u o t of t h i s s o l u t i o n is b u f f e r e d a t a pH of 5 . 3 by t h e a d d i t i o n of a phosphate b u f f e r . Then 5-ml of a bromocresol green s o l u t i o n i n t h e phosphate buff e r i s added, a f t e r which t h e aqueous s o l u t i o n is e x t r a c t e d w i t h two 25-ml p o r t i o n s of chloroform. The combined chloroform e x t r a c t s a r e d i l u t e d t o 100 m l and t h e absorbance determined a t about 420 nm. The q u a n t i t y of t r i m e t h a phan camsylate p r e s e n t i s c a l c u l a t e d by comparison w i t h a

6.5

561

Figure 8

z
z?!lLI
v)

23

4 -

k $

0 - s
3-

"

z:

PHASE SOLUBILITY ANALYSIS Sampte : Trimethophan Camsylate Solvent : Acetone Slope : 0.19 Ole Equilibration : 2 0 hrs at 25'C Extrapolated Solubility : 3.72 m g / q

z ' -;I z

= u
2 -

OE: + 3 L -lo

-:

TRIMETHAPHAN CAMSY LATE

known c o n c e n t r a t i o n of r e f e r e n c e s t a n d a r d material similarl y prepared and measured (6). Non-Aqueous T i t r a t i o n The non-aqueous t i t r a t i o n d e s c r i b e d i n t h e USP XVIII is t h e accepted method f o r t h e a n a l y s i s of t r i m e t h a phan camsylate i n t h e b u l k form ( 6 ) . The sample i s d i s solved i n a c e t i c anhydride and t i t r a t e d w i t h 0.1N H C l O 4 Each m l of 0.1N u t i l i z i n g a p o t e n t i o m e t r i c end-point. g HC104 is e q u i v a l e n t t o 59.68 m of trimethaphan camsylate. Acknowledgment The a u t h o r s wish t o acknowledge t h e a s s i s t a n c e of t h e Research Records O f f i c e of Hoffmann-La Roche I n c . f o r t h e i r l i t e r a t u r e search. 7.

6.6

563

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

8. References 1 Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal . Communication. 2 . Johnson, J . H., Hoffmann-La Roche Inc., Personal Communication. 3. Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. 4 . Boatman, J., Hoffmann-La Roche Inc., Personal Communication. 5. Benz, W., Hoffmann-La Roche Inc., Personal Communication. 6 . The United S t a t e s Phamacopeia XVIII, p p . 755-757

Toome, V., Hoffmann-La Roche Inc., Personal Communication. 8. Moros, S., Hoffmann-La Roche Inc., Personal Communication. 9 . MacMullan, E., Hoffmann-La Roche Inc., Personal Communication. 1 0 * Hagel, R., Hoffmann-La Roche Inc., Personal Communication. 11. United States Patent 2 , 4 8 9 , 2 3 2 - 2 , November, 1 9 4 9 . 1 2 . United States Patent 2 , 4 8 9 , 2 3 8 , November, 1 9 4 9 . 13. United States Patent 2 , 5 1 9 , 7 2 0 , August, 1 9 7 0 . 1 4 . Rubin, S., Hoffmann-La Roche Inc., Unpublished Data. 1 5 . Sternbach, L., Hoffmann-La Roche Inc., Unpublished Data. 16. Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. 1 7 . Sokoloff, H., Hoffmann-La Roche Inc., Unpublished Results 1 8 . Keller, C., Hoffmann-La Roche Inc., Personal Communication. 1 9 . Scurr, C. F. and Wyman, J . P., Lancet, 266, 338
7.

(1970).

(1954)

5 64

TROPICAMIDE

Kenneth W. Blessel, Bruce C. Rudy, and Bernard Z. Senkowski

KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD Z . SENKOWSKI

INDEX Analytical P r o f i l e

- Tropicamide

1.

Description 1.1 N a m e , Formula, Molecular Weight 1.2 Appearance, Color, Odor 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 F l u o r e s c e n c e Spectrum 2.5 Mass Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 D i f f e r e n t i a l Scanning Calorimetry 2.9 Thermogravimetric A n a l y s i s 2.10 S o l u b i l i t i e s 2 . 1 1 X-ray C r y s t a l P r o p e r t i e s 2 . 1 2 D i s s o c i a t i o n Constant Synthesis S t a b i l i t y Degradation Drug Metabolic P r o d u c t s Methods of Analysis 6.1 Elemental A n a l y s i s 6.2 Phase S o l u b i l i t y Analysis 6.3 Thin Layer Chromatographic Analysis Direct Spectrophotometric A n a l y s i s 6.4 6.5 Non-Aqueous T i t r a t i o n Acknowledgments References

2.

3.

4.
5.
6.

7.
8.

5 66

TROPICAM I DE

1. Description
Name, Formula, Molecular Weight Tropicamide is N-ethyl-2-phenyl-N-(4-pyridylmethyl)-hydracrylamide. OH

1.1

C17H20N2N2 Molecular Weight: 284.36 1.2 Appearance, Color, Odor Tropicamide is a white crystalline odorless powder.
2.

Physical Properties

Infrared Spectrum The infrared spectrum of a sample of reference 1 . The standard tropicamide is shown in Figure 1 ( ) spectrum was recorded on a KBr pellet containing 0.5 mg of tropicamide and 300 mg of KBr, using a Perkin Elmer 6 2 1 Spectrophotometer. The following assignments have been made of the bands in Figure 1 (I).

2.1

Band
3396 cm- 1620 cm-1 1595 and 1493 cm-l 810 cm-1

Assignment

OH stretch CEO stretch Aromatic Ring Vibrations Monosubstituted Pyridine

Ring Monosubstituted Benzene Ring

760 and 707 cm-l

2.2

Nuclear Magnetic Resonance Spectrum (NMR) The NMR spectrum of a sample of reference stan2 . The sample dard tropicamide is shown in Figure 2 ( ) solution contained 62.5 mg of tropicamide per 0.5 ml o f C D C l 3 . Due to the complex spectrum observed, extensive spin decoupling experiments were carried out in order to evaluate the coupling constants and chemical shifts. Consecutive irradiations were performed at 58.8 Hz, 6 5 . 4 Hz, 196 Hz, and 200 Hz which simplified the spectrum to the
561

5 68

569

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD Z. SENKOWSKI

e x t e n t t h a t t h e f o l l o w i n g assignments could b e made ( 2 ) .

Table I

NMR S p e c t r a l Data f o r Tropicamide*

No. o f Chemical Each Shift Multiplicity C5 p r o t o n s
C

3
2

0.98,1.09 3.27
3.60

(2)
t r i p 1e ts

J [ CH3-CH2-N] = 7Hz
J[N-CHZ-CH~]
=

protons

octet s i n g l e t (broad) multiplet

multiplet

7Hz

OH-pro ton c(5 p r o t o n s

C 1 and C2 p r o t o n s

1
2

3.75 Q3.8-4.9 7.05 7.35 8.50

cg and Cl1 protons 2

ar0rnati.c p r o t o n s on t r o p i c a c i d moiety
Cg and Cl0 p r o t o n s 2

triplet

JQ-H~ 5 ~ z =

doublet

J H ~ -= 5Hz H~

*A r b i t r a r y
2.3

numbers were a s s i g n e d t o t h e atoms i n t h e s t r u c t u r e f o r ease i n p r e s e n t a t i o n of d a t a .

U l t r a v i o l e t Spectrum The u l t r a v i o l e t spegtrum of tropicamide i n t h e r e g i o n of 200-400 n is shown i n F i g u r e 3- ( 3 ) . The m spectrum shows a maximum a t 254 n ( E = 5.1 x 103) and a m minimum a t 235-237 nm. The s Q l u t i o n c o n c e n t r a t i o n was 0.025 mg/ml i n 0.1N H C 1 .

TROPICAM I DE

Figure 3 Ultraviolet Spectrum of Tropicamide

210

250 300 NANOMETERS

350

571

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

Fluorescence Spectrum A e x c i t a t i o n axid emission s c a n were c a r r i e d o u t n with a methanol s o l u t i o n of r e f e r e n c e s t a n d a r d t r o p i c a m i d e . There w a s , however, no f l u o r e s c e n c e observed (4).

2.4

Mass Spectrum The l o w - r e s o l u t i o n mass spectrum of t r o p i c a m i d e is shown i n F i g u r e 4 ( 5 ) . The spectrum w a s o b t a i n e d u s i n g a CEC 21-110 s p e c t r o m e t e r w i t h a n i o n i z i n g v o l t a g e of 70 eV, which was i n t e r f a c e d w i t h a V a r i a n d a t a system 100 MS. The d a t a system a c c e p t e d t h e o u t p u t of t h e s p e c t r o meter, c a l c u l a t e d t h e masses, compared t h e i r i n t e n s i t i e s t o t h e b a s e peak and p l o t t e d t h i s i n f o r m a t i o n as a series of l i n e s whose h e i g h t s were p r o p o r t i o n a l t o t h e i n t e n s i t i e s . The molecular i o n was measured a t m / e 284. The b a s e peak a t m / e 254 a r i s e s due t o t h e l o s s of CH2 = 0 from t h e molecular i o n by a McLafferty r e a r r a n g e ment. The i o n a t m / e 225 probably arises v i a a s k e l e t a l rearrangement of m / e 254 l e a d i n g t o t h e loss of HCO. Cleavage between t h e benzyl carbon and t h e carbonyl group g i v e s r i s e t o t h e i o n a t m / e 163. The i o n a t m / e 92 is t h e n i t r o g e n - c o n t a i n i n g tropylium c a t i o n , C6H614+ (5).
2.5

2.6

Optical Rotation Tropicamide does n o t e x h i b i t o p t i c a l a c t i v i t y .

Melting Range The m e l t i n g r a n g e r e p o r t e d i n t h e United S t a t e s Pharmacopeia X V I I I f o r t r o p i c a m i d e i s 96-1OO0C when a Class I procedure is used ( 6 ) . D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) The DSC s c a n of a sample of r e f e r e n c e s t a n d a r d t r o p i c a m i d e is shown i n F i g u r e 5 ( 7 ) . The t e m p e r a t u r e w a s r a i s e d a t a r a t e of 10C/min. i n an atomosphere of f l o w i n g n i t r o g e n . A s i n g l e endotherm w a s o b s e r v e d , t h e e x t r a p o l a t e d o n s e t of which w a s 95.5 & 0.2OC. The peak of t h e m e l t i n g endotherm w a s observed a t 98.6 5 0.2OC. All t e m p e r a t u r e s are c o r r e c t e d . The v a l u e of AHf f o r t h e m e l t i n g endotherm was c a l c u l a t e d t o b e 8.8 kcal/mole. Thermogravimetric Analysis (TGA) The r e s u l t s of a TGA s c a n of t r o p i c a m i d e i n d i c a t e d t h a t no weight loss o c c u r r e d from ambient t e m p e r a t u r e 2.9 2.8

2.7

572

Figure 4

Mass Spectrum of Tropicamide

KENNETH W. BLESSEL, BRUCE C. RUDY,AND BERNARD 2. SENKOWSKI

Figure 5 DSC Curve f o r Tropicamide

I I I AHf = 8.8 kcal / mole

I
80

I
90
OC

I too

I
10 1
I :0

574

TROPICAM I DE

t o 15OoC. A s i n g l e w e i g h t l o s s w a s o b s e r v e d b e g i n n i n g a t 15OoC and c o n t i n u i n g t o 334OC a t which p o i n t 100% of t h e sample w e i g h t had been l o s t ( 7 ) . Solubility The s o l u b i l i t y d a t a shown i n T a b l e IIwas o b t a i n e d f o r a sample of r e f e r e n c e s t a n d a r d t r o p i c a m i d e a t a tempera t u r e of 25OC ( 8 ) . T a b l e I1 S o l u b i l i t i e s of Tropicamide Solvent 3A a l c o h o l benzene chloroform 95% e t h a n o l diethyl ether 2 - propanol methanol p e t r o l e u m e t h e r (30-60') water S o l u b i l i t y (mg/ml) 235.0 25.9 >500. 321.5 3.9 112.4 >500. 0.2 5.7 2.10

Crystal Properties T a b l e I11 g i v e s i n t e r p l a n a r s p a c i n g s from x-ray powder d i f f r a c t i o n d a t a f o r t r o p i c a m i d e ( 9 ) . The o p e r a t i n g p a r a m e t e r s of t h e i n s t r u m e n t are g i v e n below. Instrumental Conditions: G e n e r a l E l e c t r i c Model XRD-6 S p e c t r o g o n i o m e t e r Generator: Tube t a r g e t : Radiation: optics: 50 KV, 12-112 MA Copper Cu Ka = 1.542 8 01 .' D e t e c t o r s l i t 3' B e a m s l i t 0.0007" N i f i l t e r 4 take off angle O Scan a t 0.2' 28 p e r m i n u t e

2.11

Goniometer:

575

KENNETH W. BLESSEL. BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

Detector:

Recorder: Samples :

Amplifier g a i n 1 6 course, 8.7 f i n e Sealed p r o p o r t i o n a l counter t u b e and DC v o l t a g e a t plateau P u l s e h e i g h t s e l e c t i o n EL 5 volts Eu out Rate meter T.C. 4 2000 c/s f u l l scale Chart speed 1 inch p e r 5 minutes P r e p a r e d by g r i n d i n g a t room temper a t u r e

T a b l e 111 I n t e r p l a n a r Spacings from Powder D i f f r a c t i o n Data I/Io28 28 d* d* I/TV** 10.54 9 29.74 3 .OO 1 1 8.39 13.76 30.06 75 6.44 2.97 12 31.42 14.60 6.07 2.85 4 63 18.14 32.00 4.89 2.80 8 27 18.94 33.06 2.71 5 47 4.69 19.84 12 34.46 4.47 2.60 9 20.40 35.18 2.55 8 67 4.35 20.88 35.52 4.25 2.53 15 72 2.43 4 21.88 37.04 4.06 100 2.40 4 22.60 28 37.54 3.93 23.22 39.00 3.83 2.31 7 13 24.30 39.82 3.66 2.26 4 19 40.22 25.38 3.51 2.24 12 8 40.62 26.10 3.41 4 2.22 4 2.17 2 26.46 41.66 4 3.37 42.24 2.14 4 27.54 3.24 30 42.70 28.00 3.19 5 2.12 3 1.99 6 28.94 45 * 54 3.09 41

- - **

*
**

d = ( i n t e r p l a n a r spacing)

nX 2 Sin 8
i n t e n s i t y of 100)

I/Io = r e l a t i v e i n t e n s i t y ( b a s e d o n h i g h e s t

576

TROPICAMIDE

Dissociation Constant The pKa of t r o p i c a m i d e was d e t e r m i n e d by 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 and by a p o t e n t i o m e t r i c t i t r a t i o n . The v a l u e o b s e r v e d w a s 5 . 2 by s p e c t r o p h o t o m e t r y and 5 . 3 by p o t e n t i o m e t r y ( 1 0 ) . Synthesis Tropicamide may b e p r e p a r e d by t h e c o n d e n s a t i o n o f e t h y l - ( y - p i c o l y l ) -amine w i t h t r o p i c a c i d c h l o r i d e , i n the p r e s e n c e o f b a s e , c a r r i e d o u t i n anhydrous c h l o r o f o r m (11). S t a b i l i t y Degradation A s t u d y h a s b e e n c a r r i e d o u t i n which t h e s t a b i l i t y o f t r o p i c a m i d e i n o p t h a l m i c s o l u t i o n was d e t e r m i n e d ( 1 2 ) . A 3% s o l u t i o n of t r o p i c a m i d e i n o p t h a l m i c s o l u t i o n w a s maint a i n e d a t t e m p e r a t u r e s r a n g i n g from OC t o 45OC f o r p e r i o d s of t i m e up t o 1 2 weeks. I n order t o gain information about p o s s i b l e breakdown p r o d u c t s of t r o p i c a m i d e , pH measurements, t u r b i d i t y d a t a and a d i r e c t s p e c t r o p h o t o m e t r i c a s s a y was performed a t t h e s t a r t and a f t e r 3 , 6 and 1 2 weeks. No e v i d e n c e o f d e c o m p o s i t i o n w a s found a t e r p e r i o d s o f up t o 1 2 weeks a t each of t h e above t e m p e r a t u r e s ( 1 2 ) . Drug M e t a b o l i c P r o d u c t s Tropicamide i s used e x c l u s i v e l y o r o p t h a l m i c s o l u t i o n s i n t h i s c o u n t r y , and is a p p l i e d t o p i c a l l y . F o r t h i s r e a s o n no m e t a b o l i c s t u d i e s h a v e b e e n p u r s u e d . 6. Methods of A n a l y s i s

2.12

3.

4.

5.

Elemental Analysis The r e s u l t s o f a n elemental a n a l y s i s of a sample of r e f e r e n c e s t a n d a r d t r o p i c a m i d e are p r e s e n t e d i n T a b l e I V (13). E l emen t

C

6.1

% Theory

% Found
71.87

H
N

71.81
7.09

7.13
9.93

9.85

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 e s have been c a r r i e d o u t f o r t r o p i c a m i d e t o e s t i m a t e t h e - p u r i t y of t h e sample. An

6.2

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

example is shown i n F i g u r e 6 ( 8 ) where t h e s o l v e n t u s e d w a s t o l u e n e and t h e e q u i l i b r a t i o n t i m e was 20 h o u r s a t 25%. Thin Layer Chromatographic A n a l y s i s A TLC s y s t e m h a s been developed which h a s proved t o b e u s e f u l f o r a n a l y s i s of t r o p i c a m i d e . The a d s o r b a n t f o r t h e system is s i l i c a g e l and t h e d e v e l o p i n g s o l v e n t is ch1oroform:methanol:concentrated ammonium h y d r o x i d e (90:10:2). The s o l v e n t f r o n t is allowed t o t r a v e l f o r about 1 5 cm i n a p r e - s a t u r a t e d t a n k . The p l a t e is a i r dried and t h e n s p r a y e d w i t h iodine-modified Dragendorff r e a g e n t . The approximate Rf of t r o p i c a m i d e i n t h i s s y s t e m is 0 . 6 5 (14).

6.3

Direct S p e c t r o p h o t o m e t r i c A n a l y s i s Tropicamide may b e a s s a y e d s p e c t r o p h o t o m e t r i c a l l y i n opthalmic s o l u t i o n a f t e r an e x t r a c t i o n i n t o chloroform and a back e x t r a c t i o n i n t o d i l u t e s u l f u r i c a c i d . The abs o r b a n c e of t h i s s o l u t i o n is measured a t t h e wavelength of maximum a b s o r b a n c e a t a b o u t 253 nm. The amount o f t r o p i c a m i d e i n t h e o p t h a l m i c s o l u t i o n i s c a l c u l a t e d by comparison w i t h a r e f e r e n c e s t a n d a r d sample of t r o p i c a m i d e measured i n a similar way ( 6 ) .
Non-Aqueous T i t r a t i o n The non-aqueous t i t r a t i o n d e s c r i b e d i n t h e USP X V I Z I i s t h e p r e f e r r e d method f o r t h e a n a l y s i s of t r o p i c a mide i n t h e b u l k form ( 6 ) . The sample is t i t r a t e d i n g l a c i a l a c e t i c acid with O.1N HClO4 i n acetic acid, using c r y s t a l v i o l e t as t h e i n d i c a t o r . One m l of 0.1N HClO4 is e q u i v a l e n t t o 28.44 m o f t r o p i c a m i d e . g Acknowledgments The a u t h o r s wish t o acknowledge t h e S c i e n t i f i c Literat u r e Department and t h e Research Records O f f i c e of Hoffmann-La Roche I n c . f o r t h e i r a s s i s t a n c e i n t h e l i t e r a ture search f o r t h i s analytical profile,

6.4

6.5

7.

578

'

n
# .

PHASE SOLUBILITY ANALYSIS

Sample : Tropicomide Solvent : Toluene Slope : .150/0 Equilibrotian : 2 0 hrs at 25OC Extrapolated Solubility : 12.35 mq/g

1 1 1 1 1 1 1 1 , ) 1 1 1 1

25

KENNETH W. BLESSEL, BRUCE C. R U D Y , A N D B E R N A R D 2.SENKOWSKI

8. References
1.

2.
3.

4.
5.

6.

7.

8.
9.
10.
11.

12. 13.
14.

Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. Boatman, J., Hoffmann-La Roche Inc. , Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communication. The United States Pharmacopeia XVIII, pp. 762-763 (1970) Moros, S., Hoffmann-La Roche Inc., Personal Communication. MacMullan, E., Hoffmann-La Roche Inc., Personal Communication. Hagel, R., Hoffmann-La Roche Inc., Personal Communication. Heveran, J., Hoffmann-La Roche Inc., Unpublished Results. Hoffmann-La Roche Inc., United States Patent 2,726,245 (1955) . Bollinger, A., Hoffmann-La Roche Inc., Unpublished Data. Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. Sokoloff , H. , Hoffmann-La Roche Inc. , Unpublished Results.

580

CUMULATIVE INDEX
Italic numerals refer to Volume numbers.

Acetaminophen, 3, 1 Acetohexamide, I, 1; 2,573 Alpha-Tocopheryl Acetate, 3, 111 Amitriptyline Hydrochloride, 3, 127 Ampicillin, 2. 1 Chlorprothixene, 2, 63 Chloral Hydrate, 2, 85 Chlordiazepoxide, 1, 15 Chlordiazepoxide Hydrochloride, 1, 39 Clidinium Bromide, 2, 145 Cycloserine, I, 5 3 Cyclothiazide, 1, 66 Dexamethasone, 2, 163 Diazepam, 1. 79 Digitoxin, 3, 149 Dioctyl Sodium Sulfosuccinate, 2, 199 Diphenhydramine Hydrochloride, 3, 173 Echothiophate Iodide, 3, 233 Erythromycin Estolate, I, 101; 2, 573 Ethynodiol Diacetate, 3, 253 Fludrocortisone Acetate, 3. 281 Fluorouracil, 2, 221 Fluphenazine Enanthate, 2, 245 Fluphenazine Hydrochloride, 2, 263 Nurazepam Hydrochloride, 3, 307 Halothane, I, 119; 2, 573 Iodipnmide, 3, 333 Isocarboxazid, 2, 295 Isopropamide, 2, 3 15 Levallorphan Tartrate, 2, 339

Lavatesenol Bitartrate, 1, 149; 2, 573 Meperidine Hydrochloride, 1. 175 Meprobamate, I, 209 Methadone Hydrochloride, 3, 365 Methyprylon, 2, 363 Nortriptyline Hydrochloride, 1, 233; 2, 573 Oxazepam, 3, 441 Phenazopyridine Hydrochloride, 3,465 Phenelzine Sulfate, 2, 383 Phenylephrine Hydrochloride, 3, 483 Potassium Phenoxymethyl Penicillin, 1, 249 Primidone, 2, 409 Propiomazine Hydrochloride, 2, 439 Propoxyphene Hydrochloride, I , 301 Sodium Cephalothin, 1, 319 Sodium Secobarbital, I, 343 Sulfamethoxazole, 2, 467 Sulfisoxazole, 2, 487 Tolbutamide, 3, 513 Triamcinolone, 1, 367; 2, 571 Triamcinolone Acetonide, I, 397; 2, 571 Triamcinolone Diacetate, I, 423 Triclobisonium Chloride, 2, 507 Triflupromazine Hydrochloride, 2, 523 Trimethaphan Camsylate, 3, 545 Trimethobenzamide Hydrochloride, 2, 55 1 Tropicamide, 3, 565 Vinblastine Sulfate, I, 443 Vincristine Sulfate, 1, 463

58 1