- Email: [email protected]
Isoniazid
ISONlAZID
Glenn A. Brewer
184
GLENN A. BREWER
CONTENTS
1. 2.
3. 4. 5. 6.
Description 1.1 N a m e , F o r m u l a , Molecular W e i g h t 1 . 2 A p p e a r a n c e , C o l o r , Odor, T a s t e P h y s i c a l and Chemical P r o p e r t i e s 2 . 1 Spectra 2.11 I n f r a r e d Spectrum 2.12 U l t r a v i o l e t Spectrum 2.13 Chemiluminescence 2.14 Fluorescence Spectrum 2 . 1 5 N. M. R. Spectrum 2 . 1 6 E. S. R. S p e c t r u m 2 . 1 7 Mass S p e c t r o m e t r y 2.2 P h y s i c a l P r o p e r t i e s of t h e S o l i d 2.21 Melting C h a r a c t e r i s t i c s 2 . 2 2 D.T.A. a n d D.S.C. 2.23 T.G.A. E l e c t r i c a l Moment 2.24 2.25 E l e c t r i c a l Conductivity 2.26 Crystal characteristics 2.27 X-Ray D i f f r a c t i o n 2.3 S o l u b i l i t y 2 . 3 1 Water S o l u b i l i t y 2.32 Solubility i n Solvents 2.4 Physical Properties of Solution 2 . 4 1 PH 2.42 D i s s o c i a t i o n Constant 2.43 Photolysis Constant 2.44 Oxidation P o t e n t i a l Metal Complexes History, S y n t h e s i s and Manufacturing Stability A n a l y t i c a l Chemistry 6.1 Identity T e s t s 6.2 Methods o f A n a l y s i s 6 . 2 1 General Reviews 6 . 2 2 Colorimetric Methods 6 . 2 3 S p e c t r o p h o t o m e t r i c Methods 6 . 2 4 F l u o r i m e t r i c Methods 6 . 2 5 T i t r i m e t r i c Methods 6 . 2 6 E l e c t r o c h e m i c a l Methods
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7. 8. 9.
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6 . 2 7 G r a v i m e t r i c Methods 6 . 2 8 M i c r o b i o l o g i c a l a n d E n z y m a t i c Methods 6 . 2 9 M i s c e l l a n e o u s Methods 6 . 3 C h r o m a t o g r a p h i c Methods 6 . 3 1 P a p e r Chromatography 6 . 3 2 Thin-Layer Chromatography 6 . 3 3 I o n Exchange c h r o m a t o g r a p h y 6 . 3 4 O t h e r C h r o m a t o g r a p h i c Methods 6 . 4 D e t e r m i n a t i o n of I s o n i a z i d and i t s Metabolites i n Body F l u i d s and T i s s u e s 6 . 4 1 G e n e r a l Reviews 6.42 C o l o r i m e t r i c Methods 6 . 4 3 T u r b i d i m e t r i c Method 6 . 4 4 F l u o r i m e t r i c Methods 6 . 4 5 E l e c t r o c h e m i c a l Methods 6 . 4 6 G a s o m e t r i c Methods 6 . 4 7 M i s c e l l a n e o u s Chemical A s s a y s 6.48 M i c r o b i o l o g i c a l Assays 6.49 Chromatographic Assays Drug Metabolism Biopharmaceutics References
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GLENN A. BREWER
1. Description 1.1 Name, Formula, Molecular Weiqht Generic names - Isoniazidl, Isonicotinic Acid Hydrazide, INH, Isonicotinoylhydrazine, Isonicotinyl hydrazide, Isonicotinylhydrazine, Tubazid, ISoniazidum Chemical names - 4-Pyridinecarboxylic acid hydrazide, pyridine-4-carboxyhydrazide, pyridine- y-carboxylic acid hydrazide. Chemical Abstracts Registry No. 54-85-3 2
.
2HNm0c-)+( C6H7N30
Mol. Wt. 137.14
1.2
Appearance, Color, Odor, Taste Colorless or white crystalline powder which is odorless and has at first a slightly sweet and then bitter taste3. Physical and Chemical Properties 2.1 Spectra 2.11 Infrared Spectrum The infrared spectrum of isoniazid and other hydrazides of carboxylic acid have been recorded and band assignments were made4 , Nagano et a15 in a later paper made band assignments for isoniazid, metal complexes of isoniazid and related compounds The infrared spectra of isoniazid as a solid in a KBr pellet and as a mull in mineral oil are shown in Figures 1 and 2 . The following assignments have been made by Mrs. Toeplitz6. Frequency(cm-l) A s s iqnment 3300-3000 Bonded NH and C-H 1670 c=o 1560 Amide I1 1640 NH2 deformation 1610t ring C=C and C=N 1500/ 2.
.
WAVELENGTH (MICRONS)
FRMUENCY (W')
F i g u r e 1:Infrared
spectrum of isoniazid as a KBr p e l l e t .
WAVELENGTH (MICRONS)
A
8
FREQUENCY (W')
Figure 2:Infrared
s p e c t r u m of i s o n i a z i d i n m i n e r a l o i l m u l l .
ISONIAZID
2.12
189
U l t r a v i o l e t Spectrum Numerous a u t h o r s h a v e r e c o r d e d t h e u l t r a v i o l e t s p e c t r u m o f i s o n i a z i d i n a number o f s o l v e n t s 7 , 8 ~ 9 ~ 1 0 , 1 1 ~ 1 2 . The e f f e c t of t h e p H o f t h e s o l u t i o n on t h e r e s u l t i n g s p e c t r u m h a s been noted. Zommer13 h a s r e c o r d e d t h e s p e c t r a of t h e h y d r a z o n e s of i s o n i a z i d and a c e t o n e o r p-hydroxybenza ldehyde. The u l t r a v i o l e t s p e c t r u m o f i s o n i a z i d i n d i l u t e a c i d (0.01N aqueous HC1) shows t w o a p p r o x i m a t e l y e q u a l maximima a t 213 nm ( E Y i m 437) a n d 265 nm ( E l % 4 1 7 ) . The minimum 1c m o c c u r s a t 233 nm. The s p e c t r u m i n d i s t i l l e d w a t e r shows a b r o a d peak a t 2 6 1 nm (EFgm 306) w i t h o u t a d e f i n e d minimum. T h e r e i s a s h o u l d e r a t 208 nm. I n d i l u t e a l k a l i (0.01N a q u e o u s a l k a l i ) t h e s p e c t r u m t a k e n i m m e d i a t e l y shows a s h o u l d e r a t 266 nm ( E F i m 293 a n d p e a k s a t 272 nm (EFgm 298) a n d 2 9 5 nm (Elcm % ! 2 8 4 ) . On s t a n d i n g t h e s e p e a k s s h i f t so t h a t a t 2 h o u r s t h e r e a r e p e a k s a t 256 nm ( E l % 1 7 3 ) , 262 nm ( E E m 1 7 0 ) a n d 325 nm ( E r g m 7 6 ) . 1c m A t 24 h o u r s t h e 325 nm peak d i s a p p e a r s . The same s h i f t t a k e s p l a c e w i t h h i g h e r c o n c e n t r a t i o n s of a l k a l i e x c e p t t h a t i t occurs more r a p i d l y . The u l t r a v i o l e t s p e c t r u m t a k e n i n m e t h a n o l i c r a t h e r than aqueous s o l v e n t s a r e s i m i l a r t o those i n water except t h a t the absorption maximima g e n e r a l l y o c c u r a t s l i g h t l y lower wavelengths. 2.13 Chemiluminescence C a e n l 5 h a s o b s e r v e d a weak c h e m i l u m i n e s c e n c e o f i s o n i a z i d when s o l u t i o n s a r e o x i d i z e d w i t h sodium h y p o c h l o r i t e . The lumj n e s cence i n c r e a s e s w i t h pH from 1 0 . 2 t o 13. The maximum o f t h e e m i s s i o n c u r v e i s a t 0.552 p c o r r e s p o n d i n g t o a n e n e r g y o f 5 1 K c a l , Two t h e o r i e s f o r t h e o b s e r v e d l u m i n e s c e n c e a r e o f f e r e d , b o t h of which depend on t h e p r e s e n c e o f f r e e OH a n d H 0 2 r a d i ca 1s
.
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GLENN A. BREWER
2.14
Fluorescence Spectrum I s o n i a z i d shows a n i n t e n s e f l u o r e s c e n c e s p e c t r u m when o x i d i z e d w i t h p e r o x i d e o r a f t e r cleavage of the p y r i d i n e r i n g with This fluorescence is t h e basis cyanogen bromide. o f s e v e r a l s e n s i t i v e methods t o d e t e r m i n e i s o n i a z i d i n b i o l o g i c a l m a t e r i a l s ( S e e S e c t i o n 6 . 4 ) . When a s o l u t i o n o f i s o n i a z i d a t pH 6 . 5 t o 7 . 5 w a s t r e a t e d w i t h d i l u t e p e r o x i d e a t 100°C f o r 30 m i n u t e s w e found t h e e x c i t a t i o n maximum a t 333 nm and t h e e m i s s i o n p e a k a t 415 nm 14 A f t e r i s o n i a z i d i s rea c t e d w i t h cyanogen b r o m i d e r e a g e n t i n 1 . 8 N a l k a l i n e s o l u t i o n a t room temperature w e f o u n d a n a c t i v a t i o n maximum a t 312 nm a n d a f l u o r e s c e n c e maximum a t 392 nmI4. I s o n i a z i d a l s o f l u o r e s c e s when r e a c t e d w i t h c e r t a i n a r o m a t i c c a r b o n y l compounds (Section 6.24). 2.15 N. M. R. S p e c t r u m Several authors have studied t h e n u c l e a r magnetic resonance spectrum o f t h e h draz i d e s o f c a r b o x y l i c a c i d i n c l u d i n g i s o n i a z i d y 6 9 l7 9 18. H i l l e r b r a n d and c o - w ~ r k e r s ~ gt us d i e d t h e N. M. R. spectrum o f t h e copper s a l t . The N.M.R. spectra o f i s o n i a z i d a n d *zO exchanged i s o n i a z i d a r e shown i n F i g u r e s 3 a n d 420. The 60 MHz NMR s p e c t r u m of i s o n i a z i d , i n dimethyl sulfoxide-d6 containing tetramethyl s i l a n e a s i n t e r n a l r e f e r e n c e shows t h e p r e s e n c e o f hydrazino protons resonances a t (ppm) 4 . 6 0 ( b r o a d , 2 H , e x c h a n g e d ) and 10.15 ( b r o a d , l H , exc h a n g e d ) . The a r o m a t i c p r o t o n s r e s o n a n c e s appear a s m u l t i p l e t s a t 7 . 7 3 ( 2 H ) and 8 . 7 0 ( 2 H ) . ( F i g u r e s 3 a n d 4 ) . The complex p a t t e r n of t h e resonances, o t h e r than t h e expected doublets, suggests charge d i s t r i b u t i o n i n t h e p y r i d i n e ring. However, t h e h i n d e r e d r o t a t i o n a r o u n d t h e N-C=O a s w e l l as C - a r y l b o n d s c a n n o t be r u l e d o u t . The NMR s p e c t r u m i n m e t h a n o l -d4 w a s s i m i l a r t o F i g u r e 4 , t h e h y d r a z i n o p r o t o n s h a v i n g b e e n exchanged. The a d d i t i o n of d e u t e r a t e d H C 1 d i d n o t a l t e r t h e spectrum except a downfield s h i f t of t h e aromatic
.
191
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p r o t o n s r e s o n a n c e b y 0 . 1 ppm. 2.16 E. S. R. Spectrum H i l l e r b r a n d a n d co-workers19 u s e d E l e c t r o n S p i n Resonance t o s t u d y c h a r g e t r a n s f e r i n t e r a c t i o n s between i s o n i a z i d and c o p p e r i o n s . 2.17 Mass S p e c t r o m e t r y G i l l i s 2 1 h a s d i s c u s s e d t h e fragment a t i o n p a t t e r n f o r i s o n i a z i d a n d s i m i l a r compounds. F i g u r e 5 shows t h e e l e c t r o n - i m p a c t mass s p e c t r u m o b t a i n e d on an A E I MS902 mass s p e c t r o m e t e r e q u i p p e d w i t h a d a t a a c q u i s i t i o n s y s t e m . The M+ o c c u r s a t m / e 137 and t h e f r a g m e n t i o n s r e s u l t from e i t h e r d i r e c t bond c l e a v a g e ( m / e 106, 78) o r t h r o u g h t h e e l i m i n a t i o n o f HCN from t h e p y r i d y l r i n g ( m / e 5 1 ) .
m/e
m/e
51
106
m/e
]
78
Physical Properties of t h e S o l i d 2.21 Melting C h a r a c t e r i s t i c s The m e l t i n g p o i n t o f i s o n i a z i d i s u s e d a s s p e c i f i c a t i o n i n t h e U n i t e d S t a t e s Pharma~ o p o e i aand ~ ~European Pharmacopoeia3. The m e l t i n g p o i n t o c c u r s between 1 7 0 a n d 174OC. 2.22 D.T.A. and D.S.C. D i f f e r e n t i a l thermal a n a l y s i s w a s used t o s t u d y i s o n i a z i d b e f o r e t h e t e c h n i q u e gained i t s c u r r e n t p o p u l a r i t y 2 4 3 25. P i r i s i 2 6 showed t h a t i s o n i a z i d i n t h e p r e s e n c e o f z i n c , c o p p e r and ' i r o n s a l t s and m e r c u r i c o x i d e g i v e s an a b n o r m a l D.T.A. pattern. D r . J a c o b s o n 2 7 h a s shown t h a t t h e 2.2
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GLENN A. BREWER
3793
ISONIHZID LOT 866434 1
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MFISS/CHRRGE SUM = 4 4 6 7 2 B R S E PERK 2 =24.36
Figure 5:Low-resolution mass spectrum of isoniazid.
ISON IAZlD
195
S q u i b b House S t a n d a r d of i s o n i a z i d shows a s h a r p endotherm a t 17OoC u s i n g DuPont t h e r m a l a n a l y s i s
equipment.
The p u r i t y of t h i s s t a n d a r d w a s d e t e r m i n e d t o be 99.95 mole p e r c e n t u s i n g a P e r k i n E l m e r DSC-1B d i f f e r e n t i a l s c a n n i n g colorimeter27. 2.23 T.G.A. Thermogravimetry c a n be u s e d t o d e t e r m i n e m o i s t u r e or r e s i d u a l s o l v e n t s i n i s o n i a z i d . When t h e S q u i b b House S t a n d a r d w a s t e s t e d no loss on d r y i n g was r e c o r d e d 2 7 . 2.24 E l e c t r i c a l Moment Lumbroso and Barassin’* d e t e r m i n e d t h a t t h e e l e c t r i c a l moment of i s o n i a z i d w a s 2.92 I.L. 2.25 E l e c t r i c a l C o n d u c t i v i t y The e l e c t r i c a l c o n d u c t i v i t y of a compressed t a b l e t of i s o n i a z i d w a s d e t e r m i n e d a t t e m p e r a t u r e s between 50 and 1500C29. 2.26 C r y s t a l Characteristics B h a t a n d co-workers30 h a v e r e p o r t e d t h a t i s o n i a z i d c r y s t a l s a r e o r t h o r h o m b i c , space g r o u p P 2 1 2 1 8 1 , w i t h a , 14.915 ( 1 5 ) b, 11.400 ( 1 0 ) c, 3.835 ( 5 ) ~ d , ( m e a s u r e d ) = 1.417 ( 7 ) d ( c a l c u l a t e d ) = 1.395 and 2 = 4.
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GLENN A. BREWER
2.27
X-Ray D i f f r a c t i o n The powder x-ray d i f r a c t i o n c u r v e f o r i s o n i a z i d i s shown i n F i g u r e 6 3 f The r e l a t i v e i n t e n s i t i e s f o r t h e v a r i o u s peaks a r e g i v e n below:
.
Interplanar Distances Relative I n t e n s i t i e s d (ANGSTROMS ) 0.098 8.84 0.408 7.30 0.398 6.10 0.451 5.64 1.000 5.25 0.502 4.49 0.296 3.69 0.398 3.51 0.102 3.42 0.068 3.36 0.197 3.27 0.235 3.10 0.060 3.04 0.058 3.01 2.80 0.170 0.076 2.63 0.168 2.47 0.115 2.42 0.187 2.33 2.3 S o l u b i l i t y 2 . 3 1 Water S o l u b i l i t y32 F o u r t e e n g r a m s of i s n i a z i d -re s o l u b l e i n 100 m l o f water a t 25OC. T w e n t y - s i x grams a r e s o l u b l e i n 100 m l of water a t 4OoC. 2.32 Solubility i n Solvents32~33
mf
Solvent S o l u b i l it ethanol(25OC) 2 g/100 e t h a n o l ( b o i l i n g ) 10 g/100 m l ch l o r o form 0 . 1 g/100 m l ethyl ether very s l i g h t l y soluble benzene i ns o l u b l e
104
M
,
100
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70
60
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30
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Figure 6:X-ray powder-diffraction pattern of isoniazid.
4
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20
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G L E N N A. BREWER
2.4
Physical P r o p e r t i e s of Solution 2.41 pH The pH o f a s o l u t i o n ( 1 i n 10) should b e between 6.0 and 7.523. 2.42 D i s s o c i a t i o n Constant There i s a d i s c r e p a n c y i n t h e l i t e r a t u r e o n t h e d i s s o c i a t i o n c o n s t a n t s of isoniazid. T h i s i s i n p a r t due t o t h e d i f f e r e n t methods of measurement employed. F a l l a b 3 4 determined t h e b a s i c d i s s o c i a t i o n c o n s t a n t a s 3 x 10-11 measured conductC a n i 6 and D j o r d j evi635 e s t a b l i s h e d ometrically. t h a t t h e 1st b a s i c c o n s t a n t s h o u l d be a s c r i b e d t o t h e p y r i d i n e n i t r o g e n and t h e 2nd t o t h e h y d r a z i n e group. T h i s i s c o n t r a r y t o p r e v i o u s work by Cingolani and G a ~ d i a n o ~ ~ . Nagano and c o - ~ o r k e r sdetermined ~~ t h e dissociation constants potentiometrically as PK1 = 2.13, PK2 = 3.81, PK3 = 11.03. S a l v e s e n and G l e n d r a n c ~ ed~e ~ termined t h e d i s s o c i a t i o n c o n s t a n t s i n 1 . O M sodium and K2 = c h l o r i d e s o l u t i o n a s K1 = 9.80 x 1.42 10-4. Zommer and Szuszkiewicz'l have e s t a b l i s h e d pK1 = 1 0 . 7 5 and pK2 = 11.15 and prot o n a t i o n c o n s t a n t s cf 3.57 f o r t h e p y r i d i n e N and 1.75 f o r t h e h y d r a z i d e N. Rekker and N a ~ t found a ~ ~ t h a t solu t i o n s o f i s o n i a z i d became yellow a t p H 10 and 2.7. The c o l o r i s r e v e r s i b l e on changing t h e pH. They e x p l a i n e d t h i s b e h a v i o r on t h e b a s i s o f t h e e x i s t ance o f two p o s i t i v e i o n s , a monovalent yellow p o s i t i v e i o n and a d i v a l e n t c o l o r l e s s p o s i t i v e ion. The pK v a l u e s a r e pK' = 2 . 0 0 , pK" = 3.6 and pK"' = 10.8. 2.43 Photolysis Constant S a l v e s e n and Eiki113' e s t a b l i s h e d t h e p h o t o l y s i s c o n s t a n t s f o r i s o n i a z i d a t 20°C and o ' * and 370 nm i n M NaCl s o l u t i o n a s kl = 1 . 0 0 x l k2 = 1.45 x 10-4.
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2.44
Oxidation P o t e n t i a l The o x i d a t i o n p o t e n t i a l s f o r i s o n i a z i d a t v a r i o u s pH v a l u e s w e r e d e t e r m i n e d b y vu 1t e r i n 4 0 . Solution i n Ef 1 N HC1 0.70 0.025M Na2B407 0.25 3N NaOH -0.22 3.
Metal Complexes I s o n i a z i d forms metal complexes w i t h many d i v a l e n t i o n s . T h e s e complexes h a v e b e e n used i n t h e d e t e r m i n a t i o n o f i s o n i a z i d (see S e c t i o n s 6.22, 6.25 and 6.29). T a m u r a a n d Nagano4I h a v e d e t e r m i n e d t h e c o n s e c u t i v e f o r m a t i o n c o n s t a n t s f o r t h e complex formed b e t w e e n i s o n i a z i d a n d C d ( I 1 ) . The e x p e r i ments were c a r r i e d o u t a t pH 7.2 ( a d j u s t e d w i t h NaOH) i n M NaN03 u s i n g 0.001M Cd(N03)2 a t 25OC. The d e t e r m i n a t i o n was made p o l a r o g r a p h i c a l l y . The v a l u e s d e t e r m i n e d were k l = 35, k2 = 0 . 5 7 , k3=52.5. A t high concentrations o f i s o n i a z i d yellow c r y s t a l s o f Cd ( I N H ) 2 (NO3)2-H20 p r e c i p a t e d from s o l u t i o n indicating t h a t contrary t o t h e polarographic data t h a t t h e 2 : l complex is more s t a b l e t h a n t h e 3 : l complex. By p H t i t r a t i o n t h e s t e p w i s e f o r m a t i o n c o n s t a n t s w e r e k l = 1 2 . 2 , k2 = 1 2 . 6 , a n d k 3 = 3.4. The Same a u t h o r s 4 2 s t u d i e d t h e f o r m a t i o n c o n s t a n t s o f i s o n i a z i d a n d C u ( I I ) , Zn, N i ( I I ) , C o ( I 1 ) a n d Mn (11). The complexes o f c o p p e r a n d i s o n i a z i d h a v e been e x t e n s i v e l y s t u d i e d by I ~ h i d a t e ~ ~ . 4.
History, Synthesis and Manufacturinq I s o n i a z i d w a s f i r s t p r e p a r e d b y Meyer a n d M a l 1 Y s o 0 i n 1912 b y h e a t i n g a m i x t u r e o f ison i c o t i n i c a c i d a n d h y d r a z i n e above 30OoC. The a c t i v i t y of t h e compound a g a i n s t Mycobacterium SJ. was f i r s t r e c o g n i z e d b y C h o r i n e 5 0 1 a n d b y Huant502 i n 1945. The d r u g w a s r e p o r t e d a s a u s e f u l t u b e r c u l o s t a t i c a g e n t b y F a r b e n f a b r i k e n - B a y e r , A. G., Hoffmann-LaRoche, I n c . a n d E. R. S q u i b b & S o n s , I n c .
200
GLENN A. BREWER
i n 1952503. The b a s i c method of m a n u f a c t u r e o f i s o n i a z i d i s t h e condensation of hydr azine with a y - s u b s t i t u t e d pyri d i ne H y d r a z i n e c a n be d i r e c t l y c o n d e n s e d w i t h i s o n i c o t i n i c a c i d , The w a t e r formed i n t h e rea c t i o n i s u s u a l l y removed b y a z e o t r o p i c d i s t i l l a tion44945946947, E s t e r s o f i s o n i c o t i n y l a c i d c a n be h y d r o l y z e d and t h e r e s u l t i n g a c i d condensed w i t h h y d r a z i n e . Ammonia i s u s u a l l y employed f o r t h e h y d r o l y s i s 4 8 . Y - P i c o l i n e c a n be o x i d i z e d i n 70% s u l f u r i c a c i d w i t h manganous d i o x i d e t o form i s o n i c o t i n i c acid. The c o r r e s p o n d i n g a c i d c h l o r i d e i s made with thionyl chloride. The a c i d c h l o r i d e i s t h e n r e a c t e d w i t h h y d r a z i n e i n anhydrous benzene t o y i e l d isoniazid49. I n a modification of this procedure t h e a c i d c h l o r i d e i s r e a c t e d w i t h e t h a n o l t o form t h e e t h y l ester which i s t h e n r e a c t e d w i t h h y d r a z i n e i n e t h a n o l t o form i s o n i a z i d 5 0 . I n a s i m i l a r manner o n e c a n o x i d i z e 2 , 4 d i m e t h y l p y r i d i n e w i t h s e l e n i u m and s u l f u r i c a c i d . T h e m i x t u r e i s n e u t r a l i z e d w i t h ammonia. A m i x t u r e o f i s o n i c o t i n i c a c i d , i s o n i c o t i n a m i d e and i s o n i c o t i n i c h y d r a z i d e i s forrned51.
.
Stability The s t a b i l i t y o f i s o n i a z i d h a s b e e n s t u d i e d e x t e n s i v e l y i n s o l u t i o n a n d i n v a r i o u s pharmaceut i c a l p r e p a r a t i o n s . Of p a r t i c u l a r i n t e r e s t i s t h e r e a c t i o n of t h e h y d r a z i n e g r o u p w i t h n a t u r a l l y o c c u r i n g a l d e h y d e s and k e t o n e s such a s s u g a r s o r k e t o a c i d s and the complexation o f i s o n i a z i d w i t h metal i o n s . Lewin a n d H i r ~ c h a~v e~ shown t h a t n o n - i o n i c c h e l a t i n g material can l a r g e l y p r e v e n t t h e degrad a t i o n o f i s o n i a z i d when n e u t r a l a n d a l k a l i n e s o l u t i o n s a r e a u t o c l a v e d . They n o t e d t h a t C u ( I 1 ) and Mn(I1) i o n s a c c e l e r a t e d t h e d e g r a d a t i o n o f i s o n i a z i d i n t h e p r e s e n c e o f hydrogen peroxide. P o o l e a n d M e ~ e r e~p o~r t e d t h a t i s o n i a z i d i s u n s t a b l e i n human o r r a b b i t plasma w h i l e i t i s
5.
ISON I A2 ID
201
s t a b l e f o r s e v e r a l weeks i n b u f f e r e d aqueous s o l u t i o n s a t pH v a l u e s below 8 . The i n s t a b i l i t y i n plasma i s q u i t e marked even a t r e f r i g e r a t o r temperatures. Kakemi and c o - ~ o r k e r shave ~ ~ studied t h e d e g r a d a t i o n o f i s o n i a z i d i n aqueous s o l u t i o n under a n a e r o b i c c o n d i t i o n s . A l k a l i n e h y d r o l y s i s under a e r o b i c c o n d i t i o n s y i e l d s a m i x t u r e of i s o n i c o t i n i c a c i d , i s o n i c o t i n a m i d e and 1 , 2 d i i s o n i c o t i n o y l h y d r a z i n e p l u s s m a l l amounts of u n i d e n t i f i e d products. Under a n a e r o b i c c o n d i t i o n s i s o n i c o t i n i c a c i d and 1 , 2 d i i s o n i c o t i n o y l h y d r a z i n e were t h e p r i n c i p a l p r o d u c t s . When EDTA was added t o t h e r e a c t i o n m i x t u r e only i s o n i c o t i n i c a c i d was formed. F i r s t o r d e r k i n e t i c s were followed. Inoue55 found t h a t a t pH 3 . 1 u n d e r a n a e r o b i c c o n d i t i o n s i s o n i a z i d h y d r o l y z e s t o form i s o n i c o t i n i c acid. Pseudo f i r s t o r d e r k i n e t i c s a r e followed. A t lower pH v a l u e s t h e e f f e c t of b u f f e r t y p e can be s e e n . A c t i v a t i o n e n e r g i e s were c a l c u l a t e d for t h e h y d r o l y s i s by d i f f e r e n t i o n i c s p e c i e s . Horioka and c o - ~ o r k e r sfound ~~ t h a t losses o f i s o n i a z i d were encountered when t h e drug was blended w i t h v a r i o u s a n t i a c i d p r e p a r a t i o n s . The e f f e c t of t e m p e r a t u r e , humidity and pH on t h e s t a b i l i t was determined. Haldg7 found t h a t i s o n i a z i d underwent slow o x i d a t i o n i n aqueous s o l u t i o n , b u t i n t h e p r e s e n c e of s u c r o s e t h e i s o n i a z i d r e a c t e d w i t h t h e a l d o hexoses formed on i n v e r s i o n . The r e a c t i o n w i t h s u c r o s e could b e i n h i b i t e d by t h e a d d i t i o n o f 0.3% sodium c i t r a t e . Pawelczyk and c o - ~ o r k e r sfound ~~ t h a t a s long a s c o n d i t i o n s were k e p t a n a e r o b i c t h a t t h e decompos i t i o n of i s o n i a z i d i n t h e pH r a n g e 3 t o 7 followed f i r s t order kinetics. They r e p o r t e d t h a t a 1% s o l u t i o n of t h e drug was 37 times more s t a b l e a t PH 6 t h a n a t pH 3.The e f f e c t of d i f f e r e n t b u f f e r s p e c i e s on t h e r a t e o f t h e r e a c t i o n was noted. Wu and co-workers59 i n v e s t i g a t e d t h e browning r e a c t i o n between l a c t o s e and i s o n i a z i d i n t h e
202
G L E N N A. BREWER
s o l i d s t a t e with d i f f u s e r e f l e c t a n c e spectrophotometry. T h i n - l a y e r chromatography was u s e d t o demonstrate t h e p r e s e n c e of i s o n i c o t i n o y l hydrazones of l a c t o s e and h y d r o x y m e t h y l f u r f u r a l . InoueG0 h a s e s t a b l i s h e d t h e e f f e c t of t h e p r e s e n c e of copper (11) i o n s on t h e r a t e of oxidat i o n of i s o n i a z i d i n s o l u t i o n . The r e a c t i o n p r o d u c t s were i s o n i c o t i n i c a c i d , i s o n i c o t i n a m i d e , 1,2-diisonicotinoylhydrazine, i s o n i c o t i n e carboxaldehyde and i s o n i c o t i n o y l hydrazone. The copper c h e l a t e s of i s o n i a z i d a r e degraded by a f i r s t o r d e r r e a c t i o n and t h e r a t e i s determined b y t h e r a t i o of t h e c o n c e n t r a t i o n of c h e l a t e d s p e c i e s p r e se n t Inoue and Ono61 have e s t a b l i s h e d t h e k i n e t i c s of t h e d e g r a d a t i o n of i s o n i a z i d i n t h e p r e s e n c e o f Managenese (11). Shchukin62 h a s s t u d i e d t h e r e a c t i o n of copper (11) w i t h i s o n i a z i d . Kakemi and co-workers63 s t u d i e d t h e s t a b i l i t y of t h e sodium m e t h a n e s u l f o n a t e salt of i s o n i a z i d from p H 3 t o 9. Rao and c o - ~ o r k e r shave ~ ~ demonstrated t h a t i s o n i a z i d i n s y r u p f o r m u l a t i o n s undergoes hydrazone formation w i t h t h e f r e e g l u c o s e t h a t i s p r e s e n t . Absorption of t h i s hydrazone i s r e p o r t e d t o b e impaired. The a u t h o r s s u g g e s t t h e u s e of s o r b i t o l a s a replacement f o r s u c r o s e .
.
6.
A n a l y t i c a l Chemistry 6.1 Identity Tests A l a r g- e number of i d e n t i t y t e s t s have b e e n e s t a b l i s h e d f o r i s o n i a z i d . Most of t h e s e a r e colorimetric and a r e r e p o r t e d below i n t a b u l a r form.
Reaqen t
8 w
p- Dimethylaminobenza l d e h y d e A l k a l i n e Na2Fe ( C N ) 5NO Q C F e (CN) 6J + light Dini t r o c h l o r o b e n z e n e o-ninitrobenzene R e d u c t i o n w i t h Zn/HCl and ph e n y l h y d r a z i n e 1 , 2 Naphthoquinone4 - S u l f o n i c a c i d + NaOH 1,2,4-Aminonaph t h o 1 s u l f o n i c a c i d SbC13, SbC15 o r AsC13 E p i ch l o r o h y d r i n Dimethylglyoxime E t h y 1en i c d i ca rbox y 1i c a c i d s (fumaric, maleic a c i d s , e t c . ) Naphthoqu inone-HgC12 3,5-Dini t r o s a l i c y l i c a c i d N i n h y d r in BrCN a n d NaOH Benzyl c h l o r i d e NaOH D r a g e n d o r f f ' s Reagent
Reference
rnlnr i n t e n s e yellow i n t e n s e orange pink purple violet
66,67,68,74,85,86 69,74 70 71,74,86 72
y e 1low
73
bright red orange t o red
74,75,76 77 78 79 80 81
--
red red ye 1low
--
brown r e d
r e d orange green-blue f l u o r . b l u e fluorescence red
82 83 84,85 85 85 86,94
I n a d d i t i o n t o t h e s e c o l o r r e a c t i o n s a number o f c o l o r e d p r e c i p i t a t e s can be formed o n t h e a d d i t i o n of m e t a l s a l t s o r a c i d s t o i s o n i a z i d .
Reagent Am03 S e02 HgC12 cuso4 Hg2C12 KI AuI Lead a c e t a t e + K I KBr
N
e 0
~205.12~03 Picrolonic acid Tannic a c i d V i t a l i l s reagent Mecke' s r e a g e n t Frghde' s r e a g e n t Mandelin' s r e a g e n t Alloxan D i s u I f imides M e t h y 1i o d i d e K2 C r 2 0 7 Pho sphomo 1yb d i c a c id P i c r i c Acid Reineckel s s a l t Styphnic a c i d
Color of P r e c i p i t a t e White Red White Blue White Amorphous mass Dark c r y s t a l s Yellow a c i c u l a r c r y s t a l s E f f e r v e s c e n c e followed by b l a c k and c o l o r l e s s crystals Precipitate Green-ye1 low PPt Yellow mass Rose-sienna Blue Red White p p t
-Yellow n e e d l e s
--
--
Reference 74,94 78,87,88 74 86 86 86,93,95 86 86 86 86 86,93 86 86 86 86 86 89 90 91 92 92 93,94 94 94
94 94 94
Kay's reagent Vaillef s reagent Na 2 P t B r 6
F e i g l a n d c o - w o r k e r s g 6 h a v e r e p o r t e d a s p o t t e s t i n which i s o n i a z i d i s ( C N ) 6-7 q u a t e r n i z e d a n d p y r o l y z e d w i t h Na2S203 a t 180OC. A c i d i f i e d Fe i s used f o r d e t e c t i o n .
Be
97 98 Popkov and Amelink h a v e r e p o r t e d on m i c r o c r y s t a l l i n e t e c h n i q u e s f o r t h e detection of isoniazid. 6 . 2 Methods of A n a l y s i s 6 . 2 1 G e n e r a l Reviews Deltombe99, S l o u f l O O , Robles a n d Unzueta'Ol, Yalcindaglo2, B r a n d y s l 0 3 a n d G a r c i a a n d c o - w o r k e r s l o 4 h a v e a l l p u b l i s h e d r e v i e w s on t h e q u a l i t a t i v e and q u a n t i t a t i v e d e t e r m i n a t i o n o f i s o n i a z i d . 6 . 2 2 C o l o r i m e t r i c methods A number of a u t h o r s h a v e formed h y d r a z o n e s of i s o n i a z i d w i t h v a r i o u s aldehydes and k e t o n e s and used t h e h i q h l y c o l o r e d p r o d u c t s t o determine t h e d r u g . O f t h e v a r i o u s a ldehydcs u s e d , p-dimei?hylaminobenza l d e h y d e lo8, log, I l o ,lll. Benza1dehydes7, I 6 O , e a r s t o be t h e m o s t 0 ular105,106, lo7; a n d v a n i l l i n 1 1 3 ' 197yPgghave a l s o been u s e d . The o f f i c i a l method o f t h e AOAC i s t h e r e a c t i o n of i s o n i a z i d w i t h b e n z a l d e h y d e i n sodium b i c a r b o n a t e solution. The a b s o r b a n c e o f t h e h y d r a z o n e i s m e a s u r e d a t 302 nm. The absorbance a t 375 nm ( b a c k g r o u n d ) i s s u b s t r a c t e d a s a c o r r e c t i o n 1 6 9 . Sodium 1,2-naphthoquinone-4-sulfonate r e a c t s w i t h t h e h y d r a z i d e p o r t i o n o f i s o n i a z i d i n a l k a l i n e s o l u t i o n t o p r o d u c e a n orange-red c o l o r w i t h a maximum a t 480 nm1149115. 2-3-Dichloro-1,4-naphthoquinone r e a c t s
?R
206
GLENN A. BREWER
with isonia zid t o give a b lu e co lo r i n a l k a l i n e ~ o l u t i o n ~ l ~The , ~ r~e a~c t. i o n i s u s e f u l w i t h pharmaceutical p r e p a r a t i o n s which a l s o c o n t a i n sodium a m i n o s a l i c y l a t e l l 8 . An a s s a y u t i l i z i n g 1, 4-naphthoquinone h a s a l s o been regorted1l9. I s o n i a z i d r e d u c e s phosphomol b d a t e i n a l k a l i n e s o l u t i o n t o molybdenum b l u e 1 2 0 , l Y l . In a s i m i l a r r e a c t i o n molybdophosphotungstate g i v e s a b l u e color122. An a s s a y u t i l i z i n g molybdic a c i d i n a l k a l i n e a c e t o n e s o l u t i o n h a s also been r e p o r t e d l 2 ? I s o n i a z i d r e a c t s w i t h cvano en c h l o r i d e 1 2 4 ~ 1 2 5 ~ 1 2 6 c, h l o r o r h o d a n a m i n e l 2 7 ~ 18 o r
2
cyanogen bromide129 t o form g l u t a c o n i c d i a l d e h y d e which can t h e n be condensed w i t h b a r b i t u r i c o r 2t h i o b a r b i t u r i c a c i d s t o y i e l d c o l o r e d polymethine dyes. I s o n i a z i d reduces f e r r i c y a n i d e t o f e r r o c y a n i d e . The amount o f f e r r o c y a n i d e can be determined by t h e a d d i t i o n of f e r r i c i o n t o y i e l d a b l u e colorl30,131. Sodium pentacyanoaminoferroate r e a c t s w i t h i s o n i a z i d t o g i v e a yellow chromogen 132 I s o n i a z i d r e a c t s w i t h 1-chloro-2, 4-dinitrobenzene i n a l k a l i n e s o l u t i o n t o g i v e a p u r p l e color133,74,134. l-Fluoro-2,4 d i n i t r o b e n z e n e a l s o r e a c t s i n a s i m i l a r manner 135 I s o n i a z i d forms c o l o r e d complexes w i t h many m e t a l s which can be used i n a n a l y t i c a l c a n be formed w i t h ammonium methods. vanadate f e r r i c c h l o r i d e and 2 , 2 1 b i ~ y r i d i n e l ~copper139 ~, and N i c k e l (11) and f e r r i c i0nl40. Reineckels s a l t forms a w a t e r i n s o l u b l e p r e c i p i t a t e with i s o n i a z i d . This p r e c i p i t a t e d i s s o l v e s i n a c e t o n e and t h e c o n c e n t r a t i o n of i s o n i a z i d can b e determined c o l o r i m e t r i c aiiy141, 142. The f o l l o w i n g compounds have a l s o been used i n c o l o r i m e t r i c assays for i a o n i a z i d .
.
13g~y9’~358,
Reagent Reference ch loropicrin 504 epichlorohydrin 143 ninhydrin 144 145 tripheny1tetrazolium ch loride 9-chloroacridine 146 dinitrobenzoic acid 147 p-aminosalicylate-HVO~ 148 1,2,4-aminonaphtholsulfonic acid 77 p-ni trophenyldiazonium fluoroborate 149 7-chloro-4 nitrobenzo-2-oxa-1,3-diazole 150 acid chrome dark blue 151 2-bromo-1-acetonaphthone 112 N- (4-pyridyl)pyridinium chloride 152 174 picryl chloride 6.23 Spectrophotometric Methods A number of authors have utilized the strong absorbance of isoniazid in the ultraviolet as a means of determining the concentration of the drug. In many methods the a @ : f ! 7 4 IPS in alkaline and acid solution as an identity test 1659237. Isoniazid can be determined in the presence of p-aminosalicylate by an ultraviolet assay162,163,164. 6.24 Fluorimetric Methods Although isoniazid does not have any native fluorescence several sensitive fluorometric assays have been reported for the drug. Isoniazid is coupled with 2-hydroxy-1-naphthaldehyde to give a yellowgreen fluorescence. The compound has an excitation maximum at 495 m and an
Y
y3,iQ%, ar39:'iB;
Fh
208
GLENN A. BREWER
e m i s s i o n maximum a t 534 nrn166~167. I n a n o t h e r method t h e p y r i d i n e r i n g i s c l e a v e d w i t h cyanogen b r o m i d e t o form A S c h i f f ' s base i s t h e n formed glutacondialdehyde. w i t h 4 - a m i n o b e n z o i c a c i d which has a n e x c i t a t i o n maximum a t 336 6 . 2 5 T i t r i m e t r i c Methods A large variety of titrimetric methods h a v e b e e n employed f o r the d e t e r m i n a t i o n of i s o n i a z i d i n b u l k and i n formulated products. A series of reviews have been w r i t t e n on t i t r i m e t r i c methods170, 1 7 1 9 1 7 2 3 1 7 3 ~ 202. The o f f i c i a l m e t h o d s of a n a l y s i s i n t h e U . S. P. 2 3 , B. P. 174 a n d E u r o p e a n P h a r m a c o p o e i a 3 a r e t i t r i m e t r i c methods. I n t h e U.S.P,23 a n i t r i t e titration is utilized. I n t h e B.P. 174 t h e i s o n i a z i d i s r e a c t e d w i t h bromine and t h e e x c e s s bromine i s t i t r a t e d w i t h t h i o s u l f a t e a f t e r t h e l i b e r a t i o n o f i o d i n e by t h e a d d i t i o n o f potassium iodide. I n t h e European P h a r m a c o p o e i a 3 a d i r e c t t i t r a t i o n w i t h bromate i s u t i l i z e d w i t h t h e a d d i t i o n o f ethoxychysoidine a s an i n d i c a t o r . The v a r i o u s t i t r i m e t r i c methods a r e summarized i n t h e Table.
Reaq e n t
I C 1, K I
Titrant t h i o s u 1f a t e KBr03 alkali KBrO3 mr03 t h i o s u lf a t e KIO~ thiosulfate t h i o s u 1f a t e t h i o s u l f at e t h i o s u l f at e
non-aqueous
HC lo4
non-aqueous non-aqueous
NaN02 HC 104 HClOq
mr ,m r 0 3 ,KI m r Br2
-
~ 1 0 3KI , H I , K2Cr207, K I
~ ~ 1 0 4 , N
8
non-aqueous Cd++ Cd++ cU++,
NH4SCN
CU++, N H ~ S C N
I2
Indicator starch ethoxychrysoidine pheno lphtha l ei n methyl orange p o t e n t iome t r i c starch ethoxychrysoidine starch starch starch starch thermome t r i c crystal v i o l e t or methyl v i o l e t
Sb e l e c t r o d e
Complexon 111 CaC12
g l a s s electrode potentiome tric thymol b l u e eriochrome B l a c k T methylthymol B l u e
Am03 EDTA
mu r e x i de
NaClO4 NaOMe
Reference 1 7 5 ,1 7 7 , 1 7 9 , 1 8 2 176,106,180,184 178 181,186 1 8 1 , 1 8 3 ,1 8 5 ,1 8 7 74,188,192,196 189 1 9 0 , 1 9 3 ,1 9 5 191 106,194 198 200 217,211,210,209,208, 207,206,204,205,216, 213,201,74,57,203, 2 14 233 212,276 215 2 18 2 02 219,221 220 222 2 2 3 ,224
E
% m
d
~m m a ,
d
0 0 0
a E E
U
H H
C 0
3
dd
C O D
0 a 0 mEcn NUf:
h
d
9
a,
a 0 k c,
u
a,
a,
c
a,
k
a,
a m a -ti
I
k
I
m
4J
m
E
0
.ti
5
a .ti a
I
rl
h C
a,
4
tn
0-
a
5k
d
a,
m
a,
m .d
nl
m
4J
z
c,
-
Ill
4J
o"+ cn+
C cr a, m m rn W
I
3 3
u u
E 7
m
d a, Ik
210
I
H
h
d
m
'
Nesslerl s reagent I2
-
C e (So4) 2
-
K2Cr207 isopropenyl t r i chloroacetate
Y
sodium d i e t h y l d i thiocarbama t e hydraz i n e ammonium hexani t r o c e r a t e (IV) Mohrrs s a l t C e (NO314 Mohrfs s a l t
cuso4
2 56
starch a-naphthof lavone P t electrode P t electrode diphenylamine
2 57 2 58
259 2 60 261
NaOH bromphenol b l u e 262 KOH K3Fe (CN) 6 P t electrode 263,264 K 3Fe (CN) 6, KOH, H2S04, K I t h i o s u I fa t e starch 265 6.26 E l e c t r o c h e m i c a l Methods A number of a u t h o r s have d e t a i l e d p o l a r o g r a p h i c methods f o r i s o n i a z i d . The r e d u c t i o n a p p a r e n t l y o c c u r s i n two s t e p s ( t o t a l o f 4 e l e c t r o n s ) b u t t h e s t e p s a r e n o t s u f f i c i e n t l y w e l l s e p a r a t e d t o be u t i l i z e d The h a l f wave p o t e n t i a l bea n a l y t i c a l l y , s o t h a t t h e s i n g l e wave i s used. b u t t h e h e i g h t d i d n o t change r e a t l y comes more n e g a t i v e a t h i g h e r pH v a l u e s H r a n g e s t u d i e d 266,153,267,266,269,270,271,272,273,274,275,27 ,278, over t h e 279,280,2Kl
4
A.C.Polarography h a s been used by Sato282 and Vallon and c o - ~ o r k e r s ~ ~t h~ei n a s s a y o f i s o n i a z i d . Okuda and co-workers284 rea c t e d i s o n i a z i d w i t h 1,2-naphthoquinone-4-sulfonic a c i d and have t h e n used p o l a r o g r a p h y t o measure t h e r e a c t i o n p r o d u c t . se e r p l au h o r s h ve r e r t e d c o u l o e t ' c g e t h o d s for t h e a n a l y s i s o f i s o n i a z i x w i t h ekectroccemica??y g e n e r a t e 8 c 6 i o r i n e 285,286 o r bromine286,287,288,289.290.
212
GLENN A. BREWER
6.27
Gravimetric Methods R e l a t i v e l y few g r a v i m e t r i c a s s a y s have been r e p o r t e d f o r i s o n i a z i d . T h i s i s probably because of t h e l a r g e number of c o l o r i m e t r i c , t i t r i m e t r i c and e l e c t r o c h e m i c a l methods a v a i l a b l e which a r e f a s t e r and more convenient than t h e g r a v i m e t r i c methods. Leal and Alves 234 have r e p o r t e d an a s s a y using p i c r i c a c i d t o form a w a t e r i n s o l u b l e salt. Akiyama and co-workers291 prec i p i t a t e i s o n i a z i d e a s t h e C u ( I 1 ) o r Hg(I1) s a l t s . The s a l t s a r e r e d i s s o l v e d i n h y d r o c h l o r i c a c i d and t h e metal i s then r e p r e c i p i t a t e d a s t h e s u l f i d e which i s determined g r a v i m e t r i c a l l y . The z i n c 2 9 2 and cadmium293 s a l t s can be measured by d i r e c t gravimetry. The benzyli d e n e d e r i v a t i v e can be determined e i t h e r gravimetrically o r v o l ~ m e t r i c a l l y ~ ~ I s~o .n i a z i d can b e q u a t e r n i z e d and t h e s a l t can b e then measured v o l u m e t r i c a l l y o r g r a v i m e t r i c a l l y 2 95. The phosphotungstate of i s o n i a z i d can b e determined gravime t r i c a 1 1 ~ 1 6 4 . 6.28 M i c r o b i o l o q i c a l and Enzymatic Methods S e v e r a l a g a r d i f f u s i o n microbiol o g i c a l a s s a y s u t i l i z i n g s t r a i n s of a c te rium have been r e p o r t e d f o r i s o n i a ~ i d ~ ~ ~ I s o n i a z i d i n h i b i t s many enzyme systems and a number of t h e s e might b e s e l e c t e d a s t h e b a s i s of enzymatic a s s a y s . Examples of enzyme systems which a r e i n h i b i t e d a r e pea cotyledon amine o x i d a s e , c a r r o t r o o t L-glutamic decarboxylase and wheat s e e d l i n g t r a n s a m i n a ~ e ~ The ~ ~ . inhibition i s r e v e r s e d by t h e presence of k e t o a c i d s . 6 . 2 9 Miscellaneous Methods O s c i l l o m e t r i c t i t r a t i o n s have been used t o determine i s o n i a ~ i d301. ~ ~ ~ Ijs o n i a z i d can be assayed g a s o m e t r i c a l l y a f t e r o x i d a t i o n w i t h iodate3O2 o r f e r r i c y a n i d e 3 0 3 304. Conductometric t i t r a t i o n s with sodium hydroxide o r h y d r o c h l o r i c a c i d have been J
~
~
~
u s e d t o measure i s o n i a z i d c o n t e n t 3 0 5 , 3 0 6 . The c o p p e r c h e l a t e of i s o n i a z i d i s s o l u b l e i n m e t h y l i s o b u t y l ketone. The c o p p e r c o n t e n t of t h e c h e l a t e i s d e t e r m i n e d i n t h e o r g a n i c p h a s e by a t o m i c a b s o r p t i o n s p e c t r o m e t r y 3 0 7 . I s o n i a z i d i n p u r e s o l u t i o n s can b e d e t e r m i n e d by r e f r a c t o m e t r y 3 08 6.3
5 w
Chromatographic Methods 6 . 3 1 Paper Chromatoqraphy Numerous p a p e r c h r o m a t o g r a p h i c s y s t e m s h a v e b e e n u s e d t o s e p a r a t e i s o n i a z i d from i n t e r m e d i a t e s u s e d i n t h e s y n t h e s i s , d e g r a d a t i o n p r o d u c t s and m e t a b o l i c p r o d u c t s . Since isoniazid absorbs strongly i n t h e u l t r a v i o l e t and g i v e s a number of c o l o r r e a c t i o n s 3 0 9 t h e r e i s no problem i n d e t e c t i n g o r q u a n t i t a t i n g t h e d r u g a f t e r t h e s e p a r a t i o n h a s been completed. A t a b l e of some p a p e r c h r o m a t o g r a p h i c s y s t e m s i s g i v e n below: Solvent Syst e m Detection Use Ref. Water s a t u r a t e d b u t a n o l C14 l a b e l l e d Urine metabolites 310 Isoamyl alcohol-waterCNBr ,Microb i o 1. Urine metabolites 311 a c e t i c a c i d ( 5 0 :50 :1 . 5 ) I s o p r o p a n o l - w a t e r (85 :1 5 ) Urine metabolites 312,313 Butano 1-ammonia -Derivatives 31 4 1st Dimension sec. b u t a n o l w a t e r (saturated)
--
2nd Dimension i s o a m y l alcohol-acetone-acetic a c i d - w a t e r (56:24:6: 1 4 )
CNBr- o-phenyl-
enediamine dimethylbenza l d e h y d e
Urine
Butanol-10% NH4OH(lO:2) circular Butanol-water(4:l) ascending 2,4-lutidine-isoamyl alcohol-water (5: 100: 9) Butanol-HC1-pet. ether or Butanol-HCl-H20(paper sat. with KC1 solution) (a)Butanol-ethanol-water (2:2:1) (b)Butanol-pyridine-water (16:4: 3 )
Butanol sat.ammoniaca1 Impurities with silver nitrate dimethylaminobenzaldehyde Dosage forms
3 16
methanolic dinitrochlorobenzene iodine-platinic iodide
Impurities
318
other basic substances
319
metabolites
320
metabolites metabo 1ites in urine
322,323
I
(c)Ethanol-l.5N NHqOH-water ultraviolet (17:1:2) (d)Phenol-isopropanol-water (16:1:5) 0.5 ammonium chloride u 1traviole t (a)Butanol saturated with water -(b)Propanol-water( 8 0 : 2 0 )
1
317
321
(a) 1sopropanol-25% NH20H(85: 15) (b) Isopropanol-water(85:15) (c) Isopropanol-formic acidwater (80:1O:lO) Pyridine-Water(65:35) Isopropanol-NH40H-water (7:1:2) Butanol-acetic acid-water ( 5 :1:4) ( a ) E thyl me thyl ketone-acetoneformic acid water(40:2:1:6) (b)E thy 1 me thy1 ketone-diethy lam inewater (921:2 :7 7 ) (c)Methyl isobutylketone-formic acid-water(ketone sat.with 4% formic acid) (d)Chloroform-methanol-formic acidwater(CHC13 sat.with 1 part H20 and 1 part 4% formic acid) (e)Benzene-ethylmethyl ketoneformic acid-water(9 parts benzene plus 1 part ketone sat.with 2% formic acid) (f)Benzene-formic acid-water (benzene sat. with 2% formic acid)
1 1
2 Ln
CI4 and spray reagents
1
--
FeC13 and
K 3 F e (CN)6
Me tab01ites
324
Metabolites Metabolites
325 326
327
la)ISO-propanol-water (17:3) (b)Butanol-acetic acid-water(4:1:5) (c)1.4M potassium phosphate buffer pH 7.0 Butanol-acetone-water(45:5:50)
Butanol-phosphoric acid-water(3:1:3) (a)Butanol S a t . with water in atmosphere of NH3 (b)95% ethanol-M ammonium acetate(7:3) adjusted to pH 5 6.32
N m
I
1
2,4,6 trinitrobenzene-sulfonic acid chlorani1ic acid
--
copper sulfate in ethanol then 0.1% benzidine in 50% aqueous ethanol
3 28
329 330 33 1
Thin-layer Chromatrogaphy In recent years several authors have developed thin-layer chromatographic system for isoniazid. These are presented in tabular form. System Chloroform-methanol(8:2) Chloroform-acetone-diethylamine (5:4:1) Cyclohexane-chloroform-diethylamine (4:5:1) Butanol-phosphoric acid-water(3:1:3) Acetone-methanol-NJQOH (50: 50: 1) (a)Pkthanol (b)Chloroform (c)Ethanol
\
Detection Folin-Ciocalteu or Phosphomo 1ybdate
-dimethylaminobenzaldehyde 5:l mixture 10% cuso4 and 10% NH~OH
Use separation from other drugs
Ref 332
derivatives 330 identification 333 identity test
334
ISopropanol-acetone(6:4 )
--
chloroform-methanol (6:4)
--
Chloroform-methanol(125:60) (a!Ethyl acetate-cyclohexanedioxane-methanol-waterNH40H(50:50:10:10:1.5:0.5)
UV iodine
I
separation of hydrazine separation of isonicotinic acid hydrazone with lactose
(b)same solvent but (50:50: 10: 1O:O. 5: 1.5) Ninhydrin or separation from (c)Ethyl acetate-cyclohexane0.5% H2S04 drugs of abuse NHqOH-methanol-water (70:15:2:8:0.5) (d)E thyl acetate-cyclohexaneNHqOH-methanol(56 :40:0.4:0.8) (e) same but (70:15 :5 :10) (f)E thyl acetate-cyclohexaneNH40H ( 5 0:40:0.1) Methanol-NH4OH-H20(100:1:4) KMnO4 bromothymol other drugs blue
\
335 335 59
336
337
rn 3
1
338 254 nm U.V. iron chloride(a)Chloroform-methanolhexacyanoferrate,molybdo13N ammonia (90:10: 1) phosphoric acid. Folin(b) Benzene-;oethano1Ciocalteu,potassium diethylamine (90:10:1) permanganate,ammoniacal (c)Chloroform-hexanol13N ammonia(90:10:0.2) silver nitrate,amminepenta(d)Chloroform-ethyl acetate cyanoferrate,iodoplatinate, iodine,Dragendorff, cinna13N ammonia ( 50 :50 :1) maldehyde triphenyltetra(e) chloroform-acetonezolium,dithiocarbamate acetic acid (90:10: 1) or ammonium molybdate (f)Benzene-acetonediethylamine (50:50 :1) (g) Chloroform-acetoneacetic acid(50:50:1) Nishimoto and T ~ y o s h i m a ~ ~found ' that isoniazid showed tailing on thin-layer chromatography due to trace metals in the silica gel. When the adsorbent was treated with EDTA the tailing was eliminated. Wijnne and c o - w o r k e r ~found ~ ~ ~ that isoniazid could be quantitated after thin-layer chromatography by coulometric titration. Schmidt341 showed that isoniazid could be revealed on a thin-layer plate by exposure to iodine vapor. Kawale and c o - w o r k e r ~sprayed ~~~ thin-layer plates with 1% mercurous nitrate to reveal isoniazid as black spots. 6.33 Ion exchanqe Chromatoqraphy Tsuji and Sekiguchij4j have shown that isoniazid is quantitatively adsorbed on Dowex 50 cation exchange resin in various metal forms. The strength of adsorption decreases in the following order:Cu++k Ni'+2 Hg++> H+ > Co++ > cd++k En++>Fe++ > Pb++> m++> ~ l + + + .
ISONIAZID
219
No a d s o r p t i o n o c c u r s on r e s i n i n t h e Ba++, Mg++, Ca++ o r Na+ forms. H e l l e r and c o - ~ o r k e r ss ~ e p~a ~ rated a c e t y l i s o n i a z i d from i s o n i a z i d on a column o f Dowex 1-X8 i n t h e p y r u v a t e form. 9 developed a Kakemi e t -a~~~~ c h r o m a t o g r a p h i c method f o r t h e s e p a r a t i o n o f i s o n i a z i d from some d e g r a d a t i o n p r o d u c t s . I s o n i a z i d i s a d s o r b e d on a weak c a t i o n e x c h a n g e r s u c h a s A m b e r l i t e CG-50 i n t h e hydrogen form. I s o n i c o t i n i c a c i d i s n o t a d s o r b e d and i s d e t e r m i n e d c o l o r i m e t r i c a l l y u s i n g cyanogen bromide. To determine i s o n i c o t i n a m i d e t h e sample s o l u t i o n i s o x i d i z e d w i t h a l k a l i n e f e r r i c y a n i d e and t h e n p a s s e s t h r o u g h a column o f s t r o n g a n i o n e x c h a n g e r s u c h a s Dowex 1-X8 i n t h e c h l o r i d e form. The amide i s unchanged Another degradaand i s n o t a d s o r b e d on t h e r e s i n . t i o n product,1,2-diisonicotinoyl h y d r a z i d e i s d e t e r m i n e d b y a d j u s t i n g t h e s a m p l e t o pH 8 . 9 w i t h b o r a t e b u f f e r and d e t e r m i n i n g t h e a b s o r b a n c e a t 329 nm. P e t e r s a n d c o - ~ o r k e r s 347 ~ ~ ~w ,e r e a b l e t o s e p a r a t e and q u a n t i t a t e a l a r g e number o f m e t a b o l i t e s of i s o n i a z i d u s i n g Dowex AG-50-X4 r e s i n i n t h e hydrogen and ammonium forms. S e l e c t i v e c o l o r r e a c t i o n s were u s e d t o d i f f e r e n t i a t e t h e g r o u p s of m e t a b o l i t e s . Fan and Wald348 s e p a r a t e d p - a m i n o s a l i c y l i c a c i d from i s o n i a z i d u s i n g a Dowex 2 - X 8 column. I n o u e and c o - w ~ r k e r su~s e~d ~ a s y s t e m s i m i l a r t o t h a t o f Kakemi e t a 1 3 4 3 t o s e p a r a t e i s o n i a z i d from i t s d e g r a d a t i o n p r o d u c t s . Lewandowski and S y b i r ~ k a ~ ~ O s e p a r a t e d i s o n i a z i d from i s o n i c o t i n i c a c i d b y p a p e r chromatography u s i n g b u t a n o l s a t u r a t e d w i t h water. The p a p e r was c o n n e c t e d w i t h an i o n exchange p a p e r i n t h e a c i d form. The s p o t s were e l u t e d w i t h d i o x a n e . The s h a r p z o n e s on the i o n exchange p a p e r were v i s u a l i z e d w i t h i c r y l c h l o r i d e Darawy a n d Mobarak3" chromatog r a p h e d s e v e r a l d r u g s on CM-82 c a r b o x y m e t h y l c e l l u l o s e c a t i o n exchange p a p e r u s i n g a w a t e r -
-
220
GLENN A. BREWER
acetone-foramide(l0:l:l) solvent system. 6.34 Other Chromatoqraphic Methods Barreto and Sabin0352 used a anhydrous sodium sulfate column eluted with chloroform-diethylamine(9:1) to concentrate metabolites of isoniazid from serum or urine. Smolarek and Dlugosch353 separated isoniazid and p-aminosalicylic acid by paper electrophoresis in barbital buffer, pH 8.5. B a r r e t ~used ~ ~ ~ two dimensional electrophoresis to separate the metabolites of isoniazid. also used paper electrophoresis to separate several acyl hydrazides. A pH 2.0 acetate buffer was used. Isoniazid was separated from several antituberculosis drugs by gas chromatography356,357, A silanized chromosorb G coated with 6% QF1 was used. Gas chromatography was used to separate the products of oxidation of hydrazides with Fehling’s solution358. 6.4 Determination of Isoniazid and its Metabolites in Body Fluids and Tissues The methods described in this section were specifically developed for the determination of isoniazid in body fluids and tissues. Many of the methods are similar to other general analytical methods described in Section 6.2 perhaps differing only in the extraction procedure. 6.41 General Reviews Terze and D a d i ~ t o u ~studied ~’ a number of color reactions to determine their application to blood level assays. Ginoulhiac360 also made a literature review of blood level methods, A critical review of methods for isoniazid determination has been written364 6.42 Colorimetric Methods Colorimetric methods are most popular for the determination of isoniazid in biological samples. The methods are listed in tabular form.
.
Reaqent Dimethylaminobenzaldehyde
Pretreatment of sample acid hydrolysis
Dimethylaminobenzaldehyde Dimethylaminobenzaldehyde
none extraction into isoamyl alcoholether from alkaline solution deproteinization with ~ ~ 1 0 ~ deproteinization with trichloroacetic acid none
Dimethylaminobenzaldehyde Dimethylaminobenzaldehyde N
5
Vanillin Vanillin Vani 11in Van i11in Cinnama ldehyde Cinnamaldehyde
Type of specimen Ref. serum & urine 361,370, 375 urine 362,363 plasma and 365,366, urine 367,3 68, 369. serum
37 1
serum and tissues
372
serum
373,376, 377 374,432, 433,434 375
deproteinization serum with trichloroacetic acid extraction with serum organic solvent extraction with milk propanol deproteinization serum with trichloroacetic acid extraction with serum butanol-chloroform
3 78 379,380, 429,430 38 1
o-Nit robenza ldehyde Sa 1icyla 1dehyde- FeC1 S a licy la ldehyde
Salicylaldehyde Glutaconic aldehyde @-diketone N
E
Catecho1 Catecho1 H2 0 2 CNBr
-
CNBr A Ikaline
hydrolysisCNBr NHqV03-H2S04 KCN, Chloramine Tbarbituric acid
deproteinization with trichloroacetic acid extraction into isoamyl alcohol-ether from alkaline solution none extraction with acetone deproteinization with trichloroacetic acid none deproteinization with trichloroacetic acid automated method deproteinized serum deproteinized trichloroacetic acid deproteinized trichloroacetic acid acid hydrolysis
serum
382
serum
383
bio logica1 fluids cadavers
3 84
plasma
386
biological ma teria Is citrated blood serum serum & urine biological fluids urine
387
urine
385
388 389 390 391,404, 411,412 392
393,394,395, 396,397,398, 399,370,435 plasma,urine 400,401 tissues,serum
1-amino-2-naphthol-4sulfonic acid Naphthoquinone-4sulfonic acid Naphthoquinone-4sulfonic acid 2,4,6-trinitrobenzenesulfonic acid Dinitrochlorobenzene Dinitrochlorobenzene
rJ .
K3Fe (CN)6 Sodium pentacyanoaminoferroate K3Fe (CN)6 Ni tropentacyanoferroate Na ph thoquinone Na ph thoq inone H202,CNBr, aniline
Picryl chloride KMn04,BrCN,NH3
urine biol. fluids urine deproteinization Zn (OH) 2 extraction methyl isobutyl ketone deproteinized serum deproteinized tissue deproteinized with sodium tungstate deproteinized with phosphoric acid
--
trich loracetic acid tungstic acid extraction BuOH,Et20 Py tein-free fiPtrate
402
urine
403,404,405, 406,407 408
whole blood
409,410
urine serum
411 412,413
serum tissue, urine spina1 fluid serum
414 415,416,417
spinal fluid urine blood blood urine plasma urine spinal fluid plasma
418
419 420 421 422 423,424 425
3 P
4-pyridylpyridinium trichloracetic acid plasma 426 dichloride,NaOH,HCl filtrate KBrO3+ methyl o r a n g e acid tungstate blood 427 Zn powder + h e a t -urine 428 6 . 4 3 T u r b i d i m e t r i c Method I s o n i a z i d r e d u c e s K2Hg14 t o form HgI w h i c h i s i n s o l u b l e . The r e s u l t i n g t u r b i d i m e t r y c a n be measuEed t o d e t e r m i n e t h e amount o f i s o n i a z i d Wagner a n d co-worker-431 h a v e a p p l i e d t h i s method t o b l o o d f o l l o w i n g present. d e p r o t e i n i z a t i o n w i t h b a r i u m h y d r o x i d e and z i n c s u l f a t e . 6 . 4 4 F l u o r i m e t r i c Methods A number of f l u o r i q e t r i c m e t h o d s € o r i s o n i a z i d h a v e b e e n reported. H e d r i c k a n d c o - ~ o r k e r sa~b s~o ~ r b e d a p r o t e i n f r e e f i l t r a t e of s e r u m on p H 6 . 5 A m b e r l i t e XE-64 i o n e x c h a n g e r e s i n . T h e i s o n i a z i d was e l u t e d w i t h d i l u t e a c i d and t h e n r e a c t e d w i t h hydrogen p e r o x i d e i n pH 8.7 b u f f e r . The o x i d a t i o n p r o d u c t f l u o r e s c e s a t 415 nm when a c t i v a t e d by u l t r a v i o l e t l i g h t a t A s l i t t l e a s 0.05 y/m1 o f s e r u m c a n be d e t e r m i n e d . 320 nm. S c o t t and Wright437 r e a c t e d s a l i c y l a l d e h y d e w i t h i s o n i a z i d and reduced t h e r e s u l t i n g hydrazone. The r e s u l t i n g compound w a s h i g h l y f l u o r e s c e n t . R e i s s , Morse a n d P u t s c h 4 3 8 a s s a y e d i s o n i a z i d f l u o r i m e t r i c a l l y a f t e r a b s o r p t i o n a n d e l u t i o n from i o n e x c h a n g e r e s i n and t r e a t m e n t w i t h a l k a l i n e c y a n o g e n bromide. Wilson, Lever and u t i l i z e d t h e f l u o r e s c e n c e of t h e z i n c c h e l a t e of t h e hydrazone of i s o n i a z i d w i t h pentane-2,4-dione i n an a s s a y i n serum. E l l a r d , Gammon a n d W a l l a c e 4 4 0 h a v e d e v e l o p e d s p e c i f i c f l u o r i m e t r i c a s s a y s f o r i s o n i a z i d , a c e t y l i s o n i a z i d , mono-and d i a c e t y l h y d r a z i n e , i s o n i c o t i n i c a c i d a n d i s o n i c o t i n y l g l y c i n e i n serum a n d u r i n e . Boxenbaum a n d Riegelman441 h a v e a l s o d e v e l o p e d a s s a y s f o r i s o n i a z i d and i t s m e t a b o l i t e s i n whole blood. M i c e l i , O l s o n a n d Weber442 h a v e e s t a b l i s h e d a micro method f o r
ISONlAZlD
225
t h e f l u o r i m e t r i c d e t e r m i n a t i o n of i s o n i a z i d i n serum. A s l i t t l e a s 2 5 p1 o f s e r u m can be u s e d i n t h e assay. P e t e r s , Morse a n d Schmidt 443 and 0' B a r r , K e i t h and B l a i r 4 4 4 h a v e compared f 1 U O r i m e t r i c and m i c r o b i o l o g i c a l a s s a y s f o r i s o n i a z i d i n serum. 6.45 E l e c t r o c h e m i c a l Methods Lauermann a n d O t t o 4 4 5 h y d r o l y z e d i s o n i c o t i n i c a c i d h y d r a z i d e and i t s m e t a b o l i t e s t o i s o n i c o t i n i c a c i d w i t h a l k a l i . The h y d r o l y s i s product w a s determined p o l a r o g r a p h i c a l l y . The a u t h o r s found t h a t t h e r e s u l t s o b t a i n e d b y t h i s method i n t h e a n a l y s i s o f c a d a v e r i c f r a c t i o n s was comparable t o t h o s e o b t a i n e d when t h e method o f N i e l s c h a n d G i e f e r 4 0 1 was u s e d . The p o l a r o g r a p h i c method was l e s s t i m e consuming. Kane 446 d e t e r m i n e d i s o n i a z i d i n biological f l u i d s without p r i o r separation. 6.46 G a s o m e t r i c Methods The h y d r a z i n e g r o u p i n i s o n i a z i d c a n b e r e a d i l y decomposed i n t o n i t r o g e n g a s . Several a u t h o r s have u t i l i z e d t h i s r e l a t i v e l y s e l e c t i v e f i n i s h f o r b l o o d and u r i n e l e v e l a s s a s . S t r i c k l a n d a n d H e n t e l 1;47 r e a c t e d i s o n i a z i d w i t h sodium i o d a t e i n a l k a l i n e s o l u t i o n . The a s s a y i s n o t e f f e c t e d b y t h e p r e s e n c e o f pa m i n o s a l i c y l i c a c i d which i s o f t e n g i v e n i n conjunction with isoniazid. H a r t i n g and G e r z a n i t s 448used a l k a l i n e f e r r i c y a n i d e t o l i b e r a t e t h e nitrogen gas. I t o and c o - w ~ r k e r 7s450 ~ ~ were ~ able t o s e l e c t i v e l y u s e c o p p e r , i r o n a n d chromium azometry t o determine i s o n i a z i d and i t s v a r i o u s metabolites i n urine. 6.47 M i s c e l l a n e o u s Chemical A s s a y s V e r r o t t i and B a r d e l l i 4 5 1 d e t e r m i n e d i s o n i z i d i n cerebrospinal f l u i d by iodometric ti t r a t i o n . Schwenk a n d c o - ~ o r k e r s ~employed ~* a radioimmunoassay f o r t h e d e t e r m i n a t i o n o f isoniazid i n biological fluids.
Microbioloqical Assays Although isoniazid is readily measured in biological fluids and tissues by chemical assays, as with many antibacterial substances a number of microbiological assays for this substance have been proposed. Microorganism Type of assay S ens itiwi ty Ref. 2.5-30 y/ml 453 Mycobacterium phlei agar diffusion Koch bacilli turbidimetric -454 tubercle bacteria cord formation -455 bacilli vertical diffusion -456 Mycobacterium vertical diffusion -457 tuberculosis HV37 Mycobacterium agar diffusion -458 tubercu10s is 0.49 y/m1 459 vertical diffusion Mycobacterium tuberculosis H37Rv and H 3 7 R a assay of isoniazid 378 BGG in milk-agar diffusion 6.48
N
N m
>
Mycobacterium tuberculosis
--
vertical diffusion vertical diffusion
460 46 1
vert ica 1 di f€usion tube dilution vertical diffusion for urine
462 463,464
ISON IAZlD
227
Bartmann and F r e i s e 4 6 5 s t u d i e d t h e t i s s u e b i n d i n g of i s o n i a z i d w i t h t h e m i c r o b i o l o g i c a l assay. They found 40% b i n d i n g w i t h human t i s s u e w h i l e mice and g u i n e a p i g t i s s u e g a v e 80% binding. Incubating the t i s s u e a t an e l e v a t e d temperature d i d not r a i s e t h e recovery. N i ~ h i P~o o~l e~ and , Meyer467, T a n s i n i and c o - w o r k e r ~ ~P~e t~e ,r s and c o - w o r k e r s 433 and O ’ B a r r & a4!*a l l compared v a r i o u s c h e m i c a l a s s a y s and m i c r o b i o l o g i c a l a s s a y s . A l l w o r k e r s c o n c l u d e t h a t t h e two methods g a v e c o m p a r a b l e results 6 . 4 9 Chromatographic A s s a y s The m e t a b o l i s m o f i s o n i a z i d i s complex and many w o r k e r s h a v e s e l e c t e d chromatog r a p h i c a s s a y s t o measure t h e d r u g i n t i s s u e and biological fluids. T h e s e methods p r o v i d e t h e s p e c i f i c i t y t h a t a r e n o t g i v e n b y many c h e m i c a l methods. A l a r g e number of c h r o m a t o g r a p h i c s y s t e m s a r e g i v e n i n s e c t i o n 6.3. Many of t h e s e methods c o u l d p r o b a b l y be u s e d t o measure i s o n i a z i d i n t i s s u e s and b i o l o g i c a l f l u i d s . The methods g i v e n i n t h i s s e c t i o n h a v e been d e v e l o p e d j u s t f o r t h i s purpose. Makino and c o - ~ o r k e r sf o ~ l~ l o~w e d t h e m e t a b o l i s m of i s o n i a z i d i n l i v e r and i n u r i n e by p a p e r c h r o m a t o g r a p h y ( w a t e r s a t u r a t e d b u t a n o l , 1% ammonia-isopropanol(3:20), b u t a n o l s a t u r a t e d w i t h 0.02M p h o s p h a t e b u f f e r p H 7 . 4 , 1%ammonia s a t u r a t e d b u t a n o l and b u t a n o l - a c e t i c a c i d - w a t e r
.
(4:1:5)).
L e ~ s c h n e r ~u ~ s egd s e c - b u t a n o l s a t u r a t e d w i t h w a t e r and i s o a m y l a l c o h o l a s developing solvents. I ~ a i n s k ys e~p a~r a~t e d t h e h y d r a z o n e s o f i s o n i a z i d and p y r u v i c and a - k e t o g l u t a r i c a c i d from i s o n i a z i d w i t h p a p e r chromatography. S e z a k i 470 s e p a r a t e d i s o n i a z i d from p y r a z i n a m i d e i n u r i n e b y means of A m b e r l i t e IRA-400. B e l l e s and L i t t l e m a n 4 7 1 u s e d Dowex 50-X8 t o s e p a r a t e i s o n i a z i d from a c e t y l i s o n i a z i d . Abiko
228
GLENN A. BREWER
and c o - ~ o r k e r suse ~~~ Dowex 1-XlO to separate these as well as the hydrazone of glucuronic acid. Peters, Miller and Brown346 utilized ion exclusion chromatography to separate metabolites of isoniazid into ionized, slightly ionized and unionized groups of compounds. The individual metabolites were measured with specific colorimetric assays. Okudaira and c o - ~ o r k e r s ~ used ’~ Dowex 1 and Dowex 50 columns in tandem to separate the various metabolites of isoniazid. Paper chromatographic systems have been used to isolate the various metabolites of isoniazid474,475,352. Barreto and S a b i n have ~ ~ ~described ~ a two dimension separation of isoniazid metabolites using paper chromatography and paper electrophoresis The same authors352 have also used a sodium sulfate column developed with chloroform-diethylamine(9O:lO) to separate the metabolites of isoniazid. Fartushnyi and S ~ k h i n *have ~ ~ used TLC to determine isoniazid and other drugs in cadavers. Cattaneo, Fantoli and Ferrari478 claim that their chromatographic studies indicate that the tumorogenic effect of isoniazid in mouse lung is due to the large amount of isonicotinic acid produced in that organ. Hughes479 separated acetylisoniazid from isoniazid by counter-current distribution (butanol-ethylene dichloride- 9:1 - 2M phosphate buffer pH 5.1). Ozawa and Kiyomoto480 isolated three conjugated metabolites of isoniazid by paper used paper chromatography. Cuthbertson et chromatography to determine isonicotinoylglycine. They used the following systems: Water saturated butanol Methylethy1ketone:acetic acid:water(49:1:50) Propano1:water (4:1) Zamboni and D e f r a n ~ e s c h iused ~~~ a isopropanol:water(85:15) system to separate the hydrazones of pyruvic and a -ketoglutaric acid from
i son i a z id. 7. Druq Metabolism The drug metabolism of isoniazid is unusually complicated in that it is a very reactive molecule and can undergo non-enzymatic reactions in the body. A general metabolic pattern is indicated in diagram. Non-enzymatic
Enzymatic 0
II
isonicotinoylhydrazones of glucose, a-ketoglutaric acid, aldehyde pyruvic acid etc. N ID
acetylisoniazid
or ketone
0
isonicotinamide N , N * diisonicotinoylhydrazide
230
GLENN A. BREWER
The major metabolite of isoniazid is N-acetylisoniazid. The rate of acetylation is has genetically ~ o n t r o l l e d 485. ~ ~ ~ ,It ~ ~ ~ been established that the slow acetylation is a autosoma1 recessive trait. The acylation occurs by N-acetyl transferase. Six hours after the oral administration of 4 mg/Kg of isoniazid fast acetylators have plasma concentrations of 0.2 pg/ml or less while slow acet lators have plasma levels higher that 0.4 pg/ml 48Yj In a metabolic scheme,such as the one indicated earlier,relative amounts of the various metabolites found in the urine will differ for each individual and will depend on genetic factors, previous drug history (enzyme induction) and general nutrition (availability of ketoacids). Reviews on the drug metabolism of isoniazid have been re ared b a number of authors487,488, 489,490,49!?, 462,493,i94,495,496,497
.
Toth and Shimizu have reported that the continuous administration of N-acetylisoniazid in rats has markedly increased the incidence of lung tumors in this species. Since the N-acetyl derivative is a major metabolite in man this poses some questions on the long term administration of the compound497. 8. Biopharmaceutics Kakemi and co-workers498 determined the rate of absorption of derivatives of isoniazid in the stomach and intestine. The authors report a rough correlation between degree of absorption and lipidwater partition coefficient.
ISONIAZID
231
9.
References
1.
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-
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81.
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236
91.
92. 93.
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a.
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239
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GLENN A. BREWER
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16,696-9
-
-
-
.
-
IS0 NI A 2 I D
24 1
Modrzejewski, B. and Zommer, S. :Chem.Anal. 7,659-66 (1962): C. A. 57 13889e (1957) 182. Miszczuk, B. and Taborska, H.:Przemyst Chem. 11,706-10 (1955):C.A. 52 208953.(1958). Zn$ik,F. : Einkova, 0 7 a n d m r b l , J. : 183. Collection Czechoslov. Chem.Commns. 24, 2695-8 (1959);C.A. 54 8429g (1960). 184. Laszlovszky, J. :Acta Pharm.Hung. 30,101-9 54 1 9 2 6 8 (1960). ~ (1960): C.A. 185. Vulterin, J. : Collection Czech. Chem.Commun.28, 1393-1400 (1963):C.A. 59 10767h (1963). 186. Vulterin, J.:Cesk.Farm. l2,391-3(1963): C.A. 62 15994c (1965). 187. Wihchurch,R. : J.Assoc. Off.Anal. Chem. 56, 1464-6(1973):C.A. 80 87544d(1974). 188. Canb'ick, T. : J. Pharm. Pharmacol.4 407(1952): C.A. 46 83253.(1952). 189. Domleo, A.P. : J.Pharm.and Pharmacol. 2, 117-18 (1953);C.A. 47 4251d(1953). 190. Struszygski, M. and Bellen, Z. Przemyst. Chem. 32,40-l(1953) ;C.A.G 85869. (1953). 191. Montequi, F. : Farm.nueva 18,lO-14(1953) : C.A. 47 6603d(1953). 192. Noronha da Costa, A. : Rev.brasi.1. farm.33, 343-50(1952); C.A. 47 7736d(1953). 193. Kattionis,A.: Chim.Chronika 2,78-80(1952): C.A. 47 11665i (1953). 194. Berka, A. and Zijka, J. ;Chem.Listy 50,314-16 (1956);C.A. 50 7651a (1956). 195. Spacu, P. and Teodorescu, G. :Bul. inst. politeh Bucuresti l8,47-50(1956):C.A.51176071.1 (1957). 196. Tsupikov, M. T. : Aptechn. Delo. u,55-7 (1964): C.A. 61 - 9361c (1964). 197. Laipanov, A. Kh. and Lobanov,V. I. : Farm. Zh, 2344-8 (1973);C.A. 2.B 140435f (1973). 198. Nambisan, P.N.K. and Nair, C.G.R. Indian J. Chem. 10,665-6 (1972):C.A. 7 8 37750f (1973). 199. Laipanov,A. Kh. and Lobanov,V. I. :Farmatsiya 22. 48-9(1972) ;C.A.m 23619d(1973). 200. Greenhow,E.J. :Chem.Ind. 14,697-8 (1973):C.A.79 121642a (1973). 181.
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221. 222. 223. 224. 225. 226. 227. 228.
229. 230. 231. 232. 233.
243
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244
241. 242. 243. 244. 245. 246. 247. 248. 249. 250. 251. 252. 253. 254. 255. 256. 257. 258. 259.
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Franchi,G. ;Farm.sci. e tec.Z,640-4 (1952): C.A.47 4044b (1953). Akiyama,T. :Fujiwara,M. and Ichida H. : Bull. Kyoto Coll. Pharm. fl, 18-20 (1956): C.A. 51 4650a (1957). Sbnchez, J. A. ; Rev.Asoc.bioquim.Argentina 17,324-6(1952) ;C.A.g 6822b(1953). Mitchel1,B.W. ; Haugas,E.A. and McRoe, C.S. ; J.Pharm.and Pharmaco1.?,42-5 (1957):C.A. 51 608713(1957). Kum-Tatt,L. and Yan-Hon,H. :J.Pharm.Pharmacol. 14,123-4(1962) ;C.A.X 2337b(1962). J Y Spacu,P. ;Teodorescu,Gr. and Gavanescu,D. : Bul. inst. politch. Bucuresti 18,51-4 (1956): C.A.51 176073.(1957). Ibadov,A. Yu. and Drebentsova,N. F. ;Dokl. 21 39-41(1964) :C.A. 61 Akad.Nauk Uz.SSR 10538a (1964). Nair,V. R. and Nair, C.G. R. ;Analytica chim. Acta 57,429-34(1971);A.A.23 799(1972). Pinzauti,S. ;Dal Piaz,v.and LaPorta, E. : J. Pharm. Sci. 63,1446-8(1974):C. A. 82 7701f (1975) Gowda,H. S. and Rao,G. G. ;Z.Ana 1. Chem. -19 6 5 36-8 (1959):C. A. 53 11765h (1959) Budggi'nsk$,B. :Collection Czechoslov. Chem. Communs.26,781-7 (1961):C.A.z 14174b(1961). Ra0,P.V.K. and Rao, G.B.B. ;Analyst 96, 712-15 (1971);C.A.E 6762r (1972). Rao,P. V. K. and Rao,G. B. B. ;Freseniug Z.Ana1. Chem. =,360-1(1971) :C.A.E 37475d(1974). Eremina,Z. I. and Antonchik, I.A. :Farm.Zh. (Kiev) 27,79-81(1972) :C.A.z 92916~(1972). Rao,P,V. K. :Rao,G.B.B. and Ra0,R.S. :Anal. Chim. Acta 65,227-30 (1973):C.A.E 57733b(1973). Tatsuzawa,M. :Bunseki Kagaku 10,129-33 (1961): C.A.55 23931e(1961). Panwar,K. S. :Rao,S. P. and Gaur,J.N. ;Anal.Chim. Acta 25,218-21 (1961):C.A. 55 26865d (1961). Ra0,G.G. and Rao,P.V.K. ;Tzanta ll,1489-96 (1964):C.A.62 1239h(1965) Gein,L.G. and Sumbaikina,Z.A. ;Farmatsiya l6, 41-3 (1967):C.A. 67 84919a (1967).
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-
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Mitchell,J. R. and Jollow,D.J. ;Druq Interact. 65-79(1974) :C.A.U 92730g(1975). 495. Iwamoto,T. :J. Pharm. SOC.Japan 74 36-9 (1954)i C.A. & 5362b(1954). 496. Nakazono,I. and Hirata,H. ;Kekkaku 3 4 101-6(1959) ;C.A.S 25288e(1960). 497. Toth,B. and Shimizu,H. ;Eur.J. Cancer 9, 285-9(1973) :C.A.B 101439m(1973). 498. Kakemi,K. ;Arita,T. ;Sezaki,H. and Takasugi, N. :Chem. Pharm.Bul1. JJ 551-7 (1965);C.A.Q 7511c(1965). 499. Juchau,M.R. and Horita,A.;Drug Metabolism Reviews, DiCarlo F.J. Editor, Vol. 1 pages 71-86, Marcel Dekker Inc. New York, 1973. Meyer,H. and Mally,J. ;Monatsh.Chem 2 3 . 9 500. 393(1912) ;C.A.6_ 2073(1912). 501. Chorine,V. : Compt. rend,acad. sci. 220,150 (1945). 502. Huant,E.:Gazette des Hopitaux Aug.15(1945). 503. Long,E.S.:The Chemistry and Chemotheraw of Tuberculosis.3rd ed.,The Williams & Wilkins C Co. ,Baltimore,1958. 504. Kirschbaum,A. ;Pharm.Acta Helv. 27 229-33 (1952):C.A. 47 2429b (1953). 494.
Glenn A. Brewer
184
GLENN A. BREWER
CONTENTS
1. 2.
3. 4. 5. 6.
Description 1.1 N a m e , F o r m u l a , Molecular W e i g h t 1 . 2 A p p e a r a n c e , C o l o r , Odor, T a s t e P h y s i c a l and Chemical P r o p e r t i e s 2 . 1 Spectra 2.11 I n f r a r e d Spectrum 2.12 U l t r a v i o l e t Spectrum 2.13 Chemiluminescence 2.14 Fluorescence Spectrum 2 . 1 5 N. M. R. Spectrum 2 . 1 6 E. S. R. S p e c t r u m 2 . 1 7 Mass S p e c t r o m e t r y 2.2 P h y s i c a l P r o p e r t i e s of t h e S o l i d 2.21 Melting C h a r a c t e r i s t i c s 2 . 2 2 D.T.A. a n d D.S.C. 2.23 T.G.A. E l e c t r i c a l Moment 2.24 2.25 E l e c t r i c a l Conductivity 2.26 Crystal characteristics 2.27 X-Ray D i f f r a c t i o n 2.3 S o l u b i l i t y 2 . 3 1 Water S o l u b i l i t y 2.32 Solubility i n Solvents 2.4 Physical Properties of Solution 2 . 4 1 PH 2.42 D i s s o c i a t i o n Constant 2.43 Photolysis Constant 2.44 Oxidation P o t e n t i a l Metal Complexes History, S y n t h e s i s and Manufacturing Stability A n a l y t i c a l Chemistry 6.1 Identity T e s t s 6.2 Methods o f A n a l y s i s 6 . 2 1 General Reviews 6 . 2 2 Colorimetric Methods 6 . 2 3 S p e c t r o p h o t o m e t r i c Methods 6 . 2 4 F l u o r i m e t r i c Methods 6 . 2 5 T i t r i m e t r i c Methods 6 . 2 6 E l e c t r o c h e m i c a l Methods
ISON IAZlD
7. 8. 9.
185
6 . 2 7 G r a v i m e t r i c Methods 6 . 2 8 M i c r o b i o l o g i c a l a n d E n z y m a t i c Methods 6 . 2 9 M i s c e l l a n e o u s Methods 6 . 3 C h r o m a t o g r a p h i c Methods 6 . 3 1 P a p e r Chromatography 6 . 3 2 Thin-Layer Chromatography 6 . 3 3 I o n Exchange c h r o m a t o g r a p h y 6 . 3 4 O t h e r C h r o m a t o g r a p h i c Methods 6 . 4 D e t e r m i n a t i o n of I s o n i a z i d and i t s Metabolites i n Body F l u i d s and T i s s u e s 6 . 4 1 G e n e r a l Reviews 6.42 C o l o r i m e t r i c Methods 6 . 4 3 T u r b i d i m e t r i c Method 6 . 4 4 F l u o r i m e t r i c Methods 6 . 4 5 E l e c t r o c h e m i c a l Methods 6 . 4 6 G a s o m e t r i c Methods 6 . 4 7 M i s c e l l a n e o u s Chemical A s s a y s 6.48 M i c r o b i o l o g i c a l Assays 6.49 Chromatographic Assays Drug Metabolism Biopharmaceutics References
186
GLENN A. BREWER
1. Description 1.1 Name, Formula, Molecular Weiqht Generic names - Isoniazidl, Isonicotinic Acid Hydrazide, INH, Isonicotinoylhydrazine, Isonicotinyl hydrazide, Isonicotinylhydrazine, Tubazid, ISoniazidum Chemical names - 4-Pyridinecarboxylic acid hydrazide, pyridine-4-carboxyhydrazide, pyridine- y-carboxylic acid hydrazide. Chemical Abstracts Registry No. 54-85-3 2
.
2HNm0c-)+( C6H7N30
Mol. Wt. 137.14
1.2
Appearance, Color, Odor, Taste Colorless or white crystalline powder which is odorless and has at first a slightly sweet and then bitter taste3. Physical and Chemical Properties 2.1 Spectra 2.11 Infrared Spectrum The infrared spectrum of isoniazid and other hydrazides of carboxylic acid have been recorded and band assignments were made4 , Nagano et a15 in a later paper made band assignments for isoniazid, metal complexes of isoniazid and related compounds The infrared spectra of isoniazid as a solid in a KBr pellet and as a mull in mineral oil are shown in Figures 1 and 2 . The following assignments have been made by Mrs. Toeplitz6. Frequency(cm-l) A s s iqnment 3300-3000 Bonded NH and C-H 1670 c=o 1560 Amide I1 1640 NH2 deformation 1610t ring C=C and C=N 1500/ 2.
.
WAVELENGTH (MICRONS)
FRMUENCY (W')
F i g u r e 1:Infrared
spectrum of isoniazid as a KBr p e l l e t .
WAVELENGTH (MICRONS)
A
8
FREQUENCY (W')
Figure 2:Infrared
s p e c t r u m of i s o n i a z i d i n m i n e r a l o i l m u l l .
ISONIAZID
2.12
189
U l t r a v i o l e t Spectrum Numerous a u t h o r s h a v e r e c o r d e d t h e u l t r a v i o l e t s p e c t r u m o f i s o n i a z i d i n a number o f s o l v e n t s 7 , 8 ~ 9 ~ 1 0 , 1 1 ~ 1 2 . The e f f e c t of t h e p H o f t h e s o l u t i o n on t h e r e s u l t i n g s p e c t r u m h a s been noted. Zommer13 h a s r e c o r d e d t h e s p e c t r a of t h e h y d r a z o n e s of i s o n i a z i d and a c e t o n e o r p-hydroxybenza ldehyde. The u l t r a v i o l e t s p e c t r u m o f i s o n i a z i d i n d i l u t e a c i d (0.01N aqueous HC1) shows t w o a p p r o x i m a t e l y e q u a l maximima a t 213 nm ( E Y i m 437) a n d 265 nm ( E l % 4 1 7 ) . The minimum 1c m o c c u r s a t 233 nm. The s p e c t r u m i n d i s t i l l e d w a t e r shows a b r o a d peak a t 2 6 1 nm (EFgm 306) w i t h o u t a d e f i n e d minimum. T h e r e i s a s h o u l d e r a t 208 nm. I n d i l u t e a l k a l i (0.01N a q u e o u s a l k a l i ) t h e s p e c t r u m t a k e n i m m e d i a t e l y shows a s h o u l d e r a t 266 nm ( E F i m 293 a n d p e a k s a t 272 nm (EFgm 298) a n d 2 9 5 nm (Elcm % ! 2 8 4 ) . On s t a n d i n g t h e s e p e a k s s h i f t so t h a t a t 2 h o u r s t h e r e a r e p e a k s a t 256 nm ( E l % 1 7 3 ) , 262 nm ( E E m 1 7 0 ) a n d 325 nm ( E r g m 7 6 ) . 1c m A t 24 h o u r s t h e 325 nm peak d i s a p p e a r s . The same s h i f t t a k e s p l a c e w i t h h i g h e r c o n c e n t r a t i o n s of a l k a l i e x c e p t t h a t i t occurs more r a p i d l y . The u l t r a v i o l e t s p e c t r u m t a k e n i n m e t h a n o l i c r a t h e r than aqueous s o l v e n t s a r e s i m i l a r t o those i n water except t h a t the absorption maximima g e n e r a l l y o c c u r a t s l i g h t l y lower wavelengths. 2.13 Chemiluminescence C a e n l 5 h a s o b s e r v e d a weak c h e m i l u m i n e s c e n c e o f i s o n i a z i d when s o l u t i o n s a r e o x i d i z e d w i t h sodium h y p o c h l o r i t e . The lumj n e s cence i n c r e a s e s w i t h pH from 1 0 . 2 t o 13. The maximum o f t h e e m i s s i o n c u r v e i s a t 0.552 p c o r r e s p o n d i n g t o a n e n e r g y o f 5 1 K c a l , Two t h e o r i e s f o r t h e o b s e r v e d l u m i n e s c e n c e a r e o f f e r e d , b o t h of which depend on t h e p r e s e n c e o f f r e e OH a n d H 0 2 r a d i ca 1s
.
190
GLENN A. BREWER
2.14
Fluorescence Spectrum I s o n i a z i d shows a n i n t e n s e f l u o r e s c e n c e s p e c t r u m when o x i d i z e d w i t h p e r o x i d e o r a f t e r cleavage of the p y r i d i n e r i n g with This fluorescence is t h e basis cyanogen bromide. o f s e v e r a l s e n s i t i v e methods t o d e t e r m i n e i s o n i a z i d i n b i o l o g i c a l m a t e r i a l s ( S e e S e c t i o n 6 . 4 ) . When a s o l u t i o n o f i s o n i a z i d a t pH 6 . 5 t o 7 . 5 w a s t r e a t e d w i t h d i l u t e p e r o x i d e a t 100°C f o r 30 m i n u t e s w e found t h e e x c i t a t i o n maximum a t 333 nm and t h e e m i s s i o n p e a k a t 415 nm 14 A f t e r i s o n i a z i d i s rea c t e d w i t h cyanogen b r o m i d e r e a g e n t i n 1 . 8 N a l k a l i n e s o l u t i o n a t room temperature w e f o u n d a n a c t i v a t i o n maximum a t 312 nm a n d a f l u o r e s c e n c e maximum a t 392 nmI4. I s o n i a z i d a l s o f l u o r e s c e s when r e a c t e d w i t h c e r t a i n a r o m a t i c c a r b o n y l compounds (Section 6.24). 2.15 N. M. R. S p e c t r u m Several authors have studied t h e n u c l e a r magnetic resonance spectrum o f t h e h draz i d e s o f c a r b o x y l i c a c i d i n c l u d i n g i s o n i a z i d y 6 9 l7 9 18. H i l l e r b r a n d and c o - w ~ r k e r s ~ gt us d i e d t h e N. M. R. spectrum o f t h e copper s a l t . The N.M.R. spectra o f i s o n i a z i d a n d *zO exchanged i s o n i a z i d a r e shown i n F i g u r e s 3 a n d 420. The 60 MHz NMR s p e c t r u m of i s o n i a z i d , i n dimethyl sulfoxide-d6 containing tetramethyl s i l a n e a s i n t e r n a l r e f e r e n c e shows t h e p r e s e n c e o f hydrazino protons resonances a t (ppm) 4 . 6 0 ( b r o a d , 2 H , e x c h a n g e d ) and 10.15 ( b r o a d , l H , exc h a n g e d ) . The a r o m a t i c p r o t o n s r e s o n a n c e s appear a s m u l t i p l e t s a t 7 . 7 3 ( 2 H ) and 8 . 7 0 ( 2 H ) . ( F i g u r e s 3 a n d 4 ) . The complex p a t t e r n of t h e resonances, o t h e r than t h e expected doublets, suggests charge d i s t r i b u t i o n i n t h e p y r i d i n e ring. However, t h e h i n d e r e d r o t a t i o n a r o u n d t h e N-C=O a s w e l l as C - a r y l b o n d s c a n n o t be r u l e d o u t . The NMR s p e c t r u m i n m e t h a n o l -d4 w a s s i m i l a r t o F i g u r e 4 , t h e h y d r a z i n o p r o t o n s h a v i n g b e e n exchanged. The a d d i t i o n of d e u t e r a t e d H C 1 d i d n o t a l t e r t h e spectrum except a downfield s h i f t of t h e aromatic
.
191
a,
a
.d
3
m
5
W d
h
d
E
5a, .d
0 k
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p r o t o n s r e s o n a n c e b y 0 . 1 ppm. 2.16 E. S. R. Spectrum H i l l e r b r a n d a n d co-workers19 u s e d E l e c t r o n S p i n Resonance t o s t u d y c h a r g e t r a n s f e r i n t e r a c t i o n s between i s o n i a z i d and c o p p e r i o n s . 2.17 Mass S p e c t r o m e t r y G i l l i s 2 1 h a s d i s c u s s e d t h e fragment a t i o n p a t t e r n f o r i s o n i a z i d a n d s i m i l a r compounds. F i g u r e 5 shows t h e e l e c t r o n - i m p a c t mass s p e c t r u m o b t a i n e d on an A E I MS902 mass s p e c t r o m e t e r e q u i p p e d w i t h a d a t a a c q u i s i t i o n s y s t e m . The M+ o c c u r s a t m / e 137 and t h e f r a g m e n t i o n s r e s u l t from e i t h e r d i r e c t bond c l e a v a g e ( m / e 106, 78) o r t h r o u g h t h e e l i m i n a t i o n o f HCN from t h e p y r i d y l r i n g ( m / e 5 1 ) .
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Physical Properties of t h e S o l i d 2.21 Melting C h a r a c t e r i s t i c s The m e l t i n g p o i n t o f i s o n i a z i d i s u s e d a s s p e c i f i c a t i o n i n t h e U n i t e d S t a t e s Pharma~ o p o e i aand ~ ~European Pharmacopoeia3. The m e l t i n g p o i n t o c c u r s between 1 7 0 a n d 174OC. 2.22 D.T.A. and D.S.C. D i f f e r e n t i a l thermal a n a l y s i s w a s used t o s t u d y i s o n i a z i d b e f o r e t h e t e c h n i q u e gained i t s c u r r e n t p o p u l a r i t y 2 4 3 25. P i r i s i 2 6 showed t h a t i s o n i a z i d i n t h e p r e s e n c e o f z i n c , c o p p e r and ' i r o n s a l t s and m e r c u r i c o x i d e g i v e s an a b n o r m a l D.T.A. pattern. D r . J a c o b s o n 2 7 h a s shown t h a t t h e 2.2
194
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Figure 5:Low-resolution mass spectrum of isoniazid.
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S q u i b b House S t a n d a r d of i s o n i a z i d shows a s h a r p endotherm a t 17OoC u s i n g DuPont t h e r m a l a n a l y s i s
equipment.
The p u r i t y of t h i s s t a n d a r d w a s d e t e r m i n e d t o be 99.95 mole p e r c e n t u s i n g a P e r k i n E l m e r DSC-1B d i f f e r e n t i a l s c a n n i n g colorimeter27. 2.23 T.G.A. Thermogravimetry c a n be u s e d t o d e t e r m i n e m o i s t u r e or r e s i d u a l s o l v e n t s i n i s o n i a z i d . When t h e S q u i b b House S t a n d a r d w a s t e s t e d no loss on d r y i n g was r e c o r d e d 2 7 . 2.24 E l e c t r i c a l Moment Lumbroso and Barassin’* d e t e r m i n e d t h a t t h e e l e c t r i c a l moment of i s o n i a z i d w a s 2.92 I.L. 2.25 E l e c t r i c a l C o n d u c t i v i t y The e l e c t r i c a l c o n d u c t i v i t y of a compressed t a b l e t of i s o n i a z i d w a s d e t e r m i n e d a t t e m p e r a t u r e s between 50 and 1500C29. 2.26 C r y s t a l Characteristics B h a t a n d co-workers30 h a v e r e p o r t e d t h a t i s o n i a z i d c r y s t a l s a r e o r t h o r h o m b i c , space g r o u p P 2 1 2 1 8 1 , w i t h a , 14.915 ( 1 5 ) b, 11.400 ( 1 0 ) c, 3.835 ( 5 ) ~ d , ( m e a s u r e d ) = 1.417 ( 7 ) d ( c a l c u l a t e d ) = 1.395 and 2 = 4.
196
GLENN A. BREWER
2.27
X-Ray D i f f r a c t i o n The powder x-ray d i f r a c t i o n c u r v e f o r i s o n i a z i d i s shown i n F i g u r e 6 3 f The r e l a t i v e i n t e n s i t i e s f o r t h e v a r i o u s peaks a r e g i v e n below:
.
Interplanar Distances Relative I n t e n s i t i e s d (ANGSTROMS ) 0.098 8.84 0.408 7.30 0.398 6.10 0.451 5.64 1.000 5.25 0.502 4.49 0.296 3.69 0.398 3.51 0.102 3.42 0.068 3.36 0.197 3.27 0.235 3.10 0.060 3.04 0.058 3.01 2.80 0.170 0.076 2.63 0.168 2.47 0.115 2.42 0.187 2.33 2.3 S o l u b i l i t y 2 . 3 1 Water S o l u b i l i t y32 F o u r t e e n g r a m s of i s n i a z i d -re s o l u b l e i n 100 m l o f water a t 25OC. T w e n t y - s i x grams a r e s o l u b l e i n 100 m l of water a t 4OoC. 2.32 Solubility i n Solvents32~33
mf
Solvent S o l u b i l it ethanol(25OC) 2 g/100 e t h a n o l ( b o i l i n g ) 10 g/100 m l ch l o r o form 0 . 1 g/100 m l ethyl ether very s l i g h t l y soluble benzene i ns o l u b l e
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Figure 6:X-ray powder-diffraction pattern of isoniazid.
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198
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2.4
Physical P r o p e r t i e s of Solution 2.41 pH The pH o f a s o l u t i o n ( 1 i n 10) should b e between 6.0 and 7.523. 2.42 D i s s o c i a t i o n Constant There i s a d i s c r e p a n c y i n t h e l i t e r a t u r e o n t h e d i s s o c i a t i o n c o n s t a n t s of isoniazid. T h i s i s i n p a r t due t o t h e d i f f e r e n t methods of measurement employed. F a l l a b 3 4 determined t h e b a s i c d i s s o c i a t i o n c o n s t a n t a s 3 x 10-11 measured conductC a n i 6 and D j o r d j evi635 e s t a b l i s h e d ometrically. t h a t t h e 1st b a s i c c o n s t a n t s h o u l d be a s c r i b e d t o t h e p y r i d i n e n i t r o g e n and t h e 2nd t o t h e h y d r a z i n e group. T h i s i s c o n t r a r y t o p r e v i o u s work by Cingolani and G a ~ d i a n o ~ ~ . Nagano and c o - ~ o r k e r sdetermined ~~ t h e dissociation constants potentiometrically as PK1 = 2.13, PK2 = 3.81, PK3 = 11.03. S a l v e s e n and G l e n d r a n c ~ ed~e ~ termined t h e d i s s o c i a t i o n c o n s t a n t s i n 1 . O M sodium and K2 = c h l o r i d e s o l u t i o n a s K1 = 9.80 x 1.42 10-4. Zommer and Szuszkiewicz'l have e s t a b l i s h e d pK1 = 1 0 . 7 5 and pK2 = 11.15 and prot o n a t i o n c o n s t a n t s cf 3.57 f o r t h e p y r i d i n e N and 1.75 f o r t h e h y d r a z i d e N. Rekker and N a ~ t found a ~ ~ t h a t solu t i o n s o f i s o n i a z i d became yellow a t p H 10 and 2.7. The c o l o r i s r e v e r s i b l e on changing t h e pH. They e x p l a i n e d t h i s b e h a v i o r on t h e b a s i s o f t h e e x i s t ance o f two p o s i t i v e i o n s , a monovalent yellow p o s i t i v e i o n and a d i v a l e n t c o l o r l e s s p o s i t i v e ion. The pK v a l u e s a r e pK' = 2 . 0 0 , pK" = 3.6 and pK"' = 10.8. 2.43 Photolysis Constant S a l v e s e n and Eiki113' e s t a b l i s h e d t h e p h o t o l y s i s c o n s t a n t s f o r i s o n i a z i d a t 20°C and o ' * and 370 nm i n M NaCl s o l u t i o n a s kl = 1 . 0 0 x l k2 = 1.45 x 10-4.
ISONI AZ I D
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2.44
Oxidation P o t e n t i a l The o x i d a t i o n p o t e n t i a l s f o r i s o n i a z i d a t v a r i o u s pH v a l u e s w e r e d e t e r m i n e d b y vu 1t e r i n 4 0 . Solution i n Ef 1 N HC1 0.70 0.025M Na2B407 0.25 3N NaOH -0.22 3.
Metal Complexes I s o n i a z i d forms metal complexes w i t h many d i v a l e n t i o n s . T h e s e complexes h a v e b e e n used i n t h e d e t e r m i n a t i o n o f i s o n i a z i d (see S e c t i o n s 6.22, 6.25 and 6.29). T a m u r a a n d Nagano4I h a v e d e t e r m i n e d t h e c o n s e c u t i v e f o r m a t i o n c o n s t a n t s f o r t h e complex formed b e t w e e n i s o n i a z i d a n d C d ( I 1 ) . The e x p e r i ments were c a r r i e d o u t a t pH 7.2 ( a d j u s t e d w i t h NaOH) i n M NaN03 u s i n g 0.001M Cd(N03)2 a t 25OC. The d e t e r m i n a t i o n was made p o l a r o g r a p h i c a l l y . The v a l u e s d e t e r m i n e d were k l = 35, k2 = 0 . 5 7 , k3=52.5. A t high concentrations o f i s o n i a z i d yellow c r y s t a l s o f Cd ( I N H ) 2 (NO3)2-H20 p r e c i p a t e d from s o l u t i o n indicating t h a t contrary t o t h e polarographic data t h a t t h e 2 : l complex is more s t a b l e t h a n t h e 3 : l complex. By p H t i t r a t i o n t h e s t e p w i s e f o r m a t i o n c o n s t a n t s w e r e k l = 1 2 . 2 , k2 = 1 2 . 6 , a n d k 3 = 3.4. The Same a u t h o r s 4 2 s t u d i e d t h e f o r m a t i o n c o n s t a n t s o f i s o n i a z i d a n d C u ( I I ) , Zn, N i ( I I ) , C o ( I 1 ) a n d Mn (11). The complexes o f c o p p e r a n d i s o n i a z i d h a v e been e x t e n s i v e l y s t u d i e d by I ~ h i d a t e ~ ~ . 4.
History, Synthesis and Manufacturinq I s o n i a z i d w a s f i r s t p r e p a r e d b y Meyer a n d M a l 1 Y s o 0 i n 1912 b y h e a t i n g a m i x t u r e o f ison i c o t i n i c a c i d a n d h y d r a z i n e above 30OoC. The a c t i v i t y of t h e compound a g a i n s t Mycobacterium SJ. was f i r s t r e c o g n i z e d b y C h o r i n e 5 0 1 a n d b y Huant502 i n 1945. The d r u g w a s r e p o r t e d a s a u s e f u l t u b e r c u l o s t a t i c a g e n t b y F a r b e n f a b r i k e n - B a y e r , A. G., Hoffmann-LaRoche, I n c . a n d E. R. S q u i b b & S o n s , I n c .
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i n 1952503. The b a s i c method of m a n u f a c t u r e o f i s o n i a z i d i s t h e condensation of hydr azine with a y - s u b s t i t u t e d pyri d i ne H y d r a z i n e c a n be d i r e c t l y c o n d e n s e d w i t h i s o n i c o t i n i c a c i d , The w a t e r formed i n t h e rea c t i o n i s u s u a l l y removed b y a z e o t r o p i c d i s t i l l a tion44945946947, E s t e r s o f i s o n i c o t i n y l a c i d c a n be h y d r o l y z e d and t h e r e s u l t i n g a c i d condensed w i t h h y d r a z i n e . Ammonia i s u s u a l l y employed f o r t h e h y d r o l y s i s 4 8 . Y - P i c o l i n e c a n be o x i d i z e d i n 70% s u l f u r i c a c i d w i t h manganous d i o x i d e t o form i s o n i c o t i n i c acid. The c o r r e s p o n d i n g a c i d c h l o r i d e i s made with thionyl chloride. The a c i d c h l o r i d e i s t h e n r e a c t e d w i t h h y d r a z i n e i n anhydrous benzene t o y i e l d isoniazid49. I n a modification of this procedure t h e a c i d c h l o r i d e i s r e a c t e d w i t h e t h a n o l t o form t h e e t h y l ester which i s t h e n r e a c t e d w i t h h y d r a z i n e i n e t h a n o l t o form i s o n i a z i d 5 0 . I n a s i m i l a r manner o n e c a n o x i d i z e 2 , 4 d i m e t h y l p y r i d i n e w i t h s e l e n i u m and s u l f u r i c a c i d . T h e m i x t u r e i s n e u t r a l i z e d w i t h ammonia. A m i x t u r e o f i s o n i c o t i n i c a c i d , i s o n i c o t i n a m i d e and i s o n i c o t i n i c h y d r a z i d e i s forrned51.
.
Stability The s t a b i l i t y o f i s o n i a z i d h a s b e e n s t u d i e d e x t e n s i v e l y i n s o l u t i o n a n d i n v a r i o u s pharmaceut i c a l p r e p a r a t i o n s . Of p a r t i c u l a r i n t e r e s t i s t h e r e a c t i o n of t h e h y d r a z i n e g r o u p w i t h n a t u r a l l y o c c u r i n g a l d e h y d e s and k e t o n e s such a s s u g a r s o r k e t o a c i d s and the complexation o f i s o n i a z i d w i t h metal i o n s . Lewin a n d H i r ~ c h a~v e~ shown t h a t n o n - i o n i c c h e l a t i n g material can l a r g e l y p r e v e n t t h e degrad a t i o n o f i s o n i a z i d when n e u t r a l a n d a l k a l i n e s o l u t i o n s a r e a u t o c l a v e d . They n o t e d t h a t C u ( I 1 ) and Mn(I1) i o n s a c c e l e r a t e d t h e d e g r a d a t i o n o f i s o n i a z i d i n t h e p r e s e n c e o f hydrogen peroxide. P o o l e a n d M e ~ e r e~p o~r t e d t h a t i s o n i a z i d i s u n s t a b l e i n human o r r a b b i t plasma w h i l e i t i s
5.
ISON I A2 ID
201
s t a b l e f o r s e v e r a l weeks i n b u f f e r e d aqueous s o l u t i o n s a t pH v a l u e s below 8 . The i n s t a b i l i t y i n plasma i s q u i t e marked even a t r e f r i g e r a t o r temperatures. Kakemi and c o - ~ o r k e r shave ~ ~ studied t h e d e g r a d a t i o n o f i s o n i a z i d i n aqueous s o l u t i o n under a n a e r o b i c c o n d i t i o n s . A l k a l i n e h y d r o l y s i s under a e r o b i c c o n d i t i o n s y i e l d s a m i x t u r e of i s o n i c o t i n i c a c i d , i s o n i c o t i n a m i d e and 1 , 2 d i i s o n i c o t i n o y l h y d r a z i n e p l u s s m a l l amounts of u n i d e n t i f i e d products. Under a n a e r o b i c c o n d i t i o n s i s o n i c o t i n i c a c i d and 1 , 2 d i i s o n i c o t i n o y l h y d r a z i n e were t h e p r i n c i p a l p r o d u c t s . When EDTA was added t o t h e r e a c t i o n m i x t u r e only i s o n i c o t i n i c a c i d was formed. F i r s t o r d e r k i n e t i c s were followed. Inoue55 found t h a t a t pH 3 . 1 u n d e r a n a e r o b i c c o n d i t i o n s i s o n i a z i d h y d r o l y z e s t o form i s o n i c o t i n i c acid. Pseudo f i r s t o r d e r k i n e t i c s a r e followed. A t lower pH v a l u e s t h e e f f e c t of b u f f e r t y p e can be s e e n . A c t i v a t i o n e n e r g i e s were c a l c u l a t e d for t h e h y d r o l y s i s by d i f f e r e n t i o n i c s p e c i e s . Horioka and c o - ~ o r k e r sfound ~~ t h a t losses o f i s o n i a z i d were encountered when t h e drug was blended w i t h v a r i o u s a n t i a c i d p r e p a r a t i o n s . The e f f e c t of t e m p e r a t u r e , humidity and pH on t h e s t a b i l i t was determined. Haldg7 found t h a t i s o n i a z i d underwent slow o x i d a t i o n i n aqueous s o l u t i o n , b u t i n t h e p r e s e n c e of s u c r o s e t h e i s o n i a z i d r e a c t e d w i t h t h e a l d o hexoses formed on i n v e r s i o n . The r e a c t i o n w i t h s u c r o s e could b e i n h i b i t e d by t h e a d d i t i o n o f 0.3% sodium c i t r a t e . Pawelczyk and c o - ~ o r k e r sfound ~~ t h a t a s long a s c o n d i t i o n s were k e p t a n a e r o b i c t h a t t h e decompos i t i o n of i s o n i a z i d i n t h e pH r a n g e 3 t o 7 followed f i r s t order kinetics. They r e p o r t e d t h a t a 1% s o l u t i o n of t h e drug was 37 times more s t a b l e a t PH 6 t h a n a t pH 3.The e f f e c t of d i f f e r e n t b u f f e r s p e c i e s on t h e r a t e o f t h e r e a c t i o n was noted. Wu and co-workers59 i n v e s t i g a t e d t h e browning r e a c t i o n between l a c t o s e and i s o n i a z i d i n t h e
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s o l i d s t a t e with d i f f u s e r e f l e c t a n c e spectrophotometry. T h i n - l a y e r chromatography was u s e d t o demonstrate t h e p r e s e n c e of i s o n i c o t i n o y l hydrazones of l a c t o s e and h y d r o x y m e t h y l f u r f u r a l . InoueG0 h a s e s t a b l i s h e d t h e e f f e c t of t h e p r e s e n c e of copper (11) i o n s on t h e r a t e of oxidat i o n of i s o n i a z i d i n s o l u t i o n . The r e a c t i o n p r o d u c t s were i s o n i c o t i n i c a c i d , i s o n i c o t i n a m i d e , 1,2-diisonicotinoylhydrazine, i s o n i c o t i n e carboxaldehyde and i s o n i c o t i n o y l hydrazone. The copper c h e l a t e s of i s o n i a z i d a r e degraded by a f i r s t o r d e r r e a c t i o n and t h e r a t e i s determined b y t h e r a t i o of t h e c o n c e n t r a t i o n of c h e l a t e d s p e c i e s p r e se n t Inoue and Ono61 have e s t a b l i s h e d t h e k i n e t i c s of t h e d e g r a d a t i o n of i s o n i a z i d i n t h e p r e s e n c e o f Managenese (11). Shchukin62 h a s s t u d i e d t h e r e a c t i o n of copper (11) w i t h i s o n i a z i d . Kakemi and co-workers63 s t u d i e d t h e s t a b i l i t y of t h e sodium m e t h a n e s u l f o n a t e salt of i s o n i a z i d from p H 3 t o 9. Rao and c o - ~ o r k e r shave ~ ~ demonstrated t h a t i s o n i a z i d i n s y r u p f o r m u l a t i o n s undergoes hydrazone formation w i t h t h e f r e e g l u c o s e t h a t i s p r e s e n t . Absorption of t h i s hydrazone i s r e p o r t e d t o b e impaired. The a u t h o r s s u g g e s t t h e u s e of s o r b i t o l a s a replacement f o r s u c r o s e .
.
6.
A n a l y t i c a l Chemistry 6.1 Identity Tests A l a r g- e number of i d e n t i t y t e s t s have b e e n e s t a b l i s h e d f o r i s o n i a z i d . Most of t h e s e a r e colorimetric and a r e r e p o r t e d below i n t a b u l a r form.
Reaqen t
8 w
p- Dimethylaminobenza l d e h y d e A l k a l i n e Na2Fe ( C N ) 5NO Q C F e (CN) 6J + light Dini t r o c h l o r o b e n z e n e o-ninitrobenzene R e d u c t i o n w i t h Zn/HCl and ph e n y l h y d r a z i n e 1 , 2 Naphthoquinone4 - S u l f o n i c a c i d + NaOH 1,2,4-Aminonaph t h o 1 s u l f o n i c a c i d SbC13, SbC15 o r AsC13 E p i ch l o r o h y d r i n Dimethylglyoxime E t h y 1en i c d i ca rbox y 1i c a c i d s (fumaric, maleic a c i d s , e t c . ) Naphthoqu inone-HgC12 3,5-Dini t r o s a l i c y l i c a c i d N i n h y d r in BrCN a n d NaOH Benzyl c h l o r i d e NaOH D r a g e n d o r f f ' s Reagent
Reference
rnlnr i n t e n s e yellow i n t e n s e orange pink purple violet
66,67,68,74,85,86 69,74 70 71,74,86 72
y e 1low
73
bright red orange t o red
74,75,76 77 78 79 80 81
--
red red ye 1low
--
brown r e d
r e d orange green-blue f l u o r . b l u e fluorescence red
82 83 84,85 85 85 86,94
I n a d d i t i o n t o t h e s e c o l o r r e a c t i o n s a number o f c o l o r e d p r e c i p i t a t e s can be formed o n t h e a d d i t i o n of m e t a l s a l t s o r a c i d s t o i s o n i a z i d .
Reagent Am03 S e02 HgC12 cuso4 Hg2C12 KI AuI Lead a c e t a t e + K I KBr
N
e 0
~205.12~03 Picrolonic acid Tannic a c i d V i t a l i l s reagent Mecke' s r e a g e n t Frghde' s r e a g e n t Mandelin' s r e a g e n t Alloxan D i s u I f imides M e t h y 1i o d i d e K2 C r 2 0 7 Pho sphomo 1yb d i c a c id P i c r i c Acid Reineckel s s a l t Styphnic a c i d
Color of P r e c i p i t a t e White Red White Blue White Amorphous mass Dark c r y s t a l s Yellow a c i c u l a r c r y s t a l s E f f e r v e s c e n c e followed by b l a c k and c o l o r l e s s crystals Precipitate Green-ye1 low PPt Yellow mass Rose-sienna Blue Red White p p t
-Yellow n e e d l e s
--
--
Reference 74,94 78,87,88 74 86 86 86,93,95 86 86 86 86 86,93 86 86 86 86 86 89 90 91 92 92 93,94 94 94
94 94 94
Kay's reagent Vaillef s reagent Na 2 P t B r 6
F e i g l a n d c o - w o r k e r s g 6 h a v e r e p o r t e d a s p o t t e s t i n which i s o n i a z i d i s ( C N ) 6-7 q u a t e r n i z e d a n d p y r o l y z e d w i t h Na2S203 a t 180OC. A c i d i f i e d Fe i s used f o r d e t e c t i o n .
Be
97 98 Popkov and Amelink h a v e r e p o r t e d on m i c r o c r y s t a l l i n e t e c h n i q u e s f o r t h e detection of isoniazid. 6 . 2 Methods of A n a l y s i s 6 . 2 1 G e n e r a l Reviews Deltombe99, S l o u f l O O , Robles a n d Unzueta'Ol, Yalcindaglo2, B r a n d y s l 0 3 a n d G a r c i a a n d c o - w o r k e r s l o 4 h a v e a l l p u b l i s h e d r e v i e w s on t h e q u a l i t a t i v e and q u a n t i t a t i v e d e t e r m i n a t i o n o f i s o n i a z i d . 6 . 2 2 C o l o r i m e t r i c methods A number of a u t h o r s h a v e formed h y d r a z o n e s of i s o n i a z i d w i t h v a r i o u s aldehydes and k e t o n e s and used t h e h i q h l y c o l o r e d p r o d u c t s t o determine t h e d r u g . O f t h e v a r i o u s a ldehydcs u s e d , p-dimei?hylaminobenza l d e h y d e lo8, log, I l o ,lll. Benza1dehydes7, I 6 O , e a r s t o be t h e m o s t 0 ular105,106, lo7; a n d v a n i l l i n 1 1 3 ' 197yPgghave a l s o been u s e d . The o f f i c i a l method o f t h e AOAC i s t h e r e a c t i o n of i s o n i a z i d w i t h b e n z a l d e h y d e i n sodium b i c a r b o n a t e solution. The a b s o r b a n c e o f t h e h y d r a z o n e i s m e a s u r e d a t 302 nm. The absorbance a t 375 nm ( b a c k g r o u n d ) i s s u b s t r a c t e d a s a c o r r e c t i o n 1 6 9 . Sodium 1,2-naphthoquinone-4-sulfonate r e a c t s w i t h t h e h y d r a z i d e p o r t i o n o f i s o n i a z i d i n a l k a l i n e s o l u t i o n t o p r o d u c e a n orange-red c o l o r w i t h a maximum a t 480 nm1149115. 2-3-Dichloro-1,4-naphthoquinone r e a c t s
?R
206
GLENN A. BREWER
with isonia zid t o give a b lu e co lo r i n a l k a l i n e ~ o l u t i o n ~ l ~The , ~ r~e a~c t. i o n i s u s e f u l w i t h pharmaceutical p r e p a r a t i o n s which a l s o c o n t a i n sodium a m i n o s a l i c y l a t e l l 8 . An a s s a y u t i l i z i n g 1, 4-naphthoquinone h a s a l s o been regorted1l9. I s o n i a z i d r e d u c e s phosphomol b d a t e i n a l k a l i n e s o l u t i o n t o molybdenum b l u e 1 2 0 , l Y l . In a s i m i l a r r e a c t i o n molybdophosphotungstate g i v e s a b l u e color122. An a s s a y u t i l i z i n g molybdic a c i d i n a l k a l i n e a c e t o n e s o l u t i o n h a s also been r e p o r t e d l 2 ? I s o n i a z i d r e a c t s w i t h cvano en c h l o r i d e 1 2 4 ~ 1 2 5 ~ 1 2 6 c, h l o r o r h o d a n a m i n e l 2 7 ~ 18 o r
2
cyanogen bromide129 t o form g l u t a c o n i c d i a l d e h y d e which can t h e n be condensed w i t h b a r b i t u r i c o r 2t h i o b a r b i t u r i c a c i d s t o y i e l d c o l o r e d polymethine dyes. I s o n i a z i d reduces f e r r i c y a n i d e t o f e r r o c y a n i d e . The amount o f f e r r o c y a n i d e can be determined by t h e a d d i t i o n of f e r r i c i o n t o y i e l d a b l u e colorl30,131. Sodium pentacyanoaminoferroate r e a c t s w i t h i s o n i a z i d t o g i v e a yellow chromogen 132 I s o n i a z i d r e a c t s w i t h 1-chloro-2, 4-dinitrobenzene i n a l k a l i n e s o l u t i o n t o g i v e a p u r p l e color133,74,134. l-Fluoro-2,4 d i n i t r o b e n z e n e a l s o r e a c t s i n a s i m i l a r manner 135 I s o n i a z i d forms c o l o r e d complexes w i t h many m e t a l s which can be used i n a n a l y t i c a l c a n be formed w i t h ammonium methods. vanadate f e r r i c c h l o r i d e and 2 , 2 1 b i ~ y r i d i n e l ~copper139 ~, and N i c k e l (11) and f e r r i c i0nl40. Reineckels s a l t forms a w a t e r i n s o l u b l e p r e c i p i t a t e with i s o n i a z i d . This p r e c i p i t a t e d i s s o l v e s i n a c e t o n e and t h e c o n c e n t r a t i o n of i s o n i a z i d can b e determined c o l o r i m e t r i c aiiy141, 142. The f o l l o w i n g compounds have a l s o been used i n c o l o r i m e t r i c assays for i a o n i a z i d .
.
13g~y9’~358,
Reagent Reference ch loropicrin 504 epichlorohydrin 143 ninhydrin 144 145 tripheny1tetrazolium ch loride 9-chloroacridine 146 dinitrobenzoic acid 147 p-aminosalicylate-HVO~ 148 1,2,4-aminonaphtholsulfonic acid 77 p-ni trophenyldiazonium fluoroborate 149 7-chloro-4 nitrobenzo-2-oxa-1,3-diazole 150 acid chrome dark blue 151 2-bromo-1-acetonaphthone 112 N- (4-pyridyl)pyridinium chloride 152 174 picryl chloride 6.23 Spectrophotometric Methods A number of authors have utilized the strong absorbance of isoniazid in the ultraviolet as a means of determining the concentration of the drug. In many methods the a @ : f ! 7 4 IPS in alkaline and acid solution as an identity test 1659237. Isoniazid can be determined in the presence of p-aminosalicylate by an ultraviolet assay162,163,164. 6.24 Fluorimetric Methods Although isoniazid does not have any native fluorescence several sensitive fluorometric assays have been reported for the drug. Isoniazid is coupled with 2-hydroxy-1-naphthaldehyde to give a yellowgreen fluorescence. The compound has an excitation maximum at 495 m and an
Y
y3,iQ%, ar39:'iB;
Fh
208
GLENN A. BREWER
e m i s s i o n maximum a t 534 nrn166~167. I n a n o t h e r method t h e p y r i d i n e r i n g i s c l e a v e d w i t h cyanogen b r o m i d e t o form A S c h i f f ' s base i s t h e n formed glutacondialdehyde. w i t h 4 - a m i n o b e n z o i c a c i d which has a n e x c i t a t i o n maximum a t 336 6 . 2 5 T i t r i m e t r i c Methods A large variety of titrimetric methods h a v e b e e n employed f o r the d e t e r m i n a t i o n of i s o n i a z i d i n b u l k and i n formulated products. A series of reviews have been w r i t t e n on t i t r i m e t r i c methods170, 1 7 1 9 1 7 2 3 1 7 3 ~ 202. The o f f i c i a l m e t h o d s of a n a l y s i s i n t h e U . S. P. 2 3 , B. P. 174 a n d E u r o p e a n P h a r m a c o p o e i a 3 a r e t i t r i m e t r i c methods. I n t h e U.S.P,23 a n i t r i t e titration is utilized. I n t h e B.P. 174 t h e i s o n i a z i d i s r e a c t e d w i t h bromine and t h e e x c e s s bromine i s t i t r a t e d w i t h t h i o s u l f a t e a f t e r t h e l i b e r a t i o n o f i o d i n e by t h e a d d i t i o n o f potassium iodide. I n t h e European P h a r m a c o p o e i a 3 a d i r e c t t i t r a t i o n w i t h bromate i s u t i l i z e d w i t h t h e a d d i t i o n o f ethoxychysoidine a s an i n d i c a t o r . The v a r i o u s t i t r i m e t r i c methods a r e summarized i n t h e Table.
Reaq e n t
I C 1, K I
Titrant t h i o s u 1f a t e KBr03 alkali KBrO3 mr03 t h i o s u lf a t e KIO~ thiosulfate t h i o s u 1f a t e t h i o s u l f at e t h i o s u l f at e
non-aqueous
HC lo4
non-aqueous non-aqueous
NaN02 HC 104 HClOq
mr ,m r 0 3 ,KI m r Br2
-
~ 1 0 3KI , H I , K2Cr207, K I
~ ~ 1 0 4 , N
8
non-aqueous Cd++ Cd++ cU++,
NH4SCN
CU++, N H ~ S C N
I2
Indicator starch ethoxychrysoidine pheno lphtha l ei n methyl orange p o t e n t iome t r i c starch ethoxychrysoidine starch starch starch starch thermome t r i c crystal v i o l e t or methyl v i o l e t
Sb e l e c t r o d e
Complexon 111 CaC12
g l a s s electrode potentiome tric thymol b l u e eriochrome B l a c k T methylthymol B l u e
Am03 EDTA
mu r e x i de
NaClO4 NaOMe
Reference 1 7 5 ,1 7 7 , 1 7 9 , 1 8 2 176,106,180,184 178 181,186 1 8 1 , 1 8 3 ,1 8 5 ,1 8 7 74,188,192,196 189 1 9 0 , 1 9 3 ,1 9 5 191 106,194 198 200 217,211,210,209,208, 207,206,204,205,216, 213,201,74,57,203, 2 14 233 212,276 215 2 18 2 02 219,221 220 222 2 2 3 ,224
E
% m
d
~m m a ,
d
0 0 0
a E E
U
H H
C 0
3
dd
C O D
0 a 0 mEcn NUf:
h
d
9
a,
a 0 k c,
u
a,
a,
c
a,
k
a,
a m a -ti
I
k
I
m
4J
m
E
0
.ti
5
a .ti a
I
rl
h C
a,
4
tn
0-
a
5k
d
a,
m
a,
m .d
nl
m
4J
z
c,
-
Ill
4J
o"+ cn+
C cr a, m m rn W
I
3 3
u u
E 7
m
d a, Ik
210
I
H
h
d
m
'
Nesslerl s reagent I2
-
C e (So4) 2
-
K2Cr207 isopropenyl t r i chloroacetate
Y
sodium d i e t h y l d i thiocarbama t e hydraz i n e ammonium hexani t r o c e r a t e (IV) Mohrrs s a l t C e (NO314 Mohrfs s a l t
cuso4
2 56
starch a-naphthof lavone P t electrode P t electrode diphenylamine
2 57 2 58
259 2 60 261
NaOH bromphenol b l u e 262 KOH K3Fe (CN) 6 P t electrode 263,264 K 3Fe (CN) 6, KOH, H2S04, K I t h i o s u I fa t e starch 265 6.26 E l e c t r o c h e m i c a l Methods A number of a u t h o r s have d e t a i l e d p o l a r o g r a p h i c methods f o r i s o n i a z i d . The r e d u c t i o n a p p a r e n t l y o c c u r s i n two s t e p s ( t o t a l o f 4 e l e c t r o n s ) b u t t h e s t e p s a r e n o t s u f f i c i e n t l y w e l l s e p a r a t e d t o be u t i l i z e d The h a l f wave p o t e n t i a l bea n a l y t i c a l l y , s o t h a t t h e s i n g l e wave i s used. b u t t h e h e i g h t d i d n o t change r e a t l y comes more n e g a t i v e a t h i g h e r pH v a l u e s H r a n g e s t u d i e d 266,153,267,266,269,270,271,272,273,274,275,27 ,278, over t h e 279,280,2Kl
4
A.C.Polarography h a s been used by Sato282 and Vallon and c o - ~ o r k e r s ~ ~t h~ei n a s s a y o f i s o n i a z i d . Okuda and co-workers284 rea c t e d i s o n i a z i d w i t h 1,2-naphthoquinone-4-sulfonic a c i d and have t h e n used p o l a r o g r a p h y t o measure t h e r e a c t i o n p r o d u c t . se e r p l au h o r s h ve r e r t e d c o u l o e t ' c g e t h o d s for t h e a n a l y s i s o f i s o n i a z i x w i t h ekectroccemica??y g e n e r a t e 8 c 6 i o r i n e 285,286 o r bromine286,287,288,289.290.
212
GLENN A. BREWER
6.27
Gravimetric Methods R e l a t i v e l y few g r a v i m e t r i c a s s a y s have been r e p o r t e d f o r i s o n i a z i d . T h i s i s probably because of t h e l a r g e number of c o l o r i m e t r i c , t i t r i m e t r i c and e l e c t r o c h e m i c a l methods a v a i l a b l e which a r e f a s t e r and more convenient than t h e g r a v i m e t r i c methods. Leal and Alves 234 have r e p o r t e d an a s s a y using p i c r i c a c i d t o form a w a t e r i n s o l u b l e salt. Akiyama and co-workers291 prec i p i t a t e i s o n i a z i d e a s t h e C u ( I 1 ) o r Hg(I1) s a l t s . The s a l t s a r e r e d i s s o l v e d i n h y d r o c h l o r i c a c i d and t h e metal i s then r e p r e c i p i t a t e d a s t h e s u l f i d e which i s determined g r a v i m e t r i c a l l y . The z i n c 2 9 2 and cadmium293 s a l t s can be measured by d i r e c t gravimetry. The benzyli d e n e d e r i v a t i v e can be determined e i t h e r gravimetrically o r v o l ~ m e t r i c a l l y ~ ~ I s~o .n i a z i d can b e q u a t e r n i z e d and t h e s a l t can b e then measured v o l u m e t r i c a l l y o r g r a v i m e t r i c a l l y 2 95. The phosphotungstate of i s o n i a z i d can b e determined gravime t r i c a 1 1 ~ 1 6 4 . 6.28 M i c r o b i o l o q i c a l and Enzymatic Methods S e v e r a l a g a r d i f f u s i o n microbiol o g i c a l a s s a y s u t i l i z i n g s t r a i n s of a c te rium have been r e p o r t e d f o r i s o n i a ~ i d ~ ~ ~ I s o n i a z i d i n h i b i t s many enzyme systems and a number of t h e s e might b e s e l e c t e d a s t h e b a s i s of enzymatic a s s a y s . Examples of enzyme systems which a r e i n h i b i t e d a r e pea cotyledon amine o x i d a s e , c a r r o t r o o t L-glutamic decarboxylase and wheat s e e d l i n g t r a n s a m i n a ~ e ~ The ~ ~ . inhibition i s r e v e r s e d by t h e presence of k e t o a c i d s . 6 . 2 9 Miscellaneous Methods O s c i l l o m e t r i c t i t r a t i o n s have been used t o determine i s o n i a ~ i d301. ~ ~ ~ Ijs o n i a z i d can be assayed g a s o m e t r i c a l l y a f t e r o x i d a t i o n w i t h iodate3O2 o r f e r r i c y a n i d e 3 0 3 304. Conductometric t i t r a t i o n s with sodium hydroxide o r h y d r o c h l o r i c a c i d have been J
~
~
~
u s e d t o measure i s o n i a z i d c o n t e n t 3 0 5 , 3 0 6 . The c o p p e r c h e l a t e of i s o n i a z i d i s s o l u b l e i n m e t h y l i s o b u t y l ketone. The c o p p e r c o n t e n t of t h e c h e l a t e i s d e t e r m i n e d i n t h e o r g a n i c p h a s e by a t o m i c a b s o r p t i o n s p e c t r o m e t r y 3 0 7 . I s o n i a z i d i n p u r e s o l u t i o n s can b e d e t e r m i n e d by r e f r a c t o m e t r y 3 08 6.3
5 w
Chromatographic Methods 6 . 3 1 Paper Chromatoqraphy Numerous p a p e r c h r o m a t o g r a p h i c s y s t e m s h a v e b e e n u s e d t o s e p a r a t e i s o n i a z i d from i n t e r m e d i a t e s u s e d i n t h e s y n t h e s i s , d e g r a d a t i o n p r o d u c t s and m e t a b o l i c p r o d u c t s . Since isoniazid absorbs strongly i n t h e u l t r a v i o l e t and g i v e s a number of c o l o r r e a c t i o n s 3 0 9 t h e r e i s no problem i n d e t e c t i n g o r q u a n t i t a t i n g t h e d r u g a f t e r t h e s e p a r a t i o n h a s been completed. A t a b l e of some p a p e r c h r o m a t o g r a p h i c s y s t e m s i s g i v e n below: Solvent Syst e m Detection Use Ref. Water s a t u r a t e d b u t a n o l C14 l a b e l l e d Urine metabolites 310 Isoamyl alcohol-waterCNBr ,Microb i o 1. Urine metabolites 311 a c e t i c a c i d ( 5 0 :50 :1 . 5 ) I s o p r o p a n o l - w a t e r (85 :1 5 ) Urine metabolites 312,313 Butano 1-ammonia -Derivatives 31 4 1st Dimension sec. b u t a n o l w a t e r (saturated)
--
2nd Dimension i s o a m y l alcohol-acetone-acetic a c i d - w a t e r (56:24:6: 1 4 )
CNBr- o-phenyl-
enediamine dimethylbenza l d e h y d e
Urine
Butanol-10% NH4OH(lO:2) circular Butanol-water(4:l) ascending 2,4-lutidine-isoamyl alcohol-water (5: 100: 9) Butanol-HC1-pet. ether or Butanol-HCl-H20(paper sat. with KC1 solution) (a)Butanol-ethanol-water (2:2:1) (b)Butanol-pyridine-water (16:4: 3 )
Butanol sat.ammoniaca1 Impurities with silver nitrate dimethylaminobenzaldehyde Dosage forms
3 16
methanolic dinitrochlorobenzene iodine-platinic iodide
Impurities
318
other basic substances
319
metabolites
320
metabolites metabo 1ites in urine
322,323
I
(c)Ethanol-l.5N NHqOH-water ultraviolet (17:1:2) (d)Phenol-isopropanol-water (16:1:5) 0.5 ammonium chloride u 1traviole t (a)Butanol saturated with water -(b)Propanol-water( 8 0 : 2 0 )
1
317
321
(a) 1sopropanol-25% NH20H(85: 15) (b) Isopropanol-water(85:15) (c) Isopropanol-formic acidwater (80:1O:lO) Pyridine-Water(65:35) Isopropanol-NH40H-water (7:1:2) Butanol-acetic acid-water ( 5 :1:4) ( a ) E thyl me thyl ketone-acetoneformic acid water(40:2:1:6) (b)E thy 1 me thy1 ketone-diethy lam inewater (921:2 :7 7 ) (c)Methyl isobutylketone-formic acid-water(ketone sat.with 4% formic acid) (d)Chloroform-methanol-formic acidwater(CHC13 sat.with 1 part H20 and 1 part 4% formic acid) (e)Benzene-ethylmethyl ketoneformic acid-water(9 parts benzene plus 1 part ketone sat.with 2% formic acid) (f)Benzene-formic acid-water (benzene sat. with 2% formic acid)
1 1
2 Ln
CI4 and spray reagents
1
--
FeC13 and
K 3 F e (CN)6
Me tab01ites
324
Metabolites Metabolites
325 326
327
la)ISO-propanol-water (17:3) (b)Butanol-acetic acid-water(4:1:5) (c)1.4M potassium phosphate buffer pH 7.0 Butanol-acetone-water(45:5:50)
Butanol-phosphoric acid-water(3:1:3) (a)Butanol S a t . with water in atmosphere of NH3 (b)95% ethanol-M ammonium acetate(7:3) adjusted to pH 5 6.32
N m
I
1
2,4,6 trinitrobenzene-sulfonic acid chlorani1ic acid
--
copper sulfate in ethanol then 0.1% benzidine in 50% aqueous ethanol
3 28
329 330 33 1
Thin-layer Chromatrogaphy In recent years several authors have developed thin-layer chromatographic system for isoniazid. These are presented in tabular form. System Chloroform-methanol(8:2) Chloroform-acetone-diethylamine (5:4:1) Cyclohexane-chloroform-diethylamine (4:5:1) Butanol-phosphoric acid-water(3:1:3) Acetone-methanol-NJQOH (50: 50: 1) (a)Pkthanol (b)Chloroform (c)Ethanol
\
Detection Folin-Ciocalteu or Phosphomo 1ybdate
-dimethylaminobenzaldehyde 5:l mixture 10% cuso4 and 10% NH~OH
Use separation from other drugs
Ref 332
derivatives 330 identification 333 identity test
334
ISopropanol-acetone(6:4 )
--
chloroform-methanol (6:4)
--
Chloroform-methanol(125:60) (a!Ethyl acetate-cyclohexanedioxane-methanol-waterNH40H(50:50:10:10:1.5:0.5)
UV iodine
I
separation of hydrazine separation of isonicotinic acid hydrazone with lactose
(b)same solvent but (50:50: 10: 1O:O. 5: 1.5) Ninhydrin or separation from (c)Ethyl acetate-cyclohexane0.5% H2S04 drugs of abuse NHqOH-methanol-water (70:15:2:8:0.5) (d)E thyl acetate-cyclohexaneNHqOH-methanol(56 :40:0.4:0.8) (e) same but (70:15 :5 :10) (f)E thyl acetate-cyclohexaneNH40H ( 5 0:40:0.1) Methanol-NH4OH-H20(100:1:4) KMnO4 bromothymol other drugs blue
\
335 335 59
336
337
rn 3
1
338 254 nm U.V. iron chloride(a)Chloroform-methanolhexacyanoferrate,molybdo13N ammonia (90:10: 1) phosphoric acid. Folin(b) Benzene-;oethano1Ciocalteu,potassium diethylamine (90:10:1) permanganate,ammoniacal (c)Chloroform-hexanol13N ammonia(90:10:0.2) silver nitrate,amminepenta(d)Chloroform-ethyl acetate cyanoferrate,iodoplatinate, iodine,Dragendorff, cinna13N ammonia ( 50 :50 :1) maldehyde triphenyltetra(e) chloroform-acetonezolium,dithiocarbamate acetic acid (90:10: 1) or ammonium molybdate (f)Benzene-acetonediethylamine (50:50 :1) (g) Chloroform-acetoneacetic acid(50:50:1) Nishimoto and T ~ y o s h i m a ~ ~found ' that isoniazid showed tailing on thin-layer chromatography due to trace metals in the silica gel. When the adsorbent was treated with EDTA the tailing was eliminated. Wijnne and c o - w o r k e r ~found ~ ~ ~ that isoniazid could be quantitated after thin-layer chromatography by coulometric titration. Schmidt341 showed that isoniazid could be revealed on a thin-layer plate by exposure to iodine vapor. Kawale and c o - w o r k e r ~sprayed ~~~ thin-layer plates with 1% mercurous nitrate to reveal isoniazid as black spots. 6.33 Ion exchanqe Chromatoqraphy Tsuji and Sekiguchij4j have shown that isoniazid is quantitatively adsorbed on Dowex 50 cation exchange resin in various metal forms. The strength of adsorption decreases in the following order:Cu++k Ni'+2 Hg++> H+ > Co++ > cd++k En++>Fe++ > Pb++> m++> ~ l + + + .
ISONIAZID
219
No a d s o r p t i o n o c c u r s on r e s i n i n t h e Ba++, Mg++, Ca++ o r Na+ forms. H e l l e r and c o - ~ o r k e r ss ~ e p~a ~ rated a c e t y l i s o n i a z i d from i s o n i a z i d on a column o f Dowex 1-X8 i n t h e p y r u v a t e form. 9 developed a Kakemi e t -a~~~~ c h r o m a t o g r a p h i c method f o r t h e s e p a r a t i o n o f i s o n i a z i d from some d e g r a d a t i o n p r o d u c t s . I s o n i a z i d i s a d s o r b e d on a weak c a t i o n e x c h a n g e r s u c h a s A m b e r l i t e CG-50 i n t h e hydrogen form. I s o n i c o t i n i c a c i d i s n o t a d s o r b e d and i s d e t e r m i n e d c o l o r i m e t r i c a l l y u s i n g cyanogen bromide. To determine i s o n i c o t i n a m i d e t h e sample s o l u t i o n i s o x i d i z e d w i t h a l k a l i n e f e r r i c y a n i d e and t h e n p a s s e s t h r o u g h a column o f s t r o n g a n i o n e x c h a n g e r s u c h a s Dowex 1-X8 i n t h e c h l o r i d e form. The amide i s unchanged Another degradaand i s n o t a d s o r b e d on t h e r e s i n . t i o n product,1,2-diisonicotinoyl h y d r a z i d e i s d e t e r m i n e d b y a d j u s t i n g t h e s a m p l e t o pH 8 . 9 w i t h b o r a t e b u f f e r and d e t e r m i n i n g t h e a b s o r b a n c e a t 329 nm. P e t e r s a n d c o - ~ o r k e r s 347 ~ ~ ~w ,e r e a b l e t o s e p a r a t e and q u a n t i t a t e a l a r g e number o f m e t a b o l i t e s of i s o n i a z i d u s i n g Dowex AG-50-X4 r e s i n i n t h e hydrogen and ammonium forms. S e l e c t i v e c o l o r r e a c t i o n s were u s e d t o d i f f e r e n t i a t e t h e g r o u p s of m e t a b o l i t e s . Fan and Wald348 s e p a r a t e d p - a m i n o s a l i c y l i c a c i d from i s o n i a z i d u s i n g a Dowex 2 - X 8 column. I n o u e and c o - w ~ r k e r su~s e~d ~ a s y s t e m s i m i l a r t o t h a t o f Kakemi e t a 1 3 4 3 t o s e p a r a t e i s o n i a z i d from i t s d e g r a d a t i o n p r o d u c t s . Lewandowski and S y b i r ~ k a ~ ~ O s e p a r a t e d i s o n i a z i d from i s o n i c o t i n i c a c i d b y p a p e r chromatography u s i n g b u t a n o l s a t u r a t e d w i t h water. The p a p e r was c o n n e c t e d w i t h an i o n exchange p a p e r i n t h e a c i d form. The s p o t s were e l u t e d w i t h d i o x a n e . The s h a r p z o n e s on the i o n exchange p a p e r were v i s u a l i z e d w i t h i c r y l c h l o r i d e Darawy a n d Mobarak3" chromatog r a p h e d s e v e r a l d r u g s on CM-82 c a r b o x y m e t h y l c e l l u l o s e c a t i o n exchange p a p e r u s i n g a w a t e r -
-
220
GLENN A. BREWER
acetone-foramide(l0:l:l) solvent system. 6.34 Other Chromatoqraphic Methods Barreto and Sabin0352 used a anhydrous sodium sulfate column eluted with chloroform-diethylamine(9:1) to concentrate metabolites of isoniazid from serum or urine. Smolarek and Dlugosch353 separated isoniazid and p-aminosalicylic acid by paper electrophoresis in barbital buffer, pH 8.5. B a r r e t ~used ~ ~ ~ two dimensional electrophoresis to separate the metabolites of isoniazid. also used paper electrophoresis to separate several acyl hydrazides. A pH 2.0 acetate buffer was used. Isoniazid was separated from several antituberculosis drugs by gas chromatography356,357, A silanized chromosorb G coated with 6% QF1 was used. Gas chromatography was used to separate the products of oxidation of hydrazides with Fehling’s solution358. 6.4 Determination of Isoniazid and its Metabolites in Body Fluids and Tissues The methods described in this section were specifically developed for the determination of isoniazid in body fluids and tissues. Many of the methods are similar to other general analytical methods described in Section 6.2 perhaps differing only in the extraction procedure. 6.41 General Reviews Terze and D a d i ~ t o u ~studied ~’ a number of color reactions to determine their application to blood level assays. Ginoulhiac360 also made a literature review of blood level methods, A critical review of methods for isoniazid determination has been written364 6.42 Colorimetric Methods Colorimetric methods are most popular for the determination of isoniazid in biological samples. The methods are listed in tabular form.
.
Reaqent Dimethylaminobenzaldehyde
Pretreatment of sample acid hydrolysis
Dimethylaminobenzaldehyde Dimethylaminobenzaldehyde
none extraction into isoamyl alcoholether from alkaline solution deproteinization with ~ ~ 1 0 ~ deproteinization with trichloroacetic acid none
Dimethylaminobenzaldehyde Dimethylaminobenzaldehyde N
5
Vanillin Vanillin Vani 11in Van i11in Cinnama ldehyde Cinnamaldehyde
Type of specimen Ref. serum & urine 361,370, 375 urine 362,363 plasma and 365,366, urine 367,3 68, 369. serum
37 1
serum and tissues
372
serum
373,376, 377 374,432, 433,434 375
deproteinization serum with trichloroacetic acid extraction with serum organic solvent extraction with milk propanol deproteinization serum with trichloroacetic acid extraction with serum butanol-chloroform
3 78 379,380, 429,430 38 1
o-Nit robenza ldehyde Sa 1icyla 1dehyde- FeC1 S a licy la ldehyde
Salicylaldehyde Glutaconic aldehyde @-diketone N
E
Catecho1 Catecho1 H2 0 2 CNBr
-
CNBr A Ikaline
hydrolysisCNBr NHqV03-H2S04 KCN, Chloramine Tbarbituric acid
deproteinization with trichloroacetic acid extraction into isoamyl alcohol-ether from alkaline solution none extraction with acetone deproteinization with trichloroacetic acid none deproteinization with trichloroacetic acid automated method deproteinized serum deproteinized trichloroacetic acid deproteinized trichloroacetic acid acid hydrolysis
serum
382
serum
383
bio logica1 fluids cadavers
3 84
plasma
386
biological ma teria Is citrated blood serum serum & urine biological fluids urine
387
urine
385
388 389 390 391,404, 411,412 392
393,394,395, 396,397,398, 399,370,435 plasma,urine 400,401 tissues,serum
1-amino-2-naphthol-4sulfonic acid Naphthoquinone-4sulfonic acid Naphthoquinone-4sulfonic acid 2,4,6-trinitrobenzenesulfonic acid Dinitrochlorobenzene Dinitrochlorobenzene
rJ .
K3Fe (CN)6 Sodium pentacyanoaminoferroate K3Fe (CN)6 Ni tropentacyanoferroate Na ph thoquinone Na ph thoq inone H202,CNBr, aniline
Picryl chloride KMn04,BrCN,NH3
urine biol. fluids urine deproteinization Zn (OH) 2 extraction methyl isobutyl ketone deproteinized serum deproteinized tissue deproteinized with sodium tungstate deproteinized with phosphoric acid
--
trich loracetic acid tungstic acid extraction BuOH,Et20 Py tein-free fiPtrate
402
urine
403,404,405, 406,407 408
whole blood
409,410
urine serum
411 412,413
serum tissue, urine spina1 fluid serum
414 415,416,417
spinal fluid urine blood blood urine plasma urine spinal fluid plasma
418
419 420 421 422 423,424 425
3 P
4-pyridylpyridinium trichloracetic acid plasma 426 dichloride,NaOH,HCl filtrate KBrO3+ methyl o r a n g e acid tungstate blood 427 Zn powder + h e a t -urine 428 6 . 4 3 T u r b i d i m e t r i c Method I s o n i a z i d r e d u c e s K2Hg14 t o form HgI w h i c h i s i n s o l u b l e . The r e s u l t i n g t u r b i d i m e t r y c a n be measuEed t o d e t e r m i n e t h e amount o f i s o n i a z i d Wagner a n d co-worker-431 h a v e a p p l i e d t h i s method t o b l o o d f o l l o w i n g present. d e p r o t e i n i z a t i o n w i t h b a r i u m h y d r o x i d e and z i n c s u l f a t e . 6 . 4 4 F l u o r i m e t r i c Methods A number of f l u o r i q e t r i c m e t h o d s € o r i s o n i a z i d h a v e b e e n reported. H e d r i c k a n d c o - ~ o r k e r sa~b s~o ~ r b e d a p r o t e i n f r e e f i l t r a t e of s e r u m on p H 6 . 5 A m b e r l i t e XE-64 i o n e x c h a n g e r e s i n . T h e i s o n i a z i d was e l u t e d w i t h d i l u t e a c i d and t h e n r e a c t e d w i t h hydrogen p e r o x i d e i n pH 8.7 b u f f e r . The o x i d a t i o n p r o d u c t f l u o r e s c e s a t 415 nm when a c t i v a t e d by u l t r a v i o l e t l i g h t a t A s l i t t l e a s 0.05 y/m1 o f s e r u m c a n be d e t e r m i n e d . 320 nm. S c o t t and Wright437 r e a c t e d s a l i c y l a l d e h y d e w i t h i s o n i a z i d and reduced t h e r e s u l t i n g hydrazone. The r e s u l t i n g compound w a s h i g h l y f l u o r e s c e n t . R e i s s , Morse a n d P u t s c h 4 3 8 a s s a y e d i s o n i a z i d f l u o r i m e t r i c a l l y a f t e r a b s o r p t i o n a n d e l u t i o n from i o n e x c h a n g e r e s i n and t r e a t m e n t w i t h a l k a l i n e c y a n o g e n bromide. Wilson, Lever and u t i l i z e d t h e f l u o r e s c e n c e of t h e z i n c c h e l a t e of t h e hydrazone of i s o n i a z i d w i t h pentane-2,4-dione i n an a s s a y i n serum. E l l a r d , Gammon a n d W a l l a c e 4 4 0 h a v e d e v e l o p e d s p e c i f i c f l u o r i m e t r i c a s s a y s f o r i s o n i a z i d , a c e t y l i s o n i a z i d , mono-and d i a c e t y l h y d r a z i n e , i s o n i c o t i n i c a c i d a n d i s o n i c o t i n y l g l y c i n e i n serum a n d u r i n e . Boxenbaum a n d Riegelman441 h a v e a l s o d e v e l o p e d a s s a y s f o r i s o n i a z i d and i t s m e t a b o l i t e s i n whole blood. M i c e l i , O l s o n a n d Weber442 h a v e e s t a b l i s h e d a micro method f o r
ISONlAZlD
225
t h e f l u o r i m e t r i c d e t e r m i n a t i o n of i s o n i a z i d i n serum. A s l i t t l e a s 2 5 p1 o f s e r u m can be u s e d i n t h e assay. P e t e r s , Morse a n d Schmidt 443 and 0' B a r r , K e i t h and B l a i r 4 4 4 h a v e compared f 1 U O r i m e t r i c and m i c r o b i o l o g i c a l a s s a y s f o r i s o n i a z i d i n serum. 6.45 E l e c t r o c h e m i c a l Methods Lauermann a n d O t t o 4 4 5 h y d r o l y z e d i s o n i c o t i n i c a c i d h y d r a z i d e and i t s m e t a b o l i t e s t o i s o n i c o t i n i c a c i d w i t h a l k a l i . The h y d r o l y s i s product w a s determined p o l a r o g r a p h i c a l l y . The a u t h o r s found t h a t t h e r e s u l t s o b t a i n e d b y t h i s method i n t h e a n a l y s i s o f c a d a v e r i c f r a c t i o n s was comparable t o t h o s e o b t a i n e d when t h e method o f N i e l s c h a n d G i e f e r 4 0 1 was u s e d . The p o l a r o g r a p h i c method was l e s s t i m e consuming. Kane 446 d e t e r m i n e d i s o n i a z i d i n biological f l u i d s without p r i o r separation. 6.46 G a s o m e t r i c Methods The h y d r a z i n e g r o u p i n i s o n i a z i d c a n b e r e a d i l y decomposed i n t o n i t r o g e n g a s . Several a u t h o r s have u t i l i z e d t h i s r e l a t i v e l y s e l e c t i v e f i n i s h f o r b l o o d and u r i n e l e v e l a s s a s . S t r i c k l a n d a n d H e n t e l 1;47 r e a c t e d i s o n i a z i d w i t h sodium i o d a t e i n a l k a l i n e s o l u t i o n . The a s s a y i s n o t e f f e c t e d b y t h e p r e s e n c e o f pa m i n o s a l i c y l i c a c i d which i s o f t e n g i v e n i n conjunction with isoniazid. H a r t i n g and G e r z a n i t s 448used a l k a l i n e f e r r i c y a n i d e t o l i b e r a t e t h e nitrogen gas. I t o and c o - w ~ r k e r 7s450 ~ ~ were ~ able t o s e l e c t i v e l y u s e c o p p e r , i r o n a n d chromium azometry t o determine i s o n i a z i d and i t s v a r i o u s metabolites i n urine. 6.47 M i s c e l l a n e o u s Chemical A s s a y s V e r r o t t i and B a r d e l l i 4 5 1 d e t e r m i n e d i s o n i z i d i n cerebrospinal f l u i d by iodometric ti t r a t i o n . Schwenk a n d c o - ~ o r k e r s ~employed ~* a radioimmunoassay f o r t h e d e t e r m i n a t i o n o f isoniazid i n biological fluids.
Microbioloqical Assays Although isoniazid is readily measured in biological fluids and tissues by chemical assays, as with many antibacterial substances a number of microbiological assays for this substance have been proposed. Microorganism Type of assay S ens itiwi ty Ref. 2.5-30 y/ml 453 Mycobacterium phlei agar diffusion Koch bacilli turbidimetric -454 tubercle bacteria cord formation -455 bacilli vertical diffusion -456 Mycobacterium vertical diffusion -457 tuberculosis HV37 Mycobacterium agar diffusion -458 tubercu10s is 0.49 y/m1 459 vertical diffusion Mycobacterium tuberculosis H37Rv and H 3 7 R a assay of isoniazid 378 BGG in milk-agar diffusion 6.48
N
N m
>
Mycobacterium tuberculosis
--
vertical diffusion vertical diffusion
460 46 1
vert ica 1 di f€usion tube dilution vertical diffusion for urine
462 463,464
ISON IAZlD
227
Bartmann and F r e i s e 4 6 5 s t u d i e d t h e t i s s u e b i n d i n g of i s o n i a z i d w i t h t h e m i c r o b i o l o g i c a l assay. They found 40% b i n d i n g w i t h human t i s s u e w h i l e mice and g u i n e a p i g t i s s u e g a v e 80% binding. Incubating the t i s s u e a t an e l e v a t e d temperature d i d not r a i s e t h e recovery. N i ~ h i P~o o~l e~ and , Meyer467, T a n s i n i and c o - w o r k e r ~ ~P~e t~e ,r s and c o - w o r k e r s 433 and O ’ B a r r & a4!*a l l compared v a r i o u s c h e m i c a l a s s a y s and m i c r o b i o l o g i c a l a s s a y s . A l l w o r k e r s c o n c l u d e t h a t t h e two methods g a v e c o m p a r a b l e results 6 . 4 9 Chromatographic A s s a y s The m e t a b o l i s m o f i s o n i a z i d i s complex and many w o r k e r s h a v e s e l e c t e d chromatog r a p h i c a s s a y s t o measure t h e d r u g i n t i s s u e and biological fluids. T h e s e methods p r o v i d e t h e s p e c i f i c i t y t h a t a r e n o t g i v e n b y many c h e m i c a l methods. A l a r g e number of c h r o m a t o g r a p h i c s y s t e m s a r e g i v e n i n s e c t i o n 6.3. Many of t h e s e methods c o u l d p r o b a b l y be u s e d t o measure i s o n i a z i d i n t i s s u e s and b i o l o g i c a l f l u i d s . The methods g i v e n i n t h i s s e c t i o n h a v e been d e v e l o p e d j u s t f o r t h i s purpose. Makino and c o - ~ o r k e r sf o ~ l~ l o~w e d t h e m e t a b o l i s m of i s o n i a z i d i n l i v e r and i n u r i n e by p a p e r c h r o m a t o g r a p h y ( w a t e r s a t u r a t e d b u t a n o l , 1% ammonia-isopropanol(3:20), b u t a n o l s a t u r a t e d w i t h 0.02M p h o s p h a t e b u f f e r p H 7 . 4 , 1%ammonia s a t u r a t e d b u t a n o l and b u t a n o l - a c e t i c a c i d - w a t e r
.
(4:1:5)).
L e ~ s c h n e r ~u ~ s egd s e c - b u t a n o l s a t u r a t e d w i t h w a t e r and i s o a m y l a l c o h o l a s developing solvents. I ~ a i n s k ys e~p a~r a~t e d t h e h y d r a z o n e s o f i s o n i a z i d and p y r u v i c and a - k e t o g l u t a r i c a c i d from i s o n i a z i d w i t h p a p e r chromatography. S e z a k i 470 s e p a r a t e d i s o n i a z i d from p y r a z i n a m i d e i n u r i n e b y means of A m b e r l i t e IRA-400. B e l l e s and L i t t l e m a n 4 7 1 u s e d Dowex 50-X8 t o s e p a r a t e i s o n i a z i d from a c e t y l i s o n i a z i d . Abiko
228
GLENN A. BREWER
and c o - ~ o r k e r suse ~~~ Dowex 1-XlO to separate these as well as the hydrazone of glucuronic acid. Peters, Miller and Brown346 utilized ion exclusion chromatography to separate metabolites of isoniazid into ionized, slightly ionized and unionized groups of compounds. The individual metabolites were measured with specific colorimetric assays. Okudaira and c o - ~ o r k e r s ~ used ’~ Dowex 1 and Dowex 50 columns in tandem to separate the various metabolites of isoniazid. Paper chromatographic systems have been used to isolate the various metabolites of isoniazid474,475,352. Barreto and S a b i n have ~ ~ ~described ~ a two dimension separation of isoniazid metabolites using paper chromatography and paper electrophoresis The same authors352 have also used a sodium sulfate column developed with chloroform-diethylamine(9O:lO) to separate the metabolites of isoniazid. Fartushnyi and S ~ k h i n *have ~ ~ used TLC to determine isoniazid and other drugs in cadavers. Cattaneo, Fantoli and Ferrari478 claim that their chromatographic studies indicate that the tumorogenic effect of isoniazid in mouse lung is due to the large amount of isonicotinic acid produced in that organ. Hughes479 separated acetylisoniazid from isoniazid by counter-current distribution (butanol-ethylene dichloride- 9:1 - 2M phosphate buffer pH 5.1). Ozawa and Kiyomoto480 isolated three conjugated metabolites of isoniazid by paper used paper chromatography. Cuthbertson et chromatography to determine isonicotinoylglycine. They used the following systems: Water saturated butanol Methylethy1ketone:acetic acid:water(49:1:50) Propano1:water (4:1) Zamboni and D e f r a n ~ e s c h iused ~~~ a isopropanol:water(85:15) system to separate the hydrazones of pyruvic and a -ketoglutaric acid from
i son i a z id. 7. Druq Metabolism The drug metabolism of isoniazid is unusually complicated in that it is a very reactive molecule and can undergo non-enzymatic reactions in the body. A general metabolic pattern is indicated in diagram. Non-enzymatic
Enzymatic 0
II
isonicotinoylhydrazones of glucose, a-ketoglutaric acid, aldehyde pyruvic acid etc. N ID
acetylisoniazid
or ketone
0
isonicotinamide N , N * diisonicotinoylhydrazide
230
GLENN A. BREWER
The major metabolite of isoniazid is N-acetylisoniazid. The rate of acetylation is has genetically ~ o n t r o l l e d 485. ~ ~ ~ ,It ~ ~ ~ been established that the slow acetylation is a autosoma1 recessive trait. The acylation occurs by N-acetyl transferase. Six hours after the oral administration of 4 mg/Kg of isoniazid fast acetylators have plasma concentrations of 0.2 pg/ml or less while slow acet lators have plasma levels higher that 0.4 pg/ml 48Yj In a metabolic scheme,such as the one indicated earlier,relative amounts of the various metabolites found in the urine will differ for each individual and will depend on genetic factors, previous drug history (enzyme induction) and general nutrition (availability of ketoacids). Reviews on the drug metabolism of isoniazid have been re ared b a number of authors487,488, 489,490,49!?, 462,493,i94,495,496,497
.
Toth and Shimizu have reported that the continuous administration of N-acetylisoniazid in rats has markedly increased the incidence of lung tumors in this species. Since the N-acetyl derivative is a major metabolite in man this poses some questions on the long term administration of the compound497. 8. Biopharmaceutics Kakemi and co-workers498 determined the rate of absorption of derivatives of isoniazid in the stomach and intestine. The authors report a rough correlation between degree of absorption and lipidwater partition coefficient.
ISONIAZID
231
9.
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