(2) obtained nicotinic acid amide (nicotinamide) from a coenzyme isolated from the red blood cells of the horse. This finding stimulated further studies on the.
NICOTINAMIDE
E z z a t M . Abdel Moety*, Mohammad T a r i q * * Abdullah A . Al-Bad?-*
* **
and
Department o f P h a r m a c e u t i c a l C h e m i s t r y , C o l l e g e o f Pharmacy, K i n g Saud U n i v e r s i t y , P.O. Box 2457, Riyadh-11451, Saudi A r a b i a . D e p a r t m e n t of P h a r m a c o l o g y , C o l l e g e o f Pharmacy, K i n g Saud U n i v e r s i t y , P.O. Box 2 4 5 7 , Riyadh-11451, Saudi A r a b i a .
ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 20
475
Copyright 0 1991 By Academic Press, Inc. All rights of reproduction in any form reserved.
EZZAT M. ABDEL MOETY ET AL
416
CONTENTS ____ 1. L .
INTRODUCTORY CESCRIFTION 2.7
:.
2.3 ?.? 4.
Nomenclature Formu 1 de Molecular Weight Appearance, Color, Odor and Taste
PHYSICAL CHARACTERISTICS 3.1 3.: 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11
Melting point Solubility pH-Range Optical Activity Moisture Content and Hygroscopicity Dissociation Constant Stabi 1 ity Storage Identification Spectral Properties Crystallographic Characteristics
4.
SLNTHESIS
5.
PHA,RMACOKINETICS 5.1 5.2
5.3
Absorption and Distribution Metabol ism Excretion AND SIDE EFFECTS
C.
TOXTCITY
7.
METHODS OF ANALYSIS 7.1
Qualitative Analysis (Identification) 7.11 7.12 7.13
7.2
Microchemical Tests Elemental Composition Pharmacopoeia1 Specifications
Quantitative Analysis (Determination) 7.21
Volumetry
NICOTINAMIDE
7.22
7.23 7.24
7.25 7.26
ACKNOWLEDGEMENTS
REFERENCE5
Electrochemical Methods Spectrophotometry Chromatography Gravimetry Automated A n a l y s i s
411
EZZAT M. ABDEL MOETY ETAL
47 8
NICOTINAMIDE
1.
INTRODUCTORY
Nicotinamide, a s u b s t i t u t e d p y r i d i n e d e r i v a t i v e , i s an a n t i p e l l a g r a a g e n t a n d i s c a l l e d V i t a m i n PP ( p e l l a g r a - p r e v e n t i n g ) . I t i s used f o r t r e a t i n g v i t a m i n d e f i c i e n c y i n h y p e r t e n s i v e p a t i e n t s ( 1 ) because i t l a c k s t h e v a s o d i l a t o r a c t i o n . Warburg and Associates ( 2 ) obtained n i c o t i n i c a c i d amide ( n i c o t i n a m i d e ) from a coenzyme i s o l a t e d from t h e r e d b l o o d c e l l s o f t h e horse. T h i s f i n d i n g s t i m u l a t e d f u r t h e r s t u d i e s on t h e n u t r i t i o n a l value o f n i c o t i n i c acid. L i v e r e x t r a c t s were known t o be h i g h l y e f f e c t i v e i n c u r i n g human p e l l a g r a and c a n i n e b l a c k t o n g u e . E l v e h j e m and Associates ( 3 ) prepared concentrates of l i v e r t h a t were h i g h l y e f f e c t i v e i n t h e treatment o f canine b l a c k t o n g u e , and i d e n t i f i e d t h e a c t i v e s u b s t a n c e as n i c o t i n a m i d e ( 3 ) . I t was f i r s t p r e p a r e d i n 1894 by r e a c t i n g e t h y l n i c o t i n a t e w i t h concentrated ammonia (4). The drug decomposes by h y d r o l y s i s and an obvious and e f f e c t i v e means o f s t a b i l i z a t i o n i s t o l i m i t access o f t h e d r u g t o w a t e r . I n such cases s o l i d dosage f o r m s a r e n o r m a l l y recommended (5). As t h e amide i s l a i a b l e t o h y d r o l y t i c c o n v e r s i o n t o t h e p r o t o t y p e n i c o t i n i c a c i d ; t h e a c i d can m a i n l y metabolized t o t h e corresponding amide. Nicotinarnide can a l s o be s y n t h e t i c a l l y obtained from n i c o t i n i c a c i d by s e v e r a l m o d i f i c a t i o n s . Most o f t h e p u b l i s h e d a n a l y t i c a l procedures deal with t h e s o l u t i o n o f t h e coexistance o f t h e two drug forms i n b u l k m a t e r i a l , dosage f o r m u l a t i o n s and/or i n b i o l o g i c a l specimens. T h i s demonstrates how i t i s important t o i n c l u d e b o t h t h e drug substances i n one p r o f i l e .
NICOTINAMDE
2.
479
DESCRIPTION 2.1
Nomenclature 2.11
Chemical Names
Nicotinamide: Pyridine-3-carboxamide; Nicotinic acid m i d e ; 3-Pyridine c a r b o x y l i c a c i d amide; 3-Pyridine carboxamide ( 6 ) . N i c o t i n i c acid: P y r i d i n e - 3 - c a r b o x y l i c a c i d ; 3-Pyrid i n e c a r b o x y l i c a c i d ; Pyridine-l3-carboxylic a c i d . 2.12
Generic Names
Nicotinamide: Niacinamide; midum; Vitamin PP; Vitamin P i l o n i n amide; Amide PP.
Nicotylamide; NicotinaAminocotin; Dipegyl;
B3;
N i c o t i n i c acid: Niacin; PP-(Pellagra Preventive) f a c t o r ; a n t i p e l l a g r a v i t a m i n ; Acidum Nicotinicum. 2.13
Trade Names
Nicotinamide: Nicamina; Nicobion; Nicosedine; N i c o v e l ; N i c o v i t ; Nicamindon; Nicotamide; N i c o f o r t ; Niozymin; Pelmine; B e n i c o t ; V i n i e o t y l ; Amisyn; Farmabion; N i c o t i n a m i d e ; P e l o ; A p i s a t e ( i n c o m b i n a t i o n ) and E f f i c o (6,7). N i c o t i n i c a c i d : Nico-400; Nicobid; Nicotinex; Nico-Span; N i c y l ; E q u i v e r t , P e r n i v i t , N i c o l a r , Niconacid; N i c a c i d ; Nicagin; Nicotene; N i c o t i n i p c a ; Akotin; D a s k i l ; N i c o l a r ; T i n i c ; and Wampocap. (6,7). 2.14
Chemical A b s t r a c t R e g i s t r y Number [ C A S l
Nicotinamide: N i c o t i n i c acid: 2.2
198-92-01
[59-67-61
Formulae 2.21
Empirical
N ic o t inami de: CsHsN2O N i c o t i n i c acld: CeH5N02
EZZAT M. ABDEL MOETY E T A L
480
2.22
2.23
Structural
@CONH2
@COOH
Nicotinamide
Nicotinic acid
Wiswesser
Line Notation
Nicotinamide: T6NJ CVZ ( 8 ) Nicotinic acid: T6NJ CVQ (8) 2.3
Molecular Weight
Nicotinamide: Nicotinic acid: 2.4
122.13 123.11
Appearance, Color, 0 d-x and Taste
Nicotinamide exists as colorless crystals or white crystalline powder; faint characteristic odor, salty and bitter taste ( 9 ) ; while nicotinic acid appears as needles or white t o creamy-white crystals or crystal 1 ine powder ( 7 ) . 3.
______ PHYSICAL ~CHARACTERISTICS 3.1
Melting Point
Nicotinamide: 128'-131°C ( 7 , 9 , 1 0 ) . Nicotinic acid: 236.6'C (6). 234-237 "C (7). 3.2
Solubility
Nicotinamide i s highly soluble in water and alcohols; 1 g dissolves in about 1 ml of water, in about 1 . 5 ml alcohol and in 10 ml glycerol. It is slightly soluble in ether and chloroform, while it is soluble in solutions o f alkali (9,1?,13). Nicotinic acid, 1 g,
NICOTINAMIDE
48 I
is soluble in 55 to 60 ml o f water and in 100 rnl o f ethanol, very slightly soluble in chloroform; practically insoluble in ether; soluble in alkaline solutions o f hydroxides and carbonates (7,111. 3.3
pH-Range
A 1.3% ( w / v ) aqueous solution of nicotinamide gives a pH 3-3.5; its 5% solution in water has a pH o f 6 to 7.5 and the 10% solution i n water is neutral to litmus (6,11,12). The pH o f saturated solution o f nicotinic acid is about 2 . 7 (6). 3.4
ODtical Activity
Both the drug substances are optically inactive because of the absence o f chemical asymmetry. 3.5
Moisture Content and HvgroscoPicity
Nicotinamide absorbs insignificant amounts o f moisture a t 2 5 ° C at relative humidities up to about 90% ( 4 ) . 3.6
Dissociation Constant
The dissociation constant, pK, of nicotinamide: 3.3 [ 2 O " C 1 (4); while the pK of nicotinic acid = 4 . 8 5 [25'Cl (61, pKa 2.00 (-N=), 4.80 (-COOH) [25'CI (7).
3.7
Stability
Nicotinamide is stable certain pH-changes (11). 3.8
in
air, light and at
Storage
Both drug substances shou d be stored containers ( 4 ) . 3.9
n air-tight
Identification 3.91
Color Test
To 2 ml of 0.1% solution o f nicotinamide or nicotinic acid add 6 ml o f cyanogen bromide solution and 1 ml of a 2 . 5 % v/v solution o f aniline golden yellow; i.e.
EZZAT M.ABDEL MOETY ET AL.
482
K o n i g ’ s r e a c t i o n . N i c o t i n a m i d e g i v e s brown-orange c o l o r w i t h Nessler’s reagent ( 7 ) . 3.92
Microcrystal
Test
Nicotinamide y i e l d s with gold c h l o r i d e s o l u t i o n dense r o s e t t e s ( s e n s i t i v i t y 1 i n 400) and w i t h p l a t i n i c i o d i d e s o l u t i o n a mass o f h a i r l i k e neeldes ( s e n s i t i v i t y : 1 i n 400) ( 7 ) .
-
3.10
SDectral P r o m r t i e s 3.101
U l t r a v i o l e t SDectrum
The u l t r a v i o l e t spectrum o f nicotinamide i n ethanol (using a Varian UV-vis spectrophotometer model DMS 90) i s shown i n F i g u r e 1. The spectrum o f t h e drug e x h i b i t s a maximum a t 261 nm [ A ( l % , lcm) = 451, E = 5.507 x l o 3 ] . The UV-running o f methanolic s o l u t i o n s of n i c o t i n i c a c i d e x h i b i t s a c h a r a c t e r i s t i c maximum a t about 263 nm [ A ( l % , lcm)) = 2 2 9 , E = 2.819 x 1031. 3.102
I n f r a r e d SDectrum
The i n f r a r e d spectrum o f nicotinamide, i n KBr d i s c , i s presented i n Figure 2, (recorded on Pye Unicam SP 1025 i n f r a r e d s p e c t r o p h o t o m e t e r ) . The f r e q u e n c i e s and s t r u c t u r a l assignments o f nicotinamide and n i c o t i n i c a c i d are shown below: Frequency (cm-r)
Assignment
1600- 1630
c
1700 1710
c c
3200
N
3100
0
3.103
-
C
=0
0
-H -H
( s t r e t c h , amide and a c i d ) (amide) (acid) ( s t r e t c h ; amide) (stretch, acid)
Proton Nuclear SDectrurn I’H-NHRl
The ’H-NMR runnings were undertaken f o r each drug substance. The l H - N M R spectrum o f n i c o t i n a m i d e i s shown i n Figure 3. Each drug substance was dissolved
NlCOTINAMIDE
200
Fig. 1:
250
300Wavelength 350
483
400
The ultraviolet (UV) scanning o f ethanolic solution
o f nicotinamide.
484
ioo
300
400
200
100
0
DHO
-
b
. .
I
8.0
I
.
7.0
I
.
6.0
.
.
I
5.0
.
.
I
6
4.0
.
.
.
I
.
3-0
.
.
.
t
-%/
.
.
.
.
l
1.o
2.0
~~
F i g . 3:
The proton nuclear magnetic resonance (1H-NMR) spectrum o f nicotinamide.
.
.
.
.
l
0
EZZAT M. ABDEL MOETY ETAL
486
i n deuterium oxide ( D 2 0 ) and i t s spectrum determined on a V a r i a n T 6 A NMR s p e c t r o m e t e r u s i n g DSS (sodium-2,2-dimethyl-2-silapentane-5-sulfonate) as t h e i n t e r n a l s t a n d a r d . The s t r u c t u r a l assignments a r e shown below:
Nicotinamide Chemical S h i f t (6,DDm)
MultiDlicitv
acid
Protop Assignment
7.60
mu1t ip l e t
H(3)
8.25
mult f p l e t
H(2)
8.72
:~ru1t i p l e t
H(4)
8.93
doub 1e t
H(1)
re
singlet
H ( 5 ) (amide)
9.14
singlet
H(6)
9.
3.104
The
Nicotinic
Carbon-13
(acid)
NHR SDectrum C13C-NHRI
13C-NMR spectra o f nicotinamide i n deuterium oxide
u s i n g DSS as t h e i n t e r n a l r e f e r e n c e were o b t a i n e d u s i n g J e o l FX 100 MHz s p e c t r o m e t e r a t an ambient temperature. F i g u r e 4 r e p r e s e n t s t h e 'H-decoupled spectrum of nicotinamide. (D20)
L: Fig. 4:
The carbonl3 nuclear magnetic resonance (13C-NMR) o f nicotinamide (carbon assignment see t e x t j .
~~~
~~~
EZZAT M. ABDEL MOETY ETAL.
488
Nicotinic acid
N i c o t inamide Chemical s h i f t
& PPEl
Acid+
~~
Mu1t i 01 ic it y
Carbon Assignment
A m i 3e
150.3
150.2
doublet
133.5
131.3
singlet
138.6
138.7
doublet
124.8
126.8
doublet
151.5
154.3
doub 1e t
173.8
172.0
singlet
+ E . Lecte; Bioorg. Chem.: 5 , 273 (1977); c f . M. Shamna
and D.M. Hindenlang (Eds.) i n : Carbon-13 NMR S l i g h t Assignments o f Amines and A l k a l o i d s , Plenum Press, New York (1979) p. 24. 3.105
Hass Spectrum
The e l e c t r o n impact (EI) mass spectrum o f n r c o t i n a m i d e a t 70 eL recorded on Varian Mat 311 mass spectrometer and t h e methdne d e r i v e d chemical i o n i z a t i o n ( C I ) mass spectrum s b t a i n e d with F i n n i g a n 4000 mass spectrometer a r e shown i n F i g u r e s 5 & 6 , r e s p e c t i v e l y . The EI spectrum shows 8 molecular ion peak a t m/e 122 (base p e d k ) . Othe: f r a g m e n t s a r e a t m/e 106 and 78 w h i c h correspond t o M-NHr and M-CONH2 r e s p e c t i v e l y . The C I spectrum, i s simple, t h e base peak (MH) i s t h e o n l y
NICOTINAMIDE
SCHEME
1
49 1
0
0
II
II
Nicotinic acid
-~ Nicotinamide
peak and i s a t m/e 123. Scheme 1 i l l u s t r a t e s t h e p o s s i b l e f r a g m e n t a t i o n p a t t e r n s o f n i c o t i n a m i d e and n i c o t i n i c acid. 3.11
CrYstallosraDhic Characteristics 3.11 1
X-Ray Powder D i f f r a c t i o n P a t t e r n s
The X-ray powder d i f f r a c t i o n p a t t e r n o f n i c o t i n a m i d e were determined u s i n g P h i l i p s f u l l y automated X-ray d i f f r a c t i o n spectrogoniometer equipped w i t h PW1730/10 generator. R a d i a t i o n was p r o v i d e d by a copper t a r g e t (Cu anode 2000W, I: = 1.5480 A ) h i g h i n t e n s i t y X-ray tube operated a t 40 KV and 35 MA. The monochromator was a c u r v e d s i n g l e c r y s t a l one (PW1752/00). D i v e r g a n c e s l i t and t h e r e c e i v i n g s l i t were 1 and 0 . 1 nm, r e s p e c t i v e l y . The s c a n n i n g speed o f t h e goniometer (PW1050/81) used as 0.02-28 p e r second. The instrument was combined w i t h P h i l i p s PM8210 p r i n t i n g recorder and connected w i t h b o t h analogue recorder and d i g i t a l p r i n t e r . The g o n i o m e t e r was a l i g n e d u s i n g s i l i c o n sample b e f o r e use ( 1 4 ) . The X-ray p a t t e r n i s demonstrated i n F i g . 7 .
EZZAT M. ABDEL MOETY ETAL
492
7065
Fig. 7:
55
45
35 2%
25
I5
5
The X-ray powder d i f f r a c t i o n p a t t e r n o f n i c o t i n a m i d e .
NICOTINAMIDE
Table 1:
493
The X-ray d i f f r a c t i o n a l p r i n c i p a l l i n e s o f nicotinamide
29
d(A)
5.351 11.052 11.215 14.731 19.045 19.521 19.867 22.197 22.653 23.203 24.659 25.286 25.664 27.138 21.364 29.331 30.093
16.5148 8.0057 7.8892 6.0133 4.6598 4.5473 4.4689 4.0048 3 9252 3.8334 3.6101 3.5221 3.4711 3.2857 3.2592 3.0450 2.9695 3.112
I/IoX100 10.62 3.33 4.10 100.00 6.35 8.50 9.85 35.04 3.32 2.77 4.10 8.22 21.02 15.50 9.70 2.25 3.20
20 32.478 33.586 34.122 34.459 35.716 36.947 38.625 39.097 39.618 40.134 40.955 41.372 41.554 48.740 49.188 50.346 52.250
d(A)
2.7565 2.6682 2.6275 2.6026 2.5139 2.4329 2.3310 2.3039 2.2748 2.2467 2.2036 2.1823 1.9121 1.8683 1.8523 I .8124 1,7507
D i f f e r e n t i a l Thermal Scanning
I / I0x 100 3.61 6.80 2.58 4.28 2.30 14.68 11.92 2.08 2.60 2.42 5.25 3.84 2.84 2.82 2.18 3.90 2.71
0
The DSC-curves f o r n i c o t i n a m i d e and n i c o t i n i c a c i d were obtained s e p a r a t e l y on a DuPont TA-9900 Thermal Analyzer attached t o a d a t a processing u n i t . F i g u r e 8 shows t h e DSC-curve o f nicotinamide. The runnings a r e between 120-270°C a t a h e a t i n g r a t e o f 10’C.min-l. The heat o f a c t i v a t i o n and t h e p u r i t y o f t h e samples were d e t e r m i n e d u s i n g p u r i t y program. The A H - v a l u e s a r e 22.7 and 25.6 KJ.mole-1 f o r n i c o t i n a m i d e and n i c o t i n i c a c i d , i n order. 4.
SYNTHESIS
Various s y n t h e t i c pathways have been demonstrated f o r p r e p a r a t i o n o f t h e n i c o t i n i c a c i d and/or i t s amide u t i l i z i n g v a r i o u s s t a r t i n g m a t e r i a l s and procedures. The p r o t o t y p e a c i d , i . e . n i c o t i n i c a c i d , can be o b t a i n e d f r o m q u i n o l i n e v i a o x i d a t i o n f o l l o w e d by decarboxylation. N i c o t i n a m i d e can be p r e p a r e d by conversion o f n i c o t i n i c a c i d through t h e treatment e i t h e r w i t h u r e a (15-18) o r ammonia (19-23). The n i c o t i n i c a c i d e s t e r s can be converted i n t o t h e amide
200
210
220
230
240
Temperature
F i g . 8:
Cc')
250
260
The DSC-scanning o f nicotinamide.
270
NICOTINAMIDE
495
by r e a c t i o n w i t h ammonia (4, 24). N i c o t i n a m i d e can a l s o be synthesized from t h e ammonium n i c o t i n a t e ( 2 5 ) o r a c e t y l p y r i d i n e (26) by t h e treatment w i t h ammonia. Reaction o f asparagine with glutamic a c i d can y i e l d n i c o t i n a m i d e (27). The v i t a m i n amide can be o b t a i n e d from n i c o t i n o n i t r i l e by adopting v a r i o u s h y d r o l y z i n g reagents and c o n d i t i o n s . Among t h e r e p o r t e d reagents are water ( 2 8 ) , aqueous NaOH (29-31), a l k a l i n e H202 (32-37), ammonia (38-40), d i l u t e a c i d (41 8, 4 2 1 , c o n c e n t r a t e d a c i d s (43-461, a n d o n m o i s t e n e d v i z i o n i t e AB-17, OH-form ( 4 7 ) . anion-exchanger; N i c o t i n i c a c i d and i t s amide can be s y n t h e t i c a l l y p r e p a r e d by vapor-phase o x i d a t i v e ammonolysis o f m i x t u r e s o f 3 - c y a n o p y r i d i n e and q u i n o l i n e ( 4 8 ) . Different pyridine derivatives including nicotinic a c i d and n i c o t i n a m i d e can be obtained s t a r t i n g from methane, ammonia and water by h i g h frequency e l e c t r i c discharge f o l l o w e d by TLC-separation on s i l i c a g e l G u s i n g AcOEt-MeOH-HCOnH, 75:20:5, by volumes ( 4 9 ) . Some o t h e r miscellaneous procedures have been described f o r t h e p r e p a r a t i o n o f a n t i p e l l a g r a a c i d / o r amide (50-54). 5.
PHARMACOKINETICS 5.1
AbsorDtion and Distribution
Nicotinamide i s r e a d i l y absorbed from a l l p o r t i o n s o f gastrointestinal t r a c t (2). Nicotinic acid i s also absorbed from t h e food c o n t e n t s i n t h e g a s t r o i n t e s t i n a l t r a c t , which a f f e c t s t h e b i o a v a i l a b i l i t y o f o r a l l y administered n i c o t i n a m i d e ( 5 5 ) . I n u n t r e a t e d human s u b j e c t s n i c o t i n i c a c i d plasma l e v e l s g e n e r a l l y r a n g e f r o m a b o u t 0.1-0.3 mg% ( 5 6 ) . Continuous biochemical i n t e r a c t i o n s a t v a r y i n g r a t e s between nicotinamide, n i c o t i n i c a c i d , t h e i r complexes and t h e i r m e t a b o l i t e s may predominate over t h e r a t e and completeness o f g a s t r o i n t e s t i n a l (GI) a b s o r p t i o n o f n i c o t i n a m i d e (57). The peak l e v e l i s achieved i n about 30 min f o l l o w i n g o r a l a d m i n i s t r a t i o n , and t h e i s 5 . 3 h o u r s and t h e d r u g b l o o d h a l f l i f e (T$) disappeared from t h e l i v e r w i t h a h a l f - l i f e o f 4 . 4 hours ( 4 ) . Nicotinamide l e v e l has been r e p o r t e d t o be h i g h e s t i n t h e k i d n e y and i n d e c r e a s i n g o r d e r , t h e 1 iv e r , muscle, i n t e s t i n e , h e a r t , s p l e e n and 1ungs
(58).
EZZAT M. ABDEL MOETY E T A L
496
a Y 0
SCHEME 2 --
OII
N ico t i n a m i d e &oxide
at-o~
0
Nicotinamiae
C-N&
ti 6-hyd rox y
HO 6- hyaroxy
n i c o t i n i c Zcid
C-NH2
nico t in a m I ae
CH3 N-met hyl nicotinamide
C-NH2 I
I
N-me thy[--3 -carboxamide 4 p y r idone
-
.
CY
O
..
C-NH2
I
CH3
N-me?hyI -2-pyridone-5-carboxamide ( 6 - pyr i done 1
NICOTINAMIDE
5.2
497
Metabolism
The metabolism o f n i c o t i n a m i d e i n human body t a k e s place i n t o f o u r pathways as mentioned i n Scheme 2 . (I) Formation o f n i c o t i n i c a c i d and 6-hydroxy n i c o t i n i c a c i d , (11) Formation o f N-methyl n i c o t i n a m i d e and s u b s e q u e n t c o n v e r s i o n o f t h i s compound t o N-methyl-3-carboxamide 4-pyridone and N-methyl-2-pyri(111) Formation o f done-5-carboxamide 6-pyridone, 6-hydroxy nicotinamide and ( I V ) Nicotinamide N-oxide. I n animals most o f the nicotinamide i s metabolized t o nicotinamide N-oxide, i n a d d i t i o n t o N-methylnicotinam i d e , 6 - p y r i d o n e and 4 - p y r i d o n e . The l e v e l o f n i c o t i n a m i d e i n l i v e r and t i s s u e c e l l s a r e under feedback c o n t r o l and mainly depends on d i e t a r y i n t a k e and metabolic u t i l i z a t i o n (55). Nicotinamide l e v e l s i n serum a r e b u f f e r e d by t h e l i v e r by b e i n g c o n v e r t e d i n t o storage form o f NAD. I n c e l l , t h e t u r n over o f nicotinamide i s proportional t o t h e formation o f poly-ADPR as shown i n scheme 3. There i s indeed deamination, since f r e e 6-hydroxynicotinic a c i d and n i c o t i n i c a c i d a r e seen i n t h e mouse and r a t a f t e r i n j e c t i o n o f nicotinamide (59-63). 5.3
Excretion
When t h e r a p e u t i c doses a r e a d m i n i s t e r e d o n l y s m a l l amounts o f the unchanged nicotinamide appear i n t h e u r i n e (61 & 62). When e x t r e m e l y h i g h doses o f t h i s v i t a m i n i s given, the unchanged v i t a m i n represent the major component i n the u r i n e (64). Chaykin e t a1 (65) have reported t h a t a minimum o f 98% o f r a d i o a c t i v i t y was present i n mouse u r i n e a f t e r t h e a d m i n i s t r a t i o n o f nicotinamide-7- 1 4 C , i t was found i n t h e form o f the f o l l o w i n g m e t a b o l i t e s : N1-methylnico- t i n a m i d e , n ic o t in i c a c i d , n ic o t inamide-N-ox i d e , N1 -methyl -4pyridine-3-carboxamide, N 1 - m e t h y l - 4 - p y r i d i n e - 3 - c a r boxamide, N1-methyl-2-pyridone-5-carboxamide and nicotinamide.
6.
TOXICITY &ND SIDE EFFECTS Nicotinamide has a LDso o f 1.68 g/kg body weight f o r r a t s (66-68) and 1.75 g/kg f o r mice. The t o x i c i t y does not vary g r e a t l y w i t h the route o f a d m i n i s t r a t i o n . I t has a stimulant a c t i o n on t h e c e n t r a l nervous system and does not increase the work output o f the perfused
L.
c
a
-a Z
.-V .-C
a
c
C
.-
0:
W
> 1 I
I I
I
I
I
I
I I
I I I
I
NICOTINAMIDE
499
f r o g g a s t r o i n e m i u s muscle (69). L e t h a l doses cause c o n v u l s i o n s (67) and s t i l l h i g h e r doses (0.5-1.0 mg/kg) i n j e c t e d i n t r a v e n o u s l y o r i n t r a p e r i t o n e a l l y cause a f a l l o f b l o o d pressure and a r i s e i n t h e h e a r t r a t e and increase t h e r a t i o and amplitude o f b r e a t h i n g The p a r e n t r a l and sometimes i n p u l s e r a t e ( 7 0 ) . a d m i n i s t r a t i o n o f n i c o t i n a m i d e i n l a r g e doses may cause f u r u n c u l o s i s and o t h e r s k i n l e s i o n s , malaise, a c t i v a t i o n o f p e p t i c u l c e r , j a u n d i c e and impairment o f l i v e r f u n c t i o n ( 6 4 ) . N i c o t i n a m i d e i s suspected o f being t e r a t o g e n i c i f taken i n excess by pregnant women i n the f i r s t trimester (1). 7.
METHODS
7.1
OF ANALYSIS
Q u a l i t a t i v e Analvsis ( I d e n t i f i c a t i o n ) 7.11
m r o c h e m i c a l Tests
G a u t i e r ( 7 1 ) i n v e s t i g a t e d a p r e c i p i t a t i n g spot t e s t u s i ng mercur ic c h l o r i d e t o d i f f e r e n t ia t e n ic o t inamide from t h e p r o t o t y p e n i c o t i n i c acid. The p r e c i p i t a t e d n i c o t i n i c a c i d o r i t s amide can be e a s i l y regained by microsublimation. Sandri ( 7 2 ) recommended b r o m o t h a l l i c a c i d as a p r e c i p i t a t i n g r e a g e n t t o d i f f e r e n t i a t e between s e v e r a l bases i n c l u d i n g n i c o t i n a m i d e . Ludy-Tenger ( 7 3 ) described what he c a l l e d " B i l e 1.5" r e a g e n t f o r c h a r a c t e r i z a t i o n of n i c o t i n a m i d e and n i c o t i n i c acid. Nicotinamide g i v e s c h a r a c t e r i s t i c and reproduc ib l e r e a c t i o n s w i t h d i f f e r e n t aldehydes , p a r t i c u l a r l y v a n i l l i n and benzaldehyde. Comparative reconimenda t ion o f b romot ha 1 1 ic ac id , b romop 1 a t in ic a c i d , bromoauric a c i d and sulfocyanides o f some metal ions was discussed f o r microchemical i d e n t i f i c a t i o n o f nicotinamide ( 7 4 ) . Konig's r e a c t i o n , i n v o l v i n g t h e r e a c t i o n of p y r i d i n e d e r i v a t i v e s w i t h cyanobromide and a n i l i n e , was s t u d i e d e x t e n s i v e l y on t h e example o f n i c o t i n i c a c i d and i t s amide by Ciusa and B a r b i r o l i (75). H i g h a b s o r p t i o n d a t a were shown i n aqueous media i n case of n i c o t i n i c a c i d and n i c o t i n a m i d e . Quantification o f the Konig's reaction f o r d e t e r m i n a t i o n o f n i c o t i n a m i d e i n u r i n e ( 7 6 ) and pharmaceutical f o r m u l a t i o n s (77-79) was evaluated t o o . Reaction o f n i t r i c a c i d with nicotinamide provides r a p i d p r e c i p i t a t i o n o f t h e n i t r a t e s a l t (m.p. 223-5'C) c h a r a c t e r i s t i c f o r t h e drug ( 8 0 ) . Zivanov e t a1 ( 8 1 ) r e p o r t e d t h a t on h e a t i n g n i c o t i n a m i d e w i t h m e t h y l
EZZAT M. ABDEL MOETY ET AL
500
i o d i d e [55-60:C, 40 m i n . ) o r e t h y l i o d i d e (80-85’C, 2 h r s ) i t g i v e s t h e quaternary s a l t s . The p r e c i p i t a t e d nicotinamide-Me1 (yellow, m.p. 201-6’C) and n i c o t i n a m i d e - E t I (yellow-green, m.p. 146-8’C) can be used f o r g r a v i m e t r i c q u a n t i f i c a t i o n o f t h e drug (81). Van d e r Wegen ( 8 2 ) d e s c r i b e d s e v e r a l m i c r o c h e m i c a l s p o t p r e c i p i t a t i n g r e a g e n t s f o r some b a s i c d r u g substances i n c l u d i n g n i c o t i n a m i d e . D o b r y (83) suggested R o u s s i n ’ s s a l t , [KFed(N0)7S2)] ( i r o n riitrosyl sulphidej, t o form c h a r a c t e r i s t i c p r e c i p i t a t i o n s w i t h many a m i n e s o f b i o l o g i c a l i n t e r e s t s including nicotinamide. Inorganic p r e c i p i t a t i o n can be used f o r g r a v i m e t r i c d e t e r m i n a t i o n s and as t h e p r e c i p i t a t e s a r e d e e p l y $ c o l o r e d , s u c h amines can be c o l o r i r n e t r i c a l l y determined; as l i t t l e as 106 1-(g o f n i c o t i n a m i d e can be determined by t h e proposed method (83). 7.12
Elemental C m D o s i t i o n C
H
H
(XI
(X)
(X)
0
- - - (XI
123.11):
58.53
4.09
11.38
25.99
(CBH8N20, 1 2 2 . 1 3 ) :
59.00
4.95
22.94
13.10
N i c o t i n i c a c i d (C6H5N02,
:iicotinamide
7.13
Pharmacopoeia1 S p e c i f i c a t i o n s
The USP X X I (10) s p e c i f i e s t h e comparison o f t h e I R - & UV-spectra o f n i c o t i n i c a c i d and n i c o t i n a m i d e samples w i t h t h e USP-reference s t a n d a r d o f t h e a c i d o r i t s amide, i n addition t o the identity test with 2,J-dinitrochlorobenzene and a l c . KOH (deep r e d t o wine r e d c o l o r a t i o n ) and t e s t w i t h copper sulphate on t h e n e u t r a l s o l u t i o n ( b l u e p r e c i p i t a t i o n ) i n case o f n i c o t i n i c acid. The BP-1988 (8 4 ) a l s o recommends t h e concordance o f t h e sample o f n i c o t i n i c a c i d o r i t s amide w i t h t h e corresponding I R - and CJV-spectra of t h e BP-reference drug substance. Color r e a c t i o n w i t h cyanogen bromide and a n i l i n e , i . e . K o n i g ’ s r e a c t i o n , (gives yellow coloration) i s also described f o r both drug substances, w h i l e t h e e v o l u t i o n ” o f ammonia vapours on h o t r e a c t i o n w i t h sodium hydroxide i s mentioned f o r the amide o n l y .
501
NICOTINAMIDE
7.2
Q u a n t i t a t i v e Analysis m t e r m i n a t l d 7.21
Volumetry 7 . 2 1 1 Determination i n b u l k m a t e r i a l
Krewson ( 8 5 ) d e s c r i b e d an a c i d - b a s e r e s i d u a l t i t r i m e t r i c method by i n v o l v i n g a c i d s p l i t t i n g o f t h e amide bonding t h e n f o l l o w e d by a l k a l i n i z a t i o n and d i s t i l l i n g the equivalent NH3 i n t o a known excess o f standard acid. The u n r e a c t e d a c i d c o u l d be a c i d i m e t r i c a l l y determined u s i n g methyl r e d as a suitable indicator. S r e e r a m and N a i d u (86) i n v e s t i g a t e d the non-aqueous t i t r a t i o n o f nicotinamide and n i c o t i n i c a c i d w i t h chlorosulphuric a c i d i n dioxan using c r y s t a l v i o l e t and photometric t i t r a t i o n a t 610 nm. B i a n c h i ( 8 7 ) d e s c r i b e d a s i m i l a r non-aqueous photometric t i t r a t i o n u t i l i z i n g 0.2% malachite green o r n i l e blue i n a c e t i c a c i d as i n d i c a t o r s . T i t r i m e t r i c q u a n t i f i c a t i o n o f n i c o t i n a m i d e i n nonaqueous medium has been i n v e s t i g a t e d by using HC104 as the standard and F e t t b l a u as the s u i t a b l e i n d i c a t o r (88). Helmstaedter ( 8 9 ) suggested a t i t r i m e t r i c procedure f o r q u a n t i t a t i v e determination o f n i c o t i n a m i d e v i a i t s p r e c i p i t a t i o n from aqueous By d i s s o l v i n g t h e s o l u t i o n s b y e x c e s s HgC12. p r e c i p i t a t e d mercury s a l t of t h e d r u g i n HC1, t h e t i t r a t i o n was then c a r r i e d out i n d i r e c t l y w i t h EDTA a f t e r rendering ammonical and using standard ZnS04 t o d e t e r m i n e t h e r e s i d u a l amount o f t h e c h e l a t i n g standard. To q u a n t i f y n i c o t i n i c a c i d by f o l l o w i n g t h e suggested procedure, CuSO4 was a d v i s e d f o r p r e c i p i t a t i o n instead o f HgCl2 i n case o f t h e amide. N e u t r a l i m e t r y w i t h 0.1 M NaOH was d e s c r i b e d by t h e BP-1988 ( 8 4 ) f o r n i c o t i n i c a c i d i n b u l k m a t e r i a l (using phenolphthalein) and i n t a b l e t s (using phenol red). 7.212 Determination Preparations
Pharmaceutical
A l l t h e v o l u m e t r i c methods d e s c r i b e d f o r t h e q u a n t i f i c a t i o n o f nicotinamide i n dosage formulations a r e c a r r i e d o u t i n non-aqueous media u t i l i z i n g standard H C l O 4 b u t d i f f e r e n t non-aqueous solvents and indicators. Table 2 summarizes t h e s o l v e n t s ( f o r
Table 2:
NOn-aQUeOUS determination ~~~
of nicotinamide
~~
Solvent Standard
Drug
Indicator (solvent)
Dosage form injections
Reference
Glacial acetic acid
Methyl violet (chlorobenzene)
Tablets
Dioxane CHCl3 (hot)
Methyl red
Dragees
91
Glacial acetic acid
Crystal violet (acetic acid)
Injections Tablets
84
&
90
92
503
NICOTINAMDE
s t a n d a r d and d r u g ) and non-aqueous procedure.
the
indicators
i n eacn
for The use o f 1,3-dibromo-5,5-dimethylhydantoin d i r e c t d e t e r m i n a t i o n o f n i c o t i n a m i d e i n some dosage f o r m u l a t i o n s by u s i n g m e t h y l r e d , m e t h y l orange. Vat Blue 1 ) o r amaranth (C.I. Acici Red indigo (C.I. 2 7 ) was compared w i t h i n d i r e c t i o d i m e t r y and those o f N-bromosuccinimide (NBS) a s w e l l as an o f f i c i a l methods ( 9 3 ) . 7.22
Electro.chemica1 Methods
7 . 2 2 1 0s.ci ll-lletry VaJYand and Pastor ( 9 4 ) recommended an o s c i l l o m e t r i c t i t r a t i o n procedure f o r q u a n t i f i c a t i o n o f some o r g a n i c amines i n c l u d i n g n i c o t i n a m i d e i n g l a c i a l a c e t i c a c i d . Reproducible r e s u l t s c o u l d be obtained i n case o f t h e H C l - s a l t o f t h e drug. 7.222 Potent-iw-t.
Nicotinamide can be p o t e n t i o m e t r i c a l l y t i t r a t e d u s i n g
H C l O 4 s o l u t i o n i n a c e t o n i t r i l e c o n t a i n i n g 1-30% water
as s t a n d a r d and g l a s s e l e c t r o d e , i . e . indicator electrode, w i t h a reference electrode containing Ag”/AgCl ( s ) / C l - . The e q u i 1 i b r i u m p o t e n t i a l of t h e c e l l reached i n 10-15 min. showed h i g h e r accuracy w i t h low p H - d e v i a t i o n t h a n when t h e t i m e was i n 1 min. (95). Grrybowska ( 9 6 1 i n v e s t i g a t e d a p o t e n t i o m e t r i c method f o r d e t e r m i n a t i o n o f n i c o t i n a m i d e a f t e r c o n v e r s i o n t o t h e p r o t o t y p e n i c o t i n i c a c i d by r e f l u x i n g w i t h NaOH t h e n a c i d i f y i n g w i t h HC1. The l i b e r a t e d a c i d i s d i s s o l v e d i n acetone and t i t r a t e d against standard e t h a n o l i c KOH-solution. P o t e n t i o m e t r i c a c i d i m e t r y , u s i n g s t a n d a r d HC1, was suggested by Subert e t a1 (97) f o r d e t e r m i n a t i o n o f n i c o t i n a m i d e i n medium o f MgCl2 o r NaC1. Tungsten rod c o u l d be used a s i n d i c a t o r e l e c t r o d e i n t h e p o t e n t i o m e t r i c t i t r a t i o n of s m a l l a m o u n t s of n i c o t i n a m i d e i n a m i x t u r e c o n t a i n i n g a c e t i c a c i d and a c e t i c anhydride ( 1 t 6 , v / v > ( 9 8 ) . Membrane g r a p h i t e e l e c t r o d e s , p r e t r e a t e d w i t h a c i d i f i e d KMn04 t o a t t a i n maximal improvement, was described f o r q u a n t i f i c a t i o n o f n i c o t i n a m i d e i n a c e t i c a c i d medium u s i n g standard The u t i l i t y o f t h e HC104 as t h e t i t r a n t ( 9 9 ) .
504
EZZAT M. ABDEL MOETY ETAL.
h a l f - n e u t r a l i z a t i o n p o i n t i n t h e p o t e n t i o m e t r y was investigated f o r the q u a n t i f i c a t i o n o f nicotinamide i n a medium o f c o n s t a n t i o n i c s t r e n g t h , 1 M aqueous s o l u t i o n i n NaC1, where t h e i o n i c s t r e n g t h o f t h e t i t r a n t was t o be 0.01M - 0.5M-HC1 (100). Abou-Ouf e t a1 (9 3 ) showed t h e a p p l i c a b i l i t y o f u s i n g t h e h a l o g e n a t i n g agent 1,3-dibromo-5,5-dimethylhydant o i n i n d i r e c t Dotentiometric determination o f n i c o t i n a m i d e i n some pharmaceutical p r e p a r a t i o n s .
7.223 Polarography S h i k a t a and T a c h i ( 1 0 1 ) f i r s t i n v e s t i g a t e d t h e behaviour o f n i c o t i n i c a c i d a t t h e dropping mercury e l e c t r o d e and o b t a i n e d a r e d u c t i o n s t e p w i t h a E t v a l u e o f - 1 . 6 V vs s a t u r a t e d c a l o m e l e l e c t r o d e (S.C.E.) from a 0.1 M sodium b i c a r b o n a t e s o l u t i o n o f pH 8.4. More p o s i t i v e p o t e n t i a l s were observed as t h e n i c o t i n i c a c i d c o n c e n t r a t i o n was increased. Tompkins and Schmidt ( 1 0 2 ) i n v e s t i g a t e d t h e b e h a v i o u r o f r i i c o t i n i c a c i d f r o m s e v e r a l base e l e c t r o l y t e s , b u t these authors recommended a b o r a t e b u f f e r o f pH 8 t o 9 also; however, t h e d i f f u s i o n c u r r e n t was n o t p r o p o r t i o n a l t o t h e n i c o t in i c a c i d c o n c e n t r a t i o n unless t h e b u f f e r c o n c e n t r a t i o n was a t l e a s t 0.6 M. F r o m a 0 . 6 M sodium b o r a t e s o l u t i o n t h e n i c o t i n i c a c i d step has a half-wave p o t e n t i a l o f - 1.66 V vs S.C.E. Nicotinamide was r e p o r t e d by Wenig and Kopecky ( 1 0 3 ) LO reduce a t t h e dropping mercury e l e c t r o d e from a 0.1 M sadium hydroxide s o l u t i o n producing a s t e p w i t h an E-, v a l u e o f - 1 . 8 2 V v s S . C . E . L a t e r Tompkins and Schmidt ( 1 0 4 ) observed a s t e p w i t h an E+ o f - 1 . 6 V vs t h e m e r c u r y - p o o l anode f r o m a 0 . 1 M t e t r a m e t h y l ammcmiun! bromide base s o l u t i o n and, i n general, t h e y obtained w e l l - d e f i n e d steps f o r nicotinamide from s o l u t i o n s w i t h ptl values i n t h e r e g i o n o f 8, whereas :.It 'uwet. pH v a l u e s t h e y e n c o u n t e r e d c o n s i d e r a b l e i n t e r f e r e n c e from t h e hydrogen step. The E+ values i n V 7s S . C . E . r e p o r t e d for- t h e n i c o t i n a m i d e s t e p s from v d i . 1 0 ~ base ~ s d l u t i o n s a r e as f o l l o w s : 0.1 M sodium a c e t a t e (pH 7 . 5 ) - 1 . 6 5 V ; 0.1 M sodium c i t r a t e (pH 7.9) - 1 . 6 7 V ; 0 . 1 M sodium b i c a r b o n a t e (pH 9 . 2 ) 1 . 4 0 ' J : 3 . 1 M sodium b o r a t e (pH 8 . 7 ) - 1.56 V . These lhali-wave p 3 t e n t - i a i values c l e a r l y v a r y w i t h t h e pH arid a l s o ! : l i g h t l y w i t h t h e n a t u r e o f t h e b u f f e r
NICOTINAMIDE
505
constituents. I n a1 1 cases, however, t h e step-hei ght p r o v e d t o be p r o p o r t i o n a l t o t h e n i c o t i n a m i d e concentration and independent o f t h e b u f f e r capacity o f the solution. Knobloch ( 1 0 5 ) observed a c o n s i d e r a b l y l a r g e r s t e p f o l l o w i n g t h e n i c o t i n a m i d e s t e p f r o m n e u t r a l and a c i d i c b u f f e r s o l u t i o n s , due t o t h e c a t a l y t i c e v o l u t i o n o f hydrogen a t t h e d r o p p i n g mercury electrode. Using a 5 X 10-5 M s o l u t i o n o f t h e amide t h e c a t a l y z e d s t e p w i t h a v e r y pronounced maximum f i r s t appeared a t about pH 7 . 2 4 and the h e l g h t o f t h i s s t e p i n c r e a s e d as t h e pH o f t h e f i n a l s o l u t i o n was decreased. A t pH 4 . 7 8 t h e maximum on t h e s t e p disappeared completely, r e s u l t i n g i n a well-defined c a t a l y t i c step, b u t a t lower pH values i t coalesced w i t h t h e s t e p owing t o t h e normal r e d u c t i o n o f hydrogen and i t consequently proved i m p o s s i b l e t o measure the height o f the c a t a l y t i c step under these circumstances (106). Cernatescu e t a1 ( 1 0 7 ) d e s c r i b e d t h e p o l a r o g r a p h i c determination o f nicotinamide ( E q = 1.6 V) alone and 1.2 V) i n i n t h e presence o f n i c o t i n i c a c i d ( € 3 = 0.1M NaCl o r KC1 s o l u t i o n s as t h e s u p p o r t i n g electrolytes. I n a l k a l i n e s o l u t i o n s t h e wave due t o t h e a c i d w i l l disappear a l l o w i n g the determination o f the amide i n the mixture. O s c i l l o g r a p h i c polarography w i t h an e l e c t r o n i c polaroscope and a streaming- o r dropping-Hg e l e c t r o d e d i f f e r e n t i a t e between nicotinamide, n i c o t i n i c acid, p i c o l i n i c , d i p i c o l i n i c , d i n i c o t i n i c , isocinchomeronic acids, n i c o t i n e , t r i g o n e l l i n e and isoniazide, i n i n d i v i d u a l form o r i n t h e i r mixtures (108). L i n d q u i s t and Farroha ( 1 0 9 ) i n v e s t i g a t e d t h e a p p l i c a b i l i t y o f d i f f e r e n t i a l pulse (D.P.P.) p o l a r o g r a p h y f o r d e t e r m i n a t i o n o f some v i t a m i n s i n c l u d i n g n i c o t i n a m i d e and n i c o t i n i c a c i d w i t h low d e t e c t i o n l i m i t s ( 0 . 0 1 - 1 p.p.m.; i.e. There a r e s e v e r a l o t h e r p o l a r o g r a p h i c pg, L - i 1. methods f o r i d e n t i f i c a t i o n and q u a n t i f i c a t i o n o f nicotinamide and n i c o t i n i c a c i d , the f o l l o w i n g t a b l e 3 c o l l e c t s such procedures.
-
la&
'J: Poiarograuhic charecteriratlon ancl aeternination
Analite forn
Elect rode
oi nlcotina~ioeand nicOtinlC acid
KIHI
Electrolyte
Scanning range
Id,
Rau materials N icot 1nan 1Ue Nicotinic acia
Dropping-Hg
Reference
cell
Conc. range
Reference
\)
0.111 NaOH containing geltin
110
Ni cot inanide Nicotinic acia
Buffer 1 1 2 , amide ano 0 . 7 , acid!
111
N1co:inanide Cicoti!ic acid
o.in HCI
(-
n 1coi inah I de
o.in LIOH
(- 1.5)
I > 1-10,
o.in
KOH
112
(- 1.8)
1.11
Ag/AgCi[S)jCl-
0.1-14 igx
113
- 10 1gx
114
Pharmaceutical preparations 1.5 to - 2 . 1 )
Nicot inanide
0.c. polarography With H-ceil.
0.M NaOH uith 0.0021 gelatin
(-
N 1cot in ani ae+
Cathoae-ray oolaro9raDhy
0.M KOH 8 0.05n KCI 15.1-61
I - 1.651
Table 3 Contined
..,
Nicotinaide'
CrODOing-Hg
O.IM WaOh
Nlcot 1nanide'
dropping-Hg with Hcei)
LC1-solution
(-1.5 to
-
Phosphate buffer PH 6.8 and ?.5n HaOH (12-131
I-
NiCotlnamidE+
31; KOH, Kolthoff buffer pH 8.6
I- 1.80
Nicotic acia
Kolthoff buffer PH 8.6
1-
Nicotinamide'
d i f f . puis poiarograpnv !OPP,
t i n arult ;v ;tamin nreparations.
1.91
117
alk. Ha-citrate (12)
H i cot inamine+
-
0.05 to
-
1.5)
0.8 to - 1.51
1.55)
- 1.55)
S.C.E.
118
Ag/AgGl(s)/CI- (sat)
119
120
EZZAT M. ABDEL MOETY ETAL.
508
7.23
@ectroDhotometry
7.2311
col>rimetric
Methods
D i Fferent
chromogens have been i n v e s t i g a t e d f o r c o l o r i m e t r i c q u a n t i f i c a t i o n o f n i c o t inamide and/or n i c o t i n i c acid. A l t h o u g h t h e c o l o r development i n most o f t h e q u a n t i t a t i v e procedures can be recommended f o r i d e n t i f i c a t i o n o f t h e d r u g substance, a l l t h e c o l o r spot t e s t s a r e n o t s u i t a b l e i n most cases f o r quantification. The most adopted c o l o r r e a c t i o n f o r q u a l i t a t i v e and q u a n t i t a t i v e analyses o f n i c o t i n a m i d e and n i c o t i n i c a c i d i n p u r e f o r m s , i n dosage f.zrmulations and i n b i o l o g i c a l f l u i d s i s t h e Konig’s r e . j c t i o n . The c o l o r f o r m a t i o n i n t h e Konig’s r e a c t i o n depends Gn o x i d a t i o n o f t h e p y r i d i n e - b a s e d d r u g s i n c l u d i n g n i c o t i n a m i d e and i t s p r o t o t y p e a c i d by cyanogen bromide (CNBr) t o t h e corresponding aldehyde f c l l o w e d by c o u p l i n g with a base l i k e a n i l i n e t o g i v e t l ! e c o l o r e d p r o d u c t s w h i c h can be e s t i m a t e d color imet r ic a l l y . There a r e d i f f e r e n t m o d i f i c a t i o n s o f K B n i g ’ s r e a c t i o n by changing t h e o x i d i z i n g agent and/or t h e coup1 irig base-species. Abdoh and Taufel ( 1 2 1 ) were t h e f i r s t , who a p p l i e d Koni’g’s c o l o r r e d c t i o n w i t h CNBr and a n i l i n e f o r t h e q u a n t i t a t i v e deterniination o f n i c o t i n a m i d e and n i c o t i n i c a c i d i n p u r e form and i n p l a n t o r a n i m a l t i s s u e s e x t r a c t s containing e i t h e r o f t h e t w o d r u g s . Since n i c o t i n a m i d e gives l e s s c o l o r i n t e n s i t y than n i c o t i n i c a c i d i f they where t r e a t e d according t o t h e Konig’s p r i n c i p l e u t i l i z i n g CNBr and a n i l i n e , t h e amide should be f i r s t hydrolyzed by h e a t i n g w i t h 2 . 5 M HC1 f o r 1.5 h r s a t 9 0 - 9 5 °C b e f o r e d e v e l o p i n g t h e c o l o r . Such m o d i f i c a t i o n enables accurate q u a n t i f i c a t i o n o f t h e d r u g i n u r i n e , t i s s u e s and blood ( 1 2 2 ) . The colored hydrazides formed by adding b e n z i d i n e t o t h e o x i d a t i o n products o f n i c o t i n a m i d e o r n i c o t i n i c a c i d w i t h CNBr were s t u d i e d f o r q u a n t i t a t i o n o f these drugs ( 1 2 3 ) . Spectrophotometric s t u d i e s on t h e a c t i o n o f CNBr on t h e p y r i d i n e nucleus i n n i c o t i n a m i d e and n i c o t i n i c a c i d i n t h e presence o f q u i n o l i n e showed t h e presence o f t y p i c a l f u n c t i o n a l grouping ( 1 2 4 ) . Q u a n t i t a t i v e m o d i f i c a t i o n o f Konig’s c o l o r i m e t r i c
NICOTINAMIDE
509
reaction has been investigated by coup1 ing barbituric acid for determination of nicotinamide in the presence of nicotinic acid (125). Ciusa and Barbiroli (75) compared the extinction coefficients (E) at 460-465 nm, of the Konig’s reaction products of nicotinamide and nicotinic acid in aqueous and ethanolic media. The €-values obtained in ethanol are depressed with respect to those in water for both d r u g substances. Mesnard (126) discussed the applicability of coupling 2-thiobarbituric acid to the oxidation products of nicotinamide and nicotinic acid with CNBr for the drug assay. The Konig’s reaction could be applied for determination o f n i c o t i n a m i d e i n urine a f t e r chromatographic separation from nicotinic acid and nicotinuric acid by ion-exchanging utilizing Amberlite IRA-400 (OH-form) and elution with acetone-Hz0 (2: 1, v/v) (127). Automation of Konig’s color reaction has been undertaken for vitamin analysis of nicotinamide at a flow-cell measuring at 4 2 0 nm (128). Nicotinamide contents in tablets could be accurately ( - 102%) determined by following the Konig’s reaction (75). A similar method to that o f Jeffus and Kenner (129) was applied for direct determination of nicotinamide in multivitamin preparations (130). Modified Konig’s reaction was carried out by using an excess of yeast nicotinamide deaminase to yield the nicotinic acid giving rise to relatively consistent quantitative results (131). Dessouky e t a1 (132) suggested the use o f 3-methyl-1-phenylpyrazolin-5-one as coupling reagent for the quantitative determination o f nicotinamide and nicotinic acid in tablets. Nicotinamide and nicotinic acid have been quantified in pharmaceutical formulations by automating the Konig’s reaction using barbituric acid and sulphanilic acid as the coupling reagents (133). The manual AOAC-colorimetric method No. 43.044, involving Konig’s reaction with CNBr and sulphanilic acid, was automated for determination o f nicotinamide and nicotinic acid in cereal products (134). Farsam and Mahmoudian (135) discussed the mechanistic possibility of the Konig’s reaction using sodium l-phenyl-2,3-dimethyl-5pyrazolone-4-methylaminomethansulphonate (dipyrone) in case o f nicotinamide. The possibility of coupling TLC-separation with spectrodensitometric determination of the color formed according to the Konig’s reaction gives good analytical way for quantitative separation and determination of nicotinamide in some multivitamin
510
EZZAT M. ABDEL MOETY ET AL
p r e p a r a t i o n s (77,781. K r i n g s t a d and Naess (136) advised t h e phosphate b u f f e r , pH 6.1, f o r best s t a b i l i z a t i o n o f t h e c o l o r maximally formed a f t e r 7 min. f o r c o l o r i m e t r i c determination o f nicotinamide and n i c o t i n i c a c i d according t o t h e Konig’s procedure. There are some other c o l o r i m e t r i c methods, r a t h e r than t h e Kljnig’s r e a c t i o n , adopted f o r q u a n t i f i c a t i o n o f n i c o t i n a m i d e and n i c o t i n i c a c i d . K h a t t a b ( 1 3 7 ) recommended t h e bromination o f nicotinamide by using aqueous bromine, where t h e formed N-bromoderivative was determined by r e a c t i o n w i t h starch-CdIe a t 600 nm. Pauer (138) d e t e r m i n e d t h e c y a n i d e c o n t e n t s i n n i c o t i n a m i d e and n i c o t i n i c a c i d by s e p a r a t i o n f o l l o w i n g m i c r o d i f f u s i o n and then determination w i t h 2% t a r t a r i c acid, 0.5% chloramine-T s o l u t i o n followed b y p y r i d i n e - p y r a z o l o n e r e a g e n t a t 6 2 0 nm. Dimethylglyoxine (DMG)-Fe2+ c h e l a t e was recommended as a c o l o r i m e t r i c reagent f o r q u a n t i f i c a t i o n o f n i c o t i n a m i d e (139). Reaction o f nicotinamide w i t h I-chloro-2,4-dinitrobenzene y i e l d s p y r i d i n i u m d e r i v a t i v e which y i e l d s on a l k a l i n e decomposition a measurable ye1 l o w i s h r e d c o l o r o f glutaconaldehyde (140). Nudelman and Nudelman (141) recommended a n e a r l y i d e n t i c a l method t o t h a t of Karrer and K e l l e r (140) by u t i l i z i n g l-~hloro,2,4,-dinitrobenzeneas t h e chromogen i n t h e presence o f a l c o h o l i c s o l u t i o n o f NaOH w i t h o u t i n t e r f e r e n c e s t h a t may be due t o n i c o t i n i c a c i d and some other vitamins. Nicotinamide i s the p r i n c i p a l metabolite of n i c o t i n i c acid i n u r i n e , which could be c o l o r i m e t r i c a l l y assayed a f t e r c o n v e r s i o n t o t h e amino-compound by h e a t i n g w i t h hypobromite, followed by d i a z o t i z a t i o n and coupling w i t h N-(1-naphthyl I e t h y l e n e d i a m i n e . 2HC1 t o g i v e t h e azodye (142). The method could be s u c c e s s f u l l y applied t o determine t h e u r i n a r y e x c r e t i o n o f nicotinamide i n t h r e e a d u l t volunteers. Walksmundzki and Romanowski (143) showed t h e s p e c i f i c i t y o f e p i c h l o r o h y d r i n as an a n a l y t i c a l reagent f o r d e t e c t i o n and determination o f nicotinamide i n t a b l e t s . Indemans and Rademakers (144) described a c o l o r i m e t r i c procedure by t r e a t i n g w i t h hypobromite-phenol, sulphamat and N-(1-naphthyl1ethylenediamine.2HCl t o y i e l d t h e diazonium s a l t measurable a t 500 nm. Domnas ( 1 4 5 ) recommended a mixture o f hypochlorite-phenol reagent f o r determination o f nicotinamide i n raw m a t e r i a l s and i n uptake studies o f the drug substance d u r i n g metabolism i n yeast. Nicotinamide, R, could be c o l o r i m e t r i c a l l y
Table 4: Coloriuetric Deteraination Substance H i Cot i n mi@ nicotinic acia
Specimen pure form. plant a
a nicotinamide and nicotinic acid in pure forr, dosage forrs and in biological fluids Chroaogen (Reagent! C#Br t aniline
Sjecial treatment
KIl
(nu)
Sensitivity range
l6'C/23 min. using
iiefetence i21
POI-buffer pH 6.16.j
Hi cot inanide Nicotinic acid
bulk form
CNBr t benr i d 1ne
Nicotinaaide
ray uaterials
CNEr t barbituric acii
Nicot inaaide Nicotinic acid
ram materiais
CNBR t 2-thiobarbit u f i t atid
Hicotinamide
biological fluids
CHBr t aniline
2 0 ' 9 2 0 #in.. uY
CNBr + H,I-oiethylD-pneny lenedianine
ambient, 2% tiaOH amofiia buffer
iuriner H i cot 1nami de
vitamins mixtures
125
20-i2'6/30
min.
using
PO4 - bu f fer M/ 15, vH 1 . 2 )
513
- i pg.nl-'
125
126
460
6.5-1
308
Recovery from v i n e . 91 18.91
12;
420
ionrinuea
I..
.
hico:!nasiaf
tabisrs
CWBr
b c f b i t u r i c acid
roam temDereure 3b
5.0
room teip.125-30 nin,
550
Bin, 0.ia KHzPOI
mu it ivi tam preparation
thBr t satc. o a r a i t u r i c acld i n j%KH?POt
Hicotinamide i Icot.:lic iC'0
miets S carwies
CNEr t D a r b m m c am 8 julphan;lic acid
s;ajle oiscretesanple autoiateo anaiys!s
:iico t :n am; oe
ray na:eriais
CNBr t sodium 1-pnenyl2, I-diaetnyi-5-pyrazok~ne-4-netnviaainomet hanesul DnaP Lie
anoientl5D nln.
Hi cot inanioe
j%I H z P G i
raw s a t e r i a l s ray materials
N
- HCN t ?I t a r t a r i c
aclE
t
0.5: chioramine-
1 + Pyridine pyrazolone
iicroaiffusioP (20 nri, reaction 30 nin.
rea;ent
raw Baterials
3inethyiglvoxine IONi- pH i.4-1:. c o l o r Fe" s t a b i l i t y t 30 min.
j90
Recovery
:
. 1028
5 ug.arl-;; recovery loo.!%
8-33 ug.mi-'
119
130
135
labie 4 Continued
... l-chiorc-2,4-Cinitrobenzene t 20% KOH
JO'Cil hr.-ethanol
tablets. capsules, syrups & eiexir
I-clloro-!,j-a!nitrobenzene t ln NaOH
8 0 ' W hrs.-ethanol
dr!ne
- NHZ, diarotization B
nicotinanide
ran
Hicotinamiae y".l r v i
,nanide
material:
504
couciing vitn h-ilbaphthy i i-etnyienedi-
5-10 pg,nl-i
140
. 4 ug.rni-1
141
1-50 kg
142
aiine
cn w
N icot i n a i iae
tab ieis i so iu: i ons eo 1 ch loroh ydr 1n
60'C/5 nrs.
H~COtinamioe
mu i t 1v it am1 n
heating
oreparations
NaOBr, CsHsOH, oiazotization 8 coupling With A-i 1-ncphtnyl
lened :aiine
-
'
10 pg.nl-I
143
500
*
10 pg.ai-l
1l a
655
lii-50 ug.ri-1
145
ii5
3-40 Lg.ni -1
I40
retng-
Yicoiinamide
raw paterials 3 uptake stucies
HaOGl
Hic3t:namiae
rac nateriais
cooait t hiocyanate conpiex, HziColSCH,rj
GrH5i)H
rOON
teRD./i hrs.,
G H C i 3 , or 2 4 hrs.
nitroso-r? sait soiut ion
530
Cant ined i , . . ,
Table 4 Conieuec Hicotinamifle
Micctlnanide
. . #
~narmaceutical m a r a t ions
H'-nydfOlpiS Irefiux. 1 hr. I t Ha 1,f-naphtnaouinone-4-suiphsnate t Ha?S?O?.
pnarnaceitical
4-diaethy1aminoben;aldenvne IPDAB!
preparations
80'611 hr. acetone
540
- 15 !g.nl-'
147
445
80-200 uq.al-'
148
NICOTINAMIDE
515
q u a n t i f i e d by u s i n g c o b a l t t h i o c y a n a t e complex, HZ [Co(SCN)4] t o g i v e (RH)2-[Co(SCN), I which e x e r t s t h e c o l o r (146). A c i d h y d r o l y s i s o f n i c o t i n a m i d e f o l l o w e d by r e a c t i o n w i t h sodium 1,2-naphtha-quinone4-sulphonate a t 80’C f o r 1 hr. g i v e s p u r p l e c o l o r a t i o n on adding t h i o s u l p h a t e ( 1 4 7 ) . The absorbance o f t h e complex formed between 3-aminopyridine, t h e p r o d u c t o f H o f f m a n n ’ s r e a c t i o n between n i c o t i n a m i d e and 4-dimethylaminobenzaldehyde was used f o r q u a n t i t a t i v e determination o f the drug i n pharmaceutical p r e p a r a t i o n s (148). Table 4 c o l l e c t s and compares i n a summa r ized form t h e d if f e r e n t c o l o r i m e t r i c procedures f o r i d e n t i f i c a t i o n and q u a n t i f i c a t i o n o f t h e mentioned drug substances. M a r s h a l and Kenner ( 1 4 9 ) compared t h e d i r e c t s p e c t r o p h o t o m e t r y a t 262 nm o f a c i d e x t r a c t s o f n i c o t i n a m i d e s e p a r a t e d f r o m c a p s u l e s , s y r u p s and e l i x i r s , w i t h o t h e r two c o l o r i m e t r i c methods by u s i n g e i t h e r bromothymol b l u e , measured a t 430 nm, o r w i t h c y a n o g e n b r o m i d e and b a r b i t u r i c a c i d s o l u t i o n s , measured a t 550 nm. T a b l e 4 c o l l e c t s t h e v a r i o u s c o l o r i m e t r i c methods described f o r d e t e r m i n a t i o n o f n i c o t i n a m i d e and/or n i c o t i n i c a c i d i n dosage f o r m u l a t i o n s and i n b i o l o g i c a l specimens. 7.2312
UV-SDectrgphotometrv
The USP X X I ( 1 0 ) recommends t h e UV-measurement a t 262 nm f o r n i c o t i n a m i d e and n i c o t i n i c a c i d i n b u l k forms GI- i n i n j e c t i o n s and t a b l e t s , w h i l e t h e BP 1988 ( 8 4 ) s p e c i f i e s t h e same wavelength f o r t h e q u a n t i f i c a t i o n o f o n l y nicotinamide i n i t s t a b l e t s . Spectrophotonietr i c determination o f nicotinamide i n admixtures with thiamine, r i b o f l a v i n and p y r o d i x i n e , is undertaken by measuring A a t 267 nm f o r s o l u t i o n s a t pH 2 & 7 ( 1 5 0 ) . Estimation o f nicotinamide i n a d m i x t u r e s w i t h t h i a m i n e and p y r i d o x i n e by d i r e c t measurement a t 2 4 7 nm, 260 nm and 290 nm was o n l y p o s s i b l e a f t e r removal o f any r i b o f l a v i n by t h e use o f ion-exchange r e s i n ( D e c o l o r i t e anion-exchanger) (151 & 152), presence o f panthenol does n o t i n t e r f e r e because o f i t s n u l l a b s o r p t i o n s a t t h e wavelengths mentioned I n d i r e c t spectrophotometric a n a l y s i s o f above ( 1 5 3 ) . n i c o t i n a m i d e and a s c o r b i c a c i d i n e t h a n o l c o n t a i n i n g cysteine.HC1 t o p r o t e c t t h e a c i d c o u l d be c a r r i e d o u t a t 2 6 3 nm and 245 nm, i n o r d e r ( 1 5 4 ) . S i m i l a r method
5 16
EZZAT M. ABDEL MOETY ETAL.
was described f o r q u a n t i f i c a t i o n o f n i c o t i n a m i d e a t 2 6 2 nm i n t h e presence o f t h i a m i n e and p y r i d o x i n e (155). Nicotinamide i n a quaternary m i x t u r e w i t h thiarnine-HCI, r i b o f l a v i n phosphate and pyridoxine-HC1 and chloramphenicol c o u l d be i n v e s t i g a t e d by a d o p t i n g i n d i r e c t spectrophotometry (156). Resolved n i c o t i n i c a c i d and i t s amide on s i l i c a g e l G, u s i n g a c e t i c (5:5:20:70), c o u l d be acid-acetone-methanol-benzene determined i n t h e presence o f thiamine.HC1, pyr-idoxine.HC1 and p-aminobenzoic a c i d by means o f UV-ref lectance spectrometry ( 157 I . Nicotinic acid c o u l d be q u a n t i f i e d a t 2 6 2 nm a f t e r i t s s e p a r a t i o n from dosage f o r m u l a t i o n s on a c t i v a t e d polyamide powder i n a microcclumn ( 1 5 6 ) . Among o t h e r 30 d i f f e r e n t ci~mpounds w i t h p y r i d i n e chromophores, t h e e f f e c t of s u b s t i t u t i o n and s o l v e n t on t h e U V - s p e c t r o photomet r - i i: behaviour o f n i c o t inamide was s t u d i e d f o r clrug c c n t r o l ( 1 5 9 ) . D i r e c t UV-measurement o f rlicot;namidi! a t 2 6 2 nm c o u l d be u n d e r t a k e n i n admixtures w i t h i n o s i n e and u r i d i n e 5-phosphate a f t e r TLC-separation on s i l i c a g e l G F 2 5 4 u s i n g b u t a n o l - a c e t i c acid-5% ammonia s o l u t i o n ( 7 : 2 : 2 ) (160). P a r t i t i o r i chromatography, between a diatomaceous earth-water column and c h l o r o f o r m , was a p r e l i m i n a r y s t e p f o r s e p a r a t i o n o f n i c o t i n a m i d e from oharmaceutical p r e p a r a t i o n b e f o r e i t s UV-measurement a t 2 6 2 nm ( 1 4 9 ) . Paczek and Wardynska ( 1 6 1 ) a l s o adopted t h e chromatographic s e p a r a t i o n o f n i c o t i n a m i d e from some o t h e r water-soluble v i t a m i n s i n m u l t i v i t a m i n drageks before i t s q u a n t i f i c a t i o n v i a d i r e c t D i r e c t UV-measurement a t i-Yd-measurement a t 262 nm. ; 4 t nm & 2h2 nm a t pH = 2 has been d e s c r i b e d f o r d e t e r r n i n a t l a r i o f n i c o t i n a m i d e w i t h some o t h e r w a t e r - s o l u b l e v i t a m i n s (,162).
7.23 13
Spect r o f luor i&.Lry
Scudi ( l b 3 ' ) described t h e a d d i t i o n o f an a l k a l i t o t h e s o l u t i o n o f n i c o t i n a m i d e and cyanogen b r o m i d e t o A q u a n t i f y t h e d r u g spectrofluorimetrically. f l u o r i m e t r i c method f o r q u a n t i f i c a t i o n o f 1 - m e t h y l n i c o t i n a m i d e and n i c o t i n a m i d e i n serum was recammended by C l a r k e t a7 ( 1 6 4 ) by d e v e l o p i n g t h e fluorescence i n 9% f o r m i c a c i d ; EX: 370 nm, EM: 450 nm. M o d i f i e d Hankder s p e c t r o f l u o r i m e t r i c procedure Itas been recommended by M e s t r e s ( 1 6 5 ) t o d e t e r m i n e q i c s t i n a m i d e ; t h e fluorescence f o r m a t i o n t a k e s p l a c e
NICOTINAMIDE
517
by KCN-buffered s o l u t i o n and chloramine-T, EM: 462 nm. N i c o t i n a m i d e can be d e t e r m i n e d i n a c i d e x t r a c t s o f homogenized f o o d p r o d u c t s by measuring t h e Mohran ( 1 6 7 ) d e s c r i b e d fluorescence a t 426 nm ( 1 6 6 ) . a s p e c t r o f l u o r i r n e t r i c procedure f o r e s t i m a t i n g n i c o t i n a m i d e i n t a K e , based on t h e d e t e r m i n a t i o n o f plasma l e v e l s o f one t o i t s m e t a b o l i t e s , namely N’-methyl-4pyridone-3-carboxamide, w h i c h causes fluorescence on U V - i r r a d i a t i o n . N i k o l i c e t a7 ( 1 6 8 ) demonstrated fluorescence r e a c t i o n s f o r d e t e r m i n a t i o n o f a v a r i e t y o f n a t u r a l and s y n t h e t i c drugs i n c l u d i n g n i c o t inamide. I n s i t u s p e c t r o f 1u o r i m e t r y a f t e r TLC-separation was adopted by F r e i e t a 1 ( 1 6 9 ) f o r q u a n t i f i c a t i o n o f t r a c e s o f n i c o t i n i c a c i d and i t s amide. S p e c t r o f l u o r i m e t r i c q u a n t i f i c a t i o n o f n i c o t i n a m i d e and I t s methyl d e r i v a t i v e i n b i o l o g i c a l f l u i d s was r e c o m m e n d e d b y W i l l i a m s ( 1 7 0 ) . S p e c t r o f l u o r i m e t r i c measurement, EX: 436 nm, o f t h e S c h i f f ’ s base formed by r e a c t i n g n i c o t i n i c a c i d o r i t s amide w i t h cyanogen b r o m i d e , a c e t o n e and 4-aminobenzoic a c i d has been i n v e s t i g a t e d by u s i n g 3,5-diacetyl-1,4-dihydrolutidine as comparing standard f o r q u a n t i f i c a t i o n o f each d r u g substance ( 1 7 1 ) . 7.24
Chromatography 7.241 Paper Chromatography (pc)
PC-technique u s i n g d i f f e r e n t paper t y p e s i n v a r i o u s s o l v e n t systems has been i n v e s t i g a t e d f o r r e s o l u t i o n , i d e n t i f i c a t i o n o r d e t e r m i n a t i o n o f n i c o t i n i c a c i d and i t s amide. The s e p a r a t i o n o f t h e a n t i p e l l a g r a drugs can be achieved i n one d i r e c t i o n , two-dimensions, o r circular diffusion utilizing different visualizing Table 5 summarizes c o l l e c t i v e l y regents (172-193). t h e d i f f e r e n t P C - p o s s i b i l i t i e s f o r i d e n t i f i c a t i o n and q u a n t i f i c a t i o n o f n i c o t i n i c a c i d and/or n i c o t i n a m i d e . 7.242 T h i n Laver ChromatograDhy (TLC)
D i f f e r e n t adsorbents and developing s o l v e n t s have been described f o r f r a c t i o n a t i o n o f n i c o t i n i c a c i d and/or n i c o t i n a m i d e f r o m a d m i x t u r e s and i n m u l t i v i t a m i n preparations. Color development o r U V - v i s u a l i z a t i o n are among t h e v a r i o u s methods recommended f o r l o c a t i o n (194-213). The f o l l o w i n g t a b l e 6 c o l l e c t s t h e v a r i o u s
VI-m 0
-
0
N ‘ q O
518
L P)
-.
0) c
.-
-
I -
519
-. c ~
0
I
N
,
.-
.-.
=.-.
r
I -
.. . -.. -
CT
e
I . 0 cc "-orc
u .. , ,.,.eIal *
0 - o u
r
d
n
=IL - - . a m
Y)
521
Tinle f Continueo
... then u i t h 3% YaOH i n lleoH and HHsvapors.
C i c o t i n i c acid Wicot irlamide
Uv25t
sheets
PrOH-ail. NHj 11$:11 o r i 7 H t i I CH3 JCO 1 3 . 2 )
- CHtlj
2% 4-aminobenzoic acid i n 0.15 M HCIEtOH (3:1), then vapors o f CNBrll hr.
lyellou-orange)
201
Quantification by measuring Azrl ns
--
202
s i l i c a gel G
EtOH 1982, 15:121
Nlcot1oat:de i i n mu 1t iv it a n 1n Dreuarat i c n s i
s i l i c a gei I f i i o r e s cent. 254 nnl
Hz D
Clz-atRosuhere I20 #in. I . then spraying u i t h o - t o l i a i n e (0.03% i n aq. IcOii)
--
203
Nicotinamlde acid. nicotinanide [dosage forms
s11ufol & silufol 3;r 154
a i f f e r e n t soivent systets
Uv-iight an0 c o l o r i # e t r i c a i i y
--
204
Y 1cot 1na@ide
cellulose HM300 HR
BuOH-AcOH-Hz0 i8:l:lll
iodoplatinic acid
--
205
Nicot,inic acid. Nicotinaride
s i l i c a gel
66H1-He0H-lCHo JzCO-ACOH
d i f f e r e n t spraying reagents
_-
206
N i c o t i n i c acid, Nicot inaniee
s i l u i o i UYN c e l lu iose
UV-light 1254 nnl; q u a n t i f i c a t i o n a t 261 nn uas possible.
--
201
Nicotinanide in;ect!ons)
w N
urecoateo
:r,
l14:4: 1 : l l
or
0.1 U H C l
-----------------Continued /....
,--
= c l
a a *I c u 0
*-.=I
P
aJ
w
-
c
.cz
524
c -. &i
N
Ill
6 L
c .c_
-
c
-4 6
.c.
E
.-IL
525
526
EZZAT M. ABDEL MOETY ET AL.
TLC-methods f o r f r a c t i o n a t i o n , i d e n t i f i c a t i o n and/or q u a n t i f i c a t i o n o f n i c o t i n i c a c i d and n i c o t i n a m i d e . 7.243 gas-Liouid Chromatography (GLC)
Some G L C - p r o c e d u r e s ( 2 1 4 - 2 1 6 ) h a v e o f t e n been recommended, b u t do n o t t a k e i n t o a c c o u n t t h e 1 i k e l i h o o d o f e n c o u n t e r i n g n i c o t i n i c a c i d and i t s r e l a t i v e s i n a samples ( 2 1 7 ) . The methods a r e ranging from t h e isothermal t o t h e programming technique a t d i f f e r e n t temperature ranges. Different polarities have been s p e c i f i e d f o r i d e n t i f i c a t i o n o f n i c o t i n i c a c i d and i t s amide i n raw m a t e r i a l s , b i o l o g i c a l f l u i d s and pharmaceutical f o r m a t i o n s (218-2251. The f o l l o w i n g t a b l e 7 compares t h e v a r i o u s GLC-procedures. 7.244
Hish
Performance (Pressure) L i a u i d
Chromatosraphy (HPLC)
The d e t e r m i n a t i o n o f n i c o t i n i c a c i d a n d / o r n i c o t i n a m i d e has been performed u s i n g HPLC on v a r i o u s normal and reversed-phase columns. The HPLC technique has advantage over t h e GLC one i n r e q u i r i n g almost no d e r i v a t i z a t i o n , such as s i l y l a t i o n o r conversion t o the corresponding a l k y l esters, prior to The f o l l o w i n g t a b l e 8 q u a n t i f i c a t i o n (226-241). summarizes t h e d i f f e r e n t HPLC-methods recommended f o r 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 analyses o f n i c o t i n i c a c i d and i t s amide. Guilleman e t a7 ( 2 4 2 ) d e s c r i b e a h i g h performance, a t moderate pressure (10-30 b a r ) , l i q u i d chromatographic procedure f o r s e p a r a t i o n o f n i c o t i n a m i d e and some o t h e r o r g a n i c compounds on columns packed w i t h h i g h s p e c i f i c a r e a and consequently s h o r t columns. Two grades o f S p h e r o s i l , v i r , XOA 600 and XOA 800 ( w i t h s p e c i f i c areas 600 ti 800 m2.g-1, r e s p e c t i v e l y ) w i t h mean p a r t i c l e diameters E f f i c i e n c i e s up t o 800 t h e o r e t i c a l p l a t e s o f 5-7 pm. c o u l d be cm-1 w i t h a n a l y s i s t i m e s o f 5-15 min. obtained. I o n - p a i r , reverse-phase HPLC f o r s e p a r a t i o n o f n i c o t i n a m i d e i n admixtures w i t h o t h e r water s o l u b l e v i t a m i n s has been i n v e s t i g a t e d by Coleman ( 2 4 3 ) . The e f f e c t s o f pentane, hexane and heptane s u l f o n a t e s , as ion-pai r reagents a t v a r i o u s water-methanol r a t i o s on t h e r e t e n t i o n times on a RP-column were i n v e s t i g a t e d .
Table E: HPLC-Determination of nicotinic acid and nicotinaaide Substance isuecimen)
Column/tertp. ( 'GI
Developing solvent loobile pnaseliflou rate-int. Std.
Retention Detection ti#e I ;, nn
Sensitivitv
Reference
Imin.)
Nicotinic acid
6 icot inaaide
1% SCI lsulfonated fluorocarbon! Zipar. glass bead/
(27)
N icot inaa ide Inultivitaain preparations 1
ilS Pellicriex SCX
Nicotinic acid ruith otner J 1tam1 ns 1 Hi cot inam 1de
Aoinex A-5 resin
Nicotinic acid, nicot inamide (dosage formulations) Nicotinic acid. nicotinaaide ifooo)
0.1
n naNO1-0.1 MbPWl.:
ml.min-:-piccl!naride.
i
0.01%
150 ng-5 u g i
226
2?i
0.03 H KCiin 0.1 !I POI-ouffer IDH 8) linear gradient of oH and ionic strmgtn at acid pH-value isocraticalij rk 2.5.
!00-251 ng
228
p-Bondapak Cl8
0.52 H Ha CiOCtyi suiphasuccinate pH 2.5 - CHiOH ! l : l ) i 2.5 a 1 . m - I - acetophenone
25 ug.ml i.5 ug.al-'
?29.!SIl
p-8ondapak C18
Detto
Aminex 1-11 resin
)
0.1 U9.9.'
231
Phospnare buffer pH 5 Nicotinarioe 1 o lasnal
H2 d l o c t y lsui fosucci ph ? . $ . nemi 1 1 . 4 : ml.min-'-isonicotina-
0.3 1% nzte!
1 li2
Ride. 'il
crit inai7lae
;re;Eratianr!
Nicotinic acid
cotinamioe
H 1 c o t : n ' c acid nicotinaaice I piasma
N i c o t i n i c acid
inawide ilirinei
235
CsHiiSOiH as ion-pair reagent
VI W N
Hicot
232
Me3h-H20 i D to BOX iinear yaaient ~2 M i .min-1-0.025 H
1 muit i\. itanin
nl
251
O.U W Ph-buffer. guinaidic acia
Ultrasphere ODS/24.5:C iph 5.301
pH 2.6-
iMeCN-0.OI
1 CjHltS03t!. k
:5HiiSO~H
B kHzPOi) 1 0 to
~Etl3NHClI-lO.01 II
UV 12541
1-10 ng
234
UV 12601
0.1-2 ug mi-' 1-10 vg 11-i
235
236
6% linear gradientl.
------------------Continued :....
Table 8 lontinued
... neOH
-
buffer
NHIPOI
Hicotinaaioe I u i t h oiner vitahias:
C18-bonaeu phase
Wicotinanide i u i t h Other i i t a u i n s i n dosage forms)
FAST-LC-8 I5 urn)
Tetranydrofuran-tla C7 H13S03-~ H z P O I - ~ I I P O -I ( Et Ian IpH 2 . 5 I / 2 or 4 n1.m-'.
Nlcotinamide i u i t n
S i l i c a gel sitanired with
other vitatiins)
MezCtH17 siiane.
0.3: 1Bu:cNOH i n 0.015 4I PO& buffer. pH 3 - neOH
--
--
--
231
Igraa:ent, 0 t o 70%) --
uv (2721
--
238
__
UP 12301
--
239
-_
UY 1280)
-_
210
--
uv
Ill : 8 , / l hl.Blfi-'-C6H~00H
Nicotinanide i v i t h VI N \o
CB-HPLC
WeOH-0.04 II HaE7HtrSOs- i n
2% lAcOH (1:9).
other vitamins, dosage forms J Hicotinamiae inultivitaninninersl preparations)
colilnn/ i 2 5 ' C I
AP-C18
neOH I Hz0 13:17J-0.005 H h H 1 5 S ~ l H6
0.5% IEthN, pH
3 . 6 1 2 ml.min-l
(280)
--
241
530
EZZAT M. ABDEL MOETY ET AL
7.245
Ton
Exchange
7.2451
Electrophoresis
Ion-Exchange ChromatograDhv
K l o t z and Poethke have separated the v i t a m i n B complex components and d e t e r m i n e d t h e n i a c i n a m i d e w i t h ion-exchange r e s i n i n a column o f W o f a t i t CP300 (H+) and W o f a t i t L150 (OH-) (244). These authors (245) used A m b e r l i t e IR-120, IRA-401, IRC-50 and XE-100 and W o f a t i t CP300 and L 1 5 0 f o r t h e i s o l a t i o n o f nicotinamide. Nicotinamide was r e t a i n e d on t h e weakly a c i d l c c a t i o n exchangers a t pH < 4, but can be e l u t e d w i t h N-HC1 o r w i t h NaOH s o l u t i o n . Sweeney and H a l l (246) used the ion-exchange r e s i n f o r the q u a n t i t a t i v e d i f f e r e n t i a t i o n b e t w e e n n i c o t i n i c a c i d and nicotinamide. The sample s o l u t i o n a t pH 5 i s passed o v e r t h e a n i o n -e x c h a n g e r e s i n IRA-400. The nicotinamide i s e l u t e d w i t h water and the n i c o t i n i c a c i d w i t h h o t 1N HC1. Both substances a r e t h e n determined by means o f Konig’s r e a c t i o n w i t h CNBr and s u l p h a n i l i c acid. Table 9 summarizes t h e ion-exchange chromatography reported i n the 1it e r a t u r e (244-252).
7.2452
Electrophoresis
N i c o t i n a m i d e , i n pharmaceutical p r e p a r a t i o n s , have been separated by gel f i l t r a t i o n on Sephadex G-50 and e l u t e d from the column by H z 0 and saturated butanol. The e l u a t e a f t e r concentration were subjected t o paper e l e c t r o p h o r e s i s ( 1 10 V; 8-10 mA) w i t h p y r i d i n e - f o r m i c acid-water (1:1:98) b u f f e r s o l u t i o n (pH 3.35). Zones were l o c a t e d under U.V. l i g h t ( 2 5 3 ) . I n an e a r l i e r method (254) nicotinamide i n a m i x t u r e o f vitamins was separated by paper e l e c t r o p h o r e s i s i n acetate b u f f e r (pH 5 . 1 ) w i t h a c u r r e n t o f 3.5 mA. Spots are developed w i t h d i a z o t i z e d paminoacetophenone. Electrophoresis 1 f i l t e r paper t o separate on Whatmann No. n i c o t i n a m i d e f r o m a number o f d e r i v a t i v e a t a p o t e n t i a l d i f f e r e n c e o f 280 V i n 0.03 M-borate b u f f e r has been reported by Sundaram e t a1 ( 2 5 5 ) . Fukuda and Mimura (256) have i n v e s t i g a t e d a paper e l e c t r o p h o r e s i s method t o separate and i d e n t i f y n i c o t i n a m i d e i n a mixture o f vitamins by using 0.5% sodium acetate, 1 % s o d i u m b o r a t e a n d 0 . 1 % ammonium c h l o r i d e a s e l e c t r o l y t e . Paper e l e c t r o p h o r e s i s technique has a l s o been employed f o r the separation and determination o f nicotinamide i n pharmaceutical preparations o f
Table %
I o n exchange chromatography
of
n i c o t i n a m i d e and/or n i c o t i n i c
Mobile phase
acid Rf
Column
Reference
2 44
A c i d i f i e d water (pH 4)
W o f a t i t CP300 (H+) W o f a t i t L150 ( H - )
1N-HC1 o r NaOH solution
A m b e r l i t e IR-120, IRA-401, W o f a t i t CP300 and L150
Water
IRA-400
AcO form C1 form ( b u f f e r e d a t pH 1 . 6 ) .
Amberlite Amberlite Amberlite Amber1 it e
Acetate b u f f e r solut i o n pH 4.62.
A m b e r l i t e WA-2 and c a r b o x y l i c a c i d r e s i n
0.1 K-NaNO3-0.1 N-H3 PO4
1% o f SCX ( a 1% sulphonated f l u o r o c a r b o n
249
Water
Strong basic anion exchangers (OH-)
2 50
A m b e r l i t e I R C , XE-50, IR-120 and LRA-401 and W o f a t i t CP-300 and KPS-200 r e s i n s
251
Acid-precipitated a l g i n i c acid
252
0.005 N HC1
IRC-50,
XE-100,
245 246
SB-2 (AcO form) SB-2 ( C 1 form) WA-2 ( b u f f e r e d a t pH 1.6) WB-2
0.5 0.52 0.18 0.02
247
0.18
248
EZZAT M. ABDEL MOETY ETAL.
532
t i i t a m i n s ( 2 5 7 1 . T a b l e 10 c o l l e c t s t h e d i f f e r e n t e l e c t r o p h o r e t i c separations o f t h e drug substances
(258-2761.
7.25
Gravimetry
Nicotinamide can be p r e c i p i t a t e d from i t s s o l u t i o n s i n amyl a c e t a t e o r e t h e r (10-100 mg) by adding excess o f 0.025 M c h l o r a n i l i c a c i d s o l u t i o n i n t h e same s o l v e n t . A f t e r s t a n d i n g f o r 30 min. (amyl a c e t a t e ) or 5 min. ( e t h e r ) f i l t r a t i o n t h r a u g h a preweighed Gooch c r u c i b l e i s advised. Weighing o f t h e p r e c i p i t a t e can be done a f t e r i t s washing and d r y i n g t i l l c o n s t a n t w e i g h t (277). Coloriniet r i c quant 1f i c a t i o n o f n i c o t inamide can be c a r r i e d o u t by d i s s o l v i n g an a l i q u o t o f t h e c v l o r e d p r - e c i p i t a t e i n water. The c r y s t a l l i z e d qicotinamide-chloranilic a c i d s a l t s sublime a t 180 C Rtid decompose a t 240-245.C, i t s aqueous s o l u t i o n has ;jH-value = 3, a t 2 3 C w i t h c o n d u c t i v i t y o f 6 X mhc.cm-1. N i c o t i n a m i d e c a n a l s o be d e t e r m i n e d g r a v i m e t r i c d l l y by p r e c i p i t a t i o n v i a h e a t i n g w i t h m e t h y l i o d i d e ( 4 0 min, 5 5 - 6 W C ) o r e t h y l i o d i d e 1 2 3rs, 8 0 - S Y C ) t o g i v e t h e c o r r e s p o n d i n g q u a t e r n a r y salts (278). The n i c o t i n a m i d e - E t I , yellow-green c r y s t a l s , m e l t a t 146-148'C, while the MeI-salt, yellow c r y s t a l s , m e l t s a t 201-206'C. 7.26
Automated A n a l y s i s
Continuous, non-segmented, f l o w i n j e c t i o n system w i t h has been ' s o l o r i m e t r i c d e t e c t i o n a t 4 2 0 nm, investigated f o r q u a n t i f i c a t i o n o f nicotinamide admixed w i t h some o t h e r water-soluble v i t a m i n s . The yellow c o l o r f o r m a t i o n c o u l d be o b t a i n e d by r e a c t i n g n i c o t i n a m i d e w i t h polymethine dye mixed w i t h NaOH and ammonia b u f f e r c o n t a i n i n g CNBr ( 2 7 9 ) . N i c o t i n i c acid s n d n i c o t i n a m i d e i n one-component and m u l t i v i t a m i n t a b l e t s and c a p s u l e s can be q u a n t i f i e d by a d o p t i n g d u t o a n a l y s i s w i t h c o l o r i m e t r y by u s i n g C N B r - s u l f a n i l i c a c i d f o r n i c o t i n i c a c i d and CNBr-barbituric a c i d f o r nicotinamide (280). A n o t h e r automated method f o r d e t e r m i n a t i o n o f n i c o t i n i c a c i d and n i c o t i n a m i d e i n (cereal products has been c o l l a b o r a t i v e l y s t u d i e d by Gross ( 2 8 1 1. The AOAC m i c r o b i o l o g i c a l method 43.121 were compared i n such study. Coverly ( 2 8 2 ) d e s c r i b e d t h e p o s s i b i l i t y o f automating an HPLC-procedure f o r q u a n t i f i c a t i o n o f n i c o t i n a m i d e i n admixtures w i t h some
Table 10: Electrophoretic separation
of
nicotinamide and/or nicotinic acid ~
Mobile phase
Column
Detector
Reference
UV 280 nm
258
Silica gel treated with dimethyl(octy1)silane.
UV 280 nm
259
Methanol-4 mM sodium heptanesulfonate in aqueous 2% acetic acid (1:9).
Silica gel CIS
UV 280 nm
? 60
Tetrahydrofuran sodium heptanesulf onate- KHn P04-H3 PO4 - t r iethy 1 ami ne (pH 2.5).
FAST-LC-8
UV 272 nm
261
[Acetonitrile with 10 mM pentane sulfonic acid - 10 mM triethylammonium chloride ( 9 : 1 ) ] in 10 mM pentae sulphonic acid - 10 mM triethylamnonium chloride and KH2PO4 in water.
Ultrasphere ODS
UV 254 nm
262
Methanol-water (3:17), 0.005M heptane sulfonic acid, and containing 0.5% triethylamine at pH 3.6.
C16
0.3% Tetrabutyl ammonium hydroxide solution in 0.015M phosphate buffer (pH 3) - methanol (17:8)
(reversed phase)
-0
-j (D
cu
E
c
cu
W
W
In (D
E
cu
.5 u)
0 0
cu
d
OD
cu
w cu
m
a
m
Y
0
c
m
> = J
Q
a r
C
x
7
> 3
>
3
X
0 03
C 0
-0
1
m
X Kl
a
K
0
.
r-
I
0
3
0
C
E cu
?
> 3
u)
h
-7
u)
c,
n a
'c 'c 2
a
L
v
a
I
-+
a
c m X
I
a
C N
'7
o m
m
-7
U
.r
N
-r
L
a
re rc 7
n a 0
L
- a
4 - I
c,
m
r
3
L
3 I
m
a
c,
InN
m
ZEE E
n.
a x z
C I
+-'N
m a
m
3-
C
a
gG
a
OD
0-
?%
I . cu cu
I -
L
0
v)
K
c,
r
.7
0
0
a,
2 7
m
n
!-
534
I 1
.
.
.
I I I
I W I C
I 0 10
I I I l
I
;
Table 10 Continued
...
2 mM-Na (dioctyl sulfosuccinatemethanol ( 1 : l ) adjusted to pH 25 with 10% form acid.
u
0.03 M-KC1 in 0.1 M K-phosphate buffer (pH 8).
Aminex A-5 resin
0.025 M Nitric acid
HS Pellionex SCX
-VI
E
Bondapak C l a
UV 254
27 1
272 UV 254 o r
280 nm
-
Spherosi 1
0.1 N Sodium nitrate-0.1 N H3P04
1% CSX Zipax glass bead
uv
0.3% Tetrabutyl ammonium hydroxide
Silica gel treated with dimethyl (octyl) silane
UV 280
solution in 0.015 M - phosphate buffer (pH 3) - methanol (17:8).
nm
273 274 275
nm
276
EZZAT M.ABDEL MOETY ET AL.
536
o t h e r water-soluble v i t a m i n s i n t a b l e t s by continuousf l o w sample p r e p a r a t i o n with UV-detection a t 272 nm. ACKNOWLEDGEMENTS
The authors would l i k e t o thank M r . Syed R a f a t u l l a h , A s s i s t a n t Researcher, Col lege o f Pharmacy, King Saud U n i v e r s i t y f o r h i s t e c h n i c a l assistance and M r . Tanvir A. B u t t , f o r t y p i n g the manuscript.
537
NICOTINAMIDE
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121.
J . Abdoh and K. T a u f e l ; &. Untersuch.-Lebensm.: 355 (1942); c f . C.A,: 37, 6594 (1943).
122.
m: 33,
123.
J. Deltombe; J. Pharm. Belg.: 1, 1054 (1954). Abstr,:
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18,
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84,
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8 , 59 (1953); cf. Anal.
545
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124.
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126.
a,617
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127.
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128.
B.E. A l b r i g h t , E.F. Degner; Automat. Anal. Chem. Technicon Symp., 1967, 3rd, 1, 461 (publ. 1968); c f . C.A.: 7l, 33460U (1969).
129.
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130.
0. P e l l e t i e r and J.A. Campbell; J- Pharm. Sci.: 926 (1961); c f . Anal. Abstr.: 9, 2498 (June 1962).
131.
L. F u l l e r and L.S. D i e t r i c h ; Analvt. Biochem.: 9, 538 (1971); c f . Anal. Abstr.: 22, 350 (1972); C.A.: 74, 839112 (1971).
132.
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133.
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71, 321 (1974);
134.
A.F. Gross; J. Ass. O f f . Anal. Chem.: 3 0 , 2F 49 (1976). c f . Anal. Abstr.:
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135.
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137.
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138.
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24,
50,
32, 587
cf.
Anal.
EZZAT M. ABDEL MOETY ET AL
546
139.
W.-K.
Lee;
C.A.:73,
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l3, 2 2 (1969);
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140.
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141.
N.S. Nudelman and 0. Nudelman; J. Pharm. Sci.: (1976) c f . Anal. Abstr.: 31, 2E33 (1976).
142. 143. 144.
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A.W.M.
56,
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&, 65
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H. Rmanowski; Acta Polon. Pharm.:
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147.
148.
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D.K.
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33,
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163
a
1, 759 (Feb.
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547
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3 2 , 185 (1964);
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156.
Idem, ibid: 3 4 , 213 (1966);
157.
H.M. Frodyma and V.T. (1967).
158.
G. Lehmann, H. Hahn and M. S c h i r r a ; Chem,: 554 (1968).
159.
J. Kracmar, J. Kracmarova and J. Zyka; m a r m a z i e : 23, 567 (1968).
160.
G. Cosi; Farmaco,
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165.
R. M e s t r e s ; Trav. got, Pharm. M o n t p e l l i e r : (1962); c f . C.A;: 58. 144319 (1963).
166.
A. Sebesta and L. Dalma; S c i . Aliment.: 14, 7 7 (1964); c f . Anal. Abstr.: 1 2 , 6128 (Nov. 1965).
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66,
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39,
Fresenius’Z.
814
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103, 567
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22, 46
EZZAT M. ABDEL MOETY ET AL
548
7th; 5 ,
167.
Y.Z. Mohran; Proc. I n t . Congr. N u t r . . lO_,35551 (1969). (1967); c f . CA.:
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169.
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48,
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13851h (19541.
1,
14,
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C. Bergamini; Sperimentale, ch.jml (1953); c f . t;..A_L: 4_8, 13851h (1954).
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573
4,
38
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175.
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177.
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__ c.A.:
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F. Leuschner; Naturwissenschaften: @, 554 (1954); (1954).
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L.D. Rodrigues and J.A. deSilva; Rev. Port. Farm.: 75 (1955); c f . C.A.: 5 0 , 1264d (1956).
5,
NICOTINAMIDE
182.
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549
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193.
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194.
H. Ganshi r t and
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K.C. Guven and 0. P e k i n ; E c z a c i h a B u l t e n i : 4, (1966); c f . Anal. Abstr.: l4, 5689 (Sept. 1967).
440 (1971);
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(1964); c f .
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119
EZZAT M. ABDEL MOETY E T A L
550
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552
227. 228.
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103,255
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169
Sarma; J-
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EZZAT M. ABDEL MOETY ETAL
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