analytical profiles of neostigmine
TRANSCRIPT
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NEOSTIGMINE
A A Al-Ba * and
Ad
lahiq
DepahAnent 6
Phanmacaficak?
Chemhin.thy
and
**Vep&men;t 6
Phatrmacotogy,
College 6
P h m a c y ,
King Saud UVLive. Lty,
P.O.
Box
2457, Riyadh-77451,
Saudi
A m b h l .
ANALYTICAL PROFILES OF DRUG SUBSTANCES
VOLUME
16
Copyright 987 by Academic Press. Inc.
All rights of reproduction in any form reserved.
03
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404
A.
A. AL-BADR AND
M
ARIQ
1. History
2.
Description
2.1 Nomenclature
2 .2
Formulae
2 . 3
Molecular Weight
2.4 Elemental Composition
2.5
Appearance Color Odor and Taste
3. Physical Properties
4. Synthesis
5. Pharmacokinetics
6 .
Therapeutic and Other Uses
7.
Toxicity
8. Methods of Analysis
8.1 Identification
8.2 Titrimetric Methods
8 . 3
Biological Method
8.4 Spectrophotometric Methods
8.5 Chromatographic Methods
8.6
Ion-Selective Electrodes Method
8 . 7 Polarographic Method
Refer n c s
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NEOSTIGMINE 405
1. His torv
As a r e s u l t o f t he ba s ic rese arch of Stedman and
a s s o c i a t e s ( 1 ,2 ,3 ) i n e l u c i d a t i n g t h e chemical b a s i s
of t h e a c t i v i t y of physost igmine, Aeschlimann and
Reiner t ( 4 )
s y s t e m a t i ca l l y i n v e s t i g a t e d a s e r i e s o f
s u b s t i t u t e d ph enyl es te rs of a l l y l ca rbami c ac i ds .
Neostigmine (P ro st ig mi n) , a most prom isi ng member o f
t h i s ser ies , was in t roduced in to therape ut i cs
i n
1931
f o r
i t s
s ti mu la n t a c t i o n on t h e i n t e s t i n a l t r a c t .
I t
was r ep o rt e d in de pe nd en tly by Remen (5) and Walker
(6)
t o be e f fe c t i v e i n t h e symptomatic t he rapy of
myas theni agra vis which
i s
due t o d e f e c t i n s y n a p t i c
t ransmiss ion
a t
neuromuscular ju nc ti o n . When t h e
pat ient i s
given an ap pro pri a te dose o f ne ost igmine,
t h e r es po ns e t o t i t a n i c s t i m ul a ti on i s
improved along
wit h symptomatic improvement i n muscle s tr e n gt h
( 7 ) .
Neostigmine was a l so found usefu l
i n
p a r a l y t i c i l l e u s ,
a tony of ur inary b ladder and
i n glaucoma ( 8,9 ).
2 . Descr ip t ion
2.1 Nomenclature
2 . 1 . 1 Chemical Names
3- (Dimethylcarbamoyloxy)
N
, N ,N-t r imethyl -
ani l inium bromide.
3- Dimethylcarbamoyloxyphenyl)trimethyl-
ammonium bromide.
Benzenaminium, 3- [ [ dimethylamino)carbonyl]
oxy]
-N
,N,N-trimethyl bromide.
m-Hydroxypheny1)timethylammonium bromide
dimethyl carbamate.
3-
[ [
Dimethylamino)carbonyl]oxy]
N , N , N ,
trimethylbenzenaminium bromide
3- [ [ (Dimethylamino) car bon yl loxy]
-N , N , N -
trimethylbenzaminium methyl sulphate .
(m-Hydroxyphenyl) t r i m e t h y l ammonium methyl
sulphate dimethylcarbamate .
3-Dimethylcarbamoxyphenyl)trimethylamonium
methyl sulphate (10-13) .
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A. A. AL-BADR AND M ARIQ
2.1.2 Ge neri c Names
Neostigmine bromide; Neostigmine DCF; N F N ;
Neoeserine; Proserine; Synstigmine;
Eust gmine ; Phi 1 0 st igmine ; Neost igmin
bromidum; Neo st igm ini i bromidum; Neostigmium
bromatum (13,14).
2 .1 .3 Trade
Names
Neost ipmine bromide
Prostigmin; Juvastigmine; Konstigmin;
Metastigmi n; Normastigmin; Pros tigm in;
Leostigmin (13,14).
Neostigmine methyl sulphate
In tras t igmina; Juvas t igmin; Mestas t igmin;
Neoe sser in; Normastigmin; Pr osti gmin ;
Hodostin; St ig ly n; Stigmosan (13,14).
2.1.4
CAS
Registry Number
59-99-4 Neostigmine
51-60-5 Neostigmine methyl su lp ha te (14)
114-80-7 Neost igmine bromide
2 .1 .5
Wiswesser
Line Notation
1 N 1 VOR
C K - &
E
(Neostigmine bromide) (15)
2 . 2
Formulae
2 . 2 . 1 Empir ica l
C H
BrN202 Neostigmine bromide
C13H22N206S Neost igmine methy l su lph a t e
1 2 19
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NEOSTIGMINE
401
2 . 2 . 2 S t r u c t u r a l
CH I
0
c
I
x
,CH3
-N
CH3
X =
B r -
Neostigmine bromide
X
=
CH3SOi
Neostigmine methyl sulphate
2 . 3 Molecular Weipht
Neostigmine bromide
303.2
Neostigmine methyl sulphate
334.4
2.4 Elemental Composition
C
47.53%,
H
6.32%,
N
9.24%,
B r
26.36%,
0
10.55%
(bromide).
C
46.69%, H 6.63%; N 8.38%, S 9.59%,
0 28.71% (methyl sulphate)
2.5 Appearance,
Color,
Odor and
Taste
O do rl es s, c o l o r l e s s c r y s t a l o r a w hi te c r y s t a l l i n e ,
s l i g h t ly hygroscopic powder w i th a b i t t e r t a s t e
(14)
*
3.
Phys ica l Proper t ies
3 . 1 Melting Point
Neost igmine bromide
Crysta ls f rom alcohol and e ther melt a t 167 with
decomposition (13) .
Neostigmine methyl sulphate
Crystals from alcohol melt between 142OC and 145OC
(13)
*
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408
A. A , AL-BADR AND
M.
TARIQ
3.2
3 . 3
3.4
3 . 5
3.6
3.7
Ext rac t ion
Neostigmine s a l t s a r e qu at er na ry ammonium compounds
and a re so l ub l e i n
water.
i s
a c i d i f i e d wit h d i l u t e a c e t i c a c i d ,
evaporated
t o dryness and ex tr ac te d with methanol . The
methanol ext ract w i l l
contain most of t he qua t e rna ry
ammonium compound and may be p u r i f i e d by pape r
chromatography (16).
The aqueous solut ion
S o l u b i l i t y
Neostigmine s a l t s a r e f r e e l y s o l u b l e i n water .
Moderately s olu ble i n chloroform and e than ol .
Inso luble
i n
e t h e r (11).
Acidi ty
Dissolve
0 .20
g in 20
m l
carbon d ioxide- f ree
wa te r and t i t r a t e a t pH
7.0
with
0.02 N
sodium
hydroxide (carbonate-free) not more than 0.2 m l
i s requi red (17) .
Moisture Content and Hygr osco pici ty
Not more than
I , determined by drying a t
1 0 5 O C
(10)
Storage
I t should be s tored
i n
a i r t i g h t c o nt a in e rs
pro tec ted f rom l igh t
(10 ) .
S p e c t r a l P r o p e r t i e s
3 . 7 . 1
Ultraviolet SDectrum
The UV spectrum of neost igmine bromide in
ethanol
i s
given in Figure
1.
I t shows two
maxima a t 260 nm and 266 nm. The spec trum
was recorded on Varian spectrophotometer
model DMS 90.
Clarke (11) reported the fol lowing:
Neostigmine methyl s u l f a t e i n
1 N
H2SO4;
maxima a t 260 nm
( E
1 ,1
cm
20) and 266 nm
(E 1 ,1 cm 18 ) .
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NEOSTIGMINE
lot
2
2SOWavelength
300
nm)
350
400
Figure I: Ultraviolet spectrumof neostigmine bromide in methanol.
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410
A. A . AL-BADR AND M. TARIQ
3.7.2 In fr ar ed Spectrum IR)
The IR spectrum of neo st ig mi ne bromide i n
K B r
d i s c
i s
presen t ed i n F i gu re 2 , and
was recorded on Pye-Unicam
I R
spect rophoto-
meter model SP 1025.
the s t ructural ass ignments as shown below:
Frequency cm-l
The frequencies and
As s ignment
CH
s t r e t c h
0 2 0 -
2900
.)
1730
C
= 0 (amide) s t re t ch
1
610
C
= C s t r e t c h ( a r o m a t i c )
1595)
1490-1450 CH bending v ibra t ions
1
030
1070)
C
-
N
v i b r a t i o n ( a l i p h a t i c )
780 and 895
CH
(aromatic) bending
v i b r a t i o n s
Clarke (11) re porte d t h e fol lowing:
Neostigmine bromide, potas sium bromide di s c
t h e pr i nc ip al peaks a r e 1711, 1215 and
1154 cm-1.
1
3. 7. 3 Proton Nuc lea r Magnetic Resonance (
H
NMR)
Spectrum
1
The
H NMR
spectrum o f neostigmine methyl
s u l p h a t e
i s
shown i n Fi gu re
3.
di sso lved in deuter ium oxide
DzO)
and i t s
spectrum was deter mined on a Var ian - T60 A
NMR
spectrometer using sodium 2,2-dimethyl
2
s i l apen t ane- 5-su lphonate (DSS) a s t h e
i n t e r n a l s t a n d a r d .
The drug was
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I
-
f
8 0
7.0
6 0
S.OPpm
6k O
3-0 2 .o
Figure 3 : Proton
NMR
spectrum
of
neostigmine methyl sulph
TMS as internal reference.
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NEOSTIGMINE 413
Assignment of t h e chemical s h i f t s t o th e
d i f f e r e n t p r o t o n s i s shown below:
Chemical
s h i f t M u l t i -
(ppm) p l i c i t y Proton assignment
0
3.04) II
singlets -C-N(CH )
1
-3
2
3.12
3.
72
s i n g l e t
4-
g 3 )
3 .80
s
i n
g
1
e
t
CH
-S
O4
7.15-7.60
mul t ip le t s Aromatic prot ons
1
7.66-8.30
3. 7. 4 Carbon-13 Nuc lea r Magnetic Resonance
(C-13 NMR) Spectrum
The C-13
NMR
s p e c t r a of neostigmine methyl
su lp ha te i n deuterium ox ide (D2O) usi ng
DSS
(sodium 2,2-dimethyl 2-si lapentane-
5-sulphonate) as an i n t e r n a l r e f e r e n c e were
obta ined us ing a J e o l FX
100 MHz
spec t rometer
a t an ambient tem per atu re. Fig ure s 4 and
5 repr esen t t he pro ton-decoupled and of f -
resonance sp ec t ra respec t ive l y .
CH3
1 2 I +
5
13
C H 3
-
S O i
1
C H
.CH3
3 0
- C - N
2
The carbon chemical s h i f t s were assi gne d on
t h e b a s i s o f t h e c hemical s h i f t t h e or y and
t h e o f f- r es o na n ce s p l i t t i n g p a t t e r n and a r e
shown below:
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414
A . A .
AL-BADR AND M
ARIQ
Figure : Proton -decou pled carbo n-I3
N M R
spectrum
Of
n-stigmine methyl wl ph ate
in D20 w it h DSS a s internal reference.
Figure 5 : Off -Resonance carbon
-13
NMR spectrum of neostigmine methyl sulphate
in
9 0
with DSS a s internal reference.
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NEOSTIGMINE
415
Chemical
s h i f t M u l t i -
(ppm) p l i c i t y Carbon assignment
3 8 . 9 q u a r t e t 1
3 8 . 8 q u a r t e t 2
1 58 .3 s i n g le t 3
149.8 s ing
e t
4
126.9 doublet
5
134.0 doub1e t
6
119.8 doublet 7
1 54 .5 s i n g le t 8
117.1 doublet 9
5 9 .6 q u a r t e t 1 0,
11
and 12
5 7.9 q u a r t e t 1 3
3 . 7 . 5
Mass
Spectrum
The electron impact ( E I ) mass spectrum
a t
70 e V was recorded on Varian MAT 311 mass
spec t romete r i s shown i n F ig u re 6 .
spectrum an
i o n
a t
m/e
428
was
observed
which most probably arises from a
recombination o f frag ment s. The spec trum
shows a
base peak
a t m/e
72. The
most prominent i ons a re shown below:
In
t he
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Figure 6: Electron impld El)mass spectrum
Of
neostigmine methy
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NEOSTIGMINE
417
72
208
356
428
Fragment
CON
( c H ~ ) ~
0-CONc H ~ $
@-
4 2 8 - C O N ( C H , ) , I +
L .
OCON CH3)
1
1
H2
The chemical ionisation ( C I ) mass spectrum
was obtained
on
Finnigan 4000 mass
spectrometer and i s shown in Figure 7 . The
spectrum shows ions
a t m/e 1 2 7
( t he base
peak) and a t m/e 303 and both are probably
arised from a recombination of fragments.
The most prominent ions are shown below:
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Figure 7 : Chemical ionization C I mass Spectrum Of neostigmine
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N OSTIGMIN
419
m e
127
Fragment
l
CH30-S-O-CH3 t
209
303
l
4
S y n th e s i s
Neostigmine bromide can be pr ep ar ed from 3-hydroxydi-
methylani l ine , which
s
made from 3-nitroanil ine by
or di na ry sy nt he ti c methods. The 3-hydroxydimethyl-
a n i l i n e
i s
d i s so lv e d in an a l c o h o l i c so l u t i o n o f t h e
ca lc ul at ed amount of pot assium hydroxide, and t h e
so l u t io n o f t h e p o tas s iu m h y drox ide , and th e so l u t i o n
o t h e p o ta s s iu m d e r iv a t iv e
so
formed
s
t r e a t e d w it h
dimeth ylc arb amoyl c h l o r i d e , Me2N.COC1 made from
dimethylamine
and
carbony l ch l o r ide ) . Th is g ives a
t e r t i a r y bas e which, by combination wit h methyl bromide,
y i e l d s t h e r eq u ir e d qu a te r na r y s a l t i . e . t h e
dimethylcarbamic
es te r
o f
3 hydroxy NNN trimethylani
lin iu m bromide:
18, 19) .
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420
A. A. AL-BADR AND
M
ARIQ
OCON (CH 3)
OCON (CH 3)
Neostigmine methylsulphate can be made in the same
way, except t h a t i n the last stage the secondary amine
i s
combined with methyl su lp ha te t o
form
t h e
s a l t
[ (R-AMe3)MeSOi] (18).
OK OCON (CH3)
0 - C - N ,
C H 3
C H 3 S 0 i
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NEOSTIGMINE
42 1
A
m o d if i c at i on o f t h i s r o u t e
i s
t o c o nv er t t h e
hydroxydimethylan i l i n e in t o the quar t e r n ar y s a l t (by
combination wi th methyl bromide o r methyl su lp ha te ) ,
and then t o
t r e a t
th e s a l t with dimethylcarbamoyl
c h l o r i d e
(18).
(CH3)2
OK
C l C O N (CH3)
-
Neost igmine can be synthesized e i t h e r d i r e c t l y from
3-hydroxydimethylaniline with phosgene t o g ive t he
carbonyl ch lo ri de and then with dimethylamine t o giv e
3-dimethylaminophenyldimethylurethane o r t h e l a t t e r
may be prepared from th e sodium s a l t of t h e s t a r t i n g
material and dimethylcarbamic chloride.
case the product i s converted t o t he methy lsu lpha te
by treatment with dimethyl sulphate (20).
I n e i t h e r
Y
ON
a
OCON (CH31
OH
COC1 2
N
2
OCOCl
CH3S0i
OCON
(CH3)
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422
A . A. AL-BADR AND M. TARIQ
5. P h a r m a c o k i n e t i c s
N e o s t i g m i n e
i s
a b s o r b e d p o o r l y
a f t e r
o r a l a d m i n i s t r a t i o n ,
s uc h t h a t much l a r g e r d o s e s a r e n ee de d t h a n b y t h e
p a r e n t e r a l r o u t e . W hereas t h e e f f e c t i v e p a r e n t e r a l
d o s e o f n e o s t i g m i n e i n man i s 0 . 5 t o 2 . 0 mg, t h e
e q u i v a l e n t o r a l d o s e may b e 30 mg o r m o r e. L a r g e o r a l
d o s e s may p r o v e t o x i c
i f
i n t e s t i n a l a b s o r p t io n i s
e n ha nc ed f o r a ny r e a s o n a n d t h e q u a t e r n a r y a l c o h o l a n d
paren t compound are e x c re t e d i n t h e u r i n e . P y r i d o s t i g -
mine and
i t s
q u a t e r n a r y a l c o h o l
a re
a l s o t h e p r e d o m i n a n t
e n t i t i e s fo u n d i n u r i n e a f t e r a d m i n i s t r a t i o n o f t h i s
d r u g t o man ( 2 1 ) .
N e o s t i g m i n e
was
r a p i d l y e l i m i n a t e d from t h e p la sm a o f
5 p a t i e n t s t o whom 5 mg o f t h e m e t h y l s u l p h a t e h a d b e e n
g iv e n t o a n t a g o n i s e r e s i d u a l n e u r o m u sc u la r b l o c k . T he
p la sm a c o n c e n t r a t i o n o f n e o s t i g m i n e d e c l i n e d t o a b o u t
8%
o f
i t s
i n i t i a l v a l u e a f t e r 5 m i n u t e s w i t h a d i s t r i -
b u t i o n h a l f - l i f e o f l ess t h a n o ne m i n u t e . E l i m i n a t i o n
h a l f - l i f e r an ge d from a b o u t 15 t o
30
m i n u t e s .
a mo un ts o f n e o s t i g m i n e c o u l d b e d e t e c t e d i n t h e p la sm a
a f t e r
o n e h o u r ( 1 4 ) .
Trace
O f n e o s t ig m i n e t h a t r e ac h e s t h e l i v e r , 98 p e r c e n t i s
m e t a b o l i z e d i n
10
m i n u t e s . ( 2 2 )
I t s
t r a n s f e r f r o m
p la sm a t o l i v e r
c e l l s
a n d th en t o b i l e
i s
p r o b a b l y
p a s s i v e i n c h a r a c t e r . S i n c e c e l l u l a r m em branes
p e r m i t t h e p a s sa g e o f p l a s m a p r o t e i n s s y n t h e s i z e d i n
l i v e r i n t o t h e b lo od
stream
t h r o u g h c a p i l l a r y
walls
o r l y m p h a ti c v e s s e l s , t h e y may n o t p r e s e n t
a
b a r r i e r
t o t h e d i f f u s i o n o f q u a t e r n a r y a m in es s uc h
as
n e o s t i g -
m in e. P o s s i b l y t h e r a p i d h e p a t i c m e ta bo li sm
of
n e o s t i g m i n e p r o v i d e s a d o w n h il l g r a d i e n t f o r t h e
c o n t i n u a l d i f f u s i o n of t h i s com pound 2 3 ) . A c e r t a i n
am ount may b e h y d r o l y z e d s l o w l y b y p l a s m a c h o l i n e s -
t e r a s e .
p a t i e n t s w i t h m y a s t h e ni a g r a v i s
less
t h a n 5
was
f o u n d
u nc ha ng ed i n t h e u r i n e ( 1 4 ) . F o l l o w i n g i n t r a m u s c u l a r
a d m i n i s t r a t i o n a b o ut 65 o f a d o s e i.s e x c re t ed i n t h e
u r i n e u nc ha ng ed ( 1 0 ) .
20
o f a n o r a l d os e
i s
e x c r e t e d i n t h e u r i n e and
50
i n t h e faeces;
less
t h a n
5%
o f t h e o r a l d os e
i s
e x c r e t e d i n t h e u r i n e un ch an ge d.
When ne os t i gm in e was g i ve n by mouth t o 2
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NEOSTIGMINE
423
6.
Theraneutic and Other
Uses
Neostigmine
i s a
quaternary ammonium anticholinesterase.
I t
a c t s
a t
t h e e s t e r a t i c s i t e of t h e enzyme t o form t h e
i n a c t i v e dimethylcarbamoyl enzyme. Neostigmine ha s
wid esprea d a c t io n s , b u t fo r t u n a t e ly i t s e f f e c t s a r e
more prominent on ce r t a i n s t r u c t ur e s than on o t he rs ,
b ei ng p a r t i c u l a r l y e f f e c t i v e on t h e bowel, u r i n a r y
b l a dd e r , and s k e l e t a l mu sc le , t h e p u p i l , t h e h e a r t ,
b lood p ressure ,
and s e c r e t i o n s b e in g a f f e c t e d t o
a
much lesser e x t e n t i n do se s t h a t are o r d i n a r i l y
e f f e c t i v e on r e l a t e d s t r u c t u r e s ( 19 ).
The dr ug i n h i b i t s c h o l i n e s t e r a s e a c t i v i t y and p r ol o ng s
and i n t e n s i f i e s t h e m us c ar i ni c and n i c o t i n i c e f fec t s
o f a c e ty l c h o l in e .
I t
p ro ba bl y a l s o h as d i r e c t e f fec t s
on s k e l e t a l musc le f i b r e s . The a n t i c h o l in e s t e r a se
a c t io n s o f n e os t ig min e
are
r e v e r s i b l e .
I t i s
used
main ly fo r
i t s
ac t io n on sk e l e t a l musc le, and
less
f re qu en tl y t o i n c r e a s e a c t i v i t y
of
smooth muscle (1 4) .
Neostigmine methylsulphate i s used i n t h e t r e a tme n t o f
myas then ia g rav is in usua l doses o f 1 t o 2 .5 mg dai ly
given i n d iv ided doses by subcutaneous , in t ramuscul ar ,
o r i n tr a ve n ou s i n j e c t i o n a cc or di ng t o t h e s e v e r i t y o f
t h e cond i t ion (14).
Neostigmine i s use d i n c o n d i t i o n s o f u r in a ry b l a d d e r
a to ny due t o p o s t a n e s t h e t i c d e pr e ss i on o r t o n e u ro l o gi c a l
d i so rd e r s (1 9 ) .
I t i s
a l so u se d in t h e t r ea tmen t o f
p a r a l y t i c i l e u s po s to pe r at i ve u r i n a r y r e t e n t i o n , i n
d o s e s o f 0 . 5 t o
1
mg. For e x p e l l i n g in t e s t i n a l f l a tu s
p r io r t o r a dio gra ph y o f t h e g a l l -b l a d d e r , k id ne ys o r
u r e t e r s ,
a
s i n g l e d o s e o f
500
ug has sometimes been
used (14).
Neostigmine can be employed
as a
d i a g n o s t i c
t e s t ,
e s p e c i a l l y a f t e r symptoms have been purposely
a c c en tu at e d by t h e a d min i s t r a t i o n o f q u in in e .
mine
i s
used as d i a g n o s t i c t e s t agent i n myotonia
congeni ta , i n which condi t ion neost igmine aggravates
t h e symptoms.
f o r e a r l y pregnancy o r t o
t r e a t
delayed menstruation:
g iven in t ramuscular ly on three success ive days
i t
w i l l
induce menst rua tion wi th in 72 hours a f t e r t he l a s t dose
u n le s s t h e p a t i e n t
i s
pregnant.
t o p i c a l l y t o t r e a t primary open-angle glaucoma and in
Neost ig-
I t
may be used
as a
d ia g n o s t i c a g e n t
Neostigmine i s used
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424
A . A. AL-BADR
AND
M ARlQ
the emergency treatment of primary acute-angle glaucoma.
Miosis l a s t s 1 2 t o
36
hours . Longer-act ing ant ic hol i -
nes terases are preferred for the t rea tment of accommo-
da t ive e so t rop i a
(19).
7 . Toxici ty and Side Eff ec ts
Adverse and s ide effects of neost igmine include
sal ivat ion, anorexia , nausea and vomit ing , abdominal
cramps and diarrhoea.
sweating, lachrymation, watery nasal discharge,
e r u c t a t i o n, involuntary defaecat ion and ur inat ion
f lushing, miosis , conjunct ival congest ion, c i l i a spasm,
brow ache, nystagmus, res t lessness , agi t ion,
f e a r ,
excess ive dreaming, increased broc hia l se cre t i on
combined with bronchoconstriction, bradycardia and
hypotension, muscle cramps, sc at te re d fa s i cu la t i on s
and eventual severe weakness and paralysis , convulsions,
and coma.
e f fec t cou ld occur due to in te rac t ion be tween n ico t in ic
and muscarinic act ions; a consequence of t h i s could be
some evidence of an acceleration pulse-rate and
el ev at io n of blood pre ssu re. Death may foll ow due
t o c a rd i ac a r r e s t o r c e n t r al r e s p i r a t o r y pa r a l ys i s
and pulmonary oedema.
in myasthena gravis
i s inc rea sed muscular weakness (14) .
I t i s
c o n tr a i n di c a t ed i n a s th m at i c p a t i e n t s . I t should
not be employed along with choline es ters excep t fo r
ophthalmologic use.
of neostigmine (19 ) .
Symptoms
of
overdosage include
I t has a l so been s t a t ed t h a t pa radox ica l
The major symptom of overdosage
Quinidine i n t e r f e r e s wi th t h e ac t i on
Neost igmine methyl sulphate , by increas ing in tes t inal
mo t i l i t y , may cause d i s rup t ion o f i n t e s t i n a l su tu re
l i n e s ( 1 0 ) .
8. Methods of Analysis
8 . 1
I d e n t i f i c a t i o n
a )
To 0 . 1 m l
of a 1 per cent w/v s ol ut io n, add
0.5 m l
of
sodium hydroxide solution and evaporate
t o dryness on a water-bath.
o i l -ba th t o about
250'
and m ai nt ai n a t t h i s
temperature f o r about t h i r t y seconds.
d i s so lve t he r e s idue i n
1
m l o f
water,
c o o l i n
i c e water, and add
1
m l of diazoaminobenzene-
Heat qu ic kl y on an
Cool,
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NEOSTIGMINE
425
su l phon i c ac i d ;
a
che r ry - red co l ou r
i s
produced (15).
Crys t a l
t e s t s .
Micro: l ead iod id e so lu t ion
-
b r an c hi n g n e e dl e s ( s e n s i t i v i t y :
1
in 400)
;
p l a t i n i c i od i de s o l u t io n - bunches of curved
( s e n s i t i v i t y
:
1
i n 400) (11).
The in f r ar e d abso rpt io n spectrum of a potassium
bromide dispers ion of
i t ,
p r e v i o u s l y d r i e d a t
1 0 5 O f o r
3
hours , ex h i b i t s maxima on l y a t t h e
same wavelengths
as
t h a t o f a s i m i l a r
p repa ra t i on o f
USP
Neostigmine Bromide
Reference Standard (12).
A s o l u t i o n
(1 i n
50) r es po nd s t o t h e
t e s t
of
bromide (neo stigmi ne bromide) (12).
8 .2 T i t r i me tr ic Methods
8.2.1 Aqueous T i t r a t i o n
B r i t i s h Pharmacopoeia (1973) (17) re po rt ed
t h e fo l lowing p rocedure f o r t h e a s say
of
neost igmine methyl sul pha te:
Disso lve 0.15 g in 2 0
m l
o f w a te r , t r a n s f e r
t o a semimicro ammonia d i s t i l l a t i o n
apparatus, and add 20 m l of a 50 per cen t
w/v so lu t i on o f sodium hydroxide.
Pass a
curr en t o f s team through th e mix ture ,
c o l l e c t t he d i s t i l l a t e
i n
50
m l
of 0 .1
N
s u l p h u r i c a c i d u n t i l t h e t o t a l v o l u m e
reaches about
200 m l ,
and t i t r a t e t h e e xc es s
o f ac i d w i t h 0.02
N
NaOH us ing me th yl r ed
s o l u t i o n
as
i nd i c a t o r . Repeat t h e ope ra t i on
wi thout t h e substance being examined; t h e
d i f f e r e n c e between t h e t i t r a t i o n s
represents
t h e amount o f a c i d re q u ir e d t o n e u t r a l i s e t h e
dimethylamine formed from t h e neo sti gmi ne.
Each m l
o f
0.02
N
s u l p h u r i c a c i d
i s
equiva-
le n t t o 0 .006688 g of Cl3HZ2N2O6S.
Huang
t
a1
(24) de scr i bed a s imple , r ap id
and
accurate
method using t h e app l ic at i on
of
a l t e r n a t i n g - c u r r e n t o s c i l l o p ol a r o g r a p h i c
t i t r a t i o n i n p ha rm ac eu tic al a n a l y s i s . I n
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426
A. A. AL-BADR AND M ARIQ
t h i s method neostigmine methyl sul pha te i n
i n j e c t i o n s
was
t i t r a t e d wi th sodium
te t r apheny l bo ra t e . To th e i n j e c t i on
s o l u t i o n
( 2 0
ml) conta inin g 10 mg of
neostigmine methyl sul ph ate
were
added 10
m l
of 0.01 N sodium tetraphenyl borate and
10 m l o f a c e t a t e b u f f e r s o l u t i o n
(pH
5 . 3 ) .
The so lu t ion was d i l u t ed t o 50
m l
with
water ,
a f t e r
5 minutes t h e p r e c i p i t a t e
was formed, t h e co nt en ts
were
f i l t e r e d .
A
25
m l
por t ion of
f i l t r a t e
was t r ea te d wi th
8
drops of
4 0
sodium hydroxide solution and
t i t r a t e d w it h
0.01 N
TI2S04, t h e di s-
appearance of t h e in ci s io n of sodium
tetrapheny l bor ate on th e curve of d /E/dt vs
E
be ing used t o in d i ca te th e end po i n t . The
re su l t s were co r r ec t ed fo r
a
blank value.
The recovery range from
99 .3
- 100.2% and
th e coe f f i c i e n t o f v a r i a t i on was 0 .42%.
Tsubouchi e t a1
(25)
reported a method of
ana lys i s o f neos t igmine us ing the
application of one-phase end-point change
system i n two phase t i t r a t i o n t o amine
drug an al ys is . Neostigmine i n aqueous
s o l u t i o n 5
o r
10
mM)
was determined by
t i t r a t i o n with aqueous 0 .01 M t e t r apheny l
borate wi th te t rabromo phenolphthal ien
e thy l es te r a s an i n d i c a t o r
i n
t h e o r ga n ic
phase (1,2-dichloroethane)
,
i n b o r a t e
phosphate b u f f e r medium (pH 5. 5. -7 .5 ). The
end-point was det ec ted by means o f co lo r
change of t h e ind ic a tor i n the o rgan ic
phase wi thout t r a n s f e r of i ndi cat or between
pha ses . Various common io ns (i nc lu di ng
ca rbona te , a ce t a t e , c i t r a t e , and t anna te )
d id no t i n t e r f e r e bu t t h i amine
,
papaver ine
dodecyle sulphate and mercury caused
i n t e r f e r e n c e .
Diamandis and Christopoulos
2 6 )
developed
a p ot en ti om et ri c t i t r a t i o n
o f
neostigmine
bromide i n phar maceut ical compounds by
t i t r a t i n g them with sodium te t raphenyl
bo ra t e . 25
M 1
of aqueous solution (0 .2 -
1 mM)
of neostigmine bromide and
5 m l
of
th e appropr ia te bu f f e r so l u t io n was added .
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NEOSTIGMINE
421
The so l u t i on was t i t r a t e d po t en t i ome t r i ca l l y
a t 0 .36 m l per minute with 0.01
M
sodium
t e t r a p h e n y l b o r a t e a t 2 2 O 5 20 with
cont inuous s t i r r i n g . The end-point was
d e t e c t e d by a t e t r a p h en y l b o r a t e s e l e c t i v e
el ec tr od e. Thi s method was adopted f o r
determinat ion
o f
neostigmine in powders,
in je c t io ns , drops and syrups .
United S t a t e s Pharmacopeia
X I X
(1980) ( 1 2 )
desc r i bed t he fo l lowi ng procedure f o r t h e
assa y of neost igmine methyl s ulp hat e:
Place
about 100 mg of neost igmine methyl
su lp ha te , accu ra te ly weighed , in a
500
m l
Kj e l dah l f l a sk , d i s so l ve i n 150 m l
of
water ,
and add
4 0
m l of sodium hydroxide s ol ut io n
(1 i n 10) . Connect t h e fl a s k by means of
a d i s t i l l a t i o n t r a p t o a w el l- co ol ed
condenser t ha t d i ps i n t o
2 5 m l
o f b o r i c a c i d
s o l u t i o n ( 1 i n 2 5 ), d i s t i l abo ut 1 50 m l of
t h e c o n t e n t s of t he f l ask , add methyl
pu rp l e TS t o t he so l u t i on i n t he r ece i ve r ,
and t i t r a t e w it h 0 .0 2
N
su l phur i c ac i d .
Perform a blank determination, and make the
necessa ry co r rec t i on .
s u l p h u r i c a c i d i s equ i va l en t t o 6 . 688
mg
of
C13H22N206S'
B r i t i s h Pharmacopoeia (1973) ( 1 7 ) r e p o r t e d
t h e fo l lowi ng p rocedure f o r t h e a s s ay o f
neos t igmine t a b le t s
:
Weigh and powder 2 t a b l e t s . T r a n sf e r a
qua nt i ty of t he powder, equiva lent t o 0 .15
of neo stigm ine bromide, t o a semi-micro
ammonia d i s t i l l a t i o n appara tus , add 20 m l
o f a 50% w/v s o lu ti o n of sodium hydr oxide
and 0.5 m l of a 2% so lu t io n of octan-2-01
in l iq u id pa raf f in and comple te th e assay
desc r ibed under neost igmine methyl sul pha te .
(above) beg inin g a t th e words Pass a
c u r r e n t o f
steam
. . . . I . Each m l o f
0.02
N
s u l ph u r ic a c i d
i s
equi valen t t o 0 .006064 g
o f
C 1 2 H 1 9 B r N 2 0 2 .
Each
m l
of
0 . 0 2
N
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A. A. AL-BADR AND
M
ARIQ
8.2.2 Ti t r imet r i c Methods
Non-aqueous Titration
United S t a t e Pharmacopeia X I X (1980) (12)
desc r i bed t he fo l l owi ng p rocedure fo r t he
assay of neostigmine bromide:
Di ss ol ve abou t 750 mg of neostigmine bromide
ac cu ra te ly weighed, i n a mixtur e of 70 m l
of
g l a c i a l a c e t i c a c i d and 20 m l of mercur ic
ac e ta te TS, add 4 d r op s o f c r y s t a l v i o l e t TS,
and t i t r a t e wi th 0 . 1
N
p e r c kl o r ic a c i d t o
a
blue end- point . Perform a blank determina-
t i o n , and make any ne ces sa ry cor re ct io n.
Each m l of 0 .1 N p e r c h l o r i c a c i d
i s
equ iva len t t o 30.32 mg
of
C 1 2 H 1 9 B r N 2 0 2 (12) .
Bayer and Posgay (27) su gge ste d a p e r ch lo r ic
ac i d t i t r a t i on me t hod
f o r
t h e de t ermi na t ion
of
neost igmine in pharmaceut ica l p r epar a t ion s .
The neost igmine sol ut i on was t i t r a t e d wi th
0 . 1
N
perchlor ic acid which has been
sta nda rdi zed with anhydrous potassium
carbona te w it h 0 .1% gen t i an v i o l e t i n
ac e t i c ac i d con t a i n ing 3% mercu r i c ac e t a t e
a s i n d i c a t o r .
Surmann e t a1 ( 2 8 ) r e p o r t e d a t i t r a t i o n
method f o r t h e pharmaceutical hydroc hlorides
and hydrobromides i n non-aqueous media.
T h is method i n vo lv e d i r e c t t i t r a t i o n o f
d r u g w i t h p e r c h l o r i c a c i d i n a c e t i c
anhy dride medium, wit h na pht hol benz ein
o r
Sudan Red B an i nd i ca tor f o r hydro-
ch lo r ide s f o r hydrobromides re spe c t i ve l y .
Kracmarova and Kracmar (29) reported the
following non-aqueous t i t r a t i o n :
Evaporate t h e sample of t h e eye drops (5 ml)
on a water ba th t o dryness , d i ss o lve th e
res idue in anhydrous ac e t i c ac id (2 x 20 ml),
add c r ys t a l v i o l e t (0 .2%
i n
anhydrous
ac e t i c ac i d ) (2 d rops ) a s i nd i ca t o r , and
t i t r a t e w ith 0 .1 N p e r c hl o r ic a ci d t o a
blu e end- point , add 5% HgII ace t a t e so l u t i on
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NEOSTIGMINE
429
(5 ml), m i x and t i t r a t e ag ai n t o
a
blue end-
po i n t ; t h e d i f f e rence between t h e t i t r e s
corresponds t o neost igmine bromide.
8. 2. 3 Io di me tr ic Methods
Koka (30) developed an io di me tr ic deter mina-
t i o n of p ros er in e (neos t igmine methyl
sulph ate) i n drug form ulat i ons. The drug
sample
i s
d i s s o l v e d i n
water
and the
so l u t i on
i s
t r e a t e d w it h 2 - 3
m l
o f d i l u t e
su lph ur ic ac id , 2-3 m l of 10% potassium
iodide so lu t ion and 5 m l of 0 . 1 N i od i ne ,
d i l u t e d t o 25
m l
wi t h wa t e r and f i l t e red ;
10
m l
of f i l t r a t e
i s
t i t r a t e d w i t h 0 . 1
N
sodium thiosulphate .
Mitchenko and Kirichenko (31) reported the
use
of
i od im et r i c method f o r th e de termina-
t i o n of neost igmine methyl su lph ate and
p i l oca rp i ne HC1 i n eye-drops. Neostigmine
methyl su lph ate and pi l oc arp ine HC1 a r e
de termined by reac t ion wi th iod ine so lu t ion
i n d ar k, f i l t e r i n g t h e m ix tu re and t i t r a t i n g
unreac ted io d ine wi th sodium th io su lp ha te
so lu ti on . When bot h a r e pr es en t th e sum
of the two i s determined by above method
and only pi locarpine
i s
determined a l k a l i -
m e t r i c a l l y , a r g e n t i m e t r i c a l l y o r m e rc u ri -
me tr ic al ly . Neost igmine methyl sul pha te
i s
determined by th e di f fer enc e. The
r e l a t i ve
e r r o r i n de termining the two drugs
in eye-drops i s 3.56 and 2.85 respect ively .
8. 3 Bi ol og ic al Method
B u c k l e s
and Bullock (32) reporte d th e ap pl ic at io n
of enzyme i nh i b i t i o n t o t h e e s t i ma ti on o f sma ll
q u a n t i t i e s o f d r ug s po s s es s i ng a n t i c h o l i n e s t r a s e
a c t i v i t y . The method
i s
based on t h e i n h i b i t i on
o f t h e p se u d oc h o li n e st e r as e a c t i v i t y o f h o r s e
serum.
neost igmine
i s
mixed with 1
m l
of horse serum,
1
m l
of
0.2 cresol
red so lu t i on and 37
m l
of
water .
a d j u s t e d t o 7.9. After 15 minutes add 5 m l of
3%
ace t y l cho l i ne pe rch l o ra t e so l u t i on and read j us t
5 M 1 of sample containing 5 ug
of
The mixture i s h e a t e d t o 4OoC and pH
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430
A. A. AL-BADR AND M.
TARIQ
t h e pH t o 7 . 9 .
7 .8 and 8. 0 du ri ng 15 minut es by dropwise
add i t ion o f 0 .025
N
sodium hydroxide and record
th e volume of a l k a l i used. Correc t f o r non-
enzymic hydrolysis by conducting an experiment
with 1 m l buf fe r in s tead o f ho rse se rum and ca r ry
out a blank with 5
m l
water i ns te ad of sample.
The pe rcen tage inh ib i t ion i s a l i n e a r f u n ct i on o f
t h e log concentra t ion of neostigmine methyl
su lp h a te .
Maintain the pH between
8. 4 Spec trop hotom etri c Methods
8.4 .1 Colo rime tri c Methods
Belal
e t
a l , (33) descr ibed a sp e c t ro -
photometr ic de terminat ion of neost igmine
in t ab l e t s and ampoules . Tab le t p repara t io ns
conta in ing neost igmine methyl su lp hate were
powdered and di ss ol ve d i n
50 m l
of wa te r
and
1
m l o f s o l u t i o n was mixed with 3 m l o f
c i t ra te -phospha te buf fe r so lu t ion (pH 7.8)
and
2
m l of a
0.1%
solution of bromothymol
b lu e in 0 .01
N
sodium hydroxide.
mixture was ex t rac te d wi th ch loro fo rm befo re
measurement
o f
i t s absorbance a t 416 nm
v s a reagent b lank .
o f chlo ro form ex t r ac t
was
t r e a t e d w it h 10 ml
of
0 . 0 1
N sodium hydroxide and the aqueous
l a y e r
was
se pa ra te d and made up t o 25 m l
with water, i t s absorbance being measured
a t 615 nm as a reagent b lank . The ca l i br a-
t i o n g ra p h were r e c t i l i n e a r f o r 0 .2-1 .6 mg.
The re cover y by t h i s method
was
100.5%.
Belikov e t a1 ( 3 4 ) have used sulphoneph-
th a l e in d y e s as e x t r a c t i o n r e ag e nt s f o r
th e e xt r a ct i o n of qu at er na ry ammonium
compounds.
va lue , Xmax, d i s t r i bu t i o n co ef f i c i en t and
s e n s i t i v i t y o f r e a c t i o n s f o r t h e i on
as so ci at io n complexes of neos tigm ine methyl
su lp h a te .
determining the quaternary ammonium
compounds, 0 . 5 m l of 0 .01% so l u t ion o f
compound is added t o 5 m l o f a b u f f e r s o l u -
t i o n o f app rop ri ate pH, 0 .5
m l
of 0.1% dye
The
A l t e r n a t i v e l y
10
m l
They re po r t e d t h e optimum pH
In a genera l procedure for
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NEOSTIGMINE
43
s o l u t i o n
i s
added, the complex i s e x t r a c t e d
i n t o
5
m l of chloroform and absorbance of
organic phase i s measured a t 400-434 nm.
Rela t ive
e r r o r
i s l e s s
than 2 .
Tsubouchi (35) reported
a
spec t ropho tomet r ic
de te rmina t ion o f o rgan ic c a t i ons by
so lven t ex t r ac t i on wi th te trabromopheno l -
ph th al ie n et hy l e s t e r . 5 1.11 of the sample
c o n ta in in g
less
than 0 .01
WM
of neostigmine
was e x t r a c t e d w i t h 2
m l
of 0.07% e t h a n o l i c
tetrabromophenolphthalien e t h y l e s t e r
(p o ta s s iu m sa l t )
and m l o f b o r a t e -
phosphate buffer (pH
l o ) ,
d i l u t e t o 25
m l
with
water
and shake for 2 minutes with
1 0
m l
o f 1 ,2 ,d i c hlo ro eth a ne . Se p ara t e and f i l t e r
the organic phase and measure i t s
absorbance a t 615 nm aga in s t reagen t b lank .
The c o e f f i c i e n t o f v a r i a t i o n was
1.11 .
8.4.2 U l t r a vi ol e t SDectronhotometric Method
Rita (36) developed a u l t rav io le t
spec t ropho tomet r ic a ssay o f neost igmine
methyl sulphate
as
t h e a l k a l i n e h y d ro l ys i s
product , 3- (dimethy1amino )phenol. An
a l i q u o t o f i n j e c t i o n pr e p ar a t i on e q u iv a l en t
t o 5 mg o f neost igmine methyl su l phat e was
a ci d i f i e d and any phenol o r p-hydroxy
b en z oa te p r e se r v a t i v e p r e se n t was
ex t ra c te d wi th chlo ro form. The res idu a l
aqueous so lu t ion
was
made alkaline and
heat ed on steam bat h f o r 30 minutes and
t h e
3-
(dimethylamino) phenol formed i s
determined by UV-spectrophotometry.
Kracmarova and Kracmar (29) described a
spec trop hoto metr ic method
f o r
eva lua t ion o f
neost igmi ne bromide and neosti gmin e methyl
su lp h a te wi th r e g a rds t o t h e d e g ra d a tio n
products .
2
m l
o f 0.05
N
H2SO4 was added.
was
d i l u t e d t o 10 m l with water. The
absorbance was recorded a t 261 nm f o r
neostigmine bromide o r a t
260 nm f o r
neost igmine methyl su l phat e o r a t 266 nm
for both of the compounds.
To 5 ml o f i n j e c t i o n s o l u t i o n ,
The mixture
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432
A. A . AL-BADR AN D M. TARIQ
Mytchenko and Kyrychenko (37) r e p o r t e d a
spectrophotometr ic de terminat ion of
pro ser ine (neost igmine methyl sulp hate )
i n med ic inal fo rmula t ions . The t a b l e t s
are grinded in water and shaked.
s o l u t i o n i s f i l t e r e d and th e a b so rb an ce o f
f i l t r a t e i s d i r e c t l y r ec or de d
a t
260 nm.
This method can a ls o be used f o r an al ys is
of neost igmine i n eye drops (conta in i ng
al so pi lo ca rp in e HC1) a f t e r d i lu t io n . Th e
accuracy of method i s with in ? 0.66%.
0.5%
8.4 .3 In fra red Spectrophotom etric Methods -
I R
Mynka t
a1
(38) r e p o r t e d t h e i n f r a r e d
spectroscopy of drugs o f the amide class.
The
I R
spectrum
of
neostigmine methyl
su lp h a te
was
determined and in terpre ted
by measurement
a t
1732 cm-l,
be done with a maximum e r r o r o f 1 .66%.
Analysis can
Kracmarova and Kracmar (29) re po r t e d
spectrophotometr ic an al ys is of neos t igmine
i n i n f r a r e d r e g i o n .
w it h l i q u i d p a r a f f i n ( 1 2 drops) and record
the spectrum in a sodium chloride c e l l i n
the range
3-15 1 1.
C h a r a c t e r i s t i c band f o r
neostigmine bromide and neostigmine methyl
su lpha te a re obse rved .
Grind the sample
8. 4. 4 Phot ometr ic Method
Kracmarova and Kracmar (29) r e po r t e d a
photometric method or t h e eval uat io n of
neos tigm ine bromide and neostig mine
methyl sulphate as fo l lows :
To th e i n j ec t i on so lu t i on (1 .5 ml) add
buffer so lu t ion (pH 4.6) (5 ml), bromo-
c re s o l p urp l e so lu t i o n (2 g in 15 m l o f
water
and
3 . 2 m l
o f 0 . 1
N
- potassium
hydrox ide d i lu te d t o 250
m l
with water)
(S ml) and chloroform (25 ml) . Shake f o r
1 minute in a se par a t i ng funne l , and f i l t e r
t h e c hloroform l a y e r ; r e p e a t t h e e x t r a c t io n
with
2 m l
of ch lo rofo rm and d i l u t e th e
combined e x t r a c t s wi th ch loroform t o
50
m l .
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NEOSTIGMINE
433
8.5
To
a 25
m l
por t ion add 0 .1
N
potassium
hydroxide (20 m l ) , shake for 30 seconds,
f i l t e r t h e aqueous l a y e r , d i l u t e t o 100 m l
with water and measure the ex t inc t ion
a t
570 nm.
Chromatographic Methods
8 . 5 . 1 Pape r Chromatographic Method
Giebelmann (39) developed a pa pe r chromato-
graphic detection for phenoxy compounds
having
a
phenyl e s t e r funct ional group
u s in g th e fo l lo win g so lv e n t s
-
butanol-20%
formic ac id ( 4:1) , methanol acetone-
t r ie thano lamine (100: 100: 3) and bu ta no l -
water
( 6 : l ) . As
l i t t l e
as
2.5-10 1-16 of
neost igmine can be de tec t ed with Mil lon 's
r e a g e n t .
S c o t t t
a l ,
(40) reported an i n v e s t i g a t i o n
on t h e metabolism of neost igmine i n
pa t i en ts wi th myas thenia g ra v is . Neost ig -
m i
n
e
an
d
m
-
h y d roxyphen y
1
i m
e
t
h y
1
mmon
rn
bromide
are
p re c ip i t a t e d from u r in e wi th
aqueous bromine. The p r e c i p i t a t e
i s
ex t ra c te d wi th 50% methano l. Th is ex t ra c t
is a p p l i e d t o Amb er li te
CG-50
r e s i n
a t
pH
6.86.
Ten bases are e l u t e d w i th
0 . 2
N
hydroch lo r ic ac i d and submi t ted t o
chromatography on Whatman
wi th
butanol-ethanol-water-acetic
a c i d
(32: 8: 1 2 : 1) as so lvent . The
R f
va lue o f
neo sti gmi ne was between 0.5
-
0 .3 .
N O
541 paper
Kracmarova and Kracmar
( 2 9 )
descr ibed
p roc e du res fo r t h e d e t e rm in a t io n o f
neo sti gmi ne bromide and neo st igmine methyl
su lpha te in va r ious pharmaceu t ica l p repara -
t i o n s , a s w e ll as
f o r
the chromatographic
c o n t ro l o f t h e d e g ra d a t io n p ro d u c t s ;
3 dimethylaminophenol and (3-hydroxyphenyl)
trimethylammonium s a l t s , wi th t h e u se o f
Whatman
No.
1
paper, butanol-conc-aqueous
ammonia-water (3:
1
:4) as so lvent , and
Dragendorff reagent as t h e d e t e c t i n g a g e n t .
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434
A. A . AL-BADR AND M. TARIQ
Clarke (11) descr ibed the fol lowing:
(1) Paper: Whatman
No. 1,
sh eet 14 x 6 i n ,
buffered by dipping in a
5%
so lu t ion o f
sodium hydrogen ci trate, blott ing, and
d r y i n g a t
2 5 0
f o r
1
hour.
stored immediately.
Sample:
2 .5 1-11
of
a
1
s o l u t i o n ; i n
2 N
a c e t i c a c id
i f
poss ib le , o therwise i n
2
N
hydrochlor ic ac id , 2 N sodium hydroxide,
o r e thano l .
So lvent : 4 .8 g o f c i t r i c ac id i n a mixtu re
of 130 m l of water and 870 m l of n-butanol .
This may be used f o r se ve ra l
weeks i f water
i s
added from time t o
time
t o keep t h e
sp ec i f i c g r av i ty a t 0 .843 t o 0 .844.
Development: Ascending, i n ta nk
8
x
11
x 15% i n ;
4
shee ts being run a t a
time. T i m e
of run,
5
hours .
Loca t ion unde r u l t r av io l e t l i gh t , abso rp t ion ;
loca t ion reagen ts
:
weak re ac ti on ; bromocresol green sp ra y,
weak reac t ion . Rf
0 . 2 0 .
I t
can be
I
odopla t na te spray
;
( 2 ) Paper: Whatman
No. 1
o r
No.
3,
sheet 17
x
19
cm,
implegnated by dipping
i n a 10% s o l u t i o n o f t r i b u t y r i n i n
acetone and dry ing in a i r .
Sample:
ethanol or chloroform.
Solvent: Acetate b u f f e r (pH 4.58).
Equi l ibr a t io n: The beaker conta inin g t h e
so lven t i s e q u i l i b r a t e d i n a t h e r m o s t a t i c a l l y
cont ro l led over
a t
95O f o r about 15
minutes.
Development: Ascending, t h e pap er
i s
folded
i n t o
a
cy l inder and c l ipped , th e cy l inder
i s
i n se r t e d in t he beake r con ta in ing th e
solvent which
i s
not removed from the oven.
A
pla t e -g l ass d i sk t h i ck ly smeared wi th
s i l i co ne grease may ser ve
as
a cover. T i m e
of
run , 15 t o
20
minutes.
5 p1 of
1
t o 5 s o l u t i o n s i n
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NEOSTIGMINE
435
Locat ion: Under u l t r av io l e t l i g h t ,
absorp t ion ,
R f
1.00.
(3) Paper and sample a s i n 2 above.
Solv ent: Phosphate bu ff er (pH 7. 4) .
Equi l ibr a t io n: The peak conta inin g t h e
so lven t
i s
equ i l i b r a t ed in a t he rmos ta t i -
c a l l y co nt rol le d oven a t 86' f o r about
15 minutes.
Development: as in 2 above.
Locat ion: Under u l t r av io l e t l i gh t ,
absorp t ion ,
R f
0.66.
8 .5. 2 Thin-Layer Chromatography (TLC)
Anand (41) reported a thin - lay er chromato-
graphic and spectrophotometr ic invest iga-
t i o n on t h e i d e n t i f i c a t i o n o f
a
phenol ic
impu rity i n neos tigm ine. The samples
of
neostigmine
were
app lie d on
TLC
p l a t e s
coated with alumina-D and were
developed
for 30 minutes with chloroform-benzene-
methanol (5:4:1) t o giv e
a
good separat ion.
Neostigmine was det ec te d with i od in e
vapours o r wi th Dragendorff ' s re agen t .
u l t r a v io l e t spec t ropho tomet r ic t e chnique
i s
a l s o used f o r q u a n t i t a t i v e e st i m a ti o n .
Neostigmine has a maxima a t 261 nm.
Po rs t and Kny (42) de sc ri be d an al ys is of
neostigmine eye drops using
TLC
system
and ab sor pti on spec tros copy . The drug
i s
de te ct ed and determined by
TLC
on
c e l l u l o s e and u l t r a v i o l e t o r v i s i b l e
spectrophotometry
i s
done a f t e r fo rmat ion
of colored compound with 3,S-dichloro-p-
benzoquinonechlorimine
o r
sodium n i t r i t e
o r 4-dimethylaminobenzaldehyde.
The
Clarke
(11) descr i bed th e fol lowing:
Pla te :
Glass p l a t e , 20
x 20
cm,
coated wi th
a s l u r r y c o ns i st i ng o f 30
g
o f s i l i c a ge l
G
i n
60 m l
o f w at er t o g i v e - a l a y e r 0. 25 mm
l i k e and d r i ed
a t
110 for 1 hour.
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436
A. A. AL-BADR AND M. TARIQ
Sample:
ac id .
Solv ent: Stro ng ammonia so lu ti on :
methanol (1.5
:
100).
I t
should be
changed a f t e r two runs.
Equi l ibr a t io n: The solv ent
i s
allowed t o
s t an d i n t h e t an k f o r 1 hour .
Development: Ascending, i n a ta nk ,
2 1 x
21 x 1 0
cm,
th e end of th e tank be ing
c ov er ed w it h f i l t e r pap er t o ass i s t
ev apo rat io n. Time of run,
30
minutes.
Locat ion reagen t : Ac id i f ie d iodo pla t ina t e
s p ra y , p o s i t i v e r e a c t i o n ,
R f
0.02,
8.5 .3
Gas Liquid Chromatographic Methods
1 u 1
of a
1
o l u t i o n i n
2 N
a c e t i c
Chan e t a1
( 4 3 )
developed a s e n s i t i v e and
se l e c t iv e ana ly t i ca l method fo r t he
measurement o f neos tigmine i n human plasma
us in g gas chromatography. Plasma
conta ining neost igmine i s washed with
ethyl e ther and buffered wi th potass ium
iodide-g lyc ine so lu t ion , then iod ide-
glycine-drug complexes
are
e x t r a c t e d i n t o
dichloromethane. The e x t r ac t
i s
evaporated
and the res idue
i s
dissolved in methanol.
2 - 5 1-11 of methanol ic so lu t ion
was
i n j e c t e d
i n t o gas chromatographic g l a s s column
( 2 m
x
0 . 2 5
inches
O.D.)
packed with
Diatomite
C Q
coated wi th
3
of
O V - 1 , 3
of
OV-17
o r
3 . 8 of
SE-30
and s i l an i sed ;
a
ni t rogen se le c t iv e de t ec to r was used.
Pyridostigmine bromide
was
used a s i n t e r na l
sta nda rd . The temp erat ure was main tain ed
a t 205OC and th e fl ow
r a t e o f
nitogen was
30
m l
pe r minute. The de tec tio n l i m i t
was 5 ng/ml and t h e r e
was
no in t e r f e r ence
wi th o ther bas ic d rugs .
Ward e t a1 ( 4 4 ) desc ribe d a simple G L C
assay
of
neost igmine us ing die thyl
ana log of neos t igmine as in te rna l s tandard .
Neostigmine and i t s analog were dissolved
in 25
1.11 of
chloroform. 2
1.r1
of
chloroform solution was i n j e c t e d i n t o
a
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NEOSTIGMINE
437
gas chromatograph equip ed wi th a flame
i on i sa t i on de t ec t o r and
1 . 2
m x
2 mm I . D .
glass column packed with 5% O V - 1 7 on
Gas-Chrom
Q
i s
used a t 210' wit h helium
(40 ml/minute) a s a car r ie r gas and flame
i on i za t i on de t e c t o r . For de t e rmi nat i on o f
n eo st ig mi ne i n b i o l o g i c a l f l u i d s , t h e dr ug
i s
ext r ac ted in to ch loroform wi th added
3-diethylcarbamoyloxy- N-trimethylanil
inium
iodide as t he i n t e rn a l s t anda rd . The
r e l a t i v e r e t e n t i o n time of t h e drug and the
s t anda rd i s
1
t o 1 .3 5.
De Ruyte r
t a1
(45) developed a reversed-
phase, ion -pa i r l i q u id chromatography
o f
quarternary ammonium compounds
or
t h e
determinat ion of pyridost igmine,
neost igmine, and edrophonium i n bi ol og ic al
f l u i d s . The method i s based on ext ract ion
of neost igmine in to wate r -sa tura t ed
dichloromethane, then back-ext ract ion
i s
done with tetrabutyl ammonium hydrogen
sulp ha te t o enhance th e recovery t o more
than 86%. The e x t r a c t s a r e submi t ted
t o
chromatography on a v a r i e t y of reve rsed -
phased columns f i t t e d with 214
nm
d e t e c t o r s . For b i o l o g i c a l e x t r a c t s a
column (15 cm x 4.6 mm) of Ul t raspher e
o c t y l 5 pm) p ro vi de d wi th an RP-2
pre-column was used. F o r neost igmine
e l u t i o n was c a r r i e d o ut w it h a s o l u t i o n
cont ainin g sodium hep tanesulphonate
(0.01 M ) ,
sodium hydrogen phosphate
(0.01
M)
and tetramethyl ammonium chloride (2.5 mM)
i n aqueous 20% ac e t on i t r i l e . The mobi le
phase (2
m l
per minute)
was
a d j u s t e d t o
pH 3 wit h s u lp h ur ic a ci d , edrophonium was
used as an in te rn a l s t andard . The
c a l i b r a t i o n g r a p h s were r e c t i l i n e a r f o r
upto 400 ng
per
m l . The
l i m i t
of
detect ion was 5 ng per
m l .
o f
v a r i a t i o n
was 1 .5%.
The coe f f i c i en t
Davison
e t
a1 (46) described a method for
simultaneous monitoring of plasma level
of
neostigmine and pyridostigmine in man.
The assay involve pre l iminary ion p a i r
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438
A . A. AL-BADR AND M . TARIQ
ex t r ac t i on o f t h e d rugs and in t e r na l
st an da rd s (3-d ipro ypl carbamoyloxy) 1-methyl
pyridinium bromide) with the use of potassium
iodide and of g lycin e buf fer .
i s
analysed by
G L C
(10% of OV-17 on
Chromosorb
W-AW (100-120
mesh) with
a
ni t rogen -sens i t i ve de te c tor . The
ca l i -
bra t ion g raphs a r e r e c t i l i ne a r and
reproduced over the range 5-100 ng per
m l
of e i t h e r d rugs i n 3
m l
samples.
The extract
Pohlmann and Cohan (47) developed a simpli-
f i e d de te ct io n of qu ar te rn ar y ammonium
compounds by ga s chromatography. The
method has been app lie d t o pyri dost igmi ne,
neostigmine and ac et yl ch oli ne. The
qua r t e rna ry
s a l t s
i n t h e serum o r u r i n e
a r e
f i r s t
converted with potassium
t r i i o d i d e
(K13) i n t o t h e i r io di es which
ar e ex tra ct ed in to chloroform. The
e x t r a c t i s evaporated in vacuum and the
res idue i s d i s so lved in
water o r
hexane.
The solution
i s
i n j ec t e d on t o a column
(1 .8
m
x 2 mm) of Porapak
Q
(800-100 mesh),
Chromosorb 101 (80-100 mesh)
o r
Chromosorb
105 (80-100 mesh), opera ted a t 14SoC-17O0C
with helium o r n i t rogen a s c a r r i e r gas
(20 m l per ml) .
qua rte rna ry ammonium io di de s a r e the rma lly
decomposed, and th e methyl io di de re le as ed
i s measured with a 63Ni electron-capture
d e te c to r . E xt ra ct io n y i e l d s a r e a t l e a s t
95% and down t o
1 0
f-mol of qua r t e rna ry
ammonium compound can be detected with
good reproducibi l i ty .
On the column the
Chan and Dehghan (48) descr ibed a
GLC
method
f o r
i s o l a t io n and de te rmina t ion of
neost igmine, pyr idost igmine and thei r
metabol i te s i n human bio lo gic al f l u i ds .
The method involves
a
pre l iminary ,
s e l e c t i v e i o n -p a i r e x t r a c t i o n o f t h e d ru gs
and t h e i r metabol i t es in to d ich loromethane
and in
dichloromethane-acetone)
,
resp ect i vely . The an aly s i s of ext ra ct was
done by G L C on a column of
3%
(drug) o r
10% (me ta bo l it e) o f OV-17 on Chromosorb AW,
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NEOSTIGMINE
439
with a ni t rogen-sens i t ive de tec to r .
For
the determination of 3-hydroxytrimethyl
ani l i nium ion; a major metabo l i te of
neostigmine,
3-hydroxy-N-methylpyridinium
ion was used as a in te r na l s tanda rd.
As
l i t t l e as
3 ng/ml of neost igmi ne and upto
50 ng/ml of i t s metabolite can be
determined i n bi ol og ic al samples.
8. 6 Ion-S electi ve Ele ctr ode s Method
Kina t a1
(49)
described a method of neostigmine
ana lys is based on ion-se le c t ive e lec t rodes
s e n s i t i v e t o some orga ni c compounds used a s drugs.
Liquid membrane ele ctr ode s se ns i t iv e t o neo st i g-
mine have been made by the ion-association
e x t r a c t i on method. The exchange components used
were
cr ys ta l v io le t , d ip icrylamine , sodium
tet rap ent yl bora te , l , lO-phenanthroline , 4 ,7-
diphenyl- 1,lO-phenanthroline
for these compounds were 1,Z-dichloroethane and
nitrob enzen e. The choic e of s o l ve n t s a f f e c t e d
with s e l e c t i v i t i e s of t he membranes but th e choice
of ion-exchange components d id no t.
s e l e c t i v i t i e s r e l a t i v e t o un iv al en t c a t i on s
were
mostly bet ter than 10-4.
range of electrodes was 10 V M t o
0 . 1
M with a
response of 59 m V per decade change in the
concent ra t ion of the drug.
be used i n an al ys is of mixed pharmaceutical
prepara t ions .
Diamandis and Christopoulos (26) reported a
po ten t iomet r ic t i t r a t i o n o f neost igmine and o t he r
pharmac eutic al compound i n pharmac eutical formu-
l at io n with sodium tet rah ydr obo rat e. (See
Sect ion 8 . 2 . 1 . ) .
tetraphenylborate-selective
e lec t rode .
The so lv en ts used
The
The useful concentrat ion
The electrodes could
The end po in t was det ec te d by a
8. 7 Po la ro gr aphi c Method
Novotny (50) devised a pola rog rap hic method f o r
the determination of neostigmine in pharmaceutical
prepa ra t io ns having a concent ra t ion o f
0 .5
mg/ml
of the drug.
hydrolys is of neost igmine and n i t r a t io n of
re su lt in g phenol, followed by polarography of
This method i s based on the
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A . A. AL-BADR AN D M. TARIQ
r e s u l t i n g d e r i v a t iv e . The n eo s tig min e so l u t io n
( 0 . 2
t o
0 . 6
m l of
0.1%) i s
evapora ted t o d ryness
in a
50 m l
beaker on a water b a th .
1
bll of KOH
s o l u t i o n
( 2 0 ) i s
added and warmed on t h e ba th
f o r
2 0
minutes and again evaporated t o dryness.
0 . 5 M 1
of
water and 2 m l of conc.
HN03
(65%)
i s
added and warmed fo r
20
minu tes and cooled. 10
M 1
of
KOH s o l u t i o n
(20%)
is added and the volume
made upto 14.5 m l with water. The est imation
i s
carr ied out by polarography, wi th
a
galvanometer
s e n s i t i v e t o
10-9
amp.
concen t ra t ion
of
neostigmine in an unknown
s o l u t i o n , s o l u t i o n o f t h e unknown conce nt rat ion of
neostigmine
i s
t r e a t e d
as
described above, both
with and without
a
known amount of neost igmine .
To determine the
Acknowledgements
The authors would l i k e t o thank Mr. Syed A l i Jawad, College
of Pharmacy,
King Saud Univers i ty fo r h i s t e chn ic a l assis-
tance and Mr. Tanvir
A.
Bu tt or h i s s e c r e t a r i a l a s s i s t a n c e
in typ ing the manuscr ip t .
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NEOSTIGMINE
441
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