Analytical Profiles of Neostigmine

7/26/2019 Analytical Profiles of Neostigmine

1/42

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


3/42

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


5/42

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


2 . 3 Molecular Weipht

Neostigmine bromide

303.2


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) .


Crystals from alcohol melt between 142OC and 145OC

(13)

*


6/42

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 ) .


7/42

NEOSTIGMINE

lot

2

2SOWavelength

300

nm)

350

400

Figure I: Ultraviolet spectrumof neostigmine bromide in methanol.


8/42

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


9/42


10/42

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.


11/42

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:


12/42

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.


13/42

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


14/42

Figure 6: Electron impld El)mass spectrum

Of

neostigmine methy


15/42

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:


16/42

Figure 7 : Chemical ionization C I mass Spectrum Of neostigmine


17/42

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) .


18/42

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


19/42

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)


20/42

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


21/42

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


22/42

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,


23/42

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


24/42

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 .


25/42

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


26/42

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


27/42

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


28/42

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


29/42

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


30/42

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 .


31/42

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 .


32/42

434


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


33/42

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.


34/42

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


35/42

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


36/42

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,


37/42

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


38/42

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 .


39/42

NEOSTIGMINE

441

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J . ,

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(1925).

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M .

Reinert .

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Pharmacol. Exp.

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7 .

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L .

Remen. Dtsch.

Z .

erv

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M . B .

Walker. Lancet, 1, 1200 (1934).

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B . Schwartz.

N e w

England J . Med., 90, 182 (1978).

The P har mac eutic al Codex , 11 th Ed., The Pha rma ceu tica l

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Documents

Analytical Profiles of Neostigmine