Analytical Letters

ISSN: 0003-2719 (Print) 1532-236X (Online) Journal homepage: http://www.tandfonline.com/loi/lanl20

Extraction and Spectrophotometric Determination

of Neomycin Sulfate in Pharmaceutical Oral
Suspensions

M. R. C. Marques , E. R. M. Hackmann & T. Saito

To cite this article: M. R. C. Marques , E. R. M. Hackmann & T. Saito (1990) Extraction and
Spectrophotometric Determination of Neomycin Sulfate in Pharmaceutical Oral Suspensions,
Analytical Letters, 23:6, 1005-1016, DOI: 10.1080/00032719008053442

To link to this article: http://dx.doi.org/10.1080/00032719008053442

Published online: 23 Oct 2006.

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 EXTRACTION AND SPECTROPHOTOMETRIC DETERMINATION OF
NEOMYCIN SULFATE IN PHARMACEUTICAL ORAL SUSPENSIONS.

KEY WORDS: neomycin sulfate, spectrophotometry, oral

suspension, extraction.

M.R.C. Marques, E.R.M. Hackmann, T. Saito.

Faculdade de Cigncias FarmacGuticas, Universidade de
Sao Paulo, Caixa Postal 30786, 01000, Sao Paulo, Brazil.

ABSTRACT
The conditions of neomycin extraction were studied
in pharmaceutical oral suspensions to permit its deter-
mination by spectrophotometry after reaction with 2,4-

dinitrofluorobenzene. The best recovery of neomycin was

obtained with centrifugation employing water as the ex-
traction agent. The spectrophotometric analysis was
carried out in 0.02 M borate buffer (pH 9.0). under am-
bient conditions after 50 minutes of reaction. The ab-
sorbance of the yellow product was measured at 365 nm.

INTRODUCTION
Neomycin is a broad spectrum antibiotic used in
gastrointestinal infections mainly in child diarrhea
caused by Escherichia coli' ' 2 . In general, this antibi-
otic is formulated in oral suspensions with pharmaceu-

1005

Copyright 0 1990 by Marcel Dekker, Inc.

1006 MARQUES, HACKMANN, AND SAITO

t i c a l a g e n t s s u c h as k a o l i n , p e c t i n and aluminium hy-

d r o x i d e . These s u b s t a n c e s a r e u t i l i z e d as a n t a c i d , an-
t i f l a t u l e n t and a n t i d i a r r h e a l . They a d s o r b b a c t e r i a and
t o x i n s r e s p o n s i b l e f o r t h e o n s e t of d i a r r h e a , vomiting,
n a u s e a and cramps i n v a r i o u s i n t e s t i n a l i n f e c t i o n s and
by c o a t i n g t h e i n f l a m e d mucous membrane o f t h e intes-
t i n a l t r a c t 3 . However, t h e s e e x c i p i e n t s are capable of
a d s o r b i n g v a r i o u s a n t i b i o t i c s s u c h a s neomycin 3,495
Some suggested t h e u s e of b u f f e r s to
i n c r e a s e t h e l i b e r a t i o n o f a d s o r b e d neomycin. On the
o t h e r h a n d , Harris7 d e m o n s t r a t e d t h a t t h e interaction
between neomycin and p e c t i n may be i n h i b i t e d by t h e ad-
d i t i o n o f e l e c t r o l y t e s . T h e r e f o r e , a n a t t e m p t was made
t o establish the best conditions f o r the release Of the
a d s o r b e d neomycin and f o r i t s q u a n t i f i c a t i o n by s p e c t r o -
photometry a f t e r r e a c t i o n w i t h 2,4-dinitrofluorobenzene
(DNFBI8. The r e s u l t s were compared w i t h t h o s e obtained
u s i n g t h e o f f i c i a l m i c r o b i o l o g i c a l method
9
.
EXPERIMENTAL
Apparatus
The a b s o r b a n c e s were measured w i t h a V a r i a n model
634 s p e c t r o p h o t ome t e r .
Reagents
A l l of t h e c h e m i c a l s used were o f a n a l y t i c a l gra-
d e . Aqueous s o l u t i o n s were p r e p a r e d i n d i s t i l l e d w a t e r .
A 0.25% m e t h a n o l i c s o l u t i o n o f DNFB w a s freshly pre-
p a r e d d a i l y . The b u f f e r s used were 0 . 1 M p h o s p h a t e (pH
8 . 0 ) and 0.02 M b o r a t e (pH 9 . 0 ) . The e x c e s s o f DNFB w a s
DETERMINATION OF NEOMYCIN SULFATE 1007

d e c o l o r i z e d w i t h 1.0 M HC1. The a q u e o u s e l e c t r o l y t e so-

l u t i o n s employed were 0 . 1 M sodium o x a l a t e and 0.1 M so-
dium c h l o r i d e .

Pharmaceutical Suspensions
Formula A
neomycin s u l f a t e . . . . . . . . . . . . . . . . . . . 2.00 g
kaolin............................. 10.00 g
aluminium h y d r o x i d e . . . . ............ 1.00 g
d i s t i l l e d water....................to 100 m l

Formula B
neomycin s u l f a t e . . , . . . . . . . . . . . . . . . . 1.20 g
kaolin............................. 2.00 g
c i t r i c pectin..... ................. 4.00 g
sucrose............................. 75.00 g
d i s t i l l e d water....................to 200 m l

Formula C
neomycin s u l f a t e . . . . , . . . ........... 1.00 g

kaolin............................ . 20.00 g
c i t r i c pectin. ..................... 0.44 g
d i s t i l l e d water.............. ......t o 100 m l

PROCEDURE
Ex c r a c t i on p r o c e s s
A 5 m l a l i q u o t o f t h e Formula A w a s t r a n s f e r r e d t o
a 100 m l c a l i b r a t e d f l a s k and made up t o volume with
d i s t i l l e d water. A f t e r h o m o g e n i z a t i o n , the mixture was
f i l t e r e d t h r o u g h two Whatman no 1 f i l t e r paper sheets
1008 MARQUES, HACKMANN, A N D SAITO

and c o t t o n . A 3 m l a l i q u o t o f f i l t r a t e was transferred

t o a 100 m l c a l i b r a t e d f l a s k and d i l u t e d t o volume w i t h
0 . 0 2 M b o r a t e b u f f e r (pH 9 . 0 ) . The end concentration
was 30 bg/ml o f neomycin s u l f a t e .
A l i q u o t s ( 3 m l ) o f Formula A were p l a c e d i n 15 m l
c e n t r i f u g e - t u b e s and d i l u t e d t o 1 2 m l w i t h e a c h one of
t h e f o l l o w i n g s o l u t i o n s : 0 . 1 M aqueous sodium oxalate
s o l u t i o n , 0 . 1 M a q u e o u s sodium c h l o r i d e s o l u t i o n , 0.02
M b o r a t e b u f f e r (pH 9 . 0 ) and d i s t i l l e d water. The t u b e s
were c e n t r i f u g e d a t 2400 rpm f o r 15 m i n u t e s . The s u p e r -
n a t a n t s were removed by a s p i r a t i o n and c o l l e c t e d in a
100 m l c a l i b r a t e d f l a s k . The e x t r a c t i o n p r o c e d u r e was
r e p e a t e d by f o u r more t i m e s . The s o l u t i o n s were made up
t o t h e mark w i t h t h e i r r e s p e c t i v e d i l u e n t . A 3 m l ali-
quot o f each s o l u t i o n w a s t r a n s f e r r e d t o a 100 m l c a l -
i b r a t e d f l a s k and t h e n made up t o volume with 0.02 M
b o r a t e b u f f e r (p H 9 . 0 ) .

Influence of e x c i p i e n t s
Using m i x t u r e s o f 100 mg o f neomycin s u l f a t e and
e a c h of t h e f o l l o w i n g e x c i p i e n t s a l o n e : k a o l i n (500 m g ) ,
aluminium h y d r o x i d e ( 5 0 m g ) , p e c t i n (500 rng), sucrose
(500 m g ) , a l l e x t r a c t i o n s , s a v e t h a t w i t h sugar, were
c a r r i e d out using c e n t r i f u g a t i o n and distilled water
a s e x t r a c t i o n l i q u i d i n agreement w i t h t h e p r o c e s s p r e -
v i o u s l y d e s c r i b e d . The l a s t d i l u t i o n s ( 3 0 kg/ml o f neo-
mycin s u l f a t e ) were made w i t h 0.02 M b o r a t e b u f f e r (pH
9.0).
DETERMINATION OF NEOMYCIN SULFATE 1009

S p e c t r o p h o t o m e t r i c method
The r e a c t i o n between neomycin s o l u t i o n s and DNFB
was made i n t r i p l i c a t e . From e a c h t e s t s o l u t i o n and t h e
b l a n k s , 5 m l were t r a n s f e r r e d t o 2 0 m l g l a s s - t u b e s . The
c o l o u r was d e v e l o p e d by t h e a d d i t i o n o f 2 m l o f 0.25%
m e t h a n o l i c s o l u t i o n o f DNFB. A f t e r 50 m i n u t e s u n d e r am-
b i e n t c o n d i t i o n s , 1 . 5 m l o f 1 . 0 M HC1 was added t o each
t u b e . The a b s o r b a n c e s were measured a t 365 nm. The re-
s u l t s were compared w i t h t h o s e from t h e s t a n d a r d solu-
t i o n c o n t a i n i n g 30 wg/rnl o f neornycin s u l f a t e i n 0.02 M
b o r a t e b u f f e r (pH 9 . 0 ) .

M i c r o b i o l o g i c a l method
The m i c r o b i o l o g i c a l a s s a y by agar diffusion was
c a r r i e d o u t a c c o r d i n g t o t h e USP XX19 w i t h t h e same ex-
t r a c t i o n procedure, b u t t h e e x t r a c t i o n l i q u i d used was
0 . 1 M p h o s p h a t e b u f f e r (pH 8 . 0 ) . A t t h e same time t h i s
method was f o l l o w e d w i t h o u t e x t r a c t i o n o f neomycin from
t h e samples.

RESULTS AND DISCUSSION

A c c o r d i n g t o t h e l i t e r a t u r e 5 , one of t h e most ef-
f i c i e n t b u f f e r s t o e x t r a c t a d s o r b e d neomycin seems to
be p h o s p h a t e . ' T h i s b u f f e r r e l e a s e s a p p r o x i m a t e l y 50% o f
t h e adsorbed a n t i b i o t i c . But i n a n o t h e r s t u d y 8 it was
o b s e r v e d t h a t p h o s p h a t e b u f f e r (pH 8.0), which i s u s e d
9
i n t h e p h a r m a c o p e i a l m i c r o b i o l o g i c a l a s s a y , h a s a neg-
a t i v e e f f e c t on t h e a b s o r p t i o n s p e c t r u m o f 2 , 4 - d i n i t r o -
benzene-neomycin. Because 0.02 M b o r a t e b u f f e r (pH 9 . 0 )
1010 MARQUES, HACKMANN, A N D SAITO

gives the best absorbance values of 2,4-dinitrobenzene-

8
neomycin , it was the buffer employed in this study.
The results of Table 1 show that filtration is in-
effective in recovering all the neomycin contained in
the samples. The extraction by centrifugation, although
more satisfactory, was not sufficient to obtain 100% of
the neomycin content.
Harris' demonstrated clearly that electrolyte so-

lutions markedly inhibit the interaction between neorny-

cin and other agents such as pectin or amaranth. This
inhibition increased the quantity of free neomycin. When
1.0 M aqueous solution of sodium oxalate or sodium chlo-
ride and 0.02 M borate buffer were used as extraction
liquids the results were similar. The best values were
surprisingly obtained with the use of distilled water.
With the study of excipient influence (Table 2 1 ,
employing water as extraction liquid, it can be seen
that it is possible to release all neornycin adsorbed
when the excipient is aluminium hydroxide, but the same
will not occur with kaolin. This result is in accordance
with the observations of Zedan et al.3. They concluded
that aluminium hydroxide showed the lowest adsorption
coefficient indicating that its interaction with neomy-
cin is very weak.
When the first extraction step was performed with
pectin-neomycin, the mixture in water formed an insolu-
ble opaque mass that made impossible the continuation
of the extraction procedure and allowed the recuperation
of only 38.2% of neomycin.
DETERMINATION OF NEOMYCIN SULFATE 1011

m m I-
? Ln

m 0 0 0 rl
b m m m 01

m 03 N 03
co (v N N w
W P I- l- I-
N N N N N

0 0 O 0 0
0 0 0 0 0
0 0 0 0 0
m m m m m
a,
Li
3
a
a,
u
0
Lc
a I:
L E
I-I
.-I N
0 0
0;
W
co c n
3w 3 w xd p:
W
E*
3 Ej HB H H
a a
0 0
mel
X
n w
$ 02 s
0 u m

2 z z 2
0, 2H 0, 2
z
k
33 33
k
33 E
I
E cr
c3
3
rl

e, 3 k
H
e: 2B 2p: 5
a
rl k k B k
nm 4 z z z z
H
k
w
V
w
V
W
V
W
u
H
1012 MARQUES, HACKMANN, AND SAITO

0
f-l rl N 0

In 0 co 0
ul 0 m 0
f-l f-l

m m \D 0
In 0 w 0
w 0 rl 0
N m d m

0 0 0 0
0 0 0 0

0 0 0 0
m m m m

z z
ElB 0
H
B
53 53
Er Er Er
H H H
p: p: p:
H B H
z z z
W w w
U
V U

Y,
h rl
0 v
In
.-I
v

z
H
A
0
2
 tl
m
cj
W
m
I
H
z
P
+I
Table 3 - Microbiological and spectrophotometric determination of neomycin sulfate H
0
z
in pharmaceutical suspensions. 0
%
z
W
0
I
.<
n
H
MICROBIOLOGICAL SPECTROPHOTOMETRIC z
FORMULA THEORETICAL VALUE C
P
(mg/ml) Zl
ASSAY VALUE % RECOVERY ASSAY VALUE % RECOVERY >
cl
(mg/ml) (mg/ml) W

A 200 .oo 180.60 90.3 183.60 91.8

B 60 -00 54.48 90.8 52.62 87.7

C 100 .oo 83.00 83.0 69.90 69.9

 F
0
Table 4 - P e r c e n t a g e of recovery of n e o m y c i n s u , l f a t e f r o m pharmaceutical s u s p e n s i o n s .
V a l u e s expressed as neomycin base.

MICROBIOLOGICAL SPECTROPHOTOMETRIC
FORMULA THEORETICAL VALUE
(ug/ml) ASSAY VALUE % RECOVERY ASSAY VALUE % RECOVERY
(vg/mli (ugh11

14.36 12 85
- 89 - 5 0 9 -42 65.60
A
21.54 20.79 96.50 19.64 91.20
28.72 28.23 98.30 28.92 100.70

14.36 12.85 89.50 10.68 75.80 z

>
P
62
B 21.54 19.62 91.12 19.90 92.40 C
m
v)
28.72 27.71 96 .50 27.57 96 .OO "
2
P
n
3:
14.36 11.81 82.25 12.30 85.70 z
*z
C 21.54 19.27 89.50 17.53 81.40 z
28.72 26.17 91.12 25.60 89 .oo r
z
c)
rA
>
H
DETERMINATION OF NEOMYCIN SULFATE

Although the association glucose-neomycin forms a

true solution, there probably occurs an interaction be-
tween these two molecules because the neomycin percen-
tage obtained was low.
The pharmacopeial microbiological assay of neomy-
cin9 does not indicate an extractive method when the
samples are solids or suspensions. Therefore, the re-
sults obtained using the microbiological assay with-
out extraction and the spectrophotornetric assay with
extraction were compared.
The results in Table 3 show large values in the
microbiological assay without any extraction, with the
exception of Formula A. Probably because the interaction
between kaolin-aluminium hydroxide-neomycin was weaker
than with the others excipients and the diffusion forces
are probably greater than the adsorption forces.
The recovery of neomycin in each of the suspension
formulas was examined by the addition of different a-
mounts of neomycin to a placebo of each formula. The mi-
crobiological and spectrophotomet ric methods we re car-
ried out before extraction with water. As shown in Ta-
ble 4, the best recovery values were obtained with the
microbiological assay, i t indicates that the microbiolo-
gical method has a greater sensibility than the spectro-
photometric method.
So, in the case of suspensions containing neomycin,
a complex study of excipient influence need to be done
after choosing a suitable analytical method.
1016 MARQUES, HAGKMANN, AND SAITO

REFERENCES

1. Goodman, L. S . & G i l m a n , A . "The p h a r m a c o l o g i c a l ba-

s i s of t h e r a p e u t i c s " , M a c m i l l a n , N e w Y o r k , p . 1150 -
1166 ( 1 9 8 5 ) .
2. Wade, A . " M a r t i n d a l e ' s e x t r a p h a r m a c o p e i a " , 2 7 t h e d . ,
P h a r m a c e u t i c a l P r e s s , London, p . 1135-6, p . 1155 -
1160 ( 1 9 7 8 ) .
3. Zedan, H . H . , S a l a m a , M . & Abdel-Rahman, W . M . E g y p t .
J . Pharm. S c i . , = ( 1 - 4 ) , 19 (1982).
4. Heyd, A . , J . Pharm. S c i . , = ( 9 ) , 1 3 4 3 ( 1 9 7 1 ) .
5. F l o r e y , K . " A n a l y t i c a l p r o f i l e s of drug s u b s t a n c e s 1 ' ,
v . 8 , Academic P r e s s , N e w York, p . 399-488 ( 1 9 7 9 ) .
6. Ghazy, F. S., K a s s e n , A . A. & S h a l a b y , S. H . , Pharma-
z i e , 39, 8 2 1 ( 1 9 8 4 ) .
7. H a r r i s , W . A . , A u s t r a l a s . J . P h a r m . , 52, 569 ( 1 9 7 1 ) .
8. Marques, M . R . C . , Hackmann, E . R . M. & S a i t o , T .
Anal. L e t t . , 3 ( 3 ) , 621, ( 1 9 8 9 ) .
9. I'United S t a t e s P h a r m a c o p e i a " , 2 1 t h e d i t i o n , U n i t e d
S t a t e s Pharmacopeial Convention, R o c k v i l l e , MD, p.
712, 1160 ( 1 9 8 5 ) .

Received August 1. 1989

Accepted April 6 , 1990