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Determination of Allantoin
in Drug Preparations
by Quantitative High
Performance TLC
a a
Joseph Sherma & Patricia S. Cortelyou
a
Department of Chemistry , Lafayette College ,
Easton, Pennsylvania, 18042
Published online: 19 Dec 2006.

To cite this article: Joseph Sherma & Patricia S. Cortelyou (1986) Determination
of Allantoin in Drug Preparations by Quantitative High Performance TLC, Journal
of Liquid Chromatography, 9:16, 3415-3421, DOI: 10.1080/01483918608077791

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 JOURNAL OF LIQUID CHROMATOGRAPHY, 9(16), 3415-3421 (1986)

DETERMINATION OF ALLANTOIN IN DRUG

PREPARATIONS BY QUANTITATIVE HIGH
PERFORMANCE TLC

Joseph Sherma and Patricia S . Cortelyou

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Department of Chemistry
Lafay ette College
Easton, PennsyZvania 18042

ABSTRACT

A densitometric TLC method was developed for the quantifica-

tion of allantoin in creams and suppositories used to treat vaginal
infections. Allantoin was extracted with water at elevated tempera-
ture, diluted to a known volume, and separated by HP silica gel
TLC. Allantoin was detected by spraying with E-dimethylamino-
benzaldehyde reagent. The absorption of standards and samples was
compared by in situ scanning. Recoveries of allantoin from
authentic samples ranged from 96-102%, except for one sample that
assayed high. The accuracy of the method was demonstrated by
standard addition analysis of this sample.

INTRODUCTION

In earlier papers we reported quantitative TLC methods based

on fluorescence and fluorescence quench densitometry for the deter-

mination of aminacrine hydrochloride ( 1 ) and the sulfa drugs sulfan-

ilamide and sulfisoxazole ( 2 ) in pharmaceutical preparations used

to treat vaginal infections. A l l of these products also contain

3415

Copyright 0 1986 by Marcel Dekker, Inc. 0148-39 19/86/09 16-3415$3.50/0

 3416 SHERMA AND CORTELYOU

a l l a n t o i n [2,5-dioxo-4-imidazol dinyl urea] as a therapeutic

component t h a t promotes h e a l i n g

Most a n a l y s e s f o r a l l a n t o i n have been based on n o n s p e c i f i c

s o l u t i o n c o l o r i m e t r y a f t e r h y d r o l y s i s (e.g., 3-5). Determination

by HPLC i n c o s m e t i c s and p h a r m a c e u t i c a l p r o d u c t s (6-9) and i n

b i o l o g i c a l f l u i d s (10,11)h a s a l s o been r e p o r t e d .
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T h i s paper d e s c r i b e s t h e d e t e r m i n a t i o n of a l l a n t o i n i n v a g i n a l

p r o d u c t s by s c a n n i n g t h e yellow zones produced from samples and

s t a n d a r d s w i t h t h e d e t e c t i o n r e a g e n t 1-dimethylaminobenzaldehyde on

p r e a d s o r b e n t h i g h performance s i l i c a g e l l a y e r s . The method i s

a d a p t e d from a p r e v i o u s l y p u b l i s h e d TLC procedure ( 1 2 ) f o r e s t i m a -

t i o n of a l l a n t o i n i n serum, lymph, and u r i n e .

EXPERIMENTAL

Standard S o l u t i o n

A l l a n t o i n s t a n d a r d was purchased from A l d r i c h (No. A2,839-2).

P r e p a r a t i o n of a 1.00 p g / p l d i s t i l l e d w a t e r s o l u t i o n i n a 25 m l

v o l u m e t r i c f l a s k r e q u i r e d g e n t l e h e a t i n g on a h o t p l a t e t o a f f e c t

complete d i s s o l v i n g .

Sample P r e p a r a t i o n

Creams. About 1.25 g of sample was a c c u r a t e l y weighed i n t o a

25 m l b e a k e r , 15 m l of d i s t i l l e d w a t e r was added, and t h e beaker

was h e a t e d a t low t e m p e r a t u r e on a h o t p l a t e f o r 15 minutes w i t h

occasional s t i r r i n g . A f t e r c o o l i n g t o room t e m p e r a t u r e , t h e

s o l u t i o n was t r a n s f e r r e d q u a n t i t a t i v e l y t o a 25 m l v o l u m e t r i c
 ALLANTOIN IN DRUG PREPARATIONS 3417

flask, which was filled to the line with distilled water. Cloudy

solutions were filtered hot while being transferred into the

volumetric flask. For a sample of exactly 1.25 g containing 2.0%

of allantoin, the theoretical concentration of the final solution

is 1.00 pg/pl.

Suppositories. Samples of suppositories were prepared as

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previously described (l), except that water was used instead of

acidic ethanol and samples were heated for 15 minutes rather than

10. The theoretical concentrations of the final solutions for

suppositories containing 140 mg and 120 mg of allantoin were 0.560

and 0.480 pg/pl, respectively.

TLC Determination

TLC was carried out on 10 X 20 cm Whatman high performance

silica gel plates containing a preadsorbent spotting area and

scored into 8 m wide lanes using procedures described earlier

(1,2). Duplicate aliquots of t h e final sample solution (5 p1 for

creams and 9 p1 for suppositories) and 3.00, 5.00, and 7.00 pg

standards ( 3 , 5, and 7 pl of the 1.00 pg/pl solution) were

applied to adjacent lanes using a 10 pl Drummond microdispenser.

The initial zones were dried thoroughly with a ha rdrier and the

plates developed in a saturated TLC chamber with methyl ethyl

ketone-acetone-formic acid-water (40:2:1:6 v/v). The plate was

air-dried in a fume hood, sprayed heavily (but not soaking) with

-
p-dimethylaminobenzaldehyde detection solution (0.25 g reagent in

38 ml methanol and 12 ml conc. HCl), and heated in an oven at

100°C f o r 5 minutes. Allantoin zones were measured with a Kontes

 3418 SHERMA AND CORTELYOU

Chromaflex f i b e r o p t i c s s c a n n e r a s d e s c r i b e d e a r l i e r ( 1 , Z ) . A

c a l i b r a t i o n e q u a t i o n was computer c a l c u l a t e d from t h e scan a r e a s

of t h e s t a n d a r d s , t h e amounts of a l l a n t o i n i n t h e sample a l i q u o t s

were i n t e r p o l a t e d and a v e r a g e d , and c o n t e n t s of t h e p r e p a r a t i o n s

were c a l c u l a t e d and compared t o t h e l a b e l v a l u e s .

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RESULTS AND DISCUSSION

1-Dimethylaminobenzaldehyde r e a g e n t d e t e c t e d a l l a n t o i n as a

compact yellow zone w i t h an % value of 0.35. Development w i t h

t h e mobile phase f o r a d i s t a n c e of 7 cm beyond t h e preadsorbent-HP

s i l i c a g e l j u n c t i o n r e q u i r e d 30 m i n u t e s . The l i n e a r i t y c o r r e l a t i o n

c o e f f i c i e n t f o r t h e s c a n a r e a s of t h e t h r e e s t a n d a r d s v e r s u s micro-

grams s p o t t e d was u s u a l l y 0.995 or h i g h e r .

Eleven d i f f e r e n t samples were a n a l y e d by t h e TLC p r o c e d u r e

and t h e r e s u l t s a r e shown i n Table 1. The samples r e p r e s e n t

d i f f e r e n t brands and d i f f e r e n t l o t s of t h e same b r a n d , w i t h t h r e e

combinations of a c t i v e i n g r e d i e n t s . A s can be s e e n i n Table 1,

r e s u l t s ranged from 96 t o 1022, e x c e p t f o r Sample 5. Calculations

f o r each t r i a l were based on a v e r a g e a r e a s of d u p l i c a t e sample

a l i q u o t s , which had a p e r c e n t a g e d i f f e r e n c e t h a t was always below

6 % and u s u a l l y l e s s t h a n 2 % .

T r i p l i c a t e a n a l y s i s of cream Sample 5 y i e l d e d c o n s i s t e n t l y

high r e s u l t s . F i g u r e 1 shows s c a n s of t h e t h r e e s t a n d a r d s and

d u p l i c a t e sample a l i q u o t s f o r t h e f i r s t a n a l y s i s of t h i s product

shown i n Table 1. A s t a n d a r d a d d i t i o n a n a l y s i s was c a r r i e d o u t

 ALLANTOIN IN D R U G PREPARATIONS 3419

TABLE I

Analyses of Commercial Vaginal Creams

and Suppositories for Allantoin

Sample No. Dosage form Dosage level % Label claima

b
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1 cream 2.0% 100

96.0
C
2 cream 2.0% 105
b
3 cream 2.0% 100
b
4 cream 100
C
5 cream 2.0% 115
119
115
d
6 cream 2.0% 101
b
7 suppository 140 mg 100
d
a suppository 120 mg 102
102
d
9 suppository 120 mg 96.0
99.5
b
10 suppository 140 mg 97.0
99.3
b
11 suppository 140 mg 102
100

aReplicate analyses of certain samples are given in this column.

bAlso contained sulfanilamide and aminacrine.

‘Also contained sulfanilamide, aminacrine, and dienestrol.

dAlso contained sulfisoxazole and aminacrine.

 3420 SHERMA AND CORTELYOU

A B 3w 5w 7P9
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Figure 1. Scans of 3.00, 5.00, and 7.00 pg standard zones and

duplicate aliquots of cream Sample 5 (Table 1) representing 114%
( A ) and 116% (B) compared to the label value. The attenuation
setting on the Kontes densitometer was X50.

by weighing two 1.25 g portions of Sample 5, adding 25.0 mg of

allantoin standard to one portion to double the label value, and

analyzing the samples i n an identical manner except that 2.5 p l of

the fortified sample and the usual 5 p l of the unfortified sample

were spotted f o r TLC. The difference in assay values represented

99.5% of the added standard, confirming the accuracy of the method

and the unexplained high results obtained for Sample 5 .

In addition to allantoin, the pharmaceutical products tested

contained a variety of the ingredients listed in Table 1 and

Reference 2. The only compounds that are detected with the p-di-

methylaminobenzaldehyde reagent are the sulfa drugs (Rf > 0.8)

and aminacrine (R
F
< 0.1). The sulfas become yellow immediately

upon spraying, while aminacrine requires brief heating. None of

these spots interfere with measurement of allantoin zones.

The described quantitative TLC method proved to be accurate,

reproducible, and selective for the determination of allantoin in a

 ALLANTOIN IN DRUG PREPARATIONS 342 1

number of commercial d r u g f o r m u l a t i o n s . The a b i l i t y t o a n a l y z e

m u l t i p l e samples u s i n g a common s e r i e s of s t a n d a r d s on a s i n g l e

p l a t e a l l o w s h i g h sample throughput t o be a c h i e v e d .

ACKNOWLEDGMENT
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We thank E l a i n e A. Bunch, FDA, S e a t t l e , WA, f o r s u p p l y i n g t h e

samples used t o t e s t t h e method.

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