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Simultaneous Determination ofTinidazole and Furazolidone inSuspension by HPTLC and HPLCN. M. Tendolkar a , B. S. Desai a , J. S. Gaudh a & V.M. Shinde aa Analytical Laboratory , The Institute of Science ,15 Madam Cama Road, Bombay, 400 032, IndiaPublished online: 23 Aug 2006.

To cite this article: N. M. Tendolkar , B. S. Desai , J. S. Gaudh & V. M. Shinde (1995)Simultaneous Determination of Tinidazole and Furazolidone in Suspension by HPTLCand HPLC, Analytical Letters, 28:9, 1641-1653, DOI: 10.1080/00032719508002771

To link to this article: http://dx.doi.org/10.1080/00032719508002771

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ANALYTICAL LETTERS, 28(9), 1641-1653 (1995)

SIMULTANEOUS DETERMINATION OF TINIDAZOLE AND

FURAZOLIDONE IN SUSPENSION BY HPTLC AND HPLC

KEY WORDS : Tinidazole, Furazolidone, HPTLC, HPLC

N,M.Tendolkar, B. S-Desai. J. S. Gaudh and V. M. Shlnde*

Analyt ical Laboratory, The Inst i tu te o f Science, 15, Madam Cama Road,

Bombay 400 032, India.

ABSTRACT

High performance t h i n layer chromatographic (HPTLC)

and h i g h performance I i q u i d chromatographic (HPLC) methods

were developed f o r the simultaneous determination of Tinidazole

and Furazol idone i n suspension.

In the HPTLC method the separation o f Tinidazole

and Furazolidone was c a r r i e d out on s i l i c a gel 60F HPTLC

glass p la te using chIoroform:rnethanol:ammonia (9:l:0.1 v l v )

as a mobile phase. R f values obtained were 0.63 and 0.79

f o r Furazol idone and Tinidazole respectively. Densitometric

evaluation was done a t 335 nm. L i n e a r i t y was obtained w i t h i n

the concentration range 10-50 y g l m l and 3.5-17.5 p g l m l f o r

Tinidazole and Furazol idone respectively.

254

The second method i s based on h i g h performance l i q u i d

chromatography on a reversed phase column ( p Bondapak C18)

1641

Copyright 0 1995 by Marcel Dekker, Inc.

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1642 TENDOLKAR ET AL.

using a mobile phase comprised o f water : acetoni t r i te : t r ie thy lamine (80:20:0.1 v l v ) adjusted t o p H = 3.0 w i t h dil.

phosphoric acid. Retention times were 5.24 and 7.82 min

f o r Tinidazole and Furazolidone respec t ive ly a t a f low ra te

o f 1.5 mllmin. Detection was done a t 335 nm. L inear i ty

was obtained w i t h i n the concentration range 30-180 Pg lml

and 10.5-63 p g l m l f o r Tinidazole and Furazol idone resp.

INTRODUCTION

T in idazole [ 1 - [ 2- (E thy I su I phony I ) e t h y l I -2-methy I-5-

nitro-1 H-imidazolel i s a antiprotozoal and it has ant imicrobia l

action and i s used i n the treatment o f susceptible protozoal

infections.

Furazolidone [3- [ [ (5-Nitro-2-furanyl )methylenel - amino]-

2-oxazolidinone] i s a n i t ro furan d e r i v a t i v e w i t h antiprotozoal

ac t i v i t y . It i s ac t i ve against the protozoan g ia rd ia intest inal i s

and against a range o f enteric bacteria.

2 Tinidazole i s o f f i c i a l i n 1.P' and J.P . Furazolidone

3 4 5 i s o f f i c i a l i n 1.P , 6.P and U.S.P . methods

are repor ted f o r Tinidazole. S imi la r ly spectrophotometric ,

HPLC17-18, G.C1', TL?

S p e c t r o p h ~ t o m e t i c ~ - ~ , HPLC8-11, G . c1 2-1 3

14-16

0 methods are repor ted f o r Furazol idone but

the simultaneous determination o f bo th these drugs i s not

ye t reported. The simultaneous analysis o f Tinidazole and

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TINIDAZOLE AND FURAZOLIDONE 1643

Furazolidone i s requ i red f o r the purpose o f q u a l i t y control

of these drugs.

I n t h i s communication we propose HPTLC and HPLC

methods f o r the simultaneous determination o f b o t h the drugs

i n suspension form.

EXPERIMENTAL

Instrumentation

HPTLC System :-

Camag Linomat I V sample applicator, Camag TLC

Scanner I I (Densitometer), Cats 3.15 v. software, Camag t w i n

trough chamber, Merck 6OFz5,, HPTLC glass plates.

HPLC System :-

Perk in Elmer lsocrat ic L C 250 pump, Perk in Elmer

L C 290 UV detector, Perk in Elmer PE Nelson Integrator,

Rheodyne 7125 v a l v e injector, p Bondapak C,* column (Waters,

India).

Mater ia ls

Authentic samples o f Tinidazole and Furazol idone were

k i n d l y donated by Kopran ( I ) Ltd. Suspension (T in i -F) samples

were procured from market.

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1644 TENDOLKAR ET AL.

Reagents

Chromatographic grade water, acetoni t r i le,

chloroform, t r ie thy lamine (E.Merck, Ind ia) and

reagent grade ammonia were used.

Standard solut ion

Tinidazole (1OOmg) and Furato l idone (35mg) were

methanol

analyt ical

accurate1 y

weighed into a 100 ml volumetric f lask, d isso lved in

acetoni t r i le w i t h ultrasonication and d i l u t e d to volume w i t h

acetonitr i le.

Procedure f o r HPTLC

Cal ibrat ion

Cal ibrat ion solutions were prepared by taking vary ing

volumes (0.5-2.5 m l ) o f standard solution i n 50 ml f lasks

and d i l u t e d up to the mark w i t h acetonitr i le. 1 0 micro l i t res

of these solutions were app l ied on s i l icagel 60F254 HPTLC

glass p la te i n 6 mm w i d t h using sample applicator. The

p la te was developed u p t o 60 mm i n a t w i n trough chamber

containing chloroform : methanol : ammonia (9 : l :0.1 v l v )

as a mobile phase.

Densitometric evaluation was done by TLC scanner

and absorbance was measured a t 335 nm.

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TINIDAZOLE AND FURAZOLIDONE 1645

Calibrat ion curves were constructed by p lot t ing areas

of drugs vs. concentration o f drugs fo r both Tinidazole

and Furazol idone.

Sample preparation

After determining mass per m i l l i l i t r e , an accurately

weighed amount equivalent to 100 mg of Tinidazole (35 mg

of Furazol idone) was transferred into 100 ml ca l ibrated

flask, 50 ml of acetonitr i le was added, ultrasonicated fo r

5 minutes and f i na l l y d i lu ted up to the mark w i t h acetonitri le.

The result ing solution was f i l t e r e d through Whatman paper

no.1 (solution A).

Then 1 ml of solution A was p ipet ted into a 50 ml

ca l ibrated f lask and d i lu ted up to the mark w i t h acetonitri le.

10 micro l i t res o f the f ina l solution was appl ied on

s i l i ca gel 6OFz5,, HPTLC glass p la te and the p la te was

developed, dried, scanned and peak areas were recorded

as repor ted i n ca l ibrat ion procedure.

Procedure f o r HPLC

Cal i brat ion

Calibrat ion solutions were prepared by taking vary ing

volumes o f standard solution (1.5-9 ml ) i n s i x 50 ml standard

f lasks and d i lu ted up t o the mark w i t h mobile phase.

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1646 TENDOLKAR ET AL.

20 m i c r o l i t r e s of the above solutions were injected

into chromatograph.

The mobile phase consisted o f water:acetonitri le:

t r ie thy lamine (80:20:0.1 v l v ) adjusted t o pH = 3.0 w i t h

d i l u t e phosphoric acid. Th is was f i l t e r e d and degassed

through 0.22 Whatman paper. Flow ra te was kept a t

1.5 mllmin. w i t h an average operating pressure 2200 p s i

and the UV response was monitored a t 335 nm.

Cal ibrat ion curves were constructed by p lo t t ing peak

areas o f drugs vs. concentration.

Sample preparat ion

3 m l o f solution A (prepared as above1 was t ransferred

into 50 ml ca l ib ra ted f lask and d i l u t e d to the mark w i t h

the mobile phase. 20 mic ro l i t res o f t h i s solution was injected

into chromatograph and peak areas were recorded f o r both

the drugs as i n the ca l ib ra t ion experiment.

Recovery Studies

To study the accuracy, r e p r o d u c i b i l i t y , prec is ion

and to check the interference from excepients used i n the

formulation, recovery experiments were c a r r i e d out by the

standard add i t ion method f o r both HPTLC and HPLC methods.

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TINIDAZOLE AND FURAZOLIDONE 1647

Known amount o f standard Tinidazole and Furazol idone were

added to the f i x e d amount o f sample solution a t f i v e concentra-

t ion leve ls and from tota l amount o f drug found, % recovery

was calculated.

RESULTS AND DISCUSSION

HPTLC :-

To c a r r y out separation o f Tinidazole and Furazol idone

by TLC, various mobile phase mixtures were t r i e d on

s i l icagel 60F254 HPTLC glass plate. A mix tu re o f chloroform:

methano1:ammonia (9:l:O:l v l v ) gave a good resolut ion o f

the drugs. R f values obtained were 0.63 and 0.79 f o r

Furazolidone and Tinidazole respectively.

Scanning of the p la te was done a t 335 nm as a t t h i s

wavelength bo th Tinidazole and Furazol idone show good response

and there was no interference o f the bands o f inact ive

ingredients.

For quant i ta t ive application, l inear ca l ib ra t ion graphs

were obtained w i t h i n the concentration range 10-50 y g l m l

and 3.5-17.5 Fg/ml f o r Tinidazole and Furazol idone respectively.

The ca l ib ra t ion curves could be represented by fo l lowing

regression equations.

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1648

Tab le 1

TENDOLKAR ET AL.

Determinat ion o f T in idazo le and Furazo l idone by HPTLC

L a b e l c l a i m :- T in idazo le (100 mg/5 m l ) Furazo l idone (35 mg/5 m l )

1

2

101.72

100.02

34.01

34.25

3 99.43 34.21

4

5

99.38

97.96

34.03

34.81

6 100.26 34.69

7 98.43 34.98

= 33.5860 X + 23.000 (r=0.999) ‘ (Tinidazole)

‘(Furazol idone) = 51.8000 X + 7.0800 (r=0.998)

These equat ions were used f o r d i r e c t eva lua t ion o f

drugs.

Resul ts o f t h e r e p l i c a t e ana lys i s and r e c o v e r y exper imen t

o f suspension a r e tabu la ted in Tab le 1 and Tab le 2 respec t i ve l y .

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TINIDAZOLE AND FURAZOLIDONE 1649

Table 2

Result of the recovery experiment by HPTLC.

Drug name

Amount o f Amount o f Total drug standard Amount % Recovery present added recovered* mg/5 ml mg/5 ml mg/5 ml

100 0.00 99.48 99.48 100 20.00 118.24 98.53

Tin idazole 100 40.00 140.77 100.55 100 60.00 157.89 98.68 100 80.00 179.33 99.63 ...................................

Mean Recovery :- 99.37 ................................................................ 35 0.00 34.58 98.80 35 7.00 41.92 99.81

Furazol idone 35 14.00 49.51 101.04 35 21.00 55.02 98.25 35 28.00 62.77 99.63

* .- . Average o f th ree experiments.

HPLC :-

In order to ef fect the separation o f the two drugs

components by reversed phase HPLC system, a Bondapak C18

column as a stat ionary phase and mobile phase comprising

of water: acetoni tri le: tr ie thy lami ne (80: 20 : 0.1 v / v 1 was used.

The mobile phase composition was opt imised and pH f o r

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1650

Table 3

TENDOLKAR ET AL.

Determination of Tinidazole and Furazol idone by HPLC

Labe l c la im :- Tinidazole (100mg/5 m l ) Furazolidone (35 mg/5 m l )

Samp I e no.

T in idazole Furazol idone Content obtained Content obtained (mg15 ml) ( m g l 5 m l )

1 99.11 34.54

2

3

100.54

100.18

34.23

34.18

4 98.35 34.40

5

6

98.28

100.90

35.01

34.49

7 99.89 34.64

optimum resolut ion was adjusted to 3.0 using d i l u t e phosphor ic

acid. Under the descr ibed conditions, the analyte peaks

were wel l defined, resolved and f ree from tai l ing. A t a

f low ra te of 1.5 m l l m i n the retention times were 5.24 and

7.82 min f o r Tinidazole and Furazol idone.

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TINIDAZOLE AND FURAZOLIDONE 1651

Table 4

Results o f the recovery experiment by HPLC.

Amount of Amount o f Total Drug drug standard Amount % Recovery name present added recovered*

mg/5 ml mg/5 ml mg/5 ml ................................................................ 100 0.00 100.08 100.08 100 20.00 120.45 100.38

Tinidazole 100 40.00 139.41 99.58 100 60.00 1 58.82 99.26 100 80.00 178.40 99.11

Mean Recovery :- 99.68

35 0.00 34.65 99.00 35 7.00 42.68 101.62

Furazol idone 35 14.00 48.48 98.94 35 21.00 55.92 99.86 35 28.00 63.08 100.13 ___________________---_-------------

Mean Recovery :- 99.91

* :- Average o f three experiments.

The optimum wavelength f o r detection was 335 nm

a t wh ich good detector response was obtained.

L i n e a r i t y was obtained i n the concentration range

30-180 p g l r n l and 10.5-63 F g l r n l respect ive ly . The ca l ib ra t ion

curves could be represented by fol lowing regression equation.

= 23.0323 X + 18.650 (r=0.999)

= 41.3796 X + 15.0636 (r=0.999)

’ (T i n idazole)

‘(Furazolidone)

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1652 TENDOLKAR ET AL.

These equations were used f o r d i r e c t evaluation o f

drugs. Results o f the rep l i ca te analysis and recovery exper i -

ment o f suspension are tabulated i n Table 3 and Table 4

respect ive ly .

ACKNOWLEDGEMENT

Authors are grateful t o Anchrom Enterprises f o r p rov id ing

HPTLC fac i l i t ies .

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Received December 1 2 , 1994 Accepted March 28, 1995

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