Simple Extractive Spectrophotometric Method for Determination of Clopidogrel Bisulphate by Reactive dyes from Pharmaceutical Dosage Form

 

Rajan V. Rele*

Central Research Laboratory, D.G. Ruparel College, Matunga, Mumbai – 400016.

*Corresponding Author E-mail: drvinraj@gmail.com

 

ABSTRACT:

Simple sensitive and accurate ion pair complex formation with reactive dyes, extractive spectrophotometric methods have developed for the estimation of clopidogrel bisulphate in pharmaceutical dosage form. The methods are based on the formation of ion paired coloured complexes by the drug with reactive dyes like Congo red, eriochrome black T and methyl orange in acidic medium. The ion associated complexes were formed and quantitatively extracted under the experimental condition in chloroform. The absorbance values were measured at 490nm, 500nm, and 430nm respectively. The proposed methods were validated statistically. Recoveries of methods were carried out by standard addition method. The linearity was found to be 5.0-30.0μg/ml, 2 -14 μg/ml, and 2-18μg/ml for methods respectively. The low values of standard deviation and percentage RSD indicate high precision of methods. Hence these methods are useful for routine estimation of emtricitabine in pharmaceutical dosages.

 

KEYWORDS: Clopidogrel bisulphate, Methyl orange, Congo, Red, Eriochrome black T, Chloroform.

 

 

INTRODUCTION:

In this communication a new colorimetric method is developed for assay of clopidogrel bisulphate in pharmaceutical dosage form. Clopidogrel bisulphate, chemically (+) - (S) -(2-chlorophenyl)- 6,7-dihydrothieno [3,2-c] pyridine- 5(4H)-acetic acid methyl ester sulphate is a potent oral anti-platelet agent often used in the treatment of coronary artery disease, peripheral vascular disease and cerebro vascular disease.

The mechanism of action of clopidogrel is irreversible blockade of the adenosine di-phosphate (ADP) receptor P2Y12 and is important in platelet aggregation, the cross-linking of platelets by fibrin.

 

 

The blockade of this receptor inhibits platelet aggregation by blocking activation of the glycoprotein IIb/ IIIa pathway. It Literature survey reveals the assay of Clopidogrel bisulfate in pharmaceutical formulations by various HPLC1-5, spectrophotometric6-11, TLC12 methods for assay of clopidogrel bisulphate.

 

Simple, rapid and reliable UV spectrophotometric methods are developed for the determination of Clopidogrel bisulphate. These methods can be used for the routine analysis. In the proposed methods optimization and validation of this method are reported.

 

Structure of Clopidogrel bisulphate:

 

 

 

MATERIALS AND METHOD:

Instrument and Reagents:

Spectral scan was made on a Shimadzu UV-spectrophotometer, model 1800 (Shimadzu, Japan) with spectral band width of 0.5nm with automatic wavelength corrections by using a pair of 10mm quartz cells.

 

Reference standard of Clopidogrel bisulphate was obtained from reputed firm with certificate of analysis.

 

Preparation of Standard Drug Solutions:

Stock solution of Clopidogrel bisulphate (100μg/ml) was prepared in ethanol. From this stock solution working standard (10μg/ml) was prepared by diluting 10ml stock solution to 100ml with distilled water. 0.6% w/v solution of bromophenol blue, 0.25% w/v solochrome dark blue and 0.2% w/v bromocresol green were prepared in distilled water respectively.

 

Potassium hydrogen phthalate buffer solution of pH 4.01was prepared in distilled water. Dilute hydrochloric acid was used to adjust desired pH of buffer solution.

 

Preparation of Reagent:

A 0.05% w/v Congo red, 0.25% eriochrome black T and 0.02% methyl orange solutions were prepared in distilled water respectively.

 

Potassium hydrogen phthalate buffer solution of pH 4.01was prepared in distilled water. Dilute hydrochloric acid was used to adjust desired pH of buffer solution.

 

Estimation from Tablets

Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 10mg of clopidogrel bisulphate was weighed and transferred in 100ml of volumetric flask. A 30ml of ethanol was added and sonicated for 15minutes and filtered. The filtrate and washing were diluted up to the mark with distilled water to give concentration as 100μg /ml. Such solution was used for analysis.

 

Experimental:

Method 1(with Congo red):

Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 1.5ml of buffer (pH= 3.9) and 4.0ml of 0.05 %w/v Congo red were added. 10 ml of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers. The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance (λ max 420nm).

 

Method 2(with methyl orange):

Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 2.6ml of buffer (pH = 3.8) and 3.6ml of 0.2% w/v methyl orange were added. 10ml of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers. The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance (λ max=430 nm).

 

Method 3(with eriochrome black T):

Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 2.4ml of buffer (pH= 3.5) and 3.0ml of 0.025% w/v eriochrome black T were added. 10ml of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers. The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance (λ max =500 nm).

 

Results of analysis are given in table 1.

Table 1: Values of results of optical and regression of drug

Parameter

Congo red

Methyl orange

Eriochrome black T

Detection Wavelength (nm)

490

430

500

Beer Law Limits (µg/ml)

4-24

2-18

2-14

Correlation coefficient(r2)

0.9996

0.9999

0.9993

Regression equation (y=b+ac)

 

 

 

Slope (a)

0.0501

0.025

0.0098

Intercept (b)

0.0019

0.0002

0.0056

 

RESULTS:

The extractive spectrophotometric methods are popular due to their sensitivity in assay of the drug and hence ion pair extractive spectrophotometric methods have gain considerable attention for quantitative determination of many pharmaceutical preparations. These proposed methods are extractive spectrophotometric methods for the determination of drug by using chloroform as solvent from its formulations i.e. tablets. The colour ion pair complexes formed were very stable. The working conditions of these methods were established by varying one parameter at time and keeping the other parameters fixed by observing the effect produced on the absorbance of the colour species. The various parameters involved for maximum colour development for these methods were optimized. The proposed methods were validated statistically and by recovery studies. The molar absorptivity show the sensitivity of methods while the precision was confirmed by %RSD (relative standard deviation). The optical characteristics such as absorption maxima (nm), co-relation coefficient (r) were calculated and are also summarized. Assay results of recovery studies are given in table 2 (A, B, C).

 

Table No 2: A (Congo red)

Amount of Sample Added in (µg/ml)

Amount of Standard Added in (µg/ml)

Total amount recovered

Percentage recovery (%)

Standard deviation

Percentage of relative standard deviation (C.O.V.)

4

4

4.01236

100.309

0.008953

0.223135

8

8

8.014262

100.1783

0.015093

0.188332

12

12

12.00666

100.0555

0.010167

0.084674

16

16

16.00095

100.0059

0.008953

0.055953

 

 

 

 

Mean=0.010791

Mean=0.138024

 

Table No 2: B (Methyl orange)

Amount of Sample Added in (µg/ml)

Amount of Standard Added in (µg/ml)

Total amount recovered

Percentage recovery (%)

Standard deviation

Percentage of relative standard deviation (C.O.V.)

2

0

2.006699

100.3349

0.005411

0.269651

2

2

4.008798

100.22

0.005442

0.135741

2

4

6.010948

100.1825

0.006462

0.107511

2

6

8.011098

100.1387

0.006504

0.081188

 

 

 

 

Mean=0.005955

Mean=0.14852

 

Table No 2: C (Eriochrome black T)

Amount of Sample Added in (µg/ml)

Amount of Standard Added in (µg/ml)

Total amount recovered

Percentage recovery (%)

Standard deviation

Percentage of relative standard deviation (C.O.V.)

2

0

2.016608

100.8304

0.012428

0.616294

2

2

4.037888

100.9472

0.015606

0.386496

2

4

6.033476

100.5579

0.016344

0.270888

2

6

8.029064

100.3633

0.015732

0.195933

 

 

 

 

Mean=0.015028

Mean=0.367403

Results are in good in agreement with labelled value.

 

DISCUSSION:

The percent recovery obtained indicates noninterference from the common excipients used in the formulation. The reproducibility, repeatability and accuracy of these methods were found to be good, which is evidenced by low standard deviation. The proposed methods are simple, sensitive, accurate, precise and reproducible. They are directly applied to drug to form chromogen. Hence, they can be successfully applied for the routine estimation of drug, in bulk and pharmaceutical dosage form even at very low concentration and determination of stability of drug in formulation such as tablets. The strong recommendation is made here for the proposed methods for determination of Clopidogrel bisulphate from its formulation.

 

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Received on 10.12.2025      Revised on 02.01.2026

Accepted on 20.01.2026      Published on 10.04.2026

Available online from April 13, 2026

Asian J. Research Chem.2026; 19(2):113-115.

DOI: 10.52711/0974-4150.2026.00019

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