INTRODUCTION:

Clopidogrel bisulphite¹ with a chemical name methyl (2S)-2-(2-chlorophenyl)-2-(6,7-dihydro-4H-thieno[3,2-c]pyridin-5-yl)acetate. Clopidogrel bisulphite is an effective inhibitor of platelet aggregation. The drug has been found to significantly reduce infarction size in acute myocardial infarcts and is an effective antithrombotic agent in arteriovenous fistulas, aorto-coronary bypass grafts, ischemic heart disease, venous thrombosis, and arteriosclerosis. Literature survey reveals many Chromatographic methods^2-3 for the determination of Clopidogrel bisulphite in biological fluids and in tablets and very few Spectrophotometric methods^4-5were reported. Therefore the need for fast, low cost and selective spectrophotometric method is obvious especially for routine Quality Control analysis of pharmaceutical formulation.

EXPERIMENTAL:

Instrument:

Elico double beam Ultraviolet-Visible double beam spectrophotometer SL-164 with 1 cm matched quartz cells was used for all spectral measurements.

Reagents:

All the chemicals used were of analytical reagent grade. Acid phthalte buffer pH 2.4 and pH 3 were prepared as per I.P.

i) Bromothymol Blue, BTB (0.1% w/v)-100 mg is weighed accurately and dissolved in 100 ml of acid phthalate buffer pH 2.4.

ii) Orange II (0.5% w/v): 500 mg of O-II is weighed and dissolved in 100 ml water.

iii) Metanil yellow (0.02% w/v)-20 mg of Metanil yellow is weighed accurately and dissolved in 100 ml of acid phthalate buffer pH 2.4.

iv) Chloroform AR grade

Procedure:

Standard stock solution: A standard stock solution containing 1mg/ml was prepared by dissolving 100 mg of Clopidogrel bisulphite in 100 ml of distilled water. From this, a working standard solution containing 100 mcg/ml were prepared with distilled water.

ASSAY PROCEDURE

Method A: Aliquots of the drug solution of Clopidogrel bisulphite 0.5-5.0 ml (50 mcg/ml) are taken and transferred into a series of 125 ml of separating funnel. To each funnel 5 ml of BTB reagent solution is added. Reaction mixture was shaken gently for 5 min. Then 10 ml of chloroform was added to each of them. The contents are shaken thoroughly for 5 min and allowed to stand, so as to separate the aqueous and chloroform layer. Colored chloroform layer was separated out and absorbance was measured at 440 nm against reagent blank. Calibration curve was prepared from absorbance values so obtained

Method B: Aliquots of the drug solution of Clopidogrel bisulphite 0.5-5.0 ml (50 mcg/ml) are taken and transferred into a series of 125 ml of separating funnel. To each funnel 1 ml of O-II reagent is added. Reaction mixture was shaken gently for 5 min. Then 10 ml of chloroform was added to each of them. The contents are shaken thoroughly for 5 min and allowed to stand, so as to separate the aqueous and chloroform layer. Colored chloroform layer was separated out and absorbance was measured at 490 nm against reagent blank. Calibration curve was prepared from absorbance values so obtained.

Table: I - OPTICAL CHARACTERISTICS AND PRECISION DATA.

	Method A	Method B	Method C
l _max (nm)	440	490	410
Beer’s law limits (mcg/ml)	2.5-25	2.5-25	2.5-20
Molar absorptivity (l/mol.cm)	3.755x10⁴	1.052x10⁴	1.938x10³
Sand ell’s sensitivity (micrograms/cm²/0.001 absorbance unit)	0.7548	^0.6951	^0.8956
Regression Equation* (Y) Slope (m) Intercept (c)	0.004 0.0146	0.002 -0.0017	0.006 0.0609
Correlation Coefficient(r)	0.9999	0.9998	0.9989
Precision (%Relative Standard Deviation)	0.857	0.780	0.694
Standard error of mean	0.0158	0.0063	0.0981

*Y=mx+c, where X is the concentration in micrograms/ml and Y is absorbance unit.

Fig 1: Absorption Spectrum of Clopidogrel with BTB,O-II and MY

Method C: Aliquots of the drug solution of Clopidogrel bisulphite 0.5-4.0 ml (50 mcg/ml) are taken and transferred into a series of 125 ml of separating funnel. To each funnel 2 ml of Metanil yellow reagent is added. Reaction mixture was shaken gently for 5 min. Then 10 ml of chloroform was added to each of them. The contents are shaken thoroughly for 5 min and allowed to stand, so as to separate the aqueous and chloroform layer. Colored chloroform layer was separated out and absorbance was measured at 410 nm against reagent blank. Calibration curve was prepared from absorbance values so obtained.

Preparation of sample solution:

Tablets containing Clopidogrel bisulphite were successfully analyzed by the proposed methods. Twenty tablets of Clopidogrel bisulphite (Plagril 75mg, Dr.Reddy’s Laboratories) were accurately weighed and powdered. Tablet powder equivalent to 100 mg of Clopidogrel bisulphite was dissolved in 50 ml of water and filtered and washed with water, the filtrate and washings were combined and the final volume was made to 100 ml with water. The solution was suitably diluted and analyzed as given under the assay procedure for bulk samples.

The results are represented in Table II. None of the excipients usually employed in the formulation of tablets interfered in the analysis of Clopidogrel bisulphite, by the proposed methods.

Recovery Studies:

To ensure the accuracy and reproducibility of the results obtained, adding known amounts of pure drug to the previously analysed formulated samples and these samples were reanalyzed by the proposed method performed recovery experiments. The percentage recoveries thus obtained were given in Table II.

RESULTS AND DISCUSSIONS:

In the present work three methods have been developed for the estimation of Clopidogrel bisulphite from Tablet formulation. The developed Methods A, B and C are based on formation of chloroform extractable yellow colored ion-pair complexes with BTB, O-II and Metanil yellow (MY) respectively. The conditions required for the formation of colored complexes were optimized. Statistical analysis was carried out and the results of which were satisfactory. Relative Standard Deviation values were low that indicates the reproducibility of the proposed methods. Recovery studies were close to 100 % that indicates the accuracy and precision of the proposed methods.

The optical characteristics such as absorption maxima, Beer’s law limits, molar absorptivity and Sand ell’s sensitivity are presented in Table I.

The regression analysis using the method of least squares was made for slope (m), intercept (b) and correlation obtained from different concentrations and the results are summarized in Table I.

In conclusion, the proposed methods are simple, economical, sensitive, precise reliable and reproducible for the routine estimation of Clopidogrel bisulphite in bulk as well as in tablet formulation.

ACKNOWLEDGEMENTS:

The authors are grateful to M/s Dr. Reddy’s Laboratories, Hyderabad for the supply of Clopidogrel bisulphite as a gift sample and to the Principal, Sultan-Ul-Uloom college of Pharmacy, Hyderabad, for providing the necessary facilities to carry out the research work.

REFERENCES:

1. The Merck Index, XIII edition, 2001, Merck Research Laboratories, (Monograph No: 6316) page 6320.

2. Robinson A, Hillis J, Neal C, Leary AC., J Chromatogr B Analyt Technol Biomed Life Sci. 2007.

3. Nirogi RV, Kandikere VN, Shukla M, Mudigonda K, Maurya S, Boosi R., Rapid Commun Mass Spectrom.

4. Mishra P, Dolly Archana, Indian Journal of Pharmaceutical Sciences,68 (3),365( 2006).

5. Anand Kumar K., Vetrichelvan T., Ayyappan T., Shanmugam S., Shankar A.S.K, and Nagavalli D, Indian Drugs, 44 (5),342, May (2007).

Received on 30.03.2009 Modified on 23.05.2009

Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 727-729

Sample No.	Labelled Amount (mg)	% Obtained* by proposed method			** % Recovery by the Proposed method
Sample No.	Labelled Amount (mg)	Method A.	Method B	Method C	Method A	Method B	Method C
1	75	99.8	98.9	99.5	99.8	99.2	100.5
2	75	99.3	99.2	99.6	100.6	99.6	99.8