INTRODUCTION:

Clopidogrel bisulfate, chemically (+)-(S) -(2-chlorophenyl)- 6,7-dihydrothieno [3,2-c] pyridine- 5(4H)-acetic acid methyl ester sulphate is a potent oral antiplatelet 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 estimation of Clopidogrel bisulfate in pharmaceutical formulations by various HPLC^1-7, spectrophotometric ^8-13, methods for assay of clopidogrel bisulphate. In the proposed work simple, rapid and reliable UV spectrophotometric method is developed for the determination of clopidogrel bisulphate. The method can be used for the routine analysis. In the proposed method optimization and validation of this method are reported.

Fig. 1: Chemical Structure of Clopidogrel

EXPERIMENTAL:

Instrumentation:

An potentiometric titrator was used (Lab- India-Auto titrator ) for assay method development and validation. A Shimadzu analytical balance with 0.01 mg was used.

REAGENTS AND CHEMICAL:

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

Potassium hydrogen phthalate, perchloric acid and glacial acetic acid of A. R. grade were used.

General Procedure:

Standardization of 0.1 N perchloric acid:

About 0.350 mg of potassium hydrogen phthalate (previously powdered lightly, dried at 120^oC for 2 hours) was weighed accurately into clean and dry titration jar. It was dissolved in 50 ml of glacial acetic acid. It was titrated with 0.1 N perchloric acid using auto titrator. Blank determination was performed out for necessary correction. The titration was performed in duplicate. One ml of 0.1 N HClO₄ is equivalent to 0.2042 gm of potassium hydrogen phthalate (C₈H₅KO₄)

Normality of perchloric acid = W

--------------------

B.R. x 0.2042

Where W is weight of potassium hydrogen phthalate in g.

B.R. is burette reading in ml.

Quantitative determination of clopidogrel bisulphate:

About 0.100 g. of clopidogrel bisulphate test sample was weighted accurately into a clean and dried titration jar. It was dissolved in 35 ml. of anhydrous glacial acetic acid and 15 ml of 5% (w/v) mercuric acetate. It was titrated with 0.1 N perchloric acid potentiometrically. Blank determination was also carried out for necessary correction. One ml of 1 N perchloric acid is equivalent to 0.4199 g. of clopidogrel bisulphate % (Percentage) Clopidogrel bisulphate on the dried basis was calculated as below.

B.R. x N x 0.4199 x 100

% assay = _____________________________

Where B.R. is burette reading in ml at the potentiometric end point.

N is actual normality of 0.1 N perchloric acid.

W is weight of the sample taken in g.

RESULT AND DISCUSSION:

Determination of clopidogrel bisulphate:

The objective of this work was to determine accurately the content of clopidogrel bisulphate. The assay of clopidogrel bisulphate (on the dried basis) of various batches of clopidogrel bisulphate test sample was analyzed using the above method. It was in the range of 99.00 % to 100.68 %.

Analytical Method Validation:

The method precision was checked after analyzing six different preparations of homogeneous test sample of clopidogrel bisulphate. The % RSD of results obtained was found to be 0.7516. It confirms good precision of the method. The results are presented in table 1.

Linearity:

For the establishment of method linearity ,five different weights of clopidogrel bisulphate test samples corresponding to 20 % ,40 %, 60 % , 80 % and 100 % of the about weight ( 0.100 g. ) were taken and analyzed for % ( percentage) of clopidogrel bisulphate content. The results are in table 2.

Table no. 1: Method of precision

Sr. no	Weight of clopidogrel bisulphate	Burette reading in ml	Normality of perchloric acid	% assay
1	0.500	11.9	0.09990	99.84
2	0.500	12.0	0.09990	100.68
3	0.500	11.8	0.09990	99.00
4	0.500	12.0	0.09990	100.68
5	0.500	11.9	0.09990	99.84
6	0.500	11.8	0.09990	99.00
			Mean	99.84
			Std. Deviation	0.75
			RSD	0.7516

Table no.2 : Linearity

Sr. No.	Weight of lopigrel bisulphate	Burette reading	Normality of Perchloric acid	% assay
1	0.1	2.37	0.09990	99.417
2	0.2	4.77	0.09990	100.046
3	0.3	7.15	0.09990	99.976
4	0.4	9.51	0.09990	99.731
5	0.5	12	0.09990	100.675
			Mean	99.969
			Std. Deviation	0.465
			RSD	0.465

The potentiometric titration was conducted once at each level. Linearity curve was drawn by plotting test sample weight in gram on x axis and titre values on y axis.

The values of correlation coefficient, slope and intercept are given in table 3.

Table no.3: Regression values

Parameter	Values
Slope	24
Intercept	-0.04
Coefficient of co-relation	0.9999

ACCURACY AND RECOVERY:

Accuracy was determined at five different levels i.e., 20 % ,40 % ,60 % ,80 % and 100 % of the nominal concentration. (0.100 g.) The titration was conducted in triplicate at each level and the titre value was recorded. The tire value obtained in linearity study was considered as true value during the calculation of percentage (%) recovery. The percentage recovery is calculated using following equation.

Titre value x 100

Percentage recovery = ---------------------------

True titre value

The percentage range recovery of clopidogrel bisulphate was in 99.417 to 100.396 %. It confirms the accuracy of the proposed method. (Table 4).

Table no 4 : accuracy and precision

Level no.	Weight of clopidogrel bisulphate added	Weight of clopidogrel bisulphate found	% assay	Mean % assay
1	0.10	0.0998	99.836	99.417
	0.10	0.0994	99.417
	0.10	0.0990	98.997
2	0.20	0.1997	99.836	100.046
	0.20	0.2005	100.256
	0.20	0.2001	100.046
3	0.30	0.2999	99.976	99.976
	0.30	0.2995	99.836
	0.30	0.3003	100.116
4	0.40	0.3993	99.836	99.731
	0.40	0.3989	99.731
	0.40	0.3985	99.627
5	0.50	0.5034	100.675	100.396
	0.50	0.4992	99.836
	0.50	0.5034	100.675

RUGGEDNESS:

The ruggedness of the method is defined as degree of reproducibility of results obtained by analysis of clopidogrel bisulphate sample under variety of normal test conditions such as different laboratories, different analysts and different lots of reagents. Quantitative determination of clopidogrel bisulphate was conducted potentiometrically on one laboratory. It was again tested in another laboratory using different instrument by different analyst. The assays obtained in two different laboratories were well in agreement. It proved ruggedness of the proposed method.

CONCLUSION:

The proposed method of non-aqueous potentiometric titration was found to be precise, accurate and rugged. The values of percentage recovery and standard deviation showed sensitivity. The method was completely validated. It showed satisfactory data for all the parameters of validation. Hence it can be applied for routine quality control application.

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Received on 24.12.2016 Modified on 05.01.2017

Asian J. Research Chem. 2017; 10(1):26-28.

DOI: 10.5958/0974-4150.2017.00004.9