INTRODUCTION:

Clopidogrel (CLP) which is chemically methyl 2-(2-chlorophenyl)-2-(6,7-dihydro-4H-thieno[3,2-c]pyridin-5-yl)acetate¹ is an inhibitor of ADP-induced platelet aggregation² acting by direct inhibition of adenosine diphosphate³ (ADP) binding to its receptor and of the subsequent ADP-mediated activation of the glycoprotein⁴ GPIIb/IIIa. A number of methods such as UPLC⁵, LCMS⁶ were reported for the estimation of CLP. Literature survey reveals that HPLC methods have not been reported for its quantitative determination⁷ in its pure form and pharmaceutical⁸ formulations.The molecular formula is C₁₆H₁₆ClNO₂SO₄⁹and the molecular weight is 419.

Only very few HPLC methods have been reported in the literature for the estimation of CLP present in biological fluids. There are no reported methods for the determination of CLP by HPLC in pharmaceutical dosage forms. Hence the author has made an attempt to develop a HPLC method for the determination of CLP in pharmaceutical formulations.

MATERIALS AND METHODS:

Instrumentation:

High Performance Liquid Chromatography (Shimadzu HPLC, Class VP series) with two LC-10AT VP pumps, manual injector with loop volume of 20 µl (Rheodyne), programmable variable wavelength UV detector. Chromatographic integration and processing was carried out on Spincotech software. An Inertsil ODS-3 column having 250x4.6mm i.d with 5µ particle size was used as a stationary phase. The column eluents were monitored at 240 nm.

Chemicals and reagents:

clopidogrel bisulphate was obtained as a gift sample from Cipla labs, India Pvt. Ltd. water (Milli grade), acetonitrile (HPLC grade) and Formic acid (AR grade).

Chromatographic conditions:

Mobile phase consists of acetonitrile, Formic acid in the ratio (80:20). Buffer was prepared by dissolving 0.05g of Formic acid and dissolve it in 100ml of Milli grade water. Adjust the pH to 5, filter through 0.45µm nylon membrane filter and degas.

Mobile phase preparation:

The mobile phase was pumped from the solvent reservoir to the column at a flow rate 1.2 ml/min. The column was maintained at 45°C and the volume of each injection was 20μL. Above prepared buffer and acetonitrile were mixed in the proportion of 20:80. The diluents were monitored at 240nm.

Standard Preparation:

10mg of CLP was weighed and transferred into a 10ml volumetric flask, the drug was dissolved in diluent and the volume was made up to 10ml with diluents. Stock solution was sonicated for 5 minutes.

Sample Preparation:

10 tablets were taken and their average weight was calculated. The tablets were crushed to a fine powder, dose equivalent to 100mg was transferred to a 50 ml volumetric flask, dissolved in working mobile phase and then the solution was made up to the mark with mobile phase and filtered through 0.45 μ membrane filter. 5 ml of this solution was pipetted into 20ml volumetric flask and diluted with the mobile phase to get concentration of 500 μg/ml.

RESULTS AND DISCUSSION:

Several systematic trials were performed to optimize the Chromatographic conditions for developing a sensitive, precise and accurate RP‐HPLC method for the analysis of clopidogrel bisulphate in pharmaceutical dosage forms. The present method contains mobile phase acetonitrile and Formic acid in the ratio (80:20v/v) which was found to be the most suitable as the chromatographic peak obtained with good shape and symmetry. Hence this method was finalized for the estimation of CLP at retention time of 6.4 mins.

The blank graph was given in fig – 1 and sample graph was given in fig – 3.

Figure 1 clopidogrel bisulphate blank

Figure 2 clopidogrel bisulphate standard

Figure 3 clopidogrel bisulphate sample

Linearity:

The linearity graphs for the proposed assay methods were obtained over the concentration range of 7.5-17.5µg/ml. The linearity graph was given in fig – 2. Method of least squares analysis was carried out for getting the slope, intercept and correlation coefficient values in table – 1.

TABLE NO – 1: System suitability, precision and accuracy of the proposed methods for CLP

Parameter	Results
Retention Time (t) (Min)	6.4
Theoretical Plates (n)	11831
Plates per Meter (N)	118306
Linearity range (μg/ml)	7.5-17.5
Peak asymmetry	1.11
Regression equation
Slope (b)	35.88
Intercept (a)	-4.845
Standard deviation of intercept (S_a)	3.526
Correlation Coefficient (r)	0.9999
(%)Relative standard deviation	1.724
Percentage range of errors*
0.05 level	0.0214
0.01 level	0.03367

* Average of six determinations, ** Average of three determinations.

Accuracy:

To determine the accuracy of the proposed method, different amounts of bulk samples of CLP in between the upper and lower linearity limits were taken and analyzed by the proposed method this was mentioned in table – 2.

TABLE NO – 2: Results from analysis of clopidogrel bisulphate in tablets

Pharmaceutical formulation	Amount present	Amount added	Amount recovered	% recovery
Tablets	10	2.5	12.34	99.14
Tablets	10	5	14.29	99.44
Tablets	10	7.5	17.28	99.28

Precision:

The precision of the method was ascertained from the peak area of CLP obtained by determination of six replicates of fixed amount of CLP. The percent relative standard deviation and percent range of errors (0.05 and 0.01 confidence limits) were calculated and were presented.

Robustness:

Robustness of the proposed methods was evaluated by making small changes in flow rate, buffer concentration, pH of the buffer solution, organic modifier concentration and temperature. The results were found to be not affected by these small alterations.

CONCLUSION:

From the obtained results it can be concluded that the proposed method is quite precise and accurate. The absence of additional peaks in the Chromatogram indicated that there is no interference of the common excipients used in the tablets. The proposed HPLC Method is sensitive and reproducible for the analysis of clopidogrel bisulphate in Tablet dosage forms. The method was duly validated by using required statistical parameters.

ACKNOWLEDGEMENT:

The authors greatly acknowledge Cipla labs, India Pvt.Ltd for providing the gift sample of clopidogrel bisulphate.

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