Development of Reverse Phase Liquid Chromatographic Method for Determination of (+)-(S)-(o-Chlorophenyl)-6,7-Dihydrothieno [3,2-c] pyridine-5(4H)-acetic acid,Hydrochloride and Methyl (+/-) - (o-Chloro phenyl)-4,5-Dihydrothieno[2,3-c]pyridine-6(7H)-acetate, Hydrochloride from Clopidogrel Besylate

Clopidogrel besylate contain single stereogenic center and has impurities like ((+) - (S) - (o-chlorophenyl)-6,7-dihydrothieno[3,2-c] pyridine-5(4H)- acetic acid, hydrochloride) which is known as impurity A and (Methyl (+/-) - (o-chloro phenyl)-4,5-dihydrothieno[2,3-c] pyridine-6(7H)-acetate, hydrochloride) which is known as impurity B. They are introduced during production. A simple, sensitive, precise and high performance liquid chromatographic (HPLC) method has been developed and validated for quantitative determination of impurity A and impurity B from clopidogrel besylate in bulk drug using uv detector at 220 nm. The developed method was able to separate impurity A and impurity B of clopidogrel besylate from its bulk drug within 50 min. The chromatographic separation was carried out by reverse phase chromatography using C8 column (Zorbax SB C8 250 mm x 4.6 mm x 5 μm), with mobile phase comprising of buffer solution and acetonitrile in the gradient composition, at a flow rate of 1.0 ml/min, at 25°C temperature. The limit of detection and limit of quantitation of impurity A were found to be 0.07 μg/ml and 0.20 μg/ml and of impurity B were found to be 0.10 μg/ml and 0.30 μg/ml respectively. The linearity of response of impurity A was in the range of 0.20 μg/ml to 3.0 μg/ml with r > 0.9999. The linearity of response of impurity B was in the range of 0.30 μg/ml to 4.5 μg/ml with r > 0.9995. The method was validated and found to be suitable for determination of impurity A and impurity B from clopidogrel besylate bulk drug.