Development and validation of high-throughput liquid chromatography–tandem mass spectrometric method for simultaneous quantification of Clopidogrel and its metabolite in human plasma

A simple, sensitive and reliable method is described for simultaneous quantification of Clopidogrel and its metabolite in human plasma by using HTLC–MS/MS. The analytical procedure involves on-line coupling of extraction with Cyclone P (50 mm × 0.5 mm 50 μm) HTLC column by injecting 15 μL sample and chromatographic separation is performed with Cohesive Propel C18 (5 μm, 3.0 mm × 50 mm), followed by quantification with mass detector in SRM mode using ESI as an interface. The calibration curves were linear over a concentration range of 0.1–8 ng/mL of Clopidogrel and 70 ng/mL to 6 μg/mL of its metabolite using 20 mL human plasma per batch. The total run time of analysis was 7.5 min and the lower limits of quantification were 0.1 ng/mL for Clopidogrel and 70 ng/mL for its metabolite. The method validation was carried out in terms of specificity, sensitivity, linearity, precision, accuracy and stability. The validated method was applied in bioavailability and bioequivalence study.