HPLC-MS/MS method for the simultaneous determination of clopidogrel, its carboxylic acid metabolite and derivatized isomers of thiol metabolite in clinical samples.

A fast and reproducible HPLC-MS/MS method was developed for the simultaneous determination of clopidogrel (CLP), its carboxylic acid derivative (CLPM), derivatized thiol metabolite isomers MP-H3 and the active MP-H4 in incurred human plasma. CLP, CLPM, MP-H3 and MP-H4 isomers together with the internal standard piroxicam were extracted from plasma samples using a simple protein precipitation with acetonitrile. The analytes were separated on HPLC Zorbax Plus C18 column via gradient elution with water and acetonitrile, both containing 0.1% (v/v) formic acid. Detection of the analytes were performed on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. Calibration curves of the analytes prepared in 250μL plasma were found to be linear in ranges: 0.25-5.00ng/mL for CLP, 0.25-50.00ng/mL for MP-H3 and MP-H4 isomers and 50-10,000ng/mL for CLPM. The lower limit of quantitation was 0.25ng/mL for CLP, MP-H3, MP-H4 and 50.00ng/mL for CLPM. Intra- and inter-assay precision, expressed as relative standard deviation, was ≤18.1% for CLP, ≤15.2% for CLPM, ≤10.1% for MP-H3 and ≤19.9% for MP-H4. Intra- and inter-day accuracy of the method, expressed as relative error, was ≤16%. The analytes were stable in samples stored for 6h in autosampler, in plasma samples for 24h at room temperature and for 3 months at -25°C. Resolution of CLP, CLPM and MP-H3 and MP-H4 isomers of thiol metabolite during one analytical run was reported in patient plasma. The HPLC-MS/MS method was applied for pharmacokinetic studies of CLP and its metabolites in patients treated with daily dose of 75mg CLP.