Determination of Clopidogrel Carboxylic Acid in Human Plasma by LC-MS/MS

Background: Clopidogrel, is a thienopyridine derivative labeled for use to prevent thrombosis after coronary artery stenting. Pharmacokinetics of clopidogrel is studied indirectly by quantification of carboxylic acid which is a major metabolite of Clopidogrel. Objective: The aim of this work is to develop and validate a rapid, simple and sensitive LC/MS/MS assay method for the determination of Clopidogrel carboxylic acid in human plasma using Clopidogrel-D4-carboxylic acid as internal standard. Methods: Analytes was extracted from 200 μl of plasma by a simple liquid-liquid extraction using diethyl ether – n-hexane (80:20, v/v). The chromatographic separations were achieved on a C18 column using Methanol, de-ionized water and formic acid as a mobile phase at flow rate of 0.5 ml/minute. Analysis was monitored by multiple reactions monitoring mode based on m/z transition of 308.10→113 for Clopidogrel carboxylic acid and 312.10→129 for internal standard. Result: The method had a total run time of about 4 minutes. The lower limit of quantification (LLOQ) was 25 ng/ml showing good linearity over the working range of 25 – 3000 ng/ml (r ≥ 0.999). The intra- and inter day accuracies were 90% - 98% and 92.138% - 96.889% respectively (deviation within acceptable range ≤ 10%).Conclusion: It was shown that this method is suitable for pharmacokinetic study following oral administration of Clopidogrel and can be successfully applied to the therapeutic drug monitoring of Clopidogrel in Clopidogrel-treated patients.