Assay method for the carboxylic acid metabolite of clopidogrel in human plasma by gas chromatography–mass spectrometry

Abstract

This paper describes a GC–MS method for the analysis of the carboxylic acid metabolite (SR26334, II) of methyl (+)-( S)-α-( o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetate hydrogensulfate (clopidogrel, SR 25990, I) in plasma and serum. The analytical procedure involves a robotic liquid–liquid extraction with diethyl ether followed by a solid–liquid extraction on C 18 cartridges. The derivatization process was performed using n-ethyl diisopropylethylamine and α-bromo-2,3,4,5,6-pentafluoro toluene. A structural analogue ( III) of II, was used as internal standard. The 1/ X 2; weighted calibration curve obtained in the range 5–250 ng/ml was well described by a quadratic equation. The extraction efficiency was better than 48% over the range studied; for the internal standard it averaged 51% at 50 ng/ml. Precision ranged from 3.6 to 15.8%, and accuracy was between 92 and 114%. Dilution has no influence on the performance of the method which could then be used to quantitate plasma samples containing up to 25 000 ng/ml. The limit of quantification was 5 ng/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay methods for use in pharmacokinetic studies.