Simultaneous determination of clopidogrel, 2-oxo-clopidogrel, and the thiol metabolite of clopidogrel in human plasma by LC-MS/MS.

Clopidogrel is a pro-drug which needs two-step metabolism to produce the active thiol metabolite. This study aimed to explore an efficient method to simultaneously determine the plasma clopidogrel, 2-oxo-clopidogrel (2-Oxo-CLP), and the clopidogrel active metabolite (CAM). A high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) was therefore developed. The analytes were extracted from plasma by using methyl tert-butyl ether (MTBE). Chromatographic separation was performed on a C18 column under an isocratic elution, accompanied with acetonitrile and deionized water containing 0.1% formic acid. After optimizing the condition of LC-MS/MS, a stable linearity was observed in the standard curves over the concentration ranges of 0.05 to 50.0 ng/mL for clopidogrel, 0.5 to 50.0 ng/mL for 2-Oxo-CLP, and 0.5 to 100 ng/mL for clopidogrel active metabolite derivative (CAMD). The retention time was 4.78 minutes, 3.79 minutes, 3.59 minutes, and 4.82 minutes for clopidogrel, 2-Oxo-CLP, CAMD, and internal standard, respectively. Both the relative standard deviation and the relative error were within the requirement of operating criteria. No significant degradation of clopidogrel, 2-Oxo-CLP, and CAMD occurred under different storage conditions. This method was successfully validated in 3 patients with coronary artery disease. The results showed that the current LC-MS/MS method was efficient for simultaneously detecting clopidogrel, 2-Oxo-CLP, and CAM with fine linearity, accuracy, precision, and stability.