Development and validation of a sensitive LC‐MS/MS method for simultaneous analysis of clopidogrel and simvastatin and their main metabolites in beagles: Application to pharmacokinetic drug interactions

Clopidogrel (CLP) and simvastatin (SV) are commonly used in combination therapies as anti-cardiovascular drugs. However, the effect of coadministration on the absorption and metabolism of the two drugs in vivo is not clear. This study developed and validated an LC-MS/MS method for the simultaneous determination of CLP, clopidogrel carboxylic acid (CLPCA), 2-oxo-clopidogrel (2-O-CLP), SV, and simvastatin hydroxy acid (SVA) in beagle plasma. Chromatographic separation was achieved on an InfinityLab Poroshell 120 SB-C8 column (2.1 × 100 mm, 2.7 μm) using methanol and 0.1% formic acid in water as the mobile phase at a flow rate of 0.3 mL/min in gradient mode. The lower limits of quantification are 0.1, 0.8, 0.05, 0.05, and 0.05 ng/mL for CLP, CLPCA, 2-O-CLP, SV, and SVA, respectively. The selectivity, linearity, accuracy, precision, extraction recovery, matrix effect, and stability were validated within acceptable criteria. This method was successfully applied to the pharmacokinetic drug interaction study between CLP and SV, and the results revealed that combined administration affected the metabolic rate of CLP, SV, and their metabolites. This study is the first to detect CLP, CLPCA, 2-O-CLP, SV, and SVA simultaneously.