Development and validation of a LC-MS/MS method for the enantioseparation and determination of clopidogrel bisulfate in beagle plasma and its application to a stereoselective pharmacokinetic study

Abstract

A rapid and sensitive chiral liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the enantioseparation and determination of clopidogrel bisulfate enantiomers in beagle plasma was established and successfully applied to a stereoselective pharmacokinetic study. The chromatographic separation was achieved on an Ultron ES-OVM column (150 × 2.0 mm, 5 μm) with acetonitrile-ammonium acetate (10 mM, pH 4.5) (22:78, v/v) at a flow rate of 0.3 mL/min. The mass detection was conducted using multiple reaction monitoring mode with the transition of the m/z 322.00→212.00 for clopidogrel and m/z 285.00→193.00 for diazepam (IS), respectively. The calibration curve was linear at the range of 1−800 ng/mL (r2>0.997) for each enantiomer. The intra- and inter-day precision (CV%) was within 10.9 % and the accuracy was at the range of 88.5 %–101.3 % for both enantiomers. The extraction recovery was >90.2 % and no obvious matrix effect was observed. The pharmacokinetic results showed that the R-CB have higher Cmax and AUC than that of S-CB. It is first time to find the chiral inversion between S-CB and R-CB in beagle.