Abstract:
A method of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for determining clopidogrel active metabolite (AM) in beagle dog plasma. LC separation was performed using water containing 0.1% formic acid and acetonitrile as mobile phases with the gradient elution at a flow rate of 1 mL/min. Quantitative analysis was achieved after the chromatographic separation on an Ascentis C18 column (5 cm×4.6 mm×5 μm). The electrospray ionization (ESI) source in positive mode and multiple reaction monitoring (MRM) were used for the detection of clopidogrel AM derivative (MP-AM). The sample pretreatment conditions such as the extraction method, the derivative reagents were optimized. The alkylating reagent 2-bromo-3’-methoxyacetophenone (MPB) was used to stabilize clopidogrel AM in blood. Blood samples were centrifuged immediately after collection to take plasma and MPB was then added. Protein precipitation with iced acetonitrile (containing 0.1% formic acid) as precipitation solvent was used for sample extraction. The post-extracted samples were analyzed by LC-MS/MS. The peaks are narrow and symmetrical. The linear range of MP-AM is 1-1000 μg/L, and the linear correlation coefficient is more than 0.995. The method has no matrix interference. The recoveries of MP-AM range from 92.8% to 95.1% at three spiked levels of 2.4, 24 and 800 μg/L with the relative standard deviations (RSD) less than 8.27% (
n=6). The intra-assay/inter-assay accuracy and precision are all acceptable. The method is simple, accurate and robust, which is suitable for research the pharmacokinetic of AM in beagle dog plasma.