LC-MS/MS法测定比格犬血浆中氯吡格雷活性代谢物

Determination of Clopidogrel Active Metabolite in Beagle Dog Plasma by LC-MS/MS

建立了一种定量测定比格犬血浆中氯吡格雷活性代谢物(active metabolize,AM)的液相色谱-串联质谱(LC-MS/MS)法。采血管中加入酯酶抑制剂敌敌畏,采样后立即离心处理,取上层血浆,加入2-溴-3’-甲氧基苯乙酮(MPB)进行衍生化,反应完成后直接加入含0.1%甲酸的冰乙腈沉淀蛋白,取上清液,测定氯吡格雷活性代谢物的衍生化产物(MP-AM)。以乙腈-0.1%甲酸为流动相,梯度洗脱,API4000电喷雾离子源,多反应监测(MRM)模式扫描。在优化的实验条件下,获得了峰宽较窄且对称的色谱峰;MP-AM的线性范围为1~1 000μg/L,相关系数大于0.995;生物基质效应较小,提取回收率在92.8%~95.1%之间,相对标准偏差小于8.27%。该方法的准确度和精密度均符合国家食品药品监督管理总局(China Food and Drug Administration,CFDA)的相关要求,适用于比格犬血浆中氯吡格雷的药代动力学研究。

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.