摘要:
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1～1 000 μg/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.
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 1-1 000 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.
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