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目的 :建立并验证小鼠血浆中氯吡格雷及其代谢物的LC-MS/MS检测方法 ,研究氯吡格雷在小鼠体内的药代动力学特征。方法:以吡罗昔康为内标,经乙腈沉淀蛋白后,采用电喷雾离子源(ESI)正离子扫描模式多反应监测(multiple reaction monitoring,MRM)方式检测氯吡格雷及其活性代谢物、羧酸代谢物和葡萄糖醛酸代谢物的血药浓度。色谱柱为安捷伦Poroshell120 SB-C18柱(100 mm×2.1 mm,2.7μm);流动相为水(含0.1%甲酸)和乙腈(含0.1%甲酸)并按梯度洗脱。小鼠单次灌胃给予氯吡格雷10 mg/kg,测定氯吡格雷及其3种主要代谢物的血药浓度,并研究其药代动力学特征。结果:氯吡格雷及其代谢产物在一定测定范围内均呈现良好的线性关系(r>0.99),提取回收率均大于80%,未见明显基质效应,其日内、日间精密度均较好(RSD<15%),准确度相对误差(RE)为-2.40%~5.00%,稳定性较好。在小鼠的药代动力学研究中,该方法能达到预期的实验要求。结论:所建立的LC-MS/MS分析方法能快速、准确地测定小鼠血浆中氯吡格雷及其代谢产物浓度,其专属性、回收率、基质效应、稳定性、精密度和准确度等均符合生物样品测定要求。
OBJECTIVE: To establish a LC-MS / MS method for the determination of clopidogrel and its metabolites in mouse plasma and to study the pharmacokinetics of clopidogrel in mice. Methods: Piroxicam was used as an internal standard. After the protein was precipitated by acetonitrile, multiple reaction monitoring (MRM) was performed by electrospray ionization (ESI) Plasma concentrations of metabolites and glucuronic acid metabolites. The column was Agilent Poroshell 120 SB-C18 column (100 mm × 2.1 mm, 2.7 μm); the mobile phase consisted of water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid) and eluted with a gradient. Mice were given a single oral gavage of clopidogrel 10 mg / kg, determination of clopidogrel and its three main metabolites of blood concentration, and study its pharmacokinetic characteristics. Results: Clopidogrel and its metabolites showed a good linear relationship (r> 0.99) within a certain range of determination. The recovery rates of both clopidogrel and its metabolites were all above 80%. There was no obvious matrix effect. The intra- and inter-day precision was better (RSD <15%). The relative error (RE) of accuracy is -2.40% ~ 5.00%, and the stability is better. In the mouse pharmacokinetic study, this method can achieve the desired experimental requirements. Conclusion: The established LC-MS / MS method can rapidly and accurately determine the concentration of clopidogrel and its metabolites in mouse plasma, its specificity, recovery rate, matrix effect, stability, precision and accuracy All meet the biological sample determination requirements.