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目的:建立测定人体血浆华法林高效液相色谱-质谱联用(HPLC-MS)测定方法,并用于华法林片的人体药动学研究;同时,考察CYP2C9和VKORC1基因多态性的可能影响。方法:人血浆经乙酸乙酯萃取,以Inertsil~ODS-3色谱柱(4.6 mm×150 mm,5μm)为固定相,乙腈-0.5%甲酸水溶液(60∶40,v/v)为流动相,流速为0.8 mL·min~(-1),,进样量为5μL,记录华法林经时血浓度,计算其主要药动学参数;采集肘静脉血,提取DNA,采用FISH法检测受试者CYP2C9和VKORC1基因多态性。结果:华法林在0.5~600 ng·mL~(-1)范围内呈良好线性关系(r>0.99),最低定量限为5.0ng·mL~(-1),精密度和准确度RSD均小于15%,方法稳定性良好;24例受试者分别有2例CYP2C9和3例VKORC1基因突变个体检出。结论:建立了快速测定血浆华法林HPLC-MS法,方法灵敏、稳定、重现性好、选择性好、准确度高,可用于人血浆中华法林浓度测定及华法林药动学研究;CYP2C9和VKORC1基因多态性对华法林药动学行为有一定影响,为体内过程和效应差异的重要影响因素。
OBJECTIVE: To establish a method for the determination of human plasma warfarin by high performance liquid chromatography-mass spectrometry (HPLC-MS) and to study the pharmacokinetics of warfarin tablets. Meanwhile, it is possible to investigate the possibility of CYP2C9 and VKORC1 gene polymorphism influences. METHODS: Human plasma was extracted with ethyl acetate. The mobile phase was acetonitrile-0.5% formic acid solution (60:40, v / v) with Inertsil ~ ODS-3 column , The flow rate was 0.8 mL · min -1 and the injection volume was 5 μL. The warfarin concentration was recorded and the main pharmacokinetic parameters were calculated. The elbow venous blood was collected and DNA was extracted by FISH Trials CYP2C9 and VKORC1 gene polymorphisms. Results: Warfarin showed a good linearity (r> 0.99) in the range of 0.5-600 ng · mL -1 with the lowest limit of quantitation of 5.0 ng · mL -1. The precision and accuracy of RSD Less than 15%. The stability of the method was good. In 24 subjects, 2 cases of CYP2C9 and 3 cases of VKORC1 mutation were detected respectively. Conclusion: The rapid determination of plasma warfarin HPLC-MS method is established, the method is sensitive, stable, reproducible, good selectivity, high accuracy, can be used for determination of warfarin concentration in human plasma and warfarin pharmacokinetics ; CYP2C9 and VKORC1 gene polymorphisms on warfarin pharmacokinetics have some impact on the process and effect differences in the body of an important factor.