人体血浆华法林高效液相色谱-质谱联用测定方法的建立及其应用

Determination of warfarin in human plasma by HPLC-MS and its applications

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 HPLC-MS method for determination of warfarin concentration in plasma,to genotype warfarin related CYP2C9 and VKORC1 polymorphisms and to evaluate possible influence to warfarin pharmacokinetic characteristics.METHODS Plasma samples collected from healthy Chinese subjects were extracted with after centrifugation.Samples were separated on an Inertsil ODS-3 column（4.6 mm×150 mm,5μm）,eluted with mobile phase of acetonitrile：0.5%（10∶90,v/v）at a flow rate of 0.8 mL·min-1.5μL was injected into HPLC-MS system for analysis.RESULTS The calibration curve was linear in range of 0.5-400 ng·mL^-1.Inter-and intra-day RSDs were less than 15%.Correlation of two methods was good（r〉0.99）.LLOQ for warfarin was 5.0ng·mL^-1.Stability of the methods met requirements for warfarin analysis.Two CYP2C9 and 3 VKORC1 gene mutations were detected in 24 subjects.CONCLUSION The established HPLCMS method is fast,sensitive,stable,repeatable,selective and accurate,suitable for determination of warfarin plasma concentration,and for warfarin pharmacokinetic studies.Polymorphisms of CYP2C9 and VKORC1 genotypes have certain effects on warfarin pharmacokinetics,and are important factors for difference of internal course and effects.