LC-MS/MS法同时测定人血浆中维拉帕米和代谢产物去甲维拉帕米

Simultaneous determination of verapamil and its metabolite norverapamil in human plasma by liquid chromatography-tandem mass spectrometry

建立一种简便、灵敏的同时测定人血浆中维拉帕米和去甲维拉帕米浓度的液相色谱-串联质谱(LC-MS/MS)的分析方法,并用于健康人体的药代动力学研究。血浆样品经甲醇沉淀蛋白后,使用Agilent Zorbax Eclipse C18色谱柱(50 mm×4.6 mm,5μm)分离,以0.1%甲酸-5%乙腈-10 mmol·L-1甲酸铵水溶液作为水相,甲醇-乙腈(50∶50,v/v)溶液作为有机相,0.5 m L·min-1的流速进行梯度洗脱,采用电喷雾离子源(ESI),多反应(MRM)正离子监测模式对维拉帕米、去甲维拉帕米及内标维拉帕米-d6进行定量检测,监测离子对分别为m/z 445.0→165.2、m/z 441.0→165.2和m/z 461.1→165.2。本文建立的同时测定人血浆中维拉帕米及去甲维拉帕米浓度的LC-MS/MS方法的定量下限(LLOQ)为0.1 ng·m L-1,且在0.1~50 ng·m L-1浓度内线性关系良好(r2>0.997)。维拉帕米及去甲维拉帕米的基质效应分别在99.2%~100%和101%~102%之间,该方法前处理简便快速,灵敏度高,能准确同时测定人血浆中维拉帕米及去甲维拉帕米浓度,方法验证后并经伦理委员会批准后应用于健康人体的药动学研究。

A sensitive and simple high-performance liquid chromatographic tandem mass spectrometric(LC-MS/MS)method for the determination of verapamil and norverapamil in human plasma was established and utilized in a pharmacokinetic study in healthy patients.Protein was precipitated by methanol in plasma samples,and the analytes and internal standard were separated on an Agilent Zorbax Eclipse C18 column(50 mm×4.6 mm,5μm)with a gradient procedure using methanol-acetonitrile(50∶50)as the organic phase and 0.1%formic acid-5%acetonitrile-10 mmol·L-1 ammonium formate solution as the mobile phase at flow rate of 0.5 mL·min-1.Electrospray ionization(ESI)and multiple reaction monitoring(MRM)detection modes were used for quantitative detection of verapamil,norverapamil and verapamil-d6(IS).In the mode of multiple reaction monitoring of positive-ions,the monitoring ion pairs of verapamil,norverapamil and the verapamil-d6 were m/z 445.0→165.2,m/z 441.0→165.2 and m/z 461.1→165.2,respectively.The quantitative lower limit(LLOQ)for the determination of verapamil and norverapamil concentrations in human plasma can reach 0.1 ng·mL-1 in this assay.The calibration curve concentration ranged from 0.1 to 50 ng·mL-1 with high linearity(r2>0.997).The matrix effect of verapamil and norverapamil was 99.2%-100%and 101%-102%,respectively.The recovery of verapamil and norverapamil was 86.8%-95.9%and 87.4%-94.8%,respectively.This method has good specificity and high sensitivity.The determination of the verapamil and norverapamil was not subject to the matrix effect and stable extraction recovery was achieved in this assay.This method could be used to determine the concentration of verapamil and norverapamil in human plasma and suitable for human pharmacokinetic studies after approved by ethics committee.