HPLC-MS/MS法同时测定人血清中文拉法辛和O-去甲基文拉法辛的浓度

Simultaneous determination of venlafaxine and O-desmethylvenlafaxine in human serum by HPLC-MS/MS

目的建立可同时快速测定人血清中文拉法辛和O-去甲基文拉法辛质量浓度的方法。方法以乙腈为沉淀剂,经蛋白沉淀法处理,进高效液相串联质谱系统(HPLC-MS/MS)分析,色谱柱:Agilent XDB C18(4. 6 mm×50 mm,1. 8μm),流动相:甲醇-水(90∶10,v/v,含2 mmo L·L^(-1)甲酸铵),流速:0. 7mL·min^(-1),柱温:35℃,进样量:1μL,电喷雾电离,用多反应监测模式,定量分析离子对分别为m/z 278. 15→m/z 58. 20(文拉法辛)、m/z 284. 15→m/z 58. 20(文拉法辛-D6)、m/z 264. 10→m/z 58. 10(O-去甲基文拉法辛)、m/z 270. 10→m/z 58. 10(O-去甲基文拉法辛-D6)。结果文拉法辛标准曲线方程为y=1. 55x+1. 62×10^(-4)(r=0. 999 7),线性范围为4～400 ng·mL^(-1); O-去甲基文拉法辛标准曲线方程为y=1. 42x-3. 61×10^(-2)(r=0. 999 5),线性范围为20～2000 ng·mL^(-1)。除文拉法辛定量下限批间精密度为15. 33%外,其余批间和批内精密度均在0. 83%～8. 73%,准确度均在97. 36%～101. 8%,平均提取回收率均大于95. 67%,稳定性好。结论本方法样品处理简便,灵敏度高,专属性强,适用于文拉法辛和O-去甲基文拉法辛的血药浓度监测。

Objective To develop a rapid method for determining the concentration of venlafaxine and O-desmethylvenlafaxine Simultaneously in human serum.Methods The clinical serum samples were treated by protein precipitation using acetonitrile as a precipitant,and then were determined by high performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS).The column was Agilent XDB C18(4.6 mm×50 mm,1.8μm),and the mobile phase was methanol-water(90∶10,v/v,containing 2 mmoL·L-1 ammonium formate).The flow rate was0.70 mL·min-1,the column temperature was 35℃,the injection volume was 1μL,and the electrospray ionization source was used.The multi-reaction monitoring mode was used to quantitatively analyze the ionization pairs as m/z 278.15→m/z 58.20(venlafaxine),m/z 284.15→m/z 58.20(venlafaxine-D6),m/z 264.10→m/z 58.10(O-desmethylvenla-faxine),m/z 270.10→m/z 58.10(O-desmethylvenlafaxine-D6).Results The standard curve of venlafaxine was y=1.55 x+1.62×10-4(r=0.999 7),the linear range was 4-400 ng·mL-1.The standard curve of O-desmethylvenlafaxine was y=1.42 x-3.61×10-2(r=0.999 5)with a linear range of 20-2000 ng·mL-1.Except that the inter-assay precision of the lower limit of venlafaxine was 15.33%,the rest of inter-assay and intra-assay precision were between 0.83%and8.73%.The accuracy were between 97.36%and 101.8%,and average extraction recoveries of two analytes were greater than 95.67%,and the stability was good.Conclusion The method was simple,sensitivity as well as specificity,suitable for monitoring the concentrations of venlafaxine and O-desmethylvenlafaxine in human serum.