HPLC-MS/ESI同时测定人血浆中文拉法新及其3种代谢产物

HPLC-MS/ESI determination of venlafaxine and its three metabolites in human plasma

目的:建立一种快速灵敏的反相高效液相色谱-质谱联用法同时测定人血浆中文拉法新(VEN)和其3种代谢产物:氧云甲基文拉法新(ODV)、氮去甲基文托法新(NDV)和氮、氰双去甲基文拉法新(DDV)的浓度。方法:以艾司唑仑为内标,样本碱化后用乙醚捉取2次,合并有机相,40℃氮气吹干,100μL流动相定容;用 BDS HYPERSIL C_(18)反相色谱柱(250mm×4.6mm,5μm)进行分离,以乙腈-缓冲溶液(30 mmol·L^(-1)醋酸铵+2.6mmol·L^(-1)甲酸+0.13 mmol·L^(-1)三氟乙酸)(40:60,v/v)为流动相,采用梯度洗脱程序,柱温为50℃,流速为1.0 mL·min^(-1)。采用质谱电喷雾电离源(ESI)将样品离子化,选择性离子监测(SIM)准分子离子和/或主要碎片离子峰。结果:VEN、ODV、NDV、DDV 以及内标艾司唑仑在6min 内完全分离;各物质在2～900 ng·mL^(-1)时线性关系良好,相关系数均大于0.9991;萃取回收率均大于77%;方法回收率均大于91%;最低检测浓度:VEN 0.4 ng·mL^(-1)、ODV0.2 ng·mL^(-1)、NDV 0.3 ng·mL^(-1)、DDV 0.2 ng·mL^(-1);日内日间RSD 均小于11%。结论:本方法简单快速,灵敏准确,可用于药物动力学、药物代谢机制以及血药浓度的临床监护、中毒分析的研究。

Objective： To establish a high-performance liquid chromatography-electrospray ionization mass spectrometry （ HPLC - MS/ESI） method for simultaneous determination of venlafaxine （VEN） and its three metab-olites O - desmethylvenlafaxine （ ODV ）, N - desmethylvenlafaxine （ NDV ） and N, O - didesmethylvenlafaxine （DDV） in human plasma. Method： Estazolam was used as the internal standard. The compounds and internal standard were extracted from plasma by a liquid - liquid extraction. The HPLC separation of the analytes was performed on a Thermo BDS HYPERSIL Cls （250mm×4.6mm,5μm,USA）column,using a gradient elution program with solvents constituted of acetonitrile and aqueous solution（ ammonium acetate：30 mmol·L^-1, formic acid ： 2.6 mmol·L^-1 and trifluoroacetic acid ： 0.13 mmol·L^-1） , and with a flow-rate of 1.0 mL ·min^-1. All of the analytes were eluted within 6 min. The compounds were ionized in the electrospray ionization （ESI） ion source of the mass spectrometer and were detected in the selected ion monitor （SIM） mode. Result： Calibration curves in spiked whole blood were linear from 2.0-900 ng·mL^-1 for the four analytes, all of them with coefficients of determination above 0.9991. The average extraction recoveries for all the four analytes were above 77%. The methodology recoveries were higher than 91%. The limit of detection were 0.4,0.2,0.3, and 0.2 ng·mL^-1 for VEN. ODV. NDV and DDV respectively. The intra - and inter - day RSDs were less than 11%. Conclusion： The method is accurate, sensitive and reliable for the pharmacokinetic and metabolism study of the venlafaxine as well as the therapeutic drug monitoring.