HPLC-MS/MS法同时测定肝微粒体孵育体系中地西泮及其代谢产物的浓度

Determination of diazepam and its metabolites in human liver microsomal incubation systems by HPLC-MS/MS method

目的：建立同时测定地西泮及其代谢产物质量浓度的HPLC－MS／MS方法。方法色谱柱：Kromasil C18，流动相：乙腈－2 mmol· L－1乙酸铵水溶液（70∶30），流速：0．3 mL· min－1，柱温：40℃，用正离子扫描，多反应监测方式测定样品中药物的质量浓度。考察该方法的专属性、标准曲线和定量下限、精密度与回收率、基质效应及稳定性。结果肝微粒体孵育样品中，地西泮在20～1000 ng· mL－1线性关系良好（ r＞0．99），定量下限为20 ng· mL－1。去甲地西泮、替马西泮和奥沙西泮在2～100 ng· mL－1线性关系良好（ r＞0．99），定量下限为2 ng· mL－1。地西泮、去甲地西泮、替马西泮和奥沙西泮绝度回收率分别在55．47％～68．76％，64．84％～79．11％，68．32％～78．27％和72．66％～83．82％，日内与日间精密度均小于15％。结论本研究方法简便、快速、准确，适用于测定肝微粒体孵育样品中地西泮及其代谢产物的浓度。

Objective To establish a HPLC-MS/MS method for simul-taneous determination of diazapam and its three metabolites in human liv-er microsomal.Methods Kromasil C18 column was used at 40 ℃, and the mobile phase was a mixed system consisting of acetonitrile -2 mmol· L-1 mammonium acetate （ 70 ∶30 ） and a flow rate of 0.3 mL· min -1 .The detection of the samples was made using positive ion scan and multiple reaction monitoring mode.The specificity, linearity and limit of quantification, precision and recovery, matrix effect and sta-bility were assessed.Results The sample showed good linearity from 20-1000 ng· mL-1 （ r〉0.99 ） and the lower limit of quantitation was 20 ng· mL-1 .The liner calibration curve of nordiazepam, temazepam and oxazepam obtained concentration range of 2 -100 ng· mL-1 （r〉0.99） and the low limit of quantitation was 2 ng· mL-1 .The average recovery rates of diazepam, nordiazepam, temazepam and oxaze-pam were 55.47% to 68.76%, 64.84% to 79.11%, 68.32% to 78.27%and 72.66%to 83.82%.The intra-day and inter-day relative standard deviations were both less than 15%.Conclusion The method is simple, rapid and accurate, suitable for measuring the concentration of diazapam and its three metabolites in human liver microsomal.