LC-ESI-MS-MS法同时测定大鼠血浆中甲苯磺丁脲/4-羟基甲苯磺丁脲、氯唑沙宗及其药代动力学研究

Simultaneous Determination of Tolbutamide and Its Metabolite 4-hydroxytolbutamide,Chlorzoxazone in Rat Plasma by LC-MS-MS and Application to Pharmacokinetic Study

目的:建立灵敏、快速、稳定的测定大鼠血浆中"Cocktail"探针药物甲苯磺丁脲及其代谢产物4-羟基甲苯磺丁脲、氯唑沙宗的液相色谱-电喷雾串联质谱(LC-ESI-MS-MS)方法。方法:100μL血浆样品加入内标格列齐特及乙酸铵,以乙酸乙酯进行液液萃取,离心取上清挥干复溶,LC-ESI-MS-MS检测。选用Agelient Eclipse Plus-C18色谱柱(2.1 mm×50 mm,3.5μm),乙腈和0.01%甲酸(1 mmol.L-1甲酸铵)作为流动相,梯度洗脱,流速0.3 mL.min-1,进样量10μL。采用电喷雾电离源(ESI),扫描方式为多反应离子监测模式(MRM),甲苯磺丁脲m/z 269.1/170.0;4-羟基甲苯磺丁脲m/z 285.1/186.0;氯唑沙宗m/z 168.1/132.1;格列齐特m/z 322.3/170.2。结果:甲苯磺丁脲、4-羟基甲苯磺丁脲和氯唑沙宗的线性范围分别为0.98～4 000,0.25～125,0.98～2 000μg.L-1。提取回收率均>70%,稳定性RSD<11%且无基质效应。结论:该方法快速、灵敏、重复性好,可用于血浆中探针药物甲苯磺丁脲、4-羟基甲苯磺丁脲、氯唑沙宗浓度的测定,有助于深入进行代谢酶CYP2C9和CYP2E1的研究。

Objective： To set up an LC-MS-MS method for the simultaneous determination of Cytochrome P450 probe substrates,including tolbutamide and its metabolite 4-hydroxytolbutamide for CYP2C9,chlorzoxazone for CYP2E1.Method： Probe drugs with the IS gliclazide were extracted using ethyl acetate.Gradient elution was performed on an Agelient Eclipse Plus-C18 column（2.1 mm × 50 mm,3.5 μm）.The mobile phase consisted of 0.01% formic acid（1 mmol.L-1 ammonium formate） and acetonitrile.The flow-rate was 0.3 mL.min-1,and the injection volume was 10 μL.The probe drugs were analyzed by ESI MS in MRM mode.The MS-MS reaction selected ions 269.1 /170.0 m/z for tolbutamide,285.1 /186.0 m/z for 4-hydroxytolbutamide,168.1 /132.1 m/z for chlorzoxazone and m/z 322.3 /170.2 ions for gliclazide.Result： The tolbutamide,4-hydroxytolbutamide and chlorzoxazone had good linear relationship within the range of 0.98-4 000,0.25-125,0.98-2 000 μg.L-1,respectively.The extraction recoveries for all probe dings were more than 70%.The stability RSD were less than 11% and matrix effects in plasma on the ionization of probe drugs were negligible.Conclusion： The method described in this report has accuracy,sensitivity and reproducibility.The established LC-MS-MS method was suitable for pharmacokinetic study of tolbutamide and chlorzoxazone as a cocktail probe group and could be applied to hepatic microsomal enzyme study.