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目的:建立大鼠血浆中咪达唑仑及其代谢产物1’-羟基咪达唑仑测定的高效液相色谱(HPLC)方法。方法:血浆样品以地西泮为内标,经碳酸盐缓冲液碱化后,由混合有机溶剂(正己烷:二氯甲烷=7:3,v/v)提取。采用Hypersil BDS C_(18)(250mm×4.6 mm,5μm)柱,以KH_2PO_4(0.02 mol·L~(-1))缓冲液-乙腈-甲醇(38:20:42)为流动相,柱温40℃,流速1.0 ml·min~(-1),于紫外230 nm处检测1’-羟基咪达唑仑及咪达唑仑浓度。结果:在该试验条件下,血浆中1’-羟基咪达唑仑的线性范围为8.32～832 ng·ml~(-1),最低检测限为8.32 ng·ml~(-1);咪达唑仑的线性范围为25～2 500 ng·ml~(-1),最低检测限为25 ng·ml~(-1)。其日间和日内精密度均小于8%,提取回收率为84～90%。结论:本方法专属性强、灵敏度高、重复性好,适用于实验室评价药物间相互作用研究。
Objective: To establish a high performance liquid chromatography (HPLC) method for the determination of midazolam and its metabolite 1’-hydroxy midazolam in rat plasma. Methods: Plasma samples were extracted with a mixture of organic solvents (n-hexane: dichloromethane = 7: 3, v / v) using diazepam as internal standard and basified with carbonate buffer. The mobile phase consisted of Hypersil BDS C 18 (250 mm × 4.6 mm, 5 μm) and KH 2 PO 4 (0.02 mol·L -1) buffer-acetonitrile-methanol (38:20:42) ℃, the flow rate was 1.0 ml · min ~ (-1) and the concentrations of 1’-hydroxy midazolam and midazolam were detected at 230 nm. Results: The linear range of 1’-hydroxymidazolam in plasma was 8.32-832 ng · ml -1 with the lowest detection limit of 8.32 ng · ml -1. Azole linear range of 25 ~ 2 500 ng · ml -1, the lowest detection limit of 25 ng · ml -1. The intra-day and intra-day precision were less than 8%, extraction recovery was 84 ~ 90%. Conclusion: This method is specific, sensitive and reproducible, and is suitable for laboratory evaluation of drug-drug interactions.