HPLC法测定大鼠血浆中1'-羟基咪达唑仑及咪达唑仑的浓度

摘要

Objective: To describe a sensitive determination of midazolam and its related metabolite 1' -hydroxy midazolam in rat plasma by high performance liquid chromatography ( HPLC). Method: The plasma sample was mixed with internal standard solution ( diazepam) before the addition of buffer bicarbonate to alkalify the plasma drug,and then vortex-mixed with the solvent (dichlorometh-ane: hexane =3:7,v:v). A Hypersil BDS C18 column (250 mm ×4. 6 mm,5 μm)equipped with a guard column was kept at 40 ℃.The mobile phase consisted of phosphate buffer (0. 02 mol·L-1),acetonitrile and methanol (38:20:42) and was pumped at a constant rate of 1.0 ml · Min-1. The peak was detected using a UV detector set at 230 nm. Result: In this study, the method was validated for the 1' -hydroxy midazolam range of 8. 32-832 ng·ml-1 and the lower limit of quantitation (LLOD) was 8. 32 ng·ml-1.while midazolam ranged from 25 to 2 500 ng·ml-1 and LLOD was 25 ng·ml-1. The intra- and inter-day precisions for 1' -hydroxy midazolam and midazolam were less than 8% ,and the mean recoveries of 1' -hydroxy midazolam and midazolam were 84%-90%. Conclusion: A simple, rapid and repeatable HPLC method is developed for the simultaneous determination of midazolam and its main metabolite 1' -hydroxy midazolam in rat plasma, which is applicable in modeling and description of the possible pharmacological interactions between the medicines and CYP3A enzymes.%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 C18 (250mm ×4.6 mm,5μm)柱,以KH2 PO4(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.