高效液相色谱法测定大鼠肝微粒体CYP3A酶的活性  

Determination of the activity of CYP3A in rat liver microsomes by HPLC

作　　者：邱相君[1] 王哲[2] 徐涛[2] 罗顺斌[2] 胡国新[2] 

机构地区：[1]河南科技大学医学院,河南洛阳471003 [2]温州医学院药学院,浙江温州325035

出　　处：《中国临床药理学杂志》2012年第11期852-853,856,共3页The Chinese Journal of Clinical Pharmacology

摘　　要：目的以咪达唑仑为探针药用HPLC法测定大鼠肝微粒体CYP3A酶活性。方法用ZORBAX SB-C18色谱柱分离;流动相为乙腈-水-0.1%三氟乙酸;流速为1 mL.min-1;柱温40℃;检测波长为230 nm。肝微粒体加入咪达唑仑孵育5 min后,用乙腈终止反应,加入内标溶液50μL,混匀后离心10 min,取上清进行HPLC分析;计算Km和Vmax。结果 1-羟基咪达唑仑浓度在0.06～3.00 mg.L-1内线性关系良好;日内、日间精密度均<10%,回收率>75%。咪达唑仑羟化反应的Km=7.32μmol.L-1,Vmax=0.59 nmol.min-1.mg-1pro。结论本方法稳定可靠,能准确反映CYP3 A酶的活性。Objective To develop a HPLC method for the determination of the activity of CYP3A in rat liver microsomes using midazolam as probe drug.Methods The analytical column was packed with Agilent ZORBAX SB-C18.The mobile phase was acetonitrile-water-0.1% trifluoroacetic acid and the flow rate was 1.0 mL·min-1.The UV detection wavelength was 230 nm.The incubation was performed with midazolam at 37 ℃ for 5 min,then the reaction was terminated by adding acetonitrile 200 μL and internal standard solution 50 μL.After centrifugation for 10 min,the supernatant 20 μL was injected into the HPLC system for analysis.Results Excellent liner relationship of 1-hydroxymidazolam was obtained from 0.06 mg·L-1 to 3.00 mg·L-1.Km of midazolam hydroxylation was 7.32 μmol·L-1 and Vmax was 0.59 nmol·min-1·mg-1 pro.Conclusion The method was accurate,simple,rapid and could be used to evaluate CYP3A activity assay in vitro.

关 键 词：高效液相色谱法 肝微粒体 咪达唑仑 1-羟基咪达唑仑 

分 类 号：R969.1[医药卫生—药理学] R965.3[医药卫生—药学]