Abstract: A rapid,accurate and sensitive high performance liquid chromatography method was improved for accurate quantitative analysis of probe drugs testosterone and its metabolites 6β-hydroxytestosterone in rat liver microsomes,which was used in vitro to evaluate the activity of CYP3A enzyme in rat liver microsomes.Chromatography conditions were optimized,the experiment was carried out on a Durashell C18 column(250 mm×4.6 mm,5 μm),with 10 mmol/ L Na₂H-PO₄ solution(pH 9.26)-acetonitrile-methanol(58∶32∶10,V/V/V)as the mobile phase at a flow rate of 1.0 mL/min,and the detection wavelengthwas set at 245 nm.The column temperature was 30 ℃ and the injection volume was 10 μL.Rat liver micro-- 收稿日期:2015-06-25 基金项目:河北省自然科学基金项目(H2013201203);河北大学医学学科专项资金建设资助项目(2014A1003) 第一作者:赵燕燕(1960—),女,天津市人,河北大学教授,主要从事药学以及将现代分离技术用于临床、药学、食品、环境等方面的研究工作.E-mail:zhaoyany606@tom.comsomes and testosterone standards were incubated at 37 ℃,incubation products were extracted by solid phase extraction column,then reconstituted with methanol-water(1∶1,V/V)for HPLC analysis.6β - hydroxytestosterone and testosterone showed good linearities within 0.05~10 mg/L and 0.2~20 mg/L(r>0.999 5),respectively.The detection limits for 6β -hydroxytestosterone and testosterone were 0.0034 and 0.034 mg/L(S/N≥3)and the quantitative limits for 6β -hydroxytestosterone and testosterone were 0.011 and 0.114 mg/L(S/N≥10).The recoveries were 100.1% and 99.2% and the relative standard deviation(RSD)were 1.75% and 1.13% respectively.This method met the requirements of biological sample which was suitable for the quantitation of 6β -hydroxytestosterone and testosterone in vitro assays.Furthermore,with this method we could evaluate the impact of drug on the activity of CYP3A enzyme in liver microsomes and carry out the enzyme metabolism kinetics research

Key words: rat liver microsomes, testosterone, 6β -hydroxytestosterone, HPLC