大鼠肝微粒体中睾酮及其代谢产物 6β -羟基睾酮的高效液相色谱检测方法的改进

doi:10.3969/j.issn.1000-1565.2016.01.008

摘要/Abstract

摘要： 建立快速、准确、灵敏的高效液相色谱法,对大鼠肝微粒体中的探针药物睾酮及其代谢产物6β-羟基睾酮进行准确定量分析,用于体外评价大鼠肝微粒体CYP3A酶的活性.对色谱条件进行优化,采用Durashell C18色谱柱(250 mm×4.6 mm,5 μm),以10 mmol/L Na₂HPO₄水溶液(pH 9.26)-乙腈-甲醇(58∶32∶10,体积比)为流动相,流速1.0 mL/min,检测波长245 nm,柱温30 ℃,进样量10 μL.大鼠肝微粒体与睾酮标准品在37 ℃进行温孵,用固相萃取小柱对温孵产物进行萃取,用甲醇-水(体积比1:1)复溶上机分析.6β-羟基睾酮和睾酮分别在0.05~10 mg/L和0.2~20 mg/L内线性关系良好(r>0.999 5),检出限分别为0.003 4和0.034 mg/L(S/N≥3),定量限分别为0.011和0.114 mg/L(S/N≥10),加标回收率分别为100.1%和99.2%,相对标准偏差(RSD)分别为1.75%和1.13%.该方法符合生物样品测定标准.适合体外测定6β-羟基睾酮和睾酮含量,进而用于评价药物对肝微粒体CYP3A酶活性的影响.

关键词: 大鼠肝微粒体, 睾酮, 6β-羟基睾酮, 高效液相色谱法

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

中图分类号:

O657.7

赵燕燕,柳亚飞,刘丽艳,孙东,王静. 大鼠肝微粒体中睾酮及其代谢产物 6β -羟基睾酮的高效液相色谱检测方法的改进[J]. 河北大学学报(自然科学版), 2016, 36(1): 43-51.

ZHAO Yanyan,LIU Yafei,LIU Liyan,SUN Dong,WANG Jing. Improvement on HPLC detection method of testosterone and it’s metabolite 6β -hydroxytestosterone in rat liver microsomes[J]. Journal of Hebei University (Natural Science Edition), 2016, 36(1): 43-51.

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