以睾酮为探针的HPLC法测定CYP3A1酶活性的方法学研究

Methodology Research on Determination of CYP3A1 Activity by HPLC with Testosterone as Probe

目的:建立以睾酮为探针测定大鼠CYP3A1酶活性的方法。方法:采用高效液相色谱法,以咪达唑仑为内标,样品用乙酸乙酯萃取后重溶进样,进行睾酮特异代谢物6-β-羟基睾酮(6-β-OHT)浓度测定的方法学研究。色谱柱为HypersilBDSC18,流动相为甲醇-Na2HPO4水溶液(pH8.25)(60:40),流速为1mL.min-1,检测波长为254nm。结果:6-β-OHT检测浓度线性范围为1.25～100μg.mL-1(r=0.9998),最低定量限为1.25μg.mL-1;萃取回收率为83.9%～87.4%(RSD≤8.82%),相对回收率为100.4%～103.4%(RSD≤7.05%);平均日内、日间RSD均<10%;样本室温放置4h稳定性良好。结论:所建立的方法快速、简便、重复性好,适用于体外定量测定睾酮及其代谢物6-β-OHT,也可用于体外评估CYP3A1酶的活性。

OBJECTIVE： To develop a method for the determination of CYP3A1 activities in rats with testosterone as probe. METHODS： HPLC method was adopted using midazolam as internal standard, and samples were extracted by ethyl-acetate, evapo- rated and re-dissolved. The method validation of the determination of 6-fl-hydroxytestosterone （6-β-OHT） concentration had been ex- amined. The determination was performed on Hypersil BDS C18 column with mobile phase consisted of methanol-Na2HPO4 （pH= 8.25） （60：40） at the flow rate of 1 mL.min-1. Detection wavelength was set at 254 nm. RESULTS： The linear range of 6-fl-OHT was 1.25-100tg.mL-1 （r=0.999 8）. Limit of quantitation was 1.25 ~tg.mL-t. Extraction recoveries were 83.9%-87.4% （RSD≤8.82% ）. Relative recoveries were 100.4%-103.4% （RSD≤7.05%）. The RSD of intra-day and inter-day were less than 10%. Sample were stable at room temperature for 4 hours. CONCLUSION： The method is rapid, simple and repeatable. It＇s applicable to determination of testosterone and 6-fl-OHT in vitro. Also it＇s applicable for the evaluation of CYP3A1 activities in vitro.