以睾酮为探针采用高效液相色谱法测定细胞色素CYP450 3A4的酶活性

Determination of cytochrome P450 3A4 activity with testosterone probe using high performance liquid chromatography

建立了一种快速、高效的以睾酮作为探针药物评价细胞色素P450 3A4（CYP3A4）酶活性的高效液相色谱-紫外检测方法。采用的色谱柱为Phenomenex C18柱（4.6mm×150mm,5μm）,梯度洗脱,流速1.0mL/min,紫外检测波长245nm,柱温30℃。睾酮与大鼠肝微粒体温孵后,过已活化好的C18固相萃取小柱,收集甲醇洗脱液,于37℃水浴中通N2吹干,用50%甲醇复溶后进样分析测定。研究结果表明,6β-羟基睾酮的保留时间为11.60min,线性范围为0.5-32μg/mL,最低检出质量浓度为0.02μg/mL,提取率为88.41%-92.73%,方法的回收率为99.07%-101.30%;睾酮的保留时间为19.27min,线性范围为0.5-40μg/mL,最低检出质量浓度为0.01μg/mL,提取率为89.59%-92.66%,方法的回收率为96.50%-98.03%。两者的日内、日间相对标准偏差均小于10%,温孵体系中的其他内源性物质不干扰测定。该方法快速、稳定、灵敏度高,适合体外睾酮及其代谢物6β-羟基睾酮的测定,可应用于体外CYP3A4酶活性的评价及酶动力学的研究。

A method was established for evaluating cytochrome P450 3A4 （ CYP3A4 ） activity using testosterone in vitro by high performance liquid chromatography-ultraviolet detection （HPLC-UV）. It employed a Phenomenex C18 column （4.6 mm × 150 mm, 5 μm） at 30 ℃. The mobile phase consisted of （A） methanol-acetonitrile-0.05% phosphate solution （pH 2.45） （5： 15 ： 80, v/v） and （B） acetonitrile-0.05% phosphate solution （ pH 2.45 ） （ 50： 50, v/v） using a linear gradient elution of 100%A - 100%B at 0 - 18 min, then held for 5 min and returned to A. The flow rate was set at 1.0 mL/min and the ultraviolet detector was operated at 245 nm. Firstly, testosterone was incubated with rat liver microsomes in vitro and extracted by solid phase extraction （SPE）. Then, the methanol eluant was obtained and evaporated to thoroughly dry with a mild stream of N： at 37 ℃. Finally, the residues were reconstituted with 200 μL 50% （v/v） methanol and further analyzed by HPLC. The retention time of 6β-hydroxylated testosterone was 11. 60 min, the linear range of the method was from 0.5 μg/mL to 32μg/mL, and the detection limit was 0.02 μg/mL. The method recoveries were from 99.07% to 101.30% and the extraction recoveries were from 88.41% to 92.73%. Tffe retention time of testosterone was 19.27 min, the linear range of the method was from 0.5 - 40 μg/mL, and the detection limit was 0.01 μg/mL. The method recoveries were from 96.50% to 98.03% and the extraction recoveries were from 89.59% to 92.66%. All of the intraday and interday relative standard deviations were less than 10%. The method is fast, accurate, sensitive and suitable for the evaluation of CYP3A4 activity and research of enzyme metabolism kinetics in vitro.