HPLC法测定大鼠肝微粒体中睾酮的含量

Content Determination of Testosterone in Rat Liver Microsomes by HPLC

目的:建立以高效液相色谱法测定大鼠肝微粒体中细胞色素P4503A4(CYP3A4)探针底物睾酮含量的方法。方法:色谱柱为Ultimate-XB C18,流动相为水-乙腈(50∶50),内标为地西泮,流速为1.0mL·min-1,检测波长为245nm,柱温为30℃,进样量为20μL。结果:睾酮生物样品检测浓度的线性范围为10.0～200.0μmo·lL-1(r=0.9995);低、中、高浓度的平均方法回收率分别为86.15%、103.10%、97.48%,平均相对回收率为111.15%、107.24%、100.78%,日内RSD分别为3.92%、2.46%、2.82%,日间RSD分别为2.98%、1.34%、2.41%。结论:本方法简便快速、灵敏度高、重复性好,可用于大鼠肝微粒体中CYP3A4活性的测定及相关研究应用。

OBJECTIVE： To develop a HPLC method for the content determination of cytochrome P450 3A4 （CYP3A4） probe substrate testosterone in rat liver microsomes. METHODS： The determination was performed on Ultimate-XB C18 column with mobile phase consisted of water-acetonitrile （50 ∶ 50, V/V） using diazepam as internal standard at flow rate of 1.0 mL·min-1. The detection wavelength was set at 245 nm and the column temperature was 30 ℃. The injection volume was 20 μL. RESULTS： The liner range of testosterone was 10.0～200.0 μmol·L-1 （r=0.999 5）; The average method recovery rates of testosterone with low, medium and high concentration were 86.15%, 103.10% and 97.48%. The average relative recovery rates of three concentrations were 111.15%, 107.24% and 100.78%.The intra-day RSD were 3.92%, 2.46% and 2.82% and the inter-day RSD were 2.98%, 1.34% and 2.41%, respectively. CONCLUSION： The method is simple, convenient, sensitive and reproducible for the determination of CYP3A4 activity and other fields.