Abstract:

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 N2 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 32 μg/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%. The 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.

Key words: 6β-hydroxylated testosterone, cytochrome P450 3A4 (CYP3A4), liver microsomes , testosterone, high performance liquid chromatography (HPLC)