Ethyl acetate extraction procedure and isocratic high-performance liquid chromatographic assay for testosterone metabolites in cell microsomes

摘要

An isocratic reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for separation of testosterone and its main metabolites over the nominal range 20 to 40 mu g/ml and 280 to 4600 ng/ml, respectively. Mobile phase composition (phosphate buffer-methanol-acetonitrile, 50:38.5:11.5) was optimised by studying the influence of numerous chromatographic parameters. The most critical one was the ratio CH3CN/CH3OH. Good recoveries (around 90% for all compounds) and an improved specificity were assessed by a double ethyl acetate extraction of biological samples. According to the performance criteria tested, the method could be applied to enzymatic inhibition and induction in vitro studies. (C) 1998 Elsevier Science B.V. [References: 17]