Determination of testosterone and its metabolites using liquid chromatography with elevated column temperature and flow-rate gradient

A rapid high performance liquid chromatography (HPLC) method for determination of testosterone and its metabolites in biological samples is described. The method combines a flow-rate gradient with elevated column temperature to obtain a complete separation of testosterone and its metabolites. The flow-rate gradient method dramatically reduces the analysis time (17 min) compared with isocratic elution (35 min). This method is simple and reproducible with relative standard deviations (R.S.D.) of <0.1% for retention time and 1-4% for peak area. It provides limits of detection (LOD) in the range of 0.05-0.1 μM for testosterone and its five major metabolites. We applied the method to analyze rat liver microsome samples incubated with testosterone, demonstrating a testosterone metabolic profile due to enzymatic activities in the microsome. Analysis of testosterone and its metabolites is important because metabolism of testosterone is a surrogate measure for cytochrome P-450 (CYP) enzymatic activity.