Separation of testosterone and dihydrotestosterone in semen by high performance liquid chromatography

Abstract We have established a method for the separation of testosterone and dihydrotestosterone in seminal plasma by high performance liquid chromatography. The separation column is a reversed phase column (ODS). The mobile phase is methanol-water (8:2) and the flowrate is 1.5 m1/min. It takes 12–14 min to separate a sample. The recoveries are 96.4% and 96.5% respectively by radioactivety labelled steroids. We have separated 600 seminal plasma samples with this method. The separated testosterone and dihydrotestosteroneare determined by radioimmunoassay. The result have shown that the method is satisfactory.