Extraction of testosterone and epitestosterone in human urine using 2-propanol–salt–H2O system

Based on the 2-propanol–salt–H2O system, a new and simple pretreatment method was developed for the analysis of testosterone and epitestosterone in human urine coupled with high-performance liquid chromatography (HPLC). The influence factors on partition behaviors of the steroids were studied including the type and amount of salts, the volume of 2-propanol, the concentration of the analytes, and temperature. When K3PO4, K2HPO4, K2CO3 and KOH were chosen as phase separation salts, the enrichment factors were 12, 10, 12 and 13, respectively, and the extraction efficiencies for testosterone and epitestosterone were all 80–90%. In the optimal system of 2-propanol–K3PO4–H2O, the recoveries of the spiked standards for the analytes were all 92–102% with relative standard deviation of 2.3–7.2%. Combined with a HPLC method, this extraction technique has been successfully applied to the analysis of testosterone and epitestosterone in human urine with the detection limits of 1 ng/ml and the linear ranges of 10–500 ng/ml for both compounds. Compared with conventional liquid–liquid extraction or solid-phase extraction, this novel method is much simpler and more environmentally friendly due to the direct injection of the upper phase into HPLC system, no use of harmful organic solvent, and low consumption of extractant.