High-performance liquid chromatographic optimization study for the separation of natural and synthetic anabolic steroids. Application to urine and pharmaceutical samples

An HPLC separation of a complex mixture containing 14 androgenic anabolic steroids (natural and synthetic) for screening purposes has been carried out. The applied optimization method involved the use of binary, ternary and quaternary mobile phases containing acetonitrile, methanol or tetrahydrofuran as organic modifiers. The effect of different reversed-phase packings and temperature on the separation using acetonitrile as organic modifier was studied. The optimum separation was achieved by using a water–acetonitrile (55:45, v:v) mobile phase and a Hypersil ODS (250 mm×4.6 mm) 5 μm column (30°C) in about 38 min, allowing the separation of 14 out of 14 compounds tested (when danazol is exluded, 13 out of 14 were separated in 23 min). Calibration graphs were obtained using bolasterone, methyltestosterone and canrenone as internal standards. Detection limits were in the range 0.012–0.11 μg ml−1. The optimized separation was applied for monitoring the norethindrone acetate hydrolysis from tablets and to the analysis, after liquid–liquid extraction, of urine samples spiked with steroids.