A new mixed micellar electrokinetic chromatography method for analysis of natural and synthetic anabolic steroids

A simple, rapid and low-costing new mixed surfactant MEKC method has been developed for the analysis of five neutral anabolic steroids in this paper. It was found that the bile salt coupling with Triton X-100 was a suitable bi-micellar surfactant for the separation of these anabolic steroids with similar structure. The separation conditions were optimized in detail. The five natural and synthetic anabolic steroids, such as androstenedione (AD), 19-norandrostenedione (NAD), 1,4-androstadiene-3,17-dione (ADD), methandrostenolone (MA) and methyltestosterone (MT) were separated and detected in an alkaline buffer system (pH 9.0) containing 15 mM Britton-Robinson (BR) buffer, 50 mM sodium cholate (SC) and 0.1% (v/v) Triton X-100 with detection wavelength at 241 nm and 18 kV of separation voltage. Under the optimal conditions, five coexistence neutral steroids were completely separated within 12 min with the detection limits ranged from 0.20 to 0.51 μg/mL. This method was successfully used for detection and confirmation of the anabolic steroid methandrostenolone in methandrostenolone tablets and in the real human urine, GC-MS method was applied to confirm the free methandrostenolone existence in the urine sample in order to validate the reliability of MEKC method.