Partial filling micellar electrokinetic chromatography analysis of androgens and testosterone derivatives using two sequential pseudostationary phases

Separation of anabolic and androgenic steroids by micellar electrokinetic chromatography (MEKC) has been little studied. Simultaneous separation of the endogenous α-epimers testosterone and epitestosterone has not been achieved with any electroseparation technique. Here, a partial filling micellar electrokinetic chromatographic (PF-MEKC) method is described for the analysis of three endogenous steroid hormones (androstenedione, testosterone, epitestosterone) and two synthetic anabolic steroids (fluoxymesterone, methyltestosterone). The resolution efficiency of single-isomer sulphated γ-cyclodextrins and the surfactants sodium dodecyl sulphate and sodium taurocholate was exploited. The method is based on the sequential introduction of short plugs of two different pseudostationary phases into the capillary. The separation was completed in less than 10 min. The method can be used in quantitative analysis. Linear correlation was obtained between concentration and peak area of 0.996 or better. The repeatability (RSD) of the compound peak areas ranged from 3.6% (methyltestosterone) to 6.2% (androstenedione). Limits of detection were between 73 μg/L (testosterone) and 160 μg/L (fluoxymesterone). As a demonstration of the method, androstenedione, testosterone and epitestosterone were determined in a spiked urine sample.