New methodology in analysis of steroids using solid phase extraction and micellar electrokinetic chromatography (poster)

Steroid specific methodologies for monitoring body fluids are essential goals in clinical and doping analyses. The conventional GC-MS methods are laborious and time-consuming, since deconjugation, concentration and derivatisation of the steroid analytes are required. More straightforward analysis methods are thus desired. Separation of androgenic steroids with micellar electrokinetic chromatography has been studied very little. The separation of endogenous alpha-epimers testosterone and epitestosterone has not been reported with any electroseparation techniques before. Here, a novel partial filling micellar electrokinetic chromatography (PF-MEKC) method for determination of three endogenous steroid hormones (androstenedione, testosterone, epitestosterone) and two synthetic anabolic steroids (fluoxymesterone, methyltestosterone) is introduced. Discontinuous solvent pair made of single-isomer sulphated gamma-cyclodextrins and surfactants sodium dodecyl sulphate and sodium taurocholate is exploited by using two different pseudostationary phases in the capillary. The fast steroid separation is completed in 10 minutes. The PF-MEKC method was used for pretreated human urine samples. The one of the new sample preparation methods allows determination of testosterone in male urine after enzymatic hydrolysis with beta-glucuronidase from E. Coli. It is based on a highly testosterone specific microscale immunoextraction prior to PF-MEKC. The other method allows simultaneous determination of testosterone and epitestosterone in male urine after the enzymatic hydrolysis followed by their quantification in urine samples.