Development and applications of analytical procedures for the analysis of sulphate metabolites of anabolic androgenic steroids

The World Anti-Doping Agency (WADA) includes anabolic androgenic steroids (AAS) among the substances and methods prohibited in sports. The process of steroids detection undertaken by the WADA accredited laboratories is based on GC-MS(/MS) or LC-MS(/MS) analysis of urinary samples collected from male and female athletes. The methods routinely used in steroid screening mainly focus on substances that are excreted unconjugated or as glucuro-conjugates and for this reason common procedures includes enzymatic hydrolysis with β-glucuronidase from Escherichia Coli. Despite this, for doping control purposes, the development of a confirmation procedure to identify the exogenous origin of pseudo-endogenous (e.g. dehydroepiandrosterone or androstenedione) steroids and/or their metabolites excreted primarily as sulphates could be of interest. In the first part of the work we have evaluated the performance of different preparation of sulphatases from Helix Pomatia and Pseudomonas Aeruginosa compared to chemical hydrolysis of sulphate steroids and we have studied different approaches for the selective isolation of steroids conjugates from the urine matrix. In the second part of our work we have evaluated the urinary excretion of epiandrosterone sulphate after oral administration of dehydroepiandrosterone and androstenedione respectively. Our results show that chemical hydrolysis is preferable to enzymatic methods as it results in quantitative cleavage of the sulphate moiety (even if in some cases the yields of chemical hydrolysis and enzymatic hydrolysis are similar), ion paired extraction is the more reliable method for direct isolation of sulphate steroids from urinary matrices and epiandrosterone sulphate prolongs the detectability of dehydroepiandrosterone and androstenedione when compared to routinely used steroidal target compounds.