Synthesis and in vitro metabolism studies of selected steroids for anti-doping analysis

Abstract

Steroids are naturally occurring organic compounds that can be found in all eukaryotes. Anabolic Androgenic Steroids (AAS) are compounds derived from the parent steroid testosterone that were first introduced to treat medical conditions. Later, these synthetic steroids became famous as performance enhancing drugs and were used in human and animal sports to gain an advantage. Though rules are established against the use of AAS, World Anti-Doping Agency (WADA) reports have shown that athletes still use them to gain an advantage. Therefore, advanced analytical techniques and long term markers must be developed to establish a future for sport free of AAS. The second chapter presents a method to conduct in vitro sulfation studies using inexpensive starting materials. Sulfation studies are rarely demonstrated in comparison to phase I metabolism studies and glucuronylation studies as the universal sulfate donor 3-phosphoadenosine-5-phosphosulfate (PAPS) is expensive. However, sulfate metabolites have been identified as long term markers especially for 17b-hydroxy-17a-methyl steroids. In the method developed in this thesis, sodium sulfate and adenosine 5-triphosphate (ATP) were employed to generate PAPS in situ. Therefore, this method can be used to mimic in vivo sulfation and potentially to identify new long term markers. Chapter three discusses an in vitro metabolism study of a designer steroid; d6methyltestosterone that was found in a dietary supplement; junglewarfare. Depending upon the possible phase I metabolic pathways, the synthesis of reference materials was performed alongside an in vitro metabolism study. The in vitro metabolites generated were compared with synthesised reference materials. This allowed for a number of phase I and phase II metabolites to be identified and out of those parent glucuronide metabolite and a triply reduced glucuronide metabolite were identified and confirmed using the reference materials. Chapter four describes work carried out to identify an unidentified urinary metabolite observed in an in vivo equine study of hemapolin (2a,3a-epithio-17a-methyl-5a-androstan-17b-ol). A mechanistic proposal was developed for the iv formation of the suspected urinary metabolite and based on this proposal, four reference materials were synthesised. The retention time of the urinary metabolite matched with one of the synthesised steroid sulfonates according to AORC retention time criteria. Further studies are required to complete the conformation of the identified urinary metabolite based on AORC MS criteria.