Dissertation: Optimizing the Process of Introducing New Metabolites of Anabolic-Androgenic Steroids – Method Evaluation, Synthesis, Verification

The presented thesis concentrates on the metabolism of three AAS in the human body and the synthesis of potential new long-term metabolites. It demonstrates an effective way of introducing new markers to anti-doping analysis and finally confirming the proposed structures. First, the substrate specificity of a human CYP (21A2) was elucidated to use its stereoselective hydroxylation capacity for the targeted synthesis of new metabolites of several AAS. Second, new metabolites of MD and MT were synthesized chemically, characterized, and detected in urine samples. Third, a controlled administration study with DHCMT was performed to reveal the metabolism of this doping substance and provide kinetic data of the metabolic products. Different synthesis methods were used depending on the structure: either a full chemical or a combined chemical and biotechnological approach. Several analysis techniques were used to provide sufficient information during the particular project steps. To verify intermediate products in the synthesis, gas chromatography coupled by electron ionization to single quadrupole-mass spectrometry (GC-EI-MS) was used. NMR experiments together with GC-EI-MS and gas chromatography coupled by electron ionization to quadrupole time-of-flight-mass spectrometry (GC-EI-QTOF-MS) were used for the structure elucidation of the synthesized reference material. Post administration samples were analyzed by GC-EI- QQQ-MS and LC-ESI-QTOF-MS. The results of this thesis show the ideal way of providing reference material to the anti-doping community or every other research field that is dealing with the metabolism of endogenous and exogenous substances in case that the concentrations are too low for other means of comprehensive identification. In a first step, the synthesis of reference material must be planned, and the used compounds, e.g., enzymes or reagents, must be evaluated for their potential use. Subsequently, the synthesis has to be performed, and the product needs to be fully characterized so that there is no doubt of its structure. Finally, an administration of the parent compound and subsequent analysis of the excreted metabolites confirm the previous work. In summary, this work shows how new ideas about metabolism should be dealt with. It helps to dispel the doubts that have existed for years about postulated results. At the same time, it shows that steroid metabolism still has various blind spots, whose elucidation must be the goal of further research. Although anti-doping was the all-dominant topic, the gained results are also relevant for fields in physiology and pharmacology.