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Dr. Anna Stoll

Dissertation: Investigations on Phase I Metabolism of Anabolic Androgenic Steroids and Its Influenceability as Tool to Refine Steroid Detection and Evaluation

This thesis addresses the steroid metabolism and its role in the detection of steroids. The results were achieved by performing metabolic studies in combination with different analytical techniques. Three main outcomes were achieved. First, the generation of reference material for a long-term metabolite of the synthetic steroid Oral-Turinabol (OT) was presented. The obtained results enhance unambiguous detection and quantification of this metabolite and hence help to uncover the prohibited OT administration in sports doping. Second, structural requirements and tolerated functionalities for substrates of the enzyme Cytochrome P450 subtype 21A2 (CYP21A2) were evaluated. These insights help to extrapolate known metabolic reactions to new and potentially unknown compounds. Third, investigations on steroid profile changes caused by the intake of non-steroidal anti-inflammatory drugs were described. These findings provide knowledge on substances influencing steroid metabolism, which is of high relevance for accurate steroid determination and hence the correct interpretation of analytical results. All results were obtained using mainly three models to study steroid metabolism and its influenceability. First, incubations with Schizosaccharomyces pombe strains recombinantly expressing human CYPs were used to generate reference material in small amounts and to evaluate metabolic reactions. Second, incubations with isolated recombinantly expressed human enzymes were used to examine metabolic reactions and their influenceability by other drugs. Third, in vivo drug administration was used to study steroid metabolism in humans and to investigate whether the intake of non-steroidal anti-inflammatory drugs leads to changes in steroid concentrations and concentration ratios. To subsequently evaluate the samples generated by the methods described above different analytical techniques were utilized. High-resolution mass spectrometry (HRMS) was used for quantification and tentative structure identification. Liquid chromatography and gas chromatography were both combined with HRMS. Additionally, gas chromatography coupled to single or triple quadrupole mass spectrometry was employed to identify and accurately quantify compounds where reference material was already available. Finally, fluorometric measurements carried out in real time were used to determine kinetic characteristics of enzymatic reactions. The results presented in this thesis build a solid base for the refinement of steroid detection. It was shown that combinations of in vitro and in vivo metabolic studies and investigations on metabolic reactions together with adequate analytical techniques are of high relevance for the improvement of steroid detection methods. Furthermore, the results are of relevance to enhance methods for generation of reference compounds, to identify potentially new drugs and to re-evaluate the risk potential of drugs. Further areas that can benefit from the results of this work include clinical and toxicological analysis, analysis of environmental and biological samples and endocrinology. Finally, the results are of high importance for anti-doping analysis. This field shall be mentioned separately as the results presented particularly contribute to tackle challenges relevant in this research area.