Anwendungen der Isotopenverhältnismassenspektrometrie sowie hochauflösender Massenspektrometrie hinsichtlich anaboler Steroide in der Dopinganalytik: Metabolitenidentifizierung und Herkunftsbestimmung

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Within this thesis, isotope ratio mass spectrometry (IRMS) was applied for the analysis of anabolic steroids for metabolite identification and tracing the origin for doping control purposes.
In the first project, isotope ratio mass spectrometry measuring hydrogen was used to investigate the metabolism of the synthetic anabolic steroid trenbolone. The number of previously reported metabolites is limited and most routine doping testing methods rely on targeting epitrenbolone, trenbolone glucuronide and epitrenbolone glucuronide. For that reason, the objective of this project was to probe for new metabolites of trenbolone, which potentially support the extension of the detection window. An elimination study with 5-fold deuterated trenbolone administered to one healthy male volunteer was conducted and collected urine sample were prepared by combining published protocols. Samples of interest were identified by their diagnostic deuterium content with gas chromatography/isotope ratio mass spectrometry (GC-IRMS) and metabolite characterization was performed with high performance liquid chromatography/high resolution mass spectrometry (HPLC-HRMS). In total, twenty deuterium-labelled compounds were identified including glucuronic acid, sulfo- and potential cysteine conjugates. For the four most promising metabolites, structures were tentatively assigned as a two-fold dehydrogenation product of trenbolone-diol and trenbolone-diketone excreted as glucuronic acid and sulfo-conjugates. The metabolites were detectable for 5 respectively 6 days. Structural characterization was conducted of the hydrolysed and acetylated derivatives and of the intact phase-II conjugates by mass spectrometry using parallel reaction monitoring and pseudo-MS3 experiments. Product ion mass spectra were compared to reference material which was commercially available or produced by in-house synthesis.
Naturally occurring, but synthetically derived steroids were in the focus of both other projects. Endogenous and exogenous steroids can be unambiguously distinguished by the carbon isotope ratio determined by gas chromatography isotope ratio mass spectrometry. State-of-the-art methods are very laborious and time-consuming including up to two HPLC purification steps. In order to reduce manual workload and overall batch analysis time, two alternative techniques were used to omit the HPLC purification of the established methods.
For that purpose, multi-immunoaffinity chromatography (IAC) was applied within the second project. This technique exploits specific antibody-immunogen interactions and within this method, antibodies against testosterone (T), pregnanediol and 11-ketoetiocholanolone were combined into a multi-immunoaffinity gel. Due to cross reactivities also etiocholanolone, androsterone, 5β-androstanediol and 5α-androstanediol were co-extracted and included in the testing protocol. The method was validated by determining precision, recovery and carry over. Additionally, linear mixing models were performed.
Multidimensional gas chromatography (MDGC) was used as alternative technique for the determination of the carbon isotope ratios in the third project. MDGC employs two gas chromatographs with columns of different chromatographic selectivity connected by a heated transfer line and a pressure controlled transfer device which allows cutting a selected segment on the second dimension in order to improve separation capability. Within this project, 5α-androst-16-en-3α-ol was analysed additionally. The method was also validated according to the regulations of the World Anti-Doping Agency. Moreover, a reference population comprising of 74 athletes’ samples was investigated. This was employed to deduce reference limits and to compare this approach to the established routine method. On account of matrix offset and reduced precision, Δδ13C values comprising T result in comparable high reference limits and consequently, T will be not as sensitive as other target compounds. As proof-of-concept, an elimination study with androst-4-ene-3,17-dione was examined.
Both methods achieve a substantial reduction of the overall batch analysis time compared to the established method using HPLC purification. For the IAC approach, also the required urine volume was reduced, while the MDGC assay benefits from the low manual workload. Nevertheless, the HPLC-based method was superior in purification quality and robustness. The IAC approach can be suggested as complementary method, but improved IAC material or alternative resins may enhance its utility. The MDGC method is appropriate for an application to routine doping control analysis as IRMS screening for analysing suspicious samples from the Initial Testing Procedure (Steroid Profile). For confirmation purposes, the HPLC-based method is suggested.


Original languageGerman
Place of PublicationKöln
PublisherDeutsche Sporthochschule Köln
Number of pages22
Publication statusPublished - 2020

ID: 5469504

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