Piper Thomas, Schänzer Wilhelm, Thevis Mario
German Sport University Cologne, Center for Preventive Doping Research-Institute of Biochemistry, Am Sportpark Müngersdorf 6, 50933 Köln, Germany.
German Sport University Cologne, Center for Preventive Doping Research-Institute of Biochemistry, Am Sportpark Müngersdorf 6, 50933 Köln, Germany.
J Steroid Biochem Mol Biol. 2016 Sep;162:80-91. doi: 10.1016/j.jsbmb.2015.12.013. Epub 2015 Dec 14.
The synthetic anabolic androgenic steroid 19-nortestosterone is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA) due to its performance-enhancing effects. Today, doping controls focus predominantly on one main urinary metabolite, 19-norandrosterone glucuronide, which offers the required detection windows for an appropriate retrospectivity of sports drug testing programs. As 19-norandrosterone can also be found in urine at low concentrations originating from in situ demethylation of other abundant steroids or from endogenous production, the exogenous source of 19-norandrosterone needs to be verified, which is commonly accomplished by carbon isotope ratio analyses. The aim of this study was to re-investigate the metabolism of 19-nortestosterone in order to probe for additional diagnostic long-term metabolites, which might support the unambiguous attribution of an endo- or exogenous source of detected 19-nortestosterone metabolites. Employing a recently introduced strategy for metabolite identification, threefold deuterated 19-nortestosterone (16,16,17-(2)H3-NT) was administered to one healthy male volunteer and urine samples were collected for 20 days. Samples were prepared with established methods separating unconjugated, glucuronidated and sulfated steroids, and analytes were further purified by means of high-performance liquid chromatography before trimethylsilylation. Deuterated metabolites were identified using gas chromatograph/thermal conversion/isotope ratio mass spectrometer comprising an additional single quadrupole mass spectrometer. Additional structural information was obtained by gas chromatography/time-of-flight mass spectrometry and liquid chromatography/high resolution mass spectrometry. In general, sulfo-conjugated metabolites were excreted for a longer time period than the corresponding glucuronides. Several unexpected losses of the arguably stable isotope labels were observed and characterized, attributed to metabolic reactions and sample preparation procedures. The detection window of one of the newly detected metabolites was higher than currently used metabolites. The suitability of this metabolite to differentiate between endo- or exogenous sources could however not be verified conclusively.
根据世界反兴奋剂机构（WADA）的规定，合成代谢雄激素类固醇19-去甲睾酮因其增强运动表现的作用而在体育赛事中被禁用。如今，兴奋剂检测主要集中在一种主要的尿液代谢物——19-去甲雄酮葡糖醛酸苷上，它为体育药物检测项目提供了适当追溯所需的检测窗口。由于在尿液中也能检测到低浓度的19-去甲睾酮，其来源可能是其他丰富类固醇的原位去甲基化或内源性产生，因此需要验证19-去甲睾酮的外源性来源，这通常通过碳同位素比率分析来完成。本研究的目的是重新研究19-去甲睾酮的代谢，以探寻其他诊断性的长期代谢物，这可能有助于明确检测到的19-去甲睾酮代谢物的内源性或外源性来源。采用最近引入的代谢物鉴定策略，将三重氘代的19-去甲睾酮（16,16,17-(2)H3-NT）给予一名健康男性志愿者，并收集其20天的尿液样本。样本采用既定方法制备，分离未结合、葡糖醛酸化和硫酸化的类固醇，在三甲基硅烷化之前，通过高效液相色谱法对分析物进行进一步纯化。使用配备附加单四极杆质谱仪的气相色谱/热转换/同位素比率质谱仪鉴定氘代代谢物。通过气相色谱/飞行时间质谱仪和液相色谱/高分辨率质谱仪获得了更多的结构信息。一般来说，硫酸化结合代谢物的排泄时间比相应的葡糖醛酸苷更长。观察并表征了几种可论证的稳定同位素标记的意外损失，这归因于代谢反应和样品制备过程。新检测到的一种代谢物的检测窗口比目前使用的代谢物更高。然而，该代谢物区分内源性或外源性来源的适用性尚未得到最终验证。
