Putz Marlen, Piper Thomas, Thevis Mario
Center for Preventive Doping Research, Institute of Biochemistry, German Sport University Cologne, Cologne, Germany.
Front Chem. 2020 May 20;8:435. doi: 10.3389/fchem.2020.00435. eCollection 2020.
Trenbolone is a synthetic anabolic-androgenic steroid, which has been misused for performance enhancement in sports. The detection of trenbolone doping in routine sports drug testing programs is complex as methods utilizing gas chromatography/mass spectrometry are complicated by unspecific derivatization products and artifacts, and liquid chromatography/mass spectrometry-based assays have shown to allow for comparably high limits-of-detection only. The number of previously reported metabolites in human urine is limited, and most analytical methods rely on targeting epitrenbolone, trenbolone glucuronide, and epitrenbolone glucuronide. In order to probe for the presence of additional trenbolone metabolites and to re-investigate the metabolism, an elimination study was conducted. One single dose of 10 mg of 5-fold deuterated trenbolone was administered to a healthy male volunteer and urine samples were collected for 30 days. For sample processing, published protocols were combined considering unconjugated, glucuronic acid-, sulfo- and alkaline-labile conjugated steroid metabolites. The sample preparation strategy consisted of solid-phase extractions, liquid-liquid extractions, metabolite de-conjugation, HPLC fractionation, and derivatization. Analytical methods included gas chromatography/thermal conversion/hydrogen isotope ratio mass spectrometry combined with single quadrupole mass spectrometry as well as liquid chromatography/high accuracy/high resolution mass spectrometry of the hydrolyzed and non-hydrolyzed samples. Twenty deuterium-labeled metabolites were identified including glucuronic acid-, sulfo- and potential cysteine-conjugates, and characterized by parallel reaction monitoring experiments yielding corresponding product ion mass spectra. Main metabolites were attributed to trenbolone-diol and potential trenbolone-diketone derivatives excreted as glucuronic acid and sulfo-conjugated analytes with detection windows of 5, respectively 6 days. Further characterization was conducted with pseudo MS experiments of the intact conjugates and by comparison of resulting product ion mass spectra with reference material.
群勃龙是一种合成的促合成代谢雄激素类固醇，已被滥用于提高运动成绩。在常规体育药物检测项目中检测群勃龙兴奋剂很复杂，因为利用气相色谱/质谱法的方法会受到非特异性衍生化产物和假象的干扰，而基于液相色谱/质谱法的检测方法仅显示出相对较高的检测限。此前报道的人尿中代谢物数量有限，大多数分析方法依赖于检测表群勃龙、群勃龙葡萄糖醛酸苷和表群勃龙葡萄糖醛酸苷。为了探寻是否存在其他群勃龙代谢物并重新研究其代谢情况，进行了一项消除研究。给一名健康男性志愿者单次服用10毫克5倍氘代群勃龙，并收集30天的尿液样本。对于样品处理，结合已发表的方案，考虑未结合的、葡萄糖醛酸、磺酸和碱不稳定结合类固醇代谢物。样品制备策略包括固相萃取、液液萃取、代谢物去结合、高效液相色谱分离和衍生化。分析方法包括气相色谱/热转化/氢同位素比质谱联用单四极杆质谱以及水解和未水解样品的液相色谱/高精度/高分辨率质谱。鉴定出20种氘代代谢物，包括葡萄糖醛酸、磺酸和潜在的半胱氨酸结合物，并通过平行反应监测实验进行表征，得到相应的产物离子质谱。主要代谢物归因于群勃龙二醇和潜在的群勃龙二酮衍生物，它们以葡萄糖醛酸和磺酸结合分析物的形式排出，检测窗口分别为5天和6天。通过完整结合物的伪质谱实验以及将所得产物离子质谱与参考物质进行比较，进行了进一步的表征。
