Doping Control Laboratory of Athens, Institute of Biosciences & Applications, National Center for Scientific Research "Demokritos", Neratziotissis & Amaryssias Artemidos Str, Athens, 15123, Greece.
Faculty of Biology, Schoole of Science, National and Kapodistrian University of Athens, Panepistimioupolis, Zografou, Athens, 15771, Greece.
Drug Test Anal. 2020 Nov;12(11-12):1544-1553. doi: 10.1002/dta.2887. Epub 2020 Jul 15.
Methylnortestosterone is a progestin and synthetic androgenic anabolic steroid, prohibited by WADA. Methylnortestosterone misuse is commonly detected by monitoring the parent compound and its main metabolites, 17α-methyl-5α-estrane-3α, 17β-diol (M1) and 17α-methyl-5β-estrane-3α, 17β-diol (M2), in the glucuronide fraction. In the current study, a direct detection of methylnortestosterone sulfo-conjugated metabolites after ethyl acetate extraction and analysis by LC/Q/TOF-MS in negative ionization mode was performed, detecting two main sulfate metabolites (S1, S2). For the characterization of metabolites, samples from the excretion study, were additionally analyzed by GC-MS, after solvolysis and per TMS derivatization. RT and MS data collected, were compared with RT and MS data from metabolites of 17z-methyl-5α/β-estrane-3α/β, 17z-diols structures with prefixed stereochemistry at 3 and 5 positions, synthesized through Grignard reaction from 19-noretiocholanolone, 19-norandrosterone and 19-norepiandrosterone. Confirmed sulfate metabolites were S1, 17α-methyl-5α-estrane-3α, 17β-diol 3α sulfate (detected up to 72 h) and S2, 17α-methyl-5β-estrane-3α, 17β-diol 3α sulfate (detected up to 192 h). Furthermore, applying targeted analysis based on RT and MS data of the synthesized metabolites two additional metabolites M3, 17β-methyl-5β-estrane-3α, 17α-diol and M4, 17β-methyl-5α-estrane-3α, 17α-diol were detected in the glucuronide fraction and one more metabolite (S3) 17β-methyl-5β-estrane-3α, 17α-diol was detected in the sulfate fraction in lower abundance until the end of the excretion study (192 h). Interestingly, S2 could also be detected after the direct analysis of non-hydrolyzed steroid by GC-MS/MS as artifact, following normal ProcIV anabolic steroid procedure and using diethylether as extraction solvent.
美雄诺龙是一种孕激素和合成雄激素同化类固醇，被世界反兴奋剂机构禁止使用。美雄诺龙的滥用通常通过监测母体化合物及其主要代谢物 17α-甲基-5α-雄烷-3α，17β-二醇（M1）和 17α-甲基-5β-雄烷-3α，17β-二醇（M2）在葡萄糖醛酸苷分数中进行检测。在当前的研究中，通过在负电离模式下用 LC/Q/TOF-MS 进行乙酸乙酯提取和分析，直接检测了美雄诺龙的磺酰化代谢物，检测到两种主要的硫酸盐代谢物（S1、S2）。为了对代谢物进行特征分析，对排泄研究中的样品进行了 GC-MS 分析，经过溶剂解和全 TMS 衍生化处理。收集的 RT 和 MS 数据与通过 Grignard 反应从 19-去甲雄烷酮、19-雄烯酮和 19-去甲表雄酮合成的 17z-甲基-5α/β-雄烷-3α/β，17z-二醇结构的代谢物的 RT 和 MS 数据进行了比较，这些代谢物的 3 位和 5 位立体化学预先设定。确认的硫酸盐代谢物为 S1，17α-甲基-5α-雄烷-3α，17β-二醇 3α 硫酸盐（检测到 72 小时）和 S2，17α-甲基-5β-雄烷-3α，17β-二醇 3α 硫酸盐（检测到 192 小时）。此外，基于合成代谢物的 RT 和 MS 数据进行靶向分析，在葡萄糖醛酸苷分数中检测到另外两种代谢物 M3，17β-甲基-5β-雄烷-3α，17α-二醇和 M4，17β-甲基-5α-雄烷-3α，17α-二醇，以及在硫酸盐分数中检测到一种含量较低的代谢物 S3，17β-甲基-5β-雄烷-3α，17α-二醇，直至排泄研究结束（192 小时）。有趣的是，S2 也可以在 GC-MS/MS 对未水解的甾体进行直接分析后作为人工产物检测到，这是在正常的 ProcIV 同化类固醇程序下进行的，并且使用二乙醚作为提取溶剂。
