Fragkaki A G, Angelis Y S, Kiousi P, Georgakopoulos C G, Lyris E
Doping Control Laboratory of Athens, Olympic Athletic Center of Athens 'Spyros Louis', 37 Kifisias Avenue, 15123, Marousi, Greece.
Anti-Doping Laboratory of Qatar, PO Box 27775, Doha, Qatar.
J Mass Spectrom. 2015 May;50(5):740-8. doi: 10.1002/jms.3583.
Methenolone (17β-hydroxy-1-methyl-5α-androst-1-en-3-one) misuse in doping control is commonly detected by monitoring the parent molecule and its metabolite (1-methylene-5α-androstan-3α-ol-17-one) excreted conjugated with glucuronic acid using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for the parent molecule, after hydrolysis with β-glucuronidase. The aim of the present study was the evaluation of the sulfate fraction of methenolone metabolism by LC-high resolution (HR)MS and the estimation of the long-term detectability of its sulfate metabolites analyzed by liquid chromatography tandem mass spectrometry (LC-HRMSMS) compared with the current practice for the detection of methenolone misuse used by the anti-doping laboratories. Methenolone was administered to two healthy male volunteers, and urine samples were collected up to 12 and 26 days, respectively. Ethyl acetate extraction at weak alkaline pH was performed and then the sulfate conjugates were analyzed by LC-HRMS using electrospray ionization in negative mode searching for M-H ions corresponding to potential sulfate structures (comprising structure alterations such as hydroxylations, oxidations, reductions and combinations of them). Eight sulfate metabolites were finally detected, but four of them were considered important as the most abundant and long term detectable. LC clean up followed by solvolysis and GC/MS analysis of trimethylsilylated (TMS) derivatives reveal that the sulfate analogs of methenolone as well as of 1-methylene-5α-androstan-3α-ol-17-one, 3z-hydroxy-1β-methyl-5α-androstan-17-one and 16β-hydroxy-1-methyl-5α-androst-1-ene-3,17-dione were the major metabolites in the sulfate fraction. The results of the present study also document for the first time the methenolone sulfate as well as the 3z-hydroxy-1β-methyl-5α-androstan-17-one sulfate as metabolites of methenolone in human urine. The time window for the detectability of methenolone sulfate metabolites by LC-HRMS is comparable with that of their hydrolyzed glucuronide analogs analyzed by GC-MS. The results of the study demonstrate the importance of sulfation as a phase II metabolic pathway for methenolone metabolism, proposing four metabolites as significant components of the sulfate fraction.
美替诺龙（17β-羟基-1-甲基-5α-雄甾-1-烯-3-酮）在兴奋剂检测中的滥用通常通过监测母体分子及其与葡萄糖醛酸结合排泄的代谢物（1-亚甲基-5α-雄甾烷-3α-醇-17-酮）来检测。对于母体分子，在用β-葡萄糖醛酸酶水解后，使用气相色谱-质谱联用仪（GC-MS）和液相色谱-质谱联用仪（LC-MS）进行检测。本研究的目的是通过液相色谱-高分辨率（HR）质谱法评估美替诺龙代谢的硫酸盐部分，并通过液相色谱串联质谱法（LC-HRMSMS）估计其硫酸盐代谢物的长期可检测性，同时与反兴奋剂实验室目前检测美替诺龙滥用的方法进行比较。给两名健康男性志愿者服用美替诺龙，分别在12天和26天内收集尿液样本。在弱碱性pH条件下进行乙酸乙酯萃取，然后通过液相色谱-高分辨率质谱法（LC-HRMS）以负模式电喷雾电离分析硫酸盐结合物，寻找与潜在硫酸盐结构相对应的M-H离子（包括羟基化、氧化、还原及其组合等结构改变）。最终检测到8种硫酸盐代谢物，但其中4种被认为是最重要的，因为它们含量最丰富且可长期检测到。通过液相色谱净化，然后进行溶剂解以及对三甲基硅烷基化（TMS）衍生物进行气相色谱/质谱分析，结果表明美替诺龙以及1-亚甲基-5α-雄甾烷-3α-醇-17-酮、3z-羟基-1β-甲基-5α-雄甾烷-17-酮和16β-羟基-1-甲基-5α-雄甾-1-烯-3,17-二酮的硫酸盐类似物是硫酸盐部分的主要代谢物。本研究结果还首次记录了美替诺龙硫酸盐以及3z-羟基-1β-甲基-5α-雄甾烷-17-酮硫酸盐是人尿中美替诺龙的代谢物。通过液相色谱-高分辨率质谱法检测美替诺龙硫酸盐代谢物的时间窗口与其通过气相色谱-质谱法分析的水解葡萄糖醛酸类似物的时间窗口相当。研究结果证明了硫酸化作为美替诺龙代谢的II相代谢途径的重要性，提出了4种代谢物作为硫酸盐部分的重要组成成分。
