Human Metabolism of the Anabolic Steroid Methasterone: Detection and Kinetic Excretion of New Phase I Urinary Metabolites and Investigation of Phase II Metabolism by GC-MS and UPLC-MS/MS

Methasterone is a designer anabolic steroid that is prohibited for athletes and is monitored by anti-doping laboratories. In this work, our objective is to discover new human phase I metabolites, define their excretion kinetics for 30 days and analyze their phase II metabolism (sulfate, cysteine and N-acetylcysteine conjugates). Urine samples from four volunteers were analyzed by chromatographic techniques. Through gas chromatography coupled to mass spectrometry analysis it was possible to detect methasterone and its nine phase I metabolites in the urine samples after glucuronide enzymatic hydrolysis, from which one were previously unreported. These nine compounds were not excreted in free form. The new proposed metabolite is 17β-hydroxy-2α,17α-dimethyl-5β-androstan-3-one, obtained from the epimerization at C5. The 3α-hydroxy metabolite, currently monitored by anti-doping laboratories, was the most abundant and was detected for the longest time. Furthermore, four other long-term metabolites were identified. By ultra-performance liquid chromatography coupled to tandem mass spectrometry, only the drug and a known metabolite were detected after glucuronide hydrolysis, and phase II metabolites were not found. Thus, our results contribute to elucidating methasterone metabolism, including long-term metabolites besides of the 3α-hydroxy in routine doping analysis, which is very important due to variation in human metabolism.

美他雄酮（Methasterone）是一种人工定制合成代谢类固醇（anabolic steroid），被运动员禁用且由反兴奋剂实验室实施监测。本研究旨在发现新的人体I相代谢物（phase I metabolites），明确其30天内的排泄动力学特征，并分析其II相代谢（phase II metabolism）过程，即硫酸酯、半胱氨酸及N-乙酰半胱氨酸结合物（sulfate, cysteine and N-acetylcysteine conjugates）的代谢途径。研究团队采用色谱技术对4名志愿者的尿液样本进行分析。通过对经葡萄糖醛酸苷酶水解（glucuronide enzymatic hydrolysis）的尿液样本开展气相色谱-质谱联用分析，成功检出美他雄酮及其9种I相代谢物，其中1种代谢物为首次报道。上述9种化合物均不以游离形式排泄。本次新鉴定的代谢物为17β-羟基-2α,17α-二甲基-5β-雄甾烷-3-酮，其源于5位碳的差向异构化（epimerization）反应。目前反兴奋剂实验室常规监测的3α-羟基代谢物为丰度最高且检出持续时间最长的代谢物。此外，本研究还鉴定出另外4种长效代谢物。采用超高效液相色谱-串联质谱法对葡萄糖醛酸苷酶水解样本进行分析时，仅检出原型药物及1种已知代谢物，未检测到II相代谢物。鉴于人体代谢存在个体差异，本研究结果有助于阐明美他雄酮的完整代谢过程，包括常规反兴奋剂分析中除3α-羟基代谢物之外的长效代谢物，具有重要的科研与应用价值。