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.

美他诺龙（Methasterone）是一种定制合成同化类固醇（designer anabolic steroid），属于运动员禁用物质，受反兴奋剂实验室（anti-doping laboratories）监测。本研究旨在发现其新的人体I相代谢物（phase I metabolites），明确其长达30天的排泄动力学特征，并分析其II相代谢过程（phase II metabolism），涵盖硫酸盐、半胱氨酸及N-乙酰半胱氨酸结合物（sulfate, cysteine and N-acetylcysteine conjugates）。研究收集了4名志愿者的尿液样本，采用色谱技术（chromatographic techniques）进行分析。经葡萄糖醛酸苷酶解（glucuronide enzymatic hydrolysis）后，通过气相色谱-质谱联用法（gas chromatography coupled to mass spectrometry, GC-MS）可在尿液样本中检测到美他诺龙及其9种I相代谢物，其中1种为此前未被报道的新代谢物。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，由C5位差向异构化（epimerization at C5）生成。当前反兴奋剂实验室监测的3α-羟基代谢物丰度最高，且检出持续时间最长。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（ultra-performance liquid chromatography coupled to tandem mass spectrometry, UPLC-MS/MS）分析时，仅在酶解后的样本中检测到母体药物及1种已知代谢物，未检出II相代谢物。综上，本研究结果有助于阐明美他诺龙的代谢过程，除常规兴奋剂检测中覆盖的3α-羟基代谢物外，还纳入了长效代谢物；考虑到人体代谢存在个体差异，该发现对日常兴奋剂检测具有重要意义。