Zheng Siying, Ge Yuqi, Fang Xian, Liu Mengpan, Sun Haoyi, Deng Xiaojun, Liao Lei
Shanghai University of Sport, 399 Changhai Road, Yangpu District, Shanghai, China.
Shanghai Anti-Doping Laboratory, Shanghai, China.
Anal Bioanal Chem. 2025 Aug 28. doi: 10.1007/s00216-025-06066-w.
In vitro metabolic models provide a means to circumvent the ethical concerns associated with human administration research, allowing for preliminary predictions of human metabolism while generating high concentrations of metabolites for characterization. As S1.1-class anabolic androgenic steroids on the World Anti-Doping Agency (WADA) Prohibited List, oxymetholone and methasterone have consistently appeared in the top 20 substances identified in adverse analytical findings (AAFs) in recent years, reflecting their persistent abuse patterns in sports. Given their exogenous nature, the metabolites of these steroid hormones fall within the scope of doping control, making metabolic studies a crucial aspect of anti-doping research. In this study, human liver S9 fractions were employed as a model for the characterization and metabolic profiling of oxymetholone and methasterone via gas chromatography-orbitrap-high-resolution mass spectrometry (GC-Orbitrap-HRMS). The full scan mode of GC-Orbitrap-HRMS was utilized to detect free and two conjugated fractions of metabolites, comparing these with control groups to confirm the metabolites during in vitro incubation. Possible metabolite structures were inferred from EI mass spectra, and the metabolic pathways for both drugs were discussed. In vitro, three oxymetholone and five methasterone metabolites were identified, and among them, two metabolites, OMT-M3 (2α,17α-methyl-5ξ-androstan-3α,6β,17β-triol) and MTS-M3 (2α,17α-dimethyl-5ξ-androstane-3α,12ξ,16ξ,17β-tetrol), were characterized as novel metabolites based on recent human in vivo metabolic studies. These metabolites exhibited diverse metabolic pathways, and their structures were corroborated through complementary in vitro and in vivo metabolic analyses. This study provides a comprehensive evaluation of the applicability of the human liver S9 model in the metabolic studies of anabolic steroids in vitro, verifying novel human metabolites and providing valuable insights for future research in this field.
体外代谢模型提供了一种规避与人体给药研究相关伦理问题的方法，能够在生成高浓度代谢物以进行表征的同时，对人体代谢进行初步预测。氧甲氢龙和美睾酮作为世界反兴奋剂机构（WADA）禁用清单上的S1.1类合成代谢雄激素类固醇，近年来在兴奋剂违规分析结果（AAFs）中鉴定出的前20种物质中一直名列前茅，这反映出它们在体育界持续的滥用模式。鉴于其外源性，这些类固醇激素的代谢物属于兴奋剂检测的范围，使得代谢研究成为反兴奋剂研究的一个关键方面。在本研究中，采用人肝脏S9组分作为模型，通过气相色谱-轨道阱-高分辨率质谱（GC-Orbitrap-HRMS）对氧甲氢龙和美睾酮进行表征和代谢谱分析。利用GC-Orbitrap-HRMS的全扫描模式检测代谢物的游离和两种结合组分，并与对照组进行比较，以确认体外孵育期间的代谢物。从电子轰击质谱中推断出可能的代谢物结构，并讨论了两种药物的代谢途径。在体外，鉴定出了三种氧甲氢龙代谢物和五种美睾酮代谢物，其中两种代谢物，OMT-M3（2α,17α-甲基-5ξ-雄甾烷-3α,6β,17β-三醇）和MTS-M3（2α,17α-二甲基-5ξ-雄甾烷-3α,12ξ,16ξ,17β-四醇），根据最近的人体体内代谢研究被表征为新的代谢物。这些代谢物表现出不同的代谢途径，并且通过体外和体内代谢分析的互补验证了它们的结构。本研究全面评估了人肝脏S9模型在体外合成代谢类固醇代谢研究中的适用性，验证了新的人体代谢物，并为该领域的未来研究提供了有价值的见解。
