{"title":"用GC-Orbitrap-HRMS研究了人肝脏S9模型中氧甲氧酮和美沙酮代谢物的表征和代谢谱。","authors":"Siying Zheng, Yuqi Ge, Xian Fang, Mengpan Liu, Haoyi Sun, Xiaojun Deng, Lei Liao","doi":"10.1007/s00216-025-06066-w","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":462,"journal":{"name":"Analytical and Bioanalytical Chemistry","volume":" ","pages":""},"PeriodicalIF":3.8000,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Characterization and metabolic profiling of oxymetholone and methasterone metabolites studied with human liver S9 model using GC-Orbitrap-HRMS for anti-doping purposes.\",\"authors\":\"Siying Zheng, Yuqi Ge, Xian Fang, Mengpan Liu, Haoyi Sun, Xiaojun Deng, Lei Liao\",\"doi\":\"10.1007/s00216-025-06066-w\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":462,\"journal\":{\"name\":\"Analytical and Bioanalytical Chemistry\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":3.8000,\"publicationDate\":\"2025-08-28\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Analytical and Bioanalytical Chemistry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1007/s00216-025-06066-w\",\"RegionNum\":2,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical and Bioanalytical Chemistry","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1007/s00216-025-06066-w","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Characterization and metabolic profiling of oxymetholone and methasterone metabolites studied with human liver S9 model using GC-Orbitrap-HRMS for anti-doping purposes.
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.
期刊介绍:
Analytical and Bioanalytical Chemistry’s mission is the rapid publication of excellent and high-impact research articles on fundamental and applied topics of analytical and bioanalytical measurement science. Its scope is broad, and ranges from novel measurement platforms and their characterization to multidisciplinary approaches that effectively address important scientific problems. The Editors encourage submissions presenting innovative analytical research in concept, instrumentation, methods, and/or applications, including: mass spectrometry, spectroscopy, and electroanalysis; advanced separations; analytical strategies in “-omics” and imaging, bioanalysis, and sampling; miniaturized devices, medical diagnostics, sensors; analytical characterization of nano- and biomaterials; chemometrics and advanced data analysis.