title: A novel HPLC-MRM strategy to discover unknown and long-term metabolites of stanozolol for expanding analytical possibilities in doping-control.
abstract: Stanozolol is one of the most commonly abused anabolic androgenic steroids (AAS) by athletes and usually detected by its parent drug and major metabolites. However, its metabolic pathway is complex, varied and individually different, it is important to characterize its overall metabolic profiles and discover new and long-term metabolites for the aims of expanding detection windows. High performance liquid chromatography coupled with triple quadrupole mass spectrometer (HPLC-MS/MS) was used to analyze the human urine after oral administration of stanozolol. Multiple reaction monitoring (MRM), one of the scan modes of triple quadrupole mass spectrometer showing extremely high sensitivity was well used to develop a strategy for metabolic profiles characterization and long-term metabolites detection based on typical precursor to product ion transitions of parent drug and its major metabolites. Utilizing the characteristic fragment ions of stanozolol and its major metabolites as the product ions, and speculating unknown precursor ions based on the possible phase I and phase II metabolic reactions in human body, the metabolite profiles of stanozolol could be comprehensively discovered, especially for those unknown and low concentration metabolites in human urine. Then these metabolites were further well structure identified by targeted high resolution MS/MS scan of quadrupole-time of flight mass spectrometry (Q-TOF). Applying this strategy, 27 phase I and 21 phase II metabolites of stanozolol were identified, in which 13 phase I and 14 phase II metabolites have not been reported previously. The 9 out of 48 metabolites could be detected over 15days post drug administration. This strategy could be employed effectively to characterize AAS metabolic profiles and discover unknown and long-term metabolites in sports drug testing.
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Wang
authors.affiliation: National Anti-Doping Laboratory, China Anti-Doping Agency, Beijing, 100029, PR China.
authors.forename: Xinmiao
authors.initials: X
authors.lastname: Zhou
authors.affiliation: National Anti-Doping Laboratory, China Anti-Doping Agency, Beijing, 100029, PR China.
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.affiliation: National Anti-Doping Laboratory, China Anti-Doping Agency, Beijing, 100029, PR China. Electronic address: dongying@chinada.cn.
authors.forename: Ying
authors.initials: Y
authors.lastname: Dong
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China. Electronic address: zhjl@imm.ac.cn.
authors.forename: Jinlan
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000328:Adult; D045930:Anabolic Agents / Q000032:analysis / Q000378:metabolism / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004300:Doping in Sports; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D053858:Metabolic Networks and Pathways; D013197:Stanozolol / Q000032:analysis / Q000378:metabolism / Q000652:urine*; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D055815:Young Adult
chemical_list: D045930:Anabolic Agents; D013197:Stanozolol
keywords: Doping-control; HPLC–MS/MS; Long-term and unknown metabolites; Multi reaction monitoring; Stanozolol