title: Detection of stanozolol O- and N-sulfate metabolites and their evaluation as additional markers in doping control.
abstract: Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC-MS/MS to establish potential long-term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M-I to M-XI) were detected and characterized by LC-MS/MS. This paper provides valuable data on the ionization and fragmentation of O-sulfates and N-sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long-term metabolite (epistanozolol-N-glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute, Doctor Aiguader 88, 08003, Barcelona, Spain.|Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader 88, 08003, Barcelona, Spain.
authors.forename: Georgina
authors.initials: G
authors.lastname: Balcells
authors.affiliation: Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute, Doctor Aiguader 88, 08003, Barcelona, Spain.
authors.forename: Xavier
authors.initials: X
authors.lastname: Matabosch
authors.affiliation: Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute, Doctor Aiguader 88, 08003, Barcelona, Spain.|Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader 88, 08003, Barcelona, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Ventura
mesh_terms: D000284:Administration, Oral; D045930:Anabolic Agents / Q000008:administration & dosage / Q000378:metabolism / Q000652:urine*; D002853:Chromatography, Liquid / Q000379:methods; D004300:Doping in Sports; D006801:Humans; D007267:Injections; D008297:Male; D013197:Stanozolol / Q000008:administration & dosage / Q000378:metabolism / Q000652:urine*; D015813:Substance Abuse Detection / Q000379:methods*; D013431:Sulfates / Q000008:administration & dosage / Q000378:metabolism / Q000652:urine*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D045930:Anabolic Agents; D013431:Sulfates; D013197:Stanozolol
keywords: LC-MS/MS; Stanozolol; anabolic steroids; doping analysis; sulfate metabolites