Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry |
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| Authors: | Oscar J Pozo Peter Van Eenoo Koen Deventer Leen Lootens Susana Grimalt Juan V Sancho Felix Hernández Philip Meuleman Geert Leroux-Roels Frans T Delbeke |
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| Institution: | 1. DoCoLab, UGent, Department of Clinical Chemistry, Microbiology and Immunology, Technologiepark 30, B-9052 Zwijnaarde, Belgium;2. Research Institute for Pesticides and Water, University Jaume I, E-12071, Castellón, Spain;3. Center for Vaccinology, Ghent University and Hospital, De Pintelaan 185, B-9000 Ghent, Belgium;1. Bioanalysis Research Group, IMIM, Hospital del Mar, Doctor Aiguader 88, 08003 Barcelona, Spain;2. Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader 88, 08003 Barcelona, Spain;1. Research Institute for Pesticides and Water, University Jaume I, E-12071 Castellón, Spain;2. Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute, Doctor Aiguader 88, 08003 Barcelona, Spain;3. Department of Experimental and Health Sciencies, Universitat Pompeu Fabra, Doctor Aiguader 88, 08003 Barcelona, Spain;1. School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1211 Geneva 4, Switzerland;2. Human Protein Sciences Department, University of Geneva, 1211 Geneva 4, Switzerland;3. Swiss Centre for Applied Human Toxicology, Geneva, Switzerland;4. Institut Central (ICHV), Hôpital du Valais, Sion, Switzerland;5. Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Epalinges, Switzerland;1. Bioanalysis Research Group, IMIM, Hospital del Mar, Doctor Aiguader 88, Barcelona 08003, Spain;2. Department of Biological Chemistry and Molecular Modelling, Institute of Advanced Chemistry of Catalonia, Spanish Council for Scientific Research (IQAC-CSIC), Jordi Girona 18-26, Barcelona 08034, Spain;3. Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader 88, Barcelona 08003, Spain;1. Bioanalysis Research Group, IMIM, Hospital del Mar, Doctor Aiguader 88, 08003 Barcelona, Spain;2. Department of Biological Chemistry and Molecular Modelling, Institute of Advanced Chemistry of Catalonia, Spanish Council for Scientific Research (IQAC-CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain;3. Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader 88, 08003 Barcelona, Spain;1. Research Institute for Pesticides and Water, Universitat Jaume I, E-12071, Castelló, Spain;2. Department of Clinical Chemistry, University of Liège, Liège, Belgium;3. Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute, Barcelona, Spain;4. Barcelona Antidoping Laboratory, Doping Control Research Group, Fundació IMIM, Barcelona, Spain;5. Department of Physical and Analytical Chemistry, University of Oviedo, Oviedo, Spain;6. Ghent University, Department of Clinical Chemistry, Microbiology and Immunology, Doping Control Laboratory, Zwijnaarde, Belgium;7. Norwegian Doping Control Laboratory, Oslo University Hospital, Oslo, Norway |
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| Abstract: | The applicability of LC–MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC–QTOF MS. |
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