title: Detection and characterization of betamethasone metabolites in human urine by LC-MS/MS.
abstract: Glucocorticosteroids are prohibited in sports when administered by systemic routes and allowed using other administrations for therapeutic reasons. Therefore, markers to distinguish between routes of administration through the analysis of urine samples are needed in anti-doping control. As a first step to achieve that goal, the metabolism of betamethasone (BET) was investigated in the present work. Urine samples obtained after BET intramuscular injection were hydrolyzed with β-glucuronidase and subjected to liquid-liquid extraction with ethyl acetate in alkaline conditions. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry. Common open screening methods for fluorine containing corticosteroids (precursor ion scan method of m/z 121, 147, 171, and neutral loss (NL) scan methods of 20 and 38 Da in positive ionization, and 46 and 76 Da in negative ionization) were applied to detect BET metabolites. Moreover, an NL method was applied to detect A-ring reduced metabolites of BET, which are ionized as [M+NH4 ](+) (NL of 55, 73, and 91 Da, corresponding to the consecutive losses of NH3 , HF and one, two and three water molecules, respectively). BET and 24 metabolites were detected. Six metabolites were identified by comparison with standards, and for ten, feasible structures were proposed based on mass spectrometric data. Eleven of the characterized metabolites had not been previously reported. Metabolites resulting from 11-oxidation, 6-hydroxylation, C20 or 4-ene-3-one reduction and combination of some of them were detected. Moreover one metabolite resulting from cleavage of the side chain with subsequent oxidation of carbon at C17 was also detected.
authors.affiliation: Bioanalysis Research Group, IMIM (Institut Hospital del Mar d'Investigacions Mèdiques), Doctor Aiguader 88, 08003, Barcelona, Spain.
authors.forename: Xavier
authors.initials: X
authors.lastname: Matabosch
authors.affiliation: Bioanalysis Research Group, IMIM (Institut Hospital del Mar d'Investigacions Mèdiques), Doctor Aiguader 88, 08003, Barcelona, Spain.
authors.forename: Oscar J
authors.initials: OJ
authors.lastname: Pozo
authors.affiliation: Bioanalysis Research Group, IMIM (Institut Hospital del Mar d'Investigacions Mèdiques), Doctor Aiguader 88, 08003, Barcelona, Spain.
authors.forename: Núria
authors.initials: N
authors.lastname: Monfort
authors.affiliation: Human Pharmacology and Clinical Neurosciences Research Group, IMIM, Doctor Aiguader 88, 08003, Barcelona, Spain.|Department of Pharmacology, Therapeutics and Toxicology, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), 08193, Barcelona, Spain.
authors.forename: Clara
authors.initials: C
authors.lastname: Pérez-Mañá
authors.affiliation: Human Pharmacology and Clinical Neurosciences Research Group, IMIM, Doctor Aiguader 88, 08003, Barcelona, Spain.|Department of Pharmacology, Therapeutics and Toxicology, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), 08193, Barcelona, Spain.
authors.forename: Magí
authors.initials: M
authors.lastname: Farré
authors.affiliation: Bioanalysis Research Group, IMIM (Institut Hospital del Mar d'Investigacions Mèdiques), Doctor Aiguader 88, 08003, Barcelona, Spain.|Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader 88, 08003, Barcelona, Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Segura
authors.affiliation: Bioanalysis Research Group, IMIM (Institut Hospital del Mar d'Investigacions Mèdiques), 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: D001623:Betamethasone / Q000032:analysis / Q000378:metabolism* / Q000652:urine*; D002853:Chromatography, Liquid / Q000379:methods; D005938:Glucocorticoids / Q000032:analysis / Q000378:metabolism* / Q000652:urine*; D006801:Humans; D059625:Liquid-Liquid Extraction / Q000379:methods; D008297:Male; D015813:Substance Abuse Detection / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D005938:Glucocorticoids; D001623:Betamethasone
keywords: Betamethasone; doping analysis; mass spectrometry; metabolism; urine