title: Development of an analytical method for the determination of the misuse in sports of boldenone through the analysis of urine by on-line coupling liquid chromatography-gas chromatography-combustion-isotope ratio mass spectrometry.
abstract: Boldenone (Bo), androsta-1,4-dien-17β-ol-3-one, is an anabolic androgenic steroid not clinically approved for human application. Despite this, many cases are reported every year of athletes testing positive for Bo or its main metabolite 5β-androst-1-en-17β-ol-3-one (BoM). Recently the capability of different human intestinal bacteria to produce enzymes able to modify endogenous steroids in Bo has been demonstrated. When a urinary concentration of Bo and/or BoM between 5 and 30 ng/mL is measured a complementary analysis by gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) must be carried out to discriminate the endogenous or exogenous origin. In the present work, a novel analytical method that couples LC-GC by means of the TOTAD interface with C-IRMS is described. The method is based on a first RPLC separation of unacetyled steroids, followed by acetylation and automated on-line LC-GC-C-IRMS, which includes a second RPLC clean-up of acetyl Bo and BoM, isolation of the two fractions in a fraction collector and their consecutive analysis by GC-C-IRMS. The method has been applied to the analysis of urine samples fortified at 5 and 10 ng/mL, where it has shown a good performance.
authors.affiliation: Escuela Técnica Superior de Ingenieros Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario s/n, 02071 Albacete, Spain.
authors.forename: R M
authors.initials: RM
authors.lastname: Toledano
authors.affiliation: Laboratorio de Control de Dopaje, Agencia Española de Protección de la Salud en el Deporte, Calle Greco s/n, 28040 Madrid, Spain.
authors.forename: E M
authors.initials: EM
authors.lastname: Díaz-Plaza
authors.affiliation: Facultad de Educación, Universidad de Castilla-La Mancha, Plaza de la Universidad, 3, 02071 Albacete, Spain.
authors.forename: J M
authors.initials: JM
authors.lastname: Cortes
authors.affiliation: Facultad de Educación, Universidad de Castilla-La Mancha, Plaza de la Universidad, 3, 02071 Albacete, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Aragón
authors.affiliation: Facultad de Educación, Universidad de Castilla-La Mancha, Plaza de la Universidad, 3, 02071 Albacete, Spain.
authors.forename: A M
authors.initials: AM
authors.lastname: Vázquez
authors.affiliation: Escuela Técnica Superior de Ingenieros Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario s/n, 02071 Albacete, Spain. Electronic address: jesus.villen@uclm.es.
authors.forename: J
authors.initials: J
authors.lastname: Villén
authors.affiliation: Laboratorio de Control de Dopaje, Agencia Española de Protección de la Salud en el Deporte, Calle Greco s/n, 28040 Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Muñoz-Guerra
mesh_terms: D045930:Anabolic Agents / Q000652:urine*; D002247:Carbon Isotopes / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004300:Doping in Sports; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D015813:Substance Abuse Detection / Q000379:methods; D013739:Testosterone / Q000031:analogs & derivatives* / Q000652:urine
chemical_list: D045930:Anabolic Agents; D002247:Carbon Isotopes; D013739:Testosterone; C013915:boldenone
keywords: Boldenone; Doping; On-line LC–GC–C–IRMS; Steroid analysis; TOTAD interface