title: Rapid test by liquid chromatography/tandem mass spectrometry to evaluate equine urine reactivity towards 17beta-OH steroids.
abstract: Bacteria frequently found in equine urine samples may cause degradation of 17beta-OH steroids. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to evaluate the microbiological contamination of equine urine as a marker of poor storage conditions. Norethandrolone was used as the internal standard, and the linearity, sensitivity, precision and accuracy of the method were evaluated. 17beta-OH oxidation was demonstrated for testosterone, nandrolone, trenbolone and boldenone, but did not occur in alpha-epimers such as alpha-boldenone and epitestosterone, demonstrating the stereoselectivity of the reaction. A rapid test was performed by spiking one of the four 17beta-OH steroids in samples of diluted equine urine. The steroids were transformed into their respective ketones in the presence of bacterial activity. The test allows direct injection of diluted samples into the LC/MS system, without the need for prior extraction. Results show that the best method of storage is freezing at -18 degrees C. Urine specimens should be analyzed as soon as possible after thawing. This allows bacterial degradation of equine urine to be arrested temporarily, so that the urine can be used for qualitative or quantitative analysis of 17beta-OH steroids.
authors.affiliation: U.N.I.R.E. Lab. S.r.l., Via Gramsci 70, 20019 Settimo Milanese (MI), Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Fidani
authors.forename: Eleonora
authors.initials: E
authors.lastname: Casagni
authors.forename: Marco
authors.initials: M
authors.lastname: Montana
authors.forename: Emanuela
authors.initials: E
authors.lastname: Pasello
authors.forename: Chiara
authors.initials: C
authors.lastname: Pecoraro
authors.forename: Veniero
authors.initials: V
authors.lastname: Gambaro
mesh_terms: D000818:Animals; D001419:Bacteria / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D004300:Doping in Sports / Q000517:prevention & control; D004355:Drug Stability; D006736:Horses / Q000652:urine*; D006914:Hydroxysteroids / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D009277:Nandrolone / Q000652:urine; D013048:Specimen Handling / Q000379:methods; D013739:Testosterone / Q000031:analogs & derivatives / Q000652:urine; D014204:Trenbolone Acetate / Q000652:urine; D014556:Urine / Q000382:microbiology
chemical_list: D006914:Hydroxysteroids; D013739:Testosterone; C013915:boldenone; D009277:Nandrolone; D014204:Trenbolone Acetate