title: Confirmatory analysis of stanozolol metabolites in bovine, pig and sheep urines using an optimized clean-up and liquid chromatography-tandem mass spectrometry.
abstract: This paper describes a new liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the analysis of three stanozolol metabolites (16β-hydroxystanozolol, 3'-hydroxystanozolol, and 4β-hydroxystanozolol) in urines of animal origins. The solid-phase extraction (SPE) clean-up procedure was optimized to reduce the matrix effects in the LC-MS/MS analysis and to enhance recovery. Four different approaches were tested to prepare the sample, which include anion, and cation mixed-mode ion exchange, reversed-phase and normal-phase SPE cartridges. Mixed-mode anion exchange Strata-XL-A SPE column with diethyl ether elution yielded the best values. The separation of metabolites was optimized on Kinetex XB column using isocratic elution. The best mobile phase composition was achieved at the acidic pH with 0.1% (v/v) formic acid in water and methanol composition. The main advantages of the approach applied in the present study over other known methods include the single step SPE clean-up, relatively fast separation on HPLC column packed with core-shell particles, and lowering the limit of detection of target metabolites to the range between 0.05 and 0.15μg/l. Additionally, the developed method was successfully validated for the first time for three species in accordance with the European Union (EU) 2002/657/EC decision. Finally, the efficiency of method was demonstrated by analyzing incurred samples.
authors.affiliation: National Food Chain Safety Office, Food and Feed Safety Directorate, Food Toxicology National Reference Laboratory, Mester utca 81, 1095 Budapest, Hungary. Electronic address: tolgyesi83@gmail.com.
authors.forename: Ádám
authors.initials: Á
authors.lastname: Tölgyesi
authors.forename: Virender K
authors.initials: VK
authors.lastname: Sharma
authors.forename: Jenő
authors.initials: J
authors.lastname: Fekete
mesh_terms: D000728:Androgens / Q000032:analysis; D000818:Animals; D000838:Anions; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D000432:Methanol / Q000737:chemistry; D015203:Reproducibility of Results; D012756:Sheep; D052616:Solid Phase Extraction; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization; D013197:Stanozolol / Q000652:urine*; D013552:Swine; D053719:Tandem Mass Spectrometry; D014867:Water / Q000737:chemistry
chemical_list: D000728:Androgens; D000838:Anions; D012997:Solvents; D014867:Water; D013197:Stanozolol; D000432:Methanol
keywords: Kinetex XB HPLC column; LC–MS/MS; Stanozolol metabolites; Strata solid-phase extraction; Urine