Confirmatory analysis of residues of stanozolol and its major metabolite in bovine urine by liquid chromatography–tandem mass spectrometry

A reliable method for the confirmation of the synthetic hormone stanozolol and its major metabolite, 16β-hydroxystanozolol, in bovine urine by liquid chromatography coupled with tandem mass spectrometry has been developed. [2H3]Stanozolol was used as internal standard. Sample preparation involved enzymatic hydrolysis, liquid–liquid extraction and purification on an amino solid-phase extraction column. The analytes were ionized using atmospheric pressure chemical ionization with a heated nebulizer interface operating in the positive ion mode, where only the protonated molecules, [M+H]+, at m/z 329 and m/z 345, for stanozolol and 16β-hydroxystanozolol, respectively, were generated. These served as precursor ions for collision-induced dissociation and three diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring liquid chromatography–tandem mass spectrometry. The accuracy ranged from 19.7 to 14.9% and from 18.9 to 13.2% for stanozolol and 16β-hydroxystanozolol, respectively. The precision ranged from 12.4 to 2.4% and from 13.1 to 1.8% for stanozolol and 16β-hydroxystanozolol, respectively. The limit of quantification of the method was 1 ng/ml in the bovine urine for both stanozolol and 16β-hydroxystanozolol. The developed method fulfils the European Union requirements for confirmatory methods.