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, 16 beta -hydroxy-stanozolol, in bovine urine by liquid chromatography coupled with tandem mass spectrometry has been developed. [H-2(3)]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 16 beta -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 16 beta -hydroxystanozolol, respectively. The precision ranged from 12.4 to 2.4% and from 13.1 to 1.8% for stanozolol and 16 beta -hydroxystanozolol, respectively. The limit of quantification of the method was 1 ng/ml in the bovine urine for both stanozolol and 16 beta -hydroxystanozolol. The developed method fulfils the European Union requirements for confirmatory methods. (C) 2001 Elsevier Science B.V. All rights reserved.