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 l iquid chromatography coupled with tandem mass spectrometry has been develop ed. [H-2(3)]Stanozolol was used as internal standard. Sample preparation in volved enzymatic hydrolysis, liquid-liquid extraction and purification on a n amino solid-phase extraction column. The analytes were ionized using atmo spheric pressure chemical ionization with a heated nebulizer interface oper ating 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 wer e identified for the unambiguous hormone confirmation by selected reaction monitoring liquid chromatography-tandem mass spectrometry. The accuracy ran ged from 19.7 to 14.9% and from 18.9 to 13.2% for stanozolol and 16 beta -h ydroxystanozolol, respectively. The precision ranged from 12.4 to 2.4% and from 13.1 to 1.8% for stanozolol and 16 beta -hydroxystanozolol, respective ly. The limit of quantification of the method was 1 ng/ml in the bovine uri ne 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.