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, 16beta-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 16beta-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 16beta-hydroxystanozolol, respectively. The precision ranged from 12.4 to 2.4% and from 13.1 to 1.8% for stanozolol and 16beta-hydroxystanozolol, respectively. The limit of quantification of the method was 1 ng/ml in the bovine urine for both stanozolol and 16beta-hydroxystanozolol. The developed method fulfils the European Union requirements for confirmatory methods.
Draisci
R
R
Veterinary Medicine Laboratory, Istituto Superiore di Sanità, Rome, Italy. draisci@iss.it
Palleschi
L
L
Marchiafava
C
C
Ferretti
E
E
Delli Quadri
F
F
D045930:Anabolic Agents / Q000652:urine*; D000818:Animals; D002138:Calibration; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D004350:Drug Residues / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013197:Stanozolol / Q000652:urine*
D045930:Anabolic Agents; D013197:Stanozolol