High-performance liquid chromatography/tandem mass spectrometry: Its use for the identification of stanozolol and its major metabolites in human and equine urine

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Abstract
A screening procedure for the anabolic steroid stanozolol in human and equine urine was developed based on enzymatic hydrolysis, liquid-liquid extraction and reversed-phase liquid chromatography combined on-line with tandem mass spectrometry. The column effluent was introduced into the atmospheric pressure ionization source of a triple-quadrupole mass spectrometer via a heated pneumatic nubulizer liquid chromatograph/mass spectrometer interface. Abundant protonated molecular ions were generated by corona discharge ionization. Confirmation of stanozolol and several of its hydroxylated and dihydroxylated metabolites isolated from both human and equine urine was accomplished by collision-induced dissociation of their parent ions. Interpretation of the daughter ion mass spectra gave valuable information for the structural elucidation of the detected metabolites. Using the selected reaction monitoring detection mode the presence of the urinary excretion products could be monitored in equine urine up to one day and in human urine for several days after oral administration of stanozolol. Microbore highperformance liquid chromatography/ion spray mass spectrometry of an ion-pair extract enabled the direct detection of intact sulfoconjugated hydroxy-metabolites in human urine.