Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

• Published in: Steroids 2009-10, Vol.74 (10-11), p.837-852
• Main Authors: Pozo, Oscar J., Van Eenoo, Peter, Deventer, Koen, Lootens, Leen, Grimalt, Susana, Sancho, Juan V., Hernández, Felix, Meuleman, Philip, Leroux-Roels, Geert, Delbeke, Frans T.
• Format: Article
• Language: English
• Subjects: Adult; Anabolic Agents - chemistry; Anabolic Agents - metabolism; Anabolic Agents - urine; Anabolic steroids; Animals; Biological and medical sciences; Chimeric mice; Chromatography, Liquid; Doping analysis; Doping in Sports; Fundamental and applied biological sciences. Psychology; Humans; Male; Metabolites; Mice; Mice, Transgenic; Precursor ion scan; QTOF MS; Stanozolol - chemistry; Stanozolol - metabolism; Stanozolol - urine; Tandem Mass Spectrometry; Vertebrates: endocrinology
• ISSN: 0039-128X; 1878-5867
• DOI: 10.1016/j.steroids.2009.05.004

The applicability of LC–MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC–QTOF MS.