Abstract
The misuse of the anabolic steroid methyltestosterone is currently routinely monitored in doping control laboratories by gas chromatography-mass spectrometry (GC-MS) of two of its metabolites: 17α-methyl-5β-androstane-3α,17β-diol and 17α-methyl-5α-androstane-3α,17β-diol. Because of the absence of any easy ionizable moiety, these metabolites are poorly detectable using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). In this study, the metabolism of methyltestosterone has been reinvestigated by the use of a precursor ion scan method in LC-ESI-MS/MS. Two metabolites have been detected using this method. Both compounds have been confirmed in postadministration urine samples of an urokinase plasminogen activator-severe combined immunodeficiency (uPA-SCID) mouse with humanized liver and were characterized by LC-MS/MS and GC-MS using both quadrupole and time of flight analyzers. From the detailed study of the fragmentation, these metabolites were proposed to be epimethyltestosterone and a dehydrogenated compound. Epimethyltestosterone has previously been described as a minor metabolite, whereas the occurrence of the oxidized metabolite has not been reported. Comparison with the synthesized reference revealed that the structure of the dehydrogenated metabolite is 6-ene-epimethyltestosterone. A selected reaction monitoring method including three transitions for each metabolite has been developed and applied to samples from an excretion study and to samples declared positive after GC-MS analysis. 6-Ene-epimethyltestosterone was found in all samples, showing its applicability in the detection of methyltestosterone misuse.
- WADA, World Anti-Doping Agency
- GC, gas chromatography
- MS, mass spectrometry
- LC, liquid chromatography
- MS/MS, tandem mass spectrometry
- TOF, time of flight
- uPA-SCID, urokinase plasminogen activator-severe combined immunodeficiency
- QTOF, quadripole time of flight
- HPLC, high-performance liquid chromatography
- CID, collision-induced dissociation
- ESI, electrospray ionization
- EI, electron ionization
- SIM, selected ion monitoring
- SRM, selected reaction monitoring
- TMS, trimethylsilyl
- OE+, odd electron ion(s).
Footnotes
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This study was supported by the World Anti-Doping Agency; the Belgian State [IUAP P6/36-HEPRO]; and Ghent University via the Special Research Fund and a Concerted Action Grant [01G00507].
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.028373
- Received May 4, 2009.
- Accepted August 17, 2009.
- Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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