Detection and characterization of a new metabolite of 17alpha-methyltestosterone.
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: 17alpha-methyl-5beta-androstane-3alpha,17beta-diol and 17alpha-methyl-5alpha-androstane-3alpha,17beta-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.
Pozo
Oscar J
OJ
DoCoLab, Ghent University, Department of Clinical Chemistry, Microbiology and Immunology, Zwijnaarde, Belgium. oscar.pozomendoza@gmail.com
Van Eenoo
Peter
P
Deventer
Koen
K
Lootens
Leen
L
Van Thuyne
Wim
W
Parr
Maria K
MK
Schänzer
Wilhelm
W
Sancho
Juan V
JV
Hernández
Felix
F
Meuleman
Philip
P
Leroux-Roels
Geert
G
Delbeke
Frans T
FT
D000818:Animals; D004311:Double-Blind Method; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D008777:Methyltestosterone / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D018183:Transplantation Chimera
D008777:Methyltestosterone