Investigations Into Structures of In Vitro<i>–</i>Derived Phase I Metabolites of a Novel 20‐Keto‐Steroid S42 by GC‐EI HR MS Analysis and Chemical Synthesis

S42, 4-Methyl-19-norpregna-1,3,5(10)-trien-20-one a new 20-keto-steroid, is a novel selective androgen receptor modulator (SARM). The World Anti-Doping Agency (WADA) bans the use of SARMs in sports at all times. In preparation of a sensitive detection procedure to control for S42 abuse, in vitro metabolism experiments were conducted and biotransformation products were analyzed with GC-EI MS-orbitrap instrumentation. S42-C20-OH, S42-C6ß-OH, and S42-C7α-OH were synthesized as reference material to study their exemplary EI-HR (electron ionization- high resolution) mass spectra. Additionally, S42-d7, synthesized earlier with ²H-labels at carbon atoms C1, C2, C3, C6, and C7, was used for the in vitro metabolism study. Comparison of the respective mass spectra of labeled and unlabeled reference materials and of specifically mass-shifted fragment ions provided the foundation for the structure elucidation of S42 in vitro phase I metabolites. Molecular ions of selected S42 phase I metabolites found in the in vitro experiments were submitted to higher energy collisional dissociation (HCD) MS²-product ion experiments to allow straightforward and secured assignment and interpretation of fragmentation patterns. At least eight phase I metabolites of S42 were identified in the in vitro study and analyzed as tri-methyl-silyl ether derivatives. Specifically, different singly, doubly, and triply hydroxylated metabolites of S42 were identified and analyzed with GC-EI HR MS.