title: Structure elucidation of the diagnostic product ion at m/z 97 derived from androst-4-en-3-one-based steroids by ESI-CID and IRMPD spectroscopy.
abstract: Structure elucidation of steroids by mass spectrometry has been of great importance to various analytical arenas and numerous studies were conducted to provide evidence for the composition and origin of (tandem) mass spectrometry-derived product ions used to characterize and identify steroidal substances. The common product ion at m/z 97 generated from androst-4-ene-3-one analogs has been subject of various studies, including stable isotope-labeling and (high resolution/high accuracy) tandem mass spectrometry, but its gas-phase structure has never been confirmed. Using high resolution/high accuracy mass spectrometry and low resolution tandem mass spectrometry, density functional theory (DFT) calculation, and infrared multiple photon dissociation (IRMPD) spectroscopy employing a free electron laser, the structure of m/z 97 derived from testosterone was assigned to protonated 3-methyl-2-cyclopenten-1-one. This ion was identified in a set of six cyclic C(6)H(9)O(+) isomers as computed at the B3LYP/6-311++G(2d,2p) level of theory (protonated 3-methyl-2-cyclopenten-1-one, 2-methyl-2-cyclopenten-1-one and 2-cyclohexen-1-one). Product ions of m/z 97 obtained from MS(2) and MS(3) experiments of protonated 3-methyl-2-cyclopenten-1-one, 2-methyl-2-cyclopenten-1-one, 2-cyclohexen-1-one, and testosterone corroborated the suggested gas-phase ion structure, which was eventually substantiated by IRMPD spectroscopy yielding a spectrum that convincingly matched the predicted counterpart. Finally, the dissociation pathway of the protonated molecule of testosterone to m/z 97 was revisited and an alternative pathway was suggested that considers the exclusion of C-10 along with the inclusion of C-5, which was experimentally demonstrated with stable isotope labeling.
authors.affiliation: Institute of Biochemistry-Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany. m.thevis@biochem.dshs-koeln.de
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
authors.forename: Simon
authors.initials: S
authors.lastname: Beuck
authors.forename: Sebastian
authors.initials: S
authors.lastname: Höppner
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.forename: Joseph
authors.initials: J
authors.lastname: Held
authors.forename: Mathias
authors.initials: M
authors.lastname: Schäfer
authors.forename: Jos
authors.initials: J
authors.lastname: Oomens
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
mesh_terms: D007477:Ions / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013739:Testosterone / Q000737:chemistry*
chemical_list: D007477:Ions; D013739:Testosterone