title: Synthesis, characterization, and detection of new oxandrolone metabolites as long-term markers in sports drug testing.
abstract: The discovery and implementation of the long-term metabolite of metandienone, namely 17β-hydroxymethyl-17α-methyl-18-norandrost-1,4,13-trien-3-one, to doping control resulted in hundreds of positive metandienone findings worldwide and impressively demonstrated that prolonged detection periods significantly increase the effectiveness of sports drug testing. For oxandrolone and other 17-methyl steroids, analogs of this metabolite have already been described, but comprehensive characterization and pharmacokinetic data are still missing. In this report, the synthesis of the two epimeric oxandrolone metabolites-17β-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one and 17α-hydroxymethyl-17β-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one-using a fungus (Cunninghamella elegans) based protocol is presented. The reference material was fully characterized by liquid chromatography nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. To ensure a specific and sensitive detection in athlete's urine, different analytical approaches were followed, such as liquid chromatography-tandem mass spectrometry (QqQ and Q-Orbitrap) and gas chromatography-tandem mass spectrometry, in order to detect and identify the new target analytes. The applied methods have demonstrated good specificity and no significant matrix interferences. Linearity (R(2) > 0.99) was tested, and precise results were obtained for the detection of the analytes (coefficient of variation <20%). Limits of detection (S/N) for confirmatory and screening analysis were estimated at 1 and 2 ng/mL of urine, respectively. The assay was applied to oxandrolone post-administration samples to obtain data on the excretion of the different oxandrolone metabolites. The studied specimens demonstrated significantly longer detection periods (up to 18 days) for the new oxandrolone metabolites compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone, presenting a promising approach to improve the fight against doping.
authors.affiliation: Institute of Biochemistry and Center for Preventive Doping Research, German Sport University, Am Sportpark Muengersdorf 6, 50933, Cologne, Germany, s.guddat@biochem.dshs-koeln.de.
authors.forename: S
authors.initials: S
authors.lastname: Guddat
authors.forename: G
authors.initials: G
authors.lastname: Fußhöller
authors.forename: S
authors.initials: S
authors.lastname: Beuck
authors.forename: A
authors.initials: A
authors.lastname: Thomas
authors.forename: H
authors.initials: H
authors.lastname: Geyer
authors.forename: A
authors.initials: A
authors.lastname: Rydevik
authors.forename: U
authors.initials: U
authors.lastname: Bondesson
authors.forename: M
authors.initials: M
authors.lastname: Hedeland
authors.forename: A
authors.initials: A
authors.lastname: Lagojda
authors.forename: W
authors.initials: W
authors.lastname: Schänzer
authors.forename: M
authors.initials: M
authors.lastname: Thevis
mesh_terms: D045930:Anabolic Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000652:urine*; D002853:Chromatography, Liquid / Q000379:methods; D004300:Doping in Sports; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D010074:Oxandrolone / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism* / Q000652:urine*; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D045930:Anabolic Agents; D010074:Oxandrolone