Mario Thevis1,2, Andreas Lagojda3, Dirk Kuehne3, Andreas Thomas1, Josef Dib1, Annelie Hansson4, Mikael Hedeland4,5, Ulf Bondesson4,5, Tina Wigger6, Uwe Karst6, Wilhelm Schänzer1. 1. Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany. 2. European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany. 3. Bayer CropScience AG, Alfred-Nobel-Str. 50, 40789, Monheim, Germany. 4. Uppsala University, Division of Analytical Pharmaceutical Chemistry, P.O. Box 574, SE-751 23, Uppsala, Sweden. 5. National Veterinary Institute (SVA), Department of Chemistry, Environment and Feed Hygiene, SE-751 89, Uppsala, Sweden. 6. Westfälische Wilhelms-Universität Münster, Institute of Inorganic and Analytical Chemistry, Corrensstr. 30, 48149, Münster, Germany.
Abstract
RATIONALE: Potentially performance-enhancing agents, particularly anabolic agents, are advertised and distributed by Internet-based suppliers to a substantial extent. Among these anabolic agents, a substance referred to as LGD-4033 has been made available, comprising the core structure of a class of selective androgen receptor modulators (SARMs). METHODS: In order to provide comprehensive analytical data for doping controls, the substance was obtained and characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography/electrospray ionization high resolution/high accuracy tandem mass spectrometry (LC/ESI-HRMS). Following the identification of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile, the substance was subjected to in vitro metabolism studies employing human liver microsomes and Cunninghamella elegans (C. elegans) preparations as well as electrochemical metabolism simulations. RESULTS: By means of LC/ESI-HRMS, five main phase-I metabolites were identified as products of liver microsomal preparations including three monohydroxylated and two bishydroxylated species. The two most abundant metabolites (one mono- and one bishydroxylated product) were structurally confirmed by LC/ESI-HRMS and NMR. Comparing the metabolic conversion of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile observed in human liver microsomes with C. elegans and electrochemically derived metabolites, one monohydroxylated product was found to be predominantly formed in all three methodologies. CONCLUSIONS: The implementation of the intact SARM-like compound and its presumed urinary phase-I metabolites into routine doping controls is suggested to expand and complement existing sports drug testing methods.
RATIONALE: Potentially performance-enhancing agents, particularly anabolic agents, are advertised and distributed by Internet-based suppliers to a substantial extent. Among these anabolic agents, a substance referred to as LGD-4033 has been made available, comprising the core structure of a class of selective androgen receptor modulators (SARMs). METHODS: In order to provide comprehensive analytical data for doping controls, the substance was obtained and characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography/electrospray ionization high resolution/high accuracy tandem mass spectrometry (LC/ESI-HRMS). Following the identification of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile, the substance was subjected to in vitro metabolism studies employing human liver microsomes and Cunninghamella elegans (C. elegans) preparations as well as electrochemical metabolism simulations. RESULTS: By means of LC/ESI-HRMS, five main phase-I metabolites were identified as products of liver microsomal preparations including three monohydroxylated and two bishydroxylated species. The two most abundant metabolites (one mono- and one bishydroxylated product) were structurally confirmed by LC/ESI-HRMS and NMR. Comparing the metabolic conversion of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile observed in human liver microsomes with C. elegans and electrochemically derived metabolites, one monohydroxylated product was found to be predominantly formed in all three methodologies. CONCLUSIONS: The implementation of the intact SARM-like compound and its presumed urinary phase-I metabolites into routine doping controls is suggested to expand and complement existing sports drug testing methods.
Authors: Felicitas Wagener; Sven Guddat; Christian Görgens; Yiannis S Angelis; Michael Petrou; Andreas Lagojda; Dirk Kühne; Mario Thevis Journal: Anal Bioanal Chem Date: 2021-11-04 Impact factor: 4.142