title: lmplementation of the prolyl hydroxylase inhibitor Roxadustat (FG-4592) and its main metabolites into routine doping controls.
abstract: The utility of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors as a therapeutic means of treating patients suffering from anaemia has been demonstrated for various clinical settings. However, besides this intended use, HIF stabilizers can be the subject of misuse in amateur and elite sports due to their erythropoietic properties, as recently proven by several cases of adverse analytical findings in doping control testing. Consequently, to allow for adequate and comprehensive test methods, knowledge of the drug candidates' metabolism and analytical options enabling appropriate detection windows in sports drug testing samples (i.e., blood and urine) is essential to doping control laboratories. In the present study, a novel HIF prolyl hydroxylase inhibitor referred to as Roxadustat (FG-4592) and main plasma- and urine-derived metabolites were investigated in the context of routine doping control analytical approaches. Liquid chromatography-mass spectrometry-based test methods were used to study the target analytes' dissociation pathways following electrospray ionization and collision-induced dissociation. Diagnostic precursor-product ion pairs were selected to enable the implementation of the intact drug Roxadustat and selected metabolites into multi-analyte initial testing procedures for plasma and urine specimens. The assays were validated in accordance to guidelines of the World Anti-Doping Agency (WADA) and results demonstrated the suitability (fitness-for-purpose) of the employed analytical methods with detection limits ranging from 0.05 to 1 ng/mL and 1 to 5 ng/mL for urine and plasma, respectively. Subsequently, elimination study plasma and urine samples collected up to 167 h post-administration were analyzed using the validated methods, which suggested the use of different target analytes for blood and urine analyses with FG-4592 and its glucuronide, respectively, for optimal detection windows. Additionally, a light-induced rearrangement product (photoisomer) of Roxadustat resulted in the formation of an additional compound of identical mass. Copyright © 2017 John Wiley & Sons, Ltd.
authors.affiliation: Sports Medicine Research and Testing Laboratory, 560 Arapeen Drive Suite 150A, Salt Lake City, UT, 84108, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Eichner
authors.affiliation: Sports Medicine Research and Testing Laboratory, 560 Arapeen Drive Suite 150A, Salt Lake City, UT, 84108, USA.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Van Wagoner
authors.affiliation: FibroGen, Inc., 409 Illinois Street, San Francisco, CA, 94158, USA.
authors.forename: Mitch
authors.initials: M
authors.lastname: Brenner
authors.affiliation: FibroGen, Inc., 409 Illinois Street, San Francisco, CA, 94158, USA.
authors.forename: James
authors.initials: J
authors.lastname: Chou
authors.affiliation: FibroGen, Inc., 409 Illinois Street, San Francisco, CA, 94158, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Leigh
authors.affiliation: FibroGen, Inc., 409 Illinois Street, San Francisco, CA, 94158, USA.
authors.forename: Lee R
authors.initials: LR
authors.lastname: Wright
authors.affiliation: FibroGen, Inc., 409 Illinois Street, San Francisco, CA, 94158, USA.
authors.forename: Lee A
authors.initials: LA
authors.lastname: Flippin
authors.affiliation: FibroGen, Inc., 409 Illinois Street, San Francisco, CA, 94158, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Martinelli
authors.affiliation: Institute of Biochemistry - Centre for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.|European Monitoring Center for Emerging Doping Agents, Cologne/Bonn, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Krug
authors.affiliation: Institute of Biochemistry - Centre for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
authors.affiliation: Institute of Biochemistry - Centre for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.|European Monitoring Center for Emerging Doping Agents, Cologne/Bonn, Germany.
authors.forename: Mario
authors.identifier: http://orcid.org/0000-0002-1535-6451
authors.initials: M
authors.lastname: Thevis
mesh_terms: D056352:Athletes; D002853:Chromatography, Liquid; D004300:Doping in Sports; D020719:Glucuronides / Q000737:chemistry* / Q000378:metabolism*; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D007546:Isoquinolines / Q000737:chemistry*; D057230:Limit of Detection; D064800:Prolyl-Hydroxylase Inhibitors / Q000737:chemistry*; D015813:Substance Abuse Detection; D053719:Tandem Mass Spectrometry; D016482:Urinalysis
chemical_list: D020719:Glucuronides; D007546:Isoquinolines; D064800:Prolyl-Hydroxylase Inhibitors; D005998:Glycine; C584543:roxadustat
keywords: HIF stabilizer; doping; erythropoietin; mass spectrometry; sport