title: Mass spectrometric characterization of a prolyl hydroxylase inhibitor GSK1278863, its bishydroxylated metabolite, and its implementation into routine doping controls.
abstract: Drug candidates, which have the potential of enhancing athletic performance represent a risk of being misused in elite sport. Therefore, there is a need for early consideration by anti-doping authorities and implementation into sports drug testing programmes. The hypoxia-inducible factor (HIF) or prolyl hydroxylase inhibitor (PHI) GSK1278863 represents an advanced candidate of an emerging class of therapeutics that possess substantial potential for abuse in sport due to their capability to stimulate the biogenesis of erythrocytes and, consequently, the individual's oxygen transport capacity. A thorough characterization of such analytes by technologies predominantly used for doping control purposes and the subsequent implementation of the active drug and/or its main urinary metabolite(s) are vital for comprehensive, preventive, and efficient anti-doping work. In the present study, the HIF PHI drug candidate GSK1278863 (comprising a 6-hydroxypyrimidine-2,4-dione nucleus) and its bishydroxylated metabolite M2 (GSK2391220A) were studied regarding their mass spectrometric behaviour under electrospray ionization (ESI-MS/MS) conditions. Synthesized reference materials were used to elucidate dissociation pathways by means of quadrupole/time-of-flight high resolution/high accuracy tandem mass spectrometry, and their detection from spiked urine and elimination study urine samples under routine doping control conditions was established using liquid chromatography-electrospray ionization-tandem mass spectrometry with direct injection. Dissociation pathways to diagnostic product ions of GSK1278863 (e.g. m/z 291, 223, and 122) were proposed as substantiated by determined elemental compositions and MS(n) experiments as well as comparison to spectra of the bishydroxylated analogue M2. An analytical assay based on direct urine injection using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of GSK1278863 in combination with its bishydroxylated metabolite M2. Validation parameters including limit of detection (0.5-1 ng/mL), linearity, specificity, ion suppression/enhancement (<10%), intra- and inter-day precision (6-22%) were determined, demonstrating the fitness-for-purpose of the assay for doping control screening of urine samples for the presence of the drug candidate and its main metabolite and for expanding current anti-doping efforts to this new class of therapeutics. However, administration study urine sample analysis suggested the use of M2 rather than the intact drug due to extensive metabolic conversion. Copyright © 2015 John Wiley & Sons, Ltd.
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.|European Monitoring Center for Emerging Doping Agents, Cologne, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
authors.affiliation: GlaxoSmithKline, King of Prussia, PA, 19406, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Milosovich
authors.affiliation: GlaxoSmithKline, King of Prussia, PA, 19406, USA.
authors.forename: Hermes
authors.initials: H
authors.lastname: Licea-Perez
authors.affiliation: GlaxoSmithKline, King of Prussia, PA, 19406, USA.
authors.forename: Dana
authors.initials: D
authors.lastname: Knecht
authors.affiliation: GlaxoSmithKline, King of Prussia, PA, 19406, USA.
authors.forename: Tom
authors.initials: T
authors.lastname: Cavalier
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
mesh_terms: D001463:Barbiturates / Q000652:urine*; D002853:Chromatography, Liquid / Q000379:methods; D004300:Doping in Sports; D005998:Glycine / Q000031:analogs & derivatives* / Q000652:urine; D006801:Humans; D057230:Limit of Detection; D064800:Prolyl-Hydroxylase Inhibitors / Q000652:urine*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods; D016482:Urinalysis / Q000379:methods
chemical_list: D001463:Barbiturates; C000599718:GSK1278863; D064800:Prolyl-Hydroxylase Inhibitors; D005998:Glycine
keywords: GSK1278863; GSK2391220A; HIF stabilizer; doping; mass spectrometry; sport