title: Doping control analysis of trimetazidine and characterization of major metabolites using mass spectrometric approaches.
abstract: Since January 2014, the anti-anginal drug trimetazidine [1-(2,3,4-trimethoxybenzyl)-piperazine] has been classified as prohibited substance by the World Anti-Doping Agency (WADA), necessitating specific and robust detection methods in sports drug testing laboratories. In the present study, the implementation of the intact therapeutic agent into two different initial testing procedures based on gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) is reported, along with the characterization of urinary metabolites by electrospray ionization-high resolution/high accuracy (tandem) mass spectrometry. For GC-MS analyses, urine samples were subjected to liquid-liquid extraction sample preparation, while LC-MS/MS analyses were conducted by established 'dilute-and-inject' approaches. Both screening methods were validated for trimetazidine concerning specificity, limits of detection (0.5-50 ng/mL), intra-day and inter-day imprecision (<20%), and recovery (41%) in case of the GC-MS-based method. In addition, major metabolites such as the desmethylated trimetazidine and the corresponding sulfoconjugate, oxo-trimetazidine, and trimetazidine-N-oxide as identified in doping control samples were used to complement the LC-MS/MS-based assay, although intact trimetazidine was found at highest abundance of the relevant trimetazidine-related analytes in all tested sports drug testing samples. Retrospective data mining regarding doping control analyses conducted between 1999 and 2013 at the Cologne Doping Control Laboratory concerning trimetazidine revealed a considerable prevalence of the drug particularly in endurance and strength sports accounting for up to 39 findings per year.
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Gerd
authors.initials: G
authors.lastname: Sigmund
authors.forename: Anja
authors.initials: A
authors.lastname: Koch
authors.forename: Anne-Katrin
authors.initials: AK
authors.lastname: Orlovius
authors.forename: Sven
authors.initials: S
authors.lastname: Guddat
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
mesh_terms: D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D004300:Doping in Sports / Q000517:prevention & control* / Q000706:statistics & numerical data; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D057230:Limit of Detection; D012015:Reference Standards; D015203:Reproducibility of Results; D015813:Substance Abuse Detection / Q000379:methods; D053719:Tandem Mass Spectrometry; D014292:Trimetazidine / Q000032:analysis* / Q000493:pharmacokinetics* / Q000652:urine; D014665:Vasodilator Agents / Q000008:administration & dosage* / Q000493:pharmacokinetics* / Q000652:urine
chemical_list: D014665:Vasodilator Agents; D014292:Trimetazidine
keywords: angina pectoris; doping; mass spectrometry; metabolism; sport