1) German Sport University Cologne, Center for Preventive Doping Research – Institute of Biochemistry, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany, 2) Thermo Fisher Scientific, Hanna-Kunath-Straße 11, 28199 Bremen, Germany Rapid Communications in Mass Spectrometry (2013), V27, pp1904–1912, doi: 10.1002/rcm.6648 Hydrogen isotope ratio mass spectrometry and high-resolution/high-accuracy mass spectrometry in metabolite identification studies: Detecting target compounds for sports drug testing Mario Thevis (1), Thomas Piper (1), Stevan Horning (2), Dieter Juchelka (2) and Wilhelm Schänzer (1) In sports drug testing, comprehensive studies on themetabolismof therapeutic agents with misuse potential are necessary to identify metabolites that provide utmost retrospectivity and specificity. By commonly employed approaches minor and/or long-term metabolites in urine might remain undetected. Hence, an alternative strategy to unambiguously identify the majority of urinary metabolites including low-abundance representatives is desirable. Urine samples were collected for 20 days during an elimination study with an oral dose of 5 mg of 17α-C2H3- metandienone. The specimens were processed according to established sample preparation procedures including fractionation and deconjugation) and subjected to gas chromatography/hydrogen isotope ratio mass spectrometry (GC/IRMS) analysis. Due to the deuteration of the administered drug, urinary metabolites bearing the deuterium label yield abundant and specific signals on the GC/IRMS instrument resulting from the substantially altered 2H/1H ratio. The sample aliquots were measured by gas chromatography/time-of-flight (GC/Q-TOF) mass spectrometry using identical GC conditions, allowing high-resolution/high-accuracy mass data to be obtained on all urinary metabolites previously identified by IRMS.