title: Revisiting the metabolism of 19-nortestosterone using isotope ratio and high resolution/high accuracy mass spectrometry.
abstract: The synthetic anabolic androgenic steroid 19-nortestosterone is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA) due to its performance-enhancing effects. Today, doping controls focus predominantly on one main urinary metabolite, 19-norandrosterone glucuronide, which offers the required detection windows for an appropriate retrospectivity of sports drug testing programs. As 19-norandrosterone can also be found in urine at low concentrations originating from in situ demethylation of other abundant steroids or from endogenous production, the exogenous source of 19-norandrosterone needs to be verified, which is commonly accomplished by carbon isotope ratio analyses. The aim of this study was to re-investigate the metabolism of 19-nortestosterone in order to probe for additional diagnostic long-term metabolites, which might support the unambiguous attribution of an endo- or exogenous source of detected 19-nortestosterone metabolites. Employing a recently introduced strategy for metabolite identification, threefold deuterated 19-nortestosterone (16,16,17-(2)H3-NT) was administered to one healthy male volunteer and urine samples were collected for 20 days. Samples were prepared with established methods separating unconjugated, glucuronidated and sulfated steroids, and analytes were further purified by means of high-performance liquid chromatography before trimethylsilylation. Deuterated metabolites were identified using gas chromatograph/thermal conversion/isotope ratio mass spectrometer comprising an additional single quadrupole mass spectrometer. Additional structural information was obtained by gas chromatography/time-of-flight mass spectrometry and liquid chromatography/high resolution mass spectrometry. In general, sulfo-conjugated metabolites were excreted for a longer time period than the corresponding glucuronides. Several unexpected losses of the arguably stable isotope labels were observed and characterized, attributed to metabolic reactions and sample preparation procedures. The detection window of one of the newly detected metabolites was higher than currently used metabolites. The suitability of this metabolite to differentiate between endo- or exogenous sources could however not be verified conclusively.
authors.affiliation: German Sport University Cologne, Center for Preventive Doping Research-Institute of Biochemistry, Am Sportpark Müngersdorf 6, 50933 Köln, Germany. Electronic address: t.piper@biochem.dshs-koeln.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Piper
authors.affiliation: German Sport University Cologne, Center for Preventive Doping Research-Institute of Biochemistry, Am Sportpark Müngersdorf 6, 50933 Köln, Germany.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
authors.affiliation: German Sport University Cologne, Center for Preventive Doping Research-Institute of Biochemistry, Am Sportpark Müngersdorf 6, 50933 Köln, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
mesh_terms: D000284:Administration, Oral; D000328:Adult; D045930:Anabolic Agents / Q000008:administration & dosage / Q000378:metabolism* / Q000652:urine*; D002849:Chromatography, Gas / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004300:Doping in Sports; D004962:Estranes / Q000008:administration & dosage / Q000378:metabolism* / Q000652:urine*; D006801:Humans; D008297:Male; D058573:Performance-Enhancing Substances / Q000008:administration & dosage / Q000378:metabolism / Q000652:urine; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D045930:Anabolic Agents; D004962:Estranes; D058573:Performance-Enhancing Substances; C036015:19-norandrosterone
keywords: Doping; Mass spectrometry; Metabolism; Nortestosterone; Sport