Revisiting the metabolism of 19-nortestosterone using isotope ratio and high resolution/high accuracy mass spectrometry. Issue 162 (September 2016)

• Record Type: Journal Article
• Authors: Piper, Thomas; Schänzer, Wilhelm; Thevis, Mario
• Is Part Of: Journal of steroid biochemistry and molecular biology. Issue 162(2016)
• Page Start: 80
• Page End: 91
• Publication Date: 2016-09
• Subjects: Nortestosterone -- Metabolism -- Doping -- Mass spectrometry -- Sport; Steroid hormones -- Periodicals; Biochemistry -- Periodicals; Hormones -- Periodicals; Molecular Biology -- Periodicals; Hormones stéroïdes -- Périodiques; Steroid hormones; Periodicals; 572.579
• DOI: 10.1016/j.jsbmb.2015.12.013
• Languages: English
• ISSNs: 0960-0760

Abstract

Highlights: The metabolism of nortestosterone was studied using the deuterated substance. Metabolites were identified from human urine by means of hydrogen isotope ratio mass spectrometry and, where possible, characterized by GC–MS, GC-HRMS, and LC-HRMS(/MS). Artefact formation during commonly employed sample preparation steps was identified. Elimination of deuterium atoms by metabolic reactions were observed. Established metabolites such as norandrosterone glucuronide and sulfate were confirmed as best-possible options for doping control purposes. 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.