Determination of the origin of urinary norandrosterone traces by gas chromatography combustion isotope ratio mass spectrometry

Abstract

On the one hand, 19-norandrosterone (NA) is the most abundant metabolite of the synthetic anabolic steroid 19-nortestosterone and related prohormones. On the other hand, small amounts are biosynthesized by pregnant women and further evidence exists for physiological origin of this compound. The World Anti-Doping Agency (WADA) formerly introduced threshold concentrations of 2 or 5 ng of NA per ml of urine to discriminate 19-nortestosterone abuse from biosynthetic origin. Recent findings showed however, that formation of NA resulting in concentrations in the range of the threshold levels might be due to demethylation of androsterone in urine, and the WADA 2006 Prohibited List has defined NA as endogenous steroid. To elucidate the endogenous or exogenous origin of NA, (13)C/(12)C-analysis is the method of choice since synthetic 19-nortestosterone is derived from C(3)-plants by partial synthesis and shows delta(13)C(VPDB)-values of around -28 per thousand. Endogenous steroids are less depleted in (13)C due to a dietary mixture of C(3)- and C(4)-plants. An extensive cleanup based on two high performance liquid chromatography cleanup steps was applied to quality control and doping control samples, which contained NA in concentrations down to 2 ng per ml of urine. (13)C/(12)C-ratios of NA, androsterone and etiocholanolone were measured by gas chromatography/combustion/isotope ratio mass spectrometry. By comparing delta(13)C(VPDB)-values of androsterone as endogenous reference compound with NA, the origin of NA in doping control samples was determined as either endogenous or exogenous.

Recent developments in the use of isotope ratio mass spectrometry in sports drug testing

“…These fortified urine samples were extracted and the δ 13 C values obtained were compared with the values of the standards used for fortification [58]. A comparable approach was used to check for isotopic fractionation during sample preparation of steroids not regularly found in urine such NORA and BO or degradation products [65,82,84]. It was possible to show that no significant changes in CIR were introduced by different sample preparation procedures.…”

“…A CIR method was needed to distinguish these cases from those arising from administration of nandrolone or related prohormones. Sample preparation was extended to a twofold HPLC clean-up using two different columns [65]. The first investigations on the influence of diet on CIR of endogenous steroids were published at that time [66,67] together with research on interindividual variability of CIR after T administration [68].…”

Doping im Behindertensport

“…nichtbehinderter Sportler, aber auch in der Tiermast) nicht ausschließlich für die Analytik im Behindertensport etabliert und bereits seit vielen Jahren im Einsatz. Zum einen wird mit Hilfe der Isotopenverhältnismassenspektrometrie ("isotope ratio mass spectrometry" [IRMS]) die körpereigene Herkunft von 19-Norandrosteron und Boldenon bestimmt [12], zum anderen kann durch den Einsatz isotopenmarkierter (deuterierter) Substanzen eine "Bakterienaktivität" in Urin überprüft werden [10], und der Einsatz alternativer interner Standards bei Bestätigungsana-lysen kann eine Generierung auffälliger Metaboliten des Methyltestosterons verhindern.…”

Profiling of 19-norsteroid sulfoconjugates in human urine by liquid chromatography mass spectrometry

“…In 2006, androgenic anabolized steroids (AAS) corresponded to 45.4% of all adverse analytical findings in anti-doping controls and in particular nandrolone concentration level may be directly confirmed by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) [4].…”