Hebestreit Moritz, Flenker Ulrich, Fusshöller Gregor, Geyer Hans, Güntner Ute, Mareck Ute, Piper Thomas, Thevis Mario, Ayotte Christiane, Schänzer Wilhelm
Institute of Biochemistry, German Sport University Cologne, Carl-Diem Weg 6, Cologne, Germany.
Analyst. 2006 Sep;131(9):1021-6. doi: 10.1039/b603668f. Epub 2006 Jul 28.
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.
一方面，19-去甲雄酮（NA）是合成代谢类固醇19-去甲睾酮及相关激素原最丰富的代谢产物。另一方面，孕妇会生物合成少量的NA，并且有更多证据表明该化合物具有生理来源。世界反兴奋剂机构（WADA）此前设定每毫升尿液中NA的阈值浓度为2或5纳克，以区分19-去甲睾酮滥用与生物合成来源。然而，最近的研究结果表明，导致NA浓度处于阈值水平范围内的形成可能是由于尿液中雄酮的去甲基化，并且WADA 2006年禁用清单已将NA定义为内源性类固醇。为了阐明NA的内源性或外源性来源，(13)C/(12)C分析是首选方法，因为合成的19-去甲睾酮是通过部分合成从C(3)植物衍生而来，其δ(13)C(VPDB)值约为-28‰。由于饮食中含有C(3)植物和C(4)植物的混合物，内源性类固醇的(13)C消耗较少。基于两个高效液相色谱净化步骤的广泛净化方法应用于质量控制和兴奋剂检测样本，这些样本中NA的浓度低至每毫升尿液2纳克。通过气相色谱/燃烧/同位素比值质谱法测量NA、雄酮和表雄酮的(13)C/(12)C比值。通过将作为内源性参考化合物的雄酮的δ(13)C(VPDB)值与NA进行比较，确定兴奋剂检测样本中NA的来源是内源性还是外源性。
