Determination of 13C/12C ratios of endogenous urinary steroids:: method validation, reference population and application to doping control purposes
Determination of 13C/12C ratios of endogenous urinary steroids:: method validation, reference population and application to doping control purposes
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DOI：
10.1002/rcm.3601
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发表时间：
2008-07-01
影响因子：
2
通讯作者：
Schaenzer, Wilhelm
中科院分区：
文献类型：
--
作者：
Piper, Thomas;Mareck, Ute;Schaenzer, Wilhelm
介绍了一种基于气相色谱/燃烧/同位素比值质谱（GC/C/IRMS）的尿中内源性类固醇稳定碳同位素的综合分析方法。样品制备中的关键要素是用高效液相色谱法（HPLC）对未衍生化和乙酰化的类固醇进行连续净化，这允许分离10种分析物（11 β-羟基雄甾酮，5 α-雄甾-16-烯-3 β-醇，孕甾烷酮，雄甾酮，胆甾烷酮，睾酮，表雄酮，5 α-雄甾烷-3 α，17 β-二醇，5 β-雄甾烷-3 α，17 β-二醇和脱氢表雄酮）。这些类固醇对兴奋剂控制特别重要，因为它们可以灵敏地追溯检测运动员滥用类固醇。根据生物背景，10 mL样本中所有类固醇的测定限为5至10 ng/mL。该方法通过每种类固醇的线性混合模型进行验证，其中包括重复性和再现性。通过气相色谱/质谱法（GC/MS）进一步证明了每种分析物的专属性，未观察到样品制备或分析物量对碳同位素比的影响。为了确定所有实施的类固醇的天然存在的C-13/C-12比率，测量了n=61名受试者的参考人群，以能够计算所有相关类固醇δ值的参考限值。版权所有（C）2008约翰威利父子有限公司
The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)-based method for stable carbon isotopes of endogenous urinary steroids is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography (HPLC) of underivatized and acetylated steroids, which allows the isolation of ten analytes (11 beta-hydroxyandrosterone, 5 alpha-androst-16-en-3 beta-ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5 alpha-androstane-3 alpha,17 beta-diol, 5 beta-androstane-3 alpha,17 beta-diol and dehydroepiandrosterone) from a single urine specimen. These steroids are of particular importance to doping controls as they enable the sensitive and retrospective detection of steroid abuse by athletes.Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers repeatability and reproducibility. Specificity was further demonstrated by gas chromatography/mass spectrometry (GC/MS) for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring C-13/C-12 ratios of all implemented steroids, a reference population of n=61 subjects was measured to enable the calculation of reference limits for all relevant steroidal Delta values. Copyright (C) 2008 John Wiley & Sons, Ltd.