Two‐dimensional high performance liquid chromatography purification of underivatized urinary testosterone and metabolites for compound‐specific stable carbon isotope analysis,Drug Testing and Analysis

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Two‐dimensional high performance liquid chromatography purification of underivatized urinary testosterone and metabolites for compound‐specific stable carbon isotope analysis
Drug Testing and Analysis ( IF 2.6 ) Pub Date : 2020-11-05 , DOI: 10.1002/dta.2964
Karine Lalonde ₁ , Andrew Barber ₁ , Christiane Ayotte ₁

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Testosterone doping in sports is detected through the measurement of the carbon isotopic signature (δ¹³C) of testosterone and its metabolites in urine. A critical step in achieving accurate and precise δ¹³C values during compound‐specific stable carbon isotope analysis (CSIA) is the removal of interfering matrix components. To this end, the World Anti‐Doping Agency (WADA) recommends the use of high‐performance liquid chromatography (HPLC) as a method of sample pretreatment. We provide a description of an automated two‐dimensional HPLC (2D‐HPLC) purification method for urine extracts that has made possible the CSIA of underivatized steroids, requiring only 36 min per sample. Eight urinary steroids including testosterone (T) and dehydroepiandrosterone (DHEA) and four of their metabolites as well as two endogenous reference compounds were collected during HPLC purification. Comparative GC chromatograms are used to contrast the efficiency of two‐dimensional (2D) purification to a previously established 1D‐HPLC method. The 2D purification leads to improved sample purity while simultaneously decreasing the analysis time, allowing for unprecedented sample throughput. Precision of δ¹³C for all analyzed compounds in negative and positive controls was 0.5‰ or better, which is comparable with the precision of pure reference materials at similar intensities.

中文翻译：

用于化合物特异性稳定碳同位素分析的未衍生化尿睾酮及其代谢物的二维高效液相色谱纯化

通过测量尿液中睾酮及其代谢物的碳同位素特征 (δ ¹³ C)来检测运动中的睾酮兴奋剂。实现准确和精确 δ ^{13 的}关键步骤化合物特定稳定碳同位素分析 (CSIA) 期间的 C 值是干扰基质成分的去除。为此，世界反兴奋剂机构 (WADA) 建议使用高效液相色谱 (HPLC) 作为样品预处理方法。我们描述了一种用于尿液提取物的自动化二维 HPLC (2D-HPLC) 纯化方法，该方法使未衍生化类固醇的 CSIA 成为可能，每个样品只需要 36 分钟。在 HPLC 纯化过程中收集了八种尿类固醇，包括睾酮 (T) 和脱氢表雄酮 (DHEA) 及其四种代谢物以及两种内源性参考化合物。比较 GC 色谱图用于对比二维 (2D) 纯化与先前建立的 1D-HPLC 方法的效率。二维纯化可提高样品纯度，同时缩短分析时间，实现前所未有的样品通量。δ的精度阴性和阳性对照中所有分析化合物的¹³ C 为 0.5‰ 或更高，这与类似强度的纯参考材料的精密度相当。

更新日期：2020-11-05

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