ONIRIS, USC 2013, LABERCA, Atlanpole-La Chantrerie, BP 50707, Nantes F-44307, France.
J Chromatogr A. 2010 Oct 22;1217(43):6652-60. doi: 10.1016/j.chroma.2010.04.062. Epub 2010 May 5.
The use of steroid hormones as growth promoters in cattle has been banned within the European Union since 1988 but can still be fraudulently employed in animal breeding farms for anabolic purposes. If an efficient monitoring of synthetic compounds (screening and confirmation) is ensured today by many laboratories, pointing out suspicious samples from a natural steroids abuse remains a tricky challenge due to the difficulty to set relevant threshold levels for these endogenous compounds. The development of focused profiling or untargeted metabolomic approaches is then emerging in this context, with the objective to reveal potential biomarkers signing an exogenous administration of such natural steroids. This study aimed to assess sample preparation procedures based on microextraction and adapt them to high throughput urinary profiling or metabolomic analyses based on gas chromatography-mass spectrometry measurement. Two techniques have been tested and optimised, namely solid phase microextraction (SPME) and microextraction by packed sorbent (MEPS), using five model steroid metabolites (16α-hydroxyandrosterone, 2α-hydroxytestosterone, 11-keto,5β-androstanedione, 6α-hydroxyestradiol and 7β-hydroxypregnenolone). The considered performance criteria included not only the absolute response of the targeted compounds but also the robustness of the materials, and the global aspect of the diagnostic ion chromatograms obtained. After only five successive urinary extractions, a clear degradation of the SPME fiber was observed which led to discard this method as a relevant technique for profiling, whereas no degradation was observed on MEPS sorbent. Repeatability and recovery yields were calculated from urine samples fortified at 500 μg L⁻¹ and extracted by MEPS. They were found respectively below 11% and above 60% for all model compounds. Detection limits were in the 5-15 μg L⁻¹ range depending on the compounds, and a good linearity was observed on the 10-75 μg L⁻¹ range (R² > 0.99). This methodology was applied on urine samples collected from control versus androstenedione-treated bovines, revealing a significant concentration increase for several well-known metabolites such as etiocholanolone, 5α-androstane-3β,17α-diol, 5β-androstane-3α,17α-diol and 5-androstene-3β,17α-diol. Finally, these results allowed to confirm the suitability of the developed strategy and give to this new MEPS application a promising interest in the field of GC-MS based steroid profiling and metabolomic.
自 1988 年以来，欧盟已禁止在牛中使用类固醇激素作为生长促进剂，但在动物养殖场中仍可能被滥用于合成代谢目的。如果今天许多实验室都能确保对合成化合物进行有效的监测（筛选和确认），那么指出天然类固醇滥用的可疑样本仍然是一个棘手的挑战，因为为这些内源性化合物设定相关的阈值水平非常困难。因此，在这种情况下，有针对性的分析或非靶向代谢组学方法的发展正在出现，其目的是揭示潜在的生物标志物，这些标志物表明存在外源给予此类天然类固醇的情况。本研究旨在评估基于微萃取的样品制备程序，并对基于气相色谱-质谱测量的高通量尿液分析或代谢组学分析进行调整。已经测试和优化了两种技术，即固相微萃取（SPME）和填充吸附剂微萃取（MEPS），并使用五种模型类固醇代谢物（16α-羟基雄烯二酮、2α-羟基睾酮、11-酮、5β-雄烷二酮、6α-羟基雌二醇和 7β-羟基孕烯醇酮）。所考虑的性能标准不仅包括目标化合物的绝对响应，还包括材料的稳健性以及获得的诊断离子色谱图的整体方面。经过五次连续的尿液提取后，明显观察到 SPME 纤维降解，这导致该方法不能作为分析方法，而 MEPS 吸附剂则没有观察到降解。通过 MEPS 提取并在 500μg L⁻¹下进行了尿液样品的加标，计算了重复性和回收率。对于所有模型化合物，它们分别低于 11%和高于 60%。检测限取决于化合物，范围在 5-15μg L⁻¹之间，在 10-75μg L⁻¹范围内具有良好的线性关系（R²>0.99）。该方法应用于从对照与雄烯二酮处理的牛中收集的尿液样品，揭示了几种知名代谢物的浓度显著增加，例如表雄酮、5α-雄烷-3β,17α-二醇、5β-雄烷-3α,17α-二醇和 5-雄烯-3β,17α-二醇。最后，这些结果证实了所开发策略的适用性，并为新的 MEPS 应用在基于 GC-MS 的类固醇分析和代谢组学领域赋予了广阔的前景。
