Chair of Food Science, Institute of Nutritional Science, and TransMIT Center for Effect-Directed Analysis, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392, Giessen, Germany.
Chair of Food Science, Institute of Nutritional Science, and TransMIT Center for Effect-Directed Analysis, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392, Giessen, Germany.
Anal Chim Acta. 2020 Aug 15;1125:288-298. doi: 10.1016/j.aca.2020.05.057. Epub 2020 May 26.
Xenoestrogens exert antiandrogenic effects on the human androgen receptor. In the analytical field, such antagonists block the detection of testosterone and falsify results obtained by sum parameter assays. Currently, such agonistic versus antagonistic effects are not differentiated in complex mixtures. Oppositely acting hormonal effects present in products of everyday use can only be differentiated after tedious fractionation and isolation of the individual compounds along with subjection of each fraction/compound to the status quo bioassay testing. However, such long-lasting procedures are not suited for routine. Hence, we developed a fast bioanalytical tool that figures out agonists versus antagonists directly in complex mixtures. Exemplarily, 8 cosmetics and 15 thermal papers were analyzed. The determined antagonistic potentials of active compounds found were comparable to the ones of known antagonists (in reference shown for bisphenol A, 4-n-nonylphenol and four parabens). Relevant biological/chromatographic parameters such as cell viability, culture conditions, dose response curves, limits of biological detection/quantification and working range (shown for testosterone, dihydrotestosterone, nandrolone and trenbolone) were investigated to obtain the best sensitivity of the biological detection. The developed and validated method was newly termed reversed phase high-performance thin-layer chromatography planar yeast ant-/agonistic androgen screen (RP-HPTLC-pYAAS bioassay). Results were also compared with the RP-HPTLC-Aliivibrio fischeri bioassay (applied on RP plates for the first time). As proof-of-concept, the transfer to another bioassay (RP-HPTLC-pYES) was successfully demonstrated, analogously termed RP-HPTLC-pYAES bioassay detecting anti-/estrogens (exemplarily shown for evaluation of 4 pharmaceuticals used in breast cancer treatment). The new imaging concept provides (1) detection and differentiation of individual agonistic versus antagonistic effects in the bioprofiles, (2) bioanalytical quantification of their activity potential by scanning densitometry and (3) characterization of unknown bioactive compound zones by hyphenation to high-resolution mass spectrometry. Depending on the hormonal bioassay, 15 samples were analyzed in parallel within 5 h or 6 h (calculated as 20 or 24 min per sample). For the first time, piezoelectric spraying of the yeast cells was successfully demonstrated for the planar yeast-based bioassays.
外源性雌激素对人雄激素受体具有抗雄激素作用。在分析领域，这种拮抗剂会阻断对睾酮的检测，并伪造通过总和参数检测获得的结果。目前，在复杂混合物中无法区分这种激动剂与拮抗剂的作用。在日常用品中存在的具有相反作用的激素效应，只能在繁琐的分离和个别化合物的分离之后进行区分，并且需要对每个馏分/化合物进行现状生物测定测试。但是，这种持久的程序不适合常规使用。因此，我们开发了一种快速的生物分析工具，可以直接在复杂混合物中区分激动剂和拮抗剂。例如，对 8 种化妆品和 15 种热敏纸进行了分析。所确定的活性化合物的拮抗潜力与已知拮抗剂（如双酚 A、4-壬基苯酚和四种对羟基苯甲酸酯）的拮抗潜力相当。研究了相关的生物学/色谱参数，如细胞活力、培养条件、剂量反应曲线、生物检测/定量的下限和工作范围（以睾酮、二氢睾酮、诺龙和 trenbolone 为例），以获得生物检测的最佳灵敏度。开发并验证的方法被重新命名为反相高效薄层色谱平面酵母抗/激动雄激素筛选（RP-HPTLC-pYAAS 生物测定）。结果还与反相高效薄层色谱-发光菌生物测定（首次应用于 RP 板）进行了比较。作为概念验证，成功地将其转移到另一个生物测定（RP-HPTLC-pYES），同样命名为 RP-HPTLC-pYAES 生物测定，用于检测抗/雌激素（例如，用于评估用于乳腺癌治疗的 4 种药物）。新的成像概念提供了 (1) 在生物谱中检测和区分单个激动剂与拮抗剂的作用，(2) 通过扫描密度法对其活性潜力进行生物分析定量，(3) 通过与高分辨率质谱联用对未知生物活性化合物区域进行表征。根据激素生物测定，在 5 小时或 6 小时内（计算为每个样品 20 或 24 分钟）可以同时分析 15 个样品。首次成功地为基于平面酵母的生物测定展示了压电喷雾细胞。
