To analyze a complex sample for endocrine activity, different tests must be performed to clarify androgen/estrogen agonism, antagonism, cytotoxicity, anti-cytotoxicity, and corresponding false-positive reactions. This means a large amount of work. Therefore, a six-fold planar multiplex bioassay concept was developed to evaluate up to the mentioned six endpoints or mechanisms simultaneously in the same sample analysis. Separation of active constituents from interfering matrix via high-performance thin-layer chromatography and effect differentiation via four vertical stripes (of agonists and end-products of the respective enzyme–substrate reaction) applied along each separated sample track were key to success. First, duplex endocrine bioassay versions were established. For the androgen/anti-androgen bioassay applied via piezoelectric spraying, the mean limit of biological detection of bisphenol A was 14 ng/band and its mean half maximal inhibitory concentration IC was 116 ng/band. Applied to trace analysis of six migrate samples from food packaging materials, 19 compound zones with agonistic or antagonistic estrogen/androgen activities were detected, with up to seven active compound zones within one migrate. For the first time, the S9 metabolism of endocrine effective compounds was studied on the same surface and revealed partial deactivation. Coupled to high-resolution mass spectrometry, molecular formulas were tentatively assigned to compounds, known to be present in packaging materials or endocrine active or previously unknown. Finally, the detection of cytotoxicity/anti-cytotoxicity and false-positives was integrated into the duplex androgen/anti-androgen bioassay. The resulting six-fold multiplex planar bioassay was evaluated with positive control standards and successfully applied to one migrate sample. The streamlined stripe concept for multiplex planar bioassays made it possible to assign different mechanisms to individual active compounds in a complex sample. The concept is generic and can be transferred to other assays.

中文翻译：

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区分内分泌激动剂、拮抗剂、细胞毒性和假阳性反应的六重多重平面生物测定的概念


为了分析复杂样品的内分泌活性，必须进行不同的测试来阐明雄激素/雌激素的激动作用、拮抗作用、细胞毒性、抗细胞毒性和相应的假阳性反应。这意味着大量的工作。因此，开发了六重平面多重生物测定概念，以在同一样品分析中同时评估上述六个终点或机制。通过高性能薄层色谱法将活性成分与干扰基质分离，并通过沿着每个分离的样品轨道应用的四个垂直条纹（各自的酶-底物反应的激动剂和最终产物）进行效果区分是成功的关键。首先，建立了双重内分泌生物测定版本。对于压电喷雾应用的雄激素/抗雄激素生物测定，双酚A的生物检测平均限为14 ng/带，其平均半数抑制浓度IC 为116 ng/带。对食品包装材料中的 6 个迁移样品进行痕量分析，检测到 19 个具有激动或拮抗雌激素/雄激素活性的化合物区域，其中 1 个迁移区域内多达 7 个活性化合物区域。首次在同一表面上研究内分泌有效化合物的 S9 代谢并揭示了部分失活。结合高分辨率质谱分析，分子式被初步指定为已知存在于包装材料或内分泌活性或以前未知的化合物。最后，将细胞毒性/抗细胞毒性和假阳性的检测整合到双重雄激素/抗雄激素生物测定中。 使用阳性对照标准对所得六倍多重平面生物测定进行评估，并成功应用于一份迁移样品。多重平面生物测定的简化条纹概念使得为复杂样品中的单个活性化合物分配不同的机制成为可能。这个概念是通用的，可以转移到其他测定中。