Background

Phytoestrogens are found in many plants used in traditional medicines. Increasingly, plant extracts (botanicals) are also being added to foods or marketed as dietary supplements. Especially powder formulations are susceptible to adulteration and falsification, given the global processing chain. To detect estrogen-like compounds in such multicomponent mixtures, non-target screening for hormonally active or endocrine disrupting compounds in plant products is becoming more important. Unfortunately, the current planar yeast estrogen screen (pYES) is prone to zone diffusion on the normal-phase high-performance thin-layer chromatography (NP-HPTLC) plate due to long incubation times in the aqueous bioassay.

Purpose

The present study aimed to reduce zone diffusion on NP plates, which provides the basis for extending pYES to a multiplex bioassay, offering 4 different biological activity principles, followed by targeted identification of active zones.

Study design and methods

The reduction of substance diffusion via a polyisobutyl methacrylate polymer coating was studied. After successful zone fixation (^fix), a multiplex bioassay was developed, in which a 17β-estradiol-strip was applied along each sample track to detect synergists and antagonists (A), and for verification (V), a 4-methyl umbelliferone-strip to exclude false-positives. After multiplex bioassay screening of 68 botanicals, the zones with hormonal activities were heart-cut eluted to reversed-phase high-performance liquid chromatography−diode array detection−high-resolution tandem mass spectrometry (RP-HPLC–DAD–HESI-HRMS/MS).

Results

The separated substances were successfully fixed by the chromatogram coating. The zone sharpness (achieved after the bioassay) made it possible to add two strips, the 17β-estradiol-strip for antagonistic and synergistic, and the 4-methyl umbelliferone-strip for false-positive effect detection, resulting in a multiplex bioassay. Using the 12D hyphenation NP-HPTLC^fix–UV/Vis/FLD–pYAVES–FLD heart-cut RP-HPLC–DAD–HESI-HRMS/MS, it was possible to obtain information on estrogens, antiestrogens, false-positives, and synergists, and (tentatively) assign 16 hormonally active compounds, of which only 6 have been known to affect the human estrogen receptor, while another 4 had structural similarity to common phytoestrogens and antiestrogens.

Conclusions

The streamlined 12D hyphenation including a multiplex bioassay has been shown to differentiate hormonal effects, leading to new insights and better understanding. It can generally be used to identify unknown hormonally active compounds in complex samples.

中文翻译：

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多重平面生物测定法检测雌激素、抗雌激素、假阳性和增效剂作为正常阶段的尖锐区域

背景

植物雌激素存在于许多用于传统药物的植物中。越来越多的植物提取物（植物药）也被添加到食品中或作为膳食补充剂销售。考虑到全球加工链，尤其是粉末配方容易受到掺假和伪造的影响。为了检测此类多组分混合物中的雌激素样化合物，非目标筛选植物产品中的激素活性或内分泌干扰化合物变得越来越重要。不幸的是，目前的平面酵母雌激素筛选 (pYES) 容易在正相高效薄层色谱 (NP-HPTLC) 板上发生区域扩散，因为水生生物测定中的孵育时间长。

目的

本研究旨在减少 NP 板上的区域扩散，这为将 pYES 扩展到多重生物测定提供了基础，提供 4 种不同的生物活性原理，然后有针对性地识别活性区域。

研究设计和方法

研究了通过聚甲基丙烯酸异丁酯聚合物涂层减少物质扩散。成功进行区域固定 ( ^fix ) 后，开发了一种多重生物测定法，其中沿每个样品轨道应用 17β-雌二醇条以检测协同剂和拮抗剂 (A)，并为验证 (V) 使用 4-甲基伞形酮-剥离以排除误报。在对 68 种植物进行多重生物测定筛选后，将具有激素活性的区域中心切割洗脱到反相高效液相色谱-二极管阵列检测-高分辨率串联质谱 (RP-HPLC-DAD-HESI-HRMS/MS ）。

结果

分离的物质被色谱涂层成功固定。区域清晰度（在生物测定后实现）使得添加两条条带成为可能，17β-雌二醇条用于拮抗和协同作用，4-甲基伞形酮条用于假阳性效应检测，从而实现多重生物测定。使用 12D 连字符 NP-HPTLC ^fix –UV/Vis/FLD–pYAVES–FLD heart-cut RP-HPLC–DAD–HESI-HRMS/MS，可以获得有关雌激素、抗雌激素、假阳性和增效剂的信息，并且（暂时）指定了 16 种激素活性化合物，其中已知只有 6 种会影响人类雌激素受体，而另外 4 种与常见的植物雌激素和抗雌激素具有结构相似性。

结论

简化的 12D 连字符包括多重生物测定已被证明可以区分激素效应，从而带来新的见解和更好的理解。它通常可用于识别复杂样品中未知的激素活性化合物。