Azadniya Ebrahim, Mollergues Julie, Stroheker Thomas, Billerbeck Kathrin, Morlock Gertrud E
Chair of Food Science, Institute of Nutritional Science, and TransMIT Center of Effect-Directed Analysis, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392, Giessen, Germany.
Chemical Food Safety Group, Société des Produits Nestlé SA - Nestlé Research, Vers-chez-les-Blanc, Switzerland.
Anal Chim Acta. 2020 Sep 8;1129:76-84. doi: 10.1016/j.aca.2020.06.033. Epub 2020 Jul 8.
The activity of individual biotransformation products cannot be measured in multicomponent mixtures by the current status-quo assays. A prior separation and tedious isolation of compounds is required, and often in addition, a concentration step into a solvent suitable for the cell-/enzyme-based assay. Hence, the metabolizing S9 system, mimicking the complex biotransformation reactions in the liver, was aimed to be integrated into two orthogonal methods for analysis of the acetylcholinesterase (AChE) inhibition. For the microtiter plate assay method, the evaluation of the generated fluorescence signal was impaired by the incorporated S9 system. In contrast, the metabolic activator (S9 mixture) was successfully incorporated into the high-performance thin-layer chromatography (HPTLC) method. As proof of principle, four reference AChE inhibitors were studied in complex samples with and without metabolic activation. In addition to the neurotoxic carbamate eserine and the organophosphate insecticides chlorpyrifos, quinalphos and parathion, the tris(nonylphenyl) phosphite and nonylphenol, both originating from food contact materials, were tested in isolation but also in food packaging migrate and extract. A method comparison and benchmarking pointed to multifold advantages of using this newly developed bioanalytical tool for assessment of individual neurotoxins in complex samples. The sensitive HPTLC-S9-AChE assay allowed the detection of neurotoxic chemicals with and without metabolic activation, at levels consistent with the threshold of toxicological concern of organophosphates and carbamates. This new on surface metabolism system can be applied to other toxicities and samples.
在多组分混合物中，目前的常规检测方法无法测定单个生物转化产物的活性。需要事先对化合物进行分离和繁琐的提纯，而且通常还需要将其浓缩到适合基于细胞/酶的检测的溶剂中。因此，旨在将模拟肝脏中复杂生物转化反应的代谢S9系统整合到两种用于分析乙酰胆碱酯酶（AChE）抑制作用的正交方法中。对于微孔板检测方法，所加入的S9系统会干扰对产生的荧光信号的评估。相比之下，代谢激活剂（S9混合物）成功地整合到了高效薄层色谱（HPTLC）方法中。作为原理验证，研究了四种参考AChE抑制剂在有和没有代谢激活的复杂样品中的情况。除了神经毒性氨基甲酸酯类毒扁豆碱和有机磷杀虫剂毒死蜱、喹硫磷和对硫磷外，源自食品接触材料的亚磷酸三（壬基苯基）酯和壬基酚，不仅单独进行了测试，还在食品包装迁移物和提取物中进行了测试。方法比较和基准测试表明，使用这种新开发的生物分析工具评估复杂样品中的单个神经毒素具有多重优势。灵敏的HPTLC-S9-AChE检测方法能够检测有和没有代谢激活的神经毒性化学物质，其检测水平与有机磷和氨基甲酸酯类的毒理学关注阈值一致。这种新的表面代谢系统可应用于其他毒性和样品。
