New incorporation of the S9 metabolizing system into methods for detecting acetylcholinesterase inhibition.

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.