Identification of acetylcholinesterase inhibitors in water by combining two-dimensional thin-layer chromatography and high-resolution mass spectrometry

Effect-directed analysis (EDA) is increasingly used in environmental monitoring to detect and identify key toxicants. High-performance thin-layer chromatography (HPTLC) has proven to be a very suitable fractionation technique for this purpose. However, HPTLC is limited in its separation efficiency. Thus, separated fractions could still contain many different components and identification of the effective substances remains difficult. Therefore, in this study a workflow for selective EDA with two-dimensional HPTLC in combination with high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) was developed. The aim of the workflow was the stepwise reduction of the sample complexity in order to reduce the number of signals that could be responsible for the measured effects. As a consequence, the identification of effective substances should be facilitated. The acetylcholinesterase inhibition assay (AChE assay) for the detection of potential neurotoxic compounds was applied for biotesting. The transfer of effective zones from the first to the second dimension and also to the mass spectrometric measurement was enabled by extraction. A proof of concept was performed by spiking six acetylcholinesterase inhibiting substances into three different water matrices that were investigated with the developed workflow. The successful prioritization of all spiked compounds confirmed the efficiency of the workflow, regardless of the sample matrix. Biotesting of different water samples resulted in numerous potentially neurotoxic effects, which overlapped strongly in the first separation dimension. The higher peak capacity reached by two-dimensional HPTLC, on the other hand, resulted in discrete effective zones and enabled the identification of several compounds. For the substances lumichrome, a derivate of riboflavin and paraxanthine as well as for linear alkylbenzene sulfonates that were applied as anionic surfactants in detergents, the inhibiting effect to the AChE could be confirmed.