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.

Column bleed in the analysis of highly polar substances: an overlooked aspect in HRMS.

To close the "analytical gap" in the liquid chromatographic (LC) analysis of highly polar substances, two techniques which have been suggested earlier were tested in terms of retention factors and detection limits: hydrophilic interaction liquid chromatography (HILIC) and mixed-mode chromatography (MMC). A substance mix of 55 analytes ranging from logD - 8.2 to 3.4 and 17 different LC columns, also comprising additional reversed-phase columns were used. Contrary to most reversed-phase columns, column bleed has been identified as an important factor, which may cause serious restrictions during high-resolution mass spectrometric detection (HRMS). We found that highly abundant background masses continuously eluting from the columns heavily influence ion transmission to the detector. As a result, the linear dynamic range as well as the sensitivity decreases and thus limits the HRMS applicability of some columns. We therefore recommend a thorough investigation of ion transmission during HRMS method development. This will help to maintain the high potential of HRMS in terms of qualitative and quantitative screening analysis.

Advantage of liquid chromatography with high-resolution mass spectrometry for the detection of the small and polar molecules trifluoroacetic acid and sulfamic acid.

This study addresses the selectivity of liquid chromatography with tandem mass spectrometry for the analysis of the polar analytes trifluoroacetic acid and sulfamic acid in water samples. Such analytes often lack sufficient retention in standard reversed-phase liquid chromatography and rarely produce mass spectrometry transition products of high diagnostic evidence by collision-induced dissociation. The chromatography based on an extra-dense bonding liquid chromatography column for trifluoroacetic acid and on hydrophilic interaction chromatography for sulfamic acid. This enabled sufficient retention of more than twice the void volume for both analytes. Liquid chromatography coupled to low-resolution mass spectrometry was compared with high-resolution mass spectrometry. The limits of quantification for direct injection analysis were determined to <0.05 µg/L for trifluoroacetic acid and <0.5 µg/L for sulfamic acid. Correction of matrix effects either by internal standards or the standard addition is strongly recommended as matrix effects may vary strongly. The concentrations determined for trifluoroacetic acid and sulfamic acid for real samples were comparable between low and high-resolution mass spectrometry. However, low-resolution mass spectrometry does not fulfill the identification criteria of the analytes following the European Commission council directive 96/23/EC or the SANTE/11945/2015. Since low-resolution mass spectrometry for these analytes may result in false-positive findings, such findings may be controlled by high-resolution mass spectrometry.

LC-HRMS Data Processing Strategy for Reliable Sample Comparison Exemplified by the Assessment of Water Treatment Processes.

The behavior of micropollutants in water treatment is an important aspect in terms of water quality. Nontarget screening by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) offers the opportunity to comprehensively assess water treatment processes by comparing the signal heights of all detectable compounds before and after treatment. Without preselection of known target compounds, all accessible information is used to describe changes across processes and thus serves as a measure for the treatment efficiency. In this study, we introduce a novel LC-HRMS data processing strategy for the reliable classification of signals based on the observed fold changes. An approach for filtering detected features was developed and, after parameter adjustment, validated for its recall and precision. As proof of concept, the fate of 411 target compounds in a 0.1 µg/L standard mix was tracked throughout the data processing stages, where 406 targets were successfully recognized and retained during filtering. Potential pitfalls in signal classification were addressed. We found the recursive peak integration to be a key point for the reliable classification of signal changes across a process. For evaluating the repeatability, a combinatorial approach was conducted to verify the consistency of the final outcome using technical replicates of influent and effluent samples taken from an ozonation process during drinking water treatment. The results showed sufficient repeatability and thus emphasized the applicability of nontarget screening for the assessment of water treatment processes. The developed data processing strategies may be transferred to other research fields where sample comparisons are conducted.

Selective two-dimensional effect-directed analysis with thin-layer chromatography.

There are thousands of organic trace substances in the environment that are not fully characterized, and evaluation of their relevance to the ecosystem is difficult. Effect-directed analysis (EDA) is a suitable tool to assess the effects of a substance via in-vitro bioassays, which can provide information about the relevance of the substance. High-performance thin-layer chromatography (HPTLC) has been shown to be a good method for fractionation. Environmental samples, however, often have high complexity, which is why the peak capacity of HPTLC is not sufficient. Therefore, this study focused on the development of selective two-dimensional (2D) HPTLC-EDA to increase the peak capacity and facilitate the identification of effective compounds. Thus, only effective zones were selected in the first dimension in terms of heart-cutting and were transferred to the second dimension through elution head-based extraction. Three 2D approaches were developed and validated. The best results in terms of peak capacity and orthogonality were achieved when the retardation factors of the first dimension were used to adjust the mobile phase (MP) for the second dimension. Applying the acetylcholinesterase (AChE) inhibition assay as an example EDA, analysis of spiked surface water by 2D HPTLC-EDA allowed zones with neurotoxic effects to responsible substances to be assigned. The 2D separation reduced the complexity of effective zones and thus facilitated the subsequent identification of effective compounds. Knowledge about a substancés effects enabled assessment of its relevance to the environment.

The reciprocal iso-inhibition volume concept: A procedure for the evaluation in effect-directed analysis with thin-layer chromatography - using the thin-layer chromatography-luminescent bacteria assay as an example.

In effect-directed analysis (EDA) with high-performance thin-layer chromatography (HPTLC), the effect is often detected using images. Thus, an approach to create inhibition chromatograms from these images was developed using the example of the HPTLC- bioluminescence inhibition test. A comparison between the cuvette test and the HPTLC test shows that the test on the plate is significantly more sensitive. To describe the strength of the effect, the EC50 value is determined from the dose-response relationship. However, the inhibiting compounds are generally unknown and thus their concentrations are also unknown. Therefore, instead of the concentration, the known application volumes are used. This enables the calculation of the application volume necessary to achieve 50% inhibition. Since the volume is inversely proportional to the concentration, the reciprocal value of the calculated volume is indicated and is referred to as the reciprocal iso-inhibition volume (RIV). Using this RIV-concept, it is now possible to compare inhibition bands within and between plates. The entire evaluation is described by the means of two samples from a contaminated site using the bioluminescence inhibition.

A survey on trace organic chemicals in a German water protection area and the proposal of relevant indicators for anthropogenic influences.

A comprehensive monitoring programme of trace organic chemicals (TOrC) was conducted for a German water protection area in karstic ground. The aim of this survey was to detect the potential anthropogenic influences of point sources such as wastewater treatment plants and diffuse pollution such as runoff water from roads on the raw water used for drinking water treatment. The programme comprised seven sampling campaigns within 2 years each with up to 20 sampling sites. In total, the programme included 84 anthropogenic compounds from pharmaceuticals, iodinated X-ray contrast media, sweeteners, industrial chemicals (benzotriazoles, melamines and benzothiazoles) and pesticide metabolites. Cyclamate occurred with the highest median concentration of 44 µg l-1 in untreated wastewater and acesulfame occurred with a concentration of 20 µg l-1 in treated wastewater. In runoff water from roads, the most relevant compounds were tolyltriazole with 2.3 µg l-1 and the desphenyl-chloridazon with 1.2 µg l-1. In the stream waters, the highest median concentrations were found for melamine and acesulfame both at 0.61 µg l-1. High elimination during conventional wastewater treatment was observed for 5 out of 49 compounds. These are acetyl-sulfamethoxazole, aciclovir, cyclamate, ibuprofen and saccharin. Based on the survey results, we propose a set of nine compounds to be used as indicators for wastewater, untreated wastewater and runoff water from roads for an efficient surveillance. The indicators are intended to detect anthropogenic influences in surface, ground and drinking water.

General strategies to increase the repeatability in non-target screening by liquid chromatography-high resolution mass spectrometry.

This article focuses on the data evaluation of non-target high-resolution LC-MS profiles of water samples. Taking into account multiple technical replicates, the difficulties in peak recognition and the related problems of false positive and false negative findings are systematically demonstrated. On the basis of a combinatorial approach, different models involving sophisticated workflows are evaluated, particularly with regard to the repeatability. In addition, the improvement resulting from data processing was systematically taken into consideration where the recovery of spiked standards emphasized that real peaks of interest were barely or not removed by the derived filter criteria. The comprehensive evaluation included different matrix types spiked with up to 263 analytical standards which were analyzed repeatedly leading to a total number of more than 250 injections that were incorporated in the assessment of different models of data processing. It was found that the analysis of multiple replicates is the key factor as, on the one hand, it provides the option of integrating valuable filters in order to minimize the false positive rate and, on the other hand, allows correcting partially false negative findings occurring during the peak recognition. The developed processing strategies including replicates clearly point to an enhanced data quality since both the repeatability as well as the peak recognition could be considerably improved. As proof of concept, four different matrix types, including a wastewater treatment plant (WWTP) effluent, were spiked with 130 isotopically labeled standards at different concentration levels. Despite the stringent filter criteria, at 100 ng L(-1) recovery rates of up to 93% were reached in the positive ionization mode. The proposed model, comprising three technical replicates, filters less than 5% and 2% of the standards recognized at 100 and 500 ng L(-1), respectively and thus indicates the general applicability of the presented strategies.