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Development of strategies for the prioritization of organic trace substances in water by effect-directed analysis
(2020) Stütz, Lena; Schwack, Wolfgang
The protection of the aquatic environment and the supply of clean drinking water to people all over the world are central challenges of our time. Monitoring of the aquatic environment and the input of anthropogenic trace substances into it is therefore very important. However, since aquatic environmental samples often consist of complex substance mixtures, their characterization and evaluation is very demanding. By using generic target analysis methods, selected known anthropogenic trace substances can be detected and quantified very sensitively. For the detection of previously unknown substances, non-target analysis methods have been increasingly used in recent years. However, these methods do not provide information on the relevance of the anthropogenic trace substances occurring in water. In this context, especially all those trace substances are regarded as relevant from which a harmful effect on humans or water organisms is to be expected. For the detection of such effective substances, effect-directed analysis (EDA) can be used. In EDA, a bioassay is combined with a fractionation method and subsequent chemical analysis, the aim being to identify the bioactive substance. The separation method used in this work is high-performance thin-layer chromatography (HPTLC). After chromatography, the bioassay is performed directly on the HPTLC plate. If an effective zone appears in the bioassay, a prioritization strategy is used to clarify the identity of the substance. Due to the complex aquatic samples, a large number of different substances in a zone must still be expected despite the applied HPTLC separation, which makes it difficult to identify the effective substance. Therefore, a strategy to simplify the identification of effective substances should be developed. The aim was to reduce the complexity by multidimensional separation in such a way that chemical analysis can be used to prioritize to a few candidates in the effective fraction. In the first part of the work, a selective two-dimensional HPTLC separation was developed to reduce the number of substances in a bioactive zone. After the first separation dimension (1D) the acetylcholinesterase inhibition assay (AChE assay) was performed and afterwards only the effective zones were extracted from the HPTLC plate. The selected effective zones were separated in a second separation dimension (2D) and the bioassay was performed again. With this 2D separation, the peak capacity could be increased by a factor of 7 compared to a 1D HPTLC gradient development. If real water samples are examined for their effects, an additional structural elucidation must be carried out to clearly identify the unknown bioactive substances. In this work, the developed 2D EDA was therefore connected to a high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS) and a non-target screening (NTS) was performed. Using three water samples(drinking water, surface water and purified sewage water) spiked with six effective substances, it was shown that the developed strategy is suitable for the identification of effective substances and that these can be recovered despite repeated extraction. When applying the developed methodology to real samples, it was also possible to assign and quantify the detected effect in several waters to the substance lumichrome and to linear alkylbenzene sulfonates. Genotoxicity is a crucial endpoint for the effect assessment of water samples. However, this endpoint with metabolic activation cannot yet be performed directly on the HPTLC plate. Since many of the genotoxic substances have an indirect genotoxic effect, i.e. they only acquire their activity after metabolic activation; this endpoint was investigated in the present work with the umu assay in the microtiter plate. However, separation with HPTLC, subsequent extraction of effective zones and non-target analysis of the extracts, should also be performed for this assay. Therefore the umu assay in the microtiter plate was integrated into the existing EDA-with-HPTLC concept. In laboratory experiments, sodium hypochlorite was added to the drug metformin in order to simulate the behavior of the substance during water treatment (chlorination). The metformin sample treated with hypochlorite was examined with the umu assay and a genotoxic effect was detected. After HPTLC separation of the chlorinated sample, zones were extracted over the entire retardation range. When the extracted zones were examined with the umu assay, the genotoxic effect could be clearly assigned to one fraction. Using high-resolution mass spectrometry, the genotoxic effect could be assigned to an already known transformation product of metformin. The HPTLC separation and extraction of the zones from the plate led to a reduction of the possible effective candidate masses by a factor of 10 and thus to a clear prioritization in HRMS analysis.