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Institut für Lebensmittelchemie

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Browsing Institut für Lebensmittelchemie by Subject "Bioassay"

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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.
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    Rapid screening of antibiotics in foods by HPTLC-FLD/EDA/MS
    (2015) Chen, Yisheng; Schwack, Wolfgang
    Nowadays, the usage and partly abuse of veterinary antibiotics resulted in a very pressing need to control residues in foods of animal origin. Particularly, the increasingly demanding MRL issues and the huge number of samples to be monitored raised great challenges in this field. Microbial growth inhibition assays are traditionally employed for screening purposes, while sophisticated HPLC-MS methods are alternatively used or only used for confirmation purposes. To substitute the time consuming growth inhibition assays, HPTLC as a platform hyphenated to multi detection modes was employed in this study for the development of a high throughput, sensitive and cost-efficient screening-oriented methodology for antibiotics residues. The first step was focused on tetracyclines and fluoroquinolones, which are the most problematic antibiotics in the European Union and account for the most of the used veterinary antibiotics. To prevent strong tailing effects, the separation was optimized on normal-phase silica gel plates modified with ethylenediamine tetraacetic acid (EDTA). Besides, selective and sensitive fluorescence densitometry was optimized to achieve best signal/noise ratios. Under these conditions, limits of detection (LODs) and quantitation (LOQs) were in the range 12–25 and 45–95 µg/kg, respectively. Recoveries from milk samples, spiked at 50, 100 and 150 µg/kg and extracted by a modified QuEChERS procedure, ranged from 76 to 105%. To circumvent the ion suppressions due to EDTA, HPTLC-mass spectrometry (HPTLC-MS) was optimized, allowing the selective confirmation of positive findings, also offering high sensitivity of 25 µg/kg, and meeting Commission Regulation (EU) No. 37/2010. In the second step, sulfonamides were targeted, which are the secondly most administered veterinary antibiotics in the European Union. Separation of twelve most important sulfonamides was achieved on HPTLC silica gel plates, followed by fluram derivatization and sensitive and selective quantitation by fluorescent densitometry. LODs and LOQs were determined to 15–40 and 35–70 µg/kg, respectively. Samples of bovine milk, porcine liver and kidney were extracted according to the “QuEChERS” strategy. Additionally, a confirmative detection by HPTLC-MS was optimized, offering straightforward identification of target zones. The method was validated to meet the enforced Commission Regulation (EU) No. 37/2010. Finally, a more universal screening method based on HPTLC-bioautography was developed for most of the first-line veterinary antibiotics. A comprehensive HPTLC plate test revealed that the bio-compatibility of different plate layer materials to the applied bioluminescent bacteria (A. fischeri DSM No. 7151) was surprisingly different. It was then discovered that both bright bioluminescent background and significant inhibition zones of antibiotics can only be achieved on HPTLC amino F254S plates. In this case, HPTLC was not used for the chromatographic separation of individual antibiotics extracted with acetonitrile, but in terms of planar solid phase extraction to separate bioactive matrix compounds and to focus the analytes within two distinct target zones of different polarity. Together with HPTLC-MS for identification and confirmation purposes, the developed procedure enabled the rapid, sensitive and efficient multi-class screening of antibiotic residues (16 species of 5 groups, except sulfonamides and penicillins, which only affect Gram positive bacteria). The multi-sample plate images provided the results within a few hours. Thanks to the high sensitivity and the great matrix tolerance, the established method was successfully applied to bovine milk and porcine kidney samples, each spiked at the EU MLRs.