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A frequently studied environmental contaminant is the active substance diclofenac, which is removed insufficiently in sewage treatment plants. Since its inclusion in the watch list of the EU Water Framework Directive, the concentrations in surface waters will be determined throughout Europe. For this, still, more precise analytical methods are needed. As a reference, HPLC-MS is frequently employed. One of the major metabolites is 4’-hydroxydiclofenac (4’-OH-DCF). Also, diclofenac lactam is important for assessing degradation and transformation. Aceclofenac (ACF), the glycolic acid ester of diclofenac is used as a drug, too, and could potentially be cleaved to yield diclofenac again. In various sewage treatment plant influent samples, diclofenac, 4’-OH-DCF, DCF lactam and ACF could be determined with detection limits of 3 µg/L, 0.2 µg/L, 0.17 µg/L and 10 ng/L, respectively.
Studies on the development of antibodies for the highly hydrophobic plasticizers DINCH and DEHT
(2018)
Diisononylcyclohexane-1,2-dicarboxylate (DINCH) and di-2-ethylhexyl terephthalate (DEHT), two of the most important substitutes for phthalate plasticizers, are used for a wide range of applications. Consequently, an increasing occurrence in urine and environmental samples is reported. Reliable and fast analytical methods for the quantification of these plasticizers are needed. So far, mainly GC-MS or LC-MS methods are used. We aimed to develop the first antibodies and immunoassays allowing for high-throughput analysis of samples. We designed two DINCH hapten structures and one DEHT hapten structure and employed hapten-protein conjugates for the immunization of rabbits. Sensitive competitive enzyme-linked immunosorbent assays (ELISAs) against each hapten using the produced polyclonal antibodies were established. Yet, binding of DINCH to the respective antibodies was not observed in neither direct nor indirect assay formats, even when using protein conjugates with the heterologous haptens and different carrier proteins in the indirect format. The use of surfactants and solvents in the sample buffer did not result in recognition of the plasticizers. Also, no binding of DEHT in ELISA employing the respective antibodies was detected. We speculate that the production of antibodies against these highly hydrophobic molecules is not possible via our route, however a different hapten design could overcome this obstacle.
For many heterogeneous sensor applications as well as the synthesis of hapten antigens to produce antibodies, protein conjugates of the target substance are essential. A requirement is that the target substance already offers or is modified to contain a functionality that allows for coupling to a protein, that is, an amino acid residue. Ideally, to avoid shielding of the compound by the carrier protein, a sufficient distance to the protein surface should be provided. With its carboxyl function diclofenac (DCF) allows for direct binding to lysine residues after in situ synthesis of the NHS ester. One problem is that diclofenac as free acid tends to autocondensation, which results in low yields. Here we describe the 'insertion' of a C6 spacer via synthesis of the amide with 6-aminohexanoic acid. To carry out the reaction in solution, first the methyl ester of the amino acid had to be produced. Due to otherwise low yields and large cleaning efforts, solid-phase synthesis on Fmoc Ahx Wang resin is recommended. The crude product is mainly contaminated by cleavage products from the resin which were removed by chromatography. The structure of the highly pure hapten was completely determined by nuclear magnetic resonance (NMR) spectroscopy.
Immunoassays are suitable tools for high-throughput screenings. The prerequisite for accurate determinations by these methods is the selection of an excellent antibody. The production and selection of monoclonal antibodies is usually a tedious process. In this study, new strategies for improving antibody production and characterization were applied. This includes the monitoring of the immunization progress in mice through antibodies extracted from feces, which allows a time-resolved and animal-friendly monitoring of the immune response. Additionally, fluorescence polarization immunoassay (FPIA) could be successfully applied for fast and easy examination of cell culture supernatants and the investigation of antibody/antigen interactions including kinetics and fluorescence properties. These methods simplify the selection of the optimal antibody. As a target analyte, carbamazepine was chosen. This is a widely used antiepileptic drug which also frequently occurs in the environment. The new antibody enables CBZ determination in the concentration range 0.66–110 µg L-1 within 10 min using a high-throughput microtiter plate-based FPIA, and between 1.4 and 79 µg L-1 within 5 min applying an automated cuvette-based FPIA instrument, and from 0.05–36 µg L-1 using ELISA. The measurements were performed at a non-equilibrium state which improved the sensitivity and selectivity of the assays. Due to low cross-reactivity especially towards the main CBZ metabolite and other pharmaceuticals (<1%), this antibody gives the opportunity for application in medical and environmental analyses.
Carbamazepine is an antiepileptic drug that can be used as a marker for the cleaning efficiency of wastewater treatment plants. Here, we present the optimization of a fast and easy on-site measurement system based on fluorescence polarization immunoassay and the successful application to wastewater. A new monoclonal highly specific anti-carbamazepine antibody was applied. The automated assay procedure takes 16 min and does not require sample preparation besides filtration. The recovery rates for carbamazepine in wastewater samples were between 60.8 and 104% with good intra- and inter-assay coefficients of variations (less than 15 and 10%, respectively). This automated assay enables for the onsite measurement of carbamazepine in wastewater treatment plants.
Enzyme-linked immunosorbent assay (ELISA) for the anthropogenic marker isolithocholic acid in water
(2016)
Bile acids are promising chemical markers to assess the pollution of water samples with fecal material.
This study describes the optimization and validation of a direct competitive enzyme-linked immunosorbent assay for the bile acid isolithocholic acid (ILA). The quantification range of the optimized Assay was between 0.09 and 15 mg/L. The assay was applied to environmental water samples. Most studies until now were focused on bile acid fractions in the particulate phase of water samples. In order to avoid tedious sample preparation, we undertook to evaluate the dynamics and significance of ILA levels in the aqueous phase. Very low concentrations in tap and surface water samples made a pre-concentration step necessary for this matrix as well as for wastewater treatment plant (WWTP) effluent. Mean recoveries for spiked water samples were between 97% and 109% for tap water and WWTP influent samples and between 102% and 136% for WWTP effluent samples. 90th percentiles of intra-plate and inter-plate coefficients of variation were below 10% for influents and below 20% for effluents and surface water. ILA concentrations were quantified in the range of 33-72 mg/L in influent, 21-49 ng/L in effluent and 18-48 ng/L in surface water samples. During wastewater Treatment the ILA levels were reduced by more than 99%. ILA concentrations of influents determined by ELISA and LC-MS/MS were in good agreement.
However, findings in LC-ELISA experiments suggest that the true ILA levels in concentrated samples are lower due to interfering effects of matrix compounds and/or cross-reactants. Yet, the ELISA will be a valuable tool for the performance check and comparison of WWTPs and the localization of fecal matter input into surface waters.
Diclofenac (DCF) wird als nicht-steroidales, antientzündliches Arzneimittel gegen Fieber, Entzündungen, rheumatische Erkrankungen und Schmerzen eingesetzt. 70 % werden nach Einnahme wieder ausgeschieden. Aufgrund unvollständiger Elimination in den Kläranlagen lassen sich Rückstände im Trinkwasser und im Oberflächengewässer nachweisen. Diclofenac wird in der Umweltanalytik v.a. mittels GC-MS oder LCMS nachgewiesen. Bioanalytische Methoden machen sich die spezifische Antigen-Antikörper-Wechselwirkung zunutze, so z.B. Immunoassays. Die hierfür erforderlichen Antikörper werden durch Immunisierung mit einem Konjugat aus dem Analyten und einem Trägerprotein gewonnen. Die Kopplung kann direkt über die Carboxylfunktion von Diclofenac erfolgen. Um Abschirmungseffekte zu vermeiden, kann zwischen Protein und Analy1 ein Spacer sinnvoll sein.
Zearalenone and its cis-isomer, cis-zearalenone, are nonsteroidal mycotoxins that elicit an estrogenic response upon binding to the estrogen receptor. This study compares the estrogenicity of eleven congeners including novel metabolites as 15-OH-zearalenone, zearalenone-14-sulfate, α-cis-zearalenol and β-cis-zearalenol using the E-Screen assay. Overall, a change in the configuration from trans to cis retains significant estrogenic activity. In contrast, alterations of the aromatic moiety including hydroxylation and sulfation showed a markedly decreased estrogenicity when compared to zearalenone.
For the antiepileptic drug and anthropogenic marker carbamazepine (CBZ), a fast and cost-effective immunoassay based on fluorescence polarization (FPIA) was developed. The required fluorophore conjugates were synthesized from different fluorescein and CBZ derivatives. The most suitable tracer was CBZ–triglycine–5-(aminoacetamido)fluorescein. Additionally, the applicability of the assay in tubes and on microtiter plates was tested. The first format can be performed in a portable instrument and therefore can be applied in field measurements. The measurement of an individual sample can be carried out within 4 min. This assay shows a measurement range of 2.5–1000 µg L-1 and a test midpoint (or IC50) of 36 µg L-1. The FPIA performed on microtiter plates is useful for the assay development and is suitable for a very high throughput (up to 24 samples in 20 min). The test midpoint of this assay is 13 µg L-1 and the measurement range is 1.5–300 µg L-1. Furthermore, this assay requires smaller sample volumes and less reagents, including the crucial amount of antibody. The applicability of both assays to spiked surface water samples was evaluated. The recovery rates vary between 66–110% on microtiter plates and 81–140% in tubes.
Established maximum levels for the mycotoxin zearalenone (ZEN) in edible oil require monitoring by reliable analytical methods. Therefore, an automated SPE-HPLC online system based on dynamic covalent hydrazine chemistry has been developed. The SPE step comprises a reversible hydrazone formation by ZEN and a hydrazine moiety covalently attached to a solid phase. Seven hydrazine materials with different properties regarding the resin backbone, pore size, particle size, specific surface area, and loading have been evaluated. As a result, a hydrazine-functionalized silica gel was chosen. The final automated online method was validated and applied to the analysis of three maize germ oil samples including a provisionally certified reference material. Important performance criteria for the recovery (70–120 %) and precision (RSDr <25 %) as set by the Commission Regulation EC 401/2006 were fulfilled: The mean recovery was 78 % and RSDr did not exceed 8 %. The results of the SPE-HPLC online method were further compared to results obtained by liquid&–liquid extraction with stable isotope dilution analysis LC-MS/MS and found to be in good agreement. The developed SPE-HPLC online system with fluorescence detection allows a reliable, accurate, and sensitive quantification (limit of quantification, 30 µg/kg) of ZEN in edible oils while significantly reducing the workload. To our knowledge, this is the first report on an automated SPE-HPLC method based on a covalent SPE approach.