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Organisationseinheit der BAM
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.
The present study investigates the in vitro phase I metabolism of cis-zearalenone (cis-ZEN) in rat liver microsomes and human liver microsomes. cis-ZEN is an often ignored isomer of the trans-configured Fusarium mycotoxin zearalenone (trans-ZEN). Upon the influence of (UV-) light, trans-ZEN isomerizes to cis-ZEN. Therefore, cis-ZEN is also present in food and feed. The aim of our study was to evaluate the in vitro phase I metabolism of cis-ZEN in comparison to that of trans-ZEN. As a result, an extensive metabolization of cis-ZEN is observed for rat and human liver microsomes as analyzed by HPLC-MS/MS and high-resolution MS. Kinetic investigations based on the substrate depletion approach showed no significant difference in rate constants and half-lives for cis- and trans-ZEN in rat microsomes. In contrast, cis-ZEN was depleted about 1.4-fold faster than trans-ZEN in human microsomes. The metabolite pattern of cis-ZEN revealed a total of 10 phase I metabolites. Its reduction products, α- and β-cis-zearalenol (α- and β-cis-ZEL), were found as metabolites in both species, with α-cis-ZEL being a major metabolite in rat liver microsomes. Both compounds were identified by co-chromatography with synthesized authentic standards. A further major metabolite in rat microsomes was monohydroxylated cis-ZEN. In human microsomes, monohydroxylated cis-ZEN is the single dominant peak of the metabolite profile. Our study discloses three metabolic pathways for cis-ZEN: reduction of the keto-group, monohydroxylation, and a combination of both. Because these routes have been reported for trans-ZEN, we conclude that the phase I metabolism of cis-ZEN is essentially similar to that of its trans isomer. As trans-ZEN is prone to metabolic activation, leading to the formation of more estrogenic metabolites, the novel metabolites of cis-ZEN reported in this study, in particular α-cis-ZEL, might also show higher estrogenicity.
Bile acids are relevant markers for clinical research. This study reports the production of antibodies for isolithocholic acid, the isomer of the extensively studied lithocholic acid. The IgG titer and affinity maturation were monitored during the immunizations of three mice and two rabbits. In both animal models, polyclonal antibodies with a high selectivity and affinity were produced. The development of a direct competitive ELISA with a test midpoint of 0.69 ± 0.05 µg/L and a measurement range from 0.09–15 µg/L is reported. Additionally, the crystal structure of isolithocholic acid is described for the first time.
Pure U-[13C18]-labelled cis-zearalenone (cis-ZEA) has been prepared and characterised as internal standard (ISTD) for a reliable quantification of cis-ZEA in contaminated food and feed products. The cis-isomer of the naturally trans-configurated Fusarium mycotoxin zearalenone is often neglected. However, isomerisation easily occurs by exposure of ZEA to (UV-)light. Thus, the applicability of the new cis-ZEA ISTD was demonstrated in a long-term isomerisation study comparing naturally trans-ZEA-contaminated edible oil with spiked edible oil. To estimate the benefits of the newly prepared cis-ZEA ISTD, various approaches to quantify cis-ZEA by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) were compared. As a result, a significant bias was revealed if no appropriate cis-ZEA standards are used. Furthermore, the new ISTD was applied to the analysis of 15 edible oils by stable isotope dilution analysis in combination with HPLC-electrospray ionisation-MS/MS. One of the maize germ oils showed the presence of cis-ZEA above LOD (>0.3 µg/kg), whereas two out of 15 maize germ oils were found to be contaminated with trans-ZEA (range 17.0-31.0 µg/kg).
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.
Temperature is one factor that influences the performance of immunoassays. Most commonly all incubation steps of the immunoassay are performed at ambient temperature. To systematically study the influence of temperature deviations on assay parameters like signal variation coefficients, limit of detection and measurement range, direct competitive enzyme immunoassays for the determination of two anthropogenic markers, caffeine and carbamazepine, were run at different temperatures above and below room temperature during individual assay steps. The temperature during the competition step had the greatest impact on the assay sensitivity yielding changes in test midpoints by a factor of 10 to 15. For carbamazepine, the test midpoints varied from 36 ng L-1, 108 ng L-1 to 378 ng L-1 employing 4 °C, 21 °C, or 37 °C as incubation temperature, respectively. The edge effect can be minimized resulting in a good plate homogeneity with small variation coefficients, when the assay is performed at ambient temperature.
Enzyme immunoassays with optical detection are amongst the most widely used bioanalytical tools. We defined seven parameters for the quality assessment of immunoassays that were addressed in a systematic study of direct and indirect immunoassays, using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), the chromogenic substrates 3,3',5,5'-tetramethylbenzidine (TMB) and para-nitrophenyl phosphate, and the fluorescent substrates 3-(4-hydroxyphenyl)propionic acid and 4-methylumbelliferyl phosphate. The same monoclonal antibody against caffeine was used throughout the study. The four quality parameters regarding the standard curve were the test midpoint (sensitivity), the measurement range, the relative dynamic range of the signal, and the goodness of fit of the adjusted four-parameter logistic function. All HRP immunoassays showed a higher sensitivity compared to the AP assays. On the basis of all four criteria, it was established that the direct assay format is superior to the indirect format, the immunoassay using HRP TMB fulfilling all requirements best. In a second step, caffeine concentrations in 24 beverage and cosmetics samples were determined and three more quality parameters were assessed with this application. The direct HRP TMB assay showed one of the best intra- and inter-plate precisions and the best accuracy, defined by the correlation of results with those from the chosen reference method liquid chromatography tandem mass spectrometry (LC-MS/MS). Considering all criteria, HRP TMB seems to be the enzyme substrate system of choice preferably used in the direct assay format.
The reaction pathways of amino acids and reducing sugars are now fully understood. The focus in the last few years, however, has turned to the reaction of peptides and proteins with reducing sugars. We have investigated the reaction of γ-aminobutanoic acid, the heptapeptide Nα-Acetyl-Lys-Lys-β-Ala-Lys-β-Ala-Lys-Gly, and the model protein β-casein in Maillard reactions with 1-13C arabinose. Characterization of 13C-labeled acetic acid and norfuraneol by gas chromatographymass spectrometry and nuclear magnetic resonance revealed new formation pathways. The results demonstrate significant differences in the labeling pattern of the products depending on the amine used, indicating different formation pathways of acetic acid and norfuraneol.
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.