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Monitoring carbamazepine in surface and wastewaters by an immunoassay based on a monoclonal antibody
(2009)
The pharmaceutical compound carbamazepine (CBZ) is an emerging pollutant in the aquatic environment and may potentially be used as a wastewater marker. In this work, an enzyme-linked immunosorbent assay (ELISA) for the detection of carbamazepine in surface and sewage waters has been developed. The heterogeneous immunoassay is based on a commercially available monoclonal antibody and a novel enzyme conjugate (tracer) that links the hapten via a hydrophilic peptide (triglycine) spacer to horseradish peroxidase. The assay achieves a limit of detection of 24 ng/L and a quantitation range of 0.05-50 µg/L. The analytical performance and figure of merits were compared to liquid chromatography-tandem mass spectrometry after solid-phase extraction. For nine Berlin surface water samples and one wastewater sample, a close correlation of results was observed. A constant overestimation relative to the CBZ concentration of approximately 30% by ELISA is probably caused by the presence of 10,11-epoxy-CBZ and 2-hydroxy-CBZ in the samples. The ELISA displayed cross-reactivities for these compounds of 83% and 14%, respectively. In a first screening of 27 surface water samples, CBZ was detected in every sample with concentrations between 0.05 and 3.2 µg/L. Since no sample cleanup is required, the assay allowed for the determination of carbamazepine with high sensitivity at low costs and with much higher throughput than with conventional methods.
The occurrence of carbamazepine (CBZ) and its metabolites in German and Portuguese wastewater was investigated. A total of 46 samples from influent and effluent wastewater were analyzed by liquid-chromatography (LC) tandem mass spectrometry. The five metabolites 10,11-dihydro-10,11-dihydroxy-CBZ (DiOH-CBZ), 10,11-dihydro-10-hydroxy-CBZ (10-OH-CBZ), 10,11-epoxy-10,11-dihydro-CBZ, 2-hydroxy-CBZ and 3-hydroxy-CBZ were very persistent with little to no removal during wastewater treatment. The highest concentrations were found for CBZ, DiOH-CBZ, and 10-OH-CBZ, with up to 5.0, 4.8 and 1.1 µg/L, respectively. Furthermore, the related pharmaceutical oxcarbazepine and the metabolites 9-hydroxymethyl-10-carbamoylacridan, 1-hydroxy-CBZ (1-OH-CBZ) and 4-hydroxy-CBZ (4-OH-CBZ) were detected. Explicit care was taken to achieve a good chromatographic separation of the numerous isomers that were difficult to distinguish by mass spectrometry alone. A phenylether stationary phase provided the best separation. In combination with high resolution mass spectrometry and hydrogen-deuterium exchange, this LC column enabled us to identify 1-OH-CBZ and 4-OH-CBZ in wastewater for the first time.
Carbamazepine (CBZ), caffeine and cetirizine were monitored by enzyme-linked immunosorbent assays (ELISAs) in surface and wastewaters from Berlin, Germany. This fast and cost-efficient method enabled to assess the spatial and temporal variation of these anthropogenic markers in a high-throughput screening. CBZ and cetirizine were detected by the same antibody, which selectively discriminates between both compounds depending on the pH value used in the incubation step. To our best knowledge, this is the first dual-analyte immunoassay working with a single antibody.
The frequent sampling with 487 samples being processed allowed for the repeated detection of unusually high concentrations of CBZ and caffeine. ELISA results correlate well with the ones obtained by liquid chromatography tandem mass spectrometry (LC-MS/MS). Caffeine concentrations found in surface waters were elevated by combined sewer overflows after stormwater events. During the hay fever season, the concentrations of the antihistamine drug cetirizine increased in both surface and wastewaters.
Caffeine was almost completely removed during wastewater treatment, while CBZ and cetirizine were found to be more persistent. The maximum concentrations of caffeine, CBZ and cetirizine found in influent wastewater by LCMS/MS were 470, 5.0 and 0.49 µg L-1, while in effluent wastewater the concentrations were 0.22, 4.5 and 0.51 µg L-1, respectively. For surface waters, concentrations up to 3.3, 4.5 and 0.72 µg L-1 were found, respectively.