Rapid analysis of diclofenac in freshwater and wastewater by a monoclonal antibody-based highly sensitive ELISA

Abstract

The non-steroidal anti-inflammatory drug (NSAID) diclofenac (DCF) is found worldwide in the aqueous environment. Therefore, it has raised increased public concern on potential long-term impact on human health and wildlife. The importance of DCF has been emphasized by the European Union recently by including this pharmaceutical in the first watch list of priority hazardous substances in order to gather Union-wide monitoring data. Rapid and cheap methods of analysis are therefore required for fresh and wastewater monitoring with high sample load. Here, for the first time, well-characterized monoclonal antibodies (mAbs) against DCF were generated and a highly sensitive ELISA developed. The best antibody (mAb 12G5) is highly affine (KD = 1.5 × 10(-10) M), stable to potential matrix interferences such as pH value (pH range 5.2-9.2), calcium ion concentration (up to 75mg/L), and humic acid content (up to 20mg/L). The limit of detection (LOD, S/N = 3) and IC50 of the ELISA calibration curve were 7.8 and 44ng/L, respectively. The working range was defined between 11 and 180ng/L. On average, about 10% cross-reactivity (CR) was found for DCF metabolites 5-OH-DCF, 4'-OH-DCF, and DCF-acyl glucuronide, but other structurally related NSAIDs showed binding <1% compared to the parent compound. While DCF concentrations at the low ppt range were measured in river and lake water, higher values of 2.9 and 2.1μg/L were found in wastewater influents and effluents, respectively. These results could be confirmed by solid phase extraction combined with LC-MS.