Huebner Maria, Weber Ekkehard, Niessner Reinhard, Boujday Souhir, Knopp Dietmar
Institute of Hydrochemistry and Chemical Balneology, Chair of Analytical Chemistry, Technische Universität München, Marchioninistrasse 17, 81377, München, Germany.
Institute of Physiological Chemistry, Medical Faculty, Martin-Luther-Universität Halle-Wittenberg, Hollystrasse 1, 06114, Halle, Germany.
Anal Bioanal Chem. 2015 Nov;407(29):8873-82. doi: 10.1007/s00216-015-9048-9. Epub 2015 Oct 6.
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 75 mg/L), and humic acid content (up to 20 mg/L). The limit of detection (LOD, S/N = 3) and IC50 of the ELISA calibration curve were 7.8 and 44 ng/L, respectively. The working range was defined between 11 and 180 ng/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 2.1 μg/L were found in wastewater influents and effluents, respectively. These results could be confirmed by solid phase extraction combined with LC-MS.
非甾体抗炎药双氯芬酸（DCF）在全球水环境中均有发现。因此，其对人类健康和野生动物的潜在长期影响引发了公众越来越多的关注。欧盟最近强调了DCF的重要性，将这种药物列入优先危险物质的首批观察清单，以便收集全欧盟范围的监测数据。因此，需要快速且廉价的分析方法来监测高样本量的新鲜水和废水。在此，首次制备了针对DCF的特性良好的单克隆抗体（mAb），并开发了一种高灵敏度酶联免疫吸附测定法（ELISA）。最佳抗体（mAb 12G5）具有高亲和力（KD = 1.5×10⁻¹⁰ M），对潜在的基质干扰（如pH值（pH范围5.2 - 9.2）、钙离子浓度（高达75 mg/L）和腐殖酸含量（高达20 mg/L））具有稳定性。ELISA校准曲线的检测限（LOD，S/N = 3）和IC50分别为7.8和44 ng/L。工作范围定义为11至180 ng/L。平均而言，DCF代谢物5 - OH - DCF、4'-OH - DCF和DCF - 酰基葡萄糖醛酸的交叉反应率（CR）约为10%，但与母体化合物相比，其他结构相关的非甾体抗炎药的结合率<1%。虽然在河水和湖水中测得的DCF浓度处于低皮克/升范围，但在废水进水和出水中分别发现了较高值2.9和2.1 μg/L。这些结果可通过固相萃取结合液相色谱 - 质谱法得到证实。
