Development of Fluorescence Polarization Immunoassay for the Rapid Detection of 6-Chloronicotinic Acid: Main Metabolite of Neonicotinoid Insecticides

Abstract

A fluorescence polarization immunoassay (FPIA) for the quantitative determination of 6-chloronicotinic acid (6-CNA) using polyclonal antibody was developed. The 6-CNA-protein (bovine serum albumin and soybean trypsin inhibitor) conjugates and fluorescein-labeled 6-CNA derivative (tracer) were prepared and used as the immunogens and tracer, respectively. The synthesized tracer was purified by thin layer chromatography (TLC) and showed a good binding to antiserum (73/5) which was obtained from the immunized rabbit (No. 73) with 6-CNA-BSA conjugate. The detection limit (10% inhibition) of FPIA was 4 microg/mL, and IC(50) value was 32 microg/mL. The FPIA showed a cross-reaction for 5-amino-2-chloropyridine (60%), but no cross-reaction for other pesticides was observed. Recoveries for spiked apple, urine, soil, and water samples (5, 50, and 500 ppm) averaging between 78.6 +/- 8.8 and 114 +/- 18% were reasonable and in good agreement with the amounts spiked. Although the developed FPIA possesses low sensitivity, this assay is more simple and quick than other analytical methods, such as high performance liquid chromatography and gas chromatography. Thus, the developed FPIA method could be a useful tool for express screening 6-CNA in agricultural, environmental, and biological samples.