Fluorescence Polarization Immunoassay with Modulated Selectivity for Effective Detection of the Agrochemical 4-Chlorophenoxyacetic Acid

4-Chlorophenoxyacetic acid (4-CPA), a synthetic auxin analog, is employed in agriculture both as a plant growth regulator and as a constituent of herbicide formulations. Consequently, the establishment of simple and rapid detection methods is essential for effective environmental monitoring. This study reports the first development of a homogeneous fluorescence polarization immunoassay (FPIA) for the determination of 4-CPA. The monoclonal antibody (M1), raised against 4-CPA, was evaluated as a recognition element. Furthermore, two fluorescently labeled 4-CPA tracers—with ethylenediamine fluorescein thiocarbamate and aminohexylaminocarbonylfluorescein—were synthesized and purified, and their structures were unequivocally confirmed by high-performance liquid chromatography coupled with high-resolution mass spectrometric detection (HPLC-HRMS). Optimal concentrations of monoclonal antibodies and tracers were established, yielding a limit of detection of 1.2 ng/mL. The assay demonstrated a broad dynamic range of 2.3–300 ng/mL and a rapid analysis time of 15 min. Validation via the standard addition method in authentic open water samples resulted in recovery rates of 98–112%. To address the cross-reactivity with the prevalent herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), two novel strategies were devised and successfully implemented. The first approach involves the concurrent execution of two separate FPIAs—one for 2,4-D and one for 4-CPA—followed by the mathematical resolution of two analyte concentrations from the two measured binding values. The second strategy entails the preliminary selective removal of 2,4-D from sample matrices using affinity chromatography columns with immobilized anti-2,4-D antibodies prior to FPIA for 4-CPA. These proposed methodologies appear highly promising for overcoming the inherent limitations of traditional immunoassays when faced with significant cross-reactivity among structurally analogous compounds.