Simultaneous Determination of 2,4-D and 2,4,5-T in Environmental Waters by Rapid HPLC Coupled with Native Fluorescence Detection

A simple high-performance liquid chromatography coupled with fluorescence detection (HPLC-FL) method has been developed for the simultaneous determination of the phenoxy herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Chromatographic separation was achieved on a C18 column using a methanol/phosphate buffer (80:20, v/v, pH 3.0) mobile phase under isocratic conditions, using a flow rate of 1.0 mL min⁻¹. Detection was performed at an emission wavelength of 575 nm and an excitation wavelength of 553 nm, taking advantage of the native fluorescence of both herbicides. Under the optimized conditions, 2,4-D and 2,4,5-T were completely separated with retention times of approximately 36 and 48 s, respectively. Calibration curves were linear within the concentration ranges of 55.3–442.1 mg L⁻¹ for 2,4-D and 63.9–511.0 mg L⁻¹ for 2,4,5-T, with correlation coefficients of 0.9922 and 0.9976, respectively. Limits of detection were 1.36 mg L⁻¹ for 2,4-D and 1.57 mg L⁻¹ for 2,4,5-T, whereas limits of quantification were 4.11 and 4.77 mg L⁻¹, respectively. The applicability of the proposed methodology was evaluated using three environmental water samples fortified at two concentration levels of both herbicides. Recoveries ranged from 95.2 to 111.1% for 2,4-D and from 90.2 to 110.7% for 2,4,5-T, with relative standard deviation values lower than 1%, in all cases. The proposed method provides a rapid, simple and reliable alternative for the simultaneous determination of phenoxy herbicides in environmental waters and demonstrates that both analytes exhibit measurable native fluorescence under the chromatographic conditions employed.