Cold‐Induced Phase Separation Coupled With High‐Performance Liquid Chromatography‐Fluorescence Detection for Determination of Glyphosate, Aminomethylphosphonic Acid, and Glufosinate in Environmental Water

Glyphosate (GLP) and glufosinate (GLU) are among the most widely used herbicides; however, the strong polarity of these molecules, coupled with the absence of chromophores and fluorophores, poses significant challenges for their detection. In this study, a simple and highly sensitive analytical method was developed by coupling cold-induced liquid-liquid extraction (CI-LLE) pretreatment with high-performance liquid chromatography-fluorescence detection, enabling the simultaneous determination of GLU, GLP, and its metabolite aminomethylphosphonic acid (they are denoted as GLP-based compounds) in environmental water. CI-LLE employs an acetonitrile/water mixture, which is frozen at -22°C to induce phase separation, resulting in an upper acetonitrile-rich phase and a lower water-rich phase, thereby achieving effective enrichment and purification of the target compounds. The GLP-based compounds enriched in the aqueous phase were subsequently derivatized with 9-fluoromethyl chloroformate to obtain fluorescent derivatives for detection. The parameters for CI-LLE (treatment time, concentration of chelating agents, sample pH, and acetonitrile content) as well as derivatization conditions (including pH, reagent concentration, and reaction time) were systematically optimized. The developed method exhibited excellent sensitivity (with limits of detection in the range of 0.1-0.4 µg L^-1), satisfactory recoveries (81.0%-105.2%), and repeatability (relative standard deviations <10%). Compared to the chromatogram prior to CI-LLE treatment, the significant enhancement in cleanup efficiency enabled accurate and quantitative analysis. In summary, the proposed CI-LLE method provides an efficient and accurate approach for the determination of GLP-based compounds in aqueous samples, highlighting its strong potential for environmental monitoring applications.