Validation of an Optimized HPLC-FLD Method for the Simultaneous Determination of Glyphosate and AMPA with Application to Environmental Water Samples

Abstract

This study presents the validation of an optimized analytical method for the highly sensitive simultaneous determination of glyphosate and its primary degradation product AMPA using HPLC with fluorescence detection following FMOC-Cl derivatization, and its application to environmental water samples. Derivatization conditions were optimized at pH 10.5 with a reaction time of 45 min and an FMOC-Cl concentration of 8 µM. Chromatographic separation was achieved using a mobile phase consisting of acetonitrile and sodium acetate buffer (20 mM, pH 5.2) in a 50:50 (v/v) ratio, with fluorescence detection at 265/310 nm. Method validation, performed according to ICH Q2(R1) guidelines, demonstrated excellent selectivity and linearity (r² ≥ 0.999) over concentration ranges of 0.88–56.36 ppb for glyphosate and 0.58–37.01 ppb for AMPA. Recoveries ranged from 92.38–103.92% and 87.19–105.24%, respectively, with good precision
 (RSD <2% for repeatability and <5% for intermediate precision). Limits of detection were 3.9 and 1.7 ppt, while limits of quantification were 12 and 5 ppt for glyphosate and AMPA, respectively. Application to environmental water samples confirmed the presence of both analytes in agricultural surface waters, highlighting the method’s reliability and suitability for trace-level environmental monitoring.