ORIGINAL ABSTRACT

Glyphosate (GLY) and its main degradation product aminomethylphosphonic acid (AMPA), together with the chemically related phosphinic acid herbicide glufosinate (GLU), are widely used agrochemicals. Their reliable quantification in groundwater remains analytically challenging due to complexation with divalent cations and matrix-dependent ionization. This work developed and validated a groundwater-adapted LC-MS/MS method using FMOC derivatization and isotopically labeled glyphosate (GLY*) as internal standard. The new protocol (SUBTE, from SUBTERRANEAN) introduces an acidification in the presence of excess EDTA to disrupt metal complexes prior to derivatization and was benchmarked against a conventional borate-buffer workflow (BORAX). Normalized calibration (analyte/GLY*) was linear from 0.5 to 20 µg·L-1 (R2 ≥ 0.99) and origin-compatible. Operational LOD/LOQ in matrix were 0.003/0.01 µg·L-1 for GLY, 0.01/0.04 for GLU, and 0.02/0.06 for AMPA, meeting precision criteria (≤20 % RSD at the lowest level). Across real groundwater samples spanning wide conductivity and Ca+2 + Mg+2 ranges (n = 50), SUBTE achieved mean recoveries of ∼96 % compared with ∼70 % for BORAX, demonstrating effective mitigation of divalent-cation artefacts and enhanced robustness in hard waters. No analyte exceeded the detection limit in any well at the time of sampling, consistent with the low-mobility, Ca-rich geo-pedologic setting of the Pampean plain. The SUBTE workflow thus provides a validated, matrix-resilient approach for routine monitoring of GLY, AMPA, and GLU in groundwater, ensuring reliable quantification under variable hydrogeochemical conditions.