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Validation of a Liquid Chromatography Coupled to Mass Spectrometry Method for Glyphosate and Aminomethylphosphonic Acid in Urine for Human Biomonitoring Using Combined Hybrid Anion-Exchange and Hydrophilic Interaction Liquid Chromatography

Author(s): Elisa Polledri1, Rosa Mercadante1, Silvia Fustinoni1,2
1. EPIGET—Epidemiology, Epigenetics, and Toxicology Lab, Department of Clinical Sciences and Community Health, Università degli Studi di Milano, 20122 Milano, Italy, 2. Environmental and Industrial Toxicology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy

Published By: Separations 

Issue: 10(11), 576

Year of Publication: 2023

Link: https://doi.org/10.3390/separations10110576 

Abstract: Glyphosate-based herbicides are the most widely used pesticides in the world; however, the toxicity of glyphosate (GlyP) toward humans, especially its carcinogenicity, is controversial. The aim of this work was to validate a rapid assay for measuring GlyP and its metabolite aminomethylphosphonic acid (AMPA) in urine for human biomonitoring. The analytes were purified via solid-phase extraction in the presence of isotopically labeled internal standards. An LC-MS/MS assay was developed using a column with a novel hybrid stationary phase combined with anion exchange and hydrophilic interaction liquid chromatography. Detection and quantification were performed using negative electrospray ionization in a hybrid triple quadrupole/linear ion trap mass spectrometer. The retention times for AMPA and GlyP were 1.44 and 7.24 min, respectively. Calibration curves showed a linear dynamic range of up to 40 µg/L, inter- and intra-run precisions <7.5%, and accuracies within 10% of the theoretical concentrations. The limits of quantification were 0.1 µg/L and 0.5 µg/L for GlyP and AMPA, respectively. The matrix effect bias was controlled using internal standards. Successful participation in external quality assurance exercises strengthens the validity of the method. The assay was applied to the measurement of GlyP and AMPA in the urine of 9 urban residents, 26 rural residents, and 12 agricultural workers; while AMPA was mostly not quantifiable, the median GlyP values were 0.1 and 0.34 µg/L in rural residents and workers, respectively. The assay is useful to assess GlyP and AMPA in human urine following different exposure scenarios.

Acknowledgment(s): This analysis features a Restek Polar X LC column (cat.# 9311A52), which was selected after the authors tested multiple other LC columns.

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