Validated LC-MS/MS method for the quantification of glyphosate and its metabolites in human serum: A biomonitoring study in farming intensive regions of the Western Cape, South Africa

Glyphosate, a widely used herbicide approved in over 100 countries for its broad-spectrum efficacy and low cost, has raised increasing environmental and health concerns. Monitoring inadvertent exposure requires quantification of glyphosate and its metabolites, aminomethylphosphonic acid (AMPA) and methylphosphonic acid (MPA). The Western Cape Province of South Africa, characterized by a Mediterranean climate that supports intensive agriculture, lacks biomonitoring data on these compounds. This study reports the first validated LC-MS/MS method in Southern Africa for the simultaneous quantification of glyphosate, AMPA, and MPA in human serum without derivatization, and its application to clinical samples from farming-intensive areas of the Western Cape. Calibration standards ranged from 15.0 to 760 ng/mL for glyphosate, 25.0-1600 ng/mL for AMPA, and 0.75-48 ng/mL for MPA, using Oasis MAX cartridges for extraction. Analysis was performed on a Sciex QTRAP 6500+ LC-MS/MS system with a Phenomenex Luna C8 column and gradient elution. Method validation followed SANTE/2020/12830, REV.2 guidelines, assessing linearity, limit of quantification, selectivity, matrix effects, recovery, precision, and stability. Among 258 serum samples opportunistically collected from clinics located in regions with intensive pesticide use, glyphosate and/or its metabolites were detected in 11.6% of samples, with three exhibiting quantifiable concentrations. This robust, non-derivatized LC-MS/MS method enables accurate biomonitoring of glyphosate exposure and provides the first evidence of human exposure in agricultural communities of the Western Cape.