The excessive use of phytosanitary products represents a growing concern, due to their persistence and potential environmental and toxicological impacts. Among these compounds, glyphosate, a glycine-derived chemical marketed as a broad-spectrum herbicide, is one of the most widely used pesticides worldwide. Breast milk is a complex biological matrix that can reflect environmental exposure, making it highly suitable for assessing glyphosate contamination. This study aimed to demonstrate a screening method to determine glyphosate concentrations in the breast milk of 100 postpartum women residing in Tupã, São Paulo, Brazil—90 in urban areas and 10 in rural areas—using high-performance liquid chromatography (HPLC) for rapid detection. By validation parameters, it was possible to verify, through the correlation coefficient (r), that the method is linear within the working range; the LD was 0.14 mg/L and the LQ was 0.43 mg/L. The recovery obtained by standard sample fortification was 92%. All analyzed samples presented detectable levels of glyphosate, indicating consistent exposure patterns and suggesting relevant environmental contamination routes in the region. These findings provide evidence of glyphosate presence in human milk and reinforce the importance of continuous monitoring strategies and preventive public health measures aimed at reducing exposure to agricultural contaminants.
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