## 4 Glyphosate and AMPA in Foods
### Sample Pretreatment  
Sample pretreatment prior to chromatographic separation/spectroscopic and/or antibody-based detection is a critical step to ensure the efficacy of the analysis of GLYP and AMPA, especially when addressing complex matrices such as foods. The sample pretreatment constitutes the most time-consuming phase of a methodology. Typical sample pretreatment processes encompass dissolution, homogenization, extraction, precipitation, and concentration of analytes [81]. The selection of the most appropriate pretreatment technique is essential for achieving optimal accuracy and minimizing potential interference during analysis. The physicochemical properties of glyphosate and AMPA, in conjunction with the complexity of food matrices, are challenging in developing analytical methods for GLYP detection and quantification. GLYP and AMPA are highly polar compounds, rendering them highly soluble in water but virtually insoluble in organic solvents such as methanol or acetonitrile, which complicates the use of traditional extraction solvents [82]. The QuPpe-PO (Quick Polar Pesticides) method edited by the EU Reference Laboratories for Residues of Pesticides serves as the standard guideline for the extraction and measurement of glyphosate and other polar pesticides. Various modifications of this tech  
Figure 2: Suggested workflow for analysis of glyphosate and AMPA in food matrix. nique have been implemented globally to enhance sample pretreatment in each matrix [26]. For most commodities, including dried and fresh fruits and vegetables, homogenization is essential to evaluate the efficiency of the extraction. When analyzing soybean, oat, maize, rice, and other grains, reducing the particle size constitutes a critical step in the extraction process. Larger particle size may hinder the extraction of the analyte, as the solvent may not effectively penetrate the particle and access the analyte trapped within the grain, while smaller particle size increases the surface area available for solvent interactions, thereby enhancing the efficiency of the extraction process. In this context, reducing the particle size of the food sample is preferable to improve the overall extraction yield of glyphosate [72; 82; 83]. In the pretreatment of samples with a moisture content below 80%, it is necessary to adjust the water content before the extraction, aiming to minimize analytical errors [72; 82; 83]. In analysis of honey or foods with higher sugar contents, the sugar concentration should be considered in the adjustment, as it can contribute to the volume when fully dissolved in the extraction solvent; in those cases, the water amount of the sample may be less than usual [26]. Furthermore, the presence of interfering proteins can be treated with the addition of acidified methanol. The addition of methanol to the solution reduces the solubility of proteins by lowering the dielectric constant of the sample proteins, which increases the attraction between charged amino acid residues, facilitating the intermolecular interactions and producing protein aggregates [84]. The addition of weak acids decreases the pH of the medium and provides protons that can be captured by protein amino groups, enhancing the intramolecular interactions. For instance, for LC-MS/MS analysis, volatile organic acids such as formic or acetic acid are recommended to prevent contamination into the ionization source; a low concentration of acid is enough to achieve good recovery percentages; for example, 50 mM of acetic acid [82] or 1.0% formic acid [26] could be used. The process efficiency can be enhanced by resting the sample under freezing conditions. Starch present in corn and other grains can absorb glyphosate, impeding its extraction. Since starch is also soluble in the aqueous medium, it could be dissolved in the water used to extract glyphosate; however, methanol, used to precipitate proteins, may be a helping hand, since starch can be precipitated too.