## 4 Glyphosate and AMPA in Foods
### Analytical Method Validation  
Analytical method validation is an essential process to guarantee the quality of the results obtained. In the analysis of pesticide residues in food samples, the use of validated methodologies is crucial as far as satisfactory analysis is concerned [128]. Several quality standards such as ISO/IEC 17025 and regulatory bodies like the FDA require analytical laboratories to validate all newly developed analytical methods to ensure they comply with the accuracy, precision, and applicability criteria for producing reliable results [129]. The SANTE guidelines are the standard document for analytical quality control and method validation for pesticide residue analysis in food samples. The objectives of this document include defining the procedures and criteria for method validation. SANTE requires the estimation of linearity, matrix effect, LOQ (quantitative methods), LOD (qualitative methods), specificity, recovery, precision and robustness [104]. The limit of detection (LOD) refers to the lowest concentration of analyte that can be detected by the analytical method, while the limit of quantification (LOQ) is the lowest analyte concentration that can be reliably measured with acceptable repeatability and trueness [130; 131]. It is important to note that the matrix effect can influence LOD and LOQ values. To determine the actual limits, experiments should be conducted using samples analyzed under the method conditions. While the LOD of a method describes its ability to detect low concentrations of analytes, if an analyte is not detected, this does not imply its absence in the sample but rather that the method is unable to detect it [128]. Additionally, it is important to consider that LOQ is usually higher than LOD, meaning that, in some cases, the analytes may be detected but not quantified. According to the SANTE guidelines, the LOQ is preferred over LOD to avoid errors. In the validation of analytical methods for pesticide residue analysis in food, the LOQ must be lower than the established MRL for each pesticide [104]. Accuracy is typically represented as the percentage of analyte recovered compared to the actual amount spiked into the sample. Recovery studies are usually conducted by spiking pesticide-free samples (sample blank) with solutions of known concentration. The samples are then analyzed following the established protocol, and recovery percentages are calculated [132]. According to SANTE, to achieve adequate accuracy, recovery levels between 70% and 120% must be reached as the average recovery for each spiked level tested [104]. On the other hand, precision is defined as the degree of agreement between independent analytical results when the analysis is carried out under defined conditions, on different portions of the same sample by the same analyst (repeatability), by a different analyst (reproducibility) or in a different laboratory (intermediate precision) [130; 131]. Precision studies should be conducted by repeating the experiment on different days and calculating the relative standard deviation (%RSD). In pesticide residue analysis, a %RSD value of <20% for both repeatability and reproducibility must be reached for each spike level tested to demonstrate the precision of the methodology [104]. Recent analytical studies on GLYP and AMPA analysis in food matrices reveal substantial variability in methodological rigor, particularly regarding adherence to internationally recognized validation protocols. Although most studies report essential parameters such as LOD, LOQ, accuracy, and repeatability, many fail to discuss matrix effects, uncertainty estimation, or full compliance with guidelines. This undermines reproducibility and complicates comparisons between laboratories. Table 7 summarizes the validation practices in GLYP and AMPA detection studies in food samples. Despite methodological advancements, some of the reviewed studies omitted critical aspects of method validation, particularly the matrix effect assessment and uncertainty estimation.