## 4 Glyphosate and AMPA in Foods
### Detection and Quantification
The underivatized strategies offer a faster, cheaper, and simpler analytical approach for glyphosate and AMPA determination, while the derivatization approach facilitates chromatographic separations using reversed-phase columns and provides a more specific fragmentation pattern, resulting in a more robust strategy [113]. In the underivatized methodology, negative-mode ESI is preferred since both compounds tend to lose a hydrogen ion, resulting in easier detection and producing a higher abundance. For glyphosate, the quantifier transition is 168\(\rightarrow\)150 with 63 and 79 \(m/z\) as qualifier ions. For AMPA, the quantifier transition is 110\(\rightarrow\)81 with 63 \(m/z\) as the main qualifier ion [85]. Direct analysis of polar pesticides requires the use of specialized polar columns, such as the Waters Anionic polar pesticides [114] or Luna Polar Pesticides [115]. The use of polar columns offers several advantages in underivatized methods, including higher reproducibility and resolution, and more efficient retention of both anionic and cationic analytes, showing that the same column can be used either in positive or negative modes. [PERSON] et al. [111] developed an underivatized method for detecting GLYP in foods using SPE followed by separation with a hydrophilic interaction/weak anion-exchange column and LC-MS/MS coupled with negative electrospray ionization. The authors reported acceptable LOQ (16-26 \(\upmu\)g/kg) and LOD (5-8 \(\upmu\)g/kg) values, and recoveries ranged from 83 to 100%, with good accuracy and precision. Similarly, [PERSON] et al. [116] investigated the feasibility of directly detecting and quantifying GLYP in vegetables using LC-MS/MS. They employed an Obelisc N column and SPE-Oasis HLB cartridges in their methodology. The high sensitivity of LC-MS/MS makes it particularly valuable for detecting GLYP and AMPA at trace levels, and it should therefore be considered the primary option when low-concentration quantification is required [97]. Despite its analytical power, LC-MS/MS also presents certain limitations. Its high sensitivity makes it more susceptible to matrix effects, which can introduce errors in quantification. Additionally, when analyzing complex samples, the presence of impurities may contaminate the instrument, necessitating the use of more efficient extraction and cleanup procedures. Some studies validated LC-MS/MS methods across multiple food matrices, consistent with AOAC and FDA requirements. These cases highlight the benefits of robust and guideline-driven validation in ensuring the quality of results. On the other hand, HPLC systems represent one of the most accessible alternatives for many laboratories. However, due to the lack of chromophore and fluorophore functional groups in the chemical structure of GLYP and AMPA, their detection with conventional chromatographic detectors, such as fluorescence detection (FLD) and diode array detection (DAD), is limited. This necessitates the use of derivatization processes to form chromophores/fluorophores able to be detected [117]. The derivatization approach can be carried out using reagents such as dansyl chloride [80]; nevertheless, the most used derivatizing agent is 9-Fluorylmethyl chloroformate (FMOC-CI), which provides an easier extraction and separation process. The reaction mechanism involves the nucleophilic attack of the amine group in glyphosate on the acyl chloride in FMOC-CI, with chloride as the leaving group, in which HCl is produced (Figure 3). Since free HCl could react with the alkaline amine group, adding a base is necessary to improve the reaction yield; a borate buffer solution with a pH of 9.0 is required to achieve the reaction conditions [118]. The analysis of glyphosate under derivatization can be performed on LC-MS/MS systems. In these cases, the lower polarity of the glyphosate-FMOC compound addresses the poor retention encountered with polar pesticides in routine analysis. The aryl groups in FMOC enhance both AMPA and glyphosate's capability to interact with reversed-phase columns such as C8 or C18.