## 5 Challenges and Opportunities  
The extensive and continuous use of glyphosate (GLYP) in agricultural practices has resulted in its frequent detection, along with its primary metabolite aminomethylphosphonic acid (AMPA), across a wide array of fresh and processed food products and drinking water. Although most detected concentrations remain within the maximum residue limits (MRLs) established by regulatory agencies, the potential for cumulative exposure, particularly in vulnerable populations such as infants and children, continues to raise public health concerns. In this context, the reliable analytical determination of GLYP and AMPA remains a central component of food safety assessment. However, the robustness and applicability of current methods are constrained by numerous physicochemical and methodological limitations. GLYP and AMPA exhibit high polarity, lack chromophoric groups, and exhibit metal-chelating properties. These characteristics impede their retention in reversed-phase chromatographic systems and make them undetectable using conventional ultraviolet or fluorescence detectors. Their chelation with metal ions in food matrices presents further complications regarding extraction efficiency and detector response. Derivatization methods, including those based on FMOC-CI, have enabled detection by introducing fluorescent groups, but these protocols require strict pH and temperature control, are time-consuming, and often yield unstable derivatives. Alternatively, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has enabled more sensitive and selective detection without the need for derivatization. However, its susceptibility to matrix-induced ion suppression, high operational cost, and reliance on specialized expertise limit its routine use in many laboratories. The performance of analytical methods is also highly dependent on the nature of the food matrix. In plant-derived samples, derivatization-based HPLC methods can be effective when reaction parameters are rigorously optimized. In contrast, complex food matrices, such as milk, cheese, honey, or processed foods often require LC-MS/MS methods, typically combined with solid-phase extraction (SPE) or QuEChERS to improve selectivity and recovery. These enhanced protocols have demonstrated improved accuracy and sensitivity but also require additional time, resources, and validation efforts for each matrix. The reviewed literature highlights that matrix complexity not only affects extraction and cleanup efficiency but also impacts the overall method precision, particularly in the absence of matrix-matched calibration or isotopically labeled internal standards. Some major challenges remain central to the determination of GLYP and AMPA in food samples:  
1. The high polarity of glyphosate and AMPA prevents their retention on reversed-phase chromatographic columns and limits the use of traditional extraction solvents, such as ethanol, methanol, and acetonitrile. 2. Their chelating ability facilitates the formation of complexes with metallic elements commonly found in food matrices. 3. The lack of chromophore groups on the GLYP structure hinders its detection using fluorescence and diode array detectors. 4. The low concentrations of GLYP and AMPA present in foods necessitate the implementation of more sensitive and robust methodologies, with detection and quantification limits tailored to each food group and the established maximum residue limits. 5. Compliance with international regulations for glyphosate and AMPA residue limits presents a significant analytical challenge. Solvent-based extraction methods using water or methanol are favored for their simplicity and cost-effectiveness, particularly in less complex plant matrices. However, their performance is significantly reduced in high-fat or high-protein matrices. More advanced methods like SPE and QuEChERS have been developed to address these shortcomings by improving cleanup and reducing co-extractive interference.