Hands-on protocol for preparing water-soluble fractions from agri-food samples for NMR-based metabolomics analysis

The aim of this study was to address the lack of protocols for nuclear magnetic resonance (NMR)-based metabolomics in the agri-food sector by providing a reproducible workflow for the preparation and analysis of water-soluble metabolite fractions. These fractions, rich in primary metabolites such as sugars, amino acids, and organic acids, are key to assessing agri-food products' composition, quality, as well as to monitor their manufacturing and development processes. The protocol differentiates solid and liquid matrices, optimizing extraction procedures accordingly. Representative agri-food products-strawberry leaves (solid) and wine (liquid)-were analyzed to demonstrate the method's versatility and applicability. Key steps include tailored sample preparation, optimization of NMR acquisition, and spectral quality control, ensuring high data quality and reproducibility. The proposed workflow enhances reproducibility across agri-food metabolomics studies and facilitates integration into broader food quality, traceability, and safety frameworks.