Development and Validation of an LC–MS/MS Method for Quantifying Phytohormones Related to Tomato Shelf Life

Phytohormones are key signaling molecules that regulate plant growth, stress adaptation, and fruit ripening. However, their low abundance and structural diversity complicate accurate quantification in food matrices. This study presents a validated LC–MS/MS method for the simultaneous detection of seven phytohormones in tomato fruit, including two synthetic analogs that mimic natural auxins and cytokinins. Method optimization focused on extraction efficiency, solid-phase cleanup, and mobile phase composition, achieving high recovery (85–95%) and reduced matrix effects. Chromatographic separation was performed on a C18 column, with detection by triple quadrupole mass spectrometry in MRM mode. The method demonstrated excellent linearity (R2 > 0.98), precision, and robustness, with detection limits as low as 0.05 ng/mL for abscisic acid and 6-benzylaminopurine. Validation followed US-FDA and EC 2021/808 guidelines, ensuring regulatory compliance and analytical reliability. Analysis of tomato samples from five geographic origins revealed significant differences in phytohormone profiles, particularly in abscisic and salicylic acids, highlighting the method’s ability to capture biologically and agriculturally relevant variation. This workflow offers a sensitive, transferable platform for monitoring bioactive compounds in tomatoes and other food crops, supporting post-harvest quality assessment and food metabolomics research.