Organic farming and crop quality are important concerns in the food marketplace. This study employed an untargeted, semi-quantitative metabolomics and lipidomics workflow to characterise differences in aqueous metabolite and lipid profiles between organically and conventionally cultivated lettuce (Lactuca sativa). Using UPLC-Orbitrap-MS, we identified 38 aqueous metabolites at Metabolomics Standards Initiative (MSI) levels 1 and 2 and 37 lipids at MSI level 2 that showed significant differences between cultivation systems. Organic lettuce consistently exhibited elevated relative levels of phenylalanine, tryptophan, and the phytohormone indole-3-acetic acid (IAA), alongside increased relative concentrations of eleven secondary phenolic metabolites. Network analysis of these metabolites revealed that protocatechuic acid in organic lettuce was strongly associated with triacylglycerols (TAGs) and diacylglycerols (DAGs). In contrast, conventional samples exhibited a higher degree of lipid unsaturation. These findings demonstrate that lipids show important cultivation-dependent differentiation. The integrated untargeted workflow provides a useful analytical framework for profiling cultivation-associated biochemical variation in lettuce and supports future development of authentication approaches after validation in balanced, multi-farm and multi-season studies.
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