Lettuce is an important leafy vegetable that represents a significant dietary source of antioxidants and bioactive compounds. However, the levels of metabolites in different lettuce cultivars are poorly characterized. In this study, we used combined GC × GC-TOF/MS and UPLC-IMS-QTOF/MS to detect and relatively quantify metabolites in 30 lettuce cultivars representing large genetic diversity. Comparison with online databases, the published literature, standards as well using collision cross-section values enabled putative identification of 171 metabolites. Sixteen of these 171 metabolites (including phenolic acid derivatives, glycosylated flavonoids, and one iridoid) were present at significantly different levels in leaf and head type lettuces, which suggested the significant metabolomic variations between the leaf and head types of lettuce are related to secondary metabolism. A combination of the results and metabolic network analysis techniques suggested that leaf and head type lettuces contain not only different levels of metabolites but also have significant variations in the corresponding associated metabolic networks. The novel lettuce metabolite library and novel non-targeted metabolomics strategy devised in this study could be used to further characterize metabolic variations between lettuce cultivars or other plants. Moreover, the findings of this study provide important insight into metabolic adaptations due to natural and human selection, which could stimulate further research to potentially improve lettuce quality, yield, and nutritional value.

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