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Gallotannins (GTs) are a series of hydrolyzable tannins with multiple health-promoting effects. In this study, an integrated liquid chromatography tandem mass spectrometry (LC–MS/MS) strategy was developed for unveiling the spatial distribution pattern of GTs in the emerging oilseed crops Paeonia rockii and P. ostii. According to the fragmentation behavior of the representative GT (1,2,3,4,6-penta-O-galloyl-β-D-glucose, PGG), the diagnostic neutral loss (NL) of 170 Da was chosen for the non-targeted screening of GT precursors. Simultaneously, the tandem mass spectrometry spectrum (MS/MS) information was acquired through an enhanced product ion (EPI) scan. Nine major GTs were identified in tree peony. To quantify the targeted GTs in different tissues of tree peony, we established a multiple reaction monitoring (MRM)–enhanced product ion (EPI)-based pseudo-targeted approach under the information-dependent acquisition (IDA) mode. The quantitative results show that the GT compounds were ubiquitous in tree peony plants with diverse structures. The typical GT PGG was mainly distributed in roots, leaves, and petals. This strategy can also be utilized for metabolite characterization and quantification in other substrates.

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