Classification and metabolomic profiling of walnut pellicle polyphenols using a Pseudotargeted metabolomics approach

Walnut pellicle is rich in polyphenols that enhance antioxidant capacity and health benefits, including anti-inflammatory and neuroprotective effects. Profiling these compounds has been hindered by their structural diversity, regional variability, and the limitations of traditional metabolomics approaches. This study employed a pseudotargeted metabolomics strategy, integrating ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) for untargeted profiling and multiple reaction monitoring (MRM) on a QTRAP system for semi-quantification. We analyzed 21 walnut pellicle samples from Xinjiang, Yunnan and the Taihang Mountains, identifying 406 polyphenols, including flavonoids, hydrolysable tannins, phenolic acids, coumarins, lignans and minor constituents. Multivariate analyses (PCA, PLS-DA, OPLS-DA) revealed region-specific metabolic fingerprints. KEGG pathway enrichment highlighted significant variations in flavonoid and phenylpropanoid biosynthesis across regions, with origin-specific markers like casuarinin, prunin, and ε-viniferin supporting provenance authentication. This research bridges the methodological gap in walnut polyphenol analysis and informs quality assurance, targeted breeding, and functional product development in sustainable food systems.