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

## TL;DR

Researchers used a new method to classify and analyze walnut pellicle polyphenols, revealing regional differences in their chemical makeup and biosynthesis.

## Contribution

The study introduces a pseudotargeted metabolomics approach to comprehensively profile and classify walnut pellicle polyphenols.

## Key findings

- 406 polyphenolic compounds were identified and classified from walnut pellicle samples.
- Multivariate analyses revealed region-specific metabolic fingerprints in walnut pellicle polyphenols.
- KEGG analysis showed significant regional differences in flavonoid and phenylpropanoid biosynthesis.

## Abstract

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.

•Walnut pellicle polyphenols were analyzed by pseudotargeted metabolomics method, integrating UHPLC-QTOF-MS and LC-QTRAP-MS.•A total of 406 polyphenolic compounds were tentatively identified and systematically classified.•Multivariate analyses (PCA, PLS-DA, OPLS-DA) revealed region-specific metabolic fingerprints.•KEGG enrichment analysis revealedsignificant regional differences in flavonoid and phenylpropanoid biosynthesis.

Walnut pellicle polyphenols were analyzed by pseudotargeted metabolomics method, integrating UHPLC-QTOF-MS and LC-QTRAP-MS.

A total of 406 polyphenolic compounds were tentatively identified and systematically classified.

Multivariate analyses (PCA, PLS-DA, OPLS-DA) revealed region-specific metabolic fingerprints.

KEGG enrichment analysis revealedsignificant regional differences in flavonoid and phenylpropanoid biosynthesis.