Chemical Profiling of Aboveground and Underground Parts of Pterocephalus hookeri by Integrated FBMN, Untargeted LC-MS Metabolomics, and PAD-DESI-MSI

Pterocephalus hookeri (C.B.Clarke) Höeck is a classic traditional Tibetan medicinal herb with multiple pharmacological activities. The inconsistent usage of its medicinal parts (whole herb, aboveground part (AP), and underground part (UP)) in commercial circulation severely restricts its clinical safety and quality stability. Currently, most existing chemical investigations focus on the whole herb, whereas the intraspecific chemical discrepancies between AP and UP remain poorly clarified. Herein, an integrated analytical strategy combining ultra-high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS)-based untargeted metabolomics, feature-based molecular networking (FBMN), and paper-based analytical device desorption electrospray ionization mass spectrometry imaging (PAD-DESI-MSI) was established to characterize differential metabolites and their spatial distribution in P. hookeri. A total of 101 compounds were annotated, and 12 vital differential metabolites were further screened with variable importance in projection (VIP) values > 1. The visualized distribution differences of these biomarkers were validated via heatmap and PAD-DESI-MSI analysis. Obvious differences in chemical accumulation characteristics were confirmed between AP and UP, which can guide reasonable clinical medication and rational dosage regulation referring to metabolite abundance. Moreover, optimized data filtering thresholds effectively eliminated metabolomic false positives, and FBMN exhibited excellent capacity for differential biomarker screening. This study provides a solid chemical basis for the quality evaluation and rational medicinal application of P. hookeri.