Vilmantas Pedišius

TITLE: Plantago Metabolomics by LC–Orbitrap MS: From Broad Chemical Profiling to Bioactive Candidate Discovery Plant metabolomics is challenged by chemical diversity, in-source fragmentation, and structural isomerism. This work presents an integrated UHPLC–Orbitrap MSⁿ workflow for comprehensive characterization and bioactivity-guided prioritization of metabolites from Plantago lanceolata leaves and Plantago ovata husk. Low-energy stepped HCD at 10/50/80% normalized collision energy improved grouping of in-source fragments by 55% in positive mode and 16% in negative mode, while bioassay-guided chromatographic fractionation was introduced to progressively reduce extract complexity and associate defined chromatographic regions with biological responses. Complementary ddMS², ddMS³, AcquireX™ Deep Scan, and Real-Time Library Search balanced metabolome coverage and annotation confidence, yielding 472 putative identifications in P. lanceolata leaves and 233 in P. ovata husk. The workflow was subsequently extended to solvent-selected extracts, chromatographic fractions, and HUVEC-based assays measuring VCAM-1, ICAM-1, TNF-α, IL-1β, nitric oxide, reactive oxygen species, and cell viability. LC–MS features are prioritized using biological response, endothelial relevance, identification confidence, abundance, and practical isolatability. Ongoing work focuses on targeted isolation, authentic-standard confirmation, and biological re-testing of purified candidates. This study demonstrates how advanced LC–MSⁿ metabolomics can connect broad plant chemical profiling with the early-stage discovery of biologically relevant natural products.