Organ-Resolved Salicylate-Phenylpropanoid Metabolite Profiling of Echinacea purpurea, E. pallida, and E. tennesseensis Tissues Extracted with 50% Aqueous Methanol by LC-MS/MS and LC-HRMS.

Salicylic acid (SA) is a central plant defense hormone, but its occurrence and metabolic context in Echinacea tissues have not been systematically characterized. This study aimed to characterize endogenous free SA, salicylate-associated features, and their organ-level distributions in selected specimens of Echinacea purpurea, E. pallida, and E. tennesseensis, and to evaluate free SA variability among individual E. purpurea flower heads. Targeted LC-MS/MS and retrospective LC-HRMS profiling were used to examine free SA, preservative panel analytes, salicylate-related pathway features, phenylpropanoid/caffeic acid derivatives, and flavonoid-related metabolites across roots, stems, leaves, and flowers of Echinacea purpurea, E. pallida, and E. tennesseensis. Free SA was detected in selected aerial and reproductive tissue extracts, whereas benzoic acid, dehydroacetic acid, and paraben esters were not detected. Confidence-qualified LC-HRMS yielded formula-level assignments and tentative candidates associated with salicylates, phenylpropanoids, caffeic-acid derivatives, and flavonoids. Free SA detection and broader salicylate-associated feature patterns were not identical, with candidate conjugated or pathway-related features also observed in roots. Analysis of 80 individual E. purpurea flower heads showed marked free SA variability (mean ± SD 4.72 ± 13.77 μg/g; range 0-121.48 μg/g). These findings support the endogenous occurrence of free SA in Echinacea within a broader, tissue-variable specialized-metabolite context. Future studies with greater biological replication, authentic standards, seasonal sampling, and mechanistic approaches should clarify salicylate biosynthesis, regulation, and biological significance in Echinacea.