To derive and verify the chemical structure of the new components in Epimedium, the laws of secondary metabolism and high-resolution mass spectrometry (HRMS) were combined. Based on the chemical literature of Epimedium, the secondary metabolism network of flavonols was constructed, and the possible metabolites were deduced. After the metabolites, information was imported into PeakView software, and the ions with a mass error < 5 ppm, correct isotope distribution, and containing secondary fragments were taken as the target compounds. The chemical structures of new compounds were identified and verified by combining Formula Finder, Mass Calculators, online databases (SciFinder, Reaxys, ChemSpider, etc.) and secondary fragmentation rules. In this study, a total of 4 metabolic pathways and 64 compound structures were deduced, and two new components and 12 new compounds were identified in 54 batches of Epimedium samples from 15 species by high-resolution mass spectrometry. Furthermore, the long and tedious steps of phytochemical separation were simplified, experimental costs were reduced, and a new idea and method were suggested for the analysis and identification of secondary metabolites with pharmacological activity.

Qin Weihan and Yang Yong