Structural verification of phenolic compounds in the aqueous extract of Chrysanthemum morifolium using LC-DAD-SPE-NMR method

This study demonstrated the capability of LC-DAD-SPE-NMR technique for verifying structures tentatively identified by LC-ESI-MS. Previous LC-ESI-MS analyses had characterized the major phenolic compounds in Chrysanthemum morifolium, yet the lack of complementary analytical tools left these assignments unconfirmed. Herein, the main compounds of this plant were unequivocally verified using LC-DAD-SPE-NMR, leading to the unambiguous identification of 19 compounds based on their NMR spectra. Among these, the structure of 4 flavonoid glycosides were corrected, and two novel compounds were reported for the first time. Structurally, caffeoylquinic acids were confirmed using ¹H NMR only, while aglycone and glycosidic bond in flavonoid glycosides were verified by ¹H NMR and ¹H¹H NOESY spectra. This work underscores the inherent limits of MS for structural elucidation and highlights the powerful structural elucidation and verification capability of LC-DAD-SPE-NMR for phenolic compounds. Furthermore, it establishes a robust analytical strategy for the structural verification of complex phytochemical mixtures.