Structure Identification Method Of Polyphenols Based On LC-MS And The Application In Traditional Chinese Medicine

Polyphenols are a class of compounds containing multiple hydroxyl groups.They are widely found in traditional Chinese medicines.Phenolic acids and flavonoids account for about 90% in total polyphenols.Polyphenols often exist in the forms of esters,glycosides and acylated glycosides.Polyphenols have many positional isomers,because of different positions of phenolic hydroxyl groups.Standardized MS/MS spectra for many polyphenols are not available,due to the lack of commercial standards,which is a big challenge for the structural annotation analysis of polyphenols.In the study,phenolic acids and flavonoids standards were analyzed by LC-MS analysis with multi-collision energies.The fragmentation rules induced by collision energy have been studied,especially the fragmentation characteristics of positional isomers.The in-silico database of polyphenols was established and applied to the analysis of dandelion,Erycibe obtusifolia Benth.,ginkgo leaf and Lonicera japonica Thunb..(1)Phenolic acid: Mono-and di-hydroxybenzoyl/cinnamoylquinic acid isomers were synthesized for LC-MS analysis.The chromatographic retention characteristics and fragmentation rules of positional isomers have been revealed.Mono-substituted hydroxybenzoyl/cinnamoylquinic acid isomers could be identified by the relative intensity of quinic acid characteristic fragment ions(m/z 191,173)and hydroxybenzoyl/cinnamoyl characteristic fragment ions.Di-substituted hydroxybenzoylquinic/cinnamoylquinic acid isomers could be distinguished by the relative intensity and intensity ratios of the characteristic fragment ions.An in-silico database was established based on the chemical substructure and MS/MS characteristic rules of phenolic esters,including 670hydroxybenzoyl/cinnamoylquinic acids,30 hydroxybenzoyl/cinnamoyltartaric acids,and705 hydroxybenzoyl/cinnamoyl mono-glucosides.(2)Flavonoids: Flavonoid aglycones,glycosides and their positional isomers were analyzed by LC-MS.The fragmentation rules of flavonoid aglycones,mono-glycosides,diglycosides,tri-glycosides and acylated flavonoid glycosides were analyzed.Flavonoid positional isomers could be identified by characteristic fragment ions(aglycone and sugar)and energy resolution curves.An in-silico database of flavonoid glycosides was established based on 33 flavonoid/flavonol/isoflavone aglycones,including 234 flavonoid monoglycosides,340 flavonoid di-glycosides,876 flavonoid tri-glycosides,and 6665 monoacylated flavonoid glycosides.(3)Applications: The structural identification analysis were performed out on dandelion,Erycibe obtusifolia,ginkgo leaf and Lonicera japonica Thunb..Di-pcoumaroyltartaric acid,vanilloyltartaric acid,3-O-vanilloylquinic acid,4-O-vanilloylquinic acid,5-O-vanilloylquinic acid,3-O-syringyolquinic acid,4-O-syringyolquinic acid,5-Osyringyolquinic acid,caffeoyl syringyolquinic acid,feruloyl syringyolquinic acid,quercetin-3-O-malonylglucoside,quercetin-3-O-acetylglucoside,kaempferol-3-O-acetylglucoside and sinapoylquinic acid-glucoside were firstly identified.The polyphenols in different medicinal tissues and flowering stages were further analyzed.The results showed that the composition and content of polyphenols was significantly different in different medicinal tissues and flowering stages.Through the above studies,the MS/MS database of polyphenols were enriched greatly,and the fragmentation characteristics of the position isomers were elucidated.The studies could improve the structural identification ability for polyphenols in traditional Chinese medicine,and provide solid foundation for the further deep study of polyphenols.