Study On The Mass Spectrometric Analytical Method And Correlation Spectroscopy Of LC-MS/NMR Of Phenylethanoid Glycosides And Their Complex Mixtures

For the purpose of simultaneous structural identification of natural products in crude extracts,the fragmentation behavior of phenylethanoid glycosides etc.and correlation spectroscopy of LC-MS/NMR of their complex mixtures were studied.First of all,the hydroquinone glycosides and phenylethanoid glycosides were studied systematically by electrospray ionization tandem mass spectrometry in negative ion mode in order to finding out the relationship between their structural features and the corresponding characteristic fragmentation behavior.The fragmentation rules of these known compounds were summarized.Consequently,the NMR/LC-MS PDS and NMR/LC-MS CHS of the crude extracts of Fraxinus sieboldiana and Forsythia Suspensa were established.Combined with HPLC-MSⁿ and 2D NMR spectra,total nineteen constituents in these two extracts were identified successfully,in which three compounds were new from Forsythia Suspensa extract and four compounds were found from the genus Fraxinus for the first time.1.Characteristic fragmentation behavior of hydroquinone glycosides using electrospray ionization tandem mass spectrometryThe characteristic fragmentation behavior of hydroquinone glycosides from Fraxinus sieboldiana was investigated systematically by electrospray ionization tandem mass spectrometry in negative ion mode.The characteristic fragmentation reaction of these compounds was through the homolytic and heterolytic cleavage of the O-glycosidic bond to produce radical aglycone ion([Y₀-H]^-·) and aglycone ion(Y₀^-),respectively.It was found that the relative abundance of the[Y₀-H]^-· ion and Y₀^- ion of these compounds exhibited a clear difference depending on the glycosylation position,number and position of the methoxy group.In addition,unambiguous differentiation between the mono-O-glycosides isomers which differ in glycosylation position was achieved by comparing the relative abundance of[Y₀-H]^-· and Y₀^- ions with the optimized collision energy.For hydroquinone O-diglycosides,the fragmentation behavior was characterized by cross-ring cleavage of the sugar moieties,producing a series of ions related to the sugar moieties.The results of this study can facilitate the rapid determination of hydroquinone glycosides in crude plant extracts.2.Characteristic fragmentation behavior of phenylethanoifl glycosides using electrospray ionization tandem mass spectrometry The characteristic fragmentation behavior of various type phenylethanoid glycosides involving glucosyl groups,apiofuranosyl group and caffeyl group substituents was investigated systematically by electrospray ionization tandem mass spectrometry in negative ion mode.The relationship between their structural features and the corresponding characteristic fragmentation behavior was investigated.It was found that the glycosylation position and substituent group at C-3 significantly influenced the fragmentation behavior of the phenylethanoid mono-O-glycosides.For phenylethanoid glycosides with caffeyl group,there were series of ions related to caffeyl group in their MS/MS spectra.The various type phenylethanoid glycosides could be identified by investigation of their ESI-MSⁿ spectra.And the results of this study can be useful for the rapid determination of phenylethanoid glycosides in crude plant extracts.3.NMR/LC-MS PDS and NMR/LC-MS CHS of extract of Fraxinus sieboldianaBased on the incomplete separation analysis strategy,the extract of Fraxinus sieboldiana was separated into series of fractions with the concentration dynamic variation of constituents by gel chromatography.The LC-MS and ¹H NMR data of series of fractions were acquired and then processed by MATLAB to form NMR/LC-MS PDS and NMR/LC-MS CHS.The correlation relationship between ions(m/z) and chemical shift(δ) deriving from the same molecule can be extracted simultaneously from the mixture spectra.Combined with HPLC-MSⁿ,¹H-¹H COSY,1D or 2D NOESY spectra, twelve constituents including three hydroquinone glycosides,eight phenylethanoid glycosides and one vanillic glycoside were identified successfully,in which four compounds were found from genus Fraxinus for the first time.4.NMR/LC-MS PDS and NMR/LC-MS CHS of extract of Forsythia SuspensaThe extract of Forsythia Suspensa was separated into series of fractions with the concentration dynamic variation of constituents by RP preparative chromatography.The LC-MS and ¹H NMR data of series of fractions were processed by MATLAB to form NMR/LC-MS PDS and NMR/LC-MS CHS,the correlation relationship between MS and ¹H NMR data deriving from the same molecule can be extracted from the mixture spectra. Combined with HPLC-MSⁿ,¹H-¹H COSY,2D NMR spectra,seven constituents including six phenylethanoid glycosides and one flavonol were identified successfully,in which three compounds were new.