Structural Identification Of Natural Product Mixtures Ms And Nmr Parallel Dynamic Spectrum And New Analytical Methods For Orthogonal Correlation Spectroscopy Study

The NMR/LC-MS parallel dynamic spectroscopy(NMR/LC-MS PDS) and NMR/LC-MS correlation heterospectroscopy(NMR/LC-MS CHS)were established based on incompleted separation strategy for the simultaneous structure identification of natural products in crude extract. NMR/LC-MS PDS and NMR/LC-MS CHS were formed through co-analysis of multi-spectroscopic data sets from LC-MS and ~1H NMR with signal amplitude co-variation by the routines compiled and optimized in MATLAB.Based on concentration variation of constituents in series of incompletely separated fractions,the intrinsic correlation between retention time,m/z and chemical shift data of the same constituent from mixture spectra can be discovered.The two methods were primarily designed using two series of artificial mixtures,made of known flavonoids to simulate the fractions from incompleted chromatographic separation,in which constituents had different concentration variation profiles and chromatographic overlap degree.Furthermore,NMR/LC-MS PDS and NMR/LC-MS CHS were applied successfully to identification of 12 constituents in active herbal extract(AB-8-2) involving flavonol glycosides,including two sets of coeluted isomers.Besides,a data processing and visualization tool was developed based on MATLAB software.1.Ineompleted separation analysis strategySeveral series of incompleted separation mixtures were simulated with computer, then the four factors of chromatographic resolution,relative concentration,number of constituents and signal overlap were studied with the correlation coefficient between constituents as index.The results indicated that relative concentration and number of constituents had a minor influence,signal overlap reduced the correlation coefficients, and the chromatographic resolution had a notable influence.However,compared with the resolution of 1.5,one of the strategy had a lower request on chromatographic resolution, the key was to acquire the series of fractions with concentration variation of constituents, to ensure that the correlation coefficient between constituents were smaller than that of the self-correlations.Besides,based on the moderate resolution and larger sample loading of the automatic flash column chromatography,it was used to prepare the series of fractions with different concentration variation requested in this new analytical strategy.2.The establishment of NMR/LC-MS PDS and NMR/LC-MS CHSTwo series of artificial mixtures with concentration variation of constituents and different chromatographic overlaps were made of six known flavonoids,to simulate the fractions from incompleted chromatographic separation.The LC-MS and ~1H NMR spectra of the series of artificial fractions were acquired,and then processed with routines compiled and optimized in MATLAB.NMR/LC-MS PDS spectra was formed through co-analysis of parallel visualized multi-spectroscopic data sets from LC-MS and ~1H NMR with signal amplitude co-variation,based on regular concentration variation and sample distribution of constituents in series of incompletely separated fractions. Furthermore,the correlation coefficients between the signal intensities of LC-MS and NMR spectra from all the mixture samples were calculated with chemometrics algorithms, and were visualized with a reasonable cutoff to form NMR/LC-MS CHS.On the basis of integrated analysis,the correlation relationship between ions(m/z) and chemical shifts(δ) deriving from the same molecule can be extracted simultaneously from the mixture spectra.,thus the structural information including retention time,molecular ion,fragment ion,chemical shift and couple pattern of a molecule can be obtained.With the visualization of NMR/LC-MS PDS spectrum,the fine characters of changing ranges and profiles of signal intensity can be observed,which will facilitate the reorganization and deconvolution analysis of overlapped ~1H NMR signals;with the color coded XIC data,the isomers existed in mixture can be observed intuitionally;it also can guide the grouping and peak alignment processing of ~1H NMR data.With the correlation calculation of LC-MS data,the cutoff value of correlation coefficient of each constituent in NMR/LC-MS CHS spectrum can be forecasted.3.Appling NMR/LC-MS PDS and NMR/LC-MS CHS on the study of complex active herbal extractBased on incompleted separation analysis strategy,the active herbal extract AB-8-2 with remarkable activity of resisting mental retardation and antithrombotic was separated into series of fractions with concentration variation of constituents by the automatic flash column chromatography,which had the character of moderate resolution and larger sample loading.Based on the integrated analysis of the LC-MS and 1H NMR data of the obtained series of fractions by 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.Furthermore,12 flavonol compounds with relative concentration ranged from 0.1%to 39.4%in active herbal extract were identified successfully.Besides quercetin and kaempferol,there were five quercetin-based glycosides and three kaempferol-based glycosides,as well two sets of co-eluted isomers.Moreover,other relevant spectral data, including LC-MS/MS,1D TOCSY or 1D NOESY can be integrated under the guide of the assigned signals,which will provide the complemented and comprehensive data for the unambiguous structure identification of the constituents with similar structure or isomers existed in mixture.4.Data processing routines and user interfaceWith the preprocessing of the LC-MS and ~1H NMR data by the workstation software, the raw data were converted into text format file.Then the data were processed by the routines compiled and optimized in MATLAB,including signal extraction,peak alignment and correlation calculation.NMR/LC-MS PDS spectra was formed through co-analysis of parallel visualized multi-spectroscopic data sets from LC-MS and ~1H NMR with signal amplitude co-variation,based on regular concentration variation and sample distribution of constituents in series of incompletely separated fractions; NMR/LC-MS CHS with a reasonable cutoff was formed by visualizing the correlation coefficients between the signal intensities of LC-MS and NMR spectra from all the mixture samples calculated with chemometrics algorithms.Moreover,the LC-MS data were converted into series of independent vectors,which were used in the correlation calculation with the NMR data,such a data processing mode can facilitate to distinguish the correlation information of isomers by retention time.The emphases and difficulties in routines compiling were existed in the data extraction of LC-MS data and the automation of peak alignment of ~1H NMR signals.Based on the routines,the user interface was developed,which will facilitate the data processing courses and was easy to master and operate.