Construction Of A Reversed-Phase Hydrophilic Comprehensive Two-Dimensional Liquid Chromatography And Its Application In Analysis Of Traditional Chinese Medicine

Since traditional Chinese medicine(TCM)is a typical ultra-complex system,understanding its chemical composition is the basis for in-depth study of the mechanism of TCM and the promotion of modernization of TCM.However,due to its super-complexity,a single separation method is often unable to achieve a clear analysis of its components,and exploring strategies for better separation results has become a crucial topic in the field of traditional Chinese medicine research.Two-dimensional chromatography is an excellent choice to some extent.Because far exceeding the peak capacity of one-dimensional separation technology,it has great room for development in the separation of complex systems of traditional Chinese medicine.A highly orthogonal combination of two-dimensional systems such as(reverse phase×hydrophilic chromatography combination)can provide almost completely opposite separation mechanisms in two dimensions.In theory,it can achieve efficient separation of complex systems such as traditional Chinese medicine.However,the problem of high solvent incompatibility between two-dimensional systems limits the application of this combined two-dimensional chromatography.Therefore,the combination of three auxiliary modulation techniques of first-dimension pulse gradient elution,trapping column,and at-column dilution was used in this study to construct a method that overcomes the problem of solvent incompatibility and can realize a freely adjustable reverse phase×hydrophilic interaction chromatography(RPLC×HILIC)comprehensive two-dimensional system.In order to validate the application of the constructed system in the complex analysis of traditional Chinese medicine,the representative traditional Chinese medicines Panax notoginseng,Codonopsis pilosulae and Platycodon grandiflora were selected as the objects of analysis.Based on the optimization of the separation conditions of the system and the combination with the mass spectrometer detector,the efficient separation and on-line identification of the three traditional Chinese medicine components were achieved.Specific studies are as follows:1.First,we analyzed the saponins of panax notoginseng with both hydrophilic and hydrophobic groups by adjusting the first dimensional RPLC pulse elution gradient,second dimensional HILIC separation gradient,dilution factor for transfer fractions,on-column modulation time.The second dimensional column equilibrium time and second dimensional flow rate are factors for the newly developed multi-assisted technique modulated by the RPLC×HILIC comprehensive two-dimensional system was fully optimized to verify its advantages in overcoming solvent incompatibility and two-dimensional free modulation.And under two-dimensional optimization conditions,the two-dimensional system was compared between the conventional standard modulation and fixed solvent modulation strategies for the Separation of Codonopsis extract.The results show that the two-dimensional system using the modulation method proposed in this study has significant advantages in the separation effect.Finally,under optimized conditions,the constructed RPLC×HILIC system and triple quadrupole mass spectrometry were connected to perform mass spectrometry analysis on the separated panax ginseng saponins,and the MS~1 and MS~2 data collected were compared with the reported references.In comparison,41 saponins were successfully identified.2.Most of the constituents in the radix codonopsis,such as alkynyl glycosides and phenylpropanoid glycosides,contain hydrophilic and hydrophobic groups in their structures.These amphiphilic compounds are well suited for separation using the RPLC×HILIC system.Therefore,the second part of this study was conducted to further validate the separation performance of the constructed RPLC×HILIC system using Dang Gui as a sample.By adjusting the extraction solvent,the first-dimension RPLC pulse elution gradient,the second-dimension HILIC separation gradient and dilution factor of the transfer fraction,which play a key role in the two-dimensional separation,the system was carefully optimized.Under the optimization conditions of two dimensions,the system proposed in this paper is compared with the two-dimensional system using a fixed solvent modulation strategy.The results show that the comprehensive two-dimensional system using the on-column dilution strategy has significant advantages in the separation of Codonopsis Extract.Finally,the optimized RPLC×HILIC system was coupled to triple quadrupole mass spectrometry.Online mass spectrometry analysis of the chemical constituents in Dang Gui extracts was performed and the collected MS~1and MS2 data were compared with reported.A total of 46 compounds were identified by comparing the relevant mass spectra in the reference literature.3.Oleanolane triterpenoid saponins are one of the main components of platycodi radix,with both hydrophilic groups(sugars)and hydrophobic groups(parent nucleus).The RPLC×HILIC system is also well suited for the analysis,so we conducted a systematic separation and on-line identification of the constituents of Platycodon grandiflorum with the constructed two-dimensional system.The samples to be analyzed were obtained after solid-phase extraction of the extract of Platycodon grandiflorus.By optimizing the two-dimensional separation conditions and modulation parameters,high coverage separation of amphiphilic compounds in Kratom was successfully achieved.By combining the optimized RPLC×HILIC comprehensive two-dimensional system with triple quadrupole mass spectrometry,mass spectrometry analysis of the amphiphilic chemical composition of Campanulaceae was successfully achieved.32 compounds were successfully identified by comparing the collected MS~1 and MS~2 data with the identification results in the reference literature.Through the efficient separation and online identification study of three botanicals,the results showed that the RPLC×HILIC established in this paper was used to identify the three botanicals.The comprehensive two-dimensional system is a multi-dimensional separation technique with promising applications in Chinese medicine analysis.