| The Traditional Chinese Medicine(TCM)has been the achievements of the wisdom and the practical experience of many Chinese medicine scholars for thousands of years.It has made outstanding contributions to the human health and has become a part of the Chinese culture.However,it is difficult to explain its mechanism of action comprehensively and systematically and the accurate quality control also can not be realized since the Traditional Chinese Medicine is a complex system containing many super-complex components.As a result,the factors mentioned above have hindered the globalization development of the Traditional Chinese Medicine.Therefore,the in-depth and systematic research on the complex components of Chinese medicine has become the basis for its continued development.Among the existing analytical methods,two-dimensional liquid chromatography has become the first choice for the analysis of complex substance systems in the Traditional Chinese Medicine(TCM)due to its peak capacity much higher than that of one-dimensional chromatography.In addition,the proven application of LC-MS technology provides strong support for the rapid,effective and accurate identification of separated compounds online.The advantages of the two-dimensional system of RP/RP with good mobile phase compatibility,large theoretical peak capacity and high separation degree give it great development potentialfor the separation of complex systems in the Traditional Chinese Medicine(TCM).However,due to the poor orthogonality resulted from the similarity of separation mechanisms in different RP/RP column combinations,the development of the two-dimensional system of RP/RP combination is greatly limited.Therefore,this paper proposes to solve the problem of insufficient orthogonality through the optimization and combination of separation conditions,in order to take full advantage of reversed-phase chromatography for high performance separation.Three representative active components of Traditional Chinese Medicine were selected: alkaloids(M.cordata(willd.)R.Br.),anthraquinones(Radixet rhizoma rhei)and flavonoids(Scutellaria baicalensis).Different RPLC×RPLC systems were constructed for their different properties and the separation conditions were optimized to achieve efficient separation of these compound types.1.Due to the pH dependence of the dissociation of alkaloid compounds,the change of the mobile phase pH value is expected to achieve the change of the separation behavior of such compounds.Therefore,we designed a RPLC×RPLC system that used a high pH(9.0)mobile phase combined with a pulsed gradient elution mode in the first dimension(1st D)and a low pH(2.6)mobile phase in the second dimension(2nd D).On the two-dimensional connections we used parallel column modulator to reduce the dead volume in the second dimensionalseparation.Based on the above constructed system,we systematically optimized the separation conditions including the step height,pulse width,and second dimensional separation gradient of the pulse gradient,and obtained a good separation of the M.cordata(willd.)R.Br.component.The separation effect of applying a conventional gradient versus a pulsed gradient to a first-dimensional separation gradient was also compared.The results showed that the orthogonality of pulse gradient was much higher than the conventional gradient.Finally,the constructed two-dimensional system was linked to mass spectrometry to enable online analysis of the separated components.The obtained mass spectrometry results were compared with those reported in the literature and 39 alkaloids were successfully identified.2.The anthraquinone active substance contained in Radixet rhizoma rhei is a neutral compound,and the change of mobile phase pH has a limited effect on its retention.Therefore,a RPLC×RPLC system was constructed by combining a cyano(CN)column and a C18 column with certain differences in separation mechanism.Parallel column interface was used between the two dimensions to shorten the dead time of the second dimension analysis.First,according to the results of the separation of the standard products on the C18/CN column under different mobile phases(methanol,acetonitrile),the combination order of CN-RP/C18-RP was selected.In addition,the pulse elution gradient wasused instead of the linear gradient in the first dimension to increase the tunability of the two-dimensional separation time.The pulse width and step height affecting the separation result in the pulse gradient were optimized systematically.The second dimensional separation increased the resolution of a single cyclic gradient by introducing a shift gradient instead of a full gradient.In addition,the concentration difference in the shift gradient was optimized.Compared with the full gradient mode,the separation coverage was significantly improved.Finally,the CN-RPLC×C18-RPLC system was connected to mass spectrometry to identify the separated components online.By comparing and analyzing the MS information obtained from the experiment with the results reported in the reference literature,the preliminary identification results of 43 chromatographic peaks were successfully obtained.3.The active ingredients contained in scutellaria are mainly flavonoid neutral compounds.In order to verify the separation effect of CN-RPC×C18-RP the system in other neutral compounds,this study continued to use this method for the chemical constituents in Scutellaria baicalensis.The first and two-dimension used solvent-assisted modulation of pulse gradient and moving gradient respectively.The factors influencing the experimental results,including the pulse width and step height in the first-dimension pulse gradient,and the concentration difference in the second-dimension shift gradient,were systematicallyoptimized.The optimized conditions were connected in series with the mass spectrometer detector,and 38 compounds were successfully identified. |
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