| BackgroundThe composition of traditional Chinese medicine is complex,even if it is a single herb,and the chemical components are diverse in structure types and amount to over100 species.Studying the composition and structures of chemical components of TCMs are of great significance for its quality control,development and utilization.Liquid chromatography-mass spectrometry(LC-MS)can obtain the overall chemical profile and component structure information of TCMs through little number of samples and a short analysis time.However,the peak capacity of one-dimensional chromatographic is limited,and chemical components co-eluted or trace components masked are common.The mass fragments from mass spectrometry cannot satisfy the identification of isomers with consistent fragments.Therefore,there are still some challenges in the analysis of TCMs’ components by LC-MS.Two-dimensional liquid chromatography combines columns with different separation mechanisms to improve the chromatographic separation ability and increase peak capacity.Offline two-dimensional chromatography can improve the content of trace components through beneficiation,reducing the difficulty of detection.The ion mobility technology utilizes the different migration rates of ions with different structures in the electric field to achieve separation,providing important conformational information for structural differences.Combined ion mobility with chromatography and mass spectrometry,which make isomers identification and discriminate possible.Herba Gynostemma,used as a TCM,is a whole herb of various plants in the genus Gynostemma of the Cucurbitaceae,and widely distributed in many provinces and cities in southern China.Due to its good lipid lowering activity,it has been developed into a Chinese traditional patent medicine for clinical use,but it has not been included in the2020 edition of the Chinese Pharmacopoeia,and there is no unified quality standard.The main component in Herba Gynostemma is dammarane type triterpenoid saponins.Based on the previous research in our lab,it was found that there are almost no same components in Herba Gynostemma from different origins.At the same time,the commercial sources of Herba Gynostemma are different,including various species,origins,medicinal and tea usage,as well as the different part of plant,which will affect its chemical composition.Therefore,there is an urgent need to conduct a systematic chemical composition research on Herba Gynostemma in the main production areas to clarify the use part,the species and origins,and promote the quality control and application development.Objective1.To further know the composition of Herba Gynostemma from different habitats and parts through characterization and identification of its total components.2.To establish a new method and strategy by LC-MS for the analysis of TCMs based on multi-dimensional chromatography and ion mobility mass spectrometry to solve the problems of chemical components co-eluted and isomers distinguished.Methods and Results1.Analysis of Herba Gynostemma from prolific regions of ChinaA fingerprint of Herba Gynostemma was established through UHPLC-Q-TOF/MS,and 53 batches of samples from Shaanxi,Sichuan,Fujian,Hunan,and Guangxi were evaluated using similarity evaluation and multivariate statistical analysis.The results showed that the samples of G.Longipes from Shaanxi and Sichuan in Qinba Mountains had high similarity and good consistency.And the sample of G.pentaphyllum from Fujian had high consistency of the intragroup,and there were significant differences with other origins.2.Establishment a component mass spectrometry database of Herba GynostemmaA total component mass spectrometry database of Herba Gynostemma was established through literature research.The CCS values of triterpenoid saponins in the database were predicted using the ALLCCS and CCSondemand prediction platforms.The accuracy of the predicted values was verified by the measured CCS values of 100 reference standards.At the same time,100 reference standards were systematically analyzed by liquid chromatography,mass spectrometry and ion mobility,and their chromatographic separation rules,characteristic mass fragments,correspondence between parent and aglycone ions of compounds and structures,and ion mobility separation rules of isomers were summarized.The results showed that the database summarized a total of 501 chemical components of Herba Gynostemma,including triterpenoid saponins,flavonoids,organic acids,sterols,and glycosides,among them,triterpenoid saponins were the major components,altogether 434.After prediction by two platforms,4552 CCS values predicted of 434 triterpenoid saponins with different adducts were obtained.According to the validation of 100 CCS values measured for[M+HCOO]-,the deviation between the predicted values of CCSondemand and the measured values was smaller,but there was still a certain difference between them.The chromatographic separation of the reference standards showed that the retention time of compounds with high polarity was small on C18 chromatography column.When the structure is only isomeric in the R/S configuration,the retention time of the isomer in the S configuration is shorter than that in the R configuration on the C18 column.According to the mass fragments,it can be seen that gypenosides need to analyze combine in the positive and negative ion.In the negative ion mode,the main fragments are the deprotonated molecular ion,neutral-loss fragment ions of the glycosyls,and side-chain cleavage of C-17,while in the positive ion mode,the main fragments are the characteristic aglycone ions.Combining the CCS values of parent ions and aglycone ions,the hydroxyl isomers of C2,C12,C24,C25,and C26 can be distinguished and identified.The multi-cyclic separation ability of ion mobility can be used to separate co-eluted isomers by liquid chromatography.Finally,the mass spectrum database of Herba Gynostemma was formed,containing molecular structure information,mass fragments,CCS values of parent ions and aglycone ions.At the same time,based on the systematic study of the reference standards in chromatography,mass spectrometry,and ion mobility,a grading identification strategy,identifying the possible structure through mass fragments and distinguishing isomers by ion mobility,was established for the component characterization of traditional Chinese medicine.3.Composition analysis of G.pentaphyllum produced in FujianA systematic analysis of G.pentaphyllum from Fujian was carried out through multicenter cutting two-dimensional chromatography.The components were identified through the grading identification strategy.The isomers that cannot be separated by chromatography of G.pentaphyllum from Fujian were separated using cyclic ion mobility.The results showed that the six saponins with the highest content in G.pentaphyllum were removed through online multicenter cutting two-dimensional chromatography,achieving the enrichment of trace components.Combined with the grading identification strategy,a total of 190 compounds were identified from G.pentaphyllum from Fujian,including 176 triterpenoid saponins,11 flavonoids,3 organic acids,and 35 possible new compounds.According to the regularities between the CCS values and their structures of aglycone ions,the hydroxyl isomers of C2,C12,C24,C25,and C26,as well as C24 configuration isomerism of gypenosides were distinguished and identified from G.pentaphyllum.And combined the CCS values of the parent ions and aglycone ions,a total of 50 groups of isomers were identified.The separation of FJ-50 and FJ-54 in the peak 73 was achieved by 15 cycles of cyclic ion mobility.4.Composition analysis of G.Longipes produced in ShaanxiOne-dimensional hydrophilic columns and two-dimensional C18 columns were used to optimize two-dimensional chromatographic separation conditions of the roots,stems,and leaves of G.longipes from Shaanxi province,and a systematic study was conducted on the composition identification of it.The grading identification strategy was used to identify the chemical components of different parts of the samples,and the multivariate statistical analysis was used to study the component differences of different parts.The results showed that after off-line two-dimensional chromatography analysis,peak capacity was improved,and the problem of chromatographic co-eluted of G.longipes saponins was improved.Finally,396 components were identified from three parts.According to the comparative analysis between the measured CCS value and the predicted CCS value in the database,isomers with significant differences in CCS value of G.longipes were identified,among which 94 groups of isomers were identified,and217 possible new compounds were found based on the grading identification strategy,and most of them are acidic saponins connected with acetyl and/or malonyl groups.Through HCA analysis and PCA analysis,it was clear that the roots,stems,and leaves were clustered separately,showing significant differences.Using OPLS-DA to analyze the aboveground and underground parts,36 different components were selected with a VIP value greater than 6.5,including 2 flavonoids,which were higher in the stems and leaves.Among the remaining 34 saponins,32 were mainly present in the root.These triterpenoid saponins were connected with trisaccharide or tetrasaccharide,and half of them were acidic saponins.It could be seen that the roots of G.longipes in Qinba Mountains contained more triterpenoid saponins.Conclusions1.In this paper,a systematic component analysis of Herba Gynostemma from major habitats in China was analyzed using LC-MS,similarity evaluation,and PCA analysis.The Qinba Mountains(Shaanxi and Sichuan),and Fujian were selected to analyze because of the stable component composition and high yield.The composition of G.longipes from Shaanxi and the components difference between the aboveground and underground parts were analyzed by off-line two-dimensional chromatography and ion mobility mass spectrometry,as well as the composition of G.pentaphyllum from Fujian were also analyzed.Using a grading identification strategy,firstly,the novelty of the compound was judged based on quasi-molecular ions and a self-built database.Subsequently,the aglycone was identified using the characteristic mass fragments of positive and negative ions.Then,neutral loss fragments were used to identify the substituents and composition of glycosides.Finally,the CCS value was used to distinguish and identify the isomers.The results showed that a total of 586 components were identified,and a large amount of acidic saponins were found in the original components of Herba Gynostemma for the first time.Among them,the triterpenoid saponins from G.longipes from Shaanxi Province could be divided into two structural types: straight chain and cyclization at the C17 position.When the side chain at C17 position is a straight chain,the C21 position is often substituted with hydroxyl.The structure of C17 of G.longipes from Fujian was mainly a straight chain,and hydroxyl substitutions are common in C24,C25,and C26,but rarely in C21.A systematic comparison of the differences in the components of the roots,stems,and leaves of G.longipes from Shaanxi was analyzed for the first time.It was found that there were a large number of triterpenoid saponins in the roots,which has research and development value.2.In this paper,offline two-dimensional chromatography was used to improve the co-eluted separation in a single dimensional analysis of G.longipes from Shaanxi.Through multi-center cutting two-dimensional chromatography,the trace components were enriched in G.pentaphyllum from Fujian,improving the chromatographic characterization ability,effectively.Using the multi-cycles separation ability of cyclic ion mobility,the separation of isomers was achieved for the first time,those with the same mass fragments and difficult chromatographic separation of G.pentaphyllum from Fujian.3.The comparison between the predicted and the measured CCS values in this article research was used to identify the isomers with large differences in CCS values.For the first time,compared with the CCS values of dammarane type triterpene saponin aglycone ions,and combined with the CCS values of the parent and product ions altogether,the saponins isomers of the hydroxyl linkage in the C2,C12,C24,C25,and C26 positions were identification in Herba Gynostemma,successfully.4.The grading identification strategy established in this article could be used for rapid identification of Herba Gynostemma components,and this strategy is also applicable to the characterization and identification of other TCMs’ components. |
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