Characterization Of Chemical Composition In Different Varieties And Hybrids Of Gastrodia Elata Based On Mass Spectrometry Molecular Network

Object: Gastrodia elata(G.elata),a traditional Chinese medicine,has complex varieties and hybrids,which contributes to the insufficiency of its quality control research.Therefore,more studies are in demand to provide scientific and valid support for the quality control research of G.elata.Therefore,a series of in-depth studies were performed to explore the chemical basis of G.elata,including the comprehensive characterization of compounds in G.elata and the search for the characteristic chemical differences among common G.elata varieties.Then,these studies further led to the method development of distinguishing G.elata varieties and hybrids based on their chemical characteristics.Methods:(1)A five-dimension data acquisition strategy was developed for compound discovery and characterization for G.elata based on the technique named "offline twodimensional liquid chromatography with high-resolution ion mobility mass spectrometry(offline 2D-LC-Q-TOF-IMS-DIA-MS/MS)".Briefly,an offline twodimensional liquid chromatography(2D-LC)system was established.In the 2D-LC system,the first-dimensional chromatography was for preparations of the G.elata sample fractions,and the second-dimensional chromatography was combined with the Q-TOF-IMS-DIA-MS/MS technique to collect the multi-dimensional data of the G.elata sample fractions.After evaluating the peak capacity and orthogonality of the established offline 2D-LC system,the rapid compound identification and characterization strategy were developed to characterize the potential novel compounds in G.elata comprehensively.For further distinguishment and verification of the separated isomers from the G.elata compounds,the value acquired from ion mobility mass spectrometry(IMS)was further analyzed.(2)The plant metabolomics study was performed to compare G.elata varieties based on the UPLC/Q-TOF-MS/MS method.In this step,non-targeted LC-MS data from different varieties of G.elata were collected for rapid characterizations of the common components in G.elata,and Global Natural Products Social Molecular Networking(GNPS),a web-based open-source mass spectrometry ecosystem,was applied to discover and identify the unreported and the differential compounds in different G.elata varieties.The chemical differences among G.elata varieties were demonstrated and visualized using chemometric analysis.This approach was verified by various batches of commercially available G.elata samples.Furthermore,Support Vector Machine(SVM)classification model was built based on the R language and applied to achieve effective prediction and distinguishment of G.elata varieties in the market.(3)The characteristic chromatograms were obtained using high-performance liquid chromatography-ultraviolet detector(HPLC-UV)to simplify the quality control process for G.elata variants and hybrids.To achieve this,chromatographic methods were established,optimized,and validated based on the characteristic compounds’ UV absorption spectra in G.elata variants.Then,multiple batches of G.elata samples were analyzed to establish the HPLC-UV characteristic chromatograms,which revealed the potential indicative compounds for quality control.Combined with chemometric analyses,the indicative compounds obtained from HPLCUV characteristic chromatograms could successfully distinguish different G.elata varieties and hybrids,through which the quality control of G.elata could be achieved.Results:(1)A five-dimensional data acquisition system for compounds in G.elata based on Offline 2D-LC-IMS-DIA-MS/MS of RP×HILIC columns was established.A total of 13 fractions were obtained through the first-dimensional chromatography,and their multi-dimensional data were successfully collected through the seconddimensional chromatography combined with Q-TOF-IMS-DIA-MS/MS.The established Offline 2D-LC system had a peak capacity of 4550 and an orthogonality of0.73.Moreover,the four-step rapid compound discovery and characterization strategy were developed.Two hundred seventy-two compounds in G.elata were successfully characterized,including 146 potential novel compounds;the collision cross section(CCS)database of compounds from G.elata was established for the first time.The CCS prediction and matching strategy showed a prediction error of less than 4%,and the identification results of 12 pairs of isomer compounds were validated.(2)Based on the non-targeted LC-MS data of 15 batches of different G.elata varieties,the multilateral mass defect filter strategy(MDF)was successfully established,and 115 common compounds were characterized.Through the GNPS molecular network,32 potential phospholipid compounds were discovered,as well as 47 characteristic components from two G.elata varieties-Hongtianma and Wuhongtianma.The chemometric analysis revealed the chemical differences among different varieties of G.elata.Then,the accuracy of the GNPS-identified characteristic components was further verified using 37 batches of commercially available G.elata samples.The established SVM classification model could accurately distinguish the varieties of G.elata with100% correctness and effectively predict the varieties of commercially available G.elata.(3)Under the optimized wavelength at 290 nm,the chromatographic peak information of the potentially different compounds in G.elata was well presented.The HPLC-UV characteristic chromatographic method was optimized using a single factor investigation method,demonstrating good specificity,stability,and precision.The HPLC-UV characteristic chromatograms were acquired using three major G.elata varieties and hybrids,namely Wutianma(WTM),Wuhongtianma(WHTM),and Hongtianma(HTM).The PCA discriminant model was established based on 13 common chromatographic peaks and four characteristic chromatographic peaks,which could distinguish WTM from other G.elata variants.In summary,the HPLC-UV method developed above had achieved characteristic identification of different varieties and hybrids of G.elata.Conclusion: The Offline 2D-LC-Q-TOF-IMS-DIA-MS/MS system established in this study can effectively enrich,separate,discover,and characterize the compounds in G.elata.A total of 272 compounds have been identified,which will promote the research of G.elata on its chemical basis and quality control.The combination of "UPLC/QTOF-MS/MS"-based plant metabolomics and GNPS molecular network can effectively discover the chemical differences among different varieties of G.elata.The established SVM classification model can effectively distinguish different variants of G.elata samples for quality control purposes.Different HPLC-UV characteristic chromatograms of G.elata have been developed and obtained.The PCA discriminant model was established from the 13 common chromatographic peaks and four characteristic chromatographic peaks,which can successfully distinguish Wutianma,a superior G.elata variation,from other variants of G.elata.