In Situ Ionization Mass Spectrometry Analysis Of Ginsenosides

As a well-known natural product,Panax ginseng Meyer(P.ginseng)serves as raw material of various healthcare products.Modern pharmacological researche has revealed that ginseng could modulate the cardiovascular,endocrine and immune systems and has the potential to prevent cancer,reduce obesity,and slow the aging process etc.Ginsenosides are considered to be the most important biologically active substance that affects the efficacy of ginseng,which is a type of glycoside compound composed of sapogenin and sugar.The structure of ginsenosides is complex and there are a large number of isomers.The main analysis method is liquid chromatography mass spectrometry(LC-MS).When characterizing complex natural product samples,LC-MS can provide structural information with high sensitivity,specificity and versatility,and is a powerful tool for ginsenoside analysis.In recent years,ambient ionization mass spectrometry(AIMS)analysis technology has attracted widespread attention due to its direct sampling under atmospheric pressure,no need for much sample preparation and simple operation.Among them,direct analysis in real time mass spectrometry(DART-MS)has become a mature technology that can quickly analyze various samples and has shown wide applications in many fields.This paper is based on the AIMS technology—DART-MS to analyze ginsenoside and ginseng samples.The research content is as follows:1.Established a high-efficiency extraction and direct DART-MS analysis method for ginsenosides.Based on the mechanochemical extraction(MCE)method,ginsenosides were successfully extracted from ginseng samples efficiently.The extraction solvent and MCE extraction conditions were optimized.Compared with the traditional extraction method of ginsenosides,it shows that MCE is simple to operate,fast and efficient.The direct DART-MS analysis of ginsenosides produced fragments,and no complete molecular ion peaks were observed.Ginsenosides with the same sapogenin structure will produce highly similar or identical DART-MS mass spectra,while the fragment peaks of different types of ginsenosides are significantly different.Combined with MCE,this study established a DART-MS method that can quickly distinguish the types of ginsenosides contained in each ginseng sample and compare their contents,which proves that the DART-MS method can be used as a quick method for the quality assessment of ginseng samples.2.Analysis of ginsenosides by DART-MS method combined with tetramethylammonium hydroxide(TMAH)methylation.By adding the transient derivatization reagent TMAH to assist the ionization of ginsenosides,the complete molecular ion peak of DART-MS after ginsenoside methylation can be detected within a few seconds,and then various ginsenosides of different molecular weights can be identified.This method was used to quickly detect the ginsenosides in eight ginseng samples,and the relative content of ginsenosides in each ginseng sample was compared and analyzed.At the same time,using multi-dimensional variable data analysis to visually analyze the mass spectrometry data of different ginseng samples,it was found that the content of ginsenosides in real ginseng samples differed significantly depending on the variety or origin.The DART-MS combined with the TMAH methylation method proposed in this work can be used as a powerful supplement for the analysis and identification of ginsenosides,and it is an important tool for ginseng analysis.3.Direct analysis in real time tandem mass spectrometry(DART-MS~n)combined with TMAH methylation was used to identify ginsenoside isomers.Using this method to study five groups of ginsenoside isomers,covering the identification of the same type and different types of ginsenoside isomers.The analysis found that even the same type of ginsenoside isomers with subtle structural differences will show significant differences in DART-MS signals in the full mass spectrometry.Collision induced dissociation(CID)multi-stage mass spectrometry further reveals the cleavage law of ginsenosides,and there are significant differences between their fragments.In this study,DART-MS~n analysis of ginsenoside compounds by methylation reagents provided more evidence for distinguishing and identifying ginsenoside isomers.This method is simple and fast to successfully identify saponin structural isomers.In summary,this thesis applied the MCE method to efficiently extract ginsenosides.The direct DART-MS analysis method and the DART-MS method combined with TMAH methylation were used to analyze the ginsenosides and ginseng samples.On this basis,a DART-MS~n method combined with TMAH methylation was established to analyze and identify ginsenoside isomers.The research results have certain significance for the analysis of ginseng and the scientific research of ginsenoside isomers.