Study On The Fragmentation Patterns And Quantitative Methodology Of Gelsemium Elegans With Multi-components Using Mass Spectrometory

Gelsemium elegans is a genus of flowering plants in the Loganiaease family,was commonly used as a traditional medicine for treating rheumatoid arthritis pain,treating neuropathic pain,promoting animal growth and so on.Recently,a number of studies have demonstrated that Gelsemium elegans exhibits a diverse set of anti-inflammatory,immunity,analgesic and anti-tumor activities.Alkaloids and Iridoids were regarded as the major active components of Gelsemium elegans.The chemical constituents of Gelsemium elegans are complex.Currently,more than 200 compounds have been isolated according to the known pharmaceutical research ideas,but the overall material basis of Gelsemium elegans has not been studied.Therefore,this paper uses a liquid-mass spectrometry-database technology to rapidly identify chemical constituents of traditional Chinese medicines,thus systematically clarify the structure of various chemical constituents in Gelsemium elegans,and to study key techniques for the simultaneous quantification of multi-components components of Gelsemium elegans without reference substance.Orthogonal test was used to optimize the LC-QqTOF/MS detection conditions in three aspects: mobile phase,analysis time and mass spectrum mode.After optimization,0.1% formic acid-water,50 min and Auto MS/MS positive ion mode were used to obtain the largest number of Gelsemium elegans compounds with the best response to metabolites;Then,through the matching analysis with the established Gelsemium elegans MS database,31 target compounds were identified(Gelsedine-type,11;Sarpagine-type,5;Gelsemine-type,4;Iridoids,4;Koumine-type,2;Humantenine-type,2;Phenolic acids,2;Yohimbane-type,1),and studied the law of mass spectrometry cleavage to find each class of diagnostic ions.In addition,116 non-target metabolites were detected by various mass spectrometry data analysis strategies,Gelsedine-type,41;Sarpagine-type,7;Gelsemine-type,9;Iridoids,15;Koumine-type,5;Humantenine-type,14;Phenolic acids,7;Others,18.The structural characterization of the main five types of alkaloids and Iridoid non-alkaloids were characterized,simultaneously with MEGG and Massbank databases for matching,in order to characterize the structure of the unknown metabolites.Established quantitative HPLC detection methods for gelsemine and koumine.The similarity analysis of the fingerprints of Gelsemium elegans samples from different origins and locations shows that there were at least 10 common characteristic peaks in HPLC spectrometry,and only using the two compounds to control the quality of the Gelsemium elegans has a certain limitation.Therefore,the mass spectrometric parameters of gelsemine and koumine were combined with the identification of Gelsemium elegans components were used to optimize the collision energy and separation conditions of the components in the multi-components.Finally,a LC-MS/MS method for simultaneous detection of 41 chemical constituents in Gelsemium elegans was established.The multi-components contents in different parts of Gelsemium elegans were quantitatively studied by using the standard curve of koumidine.The results showed that multi-components were significant differences in different parts of Gelsemium elegans.For example,the content of gelsemine was the highest in leaves,which reached 0.65 mg/g;the content of koumine in roots was 1.08 mg/g,but the difference in stem and leaf contents was small;the highest content of gelsenicine in leaves was 1.33 mg/g;koumidine was the content of these four constituents is the lowest,and the content in the stem was only 0.13 mg/g.Gelsemine,Gelsenicine and koumidine were significantly different at different sites,but the difference in koumine was not significant.The difference in the four compounds in the root was very significant,and there were significant differences in the four compounds in the leaves.The content of 14-Hydroxygelsenicine in roots and stems gradually accumulated with the increase of the month,and the content in stems could reach 1.198 mg/g;the highest content of koumicine in fresh vines in December was 0.666 mg/g,and 19R-Hydroxydihydrogelsevirine or 19S-Hydroxydihydrogelsevirine was the highest content compound in Gelsemium elegans.Based on the above research,a rapid analysis strategy of Gelsemium elegans multi-components identification based on liquid-mass spectrometry-database technology was established in this paper.Systematically clarified the chemical composition structure of Gelsemium elegans,and Solve the problem of quantitative analysis of Gelsemium elegans under no reference.The results of this study not only provided technical support for the quality control of Gelsemium elegans and basic research on material effects,but also lay a scientific basis for the multi-components pharmacokinetics and safety evaluation of Gelsemium elegans.