Chinese Medicine Complex Composition Of The Sample By Electrospray Ionization Mass Spectrometry

Mass spectrometry is an important instrument in the modern analysis of Traditional Chinese Medicine(TCM).In particularly,the development of electrospray ionization(ESI) source enhances the ionization ability,and shows the advantages of mass spectrometer in the widely applied area.But TCM is a very complex mixture containing up to hundreds or even thousands of components, which make it extremely difficult to analyze the bioactive components contained in it.However,it was just thought that TCM expressed its effects through multi-components and multi-targets.The multiple constituents were usually responsible for its therapeutic effects by synergistic and antagonistic interaction. So it needs to get simultaneous analysis of more targets to reflect the integrity of TCM and it also inhances the difficulties of the analysis in complex TCM system. The major problem was as follows:1 The identification problem of complex constituents in TCM(in the situation of no reference substance)2 The detection problem of metabolite fingerprint problem of TCM3 The distinguishing problem of isomers in TCM(the isomers with similar fragment pathway)4 The deviation of undetectable constiuents in TCMPart one:method study on the identification of complex constituents Rhizome Corydalts(RC)According the biosynthenic pathway of plant and the related literatures,the feature of main cores and substituted groups in different alkaloids were summaried first.By the systematic study of fragment pathways of the standard compound,the distinguished method of different type alkaloid was established.Based on it,the type of unknown compound could be characterized.Then according the feature of the alkloid structure and the fragment pathways,fourteen alkaloids in RC could be identified and one of them was found in this plant for first time.Based on the identification of the alkaloid in RC,its plasma containing constituents was detected in Auto-MSⁿ negtive mode.14 components in plasma after giving extract of RC.10 of them were original constituents and 4 of them were metabolic constituents.Part two:Study of Rat urinary metabolite profile of Aurantii FructusLiquid chromatographic-tandem electrospray ionization ion trap mass spectrometry technique(LC-MSⁿ) was used to analyze rat urinary metabolite profile of TCM Aurantii Fructus(AF).According the data of MS² and MS³ spectra, more than twenty metabolites were identified or tentatively characterized.They were derived from the flavonoid O-diglycosides of AF and the major metabolic pathways of them were hydrolization,glucuronidation and sulfation.The method based on LC-MSⁿ technique was proposed to rapidly analyze the metabolites profile in rat urine.The urine samples were collected at five different time point after the consumption of AF extract and detected by LC-ESI/MS in negative multiple reactions monitoring(MRM) mode.To our knowledge,this is the first report of the analysis of rat urinary metabolite profile of flavonoid O-diglycosides from AF.The results were very valuable to analyze the metabolic course of the bioactive constituents in AF.It also could be used for investigations of the metabolite profiles of many other TCM.Part three:The isomer distinguishing method of flavonoid-glycosides and coumarin Based on the LC-MSⁿ technique,the isomers of Psoralen and Isopsoralen, Imperatorin and Isoimperatorin,Neohesperidin and Hesperidin,Naringin and Narirutin were distinguished mainly by comparison of the relative abundance of their major fragment ions.The two pairs of coumarin isomers could be distinguished by comparing the the relative abundances of fragment ions formed by the loss of CO₂ and 2CO.For the isomers of flavonoid glycosides the relative abundance of fragment ions generated by the ring-cross cleavage of glycan was different and this could be used in distinguishing them.Although the fragmentation pathways were similar,according the tiny difference of structure, the change of relative intensity of main fragment ions could be observed.Through the further study of the fragment mechanism caused by the difference of structures, it could be used to distinguish some isomers more accurately and quickly.Part four:The derivazation of undetectable constitunts in TCMThis part described the development of efficient derivatization approaches to facilitate mass spectrometric analysis of poor-ionizable and low molecular compounds.For the low polar constituents,Strong-ionizable group was combined with the structure of them to make the derivated structure could be detected.The method of benzolytion and sulfation was compared and the better one sulfation was useful in the detection of TCM complex system.The detective trouble was also happened in the the detection of monosaccharides because of their low molecular weight.Six monosaccharides were derived to make them molecular weight increased by PMP.The derived structures have high response and more fragment ions in mass spectra.It coud provided more information for the detection of monasaccharide and make the identification of them more accurate.Furthermore,for distinguishing the isomers of derivated monosacchrides structures,the method comparing relative abundance of fragment ion which proposed in the forth chart of this paper was used.The result showed that in the differentiation of the derivated monosacchrides,the method coud be also accurate and convenient.