Study On The Mass Spectrometric Analytical Method Of An Active Fraction From Gymnadenia Conopsea R. BR. And Its Metabolism

CE was an active fraction obtained from Gymnadenia conopsea R. Br. (Orchidaceae), which mainly consisted of glucopyranosyloxybenzyl succinates. Its chemical composition and metabolism biotransformation in rat biological sample were investigated systematically using rapid resolution liquid chromatography coupled with quadrupole/TOF mass spectrometry (RRLC-MS/MS). First of all, the characteristic mass spectrometric behavior of glucopyranosyloxybenzyl succinates was investigated in detail by electrospray ionization tandem mass spectrometry in positive and negative ion modes. All these compounds presented an unusual rearrangement reaction in positive ion mode and the mechanism rationalized this reaction was discussed. Consequently, a RRLC-MS/MS method was established for the analysis of CE and its rat biological sample. A novel strategy for postacquistion data mining technology combining with the extraction of characteristic ion and mass defect filter (MDF) allowed high throughput, quality and content analysis of the metabolism of complex mixtures. The rapid identification of glucopyranosyloxybenzyl succinates in complex herbal extract and their metabolites was employed in profiling the integral metabolism of CE.1. The characteristic fragmentation of glucopyranosyloxybenzyl succinates using electrospray ionization tandem mass spectrometryThe characteristic fragmentation including an unusual large group transfer rearrangement reaction of glucopyranosyloxybenzyl succinates isolated from Gymnadenia conopsea R. Br. was investigated systematically by electrospray ionization tandem mass spectrometry in positive ion mode. On the basis of ESI-MSn experiment, alkali metal cationization, deuterium labeling, as well as accurate mass measurement, the interesting 4-methylene-2,5-cyclohexadien-1-one transfer rearrangement reaction might be rationalized by hypothesis via ion-neutral complexes. In addition, the cleavage of the O-benzyl and C-2 O-glycosidic bonds were the major dissociation pathways for [M+W]+ (W= Li, Na or K). However, the [M+NH4]+ ions underwent different fragmentation processes and the rearrangement reaction did not take place, which indicated that the alkali metal ion was crucial to trigger the rearrangement reaction. The esterification position of mono(4-β-D-glucopyranosyloxybenzyl)-2-β-D-glucopyranosyl-2-isobutyl succinate isomers were determined by comparing the difference of the product ion spectra in negative ion mode. The present study supplemented the gas-phase ion chemistry of glucopyranosyloxybenzyl succinates and should be of value in the structural identification of new analogues and metabolites.2. Rapid structural characterization of glucopyranosyloxybenzyl succinates in the active fraction (CE) from Gymnadenia conopsea R. Br. using RRLC-MS/MSThe active fraction (CE) from Gymnadenia conopsea R. Br. was analyzed in detail in positive and negative ion mode using Rapid Resolution Liquid Chromatography coupled with Quadrupole/TOF mass spectrometry. Based on the relationship between the characteristic fragmentation behaviors and the structural features of related known compounds from this plant, thirty five compounds were identified temporarily, most of which were glucopyranosyloxybenzyl succinates including seven groups of isomers. Furthermore, the results could benefit to rapidly identify new analogues in crude plant extracts and other complex samples.3. Study on the integral metabolism of glucopyranosyloxybenzyl succinates in rat biological sample after administration of CEA RRLC-MS/MS method was established for the analysis of the rat biological sample after intravenous and oral administration of CE. A postacquistion data mining technology combining with the extraction of characteristic ion and MDF allowed high throughput, quality and content analysis of the metabolism of complex mixtures. Based on the complementary information from fragmentation behavior and metabolic pathways, thirty four parent constituents and forty five metabolites which were produced by methylation, dehydration, demethylation and isomerization were identified temporarily. The metabolism experiments of Dactylorhin B further revealed the possible metabolic pathways of glucopyranosyloxybenzyl succinates in rat plasma and the biotransformation relationship of the parent constituents in CE. The developed method was effective, reliable and sensitive, which provided a basis for research into rapid screening and structural characterization of the metabolism of complex mixtures. It was of benefit in targeting potential active ingredients in complex mixtures and was valuable and dependable for the further study of the metabolism of complex systems such as herbal extracts or TCM formula.