Flavonols Activity Of The Extract As A Whole Metabolic Profiling And Structural Analysis Of Mass Spectrometry

AB-8-2 was an active fraction obtained from Gossypium herbaceam L, which mainly consisted of flavonol glycosides. The metabolism biotransformation of AB-8-2 in rat was investigated systematically using high-performance liquid chromatography/multi-stage tandem mass spectrometry (HPLC-MSⁿ). A novel strategy for the rapid identification of flavonols in complex herbal extract and their metabolic pathway was proposed and employed in profiling the integral metabolism of flavonols of AB-8-2.The characteristic mass spectrometric fragmentation behavior of flavonol O-glycosides were investigated in detail by ESI-MS/MS technique in negative ion mode using three CID mode on two types of mass spectrometer. The metabolites of flavonols in the bile and urine samples after administistration of AB-8-2 were analyzed using HPLC-MSⁿ in a single chromatographic run. In addition, metabolites in plasma of rats after a single intravenous dose of quercetin and its three glycosides, respectively, were characterized and compared for the purpose of investigating the region-selectivity of conjugation of quercetin.1. Characteristic fragmentation behavior of flavonol mono-glycosides with different CID modeThe mass spectrometric fragmentation behavior of several typical flavonol O-glycosides were investigated in detail by ESI-MS/MS technique in negative ion mode using three CID mode on two types of mass spectrometer. The typical MS² spectrum obtained on a 3D ion trap, enhanced product ion (EPI) spectrum and "trap-like" MS² spectrum obtained on Q TRAP^TM mass spectrometer were compared. The fragmentation behavior of flavonol O-glycosides under different CID conditions were not identical but can provide complementary information revealing the structural differences in glycosylation position. In negative ion mode, the glycosylation position of flavonol 3-O-glycosides, flavonol 7-O-glycosides and flavonol 3'-O-glycosides were differentiated and determined through investigating the fragmentation behavior of [M-H]^- ions resulting from the different glycosylation position. [Y₀-H]^-· and ^0,2X₀^- ion were used as diagnostic ions in differentiating 3-O, 7-O and 3'-O-glycosyl flavonol using 3D ion trap, while the relative abundance of [Y₀-H]^-· ion can be employed to characterize the three isomers without the accessorial information of ^0,2X₀^- ion using EPI spectrum. The results provided a basis for structure identification using fragmentation rule derived from different instrument.2. Fast profiling of the integral metabolism of flavonols in the active fraction of Gossypium herbaceam L. using HPLC-MSⁿ techniqueThe intergral metabolism of flavonols of AB-8-2 was profiled based on the combination of fragmentation behavior and metabolic pathways. Fifty-eight flavonols as a range of mixed sulphate, methyl, glucuronide and glycoside derivatives of quercetin or kaempferol were detected in rat bile samples, including several groups of isomers. The metabolic differences in bile samples from rats after oral and intravenous administration were compared to evaluate the influence of intestinal metabolism. The relationships between metabolites and parents were elucidated for some components. By profiling the constituents in AB-8-2 and metabolites in bile, a integral view on the biotransformation of the constituents in AB-8-2 was obtained.3. Metabolites of flavonols in urine after oral administration of AB-8-2Based on the method established, the urine sample after oral administration of AB-8-2 was analyzed. In one analytical run, 51 constituents including 35 di-glucuronidated metabolites, 12 mono-glucuronidated metabolites and 4 flavonol aglycones were characterized. Flavonol diglycosides in AB-8-2 probably remained one glycoside in the structure during the absorption process in intestinal tract. Some co-eluated glucuronide methyl ester conjugated metabolites were also identified in the urine sample, which may be produced in the process of sample preparation.4. Comparsion of the metabolites in rat plasma after intravenous administration of quercetin and its three glycosidesBy employing the HPLC-MS and HPLC-MS/MS method, the metabolites in the rat plasma were characterized after intravenous administration of quercetin, quercimeritrin, quercetin-3'-O-glucoside and hyperoside, respectively. The results indicated that quercetin occupied the most various metabolic reactions among these four compounds investigated. No glucuronidated conjugates was found in the plasma sample after intravenous administration of quercimeritrin, which suggested that the free 7-OH was an essential group for the glucuronidation. For quercetin-3'-O-glucoside, no methylation metabolite was detected in the plasma sample, which futher confirmed that catechol structure in B ring was necessary for the methylation in vivo for flavonols. As for hyperoside (quercetin-3-O-galactose), two metabolites were identidied as hyperoside monoglucuronide and methylated hyperoside, respectively.