Simultaneous Quantitation Of Flavonols Of An Active Extract From Gossypium Herbaceam L By Tandem Mass Spectrometry And Comparison Analysis For Metabolites In Vivo

For the modernization of traditional Chinese herbal medicine, quanlity control and the establishment of dependenble pharmacological assessment system for the crude materials and products are considered as the two principal challenges. And at present, it is not enough to evaluate the quality of TCM using one single component. Simultaneous quantitation of multi-components on the basis of pharmacological study has attracted the interests of researchers in the quanlity control of the TCM.AB-8-2 was an active fraction obtained from Gossypium herbaceam L, which mainly consisted of flavonol glycosides. In this study, a method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the simultaneously determination of the 9 major constituents of this active fraction. And this method was successfully applied to the quantification of nine constituents in 3 AB-8-2 samples. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two fragment ions transitions to provide a high degree of sensitivity and specificity for both quantification and confirmation. Nine constituents are rutin, isoquercetrin, quercimeritrin, quercetin-3'-glucoside, 8-methoxyl-kaempferol-7-rhamnoside, astragalin, kaempferol-3-(6"-P-coumaroyl)-glucoside, quercetin, kaempferol eta.The metabolisms in bile and urine could reflect the whole profiles of biotransformation of the parent medicine or compounds. In this paper, the metabolites of flavonols in the bile and urine samples after oral administistration of AB-8-2 were analyzed using LC-MS/MS in a single chromatographic run. Data were aquried using RRLC^TM coupled with a QTOF mass spectrometer. The Information Depended Acquirer (IDA) and MS^E (where E represents collision energy) data acquisition methods are compared and it was shown that the latter has been provide to be a powerful approach for the rapid metabolites identification.Based on the IDA method established, the urine sample after oral administration of AB-8-2 was analyzed. In one analytical run, 38 constituents including 26 glucuronidated metabolites, 5 sulfation metabolites, 4 methylation metabolites , 3 flavonol aglycones and 1 glucuronidated conjudge methylation metabolite were characterized. Flavonol diglycosides in AB-8-2 probably remained one glycoside in the structure during the absorption process in intestinal tract. In bile sample, 3 constituents including 1 glucuronidated metabolite, 2 sulfation metabolites were characterized.Based on the MS^E method established, the urine sample after oral administration of AB-8-2 was analyzed. In one analytical run, 34 constituents including 25 glucuronidated metabolites, 4 sulfation metabolites, 3 methylation metabolites , 4 flavonol aglycones and 1 glucuronidated conjudge sulfation metabolite were characterized. In bile sample, 15 constituents including 13 glucuronidated metabolites, 1 flavonol aglycones metabolites and 1 methylation metabolite were characterized.In comparision with MS^E, IDA is an artificial intelligence-based produce ion scan mode providing automatic"on-the-fly"MS to MS/MS switching. And the fragmentation spectrometry was more characteristic. However, MS^E produced the same product ion profile but more comprehensive and overall. Although since no precursor-ion selection occurs in the mass spectrometer good chromatographic separation is a critical factor in affording fragment ions that are derived predominantly from the analyte of interest, MS^E enables the almost simultaneous acquisition of both LC-MS and fragmentation data from a single experiment. Conversely, IDA has its inherent drawback that has the capacity to generate some non-compound-related MS/MS spectra or miss potential metabolites.