New Methods For Analysis Of Active Compounds In Tradition Chinese Medicine By LC/CE-IT-MS

The structure elucidation, fragmentation behaviors and pathways of active compounds in tradition Chinese medicine (TCM) have been studied by ion trap (IT) mass spectrometry (MS). Novel, sensitive and selective methods have been developed for identification and quantification of active components in TCM by liquid chromatography (LC) or capillary electrophoresis (CE) IT/MS. In addition, a fast and sensitive LC-MS method has developed for the determination of ursolic acid (UA) in rat plasma and tissues. It has been demonstated that proposed LC/CE-IT-MS methods can be well used the online structural characterization and also be very useful for quantitative analysis of various active compounds in TCM. This thesis contains following parts.(1) A LC-IT-MS method for the identification and quantification of isomeride ursolic acid (UA) and oleanolic acid (OA) in Chinese herbs is described. The UA and OA standard solutions were directly infused into IT-MS for collecting MSn spectra. The major fragment ions of UA and OA were confirmed by MSn in both negative and positive ion mode. The possible main cleavage pathway of fragment ions was studied. UA and OA provided good sensitivity corresponding to the deprotonated molecular ion [M-H]-. The method is reliable and reproducible with a detection limit of5ng/mL. The method was validated in the linearity, sensitivity, accuracy and precision, and then used to determine the content of the UA and OA in nine Chinese herbs.(2) The active alkaloids vinblastine, vindoline, ajmalicine, catharanthine, and vinleurosine in Catharanthus roseus were identified by direct-infusion IT-MS for collecting MS1-2spectra. The determinations of five alkaloids were accomplished by LC-UV and LC-IT-MS.The analytes provided good sensitibvity corresponding to the protonated molecular ions [M+H]+and product ions. Two methods were used to evaluate a number of validation characteristics (repeatability, LOD, calibration range, and recovery). MS provides a high selectivity and sensitivity for determination of five alkaloids in positive mode. Finally, the five components in Catharanthus roseus were comprehensively accomplished by LC with UV and MS detection.(3) The qualitative and quantitative analysis of active constituents in Fructus Psoraleae is presented by LC coupled with different detections. Extracts of Fructus Psoraleae were examined by LC-IT-MS and18major compounds were identified. The determination of four major constituents including bavachin, isobavachalcone, bavachinin and bakuchiol were accomplished by LC with UV, IT-MS and electrochemical detection (ECD). These methods were evaluated and validated in the linearity, sensitivity, accuracy and precision. ECD offers a high sensitivity for analysis of the four components; MS provides a high selectivity and sensitivity for determination of bavachin, isobavachalcone and bavachinin in negative mode. After optimization of the methods, separation, identification and quantification of the four components in Fructus Psoraleae were comprehensively tested by LC with UV, MS and ECD.(4) A fast and sensitive LC-MS method was developed for the determination of UA in rat plasma and tissues. Glycyrrhetinic acid was used as the internal standard (IS). Quantification was performed by selecting ion monitoring (SIM) of the transitions with (m/z)-455for UA and (m/z)-469for the IS. The method was validated in the linearity, sensitivity, accuracy, precision and stability. The detection limits were0.5ng/mL or4.0ng/g. The main pharmacokinetic parameters were obtained by3P97software. The concentrations of UA in rat lung, spleen, liver, heart and cerebellum were studied for the first time. This method was validated and could be applicable to investigate the pharmacokinetics and tissue distribution of UA in rats.(5) A CE-IT-MS method for the simultaneous determination of vinblastine and its monomeric precursors (vindoline and catharanthine) has been developed. A baseline separation for three components has been achieved by using a running buffer consisting of20mM ammonium acetate and1.5%acetic acid in less than20min. Quantification of three components was assigned in positive-ion mode at protonated molecular ion [M+H]+. The CE-MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the above components in real sample Catharanthus roseus.(6) A non-aqueous CE-IT/MS with a nanospray ionization interface method was developed for the identification and quantification of tetrandrine (TET), fangchinoline (FAN) and sinomenine (SIN) using berberine as internal standard. The TET, FAN and SIN standard solutions were directly infused into IT-MS for collecting MS1-3spectra. The major fragment ions of analytes were confirmed and possible main cleavage pathways of fragment ions were studied. A bare fused-silica capillary was used for separation of the analytes. A sheath liquid (50%aqueous methanol containing0.2% acetic acid) to the capillary effluent with a nanospray ionization interface was added. Separation buffer comprised80mM solution of ammonium acetate, in a mixture of methanol, acetonitrile, and water (70:20:10), which also contained1%acetic acid. The CE-MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the components. This method was successfully applied to determine TET, FAN and SIN in real samples radix Stephaniae tetrandrae and rhizomes of Menispermum dauricum.