New Methods For Analysis Of Active Compounds In Chinese Medicine By Capillary Electrophoresis

The active components determining pharmacological activity in traditional Chinese medicines (TCM) are complex. The quantitative analysis of active components is a very important and challenging work. Capillary electrophoresis has become an indispensable important means of analysis due to the advantages of high efficiency, fast analysis speed, wide application, less consumption of sample and solvent, et.al. One of the main drawbacks is the low detection sensitivity with UV detection. Mass spectrometry has many advantages such as high sensitivity, good selectivity, and structure analysis and identification. Therefore, the CE-MS is very suitable for the analysis of Chinese medicine. On-line solid-phase extraction coupled with capillary electrophoresis has the advantages of removing impurities and improving the sensitivity of CE-UV. The outlines of this thesis are listed as follows:Part Ⅰ Bavachin and isobavachalcone are the isomeric compounds in the Fructus Psoraleae. The fragmentation pathways of the bavachin and isobavachalcone by IT-MS in negative ion mode were elucidated for the structure identification. A novel method for determination of isomeric bavachin and isobavachalcone has been developed by capillary electrophoresis coupled with mass spectrometric detection. The effects of several factors such as concentration, pH of ammonium acetate buffer, separation voltage, the composition and flow rate of sheath liquid were investigated. Under the optimal conditions, the linear concentration ranges for bavachin and isobavachalcone were0.8-100μg/mL with the correlation coefficient of0.9962and0.9953, respectively. Relative standard deviations of migration time and peak areas were lower than5%. The limits of detection (signal/noise=3) were both60ng/mL. The proposed method can be successfully applied to the determination of bavachin and isobavachalcone in the real sample of Fructus Psoraleae and six Fructus Psoraleae-containing preparations.Part Ⅱ The Amaryllidaceae are widely distributed medical plants. Lycorine, lycoramine. lycoremine, and lycobetaine are the major active alkaloids in Amaryllidaceae plants. A nonaqueous CE-ESI-IT-MS method for separation, identification, and quantification of the Amaryllidaceae alkaloids has been developed. The MS1-3behaviors have been studied and the fragmentation pathways of main fragment ions have been proposed. The effects of several factors such as composition and concentration of buffer, applied voltage, composition, and flow rate of the sheath liquid, nebulizing gas pressure, flow rate, and temperature of drying gas were investigated. Under the optimal conditions, the linear concentration ranges of these compounds were wide with the correlation coefficient (R>0.99). RSDs of migration time and peak areas were<10%. The limits of detection (signal/noise=3) were<240ng/mL. The proposed method can be successfully applied to the determination of the related alkaloids in the Lycoris radiate roots.Part Ⅲ A simple, rapid and sensitive nonaqueous capillary electrophoresis-electrospray ionization-ion trap-mass spectrometry (NACE-ESI-IT-MS) method was developed for determination of matrine and oxymatrine in Sophora Flavescens and medicinal preparations. The conditions for NACE separation and MS detection were systematically optimized. The optimum NACE buffer contained30mM ammonium acetate,1%acetic acid, and15%acetonitrile in methanol and the applied voltage on separation capillary was set at25kV. Berberine was selected as internal standard. In order to generate a stable electrospray, a sheath liquid (isopropanol/HO2/1, v/v) was used, which could also boost the flow through the ESI needle. The matrine and oxymatrine solutions were introduced into MS detection by a syringe pump for collecting the MS1-3spectra to investigate the main fragment ions and its possible cleavage pathways. Both matrine and oxymatrine showed good linearity in the concentration ranges from0.5to400μg/mL, with linear correlation coefficient R>0.99and the limit of detection (signal/noise=3) were37.5ng/mL for matrine and50.0ng/mL for oxymatrine, respectively. The recoveries at different content of Sophora Flavescens were87.6%-110.0%for MT and87.3%-102.8%for OMT, which indicated the reliability of this method.Part IV A Poly (AA-EGDMA) monolith was synthesized and constructed as a concentrator for in-line solid-phase extraction coupling with capillary electrophoresis. The developed concentrator was used for the determination of low concentration of lycobetaine. The experimental parameters for in-line solid-phase extraction, such as composition and volume of the elution plug. pH of sample solutions and the time for sample loading, were optimized. At the optimum conditions, the preconcentration factor over1000was achieved with a loading time of15min under the injection pressure of930mbar. Good linearity was obtained in the range from10to300ng/mL (R>0.99) with the detection limit (signal/noise=3) of1ng/mL. The recovery of87.6%was obtained in human plasma spiked of10ng/mL. It has been demonstrated that this work has great potential for the analysis of lycobetaine in real samples.