| A host of modern hyphenated chromatography-mass spectrometric techniques have been developed and their performance evaluated for the identification and quantification of bioactive components of medicinal values in complex natural products. These techniques include high performance liquid chromatography- electrospray time of flight mass spectrometry (HPLC-ESI-TOF/MS), high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS), high performance capillary electrophoresis- electrospray time of flight mass spectrometry (HPCE-ESI-TOF/MS), ultra performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS/MS), etc. Centered surrounding these highly sensitive instrumental techniques, effective analytical methodology have been developed and optimized for the rapid determination of active components in selected traditional Chinese medicinal materials of both herbal and marine origins. The main studies and results in the paper are outlined as follows:In Chapter One, a brief review is given covering subjects concerning the basic concepts, theories and current advances, both in China and abroad, on traditional Chinese medicines and marine drugs, and different chromatography-mass spectrometry techniques.In Chapter Two, the recent advancement of accelerated solvent extraction (ASE), as a new extraction technique developed recently, will be described. The technique offers significant advantages over conventional extraction techniques because of its high analytical speed, low solvent consumption, ease of automation and high extraction efficiency. In recent years, ASE is gaining increasing popularity in the study of traditional Chinese medicines (TCMs). Results from these studies demonstrate that ASE is indeed a powerful tool in the preparation of herbal extracts for downstream chromatographic analysis. In the present chapter, the development of ASE extraction method for the analysis of active compounds in Semen Aesculi and Coptis chinensis Franch will be reported for the first time.Firstly, to develop ASE extraction method for escins in Semen Aesculi and compare the advantage of this method with other extraction methods. By making the single factor experiments, the optimum technological parameters for ASE extraction were obtained. Through the experiments, the most favorable conditions for ASE extraction of escins in Semen Aesculi were obtained. Secondly, the optimal parameters for ASE extraction of alkaloids in Coptis chinensis Franch were obtained by making the orthogonal experiments. Results showed that the optimal conditions are as following: 80% ethanol+ 0.5% hydrochloric acid as solvent, temperature at 130℃, 10 min duration and once extraction. It can be concluded that the ASE is a quickly and effectively extraction technique for escins in Semen Aesculi and alkaloid compounds in Coptis chinensis Franch.In Chapter three, the development of a convenient method based on HPLC and positive ion ESI-TOF/MS for the analysis and characterization of active compounds in extracts of Semen Aesculi and Semen Nelumbinis will be discussed. To begin with, a reliable HPLC method for quantification of four major saponins in Semen Aesculi was developed. The ESI-MS fragmentation patterns of four major saponins were explored, and other saponin compounds in Semen Aesculi were also identified by accurate molecule weight information and fragmentation behavior obtained by collision induced dissociation experiment combined with literature review. The results indicated that the developed quantification method is simple, accurate and reliable for the determination of four major saponins in Semen Aesculi. The [M+H]+ or [M+Na]+ ions of saponins in Semen Aesculi extract were observed by ESI-TOF/MS on-line detection, and used to obtain accurate molecular weight and molecular formula information of saponins. Fourteen saponins in Semen Aesculi extract could be primary identified. On the other hand, an ASE-HPLC-DAD-ESI-TOF/MS method was developed for the analysis of alkaloids in Semen Nelumbinis. By comparing the spectrogram and mass spectrum of the chromatogram peaks with the references value, six compounds in Semen Nelumbinis were identified. The HPLC-ESI-TOF/MS method developed in this chapter has the advantages of simple operation, rapid measurement and accurate determination, and it's a powerful tool for identification of saponins in Semen Aesculi and alkaloids in Semen Nelumbinis.In Chapter 4, the development of a HPCE technique with dual diode array assay (DAD) and ESI-TOF-MS detection for the analysis of seven protoberberine alkaloids and one aporphinoid alkaloid in Coptis chinensis Franch will be described. Three background electrolytes (BGEs) were developed for HPCE-DAD and HPCE-MS analyses, and the HPCE-ESI-TOF-MS conditions were optimized. Eight main alkaloids in Coptis chinensis Franch could be separated with base-line resolution by HPCE-DAD with these three different BGEs, and identified by ESI-TOF-MS analysis. Moreover, three major alkaloids berberine, palmatine and jatrorrhizine could be quantified accurately by HPCE-DAD with the BGE system consisting of 50: 50 v/v water and acetonitrile containing 50 mM ammonium acetate at pH=6.8. The results indicated that the developed HPCE-DAD method is simple and reliable for the determination of three alkaloids in Coptis chinensis Franch. The HPCE-MS method, as a viable alternative to HPLC-MS, is found to be suitable for the rapid identification and quantification of basic compounds in herbal or natural product applications.In Chapter five, the development of a new UPLC-MS/MS method for the identification and quantification of major alkaloids in extracts of Coptis chinensis Franch will be described. The UPLC system consisted of a dual detection system of photodiode array detector (PDA) and positive ion ESI-MS/MS in sequential configuration. A tandem quadrupole spectrometer operating in either full scan mode or in MS/MS mode for multiple reaction monitoring (MRM) was used for the identification and quantitative analysis of eight major alkaloids in Coptis chinensis Franch extracts. The samples were also analyzed on a HPLC-ESI-TOF-MS system to confirm the identification results. Three of the eight major alkaloids, berberine, palmatine and jatrorrhizine were quantified by UPLC-PDA and UPLC-MS/MS. The results indicated that both UPLC-PDA and UPLC-MS/MS methods were simple, sensitive and reliable for the determination of alkaloids in Coptis chinensis Franch. Seven Coptis chinensis Franch samples from different locations were analysed using the established methods. UPLC fingerprints based on the distribution of the eight major alkaloids can serve as a rapid and reliable method for the authentication and quality evaluation of TCM herbs.In Chapter six, HPLC-APCI-MS has been used for the determination and identification of active compounds in tobacco leaves, Ganoderma Lucidum (Leyss. Ex Fr.)Karst and Ligusticum chuanxiong Hort. Firstly, an user-friendly HPLC analysis method with two detector including UV and APCI-MS was established for the determination of solanesol in tobacco leaves from different sources. The results indicated that the developed HPLC-UV method is simple, accurate and reliable for the determination of solanesol in tabacoo leaves with a good linearity. The contents of solanesol in tobacco leaves from different producing area were distinct. Moreover, the main instrumental parameters of APCI-MS for analysis of solanesol were optimized to provide the best possible sensitivity, and a comparison with ESI-TOF/MS was conducted. In the APCI+ mode, abundant stable [M–H2O+H]+ ion (m/z at 613.5) was also observed, but can not observe other ion and fragmentation. By comparing APCI-MS and ESI-TOF/MS for the analysis of solanesol in extract of tobacco leaf, it is could be find out that the APCI mode is more sensitive than ESI mode, so APCI mode is more suited for solanesol quantitative analysis. Secondly, HPCL-APCI has been applied to analyze the triterpenoids in extracts of Ganoderma Lucidum (Leyss. Ex Fr.)Karst. The detection wavelength is 253 nm and the UV spectra of peaks were obtained with a DAD detector. On-line APCI-MS in positive and negative mode was used to get the mass spectrum of the analytes. Thirty-two components of Ganoderma Lucidum (Leyss. Ex Fr.)Karst were primarily identified by comparison of the UV spectra and mass spectrum with literatures. Finally, a HPLC fingerprint method has been established for the quality control of Ligusticum chuanxiong Hort and its extract. HPLC-APCI-MS has been applied to identify the compound in methanol extracts of Ligusticum chuanxiong Hort. On-line APCI-MS in positive and negative mode was used to get the mass spectrum of the analytes. Ten components of Ligusticum chuanxiong Hort were primarily identified by comparison of the UV spectra and mass spectrum with literatures. In summary, the HPLC-APCI-MS method developed in this chapter is a powerful tool for identification of weak polar compounds in natural products, which is a hard work for ESI-MS.In Chapter seven, the antioxidant activity of the extracts of Hippocampus japonucus Kanp (Haima) was evaluated for the first time, and the HPLC fingerprint of Haima was established for the discrimination of true or false and the quality evaluation of Haima. Firstly, antioxidant properties of Haima extracts with different solvent were assayed in terms of antioxidant activity by scavenging activities on 1,1-diphenyl-2-picrylhdrazyl (DPPH). The results indicated that the antioxidant activity of water extract of Haima is higher than all the other extracts of Haima. The effect of time and concentration of Haima extract on the antioxidant activity was also studied and the results showed that Haima can provide a good source of antioxidant. Secondly, a chromatographic fingerprint method based on HPLC was developed. The results indicated that the developed HPLC method is simple, accurate and reliable for the development of Haima fingerprint. Ten Haima samples collected from different medicine hall were analyzed and the Haima HPLC fingerprint was established. The similarity of the HPLC chromatogram was performed for authentication and quality control of Haima. The HPLC fingerprinting techniques have high potential in authentication or source-tracing types of applications.In Chapter eight, the development of a method based on the HPLC online scavenging DPPH? radical activity and ESI-TOF/MS for the rapid screening and identification of radical scavengers from complex natural products such as TCM and marine drugs will be discussed. The HPLC-ESI-TOF/MS-DPPH method developed in this chapter is simple, reliable. The method was applied to Lonicera japonica Thunb and Haima extracts. The results showed that four compounds in Lonicera japonica Thunb have scavenging radical activity, and all the four compounds were identified as organic acid compounds by ESI-TOF/MS according to literatures and data base. While one compound in Haima extract was found having scavenging radical activity, which could provide a reliable and scientific evidence for the antioxidant activity of Haima.In the present dissertation, effective analytical methodology of different modes of hyphenated"liquid chromatography- mass spectrometry"techniques were developed according to the structure of active components in natural products. Some analytical problems of several typical herbal medicines (Semen Aesculi, Semen Nelumbinis, Coptis chinensis Franch, Ganoderma Lucidum (Leyss. Ex Fr.)Karst, Ligusticum chuanxiong Hort, etc) and Haima were solved, and a series of new methods for separation, identification, screening, determination of active components in selected TCM materials of both herbal and marine origins were provided. Moreover, results from our studies demonstrate that ASE is indeed a powerful tool in the preparation of herbal extracts for downstream chromatographic analysis.
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