| Due to complicated components and also matrix, Chinese Traditional Medicines (TCMs) and biological samples, are considered as two types of complex multi-component systems which are studied frequently in the flied of pharmaceutical analysis. In this case, one technique which owns both high selectivity and high efficiency are required for the separation and the determination of these two systems. CEC is one of the most rapid developing micro-analytical separation techniques. High selectivity and high efficiency can be obtained due to the combination of differences in partitioning ratio between the stationary and the mobile phase, as in capillary liquid chromatography(CLC) and to differences in electrophortic mobility, as in CE.Commonly, packed CEC are used widely because of the well-document preparation procedures of particulate-packed column, and the packed column has good selectivity. However, some inherent problems such as the requirement of advanced skills either in the fabrication of frits or the laborious packing have been the barrier to this separation technique. In addition, the bubble formation at the frits would lead to a loss of EOF. There are two methods to solve this problem. The first one is the employment of pressurized-CEC (pCEC), and the other is the usage of new type of columns. In this study, we chose monolithic columns.Pressurized CEC, which is also called pressure-assisted CEC is a relative novel technique related to CEC. By introducing high pressure on CEC, the mobile phase is driven by two factors: the EOF generated within the column and the pressure employed at the inlet of the column. The major advantages of pCEC over regular CEC include the stability of the mobile-phase flow due to bubble suppression by the application of pressure and the increased speed of separation if under the same applied voltage. Packed columns are commercially available and are most frequently used in pCEC. Because the same type of packing material is used as in HPLC, the chromatographic behavior of these columns in pCEC is in good agreement with that of HPLC. As an alternative to the conventional packed columns, monolithic columns have attracted great attention in recent decades due to their intriguing features such as simple preparation procedure, no need for frits, the easy tuning of pore size and surface area, and functionality of columns, and the high porosity and permeability from continuous porous structure. In brief, monolithic materials can be divided into two general categories, organic monolith, e.g. silica-based and porous organic monolithm, e.g. polymer-based. Due to the simpler preparation process and larger pH adaptability range, the polymer-based monoliths exhibit more potential advantages compared to silica-based monoliths. Methacrylate-based monoliths are one of the most widely ones. In this thesis, different kinds of Chinese Traditional Medicines, such as Fructus schisandrae, Ginkgo biloba leaves, Fructus. Cnidii and Angelica dahurica were chosen as the research subjects to test the matching degree of pCEC and CEC using different types of capillary columns, such as particle-based packed capillary column, methacrylate-based monolithic column.Firstly, a pCEC method with post-column detection cell had been developed for quantifying the lignans from Fructus schisandrae extracts. The effect of different experimental conditions, such as the acetonitrile content of the mobile phase, the concentration and pH of the buffer, the applied voltage, and the supplementary pressure were studied. Five lignans (Schisandrin, Gomisin A, Schisantherin C, Deoxyschizandrin, Schisandrin B) were baseline separated using a mobile phase of acetonitrile–phosphate buffer (pH 5.4; 5 mM) (40:60, v/v) under -4 kV applied voltage. With this pCEC system, fingerprints of Fructus schisandrae were preliminarily established to distinguish two members S. chinensis (Turcz.) Baill. and S. sphenanthera Rehd. Et Wils. of Fructus schisandrae by characteristic peaks, and evaluate the quality of various sources of raw materials by determining the contents of the five lignans.There is a problem in the isocratic elution method for the determination of Fructus schisandrae by pCEC with packed column. In order to get relative better separation, more analysis time was needed. In order to solve this problem, a gradient elution pCEC method was emerged. By using this gradient elution system, the flavone glucosides in Ginkgo biloba leaves extracts has been determinated on two types of columns. A commercially available column packed with 3μm particles was investigated for the performance. Additionally, a monolithic column was included into the fingerprint study as an alternative to the conventional packed columns. The effects of experimental parameters such as the composition of the mobile phase, the concentration and pH of the buffer, and the applied voltage were studied. Under optimal gradient conditions, a total of at least 40 peaks were observed within 60 min on the commercially packed column while about 20 peaks were separated on the methacrylate-based monolithic. The results of this study indicated that pCEC with packed columns might be a suitable analytical technique for the development of fingerprints of TCMs, which could offer high separation efficiency, resolution and an acceptable repeatability. Compared with CLC, the introduction of voltage in pCEC may somewhat improve the separation of components. The commercially available particle-based columns proved to be best for fingerprint development. The monolithic columns might form an alternative due to the better permeability, but further study should examine the optimization of the polymerization-mixture composition for monoliths to obtain columns more suitable for fingerprint analysis.High voltage could not be applied due to the joult heat which could lead to the unstable baseline. The root cause is the lack of condensation system in pCEC instrument. In order to solve this problem, the commercial CE instrument was introduced for its mature condensation system technique. However, the CE instrument can not applied a high pressure which was a main driven force in the pCEC separation. Instead of pressure driven, we employed voltage driven into the experiment which could produce a higher EOF. Dut to the good permeability, methacrylate-based monolithic column has been developed for the fast separation and determination of four coumarin compounds (isopimpinelline, bergapten, imperatorin, and osthole) in Fructus. Cnidii extracts. And the effect of polymerization condition including the monomers ratio and the porogens ratio were systemly studied. A comparison of separation between CEC, pCEC and HPLC was implemented. In contrast to 25 minutes of analysis time in HPLC and 10 minutes of analysis time in pCEC, a fast separation of these analytes was achieved in less than 5 minutes in CEC. Method validation, evaluating the intra- and interday precision as well as LOD and method linearity, was developed in accordance with the analytical procedures. All these results were good.With this CEC system, the quality of F. Cnidii extracts from various resourses was evaluated by determining the contents of the four coumarins.The separation and determination of four important active compounds (imperatorin, isoimperatorin, phelloptorin and falcarindiol) from Angelica dahurica extract has been performed using CEC with methacrylate ester-based monolithic column. A fast and baseline separation of the four analytes was also achieved. The method developed is sensitive, reliable and suitable for the quality control. With this CEC system, the quality of Angelica dahurica extracts from 18 various regions was evaluated. However, the functional group introduced into monolithic column materials was C4. So the butyl methacrylate monolithic column could not have the effective selectivity to separate the other five structure similar bioactive components (byakangelicin, oxypeucedanin hydrate, xanthotoxol, 5-hydroxy-8-methoxypsoralen, and bergapten). In order to solve this problem, surfactant sodium desoxycholate (SDC) was introduced into the mobile phase as the pseudostationary to dynamically increase the selectivity of analytes instead of increasing the hydrophobicity of stationary phase. In addition, the pH of phosphate buffer has also an obvious effect on the resolution but little on retention time. Satisfactory separation of these five coumarins was achieved within 6 min under a 30:70 v/v acetonitrile–buffer containing 20mM sodium dihydrogen phosphate (NaH2PO4) and 0.25mM SDC at pH 2.51. Finally, five target coumrains from the crude extracts of A. dahurica. were separated, purificated and concentrated by D-101 macroporous resin, and were successfully separation and quantitative determination within 7 min.From the content determination of main active components in TCMs mentioned above, The results of this study indicated that CEC method with methacrylate ester-based monolithic column might be a useful and effective analytical technique for the quality control of TCMs by determinating the main active componets in them, although the less hydrophobicity of this type of column.The CEC method with methacrylate ester-based monolithic column was also used in the application of the other complex system-biological samples. Field-amplified sample injection (FASI) was employed to improve the problem of high limit of detection in CEC. On the other hand, by investigating different EOF-gererate monomer, a type of no-EOF monolithic column was chosen also for improving sensitivity. Under optimal conditions, we establish a sensitive CEC method(2ng/mL, LLOD)using no-EOF methacrylate ester-based monolithic column for the determination of mirtazapine (MRT) and its main metabolite demethylmirtazapine (DMR) in hunman plasma. The method was evaluated in term of selectivity, sensitivity, linearity, accuracy, precision and stability in accordance to the recommendations published by the FDA, and applied to the analysis of mirtazapine in clinical pharmacokinetic studies of mirtazapine tablets in healthy volunteers.The last chapter is about the investigation of polymerization composition. In order to increase the hydrophobicity and retention properties of the resulting monolithic columns, bulk monomer (octyl methacrylate, OMA) was introduced into the polymerization mixture instead of butyl methacrylate (BMA). The central composite design was used to optimize the weight content or the ratio of bulk monomer, crosslinker, 1,4-butanediol, 1-propanol and also the monomer mixture. This chapter is a preminary trial to the relative long alkyl chain preparation. |
