The Application Of Capillary Electrophoresis And Mixed-mode Chromatography In Pharmaceutical Analysis

Due to the rapid development of capillary electrophoresis(CE), new opportunities and challenges have been brought. CE has a lot of advantages, including fast analysis speed, low operating cost, high efficiency and so on. Moreover, it could be successfully used in separating a variety of large and small molecules, such as small organic molecules, inorganic ions, peptides and proteins. The authenticity and quality of the traditional Chinese medicine can be identified through the CE fingerprinting. Also, the origins and differences among different medicinal herbs can be compared. Furthermore, through the online concentration techniques(field amplified sample stacking, large volume sample stacking, sweeping, etc.), CE could be well applied to determine trace pharmaceuticals and their related impurities.Mixed-mode chromatography(MMC) can provide multiple retention mechanisms for separation. It is characteristic of good selectivity, high loading capacity and low cost, compared with single-mode chromatography. It is suitable to the complex samples.The aim of the present thesis was to develop effective method to separate complex samples based on CE and MMC. It mainly covers the following four parts:1. In chapter 1, CE fingerprinting of leeches was established. The optimized CE separation condition was as follows. Wavelength was 210 nm and separation voltage was-15 k V. The running buffer was 100 m M phosphate buffer(p H=10.0) with an additive of 0.5 m M hexadecyl trimethyl ammonium bromide(CTAB). Gravity injection was performed at a height of 12 cm for 40 s. The developed CE fingerprinting method was precise with the satisfactory relative standard deviations of intra-day and inter day in terms of relative migration time and peak areas(RSD ?5%).2. In chapter 2, a method for the separation and determination of imatinib mesylate and related compounds by CE was developed. The separation conditions were optimized in detail, such as species and concentration of selector additives,buffer p H and proportion of organic solvents. The optimized separation condition was as follows. Wavelength was 267 nm and separation voltage was 20 k V. The running buffer was the aqueous solution of sodium dihydrogen phosphate at 10 m M with a p H of 2.0. The selector additive was 2-hydroxypropyl-?-cyclodextrin and the concentration was 5 m M. The separation could be obtained in less than 20 min. To improve the sensitivity, field amplified sample injection combined with large volume sample injection was adopted. Methanol containing 0.5 m M of hydrochloric acid was optimized as the sample dilution solution. The strongest analytical signal with a suitable separation was achieved when injection was performed at a voltage of 15 k V for 60 s. The linearity ranges, the relative standard deviations of intra-day and inter day were satisfactory. The developed method was validated for its selectivity, linearity,accuracy and robustness. Due to its simplicity, effectiveness and low cost, it may be used for quality control of imatinib mesylate and similar drugs, and applicable to metabolism study of the related drugs.3. In chapter 3, a reversed-phase liquid chromatography(RPLC)/zwitterionic/hydrophilic interaction liquid chromatography(HILIC) stationary phase,SIL-PS, was prepared. Various functionalities i.e. hydrophobic alkyl chain, ionizable sulfonic and amine group, as well as polar hydroxyl and ether group existed on the SIL-PS. Different chromatographic parameters, including acetonitrile content, p H amd ionic strength of the mobile phase, and the column temperature, were systematically investigated to gain much insights into the retention mechanism of the SIL-PS. Eletrostatic absorptive/repulsive, partition and hydrogen-bonding interaction were demonstrated to be the dominating retention mechanism in the HILIC mode.Moreover, to investigate the separation selectivity, the NSAIDs,nucleobases/nucleotides and alkaloids/glycosides were separated on the SIL-PS under the HILIC conditions. Additionally, the separation of the Cortex phellodendri extract was conducted under the RPLC condition, with the traditional C18 column as a comparison. The results revealed that the SIL-PS possessed favorable hydrophilic behavior and could provide better separation selectivity for complex samples than theC18 column.4. In chapter 4, the application of CE in the field of animal pharmaceutical analysis was reviewed.