Fluorescent Labeling Reagent Preparation And High Performance Liquid Chromatography - Mass Spectrometry

High performance liquid chromatography (HPLC), due to its powerful separation capacity, has been enthusiastically and widely adopted in many fields of scientific research and application, such as chemistry, biochemistry, clinical sciences, pharmacology, neurology, environmental science and quality control. It is also one of the fastest developing and the most widely used methods in analytical chemistry. However, not all compounds are suitable to be directly analyzed by HPLC because of the limitation of the compound properties and the very low contents of the corresponding compounds. Currently, fluorescence detection is still recognized to be one of the most sensitive detection technologies. Therefore, derivatization of analytes, such as amino acids, aliphatic amines, fatty acids, with fluorescence labeling reagents has been widely adopted. It can alter the chemical and physical properties of target compounds and make them easier to analyze. In this paper, we designed and synthesized five novel fluorescence labeling reagents. The optimal derivatization and chromatographic separation conditions were investigated. At the same time, fluorescence and UV-Vis spectra of labeling reagents and the suitability of the developed method for the analysis of real samples were evaluated.Chapter one: The fluorescence theory and fluorescence labeling technology were introduced systematically. The history and basic theory about HPLC were simply introduced. The applications of fluorescence labeling reagents for the labeling of carboxyl and amino compounds by HPLC were summarized.Chapter two: The syntheses of five fluorescence labeling reagents acridone-9-ethyl-p-toluenesulfonate (AETS), 1 -[2-(p-toluenesulfonate)ethyl]-2-phenylimidazole[4,5-f]9,10-phenanthrene (TSPP), 2-(9-acridone) -acetic acid (AAA), 2-(2-phenyl-lH-phenanthro- [9,10-d]imidazole-1-yl)-acetic acid (PPIA) and l,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) were described, and followed by their structural characterization and spectralproperties. In addition, the crystal structure of TSPP was evaluated.Chapter three: Pre-column derivatization methods for the sensitive determination of fatty acids, respectively, using acridone-9-ethyl-p-toluenesulfonate (AETS) and l-[2-(p-toluenesulfonate) ethyl]-2-phenylimidazole [4,5-fJ9,10-phenanthrene (TSPP) as labeling reagents followed by HPLC-MS analysis have been developed. The optimal derivatization and chromatographic separation conditions using AETS and TSPP as labeling reagents were elucidated, respectively. The contents of free fatty acids from real samples such as soil, bryophyte, deep sea fish oil of Alaska, Swertia mussotii Franch, Lomatogonium, Gentiana straminea, Gentiana macrophylla were determined. Fluorescence spectra of representative TSPP-Cs derivative in the presence of halide ions, heavy metal ions and with different solvent polarity and temperature were also investigated.Chapter four: Pre-column derivatization methods for the sensitive determination of aliphatic amines, respectively, using 2-(9-acridone) -acetic acid (AAA) and 2-(2-phenyl-lH-phenanthro-[9,10-