Synthesis And Characterization Of The Fluorescent Labeling Reagent And Its Application In 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 industry, such as biochemistry, toxicology, clinical sciences, chemical industry, clinical medicine, neurobiology, food hygiene, 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. So it is important to enhance the sensitivity of detection. Currently, fluorescence detection is still recognized to be one of the most sensitive detection technologies. Therefore, derivatization of analytes 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 three 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, carbonyl and hydroxy by HPLC were summarized.Chapter two: The syntheses of three fluorescence labeling reagents 2-(2-(anthracen-10-yl)-1H-naphtho[2,3-d]imidazol-1 -yl)ethyl-p-toluenesulfonate (ANITS), 2-(11H-benzo[a]carbazol-11-yl) ethyl carbonylhydrazine (BCECH) and 2-(11H-benzo[a]carbazol-11-yl) isopropyl chloroformate (BCPC-C1) were described, and followed by their structural characterization and spectral properties.Chapter three: Pre-column derivatization methods for the sensitive determination of free fatty acids using2-(2-(anthracen-10-yl)- 1H-naphtho[2,3-d]imidazol-1 -yl)ethyl-p-toluenesulfonate (ANITS) as labeling reagents followed by HPLC-MS analysis have been developed. The optimal derivatization and chromatographic separation conditions using ANITS as labeling reagent were evaluated. And the feasibility of this method has been identified by analyzing rape bee pollen sample.Chapter four: Pre-column derivatization methods for the sensitive determination of steroid using 2-(11H-benzo[a]carbazol-11-yl) ethyl carbonylhydrazine (BCECH) as labeling reagents followed by HPLC-MS analysis have been developed. The optimal derivatization and chromatographic separation conditions using BCECH as labeling reagent were evaluated. And the feasibility of this method has been identified by analyzing wolf feces sample.Chapter five: Pre-column derivatization methods for the sensitive determination of estradiol using 2-(11H-benzo[a]carbazol-11-yl) isopropyl chloroformate (BCPC-C1) as labeling reagents followed by HPLC-MS analysis have been developed. The optimal derivatization condition using BCECH as labeling reagent was evaluated. And the feasibility of this method has been identified by analyzing wolf feces sample.Chapter six: Pre-column derivatization methods for the sensitive determination of multihydroxyl phenolic compounds using dansyl chloride (Dns-Cl) as labeling reagents followed by HPLC-MS analysis have been developed. The optimal derivatization and chromatographic separation conditions using Dns-Cl as labeling reagent were evaluated. And the feasibility of this method has been identified by analyzing Tibetan herbal medicine Lomatogonium rotatum.