The Synthesis Of Novel Fluorescent Reagents And High Performance Liquid Chromatography - Mass Spectrometry

High performance liquid chromatography (HPLC), has grown up from the twentieth century 60^th, 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 two 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 development history and basic theory including sorts, chromatogram instrument, application, excellency and defect about HPLC are simply introduced. The fluorescence theory and chemistry derivatization are introduced systematically. The applications of fluorescence reagents are summarized.Chapter two: The syntheses of fluorescence labeling reagents 4-(1H-phenanthro[9,10-d]imidazol-2-yl)benzoic acid (PIBA) were described, and followed by their structural characterization and spectral properties. Pre-column derivatization methods for the sensitive determination of aliphatic amines, respectively, using PIBA as labeling reagents followed by HPLC-MS analysis have been developed. The optimal derivatization and chromatographic separation conditions using PIBA as labeling reagents were evaluated, respectively. The contents of aliphatic amines from real samples such as rotten fish , were determined. Studies on derivatization conditions indicated that aliphatic amines could rapidly and smoothly react with PIBA reagents under very mild conditions with maximal derivatization yields close to 100% with excellent linearity, good repeatability and higher sensitivity.Chapter three: The syntheses of fluorescence labeling reagents 4-(1-methy-1H-phenanthro[9,10-d]imidazol-2-)benzoic acid (MPIBA) were described, and followed by their structural characterization and spectral properties. A simple and sensitive method based on the derivatization of aliphatic amines with MPIBA followed by high-performance liquid chromatography (HPLC) with fluorescence detection and post-column mass spectrometric identification has been developed. The fluorescence excitation and emission wavelengths of derivatives were set at λ_ex =260 and λ_em=446nm, respectively. Optimal derivatization, giving the corresponding stable fluorescent derivatives, was obtained by the reaction of aliphatic amines with MPIBA at 80℃ for 10 min in the presence of 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC.HCl) catalyst in acetonitrile. Separation of the derivatized amines in conjunction with a gradient elution was achieved on a reversed-phase Eclipse XDB C₈(4.6×150mm i.d., 5μm) column with a good baseline resolution. The identification of aliphatic amine derivatives was carried out by post-column online APCI/MS (atmospheric pressure chemical ionization) under in positive-ion mode. Excellent linear responses are observed with coefficients >0.9996, detection limits (at signal-to-noise of 3:1) are 10.5-53.4 fmol. The established method for the determination of amines from the extracted bean curd with odor exhibits satisfactory results.Chapter four: Pre-column derivatization method for the sensitive determination of amino acids using the labeling reagents 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed byHPLC-MS analysis has been developed.