荧光标记试剂制备及在高效液相色谱—质谱中的应用

【作者】 赵先恩；

【导师】 尤进茂；

【作者基本信息】 曲阜师范大学 ， 物理化学， 2006， 硕士

【摘要】 高效液相色谱（HPLC）因其强大的分离分析能力已被广泛应用于许多研究和应用领域,如化学、生物化学、临床医学、药物学、神经生物学及食品卫生、环保检测、质量控制等。该法也是分析化学中发展最快、应用最广泛的领域之一。然而,由于样品中被测组分本身性质及其低含量的限制,并非所有化合物都可直接用于液相色谱分析。荧光检测具有很高的灵敏度,利用荧光标记技术使本身不发荧光的化合物（如氨基酸、脂肪胺、脂肪酸等）转化成发射荧光的衍生物,能够实现高灵敏荧光检测。本文重点研究了几种新荧光标记试剂的合成和表征,考察了荧光标记试剂的光谱性质,优化了衍生反应条件、色谱分离条件,并将建立的方法应用于不同生物样品的分析中。具体工作内容如下: 第一章:介绍了荧光原理及荧光标记法。简述了高效液相色谱的发展及其基础知识。综述了荧光标记试剂在羧酸类和氨基类化合物的高效液相色谱分析中的应用。 第二章:五种荧光标记试剂:吖啶酮-9-乙基对甲苯磺酸酯（AETS）、1-[2-（对甲苯磺酸酯）-乙基]-2-苯基咪唑[4,5-f]9,10-菲（TSPP）、2-（9-吖啶酮）-乙酸（AAA）、2-(2-苯基-1-氢-菲[9,10-d]咪唑-乙酸（PPIA）、1.2-苯并-3.4-二氢咔唑-9-乙基氯甲酸酯（BCEOC）的设计合成、结构表征、光谱性质研究,以及TSPP晶体结构解析。 第三章:两种荧光标记试剂AETS和TSPP分别用于脂肪酸的高效液相色谱-质谱分析。通过对衍生化条件和色谱分离条件的优化,建立了脂肪酸的柱前衍生化高效液相色谱荧光检测法,并将方法应用于土壤、苔藓、阿拉斯加深海鱼油、川西獐牙菜、肋柱花、麻花艽、秦艽样品的测定,实现了这些样品中脂肪酸的高灵敏检测。考察了溶液中的环境因素如溶剂极性、温度、卤素离子、重金属离子对TSPP-C₈衍生物荧光光谱的影响。 第四章:两种荧光标记试剂AAA和PPIA分别用于脂肪胺的高效液相色谱-质谱分析。通过对衍生化条件和色谱分离条件的优化,建立了脂肪胺的柱前衍生化高效液相色谱荧光检测法,并将方法应用于土壤、酸奶、大鼠端更多还原

【Abstract】 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-<i]irm’dazole-l-yl)-acetic acid (PPIA) as labeling reagents followed by HPLC-MS analysis have been developed. The optimal derivatization and chromatographic separation conditions using AAA and PPIA as labeling reagents were evaluated, respectively. The contents of aliphatic amines from real samples such as soil, milk, endbrain of rat, waste water of paper mill were determined. Studies on derivatization conditions indicated that aliphatic amines could rapidly and smoothly react with AAA and PPIA reagents under very mild conditions with maximal derivatization yields close to 100% with excellent linearity, good repeatability and higher sensitivity.Chapter five: Pre-column derivatization method for the sensitive determination of neurotransmitters and amino acids using the labeling reagents l,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by HPLC-MS analysis has been developed. In this study, three mono amine neurotransmitters containing primary amine and secondary amine and two aminoacid neurotransmitters were determined using BCEOC as labeling reagent. The contents of five neurotransmitters from endbrain of rat were determined. Additionally, the contents of twenty amino acids from serum of rat, Gentiana straminea and Gentiana macrophylla were also determined using BCEOC as labeling reagent.更多还原