新型荧光试剂的合成及在高效液相色谱—质谱中的应用

【作者】 刘素娟；

【导师】 尤进茂；

【作者基本信息】 曲阜师范大学 ， 无机化学， 2007， 硕士

【摘要】 高效液相色谱法（HPLC）自20世纪60年代崛起，经过数十年的发展，在理论和实践等方面都日趋完善，应用范围已涉及医药、环保、生命科学、石油化工等几乎所有基础和应用研究领域。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实际样品的分析中。具体工作内容如下：第一章：综述了HPLC发展史、分类、色谱仪、应用及优缺点；介绍了荧光原理、影响荧光强度的因素和化学衍生法；分析总结了荧光标记试剂在HPLC中的应用概况。第二章：高灵敏荧光衍生试剂4-（1H-菲并[9，10-d]咪唑-2-）苯甲酸（PIBA）的设计合成，结构表征。并将该荧光标记试剂应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，并将方法应用于腐鱼等实际样品测定中。结果表明，衍生反应条件温和，衍生产率高，该方法的线性范围宽，重现性好，灵敏度高。第三章：设计合成高灵敏荧光衍生试剂4-（1-甲基-1H-菲并[9，10-d]咪唑-2-）苯甲酸（MPIBA），并进行了结构表征。考查了MPIBA的紫外性质及荧光性质。将MPIBA进行柱前衍生并经荧光检测对脂肪胺进行了高效液相色谱（HPLC）分离和在线质谱定性。衍生物的荧光激发和发射波长分别为λ_ex=260nm，λ_em=446nm。80℃下在四氢呋喃（THF）溶剂中用N-乙基-N’-[（3-二甲氨基）丙基]碳二亚胺盐酸盐（EDC）作催化剂，衍生反应10min后获得稳定的荧光产物。在Eclipse XDB-C₈色谱柱（4.6×150mm，5μm）上，采用梯度洗脱对12种游离脂肪胺衍生物进行了优化分离。采用大气压化学电离源（APCI）正离子模式进行在线柱后质谱定性，实现了各种脂肪胺衍生物的快速、准确测定。该方法具有良好的重现性，多数脂肪胺的线性回归系数大于0.9996，检测限在10.5-53.4fmol范围内，对实际样品臭豆腐中脂肪胺的测定结果满意。第四章：1，2-苯并-3，4-二氢咔唑-9-乙基氯甲酸酯（BCEOC）用于氨基酸的高效液相色谱-质谱分析。更多还原

【Abstract】 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.更多还原