New Fluorescent Derivatization Reagents And Their Application In Analysis Of Carboxyl Compounds

Carboxylic acids are a biochemically heterogeneous group of compounds containing a carboxylic group, they are widely distributed in nature life, and exert crucially function, involved in the organism’s metabolism, signal transduction and regulation of gene expression, and also act as important component in vivo important nutrients and organ tissue, and a marker of human disease. Some carboxyl compounds in the external environment and residues in food but also a threat to human health. Therefore establish a rapid, efficient and accurate detection analysis detection method is very important for revealing the physiological, pathological and pharmacological functions of carboxylic compound.Based on the review of the function of carboxylic acids, and the determination methods for carboxylic acids, we have developed HPLC-fluorescence detection and CE-LEF methods for the phytohormones in vegetable using6-oxy-(acetyl piperazine) fluorescein(APF) as derivatization reagent. In order to improve the performance piperzine derivatization reagent for carboxylic acids, we designed and synthesized a novel derivatization reagent by using difluoroboradiaza-s-indacene (BODIPY) as fluorophore, the fluorescent properties and derivative characteristics of this new reagent was studied in details. Using the fluorescence reagents as labeling reagents, HPLC-fluorescence detection and CE-LEF methods were developed for fatty acids in plasma, unsaturated fatty acids in edible oils samples and phytohormones in flowers. The major contents in this thesis are described as follows:1. A novel piperzine reagents using BODIPY as a fluorophore were designed and synthesized. It’s1,3,5,7-tetramethyl-8-butyricpiperzine-difluoroboradiaza-s-indacene (BODIPY-butyricpiperzine). The conditions of synthesis have been investigated and the structures of all new compounds are identified with MS and1H NMR. The fluorescent properties of new reagent and its derivatives were studied in details. The effect of environment media factors and the relationship of the molecular structures on fluorescent properties of new reagents and derivatives were investigated. The result shows the derivatives of the reagents with carboxylic compounds have good photostability, high extinction coefficient (8.10×104M-1cm-1) and high fluorescence quantum yields (0.953in acetonitrile). The spectra of derivatives of carboxylic compounds are also relatively insensitive to solvent polarity and pH because of lack of ionic charge. All of the derivatives have good fluorescent properties and are suited for fluorescent detection in HPLC. 2. A novel fluorescent derivatization reagent for carboxylic acids,1,3,5,7-tetramethyl-8-butyricpiperzine-difluoroboradiaza-s-indacene (BOD IP Y-butyricpiperzine, TMBB-P), was first applied in high-performance liquid chromatography (HPLC) to derivatize and separate fatty acids. The derivatization reaction with twelve fatty acids, n-propionic acid (C3), n-butyric acid (C4), n-valeric acid (C5), n-hexanoic acid (C6), n-heptanoic acid (C7), n-nonanoic acid (C9), n-decanoic acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), oleic acid (C18:1) and linoleic acid (C18:2), was completed at55℃within40min. The derivatives of fatty acids were separated on a C8reversed-phase column and detected at excitation and emission wavelengths of490nm and510nm. The detection limits attained were0.2-1.2nM (signal-to-noise ratio of3). The method have been applied to the analysis of lipopolysaccharide induced liver damage mice sample, the recoveries are90.7-106.1%.3. An effective approach was proposed to the derivatization of Docosahexaenoic Acid (DHA) and Arachidonic acid (ARA), using BODIPY-butyricpiperzine as a derivatization reagent. The sensitive determinations of these derivatives were achieved by fluorenscent detection. The derivative was seperated40min with gradient elution using methanol-tetrahydrofuran-100mM pH6.50H3Cit-Na2HPO4buffer solutions as the eluent. The detection limit (S/N=3) was found as low as0.6nM. The procedure was successfully applied to the determination of DHA and AHA in edible oil sample with satisfying recovery efficiencies varying from90.6to109.6%.4.1,3,5,7-tetramethyl-8-butyricpiperzine-difluoroboradiaza-s-indacene (BODIPY-butyricpiperzine) has been used as a pre-column derivatization reagent for the determination of six phytohormones, indole-3-acetic acid (IAA), indole-3-yl butyric acid (IBA), abscisic acid (ABA), jasmonic acid (JA),2-(1-naphthyl) acetic acid (NAA),2,4-dichlorophenoxyacetic acid (2,4-D), with high performance liquid chromatography (HPLC) for the first time. The derivatization reaction of TMBB-P with six phytohormones was completed at50℃for1h while the present of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). On a C8column, the derivatives could be seperated completely in30min using a mobile phase of methanol/H3Cit-Na2HPO4buffer (pH6.50)/tetrahydrofuran (THF)(60:10:7.5:22.5, v/v/v) with fluorescence detection at λex/λem=490/510nm. The detection limits could reach0.6-1.6nM (signal-to-noise=3). This reagent was applied to the determination of phytohormones in cuncumber samples with satisfying recovery efficiencies varying from93.4%-106.4%. 5. Using BOD IP Y-butyricpiperzine as a fluorescent derivatizating reagent, a new CE-LIF method has been developed for the determination of gibberellic acid (GA3), indole acetic acid (IAA), indole butyric acid (IBA), abscisic acid (ABA), jasmonic acid (JA),1-naphthalene acetic acid (NAA) and2,4-dichloro-phenoxy acetic acid (2,4-D). The derivatization reaction was carried out with the use of1-ethyl-3-(3-diethylamino-propyl) carbodiimide (EDC) as condensing reagent at50℃60min. Under optimum conditions, a baseline separation of GA3, IAA, IBA, ABA, JA, NAA,2,3-D has been accomplished within20min using a running buffer composed of40mM borate buffer (pH9.0),25mM SDS and20%(v/v) acetonitrile with22.5kV as the separation voltage. The detection limits were0.3-0.9nM (S/N=3). The proposed method has been applied to the analysis of phytohormones in different parts of chineserose with satisfying results.6. Using APF as a fluorescent derivatizating reagent, a new CE-LIF method has been developed for the determination of gibberellic acid (GA3), indole acetic acid (IAA), indole butyric acid (IBA), abscisic acid (ABA), jasmonic acid (JA),1-naphthalene acetic acid (NAA) and2,4-dichloro-phenoxy acetic acid (2,4-D). The conditions of derivatization and separation have been studied. The derivatization reaction was carried out with the use of1-ethyl-3-(3-diethylamino-propyl) carbodiimide (EDC) as condensing reagent at60℃. APF could react with phytohormones absolutely to form stable fluorescent derivatives within1h. Under optimum conditions, a baseline separation of GA3, IAA, IBA, ABA, JA, NAA,2,3-D has been accomplished within20min using a running buffer composed of25mM borate buffer (pH9.2),15mM SDS and12%(v/v) ethanol with22.5kV as the separation voltage. The detection limits were1.6-6.7nM (S/N=3). The proposed method has been applied to the analysis of phytohormones in banana with satisfying results.