Studies On Chromatographic Methods For Amino Compounds Based On A New BODIPY Fluorescent Derivatizing Reagent

Small-molecule amino compounds including amino acids, biogenic amines, aliphatic amines, are a group of important compounds that widely exist in nature. They have crucial effects on the versatile physiological functions of almost all kinds of organisms, and most of them are conventional targets in sample analysis. Since many amino compounds are coexistent with extremely low content in various practical samples, it is usually necessary to analyze them by means of highly effective separation coupled to suitably sensitive detection. As a robust and sensitive approach, high performance liquid chromatography (HPLC) combined with fluorescence detection is regarded as one of ideal analytical methods for trace amount amino compounds in complex systems. Since many amino compounds have no or poor fluorescence, chemical derivatization using excellent fluorescent reagents is highly desired.Based on the survey of the current chromagraphic methods for trace amino compounds, a novel amine-reactive fluorescent derivatization reagent,1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su), has been designed and synthesized. Using TMBB-Su as the pre-column derivatizing reagent, a series of HPLC-fluorescence detection methods with high selectivity and sensitivity have been developed for amino compounds and applied to the analyses of biological, food and environmental samples.The major contents in this dissertation are summarized as follows:(1) A novel succinimidyl ester reagent with difluoroboradiaza-s-indacene (BODIPY) as a fluorophore (TMBB-Su) has been designed, synthesized, and characterized with MS and1H NMR. The fluorescent properties of TMBB-Su and its derivative with methylamine have been systematically studied. Experimental results show that the excitation and emission wavelengths of both TMBB-Su and its derivative are495and505nm, respectively. The derivative is photostable, non-sensitive to pH and solvents, and highly fluorescent with a fluorescence quantum yield of0.94. Our studies indicate that TMBB-Su is a favorable choice in the derivatizati on-based chromatographic methods for trace amino compounds.(2) Small-molecule aliphatic amines, especially secondary amines, are main precursors of carcinogenic N-nitrosamines in body. To develop the new detection method of trace primary and secondary aliphatic amines is helpful to study the form and distribution of N-nitrosamines. Although most of the existing succinimidyl ester reagents are difficult to react with secondary amines, the reactivity of TMBB-Su with secondary amines is good due to its unique structure. Therefore, a new HPLC method has been developed for the simultaneous determination of primary and secondary aliphatic amines using TMBB-Su as a pre-column derivatizing reagent. The derivatization reaction was carried out in pH7.20H3BO3-Na2B4O7buffer solution at15℃for25min. The derivatives of TMBB-Su with thirteen aliphatic amines including dimethylamine and diethylamine were separated in40min with gradient elution using methanol-tetrahydrofuran-50mM pH6.50HAc-NaAc buffer solutions as the eluent. With fluorescent detection at λex/λem-490/510nm, the detection limit reaches0.01nM (S/N=3), which is lowest compared with the existing HPLC methods. The proposed method has been applied to the simultaneous determination of primary and secondary amines in heart, liver and kidney samples of mice.(3) Biogenic amines in foods have a close relationship with human health. Using eight biogenic amines as the subjects, such as histamine, tyrosamine, tryptamine, spermine, phenethylamine, spermidine, putrescine and cadaverine, and TMBB-Su as the fluorescent derivatizing reagent, the derivatization and separation conditions of TMBB-Su with eight biogenic amines have been optimized. The reaction was performed at20℃for20min in pH7.20H3BO3-Na2B4O7buffer. On a Cg column, the derivatives of TMBB-Su with biogenic amines were baseline separated in40min with a gradient elution. The detection limits range from0.1to0.2nM (S/N=3). The proposed method is simple, feasible, sensitive, and has been applied to the determination of biogenic amines in milks and lake water.(4) Catecholamines, including norepinephrine, epinephrine and dopamine, are important neurotransmitters and hormones in mammalian species. Catecholamine levels are associated with their physiological functions and may provide evidences for the diagnosis of diseases. As a secondary amine, epinephrine is not able to react with most of amine-reactive fluorescent reagents, which makes trouble in the simultaneous determination of three catecholamines. Labeled with TMBB-Su, the optimized derivatization conditions were as follows:35μM TMBB-Su, pH7.20H3BO3-Na2B407buffer,35℃and10min. Using methanol-tetrahydrofuran-pH3.00mixed acids-NaOH buffer as the mobile phase, a pre-column derivatization HPLC-fluorescence detection method for norepinephrine, epinephrine and dopamine in mice tissues has been established. The detection limits are in the range of0.2-0.8nM (S/N=3). The method has the advantages of mild and rapid derivatization, and high sensitivity.(5) As a highly fluorescent reagent, the derivatization of TMBB-Su with three common phosphoamino acids, phosphor-serine, phosphor-threonine and phosphor-tyrosine, was investigated. The formed derivatives were determined by HPLC with fluorescence detection. Phosphoamino acids were derivatized with TMBB-Su in H3BO3-Na2B4O7buffer (pH8.00) at25℃for25min, and a base-line separation of the derivatives were obtained within27min without the interference from amino acids. Under the optimum experimental conditions, the linear ranges are0.005-0.5μM. The detection limits are0.5nM (S/N=3). The proposed method has been applied to the analysis of phosphoamino acids in plasma samples of healthy person and diabetic patient. The method has short derivatization time, excellent selectivity and high sensitivity.(6) It is a safety and efficient selenium supplemental access for human by the ingestion of seleno-amino acids from selenium-enriched foods, and the levels of seleno-amino acids has become an important index to evaluate nourishing value of selenium-enriched foods. The current analytical methods for seleno-amino acid focus on element analysis of selenium by HPLC-inductively coupled plasma mass spectrometry (ICP-MS), whereas HPLC-fluorescence detection is seldom used. Accordingly, a new HPLC-fluorescence detection method for seleno-amino acids has been developed based on TMBB-Su labeling. With80μM TMBB-Su, the labeling reaction was performed in pH7.20H3BO3-Na2B4O7buffer at25℃for25min. The derivatives were well separated in40min using a C8column with a gradient elution. Detection limits are calculated to be0.2nM (S/N=3) with fluorescence detection at λex/λem=490/510nm, which are comparable to those with HPLC-ICP-MS methods. This economical, sensitive and selective method has been used for the determination of selenomethylcysteine and selenomethionine in plant samples.