應用微胞電動層析-雷射誘發螢光偵測法檢測生物胺

Abstract

應用微胞電動層析-雷射誘發螢光偵測法檢測生物胺 研究生：陳義芳 指導教授：謝有容 國立交通大學應用化學研究所 摘要 生物胺分析物因其在人體中具有生理活性的特質，以及在食品中扮演判定腐壞程度的依據，因此研究生物胺化合物相關主題的分析日益重要，本論文研究以毛細管電泳搭配雷射誘發螢光偵測器，分析十四種生物胺分析物。在比較眾多衍生試劑(OPA、FMOC-Cl、Fluorescamine、FITC)的優缺點後，考慮到衍生後分析物的螢光強度，是否帶有電荷等等因素下，最後選取 FITC 當作本實驗的衍生試劑。 以毛細管電泳分析 FTC-生物胺衍生物實驗中，首先針對四種衍生反應條件：衍生溶液的 pH 值，衍生溶液的濃度，衍生試劑與生物胺的比值，衍生反應時間進行研究。結果顯示在 pH 值 10.1，20 mM 的硼酸鈉溶液下，以莫耳數比(FITC/BA) 10〜25倍之間，在室溫下暗室進行反應5 小時為最佳反應條件。同時也探討了分離條件：緩衝溶液的pH 值，緩衝溶液的濃度，陰離子界面活性劑的濃度。所得到最適化的分離條件，以pH 值10.1，40 mM 的硼酸鈉溶液，添加 40 mM 的陰離子界面活性劑(SDS)，可以成功地分離十四種生物胺分析物。 真實樣品上，運用於情感性精神病患者尿液及啤酒、乳房炎牛乳中的生物胺分析，在十四種生物胺分析物中，額外地再利用標準添加的方式，在尿液中可測得七種生物胺(Put, Oct, 5-HT, E, NE, Dop, Agm)，從正常人與患者的尿液圖譜結果比對，可看出多兒酚胺類（Catecholamines）的總量，在正常人與患者之間有明顯地差異。另外在市面上啤酒的生物胺含量檢測中，可測得五種不等量的生物胺(Put, Him, Tyr, PEA, Agm)，而乳房炎牛乳中可測得一種生物胺(Put)。 在晶片電泳的實驗中，可以在 3 分鐘內成功地分離五種生物胺標準品(Put, Him, 5-HT, Dop, Agm)，其最適化的分離條件為pH 值10.1，20 mM 的硼酸鈉溶液，添加 20 mM 的 SDS。

Analysis of biogenic amines by Micellar Electrokinetic Chromatography with Laser-Induced Fluorescence Student：Yi-Fang Chen Advisor： You-Zung Hsieh Department of Applied Chemistry National Chiao Tung University Abstract Mono- or polyamines involved in biological activities and that exist in nature are sometimes called by biogenic amines. Some of them are biologically synthesized from amino acids by amino acid decarboxylase degradation and play an important role in living systems. In this study, analysis of fourteen biogenic amines by micellar electrokinetic capillary electrophoresis and laser induce fluorescence were investigated. The selected derivatizing reagent was fluorescein isothiocyanate ( FITC). In order to achieve an optimum detection sensitivity and separation efficiencies,it was necessary to optimize precolumn derivatization conditions with respect to reaction time, pH, buffer concentration and the molar ratios of FITC to biogenic amines. It must also study the optimum separation conditions. In an optimized condition of micellar electrokinetic chromatography, using 40 mM sodium borate buffer (pH 10.1) with 40 mM sodium dodecyl sufate (SDS), fourteen biogenic amines could be successful separated. To check the applicability of the method, affective disorder patient’s urine was analyzed. We additional spiked E and NE to identify the existence of E and NE in the urine. Qualitatively analysis, we can find seven kinds of biogenic amines in urines (Put, Oct, 5-HT, E, NE, Dop, Agm).Quantitatively analysis, we can find the different urinary catecholamine amounts of normal people and the patients. Another application in the beers and mastitis milk, we had also found five kinds of biogenic amines in beers (Put, Him, Tyr, PEA, Agm) and one kind of biogenic amines in milk (Put). In the capillary electrophoresis microchip, we could successfully separate five kinds of biogenic amines (Put, Him, 5-HT, Dop, Agm) within 3 minutes. The optimum running buffer was pH 10.1,20 mM Borate/20 mM SDS.