毛細管電泳結合電進樣與線上濃縮法以同步偵測分析腺苷及其代謝物

Simultaneous analysis of adenosine and its metabolites by capillary electrophoresis with electrokinetic injection and on-line preconcentration

林寶生(Bao-Sheng Lin)

指導教授 ： 曾惠芬

國立暨南國際大學/科技學院/應用化學系/碩士(2010年)

摘要

5’-核苷酸酶可以調節各組織器官中具各種生理活性的嘌呤核苷、核苷酸及兩者之類似物的濃度，以維持體內核苷酸代謝的平衡，是一種重要的磷酸酯水解酶。本研究中，利用毛細管電泳結合電進樣及線上濃縮法，以提升偵測靈敏度，分析腺苷單磷酸及其代謝之相關產物。在實驗中分別對進樣方式、添加劑四丁基氫氧化銨、電泳緩衝溶液的pH及濃度等參數進行探討及最佳化。以最佳化條件進行電泳，在8分鐘之內即可完成分析。腺苷、次黃嘌呤及肌苷的線性範圍在0.5 – 50 uM之間，而偵測極限分別可達0.33 uM、0.69 uM及0.64 uM。相較於乙腈堆積法，此方法可提升偵測靈敏度約4–7倍，並可應用於5’-核苷酸酶之活性測定。

關鍵字

毛細管區帶電泳 ； 5’-核苷酸酶腺苷肌苷次黃嘌呤

並列摘要

The 5’-nucleotidases (5’-NT) are involved in the regulation of various physiologically active purine nucleosides, nucleotides, and their analogues in organs. Therefore 5’-NT maintains balanced purine nucleotide pools, so it is one of the important phosphohydrolases. In this study, adenosine and its metabolites were determined by capillary electrophoresis with electrokinetic injection and on-line preconcentration to improve the sensitivity. Several parameters including the methods of sample injection, the concentration of the additive, tetrabutylammonium hydroxide, the pH and concentration of running buffer were optimized. Good separation of adenosine, hypoxanthine and inosine was achieved in 8 minutes under optimal conditions. The linear range of the method was 0.5–50 uM. The limits of detection of adenosine, hypoxanthine and inosine were 0.33 uM, 0.69 uM and 0.64 uM, respectively. The sensitivity of our method was enhanced at least 4 fold compared to the acetonitrile-salt stacking method. This sensitive method was successfully applied to the activity assay of 5’-NT from Hep G2 cells.

並列關鍵字

capillary electrophoresis ； 5’-nucleotidase ； adenosine ； inosine ； hypoxanthine

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