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研究生：	洪榮華 Rong-Hua Hong
論文名稱：	毛細管電泳/藍光雷射誘導螢光偵測法對尿液中乳酸及3-羥丁酸之分析研究 Determination of lactate and 3-hydroxybutyrate in Urine by Capillary Electrophoresis-Blue Laser Induced Fluorescence Detection
指導教授：	林震煌 Lin, Cheng-Huang
學位類別：	碩士 Master
系所名稱：	化學系 Department of Chemistry
論文出版年：	2013
畢業學年度：	101
語文別：	中文
論文頁數：	74
中文關鍵詞：	毛細管電泳、螢光、乳酸、3-羥丁酸
英文關鍵詞：	capillary electrophoresis, fluorescence, lactic acid, 3-hydroxybutyric acid
論文種類：	學術論文
相關次數：	點閱：65 下載：3
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乳酸和3-羥丁酸為體內正常有機代謝產物，但是當肝臟疾病或體內脂肪氧化代謝異常時，血液中乳酸和3-羥丁酸就會過度累積，而發生乳酸性中毒和酮酸中毒的現象。由於乳酸和3-羥丁酸僅有極低的紫外光吸收性質，且不容易以電噴灑質譜法進行偵測，而傳統酵素測定法偵測乳酸和3-羥丁酸則容易受到內生性物質的干擾而影響準確性。本研究以毛細管電泳/藍光雷射誘導螢光偵測法，偵測尿液中的乳酸濃度以作為臨床診斷酮酸中毒的參考數據。目前市面上沒有適合的螢光衍生試劑，因此本實驗合成4-N-(4-N-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole作為乳酸和3-羥丁酸的螢光衍生試劑。衍生過程需要使用催化劑TPP (triphenylphosphine) 和DPDS (2,2’-dipyridyl disulfide) 來幫助反應進行。若利用微波輔助衍生，可將衍生反應時間縮短為3分鐘。衍生物結構在低pH值環境下會進行質子化並放出螢光，對於分離乳酸和3-羥丁酸的衍生物而言，利用pH 值小於3的磷酸緩衝液且不需添加有機修飾劑、界面活性劑即可完全分離。當以藍光雷射為螢光激發光源時，最佳偵測條件下，偵測極限約為10 g/L。由於雷射誘導螢光檢驗法的靈敏度高，因此不需要利用線上濃縮技術。本研究選擇的真實樣品為尿液和唾液，其前處理經過去蛋白和稀釋即可進行衍生。檢測結果發現，正常人尿液中的乳酸濃度約為 39 ± 11 mg/L。藉由運動的方式增加醣類代謝和脂肪氧化速度，則尿液中代謝的乳酸濃度增加為231 ± 121 mg/L。進食前唾液樣品中乳酸濃度約為49 ± 16 mg/L，進食後唾液樣品中由於葡萄糖濃度上升增加轉醣酵素的代謝速率，代謝物乳酸濃度上升至192 ± 48 mg/L。本研究提供簡單、快速的分析技術並成功的應用在真實樣品的檢測。

Lactic acid and 3-hydroxybutyric acid are metabolites in organism. Unusual -oxidation of fatty acid and metabolism in liver will result in excessive accumulation which lead to lactic acidosis and ketoacidosis in blood eventually of those acids. Lactic acid and 3-hydroxybutyric acid are ill-suited for UV and mass detection due to their poor absorbance and low selectivity. The novel indirect UV detection for those acids is a rapid and simple determination but the enzyme exhibits a slight cross-reactivity to the endogenous compounds which could affect the accuracy and reproducibility for the determination of those acids. This study synthesized 4-N-(4-N-amino -ethyl)piperazino-7-nitro-2,1,3-benzoxadiazole as the derivative reagent which reacted with acid in the presence of triphenylphosphine (TPP) and 2,2’-dipyridyl disulfide (DPDS) at room temperature within 3 hours or by microwave derivation within 3 minutes. The derivatives which were strongly fluoresced and were positively charged at pH below 3 were separated by using phosphate buffer without organic solvent or surfactant. The limit of determination were about 10 g/L by using blue laser as the light source under optimized condition. Since the sensitivity was enough for real sample detection, there is no need to use on-line concentration. This study choose urine and saliva as real sample, of which the pretreatment were only removing protein and then diluting before derivation. The concentration of lactic acid in urine was about 39 ± 11 mg/L. The concentration rose to 231 ± 121 mg/L because of the acceleration in glycolysis after exercise. On the other hand, the concentration of lactic acid in saliva was about 49 ± 16 mg/L. The concentration rose to 192 ± 48 mg/L due to the high abundance of glucose that result in accelerating the velocity that streptococcus mutans metabolized glucose after taking food. This study provided simple and fast technique which applied to the detection of real sample.

摘要 I
Abstract II
目錄 III
圖目錄 V
表目錄 VII
第一章  緒論 1
1-1酮體簡介 1
1-1-1酮體生成 (ketogenesis) 與代謝 1
1-1-2酮體測定 3
1-1-3酮體測定的臨床意義 4
1-2乳酸與科氏循環 (Cori cycle) 5
1-2-1乳酸生成與代謝 5
1-2-2乳酸測定 7
1-2-3乳酸測定的臨床意義 8
1-3分析物簡介 9
1-4研究目的 10
第二章  分析方法與原理 13
2-1毛細管電泳法之發展 13
2-2毛細管電泳法之基本原理 14
2-2-1電泳分離與遷移率 14
2-2-2電滲流 (EOF) 15
2-3毛細管電泳層析法之分離模式 18
2-3-1毛細管區帶電泳 (CZE) 19
2-3-2微胞電動層析法 (MEKC) 20
2-4毛細管電泳線上濃縮技術 21
2-4-1毛細管電泳線上堆積法 (stacking) 21
2-4-2毛細管電泳線上掃集法 (sweeping) 22
第三章  儀器、藥品與實驗方法 23
3-1自組式毛細管電泳/雷射誘導螢光分析儀 (CE-LIF) 23
3-2儀器及周邊設備列表 25
3-3藥品列表 28
3-4衍生試劑NBD-PZ-NH2合成及純化 30
3-5 NBD-PZ-NH2衍生物製備 31
3-6唾液、尿液真實樣品前處理  32
第四章  結果與討論  33
4-1衍生試劑NBD-PZ-NH2合成  33
4-2乳酸和3 -羥丁酸衍生反應之探討  38
4-3衍生物光譜性質測量  41
4-4衍生反應條件及電泳條件確立 44
4-5真實樣品中乳酸、3-羥丁酸的測定 59
第五章  結論  68
學會發表  69
參考文獻  70

                                

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