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標題: | 建立氣相層析質譜儀分析血漿中脂肪酸代謝體圖譜之方法 Development of a gas chromatography-mass spectrometry method for metabolic profiling of fatty acid in plasma |
作者: | Sung-Jeng Tsai 蔡松政 |
指導教授: | 郭錦樺(Ching-Hua Kuo) |
關鍵字: | 脂肪酸,氣相層析質譜儀,SLB-IL82,脂質萃取,代謝體圖譜, fatty acids,GC-MS,SLB-IL82,lipid extraction,metabolic profile, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 脂肪酸是體內參與能量平衡、訊息傳遞與維持細胞膜結構的重要物質。近幾年脂質代謝體的快速發展,配合化學劑量法分析大量樣品,發現血漿中許多脂質與疾病具相關性。本研究建立氣相層析質譜儀分析血漿中脂肪酸代謝體圖譜的方法,並比較不同的衍生化試劑,希望提供快速、穩固的方法在以應用於血漿中脂肪酸代謝體分析。
本研究以Folch萃取方法萃取血漿中脂質,以血漿萃取物、25種脂肪酸標準品衍生物與37種酯化脂肪酸標準品,對不同層析管柱與溫度梯度進行最佳化與探討,之後以優化條件比較Acetyl chloride、Hydrochloric acid、Boron trifluoride、Sulfuric acid、Sodium methoxide等五種衍生化試劑的衍生化效果,針對各種衍生化方法所提供之訊號強度和一日內與異日間之再現性進行討論。最佳化層析條件以SLB-IL82作為層析管柱,配合溫度梯度,可在25分鐘內建立血漿中脂肪酸之代謝體圖譜。相對其它衍生化方法,Acetyl chloride可以提供較好的訊號強度、再現性及較低的試劑價格。以最佳化的分析條件及Acetyl chloride衍生化方法分析心跳停止復甦急救之大鼠血漿,主成份分析結果顯示本研究所建立的脂肪酸代謝體圖譜可成功區分急救前、急救當下與急救後一小時的大鼠血漿。本實驗開發出一個快速且具有穩定再現性的分析方法,可以應用在未來分析脂質代謝體中脂肪酸的變化。 Fatty acids play important roles in the energy balance and signal transduction in biological systems. Some fatty acids are important constituents of cell membrane. Recent development in Lipidomics with the advance in chemometric techniques discovered many fatty acids are associated with numerous diseases. Gas chromatography-mass spectrometry (GC-MS) is the most commonly used analytical tool for fatty acid analysis. We compared five frequently used derivatization methods, and optimized GC-MS conditions. This study aimed to establish a fast, economic and robust GC-MS method for metabolic profiling of fatty acid in plasma. We used the Folch method to extract fatty acids in plasma. Plasma extract, 25 endogenous fatty acid and 37 fatty acid methyl esters were used to optimize gas chromatographic conditions. By using a SLB-IL82 column with optimized temperature gradient, metabolic profiles of fatty acid in plasma can be constructed within 25 minutes. We used the optimal GC-MS conditions to compare the performance of five derivatization methods, including Acetyl chloride, Hydrochloric acid, Boron trifluoride, Sulfuric acid and Sodium methoxide. The performances of the derivatization method were compared in terms of signal intensity, method precision and reagent cost. Compared to other derivatization methods, Acetyl chloride provided the highest singal intensity, best method precision with minimal reagent cost, and it was selected as the derivatization reagent for metabolic profiling of fatty acid in plasma. The developed method was applied to investigate fatty acid changes in post-cardiac arrest myocardial dysfunction rats. The plasma samples were collected at three time points before and after ischemia reperfusion, and they analyzed by the developed method. The results indicated the metabolic profiles of three time points can be distinquished in the scores plot of principal component analysis. The fast and robust method appears to be applicable to detect changes in fatty acids in plasma samples. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66330 |
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顯示於系所單位: | 藥學系 |
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