隨著科技的蓬勃發展，現代人民飲食與生活習慣的改變，不再只是吃得飽，而是要吃得好、吃得巧，而辦公室生活的習慣使得人們倦於運動。因此文明病隨即產生，其主要代表為三高 (高血壓，高血糖、高血脂) 等生理現象嚴重的危害人們的健康。因此，本篇論文內容是以分析血漿中之三酸甘油酯 (Triglycerides, TGs) 以及全血中之糖化血色素(glycosylated hemoglobin, HbA1c)。
一、 利用基質輔助雷射脫附游離飛行時間質譜儀 (MALDI-TOF/MS) 快速偵測血漿中之三酸甘油酯
三酸甘油酯是脂質 (lipid) 的一種，每克約可提供 9 千卡的熱量，經由游離甘油與脂肪酸所組成，由不同脂肪酸所形成之三酸甘油酯也有不同的物理特性，若存在於植物中由於其不飽和度較高室溫下為液體因此稱為植物油 (oil)，而存在於動物體內的脂肪酸多為飽和因此在室溫下為固體則稱為脂肪 (fat)。一般醫院檢測血漿中之三酸甘油酯是以試劑 (Kit) 與血漿檢體混合形成具可見光 (520 nm) 吸收之物質，再以 UV/Vis spectrometry進行分析，搭配檢量線可得知其確切的濃度，其數值若大於 200 mg/dL 則為高血脂症候群的高危險群。本研究嘗試以基質輔助雷射脫附游離飛行時間質譜儀進行三酸甘油酯的分析，其中包含血漿溶劑選擇、稀釋倍數、基質選擇的條件探討，最後將血漿樣品以水稀釋 10 倍後 1:1 混合 DHB基質於樣品盤上乾燥結晶，再以雷射進行脫附進入飛行時間 (TOF) 管分離不同質荷比 (m/z) 之物質。而開發血漿點樣技術：晶種法 (seed layer) 減少甜點效應 (sweet spot) 造成的相同樣品圖譜差異，之後將已知不同三酸甘油酯濃度之質圖譜送入統計軟體二維波峰分佈 2D peak distribution 進行分析，可半定量不同三酸甘油酯濃度的血漿樣品。
二、利用電噴灑輔助雷射脫附游離質譜法 (Electrospray-assisted laser desorption ionization mass spectrometry, ELDI/MS) 快速偵測全血中之糖化血色素
全血中糖化血色素 (glycosylated hemoglobin, HbA1c) 比例是目前糖尿病人長期控制血糖狀態的測量指標，由於血液中高濃度的代謝物、蛋白質以及鹽類的干擾，在將樣品溶液導入進行傳統的 LC/MS 分析之前需要較嚴苛的前處理過程。電噴灑輔助雷射脫附游離質譜法是一種兩階段式大氣游離技術，其已被發展直接鑑定液體表面的分析物。全血樣品一開始先探討稀釋倍數以及電噴灑溶液的組成，最後以一滴用水稀釋 10 倍的全血放置於不鏽鋼板之上，隨即以高能聚焦脈衝式雷射轟擊，脫附後的分析物在 ESI plume (70% MeOH, 0.1% acetic acid) 中被後游離成帶電離子，接著進入質譜儀中進行分析。利用 ELDI/MS 可有效的鑑定稀釋後全血裡面的血色素，不需複雜樣品的前處理且分析時間短，可成功地獲得糖化血色素離子的訊號。定量 20 個糖尿病人血液糖化血色素比例可直接計算糖化以及未糖化之血色素的離子峰面積比例，若比對傳統光譜測量則可得一線性關係。

Due to the technology development, the diet habit has completely changed. It accompanied by the metabolite diseases relevant to blood glucose and lipids, which are dependent with the atherosclerosis and cardiovascular disease. In this study, we using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS) to characterize triglycerides in human plasma. In the other, the glycosylated hemoglobin in human whole blood was detected by liquid electrospray laser desorption ionization (Liquid ELDI/MS).
Triglycerides are energy source (9 kcal/g) in human body, derived from glycerol and three fatty acids. It is a main constituent of vegetable oil and animal fats. In clinical diagnosis, human plasma was mixed with triglyceride Kit to react to the final 520 nm UV-absorbing substance, then the concentration was quantified consistent with the calibration line by UV/Visible spectrometry. By the way, it needed Kit chemicals for one trial. MALDI-TOF/MS is a simple and easy method to operate to detect complex compounds in human plasma, only need to optimize the parameters (solvent collection, sample dilution, matrix selection, sample pretreatment ) to form a homogeneous crystals. The developed “seed layer” method can reduce the sweet spot effect and cause a lower with-in spot variation (RSD < 20%) compared to “premix” method (RSD >30%). Combined with statistic software 2D peak distribution, a semi-quantification can be observe of 24 different triglyceride concentration human plasmas.
The level of glycosylated hemoglobin (HbA1c) in whole blood is currently the most important measurement of long-term control of the glycemic state of diabetes. As a result of the interferences of high concentrations of metabolites, proteins and salts in whole blood, tedious sample cleanup procedures must be performed prior to subjecting the sample solutions to conventional LC/MS and MALDI analyses for the detection of HbA1c. Electrospray laser desorption ionization mass spectrometry (ELDI/MS), a two-step ambient ionization technique, has been developed to characterize analytes directly from the liquid sample surface. One drop of the diluted hole blood (1/10, v/v in water) was placed on the stainless steel plate. The sample droplet was irradiated with a pulse laser, the desorbed analytes were post-ionized in an electrospray (ESI) plume (ESI solution: 70% methanol in water, 0.1% acetic acid), and the analyte ions were detected by a ion trap mass analyzer.
Through this study, the protocol for efficiently characterizing HbA1c present in a drop of diluted whole blood with ELDI/MS was established. We successfully detected the ion signal of HbA1c with ELDI/MS. Quantification of the level of HbA1c in the whole blood of diabetic patients was achieved by calculating the ratio of the ion peak area of the glycosylated and non-glycosylated hemoglobin ions. A linear relationship exists for the quantitative results of HbA1c in whole blood of 20 diabetic patients obtained between ELDI/MS and that through conventional spectroscopic measurement.

論文審定書…………………………………………………………………………....... i
誌謝…………………………………………………………………………………….. ii
中文摘要………………………………………………………………………………..iv
英文摘要………………………………………………………………………………..vi
目錄…………………………………………………………………….... viii
圖 目錄………………………………………………………….…...…. x
表 目錄………………………………………..………………………... xiv
第 一 章 緒論……………………………………………… 1
1.1 前言……………………………………………………………. 1
1.2 質譜歷史………………………………………………………. 2
1.3 血液的組成與功能….………………………………………… 14
1.4 脂質的介紹……………………………………………………. 15
1.5 二維波峰分布……………………………………………….... 21
1.6 糖尿病及糖化血色素相關的介紹……………………………. 22
第 二 章 以基質輔助雷射脫附游離飛行時間質譜儀分析人類血漿之
三酸甘油酯………………………………………………....................... 29
2.1 前言………………………………………………………….... 29
2.2 論文目標………………………………………………………. 29
2.3 實驗部分…………….……………………………………….. 30
2.4 結果與討論……………………………………………………. 34
2.5 結論…………………………………………………………… 45
第 三 章 以電噴灑輔助雷射脫附游離質譜法分析人類全血中之糖化血色素……………………………………………................................... 46
3.1 前言……………………………………………………………. 46
3.2 論文目標………………………………………………………. 46
3.3 實驗部分…………….………………………………………… 47
3.4 結果與討論…………………………………………………… 52
3.5 結論…………………………………………………………… 61
參考文獻……………………………………………………….…… 62

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