本論文研究主要是進行高效能液相層析法（high-performance liquid chromatography, HPLC）與融合微滴電噴灑游離質譜法（fused-droplet electrospray ionization mass spectrometry, FD-ES/MS）的串聯。此外還將探討融合微滴電噴灑游離質譜法中使用不同電噴灑溶劑，對於分析物離子訊號的影響。
高效能液相層析法是目前最常被使用的分離技術之一。但是，傳統液相層析法在與電噴灑游離質譜法連接時，往往會遭遇兩大困擾。其一為兩者流速上的差異性，其二則是分離所使用的動相溶液將對電噴灑效果造成極大的影響。研究中所使用的融合微滴電噴灑游離質譜法，除了可以生成帶有多價電荷之離子外，還能夠直接與高效能液相層析儀連接（HPLC-FD-ES/MS），而不需要經由任何分流（split）的裝置。對於各式分析混合物的直接分離與結構鑑定上具有相當的便利性與適用性。而對於上面所提到的第二個困境，在改採融合微滴電噴灑游離質譜法進行分析時也可以獲得明顯改善。
研究中使用逆相（reverse phase, C18）高效能液相層析管柱進行分離。使用的樣品則包括了各式利尿劑、甲基酯化脂肪酸、胜肽以及蛋白質混合物。實驗結果所得到的圖譜與傳統電噴灑的質譜圖相當類似，除了證明可將HPLC與FD-ES/MS成功地串聯，同時FD-ES/MS依然仍保有傳統電噴灑游離法的各項特性與優點。
此外，由於融合微滴電噴灑游離質譜法具有將噴灑與游離分成兩個步驟進行的特性。因此與傳統高效能液相層析連接電噴灑游離質譜法中，電噴灑溶劑即為動相溶液的情形便不復出現。以高效能液相層析連接融合微滴電噴灑離離質譜法時，分析者可以任意選用電噴灑溶劑進行電噴灑。不僅不會對分離的效果造成負面影響，更可因選用適當的溶劑而獲得較好的離子訊號。
在蛋白質混合物的分析中，本研究使用不同溶劑組成進行電噴灑對於不同的蛋白質會有強弱不一的影響。甚至能將某一特定訊號壓抑，進而使得另一分析物的訊號更加突顯。分析結果顯示當電噴灑溶劑為100﹪乙腈時，細胞色素-C的訊號會有明顯被壓抑的現象。但是同樣的溶劑對於肌紅蛋白卻沒有如此明顯的影響，於是在圖譜中產生了近似於分離的效果。這些改變在傳統電噴灑游離源裝置中都是無法進行研究探討的。
所以由本研究結果顯示，HPLC-FD-ES/MS可以成功地直接分析各式混合物，對於混合分析物的分離及鑑定上有相當的便利性與適用性。倘若再選用適當的電噴灑溶劑，則可控制分析物離子訊號的強弱而達到類似分離的效果。將可使得此技術把分離與偵測完全結合，大大提昇混合分析物的分析效率。

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目 錄

論文摘要
目錄
圖目錄
壹、緒論…………………………………………………………………..1
一、 前言…………………………………………………………..1
二、 電噴灑游離質譜法原理簡介與發生機制…………………..4
1. 電噴灑游離質譜法發展的歷程………………………….4
2. 電噴灑游離質譜法的反應機構…………….……………7
（a） 經由電噴灑所形成的帶電液滴…………………….7
（b） 溶劑的揮發與液滴再爆裂………………………….9
（c） 氣相多價電荷離子的產生機制………...…………10
三、 融合微滴電噴灑游離質譜法的發展歷程…………………12
四、 高效能液相層析法與質譜儀的結合………………………20
五、 論文目標……………………………………………………26
貳、實驗………………………………………………………………...28
一、 儀器裝置……………………………………………………28
二、 使用藥品及試劑配製方法…………………………………32
三、 高效能液相層析連接融合微滴電噴灑游離質譜法的裝置組裝與實驗操作步驟…………………………………………36
1. 實驗裝置組裝過程………………………………………...36
2. 實驗操作步驟……………………………………………...40
參、結果與討論…………………………………………………………45
一、 融合微滴電噴灑游離質譜法的離子產生機制……………45
二、 以高效能液相層析連接融合微滴電噴灑游離質譜法進行利尿劑、甲基酯化脂肪酸、胜肽、蛋白質混合物的分離與偵測……………………………………………………………48
1. 利尿劑混合物的分離與偵測……………………………...48
2. 甲基酯化脂肪酸混合物的分離與偵測……………...……55
3. 胜肽混合物的分離與偵測………………………………...61
4. 蛋白質混合物的分離與偵測…………………………...…64
三、 以高效能液相層析連接融合微滴電噴灑游離質譜法配合流動注入法，探討使用不同電噴灑溶劑時分析物訊號的變化……………………………………………………………71
1. 甲基酯化脂肪酸訊號的變化……………………………..71
2. 蛋白質訊號的變化………………………………………..76
肆、結論…………………………………………………………………81
伍、參考文獻……………………………………………………………83

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