指導教授：何國榮臺灣大學：化學研究所歐育孟Ou, Yu-MengYu-MengOu2014-11-252018-07-102014-11-252018-07-102014http://ntur.lib.ntu.edu.tw//handle/246246/261285本研究利用本實驗室改良之氣相層析銜接電灑法質譜的界面設計,結合氣相 層析儀的高分離解析度以及電噴灑游離化法可獲得較高分子離子鋒訊號的優點, 對有機酸血症(organic academia)之指標代謝物-有機酸進行分析,將有機酸以三 甲基矽烷衍生化後,以正離子模式進行偵測,發現分析物之分子離子同位素訊號 比例不尋常,除了[M+H]+之外,亦存在著比例相當高之[M+3]+訊號。 經實驗證實[M+3]+訊號並非來自於衍生化試劑背景或是衍生化反應所產生之 副產物,初步猜測分析物之矽酯基上的碳氧雙鍵(-C=O)在經 GC-ESI/MS 界面時 被還原為羥基(-CH-OH)而造成[M+3]+訊號異常上昇。透過二次質譜探討碎片離 子圖譜,顯示[M+3]+之碎片離子存在[M+3-18]+之訊號(-H2O),以同全氘電噴灑 溶液進行二次質譜實驗,其產生之一系列-D2O、-DHO 及-H2O 之碎片,證實[M+3] +訊號之結構中確實有羥基之存在。進一步以高解析度離子阱-飛行時間質譜進行精 確質量之資料庫比對,發現實驗值與猜測之結構存在近 200 ppm 之誤差,與[M+3] +訊號比對最為接近之推測結構 R-C3H7O3Si 亦存在近 20 ppm 之誤差。經過探討發 現,所使用之飛行時間質譜之解析度不足以將[M+3]+訊號中含有矽-30 同位素之分 析物及未知反應生成物分離開,使得實驗所測得之[M+3]+訊號為一加權平均值, 透過加權計算證實,R-C3H7O3Si 可能為[M+3]+訊號異常上昇之原因,而並非為先 前推斷的碳氧雙鍵之還原反應。接續以更高解析度之四極桿-軌道阱質譜儀進行實 驗,將[M+3]+訊號含有矽-30 同位素之分析物及未知反應生成物解析成兩峰,確認 未知反應產物的結構式為矽烷基上一甲基被羥基所取代(R-CO2-Si(CH3)2OH),並嘗 試對該產物提出一生成之反應機制。Gas Chromatography (GC) combined with Mass Spectrometry (MS) is widely used for the analysis of metabolomics. With the use of the GC-ESI/MS interface that was developed by our laboratory to analyze the TMS (Trimethylsilyl) derivatives of organic acids, we have observed unusual high abundance of [M+3]+ ions. The abundance of [M+3]+ ions were as high as a third of the protonated molecule [M+H]+ ions. Based on the speculation of reduction of the C=O double bond (from R-CO2-Si(CH3)3 to R-H(COH)-O-Si(CH3)3), we proposed a possible structure of the unusual [M+3]+ ions. When these [M+3]+ ions were studied by Tandem Mass Spectrometry (MS/MS), a [M+3-H2O]+ product ion was observed and this ion was not detected from the fragmentations of the protonated molecules [M+H]+. With the use of deuterium solvent as the ESI solvent followed by MS/MS, the deuterated [M+5]+, and [M+4]+ ions, and the protonated [M+3]+ ions could respectively produce [M+5-D2O]+, [M+4-DHO]+, and [M+3-H2O]+ fragments, suggesting the presence of a hydroxyl group in the [M+3]+ ions. With the use of the IT-TOF mass spectrometer to identify the [M+3]+ ions, we found that the mass difference between the detected mass and the proposed structure is about 200 ppm. The most possible elemental composition R-C3H7O3Si, predicted by the computer database, was about 20 ppm from the detected mass. It was speculated that not only the unusual ions but also the 30Si isotopes of the derivatives were included in the [M+3]+ peak due to the limit of the resolution of the IT-TOF mass spectrometer. After calculation, R-C3H7O3Si was believed the reasonable elemental composition of the unusual [M+3]+ ions. The elemental composition of [M+3]+ ions were identified using a Q-Orbitrap mass spectrometer having a resolution about 100,000. Based on the elemental composition, a possible structure was proposed. The [M+3]+ ion was produced due to the exchange of a methyl group in the TMS group with a hydroxyl group. A possible mechanism for the formation of the [M+3]+ ions was also proposed.目錄 謝誌 i 摘要 ii Abstract iii 第一章 序論 1 1-1 前言 1 1-2 代謝質體學及其分析方法 2 1-3氣相層析法 3 1-3-1 氣相層析法之原理 3 1-3-2 氣相層析法的偵測方法 3 1-3-3 常見的氣相層析質譜法游離化方法 4 1-4 電噴灑游離法 5 1-4-1 電灑法的發展 5 1-4-2 電灑法原理 5 1-5 氣相層析銜接電噴灑游離質譜法 7 1-6 氣相層析電噴灑質譜應用於有機酸之分析 8 1-7 質譜儀 9 1-7-1 離子阱質譜儀（Ion-Trap Mass Spectrometer） 9 1-7-2 三段四極桿質譜儀（Triple Quadrupole Mass Spectrometer） 9 1-7-3 飛行時間質譜儀（Time of Flight Mass Spectrometer） 10 1-7-4 軌道阱質譜儀（Orbitrap Mass Spectrometer） 11 1-8 目的與方法 12 第二章 實驗部分 23 2-1 實驗藥品 23 2-2 儀器裝置 24 2-3 實驗方法 26 第三章 結果與討論 31 3-1 矽烷化有機酸 [M+3]+ 訊號之探討 31 3-1-1 電噴灑游離化的電化學 33 3-1-2 質譜端進樣錐電壓之調控 33 3-1-3 質譜端離子源之溫度調控 34 3-2 第三丁酯類之合成與分析比較 35 3-3 以氘化試劑之電噴灑溶液進行探討 37 3-4 以二次質譜對矽烷化有機酸進行分析 40 3-4-1 以全氘代電噴灑溶液進行二次質譜實驗 42 3-5 以離子阱-飛行時間質譜儀（IT-TOF MS）進行實驗 45 3-6 以四極桿-軌道阱質譜儀（Q-Orbitrap MS）進行實驗 49 3-6-1 以三乙基矽烷化有機酸進行比較 50 3-7 [M+3]+離子形成之反應機制探討 51 3-8 結論 53 參考文獻 1009263591 bytesapplication/pdf論文公開時間：2014/08/26論文使用權限：同意有償授權(權利金給回饋學校)氣相層析法電灑法質譜有機酸三甲基矽烷衍生化線上反應thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261285/1/ntu-103-R01223119-1.pdf