以混合式固相萃取法搭配液相層析質譜儀檢測水樣中五種人工代糖

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研究生：	曾英翔 Ying-Hsiang Tseng
論文名稱：	以混合式固相萃取法搭配液相層析質譜儀檢測水樣中五種人工代糖
指導教授：	丁望賢 Wang-Hsien Ding
口試委員:
學位類別：	碩士 Master
系所名稱：	理學院 - 化學學系 Department of Chemistry
論文出版年：	2014
畢業學年度：	102
語文別：	中文
論文頁數：	131
中文關鍵詞：	人工代糖、固相萃取法、液相層析質譜儀
相關次數：	點閱：21 下載：0
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近年來隨著食品工業的蓬勃發展，人工合成的食品添加劑大量的被製造，其中人工代糖(Artificial sweeteners)為人工合成食品添加劑中廣為人類使用的產品之一。雖然人工代糖已經被研發約40年，但尚未有證據指出人工代糖對環境會造成什麼後果，不過由於人工代糖有易溶於水及不易降解的特性，在歐洲及北美的環境水樣中皆已發現乙醯磺胺酸鉀(Acesulfame potassium, ACE)又稱安賽蜜、糖精(Sodium Saccharin, SAC)、環己基(代)磺醯胺酸(Cyclamate, CYC)又稱甜蜜素及三氯蔗糖(Sucralose, SCL)的殘留。而隨著人類對人工代糖的大量需求，近年來，亞太市場人工代糖的產量以每年約10%的速度增加，同時環境中人工代糖殘留將會日益嚴重，因此研究環境中人工代糖的殘留越來越受到關注。
本篇研究目標是針對水樣中上述四種人工代糖及使用最為廣泛的阿斯巴甜(Asparatame, 簡稱ASP)含量，開發出檢測方法，我們使用自行填充的HR-X及C18吸附劑以3比1的比例混合進行固相萃取法(Solid Phase Extraction, SPE)，搭配液相層析質譜儀(Liquid Chromatography Mass Spectrometry, LC-MS)。最佳化使用面中心中央合成設計(Face-Centered Center Composite Design, FCCCD)進行多因子實驗條件設定，以此方法可用較少的實驗次數觀察出實驗因子之間的交互關係。最佳化結果為：混合式吸附劑重量400 mg，進樣流速0.5 mL/min，沖提溶劑甲醇體積10 mL，回收率在81%至102%間。我們也使用Mandel’s Fitting Test及Lack of fit試驗檢量線的適用性，並進行Interday與Intraday的測試，得到此方法的相對偏差小於8%，顯示此方法穩定且具有良好的再現性。
本研究ACE、SAC、CYC、ASP及SCL的定量偵測極限(LOQ)分別為0.07 μg/L、0.07 μg/L、0.06 μg/L、0.25 μg/L和0.11 μg/L，檢測六個自來水樣品及三個瓶裝水樣品，於瓶裝水中皆無檢出，而在自來水樣中每個樣品皆有檢出ACE，有其中四個樣品檢出SCL，有三個樣品檢出CYC，也有一個樣品檢出SAC。

With the development of food industry, artificially synthetic food additives are predominately supplied worldwide in recent years. One of food additives that human used extensively is artificial (high-intensity) sweeteners (ASs). ASs have been developed for 40 years. No significant evidence shows that ASs would have any impact on the environment. Since ASs are very water-soluble and difficult to biodegradation, their residues become major target in water sample. Acesulfame potassium (ACE), Sodium Saccharin (SAC), Cyclamate (CYC), Sucralose (SCL) have been detected in Europe and North America. The volume output in total of ASs grew approximately 10% in Asia Pacific market. In the meanwhile, thire residues must be increase, so we developed this method in order to monitoring the residues of ASs.
An analytical method for the determination of above four artificial sweeteners (ACE, SAC, CYC, SCL) and the most widespread ASs, Asparatame(ASP) in various water samples were developed and validated in this study. Hybrid-SPE with HR-X and ENVI C18 was applied for extract followed by liquid chromatography-mass spectrometry (LC-MS) is presented. The factor affecting the extraction efficiency of the analytes were optimized by the method of Face-Centered Central Composite Design (FCCCD). Optimized extraction conditions were as follows: HR-X : ENVI C18 = 3:1, sorbent amount: 400 mg, sample flow rate: 2.5 mL/min and elution solvent 10 mL methanol. Hybrid-SPE provide good trueness (recovery 81-102%), precision (RSD < 10%) and limit quantification (LOQ: 0.06-0.25 μg/L). In this study, we analyzed six tap water samples and three bottled water samples. No ASs were detected in bottled water sample. However, ACE was detected in all tap water samples. SCL was detected in four tap water. CYC was detected in three water sample and there is a sample detected SAC.

摘要    I
Abstract    III
目錄    VII
圖目錄    XI
表目錄    XV
第一章  前言...................................... 1
1-1　研究緣起    1
1-2　研究目標    4
第二章  文獻回顧................................... 7
2-1　人工代糖    7
2-1-1 人工代糖性質    10
2-1-1-1乙醯磺胺酸鉀    12
2-1-1-2 糖精    12
2-1-1-3 環己基(代)磺醯胺酸    13
2-1-1-4 阿斯巴甜    14
2-1-1-5 三氯蔗糖    16
2-1-2 檢測方法的文獻回顧    18
2-2 固相萃取法    25
2-2-1固相萃取法的四大優點    25
2-2-2固相萃取法的步驟    27
2-3 高效液相層析質譜儀    31
2-3-1 電噴灑游離法(Electrospray ionization)    33
2-3-1-1 電噴灑游離法之發展史    33
2-3-1-2 電灑游離法的原理及反應機構    34
2-3-1-3 影響電灑游離法形成液滴的因素    36
2-3-2 離子阱質譜儀    38
2-4 統計實驗計劃法    40
2-4-1 統計實驗設計簡史    40
2-4-2 統計實驗設計基本原理    42
2-4-3 因子設計實驗    44
2-5 迴歸模型    47
2-5-1 迴歸模型之建構：迴歸係數    47
2-5-2 迴歸模型之檢定：變異分析    48
2-5-3 迴歸模型之診斷：殘差分析    50
2-6 基質效應(Matrix effect)    54
第三章  實驗步驟與樣品分析.................. 57
3-1　實驗藥品與設備    57
3-1-1　實驗藥品    57
3-1-2　儀器設備    59
3-2 實驗步驟    60
3-2-1 標準品的配置    60
3-2-2 人工代糖的檢測    61
3-2-3 水樣之萃取步驟    62
3-3 水樣採集    64
第四章 結果與討論............................. 65
  4-1 液相層析質譜儀對人工代糖之分析    65
  4-1-1 毛細管電壓(Capillary voltage)對待測物感度之影響    68
  4-1-2 霧化氣壓力(Nebulizer pressure)對待測物感度之影響    69
  4-1-3 乾燥氣體流速(Drying gas flow rate)與乾燥氣體溫度(Drying gas temperature)對待測物感度之影響    70
  4-1-4 檢量線製作    72
  4-1-5 二次質譜(MS/MS)定性分析    74
  4-2 固相萃取法條件之最佳化    77
  4-2-1 固相萃取吸附劑之選擇    77
  4-2-2 吸附劑比例之選擇    79
  4-2-3 固相萃取法之實驗設計最佳化    81
  4-3  Mandel test 與 Matrix effect    89
  4-3-1 Mandel’s fitting test    89
  4-3-2 Lack-of-fit    90
  4-4 真實樣品之檢測    91
  4-4-1真實樣品檢測結果    91
  4-4-2 方法確效    98
  4-4-3 基質效應檢視    99
第五章  結論................................... 101
第六章  參考資料    102

                                

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