伏馬鐮孢毒素（Fumonisins，FB）是串珠鐮刀菌（Fusarium moniliforme）所產生的二次代謝產物，主要汙染作物為玉米及其相關產品，會造成豬隻出現類似感冒症狀，使的投藥錯誤，造成巨大損失。不僅如此伏馬鐮孢毒素與人類的食道癌也極為相關。
伏馬鐮孢毒素中以FB1的毒素最強且量最大（70%），目前衛生署公告的穀物中FB1的分析方法為：使用一次性免疫親合管柱（NT$≧300元，成本較高）進行萃取且須經衍生反應後再以HPLC-FLD偵測。但此方法對於沒被汙染的農作物樣品中仍然要支出同樣高的成本，並不符合綠色環保的概念。因此，在這篇論文中，我們應用基質輔助雷射脫附游離/飛行時間質譜法（MALDI-TOF MS）開發出一個具有低成本，高靈敏度且可大量篩選FB1毒素的分析方法來輔助官方方法的不足。
本方法中的前處理只需要以甲醇和水萃取分析物後離心過濾即可以MALDI-TOF/MS來進行偵測。當只以α-CHCA為基質時，只能在穀物（玉米）中測到1 ppm的Fumonisins，因此不具可行性，但在添加Cs+ 後可增加分析靈敏度1000倍，偵測極限達到1 ppb。此法所需成本低（NT≦10）且偵測時間小於1分鐘，可節省不必要的材料及時間的浪費，相當符合當前綠色化學的環保概念。

Fumonisins are secondary metabolites of Fusarium moniliforme, mainly in corn and corn-based products. It may cause flu-like symptoms on pig and result in wrong treatment. Moreover, fumonisins are associated with human esophageal cancer.
Fumonisin B1 (FB1) is the most toxic and most natural abundant type (70%) in Fumonisins. The official analysis method for FB1 is using disposable immunoaffinity column (NT$≧300) for extraction then following derivatization before HPLC-FLD measurement. However, the cost of the specific column and the time consuming are not good for high throughput analysis, especially for the non-contaminated samples. If there is a low cost and high throughput screening methods to filter out most uncontaminated samples, a lot of time consuming and cost would be avoided. Thus, we develop a low-cost, high sensitivity screening method for FB1 by MALDI-TOF MS to achieve this goal.
In this method, the pretreatment just need solvent (MeOH/H2O) extraction following centrifugation, then detected by MALDI-TOF/MS using α-CHCA as the matrix and Cs+ to increase sensitivity to 1 ppb. The detection limit would be only 1 ppm without Cs+. The cost of sample preparation for each sample is less than NT$ 10.0 dollars and analysis time for each sample is only 1 min or less. This is in line with current environmental protection concept of green chemistry.

目錄
中文摘要……………………………………………………………I
英文摘要……………………………………………………………II
誌謝…………………………………………………………………III
目錄…………………………………………………………………IV
表目錄………………………………………………………………VII
圖目錄………………………………………………………………VIII
第一章 緒論……………………………………………………………1
1.1 前言………………………………………………………1
1.2 黴菌毒素（mycotoxin）與伏馬鐮孢毒素Fumonisins）
簡介………………………………………………………2
1.2.1 黴菌毒素（Mycotoxin） ………………………2
1.2.2 伏馬鐮孢毒素（Fumonisins）………………9
1.3 黴菌毒素（Mycotoxin）檢測技術 ……………………12
1.3.1 黴菌毒素（Mycotoxin）檢測方法比較 ………12
1.3.2 伏馬鐮孢毒素（fumonisins）檢測方法比較…15
1.4 基質輔助雷射脫附游離飛行時間質譜法………………20
1.4.1 MALDI的簡介 ……………………………………20
1.4.2 MALDI-TOF 在小分子偵測的發展與應用………23
1.4.3 基質的特性………………………………………27
1.5 研究動機與目的…………………………………………30
第二章 材料與方法……………………………………………………33
2.1 實驗材料…………………………………………………33
2.1.1 實驗藥品…………………………………………33
2.1.2 實驗樣品…………………………………………36
2.1.3 儀器設備…………………………………………36
2.2 藥品配製…………………………………………………37
2.2.1 基質溶液的配製…………………………………37
2.2.2 鹽類溶液的配製…………………………………39
2.2.3 標準品的配製……………………………………40
2.3 穀物樣品的萃取…………………………………………41
2.3.1 穀物樣品的毒素添加……………………………41
2.3.2 穀物樣品萃取……………………………………41
2.4 MALDI 配製與偵測條件…………………………………42
2.4.1 MALDI 樣品的配製………………………………42
2.4.2 MALDI-TOF MS 的操作條件 ……………………43
第三章 結果與討論……………………………………………………44
3.1 最佳的分析條件…………………………………………44
3.1.1 基質的選擇………………………………………44
3.1.2 增加分析的靈敏度………………………………63
3.2 真實樣品的檢測…………………………………………75
3.3 以 PEG (Polyethylene Glycerol)對小分子精確分子量
校正改善的探討…………………………………………83
第四章 結論……………………………………………………………84
參考文獻 ………………………………………………………………85

表目錄
表一 黴菌毒素主要來源與全球分布…………………………………6
表二 常見黴菌毒素的中毒症狀………………………………………7
表三 黴菌毒素的各國規定含量限制…………………………………7
表四 食品中黃麴毒素（Aflatoxin ）含量限制標準………………8
表五 食品中赭麴毒素 A（Ochratoxin A）含量限制標準…………8
表六 食品中棒麴毒素（Patulin ）含量限制標準…………………8
表七 食品中橘黴素（Citrinin）含量限制標準……………………8
表八 伏馬鐮孢毒素檢測方法比較 …………………………………19
表九 MALDI-TOF MS 操作參數設定…………………………………43
表十 不同基質與不同濃度的 FB1 毒素之訊號……………………62
表十一 不同濃度 FB1 添加五種鹽類的訊號及強度………………68
表十二 不同 FB1 濃度添加不同 CsCl 濃度的訊號及強度………73
表十三 本實驗檢驗方法與衛生署95年公告檢驗方法的結果比較.77

圖目錄
圖一、伏馬鐮孢毒素（fumonisin）的化學結構式…………………9
圖二、單純固體基質 α-CHCA 之 MALDI-TOF 質譜圖………………46
圖三、固體基質 α-CHCA 與不同濃度的 FB1 標準品：(a) 10 ppm (b) 1 ppm (c) 0.1 ppm 進行偵測之 MALDI-TOF 質譜圖…………46
圖四、單純固體基質 2,4-DHB 之 MALDI-TOF 質譜圖 ……………48
圖五、固體基質 2,4-DHB 與不同濃度的 FB1 標準品：(a) 10 ppm (b) 1 ppm (c) 0.1 ppm 進行偵測之 MALDI-TOF 質譜圖…………48
圖六、單純固體基質 2,5-DHB 之 MALDI-TOF 質譜圖 ……………50
圖七、固體基質 2,5-DHB 與不同濃度的 FB1 標準品：(a) 10 ppm (b) 1 ppm (c) 0.1 ppm 進行偵測之 MALDI-TOF 質譜圖…………50
圖八、單純固體基質 2,6-DHB 之 MALDI-TOF 質譜圖 ……………52
圖九、固體基質 2,6-DHB 與不同濃度的 FB1 標準品：(a) 10 ppm (b) 1 ppm (c) 0.1 ppm 進行偵測之 MALDI-TOF 質譜圖…………52
圖十、單純 Et3N-α-CHCA 離子液體基質之 MALDI-TOF 質譜圖…54
圖十一、Et3N-α-CHCA 離子液體基質與不同濃度的 FB1 標準品：(a) 10 ppm (b) 1 ppm (c) 0.1 ppm 進行偵測之 MALDI-TOF 質譜圖………………………………………………………………………54
圖十二、單純 Citrate-Capped AuNPs 金奈米粒子基質之 MALDI-TOF 質譜圖……………………………………………………………56
圖十三、Citrate-Capped AuNPs 金奈米粒子基質與不同濃度的 FB1 標準品：(a) 10 ppm (b) 1 ppm (c) 0.1 ppm 進行偵測之 MALDI-TOF 質譜圖……………………………………………………56
圖十四、單純 DDAB-Capped AuNPs 金奈米粒子基質之 MALDI-TOF 質譜圖…………………………………………………………………57
圖十五、DDAB-Capped AuNPs 金奈米粒子基質與不同濃度的 FB1 標準品：(a) 10 ppm (b) 1 ppm (c) 0.1 ppm 進行偵測之 MALDI-TOF 質譜圖……………………………………………………………57
圖十六、單純 Glucose-Capped AuNPs 金奈米粒子基質之 MALDI-TOF質譜圖 ……………………………………………………………58
圖十七、Glucose-Capped AuNPs 金奈米粒子基質與不同濃度的 FB1 標準品：(a) 10 ppm (b) 1 ppm (c) 0.1 ppm 進行偵測之 MALDI-TOF 質譜圖……………………………………………………58
圖十八、單純Bare-Capped AuNPs 金奈米粒子基質之 MALDI-TOF質譜圖……………………………………………………………………59
圖十九、Bare-Capped AuNPs 金奈米粒子基質與不同濃度的 FB1 標準品：(a) 10 ppm (b) 1 ppm (c) 0.1 ppm 進行偵測之 MALDI-TOF 質譜圖……………………………………………………………59
圖二十、單純鋁箔之 MALDI-TOF 質譜圖……………………………61
圖二十一、不同濃度的 FB1 標準品點於鋁箔上：(a) 10 ppm (b) 1 ppm (c) 0.1 ppm 進行偵測之 MALDI-TOF 質譜…………………61
圖二十二、FB1 標準品 10 ppm與 α-CHCA 混合後分別添加不同的鹽類(a)LiCl (b) NaCl (c) KCl (d) RbCl (e) CsCl ………………65
圖二十三、FB1 標準品 1 ppm與 α-CHCA 混合後分別添加不同的鹽類(a) LiCl (b) NaCl (c) KCl (d) RbCl (e) CsCl………………67
圖二十四、FB1 標準品 0.1 ppm 分別與不同濃度的 CsCl 混合 (a) 1×10-3 M (b) 1×10-4 M (c) 1×10-5 M (d) 1×10-6 M………71
圖二十五、FB1 標準品 0.05 ppm 分別與不同濃度的 CsCl 混合(a) 1×10-3 M (b) 1×10-4 M (c) 1×10-5 M (d) 1×10-6 M………72
圖二十六、FB1 標準品 0.01 ppm 分別與不同濃度的 CsCl 混合(a) 1×10-3 M (b) 1×10-4 M (c) 1×10-5 M (d) 1×10-6 M………73
圖二十七、FB1 標準品 0.001 ppm 分別與不同濃度的 CsCl 混合(a) 1×10-5 M (b) 1×10-6 M…………………………………………74
圖二十八、MALDI-TOF/MS 偵測之質譜圖：(a) 空白基質 (b) 無添加毒素真實樣品萃取的玉米基質……………………………………78
圖二十九、真實穀物萃取MALDI-TOF/MS質譜圖：(a)含有 100 ppb FB1標準品的真實樣品(玉米基質) (b)含有 50 ppb FB1標準品的真實樣品(玉米基質) (c)含有 10 ppb FB1標準品的真實樣品(玉米基質)………………………………………………………………………78
圖三十 、(a)為FB1 加上 CsCl 金屬離子的理論同位素 (b)為圖二十七含有100 ppb、50 ppb、10 ppb FB1 標準品真實樣品(玉米基質)的同位素……………………………………………………………79
圖三十一、真實穀物萃取MALDI - TOF/MS質譜圖：含有 1 ppb FB1標準品的真實樣品(玉米基質) ………………………………………79
圖三十二、MALDI-TOF/MS 偵測之質譜圖：(a)空白基質 (b)無添加毒素真實樣品萃取的豬飼料基質……………………………………80
圖三十三、真實穀物萃取MALDI-TOF/MS質譜圖：(a)含有 10 ppb FB1標準品的真實樣品(豬飼料基質) (b)含有 1 ppb FB1標準品的真實樣品(豬飼料基質) ………………………………………………80
圖三十四、MALDI-TOF/MS 偵測之質譜圖：(a)空白基質 (b)無添加毒素真實樣品萃取的小麥基質………………………………………81
圖三十五、真實穀物萃取MALDI-TOF/MS質譜圖：(a)含有 10 ppb FB1標準品的真實樣品(小麥基質) (b)含有 1 ppb FB1標準品的真實樣品(小麥基質) ……………………………………………………81
圖三十六、MALDI-TOF/MS 偵測之質譜圖：(a)空白基質 (b)無添加毒素真實樣品萃取的食品 cereal 基質……………………………82
圖三十七、真實穀物萃取MALDI-TOF/MS質譜圖：(a)含有 10 ppb FB1標準品的真實樣品(cereal基質) (b)含有 1 ppb FB1標準品的真實樣品(cereal基質) …………………………………………………82
圖三十八、PEG 校正之圖譜 …………………………………………84
圖三十九、PEG 校正後偵測 FB1 標準品100 ppb 之圖譜…………85
圖四十、PEG 四點校正其 peaks 之峰形……………………………85

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