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研究生:尤品人
研究生(外文):Pin-Jen Yu
論文名稱:QuEChERS萃取技術結合液相層析串聯質譜術於穀類產品中真菌毒素之分析
論文名稱(外文):Analysis of Mycotoxins in Grain Products by QuEChERS Combined with Liquid Chromatography-Tandem Mass Spectrometry
指導教授:李茂榮李茂榮引用關係
指導教授(外文):Maw-Rong Lee
口試委員:楊慶成賴建成
口試日期:2011-06-24
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學系所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:103
中文關鍵詞:真菌毒素液相層析串聯質譜術穀類產品
外文關鍵詞:MycotoxinLiquid Chromatography-Mass SpectrometryGrain product
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本實驗以QuEChERS萃取穀類產品中真菌毒素,再以LC/MS/MS進行分離及偵測,使用C18管柱以梯度沖提之方式進行分離,25分鐘內成功分離8種分析物。樣品處理部分,則探討不同之萃取溶劑種類、溶劑中添加酸之種類與比例、吸附劑之種類及添加量與添加鹽類比例,並對此方法進行線性範圍、線性相關係數、偵測極限、精密度與回收率之研究,最後將此方法應用於真實樣品中真菌毒素之分析。
由實驗結果顯示,以QuEChERS萃取技術結合液相層析質譜技術,使用正、負離子電灑游離法將分析物離子化後,再以選擇反應偵測(Selected reaction monitoring, SRM)進行偵測,取0.8 g穀類產品粉末加入2.5 mL含1%甲酸之乙腈:水(40:60, v/v)萃取溶劑,用震盪機以750 rpm震盪三分鐘進行萃取,加入300 mg之氯化鈉和500 mg無水硫酸鎂於離心管中,以750 rpm震盪三分鐘後,再以4000 rpm離心五分鐘後取上層液至另一離心管,加入45 mg之C18作為吸附劑和50 mg 無水硫酸鎂去除雜質,將離心管以1400 rpm震盪三分鐘,再以14000 rpm轉速離心五分鐘後,將萃取液通過0.22 μm濾膜,取20 μL注入液相層析質譜儀分析。
由實驗結果顯示,黃麴毒素與赭麴毒素檢測線性範圍在1.5~100 ng/g之間,棒麴毒素於150~800 ng/g,橘黴素則為25~1000 ng/g,線性相關係數均0.992以上,偵測極限定義為訊號三倍雜訊比,分別介於0.4 ng/g(黃麴毒素B1)至28 ng/g(棒麴毒素)之間。同日間與異日間之相對標準偏差分別為1.6%至17.1%與5.8%至19.4%,回收率介於72.5~112.4%之間,使用此方法對於一些真實樣品進行偵測,可測得橘黴素在某傳統市場穀類產品之含量為433.4 ng/g。

QuEChERS combined with LC-MS/MS was used to determine mycotoxins in grain products. The 8 mycotoxins were separated on a Waters XTerraTM MS C18 column in 25 minutes. The optimum parameters of MS/MS for eight mycotoxins and conditions of QuEChERS preparation were also discussed.
0.8g of homogenous grain product was precisely weighted and put into a 15mL centrifuge tube. Subsequently, 2.5 mL of mixed extraction solvent (acetonitrile:H2O:formic acid/40:60:1 v/v/v) was added into the same tube. After vigorously shaking for 3 min, 300 mg of sodium chloride and 500 mg magnesium sulfate were added. The tube was shaken intensely for 3 min then centrifuged at 4000 rpm for 5 min. The supernatant was transferred to 1.5 mL centrifuge tube then 45 mg octadecyl bonded silica gel and 50 mg magnesium sulfate were added. After intensely shaking for 3 min then centrifuging at 14000 rpm for 5 min, the supernatant was passed through a 0.22 μm syringe filter, then the extract was analyzed by LC-MS/MS.
The analytical linearity for aflatoxins, ochratoxins, citrinin and patulin was from 1.5 to 100 μg/kg, 25 to 1000 μg/kg and 150 to 800 μg/kg, respectively. The limits of detection were from 0.4 to 1.0 μg/kg for aflatoxins, 0.4 to 0.6 μg/kg for ochratoxins, 6 μg/kg for citrinin and 28 μg/kg for patulin. The relative standard deviations of intra-day and inter-day analysis were from 1.7% to 17.1% and from 5.8% to 19.4%. The analysis of trace mycotoxin in grain products were also determined, the concentration of citrinin was detected at 433.4 ng/g. The results demonstrate the suitability of QuEChERS combined with LC-MS/MS approach to analyze trace amount of mycotoxins in grain products.


致謝辭 I
摘要 II
Abstract III
目錄 IV
表目錄 X
圖目錄 XII

壹、緒論 1
1.1. 前言 1
1.2. 黃麴毒素 3
1.2.1. 黃麴毒素的來源及產生 3
1.2.2. 黃麴毒素之毒性 5
1.3. 橘黴素 7
1.3.1. 橘黴素的來源及產生 7
1.3.2. 橘黴素之毒性 7
1.4. 棒麴毒素 9
1.4.1. 棒麴毒素的來源及產生 9
1.4.2. 棒麴毒素之毒性 10
1.5. 赭麴毒素 11
1.5.1.赭麴毒素的來源及產生 11
1.5.2. 赭麴毒素之毒性 11
1.6. 真菌毒素之法定規範 13
1.7. 真菌毒素之偵測方法 17
1.8. 質譜儀原理 19
1.8.1.串聯質譜儀 20
1.8.2.電灑游離法 22
1.8.3.電灑游離法離子形成方式 23
1.9. QuEChERS萃取技術 25
1.10. 實驗目的 26
貳、實驗部分 28
2.1. 藥品、實驗器材和儀器設備 28
2.1.1. 藥品 28
2.1.2. 實驗器材與儀器設備 29
2.1.3. 儀器 30
2.2. 藥品配製 30
2.2.1. 真菌毒素標準品配製 30


2.2.1.1. 黃麴毒素、赭麴毒素、棒麴毒素、橘黴素標準儲存
溶液之配製 31
2.2.1.2. 黃麴毒素、赭麴毒素、棒麴毒素、橘黴素2 μg/mL
標準溶液之配製 31
2.2.1.3. 黃麴毒素、赭麴毒素、棒麴毒素、橘黴素1 μg/mL
標準溶液之配製 31
2.3. 液相層析質譜儀之最佳化參數探討 32
2.3.1. 電噴灑游離法 32
2.3.1.1. 霧化氣體壓力對黃麴素、赭麴毒素、橘黴素與棒麴
毒素偵測之影響 32
2.3.1.2. 乾燥氣體壓力對黃麴素、赭麴毒素、橘黴素與棒麴
毒素偵測之影響 32
2.3.1.3. 乾燥氣體溫度對黃麴素、赭麴毒素、橘黴素與棒麴
毒素偵測之影響 33
2.3.1.4. 介面腔溫度對黃麴毒素、赭麴毒素、橘黴素與棒麴
毒素偵測之影響 33
2.3.1.5. 探針電壓對黃麴毒素、赭麴毒素、橘黴素與棒麴毒
素偵測之影響 34
2.3.1.6. 鞘電壓對黃麴毒素、赭麴毒素、橘黴素與棒麴毒素
偵測之影響 34
2.4. QuEChERS萃取技術之條件探討 35
2.4.1. 萃取溶劑種類 35
2.4.2. 萃取溶劑添加酸之種類 36
2.4.3.萃取溶劑中添加甲酸之比例 36
2.4.4. 加入氯化鈉之比例 37
2.4.5. 添加吸附劑之種類 37
2.4.6. 添加吸附劑量 38
2.5. 分析穀類產品中黃麴毒素、赭麴毒素、棒麴毒素、橘黴素之線
性範圍、線性相關係數與極限偵測值 38
2.6. 分析穀類產品中黃麴毒素、赭麴毒素、棒麴毒素、橘黴素之精
密度 39
2.7. QuEChERS萃取技術分析穀類產品中黃麴毒素、赭麴毒素、棒
麴毒素、橘黴素之回收率 40
2.8. 真實樣品之測定 40
參、結果與討論 42
3.1. 真菌毒素標準品溶液之液相層析質譜分析 42
3.2. LC/MS正、負離子電灑游離法參數之探討 51
3.2.1. 霧化氣體壓力之探討 54
3.2.2. 乾燥氣體壓力之探討 54
3.2.3. 乾燥氣體溫度之探討 56
3.2.4. 界面腔溫度之探討 58
3.2.5. 探針電壓之探討 61
3.2.6. 鞘電壓之探討 64
3.3. MS/MS正、負離子電灑游離法參數探討 67
3.4. QuEChERS萃取技術之條件探討 71
3.4.1. 萃取溶劑種類之探討 71
3.4.2. 在萃取溶劑中添加酸種類之探討 73
3.4.3. 在萃取溶劑中添加甲酸之比例探討 73
3.4.4. 鹽類添加量之探討 75
3.4.5. 添加吸附劑之種類探討 78
3.4.6. 添加吸附劑之量探討 80
3.5. QuEChERS萃取技術分析餅乾中之真菌毒素之方法確效 83
3.5.1. QuEChERS萃取技術分析穀類產品中之真菌毒素之方法
確效線性範圍、線性方程式、線性相關係數及偵測極限
值 83
3.5.2. QuEChERS萃取技術分析穀類產品中之真菌毒素之精密
度 85
3.5.3. QuEChERS萃取技術分析餅乾中之真菌毒素之回收率與
真實樣品之偵測 91
肆、結論 96
伍、參考文獻 98

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