臺灣博碩士論文加值系統

近年來養生觀念崛起，食用紅麴食品以及保健食品的人口增加，其相
關產品種類繁多，然而紅麴菌二次代謝物會產生，具有保健功效的產物，亦會產生具有強烈腎毒性的橘黴素，因此造成人們食入橘黴素風險也跟著增加。本研究以固液萃取法，以及液相層析串聯式質譜儀，建立分析方法，
並進行方法確效與方法不確定評估。實驗結果檢量線線性範圍為0.5-500 ng/mL、迴歸係數0.9989，檢出限量為20 ng/mL (S/N ≥10)。準確度及精密度測試，將添加濃度20、40、2000 ng/mL橘黴素標準品，分別添加入各類檢體中。實驗結果準確度測試各檢體之回收率，分別為玉米檢體78.00%-110.00%、紅麴餅乾檢體72.00%-95.00%、米檢體79.84%-116.00%、燕麥檢體72.00%-118.00%。各類檢體精密度試驗結果，重覆性試驗結果各檢體變異係數，都小於10，中間精密度變異係數分析結果，除了玉米、燕麥檢體添加濃度20 ng/mL變異係數高於10%，其餘分析結果變異係數皆小於10%。各檢體之擴充不確定度評估結果，玉米3922±386.08 ng/mL、紅麴餅乾3922±310.24 ng/mL、米4018±483.50 ng/mL、燕麥3922±460.60 ng/mL。

In recent years, due to health concerns, red kojic rice has drawn a lot of attentions. A wide variety of red kojic rice related products are being sold on the market. Monascus purpureus is the fermenting mold for making red kojic rice and its secondary metabolites has been shown to provide health benefits. However, the mold also produces citrinin that exhibits strong nephrotoxicity, leading to health risks. In this study, we used solid-liquid extraction and LC-MS/MS to establish an analytical method, and assessed the uncertainty and validation of the method. Experiment results showed that the calibration curve linear range of 0.5-500 ng/mL, the regression coefficient was 0.9989, and the detection limit was 20 ng/mL (S/N≥10). Accuracy and precision of tests were run, for 20, 40 and 2000 ng/mL citrinin standards concentrations added to various types of samples. The recoveries for each specimen were 78.00%-110.00% for corn, 72.00%-95.00% for red kojic rice crackers, 79.84%-116.00% for rice, and 72.00%-118.00% for oat. Among repetitive tests, the coefficient of variation was less than 10%. In the intermediate precision coefficient of variation analysis, the variation higher than 10% for corn (20 ng/mL citrinin added), the remaining results were all less than 10% coefficient of variation. The assessment of expansion uncertainties were 3922±386.08 ng/mL for corn, 3922±310.24ng/mL red kojic rice crackers, 4018±483.50 ng/mL for rice, and 3922 ±460.60 ng/mL for oat.

目 錄
中文摘要 Ⅰ
英文摘要 Ⅱ
誌謝 Ⅲ
目錄 Ⅴ
表目錄 Ⅷ
圖目錄 Ⅹ
第一章 緒論
1.1 緒論…………………………..…………………………….....................1
1.2 研究動機及目的………………………………………………………...3
第二章 文獻回顧
2.1 紅麴菌簡介……………………………………………………...………5
2.2 紅麴菌具有價值的二次代謝物………………........……………...……6
2.3 橘黴素(citrinin)……………………………..…………………...………8
2.3.1 橘黴素的發現以及生產菌種……………………………………..8
2.3.2 橘黴素的結構……………………………………………………10
2.3.3 橘黴素的毒性……………………………………………...…….12
2.3.4 橘黴素檢測方法…………………………………………………13
2.3.5 橘黴素含量之限制………………………………………………16
2.3.6 橘黴素的去毒性方法……………………………………………17
2.4 液相層析串聯質譜儀簡介…………………………………………….20
2.5 基質效應.................................................................................................22
2.6 分析方法確效.........................................................................................24
第三章 材料與方法
3.1 藥品與材料…………………………………………………………….27
3.2 器材…………………………………………………………………….27
3.3 試劑配製……………………………………………………………….28
3.4 液相層析串聯式質譜儀分析條件設定……………………………….29
3.5 實驗流程……………………………………………………………….31
第四章 結果與討論
4.1 液相層析串聯式質譜儀條件最佳化………………………....……….34
4.2 分析方法確效之結果………………………………………….………38
4.2.1 線性(linearity)……………………………………………………38
4.2.2 基質效應(Matrix effect)…………………………………………40
4.2.3 準確度(Accuracy)…………...……………………...…………....44
4.2.4 精密度(Precision)………………………....………...…….......…47
4.3 橘黴素檢測方法量測不確定度.............................................................51
4.3.1 橘黴素檢測方法須評估不確定度之項目....................................51
4.3.2 標準品不確定度............................................................................52
4.3.3 添加、重複、 查核、樣品之不確定度評估....................................54
4.3.4 檢量線之不確定度評估................................................................56
4.3.5 各基質最終擴充不確定度結果....................................................61
第五章 結論.....................................................................................................69
參考文獻.............................................................................................................71

表 目 錄
表1-1常見主要黴菌屬及其黴菌毒素.................................................................1
表2-1紅麴菌具有功效的二次代謝物以及用途.................................................7
表2-2橘黴素動物毒性試驗之相關數值...........................................................13
表2-3各國對橘黴素含量限制...........................................................................17
表3-1液相層析儀梯度設定...............................................................................29
表3-2橘黴素之質譜多重反應偵測模式參數及定量極限...............................30
表3-3相對離子強度容許範圍...........................................................................31
表4-1檢量線與基質匹配檢量線線性迴歸數值及各樣品基質效應之結果
.............................................................................................................................41
表4-2添加回收率試驗.......................................................................................45
表4-3衛生福利部食品藥物管理署回收率之規範...........................................46
表4-4添加樣品重複性試驗...............................................................................48
表4-5添加樣品中間精密度試驗.......................................................................49
表4-6衛生福利部食品藥物管理署方法之變異係數 (CV, %) 規範..............50
表4-7微量吸管擴充不確定度以及標準不確定度...........................................53
表4-8添加樣品之相對不確定度.......................................................................54
表4-9重複樣品之相對不確定度.......................................................................55
表4-10查核樣品回收率之相對不確定度.........................................................56
表4-11標準品檢量線不確定度計算.................................................................57
表4-12基質匹配檢量線不確定度計算.............................................................58
表4-13樣品玉米中橘黴素含量檢測之組合不確定度.....................................62
表4-14樣品紅麴餅乾中橘黴素含量檢測之組合不確定度.............................64
表4-15樣品米中橘黴素含量檢測之組合不確定度.........................................66
表4-16樣品燕麥中橘黴素含量檢測之組合不確定度.....................................68

圖 目 錄
圖2-1紅麴的製作程.............................................................................................6
圖2-2橘黴素之化學結構圖...............................................................................10
圖2-3紅麴菌中橘黴素的生合成路徑...............................................................11
圖2-4免疫親和管柱吸附的過程.......................................................................15
圖2-5紅色色素與橘黴素之生合成路徑...........................................................19
圖2-6質譜儀基本結構標示圖...........................................................................21
圖2-7液相層析質譜儀不同離子源使用範圍...................................................21
圖2-8管柱後灌流系統 (Post-column infusion system)....................................23
圖3-1橘黴素檢驗固液萃取分析流程圖...........................................................33
圖4-1橘黴素質譜圖前驅離子為m/z 251.2產物離子對m/z 251.2→233.2*......
； m/z 251.2→91.2有*為定量離子標準品濃度0.5 ng/mL…..........................36
圖4-2橘黴素質譜圖前驅離子為m/z 251.2產物離子對m/z 251.2 →233.2*.....
；m/z 251.2 →91.2 *為定量離子橘黴素標準品濃度2 ng/mL..........................36
圖4-3橘黴素質譜圖前驅離子為m/z 251.2產物離子對m/z 251.2→233.2*......
；m/z 251.2→205 *為定量離子橘黴素標準品濃度0.5 ng/mL.........................37
圖4-4橘黴素質譜圖前驅離子為m/z 251.2產物離子對m/z 251.2→233.2*......
；m/z 251.2→205* 為定量離子標準品濃度2 ng/mL.......................................37
圖4-5橘黴素利用液相層析質譜儀建立的檢量線...........................................38
圖4-6橘黴素質譜圖，前驅離子為m/z 251.2產物離子對m/z 251.2→233.2*....
；m/z 251.2→205有*為定量離子，標準品濃度100 ng/mL............................. 39
圖4-7玉米基質匹配檢量線與標準品檢量線比較...........................................42
圖4-8紅麴餅乾基質匹配檢量線與標準品檢量線比較...................................42
圖4-9米基質匹配檢量線與標準品檢量線比較...............................................43
圖4-10燕麥基質匹配檢量線與標準品檢量線比較.........................................43
圖4-11橘黴素檢測方法須評估不確定度之項目.............................................51

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