臺灣博碩士論文加值系統

摘要
馬鈴薯塊莖中含有glycoalkaloids (GAs) 主要由α-chaconine 和 α-solanine 佔95% 組成，馬鈴薯過早採收或不當的貯存(如機械損傷、溫度、暴露光線)會造成 GAs 高濃度累積；，GAs 的毒性作用，一是破壞腸道細胞膜，另一是抑制神經傳導物質乙醯膽鹼?(acetylcholinesterase)，主要症狀有噁心、嘔吐、腹瀉、腹痛、和神經學病症有支氣管痙攣、精神錯亂等，嚴重時甚至死亡。
本研究係以液相層析串聯質譜儀 (liquid chromatography tandem mass, LC/MS/MS) 進行α-chaconine 和 α-solanine檢測。以2%醋酸為萃取液，移動相為0.1% formic acid：acetonitrile = 70：30 (v/v)，萃取液經離心過濾後以LC-MS/MS分析。α-chaconine以852.8 m/z為母離子，以852.8>706.5為定量離子，以852.8>398.7為定性離子；α-solanine以868.8 m/z為母離子，以868.8>398.5為定量離子，以868.8>722.8為定性離子。α-chaconine 和 α-solanine 於 0.05~1.0 ppm範圍內之線性迴歸係數分別為0.9987 和 0.9993。添加 3種不同濃度0.002 mg/g 、0.01 mg/g、0.02 mg/g，平均回收率α-chaconine 介於93.0±0.26 %~101.1±1.33%，α-solanine介於 91.7±0.83%~102.7±3.62%，GAs之同日間及異日間變異係數(cv, %)小於5.77 %以下，方法偵測極限α-chaconine為1.9 ppb (i.e ng/kg)，α-solanine為 1.8 ppb (i.e ng/kg)，本報告建立之檢驗方法，可做為日常及食物中毒案件中檢驗 glycoalkaloids 含量之檢驗方法。

ABSTRACT
More than 95% of the glycoalkaloids (GAs) found in potato are composed of α-chaconine and α-solanine. Early harvest or improper storage (ex. mechanical damages, temperature extremes and exposure to light) of potato tuber induced higher levels of GAs. The toxicities of glycoalkaloids are caused by inhibition of anticholinesterase in the central nervous system and membrane disruption in the digestive system. Symptoms include nausea, vomiting, diarrhea, abdominal pain, and neurological problems such as bronchial tube convulsion, delirium. Fatal cases caused by glycoalkaloids are also repoted.
The aim of this study is to determine the amounts of α-chaconine and α-solanine in potato by liquid chromatography tandem mass (LC-MS/MS). The samples were extracted by 2 % aqueous acetic acid and acetonitrile 30% with 0.1% formic acid as the mobile phase. The extracts were subjected directly to LC-MS/MS after centrifugal and filtration. The mass spectrometer was programmed to monitor the precursor ion [M+H]+ at m/z 852.8 and the product ion at m/z 706.5 , m/z 398.7 α-chaconine, and precursor ion [M+H]+ at m/z 868.8 and via the product ion at m/z 398.5, m/z 722.8 of α-solanine. The method detection of limit (MDL) with α-chaconine and α-solanine were 1.9 ppb (i.e. ng/kg) and 1.8 ppb (i.e. ng/kg) respectively. The coefficient of variation (cv %) of GAs were less than 5.77% for intra-day and inter-day. The present method has been successfully applied to the analysis of GAs in routine and food-poisoning outbreaks case.

目錄
中文摘要--------------------------------------------------I
英文摘要--------------------------------------------------II
目錄 ----------------------------------------------------III
圖目錄---------------------------------------------------VII
表目錄--------------------------------------------------VIII
縮寫-----------------------------------------------------IX

壹、前言 --------------------------------------------------01

貳、文獻回顧-----------------------------------------------03
一、Glycoalkaloid對人體的影響-------------------------------03
二、Glycoalkaloids 對細胞膜的影響---------------------------04
三、Glycoalkaloids抑制神經傳導物質--------------------------05
四、Glycoalkaloids臨床應用---------------------------------06
五、馬鈴薯的背景資料----------------------------------------07
六、馬鈴薯與Glycoalkaloids的關係----------------------------08
七、Glycoalkaloids的生成因素-------------------------------09
八、貯藏條件對GAs的影響 -----------------------------------10
九、烹調方式對Glycoalkaloids的影響--------------------------11
十、Glycoalkaloids的萃取方法-------------------------------12
十一、Glycoalkaloids的檢測方 ------------------------------14
十二、液相層析串聯質譜儀(LC-MS/MS)介紹 ----------------------16

參、實驗設備與研究方法--------------------------------------19
一、對照標準品---------------------------------------------19
二、藥品--------------------------------------------------19
三、實驗設備-----------------------------------------------20
四、實驗樣品-----------------------------------------------21
五、溶液配製-----------------------------------------------21
1. 混合標準原液之配製---------------------------------------21
2. 標準溶液配製--------------------------------------------22
六、樣品前處理---------------------------------------------22
七、串聯質譜儀條件最佳化------------------------------------23
1.母離子掃描-----------------------------------------------23
2.子離子掃描-----------------------------------------------23
3.串聯質譜儀參數設定----------------------------------------23
八、液相層析儀移動相最佳化-----------------------------------24
九、檢量線配製---------------------------------------------24
十、萃取方法試驗--------------------------------------------25
1.萃取液之配製---------------------------------------------25
2.萃取實驗步驟---------------------------------------------26
十一、精密度 (Precision)試驗--------------------------------27
十二、準確度 (Accuracy)------------------------------------27
十三、方法偵測極限（Method detection limit, MDL）試驗--------28
十四、真實樣品測定------------------------------------------29
十五、儀器分析---------------------------------------------30
十六、實驗流程圖--------------------------------------------31

肆、結果與討論 --------------------------------------------32
1.最適離子化條件-------------------------------------------32
2.液相層析-移動相最佳化-------------------------------------33
3.檢量線配製-----------------------------------------------34
4.萃取方法探討---------------------------------------------34
5.精密度 (Precision) -------------------------------------36
6.準確度 (Accuracy) ------------------------------------- 36
7.方法偵測極限 --------------------------------------------36
8.時間控制 ------------------------------------------------37
9.真實樣品測定---------------------------------------------38

伍、結論--------------------------------------------------40

陸、參考文獻-----------------------------------------------41

柒、圖表---------------------------------------------------50


圖目錄
圖1、α-solanine 和 α-chaconine 化學結構圖-----------------50
圖2、Glycoalkaloids 破壞細胞膜反應機制圖 -----------------51
圖3、貯藏條件對glycoalkaloids 的影響 ----------------------52
圖4、電灑噴霧離子解離界面----------------------------------53
圖5、電灑游離帶電離子形成過程----------------------------- 54
圖6、正電荷電噴灑解離一級質譜圖-----------------------------55
圖7、子離子掃描質譜圖-------------------------------------56
圖8、多重反應監控模式(multiple reaction monitoring, MRM)質譜圖--57
圖9、不同濃度比率移動相(mobile phase)比較圖-----------------58
圖10、α-chaconine 和 α-solanine標準檢量線-----------------59
圖11、不同萃取溶液比較圖-----------------------------------60
圖12、甲醇萃取液萃取加工食品MRM圖--------------------------61
圖13、儀器檢測極限(IDL)-----------------------------------62
圖14、新鮮馬鈴薯樣品質譜圖(MRM)----------------------------63
圖15、白色馬鈴薯樣品--------------------------------------64
圖16、省產馬鈴薯樣品--------------------------------------65


表目錄
表1. 烹煮方式對GAs的影響----------------------------------66
表2 質譜條件參數-----------------------------------------67
表3 三種萃取液之萃取回收率比較表---------------------------68
表4. 同日間(Intra-day)及異日間(Inter-day) 再現性分析------69
表5 α-chaconine 和 α-solanine 添加回收率分析-------------70
表6.方法偵測極限(MDL)------------------------------------71
表7.馬鈴薯加工食品中Glycoalkaloids含量--------------------72
表8.市售馬鈴薯加工半成品Glycoalkaloids含量-----------------73
表9 新鮮馬鈴薯中Glycoalkaloids含量------------------------74

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