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研究生:温景煊
研究生(外文):WEN, CHING-HSUAN
論文名稱:分散型固相萃取技術( QuEChERS )結合高效液相層析電噴灑串聯式質譜儀檢測肉類樣品中磺胺類藥物殘留之研究
論文名稱(外文):Determination of Sulfonamides in Meat Sample by Dispersive Solid Phase Extraction ( QuEChERS ) with LC-MS/MS
指導教授:傅明仁
指導教授(外文):FUH, MING-REN
口試委員:何國榮李茂榮
口試委員(外文):HER, GUOR-RONGLEE, MAW-RONG
口試日期:2016-07-12
學位類別:碩士
校院名稱:東吳大學
系所名稱:化學系
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:2016
畢業學年度:104
語文別:中文
論文頁數:112
中文關鍵詞:高效液相層析電噴灑串聯式質譜儀分散型固相萃取技術磺胺類藥物
外文關鍵詞:LC-MS/MSQuEChERSSulfonamide
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分散型固相萃取技術中的QuEChERS已經成為有效且廣泛應用的樣品前處理方法,本研究主要是修改以及優化QuEChERS方法檢測肉類樣品中九種磺胺類抗生素(磺胺嘧啶,磺胺吡啶,磺胺甲基嘧啶,磺胺二甲嘧啶,磺胺間甲氧嘧啶,磺胺異噁唑,磺胺二甲氧嘧啶,磺胺喹惡啉和磺胺苯吡唑)結合高效液相層析串聯式質譜儀進行檢測。此方法對豬肉跟牛肉基質在0.125 ng/g~12.5 ng/g之間有良好的線性關係其決定係數(coefficients of determination)均大於0.9999,在五種肉類樣品中回收率範圍在74.0 %~100.3 %,偵測極限約在0.01 ng/g~0.04 ng/g,且此方法的偵測極限與其他文獻比較相對來的好,其intra-day/inter-day之精密度以及準確度分別在1.00 %~10.51% / 0.40 %~7.97 % 以及95.2%~107.1% / 97.8 %~102.1 % 範圍之間。此開發方法利用在牛肉、豬肉、雞肉、牛肚和豬肝樣品中磺胺類藥物的檢測來證明其方法之應用性。
The dispersive solid phase extraction (QuEChERS) has emerged as effective and useful sample pretreatment. The aim of this study is to modify and optimize QuEChERS method for determination of 9 sulfonamide antibiotics (sulfadiazine, sulfapyridine, sulfamerazine, sulfamethazine, sulfamonomethoxine, sulfisoxazole, sulfadimethoxine, sulfaquinoxaline and sulfaphenazole) in meat samples with LC-MS/MS. The linear concentration range was 0.125-12.5 ng/g with coefficients of determination over 0.9999. The extraction recoveries were in the range of 74.0-100.3% from five different kinds of meat samples. The detection limits were estimated at 0.01-0.04 ng/g. The detection limits of this method proved to be much better than previously studies. The intra-day/inter-day precision and accuracy were in the range of 1.0-10.5%/0.4-8.0% and 95.2-107.1%/97.8-102.1%, respectively. The applicability of this developed method was demonstrated by analyzing the occurrence of sulfonamides in various beef, pork, chicken, beef tripe and pig liver sample.
目錄
英文摘要
中文摘要
第一章 緒論 1
第一節 磺胺類藥物之介紹 3
第二節 分析磺胺類藥物之技術上的發展 4
第三節 利用高效液相層析/電噴灑串聯式質譜儀偵測肉品中之磺胺類藥物 8
第四節 QuEChERS樣品前處理之方法與發展 10
第五節 電灑/質譜儀應用與發展 12
第六節 三段四極柱質譜儀 16
第二章 儀器與設備 19
第一節 樣品與試劑 19
第二節 儀器與設備 20
(1)高效液相層析儀 20
(2)電噴灑/質譜儀 20
(3)樣品前處理 21
(四)其他 21
第三節 實驗架構及步驟 22
(1)加速電壓和斷裂電壓對訊號強度的影響 22
(2)探討HPLC流速對磺胺類訊號的影響 24
(4)基質效應於肉類樣本之探討 25
(5)測試萃取溶劑添加不同濃度之醋酸(Acetic acid)對萃取效果的影響 26
(6)測試萃取步驟中加入不同Na2SO4量的影響 28
(7)測試萃取步驟中加入不同NaCl量的影響 29
(8)測試淨化步驟中加入不同Na2SO4量的影響 30
(9)測試淨化步驟中加入不同C18 & PSA量的影響 31
(10)測試淨化步驟中加入PSA量混合不同量之Z-sep+的影響 32
(11)探討ND lipid的淨化效果 33
(12)探討不同比例回溶溶劑之差異 33
(13)回收率之測試 34
(14)基質效應之測試 35
(15)線性關係 36
(16)精密度與準確度之測試 37
(17)真實樣品之檢測 37

第三章 結果與討論 39
第一節 三重四極柱質譜儀 39
(1)質譜儀毛細管出口端電壓及碰撞電壓對訊號強度的影響 39
(2)質譜儀碰撞電壓對訊號強度的影響 45
第二節 高效液相層析/電噴灑串聯式質譜儀 58
(1)流速對高效液相層析/電噴灑串聯式質譜儀的影響 58
(2)移動相添加劑的量對磺胺類訊號偵測的影響 58
第三節 樣品前處理 61
(1)探討萃取溶劑添加不同濃度之醋酸對萃取效果的影響 61
(2)探討萃取步驟中加入不同量氯化鈉的影響 61
(3)探討萃取步驟中加入不同量無水硫酸鈉的影響 63
(4)探討淨化步驟中加入不同Na2SO4量的影響 65
(5) 探討淨化步驟中加入不同吸附劑的淨化效果 65
(6)探討ND lipid (Non-drip lipid)的淨化效果 67
(7)探討不同比例回溶溶劑之差異 67
(8)不同基質之回收率與基質效應 72
(9)基質效應對線性的影響 76
第四節 線性關係與真實樣品 77
(1)線性關係 77
(3)真實樣品之檢測 83
第五節 結論 91
第四章 參考文獻 93


表目錄

表一 九種磺胺類藥物以及一種內標準品之結構式與分子量……………………..2
表二 文獻中分析磺胺類藥物之方法………………………………………………..7
表三 磺胺類之二次質譜斷裂離子之結果…………………………………………56
表四 磺胺類藥物在正離子模式下之MRM參數設定整理…………………...….57
表五 高效液相層析層析條件………………………………………………………62
表六 五種空白基質中添加磺胺類藥物之基質效應與回收率的比較…..……..…75
表七 基質效應對線性的影響……………………………………………………....78
表八 空白豬肉和空白牛肉基質中添加磺胺類藥物之偵測極限與線性關係……79
表九 空白牛肉基質中添加磺胺類藥物之同日間與異日間的精密度和準確度…84
表十 肉類真實樣品檢測之結果……………………………………………………85
表十一 其他文獻報告與本研究之實驗結果比較…..……………………………..92



圖目錄

圖一 高效液相層析/電噴灑串聯式質譜儀之儀器簡圖………………………….....9
圖二 電噴灑游離法之電灑現象……………………………………………………15
圖三 三段四極柱質譜儀之示意圖…………………………………………………18
圖四 四極柱質量分析器之結構圖…………………………………………………19
圖五 QuEChERS萃取方法之優化步驟……………………………………………27
圖六 毛細管出口端電壓對(a)SMM、(b)SMZ施加不同加速電壓對其特徵離子訊號強度的影響………………………………………………………………………..40
圖七 毛細管出口端電壓對(a)SPD、(b)SDM施加不同加速電壓對其特徵離子訊號強度的影響………………………………………………………………………..41
圖八 毛細管出口端電壓對(a)SIA、(b)SDZ施加不同加速電壓對其特徵離子訊號強度的影響…………………………………………………………………………..42
圖九 毛細管出口端電壓對(a)SMR、(b)SPA施加不同加速電壓對其特徵離子訊號強度的影響………………………………………………………………………..43
圖十 毛細管出口端電壓對SQX施加不同加速電壓對其特徵離子訊號強度的影響……………………………………………………………………………………..44
圖十一 (1)SMM之分子結構圖及其特徵子離子斷裂示意圖、(2)碰撞電壓對SMM訊號強度之影響.……………………………………………………………..46
圖十二 (1)SMZ之分子結構圖及其特徵子離子斷裂示意圖、(2)碰撞電壓對SMZ訊號強度之影響……………………………………………………………………..47
圖十三 (1)SPD之分子結構圖及其特徵子離子斷裂示意圖、(2)碰撞電壓對SPD訊號強度之影響……………………………………………………………………..48
圖十四 (1)SDM之分子結構圖及其特徵子離子斷裂示意圖、(2)碰撞電壓對SDM訊號強度之影響………………………………………………………………49
圖十五 (1)SIA之分子結構圖及其特徵子離子斷裂示意圖、(2)碰撞電壓對SIA訊號強度之影響……………………………………………………………………..50
圖十六 (1)SDZ之分子結構圖及其特徵子離子斷裂示意圖、(2)碰撞電壓對SDZ訊號強度之影響……………………………………………………………………..51
圖十七 (1)SMR之分子結構圖及其特徵子離子斷裂示意圖、(2)碰撞電壓對SMR訊號強度之影響……………………………………………………………………..52
圖十八 (1)SPA之分子結構圖及其特徵子離子斷裂示意圖、(2)碰撞電壓對SPA訊號強度之影響……………………………………………………………………..53
圖十九 (1)SQX之分子結構圖及其特徵子離子斷裂示意圖、(2)碰撞電壓對SQX訊號強度之影響……………………………………………………………………..54
圖二十 9種磺胺類在高效液相層析/電噴灑串聯式質譜儀中流速對的影響……59
圖二十一 9種磺胺類在高效液相層析/電噴灑串聯式質譜儀中移動相添加添加劑的影響……………………………………………………………………………......60
圖二十二 萃取溶劑添加不同濃度之醋酸對萃取效果的影響……………………62
圖二十三 萃取步驟中加入不同量氯化鈉的影響…………………………………64
圖二十四 萃取步驟中加入不同量無水硫酸鈉的影響……………………………64
圖二十五 淨化步驟中加入不同量無水硫酸鈉的影響……………………………66
圖二十六 不同吸附劑的淨化效果…………………………………………………68
圖二十七 (1)、(2)加入C18之實驗照片; (3)、(4)加入PSA之實驗照片……….69
圖二十八 ND lipid的淨化效果…………………………………………………….70
圖二十九 (a) 牛肉空白基質之SQX層析圖;(b) 標準溶液之SQX層析圖 (0.25 ng/g);(c) 過ND lipid 管柱後之SQX層析圖 (0.25 ng/g);(d) 未過ND lipid 管柱之SQX層析圖 (0.25 ng/g)………………………………………………………71
圖三十 不同比例之回溶溶劑對樣品訊號的影響…………………………………73
圖三十一 不同比例之回溶溶劑對樣品波寬的影響………………………………73
圖三十二 QuEChERS 樣品前處理步驟…………………………………………...74
圖三十三 在空白牛肉基質中添加磺胺類藥物與內標準品之層析圖……………81
圖三十四 在空白豬肉基質中添加磺胺類藥物與內標準品之層析圖……………82
圖三十五 (A)真實樣品A-2之層析圖、(B)真實樣品B-4之層析圖、(C)真實樣品C-2之層析圖……………………………………………………………………..86
圖三十六 標準溶液與真實樣品A-2之萃取離子層析圖…………………………88
圖三十七 標準溶液與真實樣品B-4之萃取離子層析圖…………………………89
圖三十八 標準溶液與真實樣品C-2之萃取離子層析圖…………………………90


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