本論文分為三部分，第一部分為建立高效液相層析串連質譜儀分析魚肌肉組織中大環內酯類與林可胺類抗生素：包括紅黴素 (erythromycin)、竹桃黴素 (oleandomycin)、北里黴素 (kitasamycin)、泰勒黴素 (tylosin)、交沙黴素 (josamycin)、史黴素 (spiramycin)、替米考星 (tilmicosin)及林可黴素 (lincomycin)等八種抗生素殘留之分析方法。均質樣品以乙腈萃取、無水硫酸鈉除水後，濃縮並以甲醇定容，經正己烷脫脂處理淨化後，以LC/MS-MS進行檢測，使用層析管柱為Agilent HC-C18（5μm, 150 mm ×4.6 mm），搭配含0.1％ 甲酸之5mM醋酸銨溶液及乙腈組成之移動相梯度沖提，串聯質譜儀部分使用電灑游離法（ESI＋）配合多重反應模式（MRM）同步檢測，10分鐘內可完成八種大環內酯與林可胺類抗生素的快速分析檢測。
第二部分為依據歐盟2002/657/EC決議書之規定進行方法確效，將藥物設定為允用藥物，依其最大殘留量 (MRL) 規定梯度添加下之回收率介於 93.64%~106.67% ，變異係數 (CV) 介於0.27%~7.17%；設定為禁用藥物時，依其檢測能力最低要求 (MRPL) 規定梯度添加下之回收率介於 96.35%~104.88% ，變異係數 (CV) 介於6.77%~13.91%。八種抗生素Decision limit (CCα) 介於 0.24~ 0.40 μg kg-1；Detection capability (CCβ)為0.33~0.49 μg kg-1。
第三部分為根據國人平常對魚肉之保存及處理方式，包括冷凍 -20℃、冷藏 4 ℃，模擬藥物在魚體內之穩定性，實驗方法為依各藥物之最大殘留量 (MRL) 規定，添加於魚肉基質中，依訂定之時間取出測試抗生素之殘留，測試時間共計四十天，另比較藥物經100 ℃高溫烹煮過後對魚肉內殘留之藥物的影響，結果顯示 erythromycin 、oleandomycin、 kitasamycin與tylosin經100 ℃高溫烹煮過後殘留量會降低到20%以下， spiramycin、josamycin、tilmicosin與 lincomycin則降到35%以下。erythromycin、josamycin、tylosin與 lincomycin若儲存在 -20 ℃條件下，藥物會穩定的存在肉品裡，未經烹煮的魚肉中若殘留這類抗生素，可能會對人體造成影響。

The main research of this thesis includes three sections. The purpose of first part is to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of 8 macrolide antibiotics and lincosamides inside fish tissue, including erythromycin (ERM), oleandomycin (OLD), kitasamycin (KIT), tylosin (TYL), josamycin (JOS), spiramycin (SPM), tilmicosin (TIL), and lincomycin (LIN). Homogenized samples are first extracted with acetonitrile, dehydrated with sodium sulphate anhydrous, and then condensed. After the residue was redissolved in methanol and the extracts were partitioned with n-hexane to remove lipids, the sample is filterced and detected by LC/MS-MS using chromatography columns of Agilent HC-C18 (5μm, 150 mm ×4.6 mm). The mobile phase A was 5mM ammonium acetate containing 0.1% formic acid, while the mobile phase B was acetonitrile. The analysis of 8 macrolide antibiotics and lincosamides can be achieved within 10 minutes with electrospray ionization-tandem mass spectrometry in positive mode using multiple reaction monitoring (MRM) for simultaneous detection.

The second part is to verify the method by regulation of European Union (EU) resolution scheme (2002/657/EC). In the case where the drug is set as allowed drug, the recovery rate under gradient addition according to MRL is between 93.64% to 106.67%, and the CV is between 0.27% to 7.17%. In the case where the drug is set as prohibited drug, the recovery rate under gradient addition according to MRPL is between 96.35%~104.88%, and the CV is between 6.77%~13.91%. As a result, the decision limit (CCα) and the Detection capability (CCβ) of the 8 macrolide antibiotics and lincosamides is between 0.24 to 0.40μg kg-1 and 0.33 to 0.49μg kg-1.

The last section is to evaluate the stability of drugs in fish body under domestic preservation and process methods on fish, including refrigeration at -20℃ and cold storage at 4 ℃. The test is implemented by adding the drug into fish tissue according to MRL and detecting the antibiotics residue after regulated 40 days. Besides, the effect on activity of drug residue in fish body after boiling at 100 ℃ is compared. The results show that the residual amount of spiramycin, josamycin, tilmicosin, and lincomycin is below 35% while that of erythromycin, oleandomycin, kitasamycin, and tylosin will be below 20%. Therefore, the drugs including erythromycin, josamycin, tylosin, and lincomycin will stay stably in fish tissue if they are stored under -20 ℃. However, it may affect human health if the fish contains such antibiotic residues is not boiled.

摘要 ..................................................................................................................................... I
Abstract .............................................................................................................................. III
目錄 .................................................................................................................................... V
表目錄 .............................................................................................................................. VIII
圖目錄 ............................................................................................................................... IX
中英文縮寫對照表 ............................................................................................................ XI
第一章 緒論 ....................................................................................................................... 1
1-1 前言 ......................................................................................................................... 1
1-2 巨環內酯類抗生素簡介 .......................................................................................... 3
1-3 林可胺類抗生素簡介 ............................................................................................. 4
1-4 藥物殘留檢查法規 .................................................................................................. 5
1-5 抗生素殘留的分析方法 .......................................................................................... 6
1-5-1 樣品前處理萃取淨化方法 .................................................................................. 6
1-5-1-1 均質震盪萃取 ................................................................................................... 7
1-5-1-2 固相萃取 ........................................................................................................... 8
1-5-1-3 加壓溶劑萃取 ................................................................................................... 9
1-5-2 動物用藥檢驗方法之探討 ................................................................................ 10
1-5-2-1 薄層層析法 .................................................................................................... 10
1-5-2-2 高效液相層析法 ............................................................................................ 11
1-5-2-3 液相層析-質譜聯用法 .................................................................................... 11
第二章 研究目的 ............................................................................................................. 14
2-1 研究動機 ............................................................................................................... 14
2-1 研究架構 ............................................................................................................... 15
第三章 材料與方法 ......................................................................................................... 18
3-1 藥品與試劑 ........................................................................................................... 18
3-1-1 抗生素標準品: ................................................................................................... 18
3-1-2 實驗用藥劑 ........................................................................................................ 19
3-1-3 藥品配製 ........................................................................................................... 19
3-2 儀器與設備 ........................................................................................................... 22
3-3 分析方法 ............................................................................................................... 23
3-3-1 實驗用空白樣品 ............................................................................................... 23
3-3-2 質譜測定條件優化 ........................................................................................... 24
3-3-2-1 樣品回溶液 .................................................................................................... 24
3-3-2-2 質譜分析參數 ................................................................................................ 24
3-3-3 層析分離條件 ................................................................................................... 25
3-3-4 萃取條件 ........................................................................................................... 26
3-3-4-1 萃取溶劑的選取 ............................................................................................ 26
3-3-4-2 萃取溶劑量對萃取率的影響 ........................................................................ 27
3-3-4-3 酸鹼條件對萃取率的影響 ............................................................................ 27
3-3-4-4 乾燥溫度對萃取率的影響 ............................................................................ 27
3-3-4-5 去脂溶劑量對萃取率的影響 ......................................................................... 28
3-4 分析方法確效 ....................................................................................................... 28
3-4-1 樣品前處理 ....................................................................................................... 29
3-4-2 大環內酯類與林可胺類抗生素之離子對 ....................................................... 29
3-4-3 層析滯留時間 ................................................................................................... 30
3-4-4 離子比 ............................................................................................................... 30
3-4-5 檢量線範圍與線性 ........................................................................................... 31
3-4-6 真值 ................................................................................................................... 32
3-4-7 方法精密度的測定 ............................................................................................ 33
3-4-8 基質干擾 ........................................................................................................... 33
3-4-9 穩定性 ............................................................................................................... 34
3-4-10 CCα 與CCβ ...................................................................................................... 35
3-5 模擬不同的儲存方式對抗生素在肉類之穩定性影響 ....................................... 38
第四章 結果與討論 ......................................................................................................... 39
4-1 質譜測定條件優化 ............................................................................................... 39
4-1-1 樣品溶劑的選擇 ............................................................................................... 39
4-1-2 分析參數之選擇 ................................................................................................ 39
4-2 層析分離條件優化 ............................................................................................... 40
4-3 萃取條件的優化 ................................................................................................... 41
4-3-1 萃取溶劑的選取 ............................................................................................... 41
4-3-2 不同溶劑量條件對萃取率的影響 ................................................................... 42
4-3-3 不同酸鹼條件對萃取率的影響 ....................................................................... 43
4-3-4 乾燥溫度對萃取率的影響 ............................................................................... 43
4-3-5 去脂溶劑量對萃取率的影響 ............................................................................ 44
4-4 分析方法確效 ....................................................................................................... 44
4-4-1 大環內酯類與林可胺類抗生素之離子對 ....................................................... 44
4-4-2 層析滯留時間 ................................................................................................... 45
4-4-3 離子比 ............................................................................................................... 45
4-4-4 檢量線 ............................................................................................................... 46
4-4-5 真值 ................................................................................................................... 47
4-4-6 方法精密度 ....................................................................................................... 48
4-4-7 基質干擾 ........................................................................................................... 49
4-4-8 穩定性 ............................................................................................................... 50
4-4-9 CCα 與CCβ ........................................................................................................ 51
4-5 不同的儲存方式對抗生素在肉類之穩定性影響 ............................................... 52
第五章 總結 ..................................................................................................................... 54
參考文獻 ........................................................................................................................... 57
附錄 ................................................................................................................................ 117

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