本研究之目的為二，一是建立以高效液相層析串聯式質譜儀檢測魚肉中安比西林抗生素殘留之方法，且方法需經過方法確效，符合歐盟標準；二為吳郭魚體內安比西林的代謝及殘留實驗。
萃取方法為分散固相萃取，將組織樣品均質後，使用甲醇:水(4:1)萃取，並加入C18吸附劑去除油脂與雜質，將萃液經38℃吹氮濃縮及水回溶，經過濾處理後，以LC/MS/MS偵測。液相層析系統為添加0.1%甲酸之10mM醋酸銨溶液與甲醇作為移動相梯度沖提，層析管柱為Agilient HC-C18(5μm, 150mm ×4.6mm)，串聯式質譜儀部分使用電灑游離法(ESI+)配和多重反應模式(MRM)進行檢測。各組織回收率均為81~86%，方法偵測極限為6.00 μg kg-1，ampicillin在魚肉的CCα為63.65 ± 7.99 μg kg-1。
在藥物殘留實驗方面，本研究使用灌食給藥於吳郭魚，口服劑量20 mg/kg．BW，血清、肝臟及肌肉均在0.5小時達到藥物最高濃度，達最高濃度分別為27.53 mg L-1、1.33 mg kg-1、66.75 mg kg-1，最高濃度為肝臟，血清次之，肌肉最低；給藥後24小時，肌肉中ampicillin濃度為0.04 mg kg-1，低於歐盟法規標準(MRL) 0.05 mg kg-1；在給藥後48小時，血清、肌肉、肝臟均無ampicillin殘留情形。本研究經給藥後72小時(3天)，檢測血清、肌肉、肝臟等組織，已無ampicillin殘留，符合台灣水產動物用藥法規規範，所規定的停藥期5天。

In this study, a LC/MS/MS method for the determination of ampicillin antibiotic in fish muscle tissue was developed and accredited according to Commission Decision 2002/657/EC. The metabolism of ampicillin in tilapia was them studied in serum, liver and muscle.
The homogenized fish tissue was first extracted with MeOH-H2O(4:1), C18 sorbent was added to remove lipids and impurities, the extract was then evaporated to dryness with a steam of nitrogen gas at 38 °C. The residue was redissolved with H2O, filtered and analyzed by LC/MS/MS equipped with an Agilient HC-C18(5μm, 150mm ×4.6mm), the mobile phase A was 10mM ammonium acetate containing 0.1% formic acid, while the mobile phase B was methanol. The determination of ampicillin was performed with electrospray ionization-tandem mass spectrometry in positive mode using multiple reation monitoring(MRM) for detection. Average recoveries were 81–86%, the limit of detection was 6.00 μg kg-1，decision limit(CCα) of ampicillin in fish muscle sample was 63.65 ± 7.99 μg kg-1.
In the metabolism study, the oral administered dose to talipia was 20 mg/kg．BW. The maximum concentration of ampicillin in each tissues was obserned at 0.5 hour after oral administration, the maximum concentration in serum, liver and muscle was 27.53 mg L-1, 66.75 mg kg-1 and 1.33 mg kg-1, respectively. The concentration of ampicillin in muscle was 0.04 mg kg-1 24 hours after oral administration, which is lower than the 0.05 mg kg-1 MRL value of European Union resolutions. No residual ampicillin was detected in tilapia 48 hours after oral treatment, which conformed to the drug regulations for aquaculture ainmals in Taiwan.

論文審定書 ........................................................ i
誌謝 ............................................................. iii
摘要 ............................................................. iv
Abstract ........................................................... v
目 錄 ............................................................ vii
圖 次 ............................................................ xii
表 次 ............................................................ xiv
第 一 章 緒 論 ................................................. - 1 -
1-1 前言 ....................................................... - 1 -
1-2 Ampicillin 介紹 ............................................. - 2 -
1-3 吳郭魚之簡介 ............................................... - 3 -
1-4 水產養殖生物藥物處理方法 ................................... - 5 -
1-4-1 血管注射法 ............................................... - 5 -
1-4-2 口服法 ................................................... - 5 -
1-4-2-1 投餵法 ................................................. - 6 -
1-4-2-2 灌食法 ................................................. - 6 -
1-4-3 藥浴法 ................................................... - 6 -
1-4-4 肌肉注射法 ............................................... - 6 -
1-5 抗生素藥物殘留之分析方法 ................................... - 7 -
1-5-1 萃取方法 ................................................. - 7 -
1-5-1-1 固相萃取 ............................................... - 7 -
1-5-1-2 分散固相萃取 ........................................... - 8 -
1-5-2 抗生素藥物之檢驗方法 ..................................... - 9 -
1-5-2-1 薄層層析法 ............................................. - 9 -
1-5-2-2 酵素結合免疫吸附法 .................................... - 10 -
1-5-2-3 高效液相層析法 ........................................ - 10 -
1-5-2-4 液相層析-質譜聯用法 ................................... - 11 -
1-6 研究目的 .................................................. - 13 -
1-6-1 研究動機 ................................................ - 13 -
1-6-2 研究架構 ................................................ - 13 -
第 二 章 材 料 與 方 法 ....................................... - 15 -
2-1 藥品與試劑 ................................................ - 15 -
2-1-1 藥品 .................................................... - 15 -
2-1-2 溶劑 .................................................... - 15 -
2-1-3 氣體 .................................................... - 16 -
2-1-4 試劑水 .................................................. - 16 -
2-2 試劑配製 .................................................. - 16 -
2-2-1 標準品溶液配製 .......................................... - 16 -
2-2-2 內部標準品溶液配製 ...................................... - 17 -
2-2-3 其他溶液配製 ............................................ - 17 -
2-3 儀器設備與器材 ............................................ - 18 -
2-3-1 儀器設備 ................................................ - 18 -
2-3-2 器材 .................................................... - 18 -
2-4 樣品前處理方法 ............................................ - 19 -
2-4-1 前處理條件 .............................................. - 19 -
2-4-1-1 萃取溶劑的選擇 ........................................ - 19 -
2-4-1-2 萃取溶劑量對回收率之影響 .............................. - 19 -
2-4-1-3 加酸萃取對回收率之影響 ................................ - 20 -
2-4-1-4 萃取次數對萃取回收率之影響 ............................ - 20 -
2-4-1-5 乾燥溫度對萃取回收率之影響 ............................ - 20 -
2-4-1-6 回溶液對萃取回收率之影響 .............................. - 20 -
2-4-1-7 去脂溶劑量對萃取回收率之影響 .......................... - 20 -
2-4-1-8 液相萃取方法 .......................................... - 21 -
2-4-1-9 分散固相萃取法 ........................................ - 21 -
2-4-2 LC/MS/MS 分析方法 ...................................... - 21 -
2-5 方法確效 .................................................. - 22 -
2-5-1 滯留時間 ................................................ - 22 -
2-5-2 離子比 .................................................. - 22 -
2-5-3 檢量線範圍與配製 ........................................ - 23 -
2-5-3-1 溶劑配製 .............................................. - 23 -
2-5-3-2 基質添加 .............................................. - 23 -
2-5-4 基質干擾 ................................................ - 23 -
2-5-4-1 標準檢量線(SCC) ....................................... - 23 -
2-5-4-2 空白基質萃取前添加之檢量線(MMCC) .................... - 23 -
2-5-4-3 空白基質萃取後添加之檢量線(MCC) ...................... - 24 -
2-5-4-4 基質效應與絕對回收率之計算公式 ........................ - 24 -
2-5-5 真值 .................................................... - 24 -
2-5-6 方法精密度的測定 ........................................ - 25 -
2-5-7 方法偵測極限與偵測極限(LOD)、定量極限(LOQ) ............ - 25 -
2-5-7-1 方法偵測極限 .......................................... - 25 -
2-5-7-2 偵測極限(limit of detection, LOD) ......................... - 25 -
2-5-7-3 定量極限(limit of quantitation, LOQ) ....................... - 26 -
2-5-8 CCα、CCβ ............................................... - 26 -
2-5-8-1 判定界限(CCα) ......................................... - 26 -
2-5-8-2 最低可測得之濃度(CCβ) ................................. - 26 -
2-6 Ampicillin 在吳郭魚體內代謝殘留研究......................... - 28 -
2-6-1 實驗用魚 ................................................ - 28 -
2-6-2 選用飼料 ................................................ - 28 -
2-6-3 給藥方式 ................................................ - 28 -
2-6-4 取樣 .................................................... - 29 -
2-6-5 樣品萃取處理 ............................................ - 30 -
2-6-5-1 肌肉樣品 .............................................. - 30 -
2-6-5-2 血液樣品 .............................................. - 30 -
2-6-5-3 肝臟樣品 .............................................. - 30 -
第 三 章 結 果 與 討 論 ....................................... - 31 -
3-1 層析與質譜條件 ............................................ - 31 -
3-1-1 質譜分析參數的選擇 ...................................... - 31 -
3-1-2 層析條件選擇與設定 ...................................... - 36 -
3-2 萃取條件最佳化 ............................................ - 39 -
3-2-1 萃取溶劑選擇 ............................................ - 39 -
3-2-2 萃取溶劑量對回收率之影響 ................................ - 41 -
3-2-3 加酸萃取對回收率之影響 .................................. - 42 -
3-2-4 不同的萃取次數對回收率之影響 ............................ - 42 -
3-2-5 乾燥溫度對萃取回收率之影響 .............................. - 43 -
3-2-6 回溶液對萃取回收率之影響 ................................ - 44 -
3-2-7 去脂溶劑量對萃取回收率之影響 ............................ - 45 -
3-2-8 液相萃取與分散固相萃取之回收率比較 ...................... - 46 -
3-3 分析方法確效 .............................................. - 49 -
3-3-1 離子比 .................................................. - 49 -
3-3-2 層析滯留時間 ............................................ - 50 -
3-3-3 檢量線 .................................................. - 50 -
3-3-4 真值 .................................................... - 51 -
3-3-5 方法精密度 .............................................. - 51 -
3-3-6 基質干擾 ................................................ - 52 -
3-3-7 方法偵測極限 ............................................ - 54 -
3-3-8 CCα、CCβ .............................................. - 55 -
3-4 Ampicillin 在吳郭魚體內代謝殘留 ............................ - 56 -
3-4-1 Ampicillin 在吳郭魚血液中的濃度變化情形 .................. - 56 -
3-4-2 Ampicillin 在吳郭魚體內肝臟中的濃度變化情形 ............... - 57 -
3-4-3 Ampicillin 在吳郭魚體內肌肉中的濃度變化情形 ............... - 58 -
第 四 章 結 論 ................................................ - 61 -
參考文獻 ...................................................... - 62 -
附錄 .......................................................... - 67 -

圖　次
圖1-1.Ampicillin之結構…………………………………………………………….. 2
圖3-1.Ampicillin標準品之母離子(Q1)質譜圖……………………………………. 32
圖3-2.Ampicillin標準品之子離子(Q3)質譜圖……………………………………. 32
圖3-3.Ampicillin N-15內部標準品之母離子(Q1)質譜圖………………………... 33
圖3-4.Ampicillin N-15內部標準品之子離子(Q3)質譜圖………………………... 33
圖3-5.Ampicillin標準品之DP圖………………………………………………….. 34
圖3-6.Ampicillin標準品之EP圖………………………………………………….. 34
圖3-7.Ampicillin標準品之CE圖…………………………………………………. 35
圖3-8.Ampicillin標準品之CXP圖……………………………………………….. 35
圖3-9.Ampicillin標準品之子離子350.2/105.8質譜層析圖……………………... 37
圖3-10.Ampicillin標準品之子離子350.2/160.0質譜層析圖……………………. 37
圖3-11. Ampicillin N-15內部標準品之子離子351.3/106.9質譜層析圖………… 38
圖3-12. Ampicillin N-15內部標準品之子離子351.3/159.5質譜層析圖………… 38
圖3-13.層析之移動相-不同有機相之比較………………………………………... 39
圖3-14.不同萃取溶劑對回收率之影響…………………………………………… 40
圖3-15.萃取溶劑量對回收率之影響……………………………………………… 41
圖3-16.加酸萃取對回收率之影響………………………………………………… 42
圖3-17.萃取次數對回收率之影響………………………………………………… 43
圖3-18.氮氣吹拂乾燥溫度對回收率之影響……………………………………… 44
圖3-19.回溶液對回收率之影響…………………………………………………… 45
圖3-20.去脂溶劑n-Hexane使用量對回收率之影響……………………………… 46
圖3-21.液相萃取與分散固相萃取之回收率比較……………………………….... 47
圖3-22. Ampicillin標準品之檢量線………………………………………………. 50
圖3-23.液相萃取法- ampicillin於吳郭魚之基質效應SCC、MCC、MMCC檢量線……………………………………………………………………………………. 53
圖3-24.分散固相萃取法- ampicillin於吳郭魚之基質效應SCC、MCC、MMCC檢量線…………………………………………………………………………………. 54
圖3-25.Ampicillin在吳郭魚體內血清之代謝殘留………………………………. 57
圖3-26.Ampicillin在吳郭魚體內肝臟之代謝殘留………………………………. 58
圖3-27.Ampicillin在吳郭魚體內肌肉之代謝殘留………………………………. 59


表 次
表1-1. Ampicillin抗生素歐盟及台灣最大殘留容許量（MRL）…………………. 14
表2-1.質譜技術中相對離子強度的最大允許限度範圍…………………………. 22
表2-2.待測物濃度範圍相對應之最大可容許真值之誤差範圍…………………. 24
表2-3.定量方法的變異係數(CV)元素品質分數的範圍…………………………. 25
表2-4.Ampicillin使用規範………………………………………………………... 29
表3-1. Ampicillin與內部標準品之層析質譜條件……………………………….. 31
表3-2.移動相條件………………………………..………………………………... 36
表3-3液萃法與分散固萃法之優缺點比較………………………………………. 47
表3-4. Ampicillin相關離子強度比……………………………………………….. 49
表3-5. Ampicillin之檢量線相關係數…………………………………………….. 51
表3-6. Ampicillin在允用條件下之真值及精密度……………………………….. 52
表3-7. Ampicillin於吳郭魚魚肉之基質效應與絕對回收率…………………….. 53
表3-8. Ampicillin在魚肉之方法偵測極限、偵測極限與定量極限…………….. 55
表3-9. Ampicillin在允用條件下的CCα…………………………………………. 55

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