臺灣博碩士論文加值系統

本研究之目的在於利用快速簡便的逆相高效液相層析法分析豬肉、豬肝及牛肉中clenbuterol殘留量及最低偵測極限之探討。檢體經0.4 N過氯酸均質萃取，上清液經乙醚淨化後濃縮，再以0.2 M硫酸定容至0.2 mL，最後利用高效液相層析儀分析定量。使用之層析管柱為Waters 5C18-MS (2.0×150 mm)(Cosmosil)微量管柱，分析管柱溫度40℃，移動相為0.05 M NaH2PO4（pH3.0）/acetonitrile（80/ 20，v/v）及紫外光檢出器波長212 nm之條件下檢測，約於6分鐘時偵測出clenbuterol之波峰。經以0.0002~0.001 μg/mL之clenbuterol標準溶液所製作標準曲線之線性迴歸係數為0.9999，顯示呈良好線性關係。分別添加0.0005、0.001、0.002、0.005及0.01 ppm之clenbuterol於豬肉、豬肝及牛肉中進行回收試驗，其回收率結果分別為80.9~88.8﹪、80.3~86.9﹪及 80.6~90.6﹪，變異係數則介於0.7~6.3﹪之間。本方法在此三種檢體中之最低檢出現量可達0.0001 ppm，此結果均能達到衛生署所規定在牛之肝、腎中0.0006 ppm及牛肉中 0.0002 ppm容許量之檢測。利用此檢驗方法分析市售豬肉、豬肝及牛肉clenbuterol之殘留量，檢體各20件，共計60件，其檢驗結果發現2件檢體有殘留現象，但並未超過衛生署於2001年所規定之「動物用藥殘標準」中容許此藥劑之殘留量。

A simple and rapid method with reverse phase high performance liquid chromatographic (HPLC) to determine clenbuterol in muscle and liver of swine and cattle. After homogenizing the tissue with 0.4N perchloric acid, the supernatant was extracted with diethyl ether, followed by back-extraction in 0.2M sulphuric acid, and finally determined by HPLC. The separation was performed with a Waters 5C18-MS (2.0×150 mm)(Cosmosil) column on 0.05M NaH2PO4 (pH3.0):acetonitrile (80:20, v/v) as a mobile phase, and clenbuterol was detected by absorbance at 212nm. The recovery of this method was studied spiking 0.0005, 0.001, 0.002, 0.005 and 0.01 ppm levels in swine muscle, swine liver and bovine muscle and the results showed recoveries at the ranges of 80.9~88.8﹪, 80.3~86.9﹪ and 80.6~90.6﹪，respectively. The coefficients variation of average recoveries was between 0.7and 6.3﹪. The limitation of detection by this method was 0.0001 ppm in these three commodities，which meets the requirement for detecting clenbuterol residues in these commodities at the officials allowed levels. Complying with this method, 60 samples were purchased at markets. The result showed two samples were detected but non of them were violated the regulation.

目錄
中文摘要 I
英文摘要 II
壹、前言 1
貳、文獻回顧...... 3
ㄧ、clenbuterol之基本特性.. 3
二、腎上腺素受體之分類..... 4
三、clenbuterol之作用...... 4
(ㄧ) clenbuterol於心臟之作用........ 4
(二) clenbuterol於肺部之作用........ 5
(三) clenbuterol於肌肉之作用........ 5
(四) clenbuterol於肝臟之作用........ 7
(五) clenbuterol於子宮之作用........ 7
(六) clenbuterol於脂肪之作用........ 8
(七) clenbuterol於氣管之作用........ 9
(八)其他. 9
四、clenbuterol藥物殘留及安全性評估. 10
五、clenbuterol之毒性與副作用....... 12
六、clenbuterol之分析方法.. 13
(ㄧ)酵素免疫分析法 13
(二)毛細管電泳分析法....... 14
(三)氣相層析質譜分析法..... 18
(四)液相層析串聯質譜分析法 21
(五)高效液相層析分析法..... 24
七、萃取方法...... 27
(ㄧ)固相萃取法.... 27
(二)液-液相萃取法 28
參、材料與方法.... 32
ㄧ、實驗材料...... 32
(ㄧ)標準品........ 32
(二)檢體來源...... 32
(三)儀器設備...... 32
(四)試藥及試劑.... 33
二、實驗方法...... 35
(ㄧ)評估檢驗方法.. 35
(二)進行市售調查.. 35
(三)最低偵測極限之探討.............36
(四)資料分析.......................36
三、實驗流程.......................36
(ㄧ)標準溶液之調製製...............36
(二)衍生化反應.....................36
(三)高效液相層析用移動相溶液之調製..37
(四)檢液之調製.....................38
(五)定量操作.......................38
(六)標準曲線之製作.................39
(七)鑑別試驗及含量測定.............39
(八)回收試驗之製作.................40
(九)最低檢出限量試驗...............40
肆、結果與討論.....................41
伍、動物用藥clenbuterol於豬肉、豬肝及牛肉中檢出 殘留量檢驗之量測不確定度評估.......................69
陸、結論...........................81
柒、參考文獻.......................83
圖目錄
圖1 clenbuterol之結構...... 3
圖2毛細管電泳系統之基本構造 17
圖3電滲流泳動圖.............17
圖4氣相層析儀之構造........ 20
圖5層析質譜法之應用........ 20
圖6串聯式層析質譜儀之構造.. 23
圖7 HPLC儀之構造........... 26
圖8 HPLC方法之應用 .........26
圖9在酸性、中性及鹼性環境下clenbuterol之離子狀態...... 31
圖10 clenbuterol經衍生化反應之結構.....................42
圖11 clenbuterol衍生物(m/z 86,262)之氣相層析質譜層析圖及其最低偵測極限圖譜........ 43
圖12 clenbuterol標準品之液相層析串聯式質譜層析圖及其最低偵測極限圖譜.............. 45
圖13未添加三乙胺之移動相溶液其紫外光吸收圖譜.......... 49
圖14添加三乙胺之移動相溶液其紫外光吸收圖譜............ 50
圖15 clenbuterol溶於0.05M NaH2PO4 (pH3.0):acetonitrile (80:20，v/v)溶液之紫外光吸收圖譜.............................. 51
圖16 clenbuterol之標準曲線圖.......................... 56
圖17檢液製備之流程圖.................................. 57
圖18 豬肉中clenbuterol最低檢出限量之高效液相層析圖譜.. 58
圖19豬肝中clenbuterol最低檢出限量之高效液相層析圖譜... 59
圖20牛肉中clenbuterol最低檢出限量之高效液相層析圖譜... 60
圖21豬肉中clenbuterol之高效液相層析回收圖譜........... 61
圖22豬肝中clenbuterol之高效液相層析回收圖譜........... 62
圖23牛肉中clenbuterol之高效液相層析回收圖譜........... 63
圖24 化學分析不確定度來源要因圖....................... 72
表目錄
表1 正相及逆相層析比較..... ...........................25
表2 豬肉、豬肝及牛肉中添加clenbuterol之回收率.........64
表3 本研究之抽樣記錄表..... ...........................65
表4 5克砝碼重複稱重試驗數據..........................73
表5 1 mL pittet重複試驗數據........ ..................75
表6 含0.001 ppm clenbuterol之豬肉重複試驗數據......... 76
表7 含0.001 ppm clenbuterol之豬肝重複試驗數據......... 76
表8 含0.001 ppm clenbuterol之牛肉重複試驗數據......... 77
表9 校正後之數據.......................................77
表10 clenbuerol量測不確定度分析之數據..................79

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