臺灣博碩士論文加值系統

Clenbuterol為擬腎上腺素性β受體致效劑，臨床用於治療馬匹的過敏性氣喘（asthma），然而有人將之作為比賽用途。基於保護動物的立場以及維持馬場競賽的公平性，因此有必要建立一套檢測clenbuterol的分析方法。本實驗計畫乃利用ELISA檢測馬匹血清及尿液中的clenbuterol，希望藉由藥物動力學和代謝情況的觀察，提出一合理的停藥期。一方面供飼主參考，另一方面令非法濫用者有所警惕。
本實驗選用四匹健康馬，連續三天口服投予治療劑量(0.8 mg/kg)的clenbuterol，在最後一次給藥之後開始採集血清和尿液。血清收集間間隔分為10分鐘、15分鐘、20分鐘、30分鐘、1小時、2小時、4小時、6小時、12小時、一天、二天，總計採集時間為七天；尿液收集時間分為停藥後20分鐘、30分鐘、1小時、2小時、4小時、6小時、12小時、一天、二天，採樣為期十四天。應用商品化酵素免疫試藥套組對收集的樣本進行分析，血清試樣並未檢出藥物陽性反應。尿液樣本中clenbuterol濃度在投藥後40分鐘開始上升，於第160分鐘藥物濃度達到最高（72.5 ng/ml），然後很快地降低，第1.5天以後檢測濃度＜2 ng/ml，此一低濃度可持續至第十四天。補助實驗中改以單次投予五倍治療劑量的clenbuterol，血清樣本中藥物方可檢出，給藥後10分鐘便可測得藥物反應，約3.5小時左右達最大濃度（約2.9 ng/ml），之後濃度亦很快降低，24小時後濃度介於（0.5-0.3 ng/ml），約一週後即無法測得。血清和尿液皆同樣觀察到複相（biphasic pattern）排清方式的表現。
利用固相萃取管柱（SPE column,certify）萃取血清或尿液樣本中的clenbuterol，尿液樣本萃取率接近80%；血清試樣由於殘留濃度過低以及血中蛋白質的干擾，萃取後並未檢出藥物反應。運用毛細管電泳分析法針對萃取後的試樣進行確認，發現和ELISA的分析結果差異不大，兩者的相關係數為0.998，故彼此間可以相互驗證檢測結果。
本研究證實利用ELISA可以檢測馬匹口服clenbuterol後，血清和尿中的藥物反應。本研究發展之固相萃取方法比其他文獻發表的萃取法優越，而且發展的毛細管電泳條件很適合於認證尿中的clenbuterol。

The objective of this study was to determine the concentrations of clenbuterol in equine urine and serum after a continuously oral dose of clenbuterol?HCl.
Horses were fed with 0.8mg of clenbuterol/kg BW twice a day (therapeutic dose). Urine and serum were collected separately after the latest treatment. Blood samples were given at 10min, 15min, 20min, 30min, 1h, 2h, 4h, 6h, 12h, 1d, 2d after dosing; urine were collected at 20min, 30min, 1h, 2h, 4h, 6h, 12h, 1d, 2d after dosing. Samples from treated animal were analysed and the clenbuterol concentrations were comparative to those obtained by ELISA and capillary electrophoresis (CE), using solid phase extraction for sample clean-up.
The highest concentration of clenbuterol found in urine was 72.5 ng/ml (at therapeutic dose) and 2.9 ng/kg in serum (5X therapeutic dose). The initial half-life of serum and urine clenbuterol residues in horses were estimated to be 3.5 and 8 h; the slower elimination for serum and urine approximately 24 and 48 h, respectively. A biphasic pattern of clenbuterol elimination is also characteristic for plasma and urine. Recoveries of clenbuterol from fortified 0.1, 1, 10 ng/ml in serum and urine samples carried through the entire procedure were about 80% relative to pure standards.
ELISA was identified in our reserch to detect clenbuterol in equine serum and urine after oral administration. The SPE method developed in our experiment were appropriate to others in higher recovery rate.

目錄
頁碼
中文摘要--------------------------------------------------------------------------Ⅱ
英文摘要--------------------------------------------------------------------------Ⅳ
表次--------------------------------------------------------------------------------Ⅴ
圖次--------------------------------------------------------------------------------Ⅵ
序------------------------------------------------------------------------------------1
第一章 文獻探討
第一節 腎上腺素接受體的分類--------------------------------------------3
第二節 Clenbuterol的基本性質--------------------------------------------4
第三節 酵素連結免疫吸附法（ELISA）--------------------------------25
第四節 毛細管電泳分析（CE）-------------------------------------------29
第五節 固相萃取（SPE）---------------------------------------------------33
第二章 材料與方法
第一節 材料------------------------------------------------------------------39
第二節 方法------------------------------------------------------------------44
第三章 結果--------------------------------------------------------------------53
第四章 討論--------------------------------------------------------------------73
參考文獻--------------------------------------------------------------------------79
表次
頁碼
表一、 Clenbuterol在不同動物的血中半衰期……………………15
表二、 試驗馬匹的基本資料…………………………………………39
表三、 採樣時間表……………………………………………………45
表四、 ELISA套組操作之準確度（accuracy）與精確度（precision）測試………………………………………………………….54
表五、 不同廠牌ELISA kits其檢測敏感範圍之比較…………….55
表六、 ELISA之anti-clenbuterol antibody與其他β受體素（Salbutamol,Terbutaline）之交叉反應………………….55
表七、 ELISA分析檢測馬匹尿液試樣之濃度………………………61
表八、 ELISA分析檢測馬匹血清試樣之濃度………………………62
表九、 馬血清中clenbuterol之萃取回收率………………………71
表十、 馬尿液中clenbuterol之萃取回收率………………………71
表十一、CE與ELISA分析差異性之比較………………………………72
圖次
頁碼
圖 一、 Clenbuterol(左)和Phenethanolamineβ受體作用劑(右)之化學結構…………………………………………………5
圖 二、 Clenbuterol的作用機制……………………………………8
圖 三、 ELISA結合原理示意圖……………………………………28
圖 四、 毛細管電泳的基本組成……………………………………29
圖 五、 電滲流泳動示意……………………………………………31
圖 六、 固相萃取四大步驟…………………………………………34
圖 七、 非極性萃取原理……………………………………………35
圖 八、 極性萃取原理………………………………………………36
圖 九、 離子交換萃取原理…………………………………………36
圖 十、 純血馬體重估算表…………………………………………40
圖十一、 微量滴定盤檢測配置………………………………………48
圖十二、 未加終止液的IDS ELISA kit之標準曲線…………………56
圖十三、 附加終止液的IDS ELISA kit之標準曲線…………………57
圖十四、 國光科技ELISA kit之標準曲線…………………………58
圖十五、 同廠牌ELISA kit添加終止液(紅)與不加終止液(黑)，標準曲線之比較………………………………………….59
圖十六、 不同廠牌ELISA kit之間標準曲線的比較………………59
圖十七、 評估母體組織對試驗套組造成之干擾……………………60
圖十八、 ELISA檢測馬匹尿液樣本clenbuterol濃度………………63
圖十九、 ELISA檢測四匹馬尿液樣本之平均濃度…………………64
圖二十、 ELISA檢測馬匹血清樣本（五倍治療劑量）濃度…………64
圖二十一、注入標準品進行毛細管電泳分析…………………………66
圖二十二、毛細管電泳分析之全光譜圖比對…………………………67
圖二十三、Clenbuterol的CE標準曲線圖……………………………68
圖二十四、試樣萃取後進行毛細管電泳分析，其母體組織（matrix）的背景值………………………………………………….69
圖二十五、利用毛細管電泳進行重複注射試驗（co-injection）……70
圖二十六、ELISA和CE對於血清和尿液試樣檢測的相關性…………72
圖二十七、Clenbuterol高劑量下馬匹呈現之副作用……………….76

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