Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/14192
標題: 馬匹血清和尿液中clenbuterol之檢測
Detection of clenbuterol in equine serum and urine
作者: 莊孟修
Chuang, Meng Shiu
關鍵字: lipolysis;脂肪分解;muscle anabolism;equine;horse;serum and urine;pharmacodynamics;residues of clenbuterol;ELISA;CE;肌肉蛋白質合成;馬匹;血清和尿液;藥物動力學;clenbuterol殘留;酵素免疫結合分析法;毛細管電泳分析法
出版社: 獸醫學系
摘要: 
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.
URI: http://hdl.handle.net/11455/14192
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