建立氟甲磺氯黴素在動物之殘留檢測方法

Abstract

台灣在加入世界貿易組織(WTO)後，禽畜水產品的進出口量明顯增加，而國際上對禽畜水產品的品質要求提高，更加重視肉品衛生安全，因此藥物殘留檢測更是不容忽視且相當重要的一環。氟甲磺氯黴素(florfenicol，FF)是屬於氯黴素類藥物，化學構造類似氯黴素(chloramphenicol，CAP)，其具有良好生物利用率，組織分佈率與抗菌範圍亦比氯黴素廣；目前普遍應用在禽類、豬及牛的疾病治療上，也被各國列為藥物殘留檢驗藥品之一。 本研究已設計開發HPLC與ELISA二套快速檢驗氟甲磺氯黴素(florfenicol)之標準流程及檢測套組，HPLC分析方式建立FF藥物殘留組織萃取方法及分析資料，組織中藥物濃度檢測敏感度為100 ng/mg，香魚肌肉回收率為81%。另一方面利用化學合成技術設計FF的半抗原、抗原、免疫原與FF酵素結合體(enzyme conjugate)，自行製備多株抗體，抗體敏感度為0.3 ng/mL；經過穩定性測試後成功建立competitive ELISA kit 雛型套組。並比對其他類抗生素與FF ELISA kit 的交叉反應度結果顯示CAP、TAP的交叉反應為9.54 %與9.53 %，與其他類抗生素交叉度皆小於1 %。利用研發套組，已完成市售吳郭魚組織藥物殘留檢測與石斑魚殘留期試驗；在市售吳郭魚檢測方面並未檢測到FF的殘留(檢測敏感度為0.3 ng/mg，回收率97 %)；10 mg/kg一次口服投與石斑魚殘留期試驗方面，停藥後48小時肌肉組織殘留濃度為379 ng/mg；其他停藥時間點(60小時、72小時、84小時、96小時)之肌肉組織殘留濃度分別為49 ng/mg、42 ng/mg、4.22 ng/mg與7.58 ng/mg (最低檢測敏感度為0.3 ng/mg，魚肉樣品回收率為97 %)。本研究所建立的兩套檢測模式可供未來藥物檢測單位之應用。

Florfenicol (FF), a derivative of chloramphenicol, is a broad spectrum antibiotic approved for use in livestock, poultry, and aquaculture. The drug is approved for use in animal for the treatment of bacterial infection, but the residues of FF in consumable meat pose potential risks for public health because of the increasing incidence of microbial resistance. We had developed ELISA and HPLC methods to examine the FF residue in animals, especially in ayu, tilapia, chicken, and pork. At HPLC, the LOD was 0.1 ug/mg, and the analytical average retrieve of FF in ayu’s muscle was 81%. When FF was giving to ayu in measuring medicine of hurl a time with 10, 20 and 50 mg/kg then the concentration distribution in muscle detected. The withdrawal times were 72, 96 and 96 hours, respectively. In order to raise the polyclonal antibody of FF for used in ELISA testing, we synthesize the hapten, antigen and immunogen of FF. The chemical structure of the synthetic hapten was confirmed by Nuclear Magnetic Resonance (NMR) (Brucker 500 MHz). The hapten-HRP conjugate was synthesized with the suitable hapten/HRP ratio. The cross-reactivity of the other analogue is determined by the florfenicol ELISA. The limit of detection (LOD) was 0.3 ng/mL. Cross reactivity with thiamphenicol, chloramphenicol, amoxicillin, penicillin G, chlortetracycline, tetracycline, gentamicin and streptomycin were 9.53%, 9.54%, <1%, <1%, <1%, <1%, <1%, and <1%, respectively. And that, the kit were applicable on the FF residual examination of fish. In conclusion, we have developed HPLC method and the sensitive florfenicol ELISA set for which FF could efficiently be detected.