| Summary: | 本實驗之目的在於嘗試以高效液相層析法開發出一種簡單,快速,精準 又可連續同步檢測鰻魚肌肉中第一代 quinolone 類抗菌劑(flumequine, nalidixic acid 及 oxolinic acid)與第二代 quinolone 類抗菌劑( enrofloxacin 及 sarafloxacin)之方法.同時依所檢測的方法探討單一劑 量 sarafloxacin 歐洲鰻(Anguilla anguilla)體內之藥物動力學. 本 試驗結果顯示以裝有保護分離管之 LiChrospher RP-18 分離管, 配合流 動相 0.001M oxalic acid-acetonitrile-methanol- tetrahydrofuran(7.5:1.5:0,5:0.5,pH=3.0),以260 nm 波長之紫外線檢 測器可精確地同時分析上述五種 quinolone 類抗菌劑.自含有上述五種 quinolone 類之抗菌劑之肌肉(1ug/g),經去離子水與 acetonitrile(1:4) 萃取後,回收率可達 68.3-84.9%,最低檢測濃度達 0.05-0.5 ug/g. 以所 建立之檢測方法檢測一個樣品所需之時間平均可於40分鐘內可完成.鰻魚 各組織(肝臟,腎臟,肌肉及皮膚)經上述萃取方法處理後,將所得之水層萃 取液及血漿分別加入於C2固相萃取管中,並以methanol-0.01M oxalic acid(7:3)作為沖洗液,將留存在固相萃取管中的 sarafloxacin 洗出,再 注入層析儀以278 nm的波長測定.而以各組織及血漿(1ug/g)5之回收率及 最低檢測濃度.實驗結果顯示最低檢測濃度均為 0.07 ug/g. 本實驗令 歐洲鰻口服單一劑量 15mg/kg b.w.之 sarafloxacin 以研究 sarafloxacin在魚體內之藥物動力學.血漿,肝臟,腎臟,肌肉及皮膚濃度對 時間之曲線下面積(AUC),分別為53.64,358.91,177.2,38.22及 35.44 ug. h/g(ml).血漿,肝臟,腎臟,肌肉及皮膚達到最高濃度時間之平均濃度( Cmax),分別為 2.64,13.39,5.53,1.82及 0.78 ug/g(ml). 試驗結果顯 示血漿於 28 小時,而肝臟及腎臟於 32 小時均可將 sarafloxacin 自魚 體內排除,並呈現快速及緩慢排除之雙相排除模式. 在分別自血漿( 第12-32小時),肝臟及腎臟 (第12-28小時)做直線求得快速期之分佈速率 常數(a),分別為 0.085,0.12,及 0.071 h/l,並且血漿,肝臟及腎臟之分佈 速率半衰期(t1/2a)分別為 8.15,5.78及 9.76小時.而自血漿(第32-96小 時),肝臟,腎臟(第40-240小時)做直線求得緩慢期之直線排除速率常數(B) 分別為0.023,0.007,0.012,0.039及 0.016 h/l,並且排除速率半衰期( t1/2B)分別為 30.13,99,57.75,17.77及 43.3小時. 依本試驗結果顯 示血漿,肝臟,腎臟,肌肉及皮膚之檢測不出時間分別為 7,14,14,7及 14天,因此建議停藥期為 16 天. The aim of this study is to develop a simple, rapid, and reliable high per-formance liquid chromatography (HPLC) method for the simultaneous determinationof the first generation of quinolone (flumequine, nalidixic acid, and oxolinic acid) and the second generation of quinolones (enrofloxacin and sarafloxacin) in eel's meat. Based on the developed method, the single dose pharmacokineticsof sarafloxacin in eel (Anguilla anguilla) was studied. The simultaneous determination of five above quinolones in eel's meat was studied by a HPLC method. Quinolones were extracted with distilled water-acetonitrile (1:4). After homogenization of fish tissues, the fat separated byextraction into organic solvents and the aqueous phase was filtered than 20 ulaliquot of the filtrate were used for HPLC analysis. The separations were per-formed on LiChrospher RP-18 columns (150 * 4.6 mm I. D.) and guard columns with 0.01 M oxalic acid solution-acetonitrile-methanol-tetrahydrofuran (7.5:1.5:0.5:0.5, pH=3.0) as the mobile phase. A UV-VIS detector was used at a 260nm.The calibration graphs were linear from 1 ng to 200 ng for tested quinolones.The recoveries and detection limitation of the drug from tissues fortified at alevel of 1ug/g were 68.3%-84.9%and 0.07-0.5ug/g,respectively.The detection time of per sample was less than 40 min. On the determination of sarafloxacin in eel's tissues(liver,kindey,muscle,and skin)and plasms were detected in this investigation.The residues of sarafloxacin in fish tissues were extracted by the above simultaneous determination method; otherwise,the tissue's aqueous phase extract solution andthe plasma sample were loaded onto the C2 sorbent cartidge.Methanol-0.01M oxalic acid(7:3) was used for eluting sarafloxacin from the cartidge then the elution was determined by HPLC.A UV-VIS detector was used at a 278 nm.Thecalibration graph was linear from 1-500ng.The recoveries of the drug from tissues and plasma at a level of 1 ug/g(ml) were 73.4-91.1%.The detection limits were 0.07ug/g(ml) in all tissues and plasma. The pharmacokinetics of sarafloxacin in eel after single oral administration at a dose of 15mg/kg b. w. at 24C.The area under concertration-time curve(AUC)in plasma, liver,kidney,muscle,and skin was 12h,12h,12h,24h,and 40h.The peak concentration(Cmax) at Tmax in plasma,liver,kidney,muscle, and skinwas 2.64,13.39,5.53,1.82,and 0.78 ug/g(ml),respectively. On withdrawal of the drug,sarafloxacin concentration in plasma, liver,and kidney declined rapidly on the 28th,32nd,and 32nd h, respectively.It was conformed to be a biomodal elimination pattern with a rapid initial phase anda slower secondary phase. The distribution rate constant(a) for the line of best fit through the initial rapid phase(sample time 12th-32nd h) in plasma was 0.085h/l,(sample time 12th-28th h) liver and kidney were 0,12 and 0.071h/l,respectively.The distribution half-life( t1/2a)in plasma,liver,and kidney was 8.15,5.78,and 9.76 h, respectively.The elimination rate constant(B) for the lineof best fit through the slower final elimination phase and the elimination half-life(t1/2b) in plasma(sample time 32nd-96th h), liver,kidney(sample time28th-230th h),and skin(sample time 40th-240th h) was(0.023 h/l,30.13 h),(0,007 h/l,99 h),(0.012 h/ l,57.75h),(0.039 h/l,17.77 h),and(0.016 h/l,43.3 h), respectively. Sarafloxacin tissue levels below detectable limits in the plasma and muscleis 7days but liver,kidney, and skin is 14days.Based on the results of this study,we suggest that the best withdrawal period of sarafloxacin is 16 days.
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