以快速簡易溶膠凝膠法製備多模板分子印模移液管尖頭整體柱結合HPLC-FLD萃取分析豬肉中三種瘦肉精之研究

A novel sol-gel based in-pipette tip monolithic multiple-templates molecularly imprinted polymer (MIP) was prepared as the sorbent to collect three β2-agonists from the pre- extraction aliquots of pork for high performance liquid chromatography- fluorescence detection (HPLC-FLD). The monolithic MIP sorbent was prepared by mixing β2-agonists (ractopamine, fenoterol, and isoxsuprine) as templetes, 3-aminopropyl-triethoxysilane (APTES) and phenyltrimethoxysilane (PTMOS) as functional monomers, and tetraethylorthosilicate (TEOS) as cross-linker in methanol, and adding appropriate amount of sulfuric acid as catalyst. After mixing thoroughly, 150-μL of the mixed solution was withdrawn in 10-mL pipette tip to achieve the polymerization for 60 min under room temperature. Then the monolithic MIP was washed by methanol to remove templates completely, prior to be used for extraction. The collection of β2-agonists was performed by pre-extraction of β2-agonists from ground-pork with acetonitrile, and mixed with 10 mLof pH 10 phosphate buffer solution containing 1 % NaCl, The mixed solution was withdrawn through the MIP sorbent in pipette tip to collect β2-agonists onto MIP sorbent. After pushed the phosphated matrix solution out and rinsed with buffer solution, threeβ2-agonists were eluted with methanol for HPLC-FLD analysis. Parameters influenced the extraction efficiencies of β2-agonists such as selection of templetes, ratio of functional monomer, sample pH, salting out effect, and solution for eluting were investigated thoroughly and optimized. Under the optimal condition, the detections were linear in 0.05 - 40 ng/mL for fenoterol, 0.02 - 40 ng/mL for ractopamine and 0.01 - 40 ng/mL for isosuxprine, with correlation coefficients above 0.9964. The detection limits (based on S/N = 3) were 0.05, 0.02 and 0.01 ng/mL, for fenoterol, ractopamine, and isosuxprine, respectively. The β2-agonists in pork were analyzed and characterized by mass spectrometry. Recoveries were ranged 66.2 - 78.9 % for fenoterol, 87.5 - 96.2% for ractopamine and 89.3 - 101.1% for isosuxprine, with the relative standard deviations (RSD) were all less than 6.3 %. Results indicated that the present approach provides a rapid, simple, low cost, sensitivity and eco-friendly method to extract three β2-agonists from pork sample for HPLC-FLD analysis.本研究以溶膠-凝膠聚合法將分子印模聚合物(MIP)製備於移液管尖頭內為整體柱吸附床，用於快速萃取豬肉中的瘦肉精，做為高效液相層析-螢光偵測器(HPLC-FLD)分析之前處理。 研究使用多種分析物為模板，藉著氫鍵及π-π作用力設計製備能同時萃取多種瘦肉精的MIP。其製備過程乃將模板(Ractopamine、Fenoterol、Isoxsuprine)、功能性單體(3-Amino- propyl-triethoxysilane (APTES)、Phenyltrimethoxysilane (PTMOS))、交聯劑(Tetraethylorthosilicate (TEOS)，以甲醇溶解均勻後，再加入適量的硫酸進行酸催化聚合成膠體溶液。再以移液管尖頭吸取膠體溶液150 μL，在常溫下聚合反應60分鐘後，以甲醇清洗整體柱，去除模板後即可使用。 瘦肉精的萃取為將豬肉先以乙腈進行液相萃取前置處理後，將萃取液吸入移液管使瘦肉精為MIP所吸附，押出基質溶液後以緩衝溶液清洗整體柱，再以甲醇脫附瘦肉精為HPLC-FLD分析。影響萃取效率的因素，如模板分子的選用、功能性單體的比例、樣品pH值、鹽析作用、與沖提溶劑進行詳盡地探討。實驗結果顯示，將前置處理萃取液加入10 mL含1 %氯化鈉之pH 10磷酸鹽緩衝溶液，通過MIP萃取後，以1 mL緩衝溶液清洗，再以1.5 mL甲醇脫附分析物，脫附液經氮氣吹乾後以100 μL甲醇回溶，注入HPLC-FLD分析，可得到最佳萃取結果。偵測訊號對濃度的線性範圍分別為Fenoterol 0.05 - 40 ng/mL、Ractopamine 0.02 - 40 ng/mL及Isosuxprine 0.01 - 40 ng/mL，線性相關係數皆達0.9964以上，偵測極限分別為0.05 ng/mL、0.02 ng/mL及0.01 ng/mL。本方法應用於真實樣品之萃取時間少於15分鐘，可重複使用20次以上，回收率分別介於Fenoterol (66.2 - 78.9%)、Ractopamine (87.5 - 96.2%)、Isosuxprine (89.3 - 101.1%)，RSD皆低於6.3 %。研究結果證明所製備之多模板MIP用於萃取豬肉中之三種瘦肉精為一高選擇性、簡易且快速的樣品前處理方法。摘要…..………….………………………………………………i Abstract…………………………………………………………iii 目錄……………………………………………………………………v 圖目錄………………………………………………………………viii 表目錄………………………………………………………………x 第一章 緒論 1.1前言...............................................1 1.2瘦肉精(Ractopamine、Fenoterol、Isoxsuprine)之簡介...2 1.3瘦肉精(β2-agonists)之相關分析技術之回顧..............5 1.4 移液管尖頭固相萃取技術..............................8 1.5分子印模聚合物之簡介與概述...........................9 1.5.1 分子印模聚合物之簡介與概述....................9 1.5.2 分子印模聚合物之原理.........................12 1.5.3 分子印模聚合物之組成試劑.....................12 1.5.4 分子印模聚合物之作用力.......................20 1.5.5分子印模聚合物之合成..........................25 1.6溶膠凝膠法.........................................27 1.7 研究目的..........................................30 第二章 研究方法 2.1藥品、器材及儀器設備................................31 2.1.1 藥品........................................31 2.1.2 器材........................................32 2.1.3 儀器設備....................................32 2.2 藥品製備..........................................33 2.2.1 0.1 %磷酸動相沖提液的配置....................33 2.2.2 緩衝溶液之配置...............................34 2.2.3 瘦肉精之標準溶液之配置.......................35 2.2.4 豬肉樣品之取樣與處理.........................36 2.2.5 添加分析物於豬肉樣品中之配置..................36 2.3 玻璃裝置之矽烷化...................................36 2.4 儀器操作之參數.....................................37 2.4.1 高效能液相層析儀之參數........................37 2.4.2 In-pipette tip MIP整體柱之微固相萃取法最佳化參數37 2.5 實驗方法與流程......................................37 2.5.1 In-pipette tip MIP整體柱之合成方法............37 2.5.2 In-pipette tip NIP整體柱之合成方法............37 2.5.3 In-pipette tip MIP整體柱之特性測試 ...........38 2.5.4 In-pipette tip MIP整體柱之微固相萃取條件最佳化.38 2.6 方法可行性之評估....................................40 2.6.1 儀器檢量線製作................................40 2.6.2 方法檢量線製作................................41 2.6.3 In-pipette tip MIP整體柱之重複使用性測試.......41 2.6.4 In-pipette tip MIP整體柱製備批次間之比較.......41 2.7 真實樣品之分析與回收率...............................42 2.7.1豬肉樣品分析...................................42 2.7.2 豬肉樣品之回收率測定...........................42 第三章 結果與討論 3.1 In-pipette tip MIP整體柱之特性測試..................44 3.1.1 In-pipette tip MIP整體柱之IR光譜..............44 3.1.2 In-pipette tip MIP整體柱之SEM................44 3.2 微固相萃取之最佳化..................................47 3.2.1功能性單體(APTES與PTMOS)之添加比例..............47 3.2.2模板分子的選擇.................................49 3.2.3水樣pH的影響..................................51 3.2.4鹽析影響......................................55 3.2.5沖提溶劑影響..................................57 3.3 方法可行性之評估...................................59 3.3.1 儀器檢量線製作...............................59 3.3.2 方法減量線製作...............................59 3.3.3 市售豬肉之測定...............................63 3.3.4 添加分析物之豬肉測定..........................63 3.3.5 In-pipette tip MIP/ NIP之吸附效率比較.........66 3.3.6 In-pipette tip MIP整體柱之穩定性與再現性......71 3.3.7分析方法之比較................................76 第四章 結論............................................78 第五張 參考文獻........................................8