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研究生:柯泰安
研究生(外文):Tai-An Ko
論文名稱:以分子印模聚合物固相萃取技術結合HPLC-PDA分析微波輔助酸水解尿液中對乙醯氨基酚代謝物
論文名稱(外文):Microwave-assisted acid hydrolysis of urinary paracetamol metabolite and using pipette-tip molecularly imprinted solid phase extraction for HPLC-PDA analysis
指導教授:鄭政峰鄭政峰引用關係
指導教授(外文):Jen-Fon Jen
口試委員:李茂榮楊慶成
口試委員(外文):Maw-Rong LeeChing-Cherng Yang
口試日期:2015-07-09
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學系所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:83
中文關鍵詞:分子印模聚合物固相萃取微波輔助水解對乙醯氨基酚
外文關鍵詞:microwave-assisted hydrolysismolecularly imprinted polymerparacetamolurine
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本研究開發以微波輔助酸水解尿液中乙醯胺基酚(paracetamol)代謝物(乙醯胺基酚葡萄糖酸共軛物),繼以分子辨識印模高分子吸附整體柱(MMIP-SPE)之前處理,結合HPLC-PDA之快速檢測技術。MMIP-SPE吸附管的製備,以乙醯胺基酚為模板分子,甲基亞克力酸(MAA)為功能性單體、ethylene glycol dimethylacrylate (EGDMA)為交聯劑、AIBN為起始劑、及製孔溶劑,經均勻溶解及脫氧後,放入移液管尖頭內,以紫外光照射下聚合4小時即完成製備。尿液樣品經加酸過濾去蛋白質後,加入鹽酸,置於微波爐中進行微波輔助酸水解乙醯胺基酚葡萄糖酸共軛物生成乙醯胺基酚,再利用MMIP-SPE對乙醯胺基酚進行萃取後,以HPLC-PDA分析。研究中除製備MMIP-SPE外,也對微波輔助酸水解及MIPSPE的條件進行最佳化探討,包含微波水解鹽酸的濃度及水解時間,MIPSPE時樣品的pH值、潤洗雜質溶液及沖提時流洗液的條件均加以詳盡探討。研究結果顯示,在pH 2的鹽酸下以700W微波的功率下水解45分鐘可得最佳的水解效果;在pH 2的樣品溶液下進行MMIP- SPE、以pH2的水溶液0.2 mL潤洗雜質,及以pH=12之50%甲醇溶液0.2 mL脫附,可得最佳的前處理效果。在最佳條件下分析尿中對乙醯氨基酚,其線性範圍介於0.005~10 mg/L,線性回歸係數為0.9977,方法偵測極限(LOD)為0.6 μg mL-1,定量偵測極限(LOQ)為1.9 μg mL-1,相對標準偏差(RSD)為3.2 %,回收率為83.5 - 95.8 %。只要60分鐘即可完成整個樣品的前處理及分析。研究結果證明了本開發方法可大幅提升水解效率、縮短水解時間、和降低分析成本,為一快速、簡單且兼具環保的尿液中樣品分析方法。

An eco-friendly method for rapid determination of paracetamol β-D-glucuronide in human urine by using microwave-assisted acidic hydrolysis, in-tip monolithic molecularly imprinted polymer solid phase extraction (MMIP-SPE), and HPLC-PDA determination, was investigated. MMIP was synthesized by using paracetamol as template, methyl acrylic acid (MAA) as monomer, ethylene glycol dimethylacrylate (EGDMA) as cross-linker, azobisisobutyronitrile (AIBN) as initiator and 15 mL of methanol/acetone/ toluene (8:2:5) as porogenic solvent. After being mixed homogeneously and deoxygenated by purging nitrogen, the solution (300 μL) was injected into a pipette-tip to achieve polymerization under UV irradiation for 4 hours. The in-tip MMIP solid phase with high recognition on paracetamol was thus obtained. After removing protein from urine sample by acidification and filtration, the urinary paracetamol β-D-glucuronide in sample solution was hydrolyzed under microwave irradiation, then the paracetamol was collected by MMIP-SPE process, prior to HPLC-PDA analysis. In addition to the preparation of MMIP-SPE sorbent, parameters affecting the microwave assisted hydrolysis such as concentration of HCl and time for hydrolysis, and MMIP-SPE such as the pH of sample solution, the composition in rinsing solution, and the composition of eluting solution and its pH were thoroughly optimized. The best hydrolysis efficiency was obtained with pH 2 (by HCl) urinary solution irradiated by 700 W microwave power for 45 min, and the maximum extraction efficiency of MMIPSPE was obtained by loading sample solution at pH 2, rinsing with 0.2 mL of pH 2 aqueous solution, and eluting by 0.2 mL of 50% methanol at pH 12 solution. P aracetamol in the collected eluent were analyzed by HPLC-PDA. Under the optimal condition, the linear range was from 0.005-10 μgmL-1 with linear coefficient for determination was 0.9977. LOD of method was 0.6 μg mL-1 and LOQ was 1.9 μg mL-1. RSD was 3.2 %. The recoveries were ranged in 83.5-95.8 % for spiked 1-4 μg mL-1 in real urine samples. Only 60 min was required to achieve the determination including sample preparation and HPLC-PDA analysis. The results showed that the present method was a simple, rapid, and low cost method for the analysis of paracetamol in urine samples.

謝誌 i
摘要 ii
Abstract iii
目錄 v
圖目錄 viii
表目錄 x
第一章 緒論 1
一、對乙醯氨基酚( paracetamol ) 1
(一) 對乙醯氨基酚之簡介 1
(二) 對乙醯氨基酚代謝機制 1
(三) 分析方法回顧及研究動機 4
二、微波及其輔助水解之概述 6
(一) 微波理論與歷史 6
(二) 微波加熱與傳統加熱之比較 8
(三) 微波加速反應原理 11
三、分子印模聚合物之概述 13
(一) 分子印模聚合物之歷史發展 13
(二) 分子印模聚合物之原理 15
(三) 分子印模聚合物吸附之作用力 17
(四) 分子印模聚合物之聚合溶液 20
(五) 分子印模聚合物前處理技術之應用 26
(六) 分子印模聚合物之製備方式 28
第二章 研究方法 30
一、 藥品、實驗器材與儀器設備 30
(一) 藥品 30
(二) 儀器設備與實驗器材 30
二、 藥品配置 32
(一) 高效能液相層析儀動相配置 32
(二) 緩衝溶液的配置 32
(三) Paracetamol標準品儲存溶液之配製 33
(四) Paracetamol β-D-glucuronide標準品儲存溶液之配製 34
三、 儀器設備 35
四、 實驗方法 35
(一) 合成溶液配置與反應 35
(二) 分子印模固相萃取流程 37
(三) 微波輔助水解裝置 39
(四) 分子印模聚合物之特性測試 40
(五) 方法可行性評估 41
(六) 真實樣品之分析 42
第三章 結果與討論 43
一、 微波輔助水解之分析條件 45
(一) 微波輔助水解條件最佳化探討 45
(二) 微波輔助水解與加熱水解法的比較 47
二、 MIP之製備與特性分析 49
(一) MIP之合成最佳化及特性測試 49
(二) 分子印模聚合物吸附量 49
(三) 分子印模聚合物之穩定性 50
(四) 分子印模聚合物之IR光譜 53
(五) 分子印模聚合物之SEM圖 53
三、 微量吸管頭內分子印模固相萃取最佳化 56
(一) 樣品溶液之pH值 56
(二) 沖提溶液之pH 58
(三) 沖提溶液之體積 60
(四) 沖提溶液之甲醇比例 62
(五) 沖洗溶液之選擇 64
四、 檢量線與真實樣品 66
(一) 儀器檢量線 66
(二) 方法檢量線 66
(三) 真實樣品之測定 68
(四) 真實樣品之質譜測定 71
(五) 分子印模固相萃取與其他方法之比較 73
第四章 結論 75
參考文獻 76

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