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系統識別號 U0002-2306201609424300
中文論文名稱 銅基礎的電化學偵測搭配高效液相層析於腎功能指標上之應用
英文論文名稱 Development of Renal Function Evaluation by HPLC with Copper Based ECD
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 104
學期 2
出版年 105
研究生中文姓名 林鈺淳
研究生英文姓名 Yu-Chun Lin
學號 603160184
學位類別 碩士
語文別 中文
口試日期 2016-05-30
論文頁數 92頁
口試委員 指導教授-林孟山
委員-傅明仁
委員-蔡東湖
中文關鍵字 腎功能    尿酸  肌酸酐  腎臟廓清率 
英文關鍵字 Renal Function  Copper based  Uric Acid  Creatinine  eGFR 
學科別分類 學科別自然科學化學
中文摘要    近幾年因食安的問題、工業重金屬的汙染及藥物的濫用,全球腎臟病人口不斷攀升,如何能準確測量尿酸及肌酸酐含量並計算腎臟廓清率,成為臨床上一重要的課題;本研究以銅電極為基礎之薄層電化學偵測器做為高效液相層析之偵測器,開發能同時偵測人體血液及尿液中尿酸及肌酸酐含量之偵測方法,並可計算腎臟廓清率,完成臨床之腎功能評估偵測方法;此研究藉由施加氧化工作電位,使電極表面形成難溶性氧化銅層,當尿酸或肌酸酐隨流動相經過電極表面時,與電極表面的難溶性氧化銅配位形成游離性錯合物,而電極表面在-0.1V的電壓下會再生成新的氧化銅層,藉由偵測所量測的氧化電流的改變,間接偵測人體血液及尿液中的尿酸及肌酸酐含量;本法其他最佳偵測條件,緩衝溶液流動相為50mM磷酸-75mM醋酸緩衝溶液,酸鹼值為pH7.4,流動相流速為1.0ml/min,樣品注射體積為20μl,利用最佳化條件,達到最佳偵測靈敏度。在最佳化的偵測條件下進行尿酸及肌酸酐的偵測,所得到的偵測分析特性之尿酸偵測極限為0.11μM,肌酸酐為0.50μM,尿酸及肌酸酐線性範圍皆為2.5-500μM,偵測靈敏度為尿酸168.06(nA/mM),而肌酸酐為11.74(nA/mM) (S/N=3);此偵測方法之線性範圍足以涵蓋人體血液中尿酸及肌酸酐含量之正常範圍,且實驗最佳化條件在真實樣品測試中,也足以使尿酸及肌酸酐與人體血液及尿液中其他雜質完全分離,證明此偵測方法能被作為臨床檢測的工具。
英文摘要    Both of uric acid(UA) and creatinine(Cr) are important indicator of renal function. For the past years, food additives, industry heavy metal pollution and drug abuse make a severe affect for human health and the population of renal failure is getting higher. For these reason, how to calculate the estimated glomerular filtration rate(eGFR) is always on critical. In this scheme, it developes a determination of HPLC-Cu-ECD to detect UA and Cr simultaneous in human serum and urine and estimate eGFR further. Some reports presented UA and Cr can be chelated with Cu(I) and Cu(II). In this determination, it supplied an oxdation voltage -0.1V(vs. Ag/AgCl) to create the rigid Cu(II) layer on electrode surface. A 50mM phosphate - 75mM acetate buffer solution was used to be a mobile phase at pH7.4 for flow rate 1.0ml/min. UA or Cr will chelate with Cu(II) of the electrode surface then desorption. A oxidative current will be generated when the copper electrode surface formation rearrangement. The detection limit of the determination are presented 0.11 and 0.50μM for UA and Cr which was calculated from 12 times blank injections. The linear range of UA is form 2.5-500μM and Cr is from 10-500μM. The linear range of UA and Cr are over the human serum normal range of UA and Cr. The analytical sensitivity of UA and Cr are 168.06nA/mM and 11.74nA/mM respectively. Both of UA and Cr can be separated other impurities in this scheme that proved it is useful on clinical determination of renal function.
論文目次 目 錄
第一章 緒論........................................................................................................ 1
1-1 腎功能........................................................................................................... 2
1-2 常規腎功能檢測........................................................................................... 5
1-3 尿酸............................................................................................................. 10
1-4 肌酸酐......................................................................................................... 12
1-5 尿酸及肌酸酐檢測方法............................................................................. 14
1-5-1 尿酸檢測方法.................................................................................. 15
1-5-2 肌酸酐檢測方法.............................................................................. 21
1-5-3 同時偵測尿酸及肌酸酐偵測方法.................................................. 25
1-6 層析法......................................................................................................... 28
1-6-1 薄層層析法...................................................................................... 29
1-6-2 氣相層析法...................................................................................... 30
1-6-3 液相層析法...................................................................................... 30
1-6-3-1 分配層析法......................................................................... 31
1-6-3-2 吸附型層析法..................................................................... 33
1-6-3-3 離子層析法......................................................................... 34
1-6-3-4 分子排除式層析法............................................................. 34
1-6-3-5 親和層析法......................................................................... 35
1-6-4 超臨界流體層析法.......................................................................... 36
1-7 研究目的..................................................................................................... 37
第二章 實驗...................................................................................................... 38
2-1 儀器............................................................................................................. 38
2-1-1 電化學實驗...................................................................................... 38
2-1-2 高效液相層析法.............................................................................. 38
2-1-3 其他.................................................................................................. 39
2-2 藥品............................................................................................................. 40
2-3 實驗前處理................................................................................................. 41
2-3-1 電極製備.......................................................................................... 41
2-3-2 管柱平衡.......................................................................................... 41
2-3-3 標準品製備...................................................................................... 41
2-3-4 尿液及血液真實樣品前處理.......................................................... 42
2-4 實驗流程設計............................................................................................. 43
2-4-1銅電極偵測機制............................................................................... 43
2-4-2銅電極工作電位探討....................................................................... 43
2-4-3流動相酸鹼值探討........................................................................... 44
2-4-4緩衝溶液種類的探討....................................................................... 45
2-4-5緩衝溶液濃度探討........................................................................... 45
2-4-6緩衝溶液修飾劑探討....................................................................... 46
2-4-7磷酸-醋酸鈉緩衝溶液探討.............................................................. 47
2-4-8實驗流速探討................................................................................... 48
2-4-9樣品迴路體積探討........................................................................... 48
2-4-10分析特性......................................................................................... 49
第三章 結果與討論.......................................................................................... 50
3-1銅電極偵測機制................................................................................... 51
3-2銅電極工作電位探討........................................................................... 56
3-3流動相酸鹼值探討............................................................................... 59
3-4緩衝溶液種類探討............................................................................... 64
3-5緩衝溶液濃度探討............................................................................... 67
3-6緩衝溶液修試劑探討........................................................................... 69
3-7磷酸-醋酸鈉緩衝溶液探討.................................................................. 75
3-8實驗流速探討....................................................................................... 77
3-9樣品迴路體積探討............................................................................... 78
3-10分析特性............................................................................................. 80

資料來源............................................................................................................ 86
附錄.A................................................................................................................. 92










圖 表
圖1-1 尿酸於人體中的代謝機制..................................................................... 12
圖1-2 肌酸及肌酸酐在人體中的代謝機制..................................................... 14
圖3-1銅電極偵測機制探討.............................................................................. 54
圖3-2銅電極於流動注射系統中的偵測........................................................... 55
圖3-3銅電極工作電位探討.............................................................................. 58
圖3-4酸鹼值對偵測背景電流值的影響.......................................................... 61
圖3-5偵測酸鹼值探討...................................................................................... 62
圖3-6尿酸與肌酸酐滯留時間.......................................................................... 63
圖3-7緩衝溶液種類探討.................................................................................. 66
圖3-8緩衝溶液濃度探討.................................................................................. 68
圖3-9醋酸根的影響.......................................................................................... 72
圖3-10緩衝溶液修試劑對訊號強度的探討.................................................... 73
圖3-11修試劑對尿酸及肌酸酐的影響............................................................ 74
圖3-12磷酸-醋酸鈉溶液探討........................................................................... 76
圖3-13樣品迴路體積探討................................................................................ 79
圖3-14尿酸檢量線與肌酸酐檢量線................................................................ 83
圖3-15真實樣品................................................................................................ 84
圖3-16相關係數................................................................................................. 85


表.1腎絲球廓清率與腎病程度分析表............................................................... 4
表.2腎絲球疾病的分類....................................................................................... 5
表.3尿液尿蛋白檢測對照表............................................................................... 7
表.4尿酸與肌酸酐於不同緩衝溶液中之訊號半峰寬..................................... 65
表.5最佳化分析條件......................................................................................... 81
表.6分析特性..................................................................................................... 82
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