- 學位論文
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
Development of Renal Function Evaluation by HPLC with Copper Based ECD
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。
並列摘要
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.