臺灣博碩士論文加值系統

在生物體之新陳代謝過程中，氧化作用會產生自由基造成蛋白質過氧化、DNA傷害與脂質過氧化等，直接導致細胞衰老、死亡，並間接使人體抵抗力變差與老化，因此測定人體中過氧化指標有其醫療上的重要性，本研究主要目的在於發展一個快速且準確的方法，測定血清和尿液中脂質過氧化作用產生之丙二醛(Malondialdehyde, MDA)。研究中藉由傳統的高效能液相層析法(High performance liquid chromatography, HPLC)為基礎，加以改良為超高效能液相層析法(Ultra performance liquid chromatography, UPLC)，於532 nm可見光下，偵測硫代巴比妥酸(Thiobarbituric acid, TBA)與MDA結合的複合物，過程中探討移動相的pH值、移動相非極性的比例、管柱流速、注射體積等分析條件對滯留時間及吸收訊號的影響，期能大幅提升靈敏度及縮短滯留時間，在最佳的條件下討論線性、偵測極限、血清和尿液等樣品的基質是否造成干擾、回收率和精密度等，建立方法後驗證其穩定性，最後進行真實樣品的檢測，證實所建立的方法確實可行。結果顯示UPLC於移動相pH值6.8，移動相非極性比例KH2PO4:Methanol為60:40(v/v)，注射體積5 μL時，即可得到和HPLC注射體積20 μL相等的訊號，且流速只要0.4 mL/min即可縮短滯留時間為2.5分鐘，比HPLC可得到較高的分離率及較短一半之滯留時間；比較水溶液、血清和尿液為基質的MDA吸收訊號，發現血清和尿液無基質干擾之問題，可利用水溶液建立的檢量線進行血清和尿液的分析；方法之線性範圍為0.61-38.88 μmol/L、偵測極限43 fg、血清及尿液同日內與異日間精密度均低於10%內、血清及尿液回收率分別為94% - 110%及81% - 110%，皆在可接受的範圍；而經結果證實，MDA於4℃與-20℃溫度環境中可穩定保存。本研究開發之UPLC測定MDA的方法比傳統HPLC方法更靈敏且可縮短大半的偵測時間，值得推廣為臨床實驗室測定氧化壓力指標之標準方法。

Free radicals produced from metabolism of organisms induces oxidative stress such as protein oxidation, DNA damage and lipid peroxidation. Oxidative stress induces not only cell aging and death but also indirect human aging and deterioration of resistance. Therefore, developing a rapid and sensitive analysis of peroxidation is an important event. In this study, we reported a novel method for rapid and sensitive determination of malondialdehyde (MDA) in serum and urine by ultra performance liquid chromatography (UPLC). MDA was reacted with thiobarbituric acid (TBA) to form MDA–TBA2, a red-colored adduct with maximum absorbance at 532 nm. The effects of UPLC parameters (pH of mobile phase, non-polar ratio of mobile phase, flow rate and injection volume) on peak area and retention time were investigated for the optimized operation conditions that could enhance the sensitivity and the rapidity. The linearity, matrixes interference of serum and urine, limit of detection, recovery, precision and the test of real samples were validated in the experimental section. Our results indicated the absorbance at mobile phase pH6.8, KH2PO4: Methanol 60:40(v/v), 5 μL of injection volume by UPLC was the same as at 20 μL of injection volume by HPLC. When the flow rate was 0.4 mL/min, the retention time was 2.5 min. Comparing to HPLC, the high separation capacity and half retention time of UPLC were found. The linear range of standard solution was from 0.61 to 38.88 μmol/L and the detection limit of MDA was 43 fg. The intra-assay or inter-assay precisions of MDA in serum and urine were all below 10%. The recoveries of MDA in serum and urine were 94-110% and 81-110%, respectively. Furthermore, the stable temperatures of MDA were 4℃ and -20℃. The results showed that there were no matrixes interferences for the determination of MDA in serum and urine. Therefore, the calibration curves for the analysis of serum and urine could be established by the aqueous standard solution. The proposed method provided sensitive and rapid measurement of MDA in serum and urine by UPLC can be easily applied to clinical laboratory.

第一章 緒論 1
第一節 自由基對生物體之影響 3
第二節 自由基與脂質過氧化作用產物MDA 9
第三節 偵測MDA之方法 11
第四節 超高效能液相層析法 15
第五節 研究動機與目的 17
第二章 材料與方法 20
第一節 材料、儀器與試劑配製 20
第二節 探討UPLC分析系統最佳化條件 23
第三節 UPLC與HPLC之比較 26
第四節 分析方法驗證 26
第五節 真實樣品測試 31
第三章 結果與討論 32
第一節 探討UPLC分析系統最佳化條件 32
第二節 UPLC與HPLC之比較 35
第三節 分析方法驗證 36
第四節 真實樣品測試 38
第四章 總結 40
第五章 參考文獻 42
第六章 表 50
第七章 圖 55

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