臺灣博碩士論文加值系統

本研究成功建立一連線固相萃取-液相層析串聯質譜儀 (on-line SPE LC-MS/MS)，搭配2,4-二硝基苯胼 (2,4-dinitrophenylhydrazine, DNPH) 衍生法，以快速準確的分析尿液中丙二醛 (malondialdehyde, MDA)。本研究的分析方法，尿液稀釋後與 DNPH 進行衍生反應，即可直接上機分析，不需再搭配其他前處理，層析時間為11分鐘。我們也利用建立的分析方法，探討 MDA 與 DNPH 衍生反應之最佳條件；發現尿液樣本中的 MDA 衍生反應要到達飽和所需添加的 DNPH 濃度約比 MDA 標準品高出60倍。此結果顯示尿液中富含其他與 DNPH 進行反應的物質，因此反應時需比標準品添加更多量的 DNPH，才足以將尿液中的 MDA 反應完全。接著我們將此方法應用於109個健康成年人的尿液樣本分析，其包含63個非吸菸者以及46個吸菸者，分析結果發現非抽菸者以及吸菸者尿液中的 MDA 平均值，分別為1.6 ± 0.9 μmol/g creatinine 以及 1.8 ± 1.8 μmol/g creatinine。兩個組別尿液中的 MDA 含量並無顯著的差異 (p > 0.05)。整體來說，本研究建立之分析方法快速準確，未來可運用於臨床高通量的樣本檢測，以評估人體內之氧化壓力。
此外，本研究也另將尿液樣本於衍生化前添加同位素內標 d2-MDA 並以 「on-line SPE LC-MS/MS」 進行分析，同時與不添加同位素內標之分析方法進行比較。其結果顯示與不添加內標的分析方法所測得的值，十分相近 (Pearson 相關係數 r = 0.9957, p < 0.001, n = 34)。

A high-throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) method coupled with an on-line solid phase extraction (SPE) system was developed to determine malondialdehyde (MDA) in urine following derivatization with 2,4-dinitrophenylhydrazine (DNPH). After derivatization, the urine samples can be directly analyzed within 11 min without further purification. The method was then applied to investigate the optimal reaction conditions for MDA derivatization using DNPH. The results showed that for a completely MDA derivatization, the additions of DNPH to MDA calibration solutions or urinary samples were extremely different. The optimal addition ratio of DNPH to urinary MDA is 60 times higher than that of MDA standard solution. This finding indicated that there were considerable interfering substances present in urine matrix that could also react with DNPH.
Furthermore, this “on-line SPE LC-MS/MS” method was applied to determine the urinary levels of MDA in 109 healthy adults, including 63 non-smokers and 46 smokers. The mean urinary MDA levels were 1.6 ± 0.9 and 1.8 ± 1.8 μmol/g creatinine for non-smokers and smokers,respectively. There was no significant difference in urinary MDA levels between the two groups (p > 0.05). Overall, this newly developed “on-line SPE LC-MS/MS” method could be applicable in both research and clinical practice for assessing oxidative stress.
In addition to this, the urine samples were also added with a stable isotope d2-MDA prior to derivatization and analyzed by on-line SPE LC-MS/MS. The results showed that the urinary MDA values obtained from both methods (with or without stable isotope) were highly correlated (Pearson correlation coefficient r = 0.9957, p < 0.001, n = 34).

摘要-I
Abstract-III
目錄-V
表目錄-VIII
圖目錄-IX
第一章 緒論
1-1 前言-1
1-2 研究動機-2
1-3 研究目標-3
第二章 文獻回顧
2-1 氧化壓力 (oxidative stress) 與脂質過氧化 (lipid peroxidation, LPO)-4
2-1-1 氧化壓力-4
2-1-2 脂質過氧化 (lipid peroxidation, LPO) 及其主要產物丙二醛 (malondialdehyde, MDA)-8
2-2 MDA 與疾病的相關性-14
2-3 MDA 的分析方法-19
第三章 材料與方法
3-1 化學藥品及試劑-25
3-2 人體尿液樣本-26
3-3 連線固相萃取-液相層析-串聯質譜儀分析方法開發-27
3-3-1 自動連線式固相萃取裝置 (automated on-line SPE)-27
3-3-2 液相層析系統 (LC)-30
3-3-3 電灑離子化串聯質譜儀 (electrospray ionization MS/MS)-30
3-4 MDA-DNPH 衍生反應最佳化-32
3-4-1 MDA 標準品與 DNPH 反應之反應曲線-32
3-4-2 尿液樣本與 DNPH 之反應曲線-33
3-5 分析方法確認-33
3-5-1 偵測極限 (limit of detection, LOD) 和定量極限 (limit of quantification, LOQ)-33
3-5-2 方法回收率 (recovery)、組內變異 (intra-day variation)-33
3-6 以 on-line SPE LC-MS/MS 分析尿液樣本-34
3-7 以 on-line SPE LC-MS/MS 搭配同位素內標稀釋法 (isotope dilution) 分析尿液樣本-35
3-8 統計分析-36
第四章、結果與討論
4-1 待測物 MDA-DNPH 的初步鑑定-37
4-2 on-line SPE LC-MS/MS 分析方法的建立-42
4-3 MDA 與 DNPH 衍生反應最佳化-44
4-3-1 MDA 標準品與 DNPH 之反應曲線-44
4-3-2 尿液樣本與 DNPH 之反應曲線-45
4-4 方法確認-47
4-4-1 偵測極限 (LOD) 和定量極限 (LOQ)-47
4-4-2 精準度 (precision) 和回收率 (recovery)-47
4-4-3 離子抑制 (ion suppression)-49
4-5 應用 on-line SPE LC-MS/MS 於一般健康成人之尿液 MDA 分析-52
4-6 on-line SPE-LC-MS/MS 搭配同位素稀釋法 (isotope dilution)-57
第五章 結論63
參考文獻-64

表目錄
表2-1 生物體 ROS 的來源-6
表2-2 MDA 與疾病之相關性-15
表2-3 各種層析方法於體液 MDA 之分析-22
表3-1 分析 MDA-DNPH 之 on-line SPE LC 管柱切換流程表-29
表3-2 待測物 MDA-DNPH 離子對選擇及其碰撞能量-32
表4-1 MDA-DNPH 以及 d2-MDA-DNPH 之離子對-41
表4-2 方法精準度與回收率-48
表4-3 分析方法比較 -50
表4-4 非吸菸者與吸菸者尿液中 MDA 的含量-53
表4-5 本研究及文獻中健康成年人尿液 MDA 的含量-54

圖目錄
圖2-1 ROS 與抗氧化系統之氧化壓力平衡圖-8
圖2-2 多元不飽和脂肪酸的脂質過氧化反應機制-10
圖2-3 常見的 MDA-DNA 共價鍵結產物-12
圖2-4 MDA 與蛋白質所形成之 Schiff base-13
圖2-5 MDA-TBA 衍生化物之形成機制-20
圖2-6 MDA-DNPH 衍生化物之形成機制-20
圖3-1 on-line SPE LC-MS/MS 之自動系統架構圖-28
圖4-1 待測物 MDA-DNPH 全離子掃描圖 (Q1 scan) 及結構圖-38
圖4-2 d2-MDA-DNPH 全離子掃描圖 (Q1 scan) 及結構圖-39
圖4-3 MDA-DNPH 子離子掃描圖 (product ion scan)-40
圖4-4 d2-MDA-DNPH 子離子掃描圖 (product ion scan)-41
圖4-5 MDA-DNPH 標準品之層析圖-43
圖4-6 尿液樣本 MDA-DNPH 之層析圖-44
圖4-7 0.2 mM MDA 與不同濃度的 DNPH 反應之反應曲線-45
圖4-8 尿液稀釋5倍與不同濃度的 DNPH 反應之反應曲線(n=3)-46
圖4-9 MDA-DNPH 方法回收率-48
圖4-10同位素內標添加前後之分析結果比較 (n=34)-58
圖4-11 僅含0.5 mM d2-MDA 內標準品之貢獻情形-60
圖4-12 僅含0.5 mM MDA 外標準品之貢獻情形-61

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