開發連線固相萃取-液相層析串聯質譜儀搭配2,4-二硝基苯胼衍生法分析尿液中丙二醛

Urinary analysis of malondialdehyde by LC-MS/MS with automated solid-phase extraction following derivatization with 2,4-dinitrophenylhydrazine

黃妤婕(Yu-Jie Huang)

指導教授 ： 趙木榮

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2010年)

摘要

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)。

關鍵字

連線固相萃取 ； 液相層析串聯質譜儀 ； 丙二醛 ； 2,4-二硝基苯胼 ； 衍生反應

並列摘要

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).

並列關鍵字

on-line SPE ； LC-MS/MS ； malondialdehyde ； 2,4-dinitrophenylhydrazine ； derivatization

參考文獻

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