運用連線固相萃取搭配液相層析串聯質譜儀同時分析尿液中 8-oxo-7,8-dihydro-2'-deoxyguanosine 與 8-oxo-7,8-dihydroguanine

Simultaneous direct quantification of urinary 8-oxodGuo and 8-oxoGua using on-line SPE LC-MS/MS method

謝樵漢(Ciao-Han Sie)

指導教授 ： 胡瓊文

中山醫學大學/健康管理學院/公共衛生學系碩士班/碩士(2009年)

摘要

摘要
本研究成功建立一液相層析串聯質譜儀搭配連線固相萃取方法，可快速準確及同時分析尿液中 8-oxo-7,8-dihydroguanine (8-oxoGua) 及 8-oxo-7, 8-dihydro-2’-deoxyguanosine (8-oxodGuo)。研究中成功合成 8-oxoGua 之同位素內標準品 15N5-8-oxoGua，尿液樣本經稀釋後，加入同位素內標準品，不需再經前處理即可直接上機分析，層析時間為15分鐘。接著，利用新開發之分析方法確認 8-oxoGua 在不同儲存環境下之穩定性。研究發現，以0.01 N NaOH 溶液溶解 8-oxoGua 標準品後，高濃度標準品 (10 ug/ml) 在4℃、pH ≤ 11條件下有最佳穩定性，其半衰期達105天；在－20℃、pH ≤ 11條件下穩定性最差，半衰期只有4天。低濃度標準品 (50 ng/ml) 在－20℃、pH < 8條件下穩定性最佳，其半衰期達578天；在－20℃、pH = 12狀況下穩定性最差，半衰期為4天。另外再運用此方法分析20個人體尿液樣本，結果發現尿液中 8-oxoGua 和 8-oxodGuo 背景值分別為5.95 ± 5.20和1.74 ± 0.84 (nmol/mmol creatinine)，比值為3.41。本研究建立之分析方法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。

關鍵字

液相層析串聯質譜儀 ； 穩定性 ； 基因氧化傷害產物

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 simultaneously determine 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxo-7, 8-dihydro-2’-deoxyguanosine (8-oxodGuo) in urine. 15N5-labeled 8-oxoGua was also successfully synthesized to serve as an internal standard in this method. After spiking internal standard, the urine sample can be directly analyzed within 15 min without further sample purification. With this newly developed method, we further investigated the stability of 8-oxoGua under several common storage conditions. The results showed that the stability of 8-oxoGua was affected by pH, temperature and its initial concentration. It was found that the high concentration of 8-oxoGua (10 μg/mL) was the most stable at 4°C with pH value ≤ 11 (the half-life was estimated to be around 105 days), while it was the less stable at -20°C with pH value of 12 (the half-life was estimated to be 4 days). On the other hand, the low concentration of 8-oxoGua (50 ng/mL) had the best stability at -20°C with pH value < 8 (half-life: 578 days), and the worst stability at -20°C with pH value of 12 (half-life: 4 days). Meanwhile, this “on-line SPE LC-MS/MS” method was also applied to determine the background levels of 8-oxoGua/8-oxodGuo in urine of healthy adults. The urinary levels of 8-oxoGua and 8-oxodGuo were 5.95 ± 5.20 and 1.74 ± 0.84 (nmol/mmol creatinine), respectively. The ratio of 8-oxoGua and 8-oxodGuo levels in urine was around 3.41. This “on-line SPE LC-MS/MS” method could be applicable for use in daily clinical practice for assessing oxidative stress in patients.

並列關鍵字

LC-MS/MS ； stability ； oxidatively damaged DNA

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