開發連線固相萃取-液相層析串聯質譜儀搭配同位素稀釋法分析尿液中硝化壓力生物指標

Urinary analysis of nitrative stress biomarkers by isotope-dilution LC-MS/MS with automated solid-phase extraction

林家宏(Jia-Hong Lin)

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(2012年)

https://doi.org/10.6834/CSMU.2012.00097

摘要

本研究成功建立連線固相萃取-液相層析串聯質譜儀 (on-line SPE LC-MS/MS) 搭配同位素稀釋法，同時分析尿液中之蛋白質硝化壓力生物指標3-nitrotyrosine (3-NTYR) 及3-nitro-4-hydroxyphacetic acid (NHPA)。尿液中基質複雜且3-NTYR 及 NHPA 極為低量，因此尿液於分析前需進行濃縮及純化，本研究使用 C18 手動固相萃取匣濃縮尿液樣本，其最佳化之手動固相萃取步驟為：以1 % formic acid 做為沖洗液；40% methanol + 1% formic acid 做為沖堤液，並運用 HPLC fractionation 純化樣本，總濃縮倍率為30倍。3-NTYR 及 NHPA 之方法偵測極限分別為17.1及17.7 fmol/ml；方法回收率分別為56.5% 及28.9%；總層析時間為30分鐘。此分析方法應用於19位健康成年人尿液樣本分析，其中包括11位抽菸者及8位非抽菸者，分析結果發現抽菸者與非抽菸者尿液中3-NTYR 分別為183 ± 128及141 ± 130 fmol/mg creatinine；NHPA 分別為261 ± 239及306 ± 180 fmol/mg creatinine。抽菸者及非抽菸者尿液中3-NTYR 及 NHPA 並無顯著的差異。本研究建立之分析方法可避免人為硝化，準確的分析人體尿液之3-NTYR 及 NHPA，未來可運用於評估人體蛋白質硝化壓力程度。
本研究亦開發連線固相萃取-液相層析串聯質譜儀搭配同位素稀釋法分析人體尿液中基因硝化壓力生物指標8-nitroguanine (8-NO2-G)。尿液中8-NO2-G 極為低量且含有大量的雜質，本研究開發2種前處理方法，分別為 (1) 運用 C18 手動固相萃取匣及 HPLC fractionation 濃縮30倍，直接量測8-NO2-G；(2) 運用6-methoxy-2-naphthyl glyoxal (MTNG) 衍生化法合成8-NG-MTNG 以增加目標物的分子量，並運用 C18手動固相萃取匣濃縮30倍。本研究開發之2種前處理方法，其方法偵測極限分別為114及1636 fmol/ml。本研究所開發之2種前處理方法皆無法順利分析尿液中之8-NO2-G，推估人體尿液中之8-NO2-G 低於114 fmol/ml urine。

關鍵字

液相層析串聯質譜儀 ； 連線固相萃取 ； 3-nitrotyrosine ； 3-nitro-4-hydroxyphenylacetic acid ； 8-nitroguanine ； 尿液

並列摘要

3-Nitrotyrosine (3-NTYR) and its metabolite 3-nitro-4-hydroxyphenylacetic acid (NHPA) are recognized to be useful biomarkers for assessing nitration of protein. In this study, a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled with an on-line solid-phase extraction (on-line SPE) system was developed to simultaneously determine 3-NTYR and NHPA in urine. After spiking the deuterium-labeled internal standards, urine was purified and concentrated by manual SPE and HPLC fractionation, followed by on-line SPE LC-MS/MS analysis. The method detection limits (MDL) of 3-NTYR and NHPA were estimated to be 17.7 and 17.1 fmol/ml urine, respectively. Total recovery of 3-NTYR and NHPA were estimated to be 56.5% and 28.9%, respectively. This method was further applied to measure the urinary levels of 3-NTYR and NHPA in 19 healthy adults, including 11 smokers and 8 non-smokers. The mean urinary 3-NTYR levels were 183 ± 128 and 141 ± 130 fmol/mg creatinine for smokers and non-smokers, respectively, while the mean urinary NHPA levels were 261 ± 239 and 306 ± 180 fmol/mg creatinine for smokers and non-smokers, respectively. There was no significant difference in urinary 3-NTYR and NHPA levels between the smokers and non-smokers (p > 0.05).
An on-line SPE LC-MS/MS method was also developed to determine the urinary level of 8-nitroguanine (8-NO2-G), which is a representative biomarker of nitrative stress. After spiking 13C2-15N-8-NO2-G as an internal standard, urine was purified and concentrated by manual SPE and HPLC fractionation, followed by on-line SPE LC-MS/MS analysis. The MDL was estimated to be 114 fmol/ml urine. Although a low MDL was achieved in this study, the urinary level of 8-NO2-G was unable to be detected for 19 healthy adults. It is therefore concluded that background urinary 8-NO2-G level must be below 114 fmol/ml in human.

並列關鍵字

LC-MS/MS ； on-line SPE ； 3-nitrotyrosine ； 3-nitro-4-hydroxyphenylacetic acid ； 8-nitroguanine ； urine

參考文獻

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