運用連線固相萃取-液相層析串聯質譜儀搭配同位素稀釋法分析尿液中硝酸鹽 及亞硝酸鹽

Urinary analysis of nitrite/nitrate by isotope dilution LC-MS/MS with automated solid-phase extraction

林柏慧(Bo-Huei Lin)

指導教授 ： 趙木榮

中山醫學大學/健康管理學院/職業安全衛生學系碩士班/碩士(2015年)

https://doi.org/10.6834/CSMU.2015.00134

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) 快速準確地分析尿液中亞硝酸鹽 (nitrite, NO2-) 與硝酸鹽 (nitrate, NO3-)。尿液樣本加入同位素內標準品後與 2,3-diaminonaphthalene (DAN) 進行衍生反應形成產物 2,3-naphthotrliazoe (NAT)，即可直接上機分析。nitrite 及 nitrate 的偵測極限分別為 5 及 6 fmol。組內變異 (intra-day) 和 組間變異 (inter-day) 之變異係數皆小於 10 %。nitrite 及 nitrate 的平均回收率為 95.3% 及 103%。本研究之分析方法需探討尿液樣本及標準品中 nitrite 與 DAN 衍生反應之最佳濃度及硝酸鹽還原酶 (將 nitrate 轉化為 nitrite) 之最佳添加濃度；尿液樣本及標準品進行 DAN 衍生反應，DAN 反應莫爾濃度比分別為 DAN/nitrite:1.21:1 及 10593:1。所需 DAN 反應莫爾濃度相差 8754 倍。另一方面標準品與尿液樣本中需要硝酸鹽還原酶濃度為 25 mU，能完全將 nitrate 轉化為 nitrite。將此方法應用於 50 個健康成年人的尿液樣本，尿液中的 nitrite 及 nitrate 背景濃度平均值分別為 0.51±0.48 mol/g creatinine 及 1404±587 mol/g creatinine。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尿液樣本檢測。

關鍵字

連線固相萃取 ； 液相層析串聯質譜儀 ； 亞硝酸鹽 ； 硝酸鹽 ； 2, 3-diaminonaphthalene ； 衍生反應 ； 尿液

並列摘要

A highly sensitive quantitative liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed for measuring urinary nitrite (NO2-) and nitrate (NO3-). After added the isotope internal standard, urine samples were directly reacted with 2,3-diaminonaphthalene (DAN) to form 2,3-naphthotrliazoe (NAT),followed by on-line solid-phase extraction (SPE) LC-MS/MS analysis.The detection limit of were estimated to be 5 fmol and 6 fmol for nitrite and nitrate, respectively. Intra- and inter-day imprecision (CV) were <10%. Mean recovery of nitrite and nitrate in urine were 95.3% and 103%,respectively. The method was applied to assess the optimal addition of DAN for derivatization reaction and the optimal addition of nitrate reductase for conversion of nitrate to nitrite, respectively. Derivatization optimization revealed that to achieve complete derivatization reaction, an excess of DAN is required for urine samples and the molar ratio required (DAN/nitrite: 10593) in urine is about 8754 times higher than that of in nitrite standard solutions (DNA/nitrite: 1.21). Optimization addition of nitrate reductase was found to be 25 mU for conversion of nitrate to nitrite for both urine samples and nitrate standard solutions. This method was also applied to measure the urine samples of 50 healthy subjects. The results showed that the mean of urinary nitrite and nitrate were 0.51±0.48 mol/g creatinine and 1404±587 mol/g creatinine, respectively. This method is capable of high-throughput quantitative analysis of urinary
nitrite and nitrate.

並列關鍵字

on-line SPE ； LC-MS/MS ； nitrite ； nitrate ； 2,3-diaminonaphthalene ； derivatization ； urine

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