| Summary: | 碩士 === 中山醫學大學 === 職業安全衛生學系碩士班 === 103 === 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.
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