Jackson Sarah J, Siervo Mario, Persson Emma, McKenna Louise M, Bluck Leslie J C
MRC Human Nutrition Research, Elsie Widdowson Laboratory, Fulbourn Road, Cambridge CB1 9NL, UK.
Rapid Commun Mass Spectrom. 2008 Dec;22(24):4158-64. doi: 10.1002/rcm.3841.
Previous gas chromatography/mass spectrometry (GC/MS) methods for determining nitrate in biological samples involve either hazardous chemicals or produce multiple isomers that can be difficult to quantitate. Modification of these methods, by the nitration of mesitylene instead of benzene and in the presence of trifluoroacetic anhydride rather than sulphuric acid, should enable simple isotopic quantitation for use in tracer studies, for example, in the measurement of nitric oxide production. Desiccated urine and saliva samples, in addition to aqueous labelled and unlabelled nitrate standards, were treated with trifluoroacetic anhydride and mesitylene at 70 degrees C for 1 h, cooled, then sequentially washed with deionised water and aqueous sodium bicarbonate. The solution of nitromesitylene in mesitylene was separated, dried and analysed by GC/MS. The full mass spectra exhibited abundant ions at m/z 165 and 166 corresponding to the unlabelled and labelled molecular species of nitromesitylene, respectively. Selected ion monitoring of these masses for a series of gravimetrically prepared standards indicated good agreement with isotopic enrichments in the range 0.0625-5 mole % excess, and at nitrate concentrations within the physiological range of 0.078-2 mmol/L. Derivatised samples were stable with respect to isotopic enrichments and nitrate concentrations at -20 degrees C for up to 21 days and exhibited excellent repeatability. Nitration of mesitylene proved to be a simple and rapid method for the measurement of isotope ratios in aqueous nitrates by GC/MS, which has applications in tracer studies and in concentration determinations by isotope dilution techniques for nitric oxide production.
以往用于测定生物样品中硝酸盐的气相色谱/质谱（GC/MS）方法，要么涉及危险化学品，要么会产生多种难以定量的异构体。通过用均三甲苯而非苯进行硝化，并在三氟乙酸酐而非硫酸存在的条件下对这些方法进行改进，应该能够实现简单的同位素定量，用于示踪研究，例如，在一氧化氮生成量的测量中。除了水性标记和未标记的硝酸盐标准品外，将干燥的尿液和唾液样品与三氟乙酸酐和均三甲苯在70℃下处理1小时，冷却，然后依次用去离子水和碳酸氢钠水溶液洗涤。将均三甲苯中的硝基均三甲苯溶液分离、干燥并通过GC/MS进行分析。全质谱图在m/z 165和166处分别显示出对应于未标记和标记的硝基均三甲苯分子物种的丰富离子。对一系列重量法制备的标准品对这些质量数进行选择离子监测表明，在0.0—5摩尔%过量的同位素富集范围内以及在0.078—2 mmol/L的生理范围内的硝酸盐浓度下，与同位素富集情况吻合良好。衍生化样品在-20℃下，在同位素富集和硝酸盐浓度方面长达21天保持稳定，并表现出出色的重复性。事实证明，均三甲苯硝化是一种通过GC/MS测量水性硝酸盐中同位素比率的简单快速方法，可应用于示踪研究以及通过同位素稀释技术测定一氧化氮生成量的浓度。
