Smythe G A, Matanovic G, Yi D, Duncan M W
Ray Williams Biomedical Mass Spectrometry Facility, Faculty of Medicine, University of New South Wales, Sydney, Australia.
Nitric Oxide. 1999;3(1):67-74. doi: 10.1006/niox.1999.0210.
The nitration of aromatic compounds by electrophilic substitution is often utilized in analyses of nitrate concentrations in physiological samples by gas chromatographic methods. Problems associated with the use of concentrated sulfuric acid, which is normally used to catalyze this reaction, led us to investigate an alternative method. We describe here a facile GC/MS assay for nitrate in plasma or urine samples which takes advantage of the ability of trifluoroacetic anhydride (TFAA) to catalyze the nitration of aromatics. Toluene, utilized as both reaction solvent and electrophile, was shown to react with nitrate in the presence of TFAA to quantitatively produce the three nitrotoluene isomers (ratio o-:m:p-, approx 57:3:40). Following the incorporation of 15N-labeled nitrate as internal standard, nitrotoluene was quantified using GC/MS by analysis of the selected the ion pairs m/z 120 and 121 (M+ -OH) for the o-isomer or m/z 137 and 138 (molecular ion, M+) for the p-isomer. The limit of detection for nitrate after TFAA-catalyzed conversion to nitrotoluene was less than 100 fmol on column (s/n; 40:1). The TFAA-based GC/MS assay was compared with that utilizing the usual catalyst, concentrated sulfuric acid. With the exception of samples containing nitroarginine analogues, good correlation was found for urine or plasma samples analyzed using either a standard sulfuric acid-catalyzed method or the TFAA-catalyzed procedure. Nitroarginine analogues, which can be present in samples following their use as nitric oxide synthase inhibitors, did not decompose under the conditions of the TFAA-catalyzed assay and, hence, do not give rise to significant interference with nitrate analysis in this procedure. In contrast, catalytic sulfuric acid caused nitroarginine analogues to decompose (essentially quantitatively) and cause spuriously high nitrate levels in samples. The use of TFAA as a catalyst for the nitration of toluene enables a facile and sensitive GC/MS analysis for nitrate which offers improved safety and sample integrity.
通过亲电取代对芳香族化合物进行硝化，常用于气相色谱法分析生理样品中的硝酸盐浓度。使用通常用于催化该反应的浓硫酸会带来一些问题，这促使我们研究一种替代方法。我们在此描述一种用于血浆或尿液样品中硝酸盐的简便气相色谱/质谱分析方法，该方法利用了三氟乙酸酐（TFAA）催化芳香族硝化反应的能力。甲苯既用作反应溶剂又用作亲电试剂，结果表明在TFAA存在下它能与硝酸盐反应，定量生成三种硝基甲苯异构体（邻位：间位：对位比例约为57:3:40）。加入15N标记的硝酸盐作为内标后，通过气相色谱/质谱分析选定的离子对（邻位异构体为m/z 120和121（M+ -OH），对位异构体为m/z 137和138（分子离子，M+））对硝基甲苯进行定量。经TFAA催化转化为硝基甲苯后，硝酸盐的检测限在柱上小于100飞摩尔（信噪比；40:1）。将基于TFAA的气相色谱/质谱分析方法与使用常用催化剂浓硫酸的方法进行了比较。除了含有硝基精氨酸类似物的样品外，使用标准硫酸催化法或TFAA催化法分析的尿液或血浆样品之间具有良好的相关性。硝基精氨酸类似物在用作一氧化氮合酶抑制剂后可能存在于样品中，在TFAA催化分析的条件下不会分解，因此在该方法中不会对硝酸盐分析产生显著干扰。相比之下，催化硫酸会使硝基精氨酸类似物分解（基本上是定量的），并导致样品中硝酸盐水平出现假性升高。使用TFAA作为甲苯硝化反应的催化剂，能够实现对硝酸盐的简便且灵敏的气相色谱/质谱分析，具有更高的安全性和样品完整性。
