University of Medicine and Pharmacy of Târgu Mureş, Gh. Marinescu Street, no. 38, Târgu Mureş 540139, Romania.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Dec 12;911:154-61. doi: 10.1016/j.jchromb.2012.11.006. Epub 2012 Nov 15.
Measurements of nitrite and nitrate are used in biomedical research to estimate the endogenous formation of nitric oxide (an important biomolecule). These anions are also toxins and their concentration is regulated in certain foodstuffs. There are many published methods for detecting nitrite and nitrate but most of them fail to detect nitrite in biological samples. A new HPLC-UV/VIS method was developed which easily detects low concentrations of nitrite and nitrate present in mammal blood, urine and in vegetal samples. The method is based on a pre-column derivatization of nitrite anion using the Griess reaction and direct determination of nitrate using its UV absorbance. A chromatographic process with detection at two wavelengths allows the determination of both anions in one run (23min with column reequilibration included). The limits of quantification in mammal blood are 2ng/ml and 200ng/ml for nitrite and nitrate, respectively. As regards vegetables, due to the need of sample dilution in the preparation steps, these limits are 3 times higher. Concentrations measured in rabbit blood samples ranged from 1.09 to 42.65μg/ml for nitrate and 15.8 to 384.6ng/ml for nitrite. Concentrations in vegetables ranged from below the limit of detection to 4g/kg for nitrate and from below the limit of detection to 369.2μg/kg for nitrite. The specificity of Griess reaction toward nitrite is under discussion since substances able to mimic this reaction were found, leading to compounds with spectral properties in visible domain indistinguishable from that of nitrite related azo dye.
亚硝酸盐和硝酸盐的测量被用于生物医学研究，以估计内源性一氧化氮（一种重要的生物分子）的形成。这些阴离子也是毒素，它们的浓度在某些食品中受到调节。有许多已发表的方法可用于检测亚硝酸盐和硝酸盐，但大多数方法都无法检测生物样本中的亚硝酸盐。我们开发了一种新的 HPLC-UV/VIS 方法，该方法可轻松检测哺乳动物血液、尿液和植物样本中存在的低浓度亚硝酸盐和硝酸盐。该方法基于亚硝酸盐阴离子与格里斯反应的柱前衍生化，以及使用其紫外吸光度直接测定硝酸盐。使用两个波长进行色谱处理可在一次运行中同时测定两种阴离子（包括柱再平衡时间为 23 分钟）。哺乳动物血液中亚硝酸盐和硝酸盐的定量限分别为 2ng/ml 和 200ng/ml。至于蔬菜，由于在准备步骤中需要对样品进行稀释，因此这些限度要高 3 倍。在兔血液样本中测量的浓度范围为硝酸盐 1.09-42.65μg/ml，亚硝酸盐 15.8-384.6ng/ml。蔬菜中的浓度范围从低于检测限到硝酸盐 4g/kg，以及从低于检测限到亚硝酸盐 369.2μg/kg。格里斯反应对亚硝酸盐的特异性正在讨论中，因为发现了能够模拟这种反应的物质，这些物质在可见光谱域中具有与亚硝酸盐相关的偶氮染料无法区分的光谱特性。
