Nitrite and nitrate can be accurately measured in samples of vegetal and animal origin using an HPLC-UV/VIS technique

Abstract

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.