Simultaneous Analysis of Nitrite and Nitrate in Whole Blood by Ion Chromatography

Abstract

A simple and modern method for simultaneous analysis of nitrite and nitrate in whole blood has been devised by ion chromatography with an autosuppressor and a conductivity detector. To our knowledge, this is the first report for such analysis dealing with whole blood as a specimen. A 1 mL volume of whole blood was mixed with 2 mL of Milli Q water, vortex‐mixed for hemolysis, and ultrafiltered to obtain a clear extract solution. It was passed through double connected mini-cartridges of silver resin and a silver absorbing material to remove a high concentration of chloride ion usually present in whole blood. An aliquot of the filtrate was analyzed on a Dionex DX‐500 instrument with a microbore IonPac® AS9‐HC column (2×250 mm). The present method gave very low backgrounds for blank whole blood at locations where nitrite should appear, and around the endogenous peak of nitrate. The spiked nitrite markedly decreased in whole blood, probably by the action of hemoglobin in vitro; it was only 10–20% 2 h after the addition. Therefore, quantitative validation had to be made only for nitrate. The recoveries of nitrate from whole blood were more than 90%; the linearity was confirmed in the range of 5–100 µg mL−1 for whole blood. Detection limit of nitrate in whole blood was about 0.5 µg mL−1. Coefficients of intra‐ and inter‐day variations were 3.66–7.14%. Using the present method, nitrite and nitrate concentrations were measured for human blood specimens after sniffing isobutyl nitrite gas in two male subjects. The endogenous nitrate concentrations without such sniffing were 1.27 to 7.60 µg mL−1 for seven subjects.