Validation of assay based on ferricyanide to stabilize nitrite in whole blood. (A) Original registration of reductive tri-iodide-based chemiluminescence after injection of 200 μL plasma, treated erythrocytes, and whole blood with or without addition of acid sulfanilamide. Loss of signal after sulfanilamide indicates specificity of the assay for nitrite. (B) Recovery and linearity of nitrite spiked in whole blood to which nitrite preservation solution was added. (C) Freeze-thaw and sample processing does not influence nitrite concentration in whole blood. Open circles represent measurements after freezing, storage at -80°C for 4 days, thawing, and deproteination. With an injection volume of 200 μL, 1 pmol nitrite could be recovered at a signal-to-noise ratio of 3:1. (D) Stability of nitrite in whole blood. Spiking of nitrite to a final concentration of 1 μM and measurement of nitrite concentration of whole blood up to 24 hours after nitrite addition. Samples were either treated with nitrite preservation solution (○) or saline (□) after addition of nitrite. The inset illustrates the disappearance of nitrite in whole blood with or without nitrite preservation solution. Samples were stored at room temperature during incubation.