Determination of the stable isotope of nitrite flux in the blood of mice by gas chromatography-mass spectrometry with selected ion monitoring.

A simple and reliable method for the determination of the stable isotope of nitrite (15NO2) flux in the blood of mice is described. It is based on the reaction of 15NO2 with 1-hydrazinophthalazine in acidic solution to form [15N]tetrazolophthalazine, a stable compound which can be extracted with an organic solvent and then determined by gas chromatography-mass spectrometry with selected ion monitoring using the pentafluorobenzoyl ester of 2,4-dinitro-6-sec.-butylphenol as an internal standard. Amounts of 0.2-10 micrograms of 15NO2 can be determined. The detection limit of 15NO2 was 0.1 microgram/ml. This is a specific method for 15NO2. The procedure for determining 15NO2 in the blood of mice involves extraction with solvent, followed by further clean-up by alumina column chromatography; the detection limit is 0.05 microgram. With the new technique we were able to perform a metabolic fate study of nitrite in the blood of mice following a single oral dose of 15NO2 or the stable isotope of nitrate (15NO3).