Gas Chromatography-Mass Spectrometry Analysis of Nitrite in Biological Fluids without Derivatization

We report on a gas chromatography-mass spectrometry (GC-MS) method for the quantification of nitrite in biological fluids without preceding derivatization. This method is based on the solvent extraction with ethyl acetate of nitrous acid (HONO, pK(a) = 3.29), i.e., HO(14)NO and (15)N-labeled nitrous acid (HO(15)NO) which was supplied as the sodium salt of (15)N-labeled nitrite and served as the internal standard. HO(14)NO and HO(15)NO react within the injector (at 300 C) of the gas chromatograph with the solvent ethyl acetate to form presumably unlabeled and 15N-labeled acetyl nitrite, respectively. Under negative ion chemical ionization (NICI) conditions with methane as the reagent gas, these species ionize to form O(14)NO(-) (m/z 46) and O(15)NO(-) (m/z 47), respectively. Quantification is performed by selected ion monitoring of m/z 46 for nitrite and m/z 47 for the internal standard. Nitrate at concentrations up to 20 mM does not interfere with nitrite analysis in this method. The GC-MS method was validated for the quantification of nitrite in aqueous buffer, human urine (1 mL, acidification) and saliva (0.1-1 mL, acidification), and hemolysates. The method was applied in studying reactions of nitrite (0-10 mM) with oxyhemoglobin (similar to 6 mM) in lysed human erythrocytes (100 mu L aliquots, no acidification).