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

摘要

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) velence 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 deg C) of the gas chromatograph with the solvent ethyl acetate to form presumably unlabeled and ~(15)N-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 (approx6 mM) in lysed human erythrocytes (100 (mu)L aliquots, no acidification).