Nitrate Analysis in Biological Fluids by Gas Chromatography–Nitrogen-Phosphorous Detection

Abstract

Nitrate analysis in body fluids is an important part of the study of nitric oxide metabolism. A sensitive, simple procedure for nitrate analysis was developed by producing nitrobenzene from the nitrate in the samples and benzene. The nitrobenzene produced was measured on a gas chromatograph (GC) equipped with a nitrogen-phosphorous detector. Cl−, which interfered with the derivitization procedure, was removed as silver chloride after precipitation with silver lactate. Rat urine samples were also analyzed after elution through a C18 reverse-phase sample preparation column to remove organic molecules that competed with benzene for the nitrate in the sample. A benzene derivative, 2-nitro-1,3-dimethylbenzene (2-nitro-m-xylene), was chosen as an internal standard for GC analysis. Recovery of nitrate was 67 ± 2% in serum and 87 ± 8% in urine. Sensitivity of the procedure was 1 μM. This procedure offers improvements in sensitivity, simplicity, and consistency over previously published procedures and makes possible the measurement of nitrate and determination of isotopic ratios by GC–MS on the same sample.