Nitrate analysis by capillary gas chromatography

A microanalytical gas chromatographic (GC) method for the analysis of nitrate in rat urine is described. The method involves the conversion of nitrate to nitromesitylene and quantitation using 3,4-dimethylnitrobenzene as an internal standard. The nitroaromatics were separated on a wide-bore capillary column and detected with a nitrogen-phosphorus thermionic detector. The method exhibited linearity over the range of 1.0 to 1000 microM nitrate, and the detection limit was 0.5 microM nitrate (200-microliters sample). The coefficient of variation (CV) range for intra-day precision was 2.2 to 5.8% (20 microM level) and 3.1 to 6.5% (200 microM level). Inter-day CVs ranged from 2.0 to 6.1% for the samples tested. The average recovery was 77% (20 microM level) and 80% (20 microM level). The accuracy of the GC method compared favorably with results obtained from a standard colorimetric nitrate assay. Interference by urinary chloride was eliminated by pretreatment of samples with saturated silver acetate. Both processed and unprocessed samples were stable for at least 60 days at -15 degrees C. The procedure was used to measure urinary nitrate in rats fed a custom low-nitrate diet.