Quantification of ¹⁵N-nitrate in urine with gas chromatography combustion isotope ratio mass spectrometry to estimate endogenous NO production

The use of stable isotope labeled substrates and subsequent analysis of urinary nitrate, forms a noninvasive test for evaluation of the in vivo NO metabolism. The present paper describes a new method for simultaneous quantification of 15N-nitrate and total nitrate with gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS). Nitrate, isolated from urine with a nitrate selective resin, was reduced to nitrite using copperized cadmium. Subsequently, Sudan I was formed by diazotation. Sudan II was added as internal standard, and both molecules were analyzed with GCC-IRMS as tert-butyldimethylsilyl derivatives. The accuracy was determined during a recovery study of two different known nitrate concentrations and two 15N- enrichments. A recovery of 101.6% and 103.9% for total nitrate and 107.6% and 91.2% for 15N-nitrate was obtained, respectively. The validated method was applied on complete 72 h urine collections after intravenous administration of 15N-nitrate and 15N-arginine in humans. On average, 51.8% (47.0-71.0%) of administered 15N-nitrate was excreted, while 0.68% (0.44-1.17%) of 15N-arginine was metabolized to nitrate. In conclusion, this method can be used for accurate simultaneous determination of 15N-nitrate and total nitrate concentrations in urine and can be applied in clinical studies for noninvasive evaluation of NO metabolism in vivo.