Quantification of ¹⁵N-Nitrate in Urine with Gas Chromatography Combustion Isotope Ratio Mass Spectrometry to Estimate Endogenous NO Production

Abstract

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 GC-C-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.

Simultaneous Measurement of [15N]Nitrate and [15N]Nitrite Enrichment and Concentration in Urine by Gas Chromatography Mass Spectrometry as Pentafluorobenzyl Derivatives

“…Certainly, unlike the method by Houben et al, our GC/ECNICI/MS method does not allow simultaneous quantification of total nitrate and the 15 N-enrichment for [ 15 N]nitrate. Also, because of the incomplete derivatization of nitrate to PFB-ONO 2 , the derivatization procedure proposed by us appears unsuitable for extension to GC/C/IRMS …”

“…Using [ 15 N]nitrate and [ 15 N]nitrite as internal standards, we demonstrated the utility of this procedure for the simultaneous quantification of nitrate and nitrite in various biological fluids by gas chromatography electron capture negative ion chemical ionization mass spectrometry (GC/ECNICI/MS) . Certainly, unlike the method by Houben et al, our GC/ECNICI/MS method does not allow simultaneous quantification of total nitrate and the 15 N-enrichment for [ 15 N]nitrate. Also, because of the incomplete derivatization of nitrate to PFB-ONO 2 , the derivatization procedure proposed by us appears unsuitable for extension to GC/C/IRMS …”

“…In a recent article appearing in this journal, Houben and colleagues reported a gas chromatography combustion isotope ratio mass spectrometry (GC/C/IRMS) method for the quantification in urine of [ 15 N]nitrate and total nitrate, i.e., the sum of [ 14 N]nitrate and [ 15 N]nitrate, which allows one to estimate endogenous nitric oxide (NO) synthesis in humans. Houben and colleagues have demonstrated that involvement of preparative steps in this GC/C/IRMS method, notably selective isolation of nitrate from urine, is highly profitable from an analytical point of view, even if such steps may make the whole method time-consuming.…”

Advancements in Preprocessing and Analysis of Nitrite and Nitrate since 2010 in Biological Samples: A Review

“…Using toluene and ethyl acetate as the extraction solvents is potentially harmful to the experimenters. Another disadvantage is that the nitrate derivatization product was unstable, resulting in unsatisfactory results for nitrate determination [94][95][96].…”

Direct Detection of Inorganic Nitrate Salts by Ambient Pressure Helium-Plasma Ionization Mass Spectrometry

“…Although techniques such as direct analysis in real time (DART) have revolutionized sample analysis by ambient-ionization mass spectrometry, because the need for a solvent spray can be eliminated, the detection of inorganic ions has remained particularly difficult because of their low volatilities. In biology and medicine, the analytical determination of NO 3 – concentration in body fluids is important to monitor the levels of the endogenous signaling molecule NO, ,, which is usually accomplished only by GC/MS or GC-IRMS after lengthy derivatization procedures.…”