Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography-mass spectrometry using their 15N-labelled analogs.

For the quantification of nitrite and nitrate, the stable metabolites of L-arginine-derived nitric oxide (NO) in human urine and plasma, we developed a gas chromatographic-mass spectrometric (GC-MS) method in which [15N]nitrite and [15N]nitrate were used as internal standards. Endogenous nitrite and [15N]nitrite added to acetone-treated plasma and urine samples were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [15N]nitrate they were reduced to nitrite and [15N]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-microliter aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [15N]nitrite ([15N]nitrate). The intra- and inter-assay relative standard deviations for the determination of nitrite and nitrate in urine and plasma were below 3.8%. The detection limit of the method was 22 fmol of nitrite. Healthy subjects (n = 12) excreted into urine 0.49 +/- 0.25 of nitrite and 109.5 +/- 61.7 of nitrate (mean +/- S.D., mumol/mmol creatinine) with a mean 24-h output of 5.7 mumol for nitrite and 1226 mumol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean +/- S.D., mumol/l) 3.6 +/- 0.8 and 68 +/- 17, respectively.

[1]  J. Frölich,et al.  Determination of endothelium-derived nitrite/nitrate by gas chromatography/tandem mass spectrometry using (15N)NaNO2 as internal standard. , 1992, Biological mass spectrometry.

[2]  T. Shono,et al.  Gas chromatographic determination of inorganic anions as pentafluorobenzyl derivatives , 1983 .

[3]  Å. Wennmalm,et al.  Dependence of the metabolism of nitric oxide (NO) in healthy human whole blood on the oxygenation of its red cell haemoglobin , 1992, British journal of pharmacology.

[4]  C. Kablitz,et al.  Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy. , 1992, The Journal of clinical investigation.

[5]  H. Schmidt,et al.  Arginine is a physiological precursor of endothelium-derived nitric oxide. , 1988, European journal of pharmacology.

[6]  D. Tsikas,et al.  L-arginine infusion decreases peripheral arterial resistance and inhibits platelet aggregation in healthy subjects. , 1994, Clinical science.

[7]  K. Krohn,et al.  Nitrogen-13-labeled nitrite and nitrate: distribution and metabolism after intratracheal administration. , 1981, Science.

[8]  Å. Wennmalm,et al.  Detection of endothelial-derived relaxing factor in human plasma in the basal state and following ischemia using electron paramagnetic resonance spectrometry. , 1990, Analytical biochemistry.

[9]  R. Furchgott.  The role of endothelium in the responses of vascular smooth muscle to drugs. , 1984, Annual review of pharmacology and toxicology.

[10]  S. Tannenbaum,et al.  Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. , 1982, Analytical biochemistry.

[11]  S. Eguchi,et al.  l‐ARGININE INFUSION INDUCES HYPOTENSION AND DIURESIS/NATRIURESIS WITH CONCOMITANT INCREASED URINARY EXCRETION OF NITRITE/NITRATE AND CYCLIC GMP IN HUMANS , 1992, Clinical and experimental pharmacology & physiology.

[12]  S. Moncada,et al.  Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor , 1987, Nature.

[13]  D. Tsikas,et al.  Determination of nitrite as pentafluorobenzyl derivative by RP-HPLC and UV detection with and without ion-pair extraction , 1992 .

[14]  R. Sievers,et al.  Microdetermination of nitrates and nitrites in saliva, blood, water, and suspended particulates in air by gas chromatography. , 1976, Journal of chromatography.