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.
Tsikas
D
D
Institute of Clinical Pharmacology, Hannover Medical School, Germany.
Böger
R H
RH
Bode-Böger
S M
SM
Gutzki
F M
FM
Frölich
J C
JC
D005260:Female; D005464:Fluorobenzenes / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D009566:Nitrates / Q000097:blood* / Q000737:chemistry / Q000652:urine*; D009573:Nitrites / Q000097:blood* / Q000737:chemistry / Q000652:urine*; D009587:Nitrogen Isotopes; D012015:Reference Standards; D015203:Reproducibility of Results; D005466:Fluorocarbons
D005464:Fluorobenzenes; D009566:Nitrates; D009573:Nitrites; D009587:Nitrogen Isotopes; C025063:pentafluorobenzyl bromide; D005466:Fluorocarbons