QUANTIFICATION OF NITRITE AND NITRATE IN HUMAN URINE AND PLASMA AS PENTAFLUOROBENZYL DERIVATIVES BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY USING THEIR N-15-LABELED 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 [N-15]nitrite and [N-15]nitrate were used as internal standards. Endogenous nitrite and [N-15]nitrite added to acetone-treated plasma and urine sample were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [N-15]nitrate they were reduced to nitrite and [N-15]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-mu l aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [N-15]nitrite ([N-15]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., mu mol/mmol creatinine) with a mean 24-h output of 5.7 mu mol for nitrite and 1226 mu mol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean+/-S.D., mu mol/l) 3.6+/-0.8 and 68+/-17, respectively.