Nitric oxide (NO) is a cell-signaling agent in physiologic processes including vasodilation, inhibition of clotting, and inflammation. A sensitive chromatography-mass spectrometry method was developed to measure two of its metabolites, nitrite (NO2- ) and nitrate (NO3- ), in biological fluids. Internal standards, 15NO2- and 15NO3-, were added to the fluid at concentrations of 80.0 and 800.0 M, respectively. Natural 14NO2- and 14NO3- plus the internal standards were then converted to volatile derivatives:

Because biological fluids are so complex, the derivatives were first isolated by high performance liquid chromatography. For quantitative analysis, liquid chromatography peaks corresponding to the two products were injected into a gas chromatograph, ionized by negative ion chemical ionization (giving major peaks for NO2- and NO3-) and the products measured by selected ion monitoring. Results are shown in the figure. If the 15N internal standards undergo the same reactions and same separations at the same rate as the 14N analytes, then the concentrations of analytes are simply
[14NO-x] = [15NO-x](R - Rblank)
where R is the measured peak area ratio (m/z 46/47 for nitrite and m/z 62/63 for nitrate) and Rblank is the measured ratio of peak areas in a blank prepared from the same buffers and reagents with no added nitrate or nitrite. The ratios of peak areas are m/z 46/47 0.062 and m/z 62/63 0.538. The ratios for the blank were m/z 46/47 0.040 and m/z 62/63 0.058. Find the concentrations of nitrite and nitrate in the urine.

F. O-NO2 NO F F mz 62 for WN m/z 63 for N Pentafluorobenzy bromide O-NO F F mz46 for N m47 for 15N -no; DNO "NO 14NO 2.6 2.8 3.0 3.2 3.4 3.6 Time (min)