Sodium nitrate (NaNO3), sodium nitrite (NaNO2), 2,3-diaminonaphthalene (DAN, the derivatizing agent), nitrate reductase (from Aspergillus niger), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (β-NADPH), and flavin adenine dinucleotide disodium salt hydrate (FAD) were obtained from Sigma–Aldrich; 15N-NaNO2 and 15N-NaNO3 were purchased from Cambridge Isotope Laboratories.
Standard stock solutions of nitrite and nitrate used to establish calibration curves were individually prepared by dissolving NaNO2 or NaNO3 in deionized water to the desired concentrations. The linear range for NO2− was 0.03 to 2.0 μM, and each calibrator contained 0.5 nmol of the stable isotope-labeled internal standard (SIL-IS) 15N-NO2−. The linear range for NO3− was 15.6 to 1000 μM, and each calibrator contained 6.25 nmol of the SIL-IS 15N-NO3−. The calibrators were processed and analyzed as described later for the PE samples.
Standard stock solutions of nitrite and nitrate used to establish calibration curves were individually prepared by dissolving NaNO2 or NaNO3 in deionized water to the desired concentrations. The linear range for NO2− was 0.03 to 2.0 μM, and each calibrator contained 0.5 nmol of the stable isotope-labeled internal standard (SIL-IS) 15N-NO2−. The linear range for NO3− was 15.6 to 1000 μM, and each calibrator contained 6.25 nmol of the SIL-IS 15N-NO3−. The calibrators were processed and analyzed as described later for the PE samples.