Determination of Nitrite and Nitrate in Cell Culture Medium by Reversed-Phase High-Performance Liquid Chromatography with Simultaneous UV-VIS and Fluorescence Detection

A reliable method for determination of nitrite and nitrate in cell culture medium has been developed. The method is based on pre-column derivetization of nitrite with 2,3- diaminonaphthalene (DAN) under acidic medium to yield 2,3-naphthotriazole (NAT). For determination of nitrate, it was converted to nitrite by nitrate reductase before the derivetization step. NAT was separated on a 5 μm C18 column (250x4.6 mm, I.D) guarded by a 10μm C18 column (20X4.6 mm, I.D), and eluted with 15 mM phosphate buffer (pH 7.5) containing 35% acetonitrile (flow rate 1.0 mL/min). Average retention time of NAT was 12.4 min. Absorbance of NAT was monitored at 369 nm and its fluorescence was monitored at 375 nm for excitation and 415 nm for emission. Calibration curves of nitrite or nitrate were linear in the range 0.03-2.0 μM for fluorescenc detection and 0.5-50 μM for UV-VIS detection. Correlation coefficients were better than 0.994 and mean recoveries were 96±7 %.