Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection

Measurement of nitrite and nitrate, the stable oxidation products of nitric oxide (NO), provides a useful tool to study NO synthesis in vivo and in ce ll cultures. A simple and rapid fluorometric HPLC method was developed for determination of nitrite through its derivatization with 2,3-diaminonaphtha lene (DAN). Nitrite, in standard solution, cell culture medium, or biologic al samples, readily reacted with DAN under acidic conditions to yield the h ighly fluorescent 2,3-naphthotriazole (NAT). For analysis of nitrate, it wa s converted to nitrite by nitrate reductase, followed by the derivatization of nitrite with DAN to form NAT. NAT was separated on a 5-mu m reversed-ph ase C-8 column (150x4.6 mm, I.D.) guarded by a 40-mu m reversed-phase C-18 column (50x4.6 mm, I.D.), and eluted with 15 mM sodium phosphate buffer (pH 7.5) containing 50% methanol (flow-rate, 1.3 ml/min). Fluorescence was mon itored with excitation at 375 nm and emission at 415 nm. Mean retention tim e for NAT was 4.4 min. The fluorescence intensity of NAT was linear with ni trite or nitrate concentrations ranging from 12.5 to 2000 nM in water, cell culture media, plasma and urine. The detection limit for nitrite and nitra te was 10 pmol/ml. Because NAT is well separated from DAN and other fluores cent components present in biological samples, our HPLC method offers the a dvantages of high sensitivity and specificity as well as easy automation fo r quantifying picomole levels of nitrite and nitrate in cell culture medium and biological samples. (C) 2000 Elsevier Science B.V. All rights reserved .