Journal of Chromatography B, 746 (2000) 199–207 www.elsevier.com / locate / chromb Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection a b a,b, * Hui Li , Cynthia J. Meininger , Guoyao Wu a Department of Animal Science and Faculty of Nutrition, Room 212, Kleberg Building, Texas A&M University, 2471 TAMUS, College Station, TX 77843-2471, USA b Cardiovascular Research Institute and Department of Medical Physiology, The Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA Received 10 January 2000; received in revised form 11 May 2000; accepted 31 May 2000 Abstract 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 cell cultures. A simple and rapid fluorometric HPLC method was developed for determination of nitrite through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite, in standard solution, cell culture medium, or biological samples, readily reacted with DAN under acidic conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). For analysis of nitrate, it was converted to nitrite by nitrate reductase, followed by the derivatization of nitrite with DAN to form NAT. NAT was separated on a 5-mm reversed-phase C column (15034.6 mm, I.D.) guarded by a 40-mm 8 reversed-phase C column (5034.6 mm, I.D.), and eluted with 15 mM sodium phosphate buffer (pH 7.5) containing 50% 18 methanol (flow-rate, 1.3 ml / min). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. Mean retention time for NAT was 4.4 min. The fluorescence intensity of NAT was linear with nitrite or nitrate concentrations ranging from 12.5 to 2000 nM in water, cell culture media, plasma and urine. The detection limit for nitrite and nitrate was 10 pmol/ml. Because NAT is well separated from DAN and other fluorescent components present in biological samples, our HPLC method offers the advantages of high sensitivity and specificity as well as easy automation for quantifying picomole levels of nitrite and nitrate in cell culture medium and biological samples.  2000 Elsevier Science B.V. All rights reserved. Keywords: Nitrite; nitrate 1. Introduction thesis [4,5]. Because NO is a free radical molecule released by cells in picomolar to nanomolar ranges The diverse physiological and pathological roles and has a very short half-life [6], a direct measure- for nitric oxide (NO) in the cardiovascular, immune ment of its production is difficult. Thus, the analysis and nervous systems [1–3] have led to the develop- of nitrite and nitrate, the stable products of NO ment of various methods for determining NO syn- oxidation, is often performed to estimate NO syn- thesis in biological systems and cell cultures [4,7]. The commonly employed methods for nitrite *Corresponding author. Tel.: 11-4098-451-817; fax: 11-4098- determination have included the Griess colorimetric 455-292. E-mail address: g-wu@tamu.edu (G. Wu). assay [8], the chemiluminescence analysis [9], and 0378-4347 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0378-4347(00)00328-5