Review the Application of Chromatography in the Analysis of Nitric Oxide-derived Nitrite and Nitrate Ions in Biological Fluids

Nitric oxide (NO), a reactive free radical, is an important messenger molecule in biological cells. It plays a critical role in regulating vascular tone, host immunity, neurotransmission, nutrient metabolism and the whole-body homeostasis. NO is metabolized into various products, including plentiful nitrite and nitrate in the circulatory and excretory in vivo, others such as peroxynitrite, nitronium ions, nitroxide ions, and S-nitroso/nitro compounds are unstable and degraded to nitrite and nitrate. The direct measurement of NO is difficult for its reactive chemical properties and short half life. The determinations of nitrite and nitrate could be used to evaluate NO level. Numerous methods are available for measurement of nitrite and nitrate, such as high performance liquid chromatography (HPLC), gas chromatography (GC), ion chromatography (IC), Griess, spectrophotometry, chemiluminescence, fluorescence, catalytic kinetic, infrared spectrophotometry and electron paramagnetic resonance. Of note, chromatography analysis is a common and sensitive method. Based on the reaction of nitrite with 2,3-diaminonaphthalene (DAN) to form N-naphthotriazole (NAT), HPLC coupled with fluorescence detector could lower the limit of detection of nitrite and nitrate, and has no interference of impurities in biological samples. Furthermore, the method involves the GC/LC-MS with the use of N-15 as a stable-isotope label. GC-MS can determine the PFB-NO2 and PFB-NO3 derived from nitrite and nitrate with PFB-Br, while LC-MS can determine NAT derived from nitrite with DAN. GC/LC-MS could not only enable specific assessment for low nitrite and nitrate, but also enable reliable investigations on the metabolic fate and reactions of NO and its metabolites S-nitro compounds in vitro and in vivo. Here, we reviewed the applications of HPLC, GC and IC on the analysis of nitrite and nitrate in various biological fluids. It will provide some suggestions for the study of NO in biochemistry, physiology, and pharmacology researches in the future.