Developed Method Application for Nitrite Ion (NO2¯ ) Analysis of Tib -186 Macrophage Like Cell Lines by Rapid Isocratic HPLC System with High Sensitive Glassy Carbon Electrochemical Detector

Abstract

A rapid isocratic method of high performance liquid chromatography system (HPLC) with a glassy carbon working electrode of electrochemical detector is set up for quantitative detection of trace amount of nitrite ion (NO2¯) in aqueous protein containing cell lysate, cell media, plasma, serum, urine and other body fluids. The solid extraction reversed phase cartridges (Sep-pak) are used for deproteinizing and purification of the samples. Nitrite ion is the only stable end product of autoxidation of nitric oxide (NO) ; which is a highly reactive paramagnetic molecule produced via the enzymatic conversion of L-arginine to L-citroline. The enzyme involved in this process is the inducible nitric oxide synthase (iNOS), the main isoform of the enzymes in macrophage and macrophage like cell lines such as Raw-264, J774, and Ic-21. Nitrite ion (NO2¯) in nanomolar concentration range is measured by the ECD detector with an amperometric cell, applied voltage of + 800 mV and Ag-AgCl as the reference electrode. Elusion buffer is 8 mM ammonium chloride containing 25% methanol, flow rate of 1 ml/min and column temperature set at 20° C. The reproducibility of sample preparation and analysis had a coefficient of variance (c.v.) less than 10 % in the cell lysates and cell media of the Tib-186 cell lines. Therefore, this will be a reliable analytical method for the nitrite ion analysis under various conditions of cytokines, LPS, irradiation, or other chemical applications for evaluation of the probable over expression of the inducible nitric oxide synthase ( iNOS ) gene in these type of cells.