چكيده لاتين :
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 (NO]) in aqueous protein containing cell
lysate, cell media, plasma, serum, urine and other body fluids. The solid extraction reversedphase
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 (NO]) in nanomolar concentration range is measured by the ECD detector with
an amperometric cell, applied voltage of + 800 mV and Ag-AgCI as the reference electrode.
Elusion buffer is 8 mM ammonium chloride containing 25% methanol, flow rate of 1 mllmin 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 ceillysates and cell media ofthe Tib-186 cell
lines. Therefore, this will be a reliable analytical methodfor the nitrite ion analysis under various
conditions of cytokines, LPS, irradiation, or other chemical applications for evaluation of the
probable over expression ofthe inducible nitric oxide synthase ( iNOS) gene in these type ofcells.
