Quantitation of nitric oxide-derived nitrite from activated macrophages using microdialysis sampling

An HPLC method for detecting nitrite in microdialysis samples obtained from activated RAW 264.7 macrophages in cell culture has been developed. Nitrite was quantified using a pre-column derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by HPLC–UV analysis of the azide product. For dialysates, the detection limit of nitrite was 750 nM and the quantitation limit was 2.5 μM. The microdialysis relative recovery of nitrite in the macrophage cell culture medium was determined to be 86±2% (n=3) at a flow rate of 0.7 μl/min. Nitrite produced from activated macrophages was measured immediately after lipopolysaccharide (LPS) stimulation using microdialysis sampling.