Determination of nitrotyrosine by HPLC-ECD and its application.

3-nitrotyrosine, a product of tyrosine nitration, is a useful indicator of oxidative damage. We modified the previously reported HPLC-electrochemical detection (ECD) method: specifically, a through-type porous carbon electrode was used as a reducing electrode instead of the mercury-gold amalgam electrode, because the response of the latter changes over time. A combination of reverse-phase HPLC and electrochemical detector passed through -800 mV reduction potential and subsequently under +250 mV oxidation potential allows measurement of 3-nitrotyrosine. The detection limit of this assay was less than 10 fmol. In mice to which lipopolysaccharide (LPS) was administered intraperitoneally, plasma 3-nitrotyrosine levels were elevated, corresponding to LPS dosage. These findings suggest that the improved HPLC-ECD method can be used as a specific and sensitive assay of biological 3-nitrotyrosine and can be applied clinically.