Determination of 3-nitrotyrosine by high-pressure liquid chromatography with a dual-mode electrochemical detector

3-Nitrotyrosine, a product of tyrosine nitration, is useful as a marker for the generation of reactive nitrogen oxide species with short half-lives su ch as peroxynitrite. A reverse-phase high-pressure liquid chromatographic m ethod using a dual-mode electrochemical detector in series with a photodiod e array detector has been developed to determine the levels of 3-nitrotyros ine in biological samples. The principle of this method involves reduction of 3-nitrotyrosine at an upstream gold amalgam electrode and oxidation of t he resulting product(s) at a downstream glassy carbon electrode. 3-Nitrotyr osine is quantified by the amount of the current generated at the downstrea m electrode, and a femtomole detection level can be achieved. The disappear ance of the corresponding peak when the electrochemical detector is used on ly in the single oxidative:mode provides additional evidence for the identi ty of 3-nitrotyrosine in the sample. Tyrosine from the same sample is deter mined by its UV absorption at 280 nm, thus eliminating the need for an inte rnal standard. With this method a dose-dependent increase of 3- to 10-fold in the levels of protein 3-nitrotyrosine was observed in the blood plasma, and a 2- to 4-fold increase in the lung cytosols, of rats treated with the lung carcinogen and nitrating agent tetranitromethane. (C) 2000 Academic Pr ess.