Determination of nitrocatecholamines in rat brain using high-performance liquid chromatography-peroxyoxalate chemiluminescence reaction detection

We have developed a method to determine nitrocatecholamines (nitroCAs: nitronorepinephrine (nitroNE), nitroepinephrine (nitroE), and nitrodopamine (nitroDA)) in rat brain using high-performance liquid chromatography-peroxyoxalate chemiluminescence (POCL) reaction detection. This method consists of in-line extraction of amines, including nitroCAs, followed by separation on an ODS column, reduction of nitroCAs with a reducer column, derivatization with ethylenediamine, and finally the POCL reaction detection. Use of the reducer column increased the sensitivity of detection of nitroCAs by 45-172%. The limits of detection (signal-to-noise ratio of 3) were 75 fmol for nitroNE, 125 fmol for nitroE, and 150 fmol for nitroDA. By using this method, not only nitroNE, but nitroE and nitroDA were determined in rat brain. The concentrations of nitroNE and nitroE in rat brain were 9.2 ± 2.0 and 13.5 ± 2.5 pmol/g tissue, respectively (n = 5).