A simple high-performance liquid chromatography assay for simultaneous determination of plasma norepinephrine, epinephrine, dopamine and 3,4-dihydroxyphenyl acetic acid.

A reversed-phase HPLC assay coupled with electrochemical detection for simultaneously measuring plasma levels of norepinephrine, epinephrine, dopamine, and 3,4-dihydroxyphenylacetic acid (DOPAC) was developed. Separation of the catecholamines and the internal standard isoproterenol was obtained by a mobile phase consisting of 7% methanol in 0.1 M citrate buffer containing 0.3 mM sodium ethylenediaminetetraacetic acid (EDTA), and 0.5 mM 1-octanesulfonic acid, operated under isocratic condition at a flow rate of 1.2 ml/min. The potential of the guard cell was set at +650 mV, the first electrode of the analytical cell at +100 mV and the second at + 350 mV. Using a signal-to-noise ratio of > 3, the minimum detection limit assessed by direct on column injection was < 10 pg for analyte. The assays were linear from basal concentrations to 400 ng/ml. The intra- and inter-assay variations were < 10 and 15%, respectively. The assay has been applied successfully to measure plasma concentrations of these catecholamines in humans, rabbits and rats.