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 simu ltaneously measuring plasma levels of norepinephrine, epinephrine, dopamine , and 3,4-dihydroxyphenylacctic acid (DOPAC) was developed. Separation of t he 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-octanesu lfonic acid, operated under isocratic condition at a flow rate of 1.2 ml/mi n. 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 co lumn injection was <10 pg for analyte. The assays were linear from basal co ncentrations to 400 ng;ml. The intra- and inter-assay variations were <10 a nd 15%, respectively. The assay has been applied successfully to measure pl asma concentrations of these catecholamines in humans, rabbits and rats. (C ) 1999 Elsevier Science B.V. All rights reserved.