Simultaneous determination of norepinephrine, dopamine and serotonin in rat brain regions by ion-pair liquid chromatography on octyl silane columns and amperometric detection.

A highly sensitive and specific ion-pair liquid chromatography-electrochemical detection method is described for the simultaneous determination of rat brain regional norepinephrine, dopamine and serotonin levels using octylsilane (C8) columns. These amines were first isolated from tissue homogenates by adsorption on Amberlite CG-50 resin followed by separation on RP-8 columns with a mobile phase consisting of 0.05 M NaH2PO4 (pH 3.0) 0.02 mM EDTA, 1 mM heptanesulphonate-methanol (92:8, v/v) at 1.8 ml/min. Using 3,4-dihydroxybenzylamine as the internal standard, tissue recoveries (mean +/- S.D.) for norepinephrine, dopamine and serotonin were 87.5 +/- 2.6%, 61.8 +/- 10.5% and 72.9 +/- 7.5%, respectively. Assay sensitivities were sufficient for reliable quantitation of at least 200 pg of these compounds in a brain sample. The procedure is readily adaptable to determination of brain epinephrine and normetanephrine levels, as well. Finally, the reversed-phase system employed is highly flexible in that the same column and mobile phase conditions may be used for assay of biogenic amine metabolites.