Simultaneous Determination of Dopamine, Norepinephrine, Tyramine and Octopamine by Reverse-Phase High Performance Liquid Chromatography with Electrochemical Detection

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Abstract
A method is described for the simultaneous separation and estimation of the catecholamines, norepinephrine and dopamine and their monohydroxy-equivalents, octopamine and tyramine. The method employs high-performance liquid chromatographic separation of the compounds on a C18 reverse-phase column with a mobile phase containing methanol as the organic modifier, octane sulphonate as an ion-pair reagent and acetic acid/ammonium hydroxide buffer. The influences of electrode potential and solvent pH on detector response were studied, and the optimal conditions identified as detector potential of 0.95 volts and pH 6.0. The technique of post-column mixing was introduced to provide optimal pH conditions for detector response without the constraint of on-column oxidation of catecholamines. The effects of buffer ionic strength on retention factors and detector response were also investigated and, on the basis of the results obtained, the optimal buffer strength was identified as 0.08–0.09 molar. The described procedure can be used for simultaneous estimation of catecholamines and monohydroxyphenolamines at concentrations between 200–5000 pg.