Automated HPLC Analysis of Tissue Levels of Dopamine, Sertonin, and Several Prominent Amine Metabolites in Extracts from Various Brain Regions

Abstract

Techniques have been developed to permit rapid and accurate automated analyses of rat brain regional extracts for dopamine, dihydroxyphenyl acetic acid, homovanillic acid, serotonin, and 5-hydroxyindole acetic acid. Separations on a C-18 reverse phase column packing were effected with the following mobile phases: one phase consisted of a solution of 1 mM EDTA pus 400 mM sodium acetate, at pH 4.0, mixed with 0.015 volume of methanol; the second phase consisted of 1 mM EDTA plus 500 mM sodium acetate, at pH 5.0. Chemical stability of the 5-hydroxyindole compounds was achieved by adding bisulfite to the perchloric acid homogenizing solution immediately prior to preparation of extracts. Time-related alterations in response of the electrochemical detector were monitored by repeated intermittent analysis of reference tissue extracts during an automated run; results obtained from analyses of the reference solutions permitted construction of detector response curves which were used to correct integrator area values to zero-time detector responsiveness.