In vivo on-line HPLC-microdialysis: simultaneous detection of monoamines and their metabolites in awake freely-moving rats.

An on-line microbore high performance liquid chromatographic--electrochemical (HPLC--EC) detection method has been developed for the simultaneous measurement of dopamine (3,4-dihydroxyphenethylamine, DA), its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (3-methoxy-4-hydroxyphenylacetic acid, HVA), as well as serotonin (5-hydroxytryptamine, 5-HT) and its metabolise 5-hydroxyindole-3-acetic acid (5-HIAA) in the striatum of awake and freely moving rats. Our method was capable of detecting DOPAC, 5-HIAA, HVA, DA and 5-HT at retention time of 3, 5, 6.5, 9, and 24 min, respectively, in repeated on-line microdialysis sampling. Analysis was performed using a 150 x 1 mm 5 microm C18 microbore column attached directly to a thin-layer radial flow electrochemical cell (UniJet) comprising a 6 mm glassy carbon working electrode. In order to accommodate signal outputs (due to a varying level in the neurochemicals under investigation), an amperometric detector was equipped with a sensitivity programmable controlling software for automatically switching sensitivity inattentively and repeatedly for each collection cycle.