High-sensitive liquid chromatographic method for determination of neuronal release of serotonin, noradrenaline and dopamine monitored by microdialysis in the rat prefrontal cortex

A high-sensitive liquid chromatographic method based on precolumn derivatization and fluorescence detection allowing simultaneous determination of serotonin (5-HT), noradrenaline (NA) and dopamine (DA) in brain microdialysis samples is described. 5-HT, NA and DA were derivatized with benzylamine and 1,2-diphenylethylenediamine in the presence of potassium hexacyanoferrate(M) and glycine. which yielded to highly fluorescent and stable benzoxazoles. The derivatized samples were separated on a microbore column (150 mm X 1.0 mm i.d., packed with C18 silica, 5 mum) within 60 min. The mobile phase consisted of acetonitrile-Briton-Robinson buffer (pH 7.2) (32:68, v/v) containing 5 mM Na(2)EDTA and 5 mM octanesulfonic acid sodium salt. The detection limits (signal-m-noise ratio of 3) for 5-HT. NA and DA were 76, 42 and 95 amol/10 mul injected on-column, respectively. Microdialysis samples were collected at 10-min intervals from the probe's implanted in the prefrontal cortex of awake rats. The basal levels of 5-HT, NA and DA were 7.3 +/- 0.7. 5.3 +/- 0.31 and 8.1 +/- 0.47 fmol/5 mul (mean +/- S.E.M., n = 5). Following 90-min perfusion with tetrodotoxin (1 muM) or calcium-free Ringer solution. the DA and NA levels level reduced to about 15 and 20%, respectively and the 5-HT levels to 45 and 60% of the basal levels. respectively. Reserpine. 12 h after a dose of 5 mg/kg i.p., reduced the extracellular 5-HT, NA and DA concentrations to about 34. 39 and 32% of the basal levels. respectively. la conclusion, the preset microdialysis/analytical method enables simultaneous monitoring of basal and pharmacologically reduced neuronal release of 5-HT, NA and DA in the rat brain. (C) 2004 Elsevier B.V. All rights reserved.