Determination of serotonin in microdialysis samples from rat brain by microbore column liquid chromatography with post-column derivatization and fluorescence detection

The present paper describes a new method for on-line determination of 5-HT in brain microdialysates from awake rats by microbore column liquid chromatography with post-column derivatization and fluorescence detection. The derivatization reagent contained I mM benzylamine and 0.5 mM potassium hexacyanoferrate (III), both dissolved in a mixture of acetonitrile and 25 mM borate buffer (pH 11.0) (1:1, v/v). The limit of detection (S/N = 3) for 5-HT was 0.5 fmol/20 mul. The samples were injected every 20 min onto a microbore column packed with C18 silica gel. The method exhibits an excellent stability over the periods of at least 12-24 h. The basal levels of 5-HT from 25 awake rats were 7.10 +/- 1.06 fmol/20 mul in the dorsal hippocampus and 4.64 +/- 0.91 fmol/20 mul (mean +/- SID) in the striatum. The 5-HT release increased to about 1500% during the perfusion with 100 mM K+ containing Ringer solution or it was reduced to 60 or 40% during the perfusion with I IM tetrodotoxin or calcium free Ringer, respectively. The new method can be used to monitor extracellular 5-HT following acute systemic drug administration. (C) 2001 Elsevier Science B.V. All rights reserved.