Determination of norepinephrine in microdialysis samples by microbore column liquid chromatography with fluorescence detection following derivatization with benzylamine

A microbore column liquid chromatographic method is described for the deter mination of norepinephrine (NE) in microdialysis samples from rat brain. Th e method is based on precolumn derivatization of ME with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in a highly fluoresc ent and stable benzoxazole derivative. Typically, a 10-mu l sample was mixe d with 10 mu l derivatization reagent containing 0.45 M Caps buffer (pH 12. 0), 0.2 M benzylamine, 10 mM potassium hexacyanoferrate(III), and N,N-dimet hylformamide (1:1:1:15, v/v). The derivatization was carried out at 50 degr ees C for 20 min. Under these conditions only NE and epinephrine produced h igh fluorescence yields at excitation and emission wavelengths of 345 and 4 80 nm, respectively, while fluorescence of other catechols and 5-hydroxyind oles was quenched by 10-100 times. The NE derivative was separated on a rev ersed-phase column (100 x 1.0 mm i.d., packed with C18 silica, 5 mu m) with in 10 min with no late eluting peaks. The mobile phase consisted of 40 mM B ritton-Robinson buffer (pH 7.5) containing 1 mM didodecyldimethylammonium b romide and acetonitrile (34%, v/v), the how rate was 40 mu l/min. The limit of detection (signal-to-noise ratio of 3) for NE was 90 amol in 10 mu l sa mple injected. Microdialysis samples were collected in 5-min intervals from the probes implanted in the hippocampus, frontal cortex, or hypothalamus o f awake rats. The basal extracellular NE levels in the respective areas wer e 4.7 +/- 0.9, 1.8 +/- 0.3, and 0.8 +/- 0.2 fmol/10 mu l (mean +/- SE, n = 7). Perfusion with a Ringer solution containing 100 mM. K+ increased hippoc ampal NE levels by 700%, while NE uptake inhibitors maprotiline and amitrip tyline administered orally or subcutaneously increased extracellular NE in the frontal cortex by about 300%. On the other hand, reserpine (5 mg/kg) re duced cortical NE levels by 40% 3 h after the administration. This new fluo rescence derivatization method provides better selectivity, sensitivity, an d speed for NE determination than the electrochemical detection since no la te-eluting compounds such as dopamine, serotonin, and their metabolites are detectable in the chromatograms of the microdialysis samples. (C) 1999 Aca demic Press.