Determination of Norepinephrine in Microdialysis Samples by Microbore Column Liquid Chromatography with Fluorescence Detection Following Derivatization with Benzylamine

Abstract

A microbore column liquid chromatographic method is described for the determination of norepinephrine (NE) in microdialysis samples from rat brain. The method is based on precolumn derivatization of NE with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in a highly fluorescent and stable benzoxazole derivative. Typically, a 10-μl sample was mixed with 10 μl derivatization reagent containing 0.45 M Caps buffer (pH 12.0), 0.2 M benzylamine, 10 mM potassium hexacyanoferrate(III), andN,N-dimethylformamide (1:1:1:15, v/v). The derivatization was carried out at 50°C for 20 min. Under these conditions only NE and epinephrine produced high fluorescence yields at excitation and emission wavelengths of 345 and 480 nm, respectively, while fluorescence of other catechols and 5-hydroxyindoles was quenched by 10–100 times. The NE derivative was separated on a reversed-phase column (100 × 1.0 mm i.d., packed with C18 silica, 5 μm) within 10 min with no late eluting peaks. The mobile phase consisted of 40 mM Britton–Robinson buffer (pH 7.5) containing 1 mM didodecyldimethylammonium bromide and acetonitrile (34%, v/v), the flow rate was 40 μl/min. The limit of detection (signal-to-noise ratio of 3) for NE was 90 amol in 10 μl sample injected. Microdialysis samples were collected in 5-min intervals from the probes implanted in the hippocampus, frontal cortex, or hypothalamus of awake rats. The basal extracellular NE levels in the respective areas were 4.7 ± 0.9, 1.8 ± 0.3, and 0.8 ± 0.2 fmol/10 μl (mean ± SE,n= 7). Perfusion with a Ringer solution containing 100 mM K+increased hippocampal NE levels by 700%, while NE uptake inhibitors maprotiline and amitriptyline administered orally or subcutaneously increased extracellular NE in the frontal cortex by about 300%. On the other hand, reserpine (5 mg/kg) reduced cortical NE levels by 40% 3 h after the administration. This new fluorescence derivatization method provides better selectivity, sensitivity, and speed for NE determination than the electrochemical detection since no late-eluting compounds such as dopamine, serotonin, and their metabolites are detectable in the chromatograms of the microdialysis samples.