Simultaneous determination of norepinephrine, serotonin, and 5-hydroxyindole-3-acetic acid in microdialysis samples from rat brain by microbore column liquid chromatography with fluorescence detection following derivatization with benzylamine

Abstract

A microbore column liquid chromatographic method for the simultaneous determination of norepinephrine (NE), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5HIAA) in microdialysis samples from rat brain is described. The method is based on precolumn derivatization of NE, 5HT, and 5HIAA with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in the corresponding highly fluorescent and stable benzoxazole derivatives. A 15-μl sample was mixed with 15 μl derivatization reagent solution containing 0.3 M 3-cyclohexylaminopropanesulfonic acid buffer (pH 12.0), 0.5 M benzylamine, 10 mM potassium hexacyanoferrate(III), and methanol (1/1/1/12, v/v/v/v). The derivatization was carried out at 50 °C for 20 min. The benzylamine derivatives of NE, 5HT, and 5HIAA were separated on a reversed-phase column (100×1.0 mm i.d., packed with C18 silica, 5 μm) within 30 min. The mobile phase consisted of 15 mM acetate buffer (pH 5.0) and acetonitrile (31%, v/v); the flow rate was 50 μl/min. The detection limits (signal-to-noise ratio of 3) for NE, 5HT, and 5HIAA in the injection volume of 20 μl were 90, 210, and 260 amol, respectively. Microdialysis samples were collected in 7.5-min intervals from the probes implanted in the hippocampus and prefrontal cortex of awake rats. The basal levels of NE, 5HT, and 5HIAA in the dialysates from the hippocampus were 4.2±0.5, 4.9±0.6, and 934.1±63.4 fmol/20 μl , and those from the prefrontal cortex were 6.0±1.2,5.51.3, and 669.1±96.0 fmol/20 μl (mean ± SE, n=25), respectively. The NE and 5HT levels were altered by perfusion of high-potassium or low-calcium solution and following antidepressant drugs imipramine and desipramine. It is concluded that the new fluorescence derivatization method in combination with microbore column liquid chromatography allows the simultaneous determination of NE, 5HT, and 5HIAA in the microdialysis samples at higher sensitivity, providing easier maintenance in routine use than that achieved by high-performance liquid chromatographic methods with electrochemical detection.