Determination of serotonin, noradrenaline, dopamine and their metabolites in rat brain extracts and microdialysis samples by column liquid chromatography with fluorescence detection following derivatization with benzylamine and 1,2-diphenylethylenediamine

A highly selective and sensitive column liquid chromatographic method for fluorescence determination of serotonin (5-HT), dopamine (DA), noradrenaline (NA) and their related metabolites 5-hydroxyindole-3-acetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC) following derivatization with benzylamine and 1,2-diphenylethylenediamine (DPE) is described. The monoamines and the metabolites (20 μl samples) were derivatized in a two-step reaction, initiated with 20 μl of 0.3 M benzylamine in 0.3 M 3-cyclohexylaminopropanesulfonic acid (CAPS) buffer (pH 10.0), (for 5-HT, 5-HIAA, 2 min, 24 °C) and followed by 20 μl of 0.1 M DPE in 0.3 M glycine buffer (pH 10.0), (for DA, NA, DOPAC, 20 min, 50 °C). Both reagents contained 0.02 M potassium hexacyanoferrate(III) and 50% (v/v) methanol. The resulting highly fluorescent and stable benzoxazole derivatives were isocratically separated on a reversed-phase column (150 mm×1.5 mm i.d., packed with C18 silica, 5 μm) within 45 min. Using fluorescence detection at ex. and em. wavelengths of 345 and 480 nm, respectively, the detection limit (signal-to-noise ratio of 3) for 5-HT, DA, NA, 5-HIAA, L-DOPA and DOPAC ranged between 0.08 and 5.65 fmol per 20-μl injection (12–847.5 pM in standard solution). The concentrations of monoamines (expressed in μg/g wet weight, mean±S.E.M., n=5) in tissue extracts from the rat striatum were: 0.45±0.05 (5-HT), 4.27±0.08 (DA), 0.27±0.04 (NA), 0.55±0.06 (5-HIAA), 1.26±0.16 (L-DOPA) and 1.62±0.11 (DOPAC). Microdialysis samples were collected in 20 min intervals from the probes implanted in the striatum of awake rats. The basal monoamine levels (in fmol/20 μl, mean±S.E.M., n=5) in the dialysates were: 4.1±0.7 (5-HT), 78.4±9.1 (DA), 6.4±0.8 (NA), 785.5±64.5 (5-HIAA) and 5504.5±136.5 (DOPAC). It is concluded that the new fluorescence derivatization protocol provides an excellent means for simultaneous determination of all three monoamines both in the complex samples (e.g. brain homogenates) and also at trace levels, such as those found in the microdialysis samples.