An isocratic assay for norepinephrine, dopamine, and 5-hydroxytryptamine using their native fluorescence by high-performance liquid chromatography with fluorescence detection in discrete brain areas of rat.

A rapid and simple isocratic chromatographic procedure for simultaneous determination of norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT) using their native fluorescence by high-performance liquid chromatography coupled to fluorescence detection (HPLC-FD) is described. Since the present procedure does not involve sample prepurification, the recovery of monoamines was more than 97% (n = 12) and within a given run, coefficient of variation was less than 3.1% (n = 12). Accordingly, use of an internal standard is not mandatory. In a single chromatographic run, levels of NE, DA, and 5-HT can be determined in less than 30 min. The minimum concentration of monoamines which could be detected by this method was found to be 250 pg for NE and DA and 100 pg for 5-HT. The validity of the method was confirmed by the estimation of levels of monoamines in the hypothalamus and striatum of rat brain following treatment with clorgyline, a monoamine oxidase inhibitor.