Simultaneous measurement of dopamine, serotonin, their metabolites and tryptophan in mouse brain homogenates by high-performance liquid chromatography with dual coulometric detection

A new rapid and sensitive high-performance Liquid chromatography (HPLC) met hod for the simultaneous determination of dopamine, 3,4-dihydroxyphenylacet ic acid (DOPAC), 3-methoxytyramine, 5-hydroxytryptamine (serotonin), 5-hydr oxyindoleacetic acid (5-HIAA), homovanillic acid and tryptophan has been de veloped and applied to mouse frontal cortex, caudate nucleus and dorsal rap he assays. A dual coulometric detector was used with detection at +0.25 and +0.55 V, which allowed the determination of tryptophan. Detection Limits f or all compounds (0.8-9.0 pg per injection, depending on the compounds) wer e useful for this application. Owing to great sensitivity of the method, th e brain tissue samples can be very small, less than 2 mg. Linearity of stan dards was excellent (r > 0.999 in all cases). Intraday and interday precisi ons for samples analytes were generally acceptable (intraday assay CV < 8.7 % and interday assay CV < 7.0% except for DOPAC and 5-HIAA, which was 11.4% for the low concentrations). Average recoveries of standard additions to s ample analytes were >90%. Attention was paid to stability of standard and s ample analytes when stored at +4 degrees C or at -70 degrees C with two dif ferent homogenizing agents (0.1 M HClO4 with 10(-7) M ascorbic acid and 0.0 5 M HClO4 without ascorbic acid). This simple, rapid and efficient method c an be used as a basic research tool for modification of brain neurotransmit ters in experimental pharmacological protocols for following psychotropic d rug treatments in animals. Copyright (C) 1999 John Wiley & Sons, Ltd.