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) method for the simultaneous determination of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine, 5-hydroxytryptamine (serotonin), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid and tryptophan has been developed and applied to mouse frontal cortex, caudate nucleus and dorsal raphe assays. A dual coulometric detector was used with detection at +0.25 and +0.55 V, which allowed the determination of tryptophan. Detection limits for all compounds (0.8-9.0 pg per injection, depending on the compounds) were useful for this application. Owing to great sensitivity of the method, the brain tissue samples can be very small, less than 2 mg. Linearity of standards was excellent (r > 0.999 in all cases). Intraday and interday precisions 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 sample analytes were > 90%. Attention was paid to stability of standard and sample analytes when stored at +4 degrees C or at -70 degrees C with two different homogenizing agents (0.1 M HClO4 with 10(-7) M ascorbic acid and 0.05 M HClO4 without ascorbic acid). This simple, rapid and efficient method can be used as a basic research tool for modification of brain neurotransmitters in experimental pharmacological protocols for following psychotropic drug treatments in animals.
Département de Biochimie-Pharmacologie-Toxicologie, Hôpital A. Mignot, Le Chesnay, France.
J C
JC
Alvarez
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D
Bothua
I
I
Collignon
C
C
Advenier
O
O
Spreux-Varoquaux
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004298:Dopamine / Q000378:metabolism*; D004563:Electrochemistry; D051379:Mice; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012701:Serotonin / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
D012701:Serotonin; D014364:Tryptophan; D004298:Dopamine