Effect of l‐Tryptophan on Mouse Brain 5‐Hydroxytryptamine: Comparison of Values Obtained Using a Fluorimetric Assay and a Liquid Chromatographic Assay with Electrochemical Detection

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Abstract
5-Hydroxytryptamine (5-HT) in mouse brain and spinal cord was assayed in the same samples using a fluorimetric assay and a high pressure liquid chromatographic (HPLC) assay with electrochemical detection. The HPLC assay was able to detect levels of 5-HT as low as 0.2-0.5 pmol. With the column (Vydac cation exchange), solvent system (acetate/citrate buffer, 0.1 or 0.2 M, pH 4.8-5.2) extraction procedure and electrode potential (+0.55 V) used, the HPLC assay was specific for 5-HT. When the electrode potential was increased to +0.9 V, tryptamine could also be detected, with a longer retention time than 5-HT. The percentage increase in mouse brain 5-HT after pargyline (75 mg/kg) and pargyline + L-tryptophan (100 mg/kg) was very similar whether measured by fluorimetry or HPLC, although the fluorimetric assay gave consistently higher absolute values (24-32%) in both control and drug-treated animals. L-Tryptophan (25, 50 and 100 mg/kg) also increased brain 5-HT with similar percentage increases with either assay method. There was a significant correlation (P < 0.001) between the values obtained with the 2 assay methods. The results confirm the use of HPLC with electrochemical detection as a sensitive and specific assay method for 5-HT and indicate its potential use for the assay of tryptamine, and the importance of determining the electroactivity and retention characteristics of any drugs used.