A rapid and simple method to determine the specific activities of serotonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophan in brain by HPLC with electrochemical detection

Abstract

The specific activities of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) have been determined in the brain of rats by HPLC using electrochemical detection. The method allows, from a single sample, the simultaneous measurement of all three compounds and collection of each peak for radioactivity determinations. Five male Wistar rats were injected i.v. with 2.0 mCi/kg ofDl-5-hydroxy-[G-³H]tryptophan (2.6 Ci/mmol) and 30 min later the animals were killed by near freezing. Whole brains were removed and homogenized in an acid medium. The content of 5-HTP, 5-HT, and 5-HIAA were determined by HPLC. Each peak of interest was immediately collected after detection in scintillation vials by use of a small dead space detector (TL-9A, Bioanalytical Systems, Inc.). The amounts of radioactivity were determined and specific activities calculated from the results. A second chromatography system (TLC) was used to check the authenticity and purity of compounds separated by the HPLC.