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.
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 of DL-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.
D006916:5-Hydroxytryptophan / Q000032:analysis*; D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006897:Hydroxyindoleacetic Acid / Q000032:analysis*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012701:Serotonin / Q000032:analysis*; D014316:Tritium
D014316:Tritium; D012701:Serotonin; D006897:Hydroxyindoleacetic Acid; D006916:5-Hydroxytryptophan