Determination of Picomole Amounts of Dopamine, Noradrenaline, 3,4‐Dihydroxyphenylalanine, 3,4‐Dihydroxyphenylacetic Acid, Homovanillic Acid, and 5‐Hydroxyindolacetic Acid in Nervous Tissue After One‐Step Purification on Sephadex G‐10, Using High‐Performance Liquid Chromatography with a Novel Type of Electrochemical Detection

Abstract

A determination of dopamine (DA), noradrenaline (NA), 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindolacetic acid (5-HIAA) in nervous tissue is described. The method is based on a rapidly performed isolation of DA, NA, DOPA, DOPAC, HVA, and 5-HIAA from one single nervous tissue sample on small columns of Sephadex G-10, followed by reverse-phase high-performance liquid chromatography with electrochemical detection. A new type of electrochemical detector based on a rotating disk electrode (RDE) was used. The rotating disc electrode was found to be a reliable and sensitive amperometric detector with several advantages over the currently used thinlayer cells. The detector appeared very useful for routine analysis. Practical details are given for the routine use of the RDE. Brain samples containing no more than 75–150 pg (DA, DOPA, DOPAC, HVA, and 5-HIAA) or 500 pg (NA) could be reproducibly assayed with high recovery (approx. 85%) and precision (approx. 5%), without the use of internal standards. Endogenous concentrations of DA, NA, DOPA, DOPAC, HVA, and 5-HIAA were determined in eight brain structures.