Use of alumina, sephadex G10, and ion-exchange columns to purify samples for determination of epinephrine, norepinephrine, dopamine, homovanillic acid, and 5-hydroxyindoleacetic acid in urine

Abstract

We investigated the usefulness of small Sephadex columns, prepared in Pasteur pipettes, in purifying samples to be analyzed for catecholamines, homovanillic acid, and 5-hydroxyindoleacetic acid. After free catecholamines in urine samples were purified on alumina followed by Sephadex G10, a reliable and simultaneous quantification of epinephrine, norepinephrine, and dopamine was achieved by using high-performance liquid-chromatography with electrochemical detection. Purification of urine on Bio-Rad prepacked ion-exchange columns followed by Sephadex G10 resulted in a reliable, fast (200 samples processed per week) method for determination of free catecholamines in urine. Analytical recoveries of both methods were between 80 and 95%, with a CV of about 3%. Single purification on Sephadex G10 sufficed to allow simultaneous determination of homovanillic acid and 5-hydroxyindoleacetic acid in urine. Analytical recovery for this method was about 90%, with a CV of about 5%. Sephadex G10 columns, which can be re-used without regeneration for at least one year, appear to have a great potential in clinical chemistry.