title: Two-dimensional high-performance liquid chromatographic determination of 5-hydroxyindoleacetic acid and homovanillic acid in urine.
abstract: The biomedically and neurochemically important compounds 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) have been simultaneously determined in human urine after reverse-phase two-dimensional high-performance liquid chromatography. A 10-fold-diluted urine sample (20 microliters) is first separated on a C18 column (30 X 0.39 cm) using an 85% pH 6.0 phosphate buffer/15% methanol solvent system. The elution volume containing both 5-HIAA and HVA (Rt approximately 3 min) is collected. Recoveries (mean +/- SD) for this purification step, which is monitored using fluorometric detection, were usually above 90%. After acidification of the approximately 2 ml collected fraction, 100 microliters is reinjected on a C18 column and separated (Rt: 5-HIAA, 4 min; HVA 5.5 min) using an 80% pH 3.5 phosphate buffer/20% methanol mobile phase. The compounds are determined by flow-through amperometry with absolute detection limits of approximately 25 pg. Both 5-HIAA and HVA are well resolved from other electroactive species present and are easily determined at normal and greatly reduced concentrations in human urine.
authors.forename: G M
authors.initials: GM
authors.lastname: Anderson
authors.forename: K R
authors.initials: KR
authors.lastname: Schlicht
authors.forename: D J
authors.initials: DJ
authors.lastname: Cohen
mesh_terms: D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006719:Homovanillic Acid / Q000652:urine*; D006801:Humans; D006897:Hydroxyindoleacetic Acid / Q000652:urine*; D010648:Phenylacetates / Q000652:urine*
chemical_list: D010648:Phenylacetates; D006897:Hydroxyindoleacetic Acid; D006719:Homovanillic Acid