Automated On-Line Solid-Phase Extraction Coupled with HPLC for Measurement of 5-Hydroxyindole-3-acetic Acid in Urine

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2005-09, Vol.51 (9), p.1698-1703
• Main Authors: Mulder, Erik J, Oosterloo-Duinkerken, Alida, Anderson, George M, De Vries, Elisabeth G.E, Minderaa, Ruud B, Kema, Ido P
• Format: Article
• Language: English
• Subjects: Acetic acid; Analytical, structural and metabolic biochemistry; Autism; Autistic Disorder - diagnosis; Autoanalysis; Automation; Biological and medical sciences; Biomarkers - urine; Carboxylic acids; Carcinoid Tumor - diagnosis; Chromatography; Chromatography, High Pressure Liquid; Excretion; Fluorometry; Fundamental and applied biological sciences. Psychology; Humans; Hydroxyindoleacetic Acid - urine; Investigative techniques, diagnostic techniques (general aspects); Liquid chromatography; Medical sciences; Metabolism; Methods; Neuroendocrine tumors; Quality Control; Serotonin; Solvent extraction; Triathlon; Urine
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2005.050062

Quantification of 5-hydroxyindole-3-acetic acid (5-HIAA) in urine is useful in diagnosing and monitoring of patients with carcinoid tumors and in the study of serotonin (5-hydroxytryptamine) metabolism in various disorders. We describe an automated method that incorporates on-line solid-phase extraction (SPE) and HPLC to measure urinary 5-HIAA. Automated prepurification of urine was accomplished with HySphere-resin GP SPE cartridges containing strong hydrophobic polystyrene resin. The analyte (5-HIAA) and internal standard [5-hydroxyindole-3-carboxylic acid (5-HICA)] were eluted from the SPE cartridge, separated by reversed-phase HPLC, and detected fluorometrically with a total cycle time of 20 min. Urinary excretion of 5-HIAA was measured in a group of patients with known and suspected carcinoid tumors (n=63) and in 20 patients with autism. The internal standard (5-HICA) and 5-HIAA were recovered in high yields (87.2%-114%). Within- and between-series CVs for the measurement of 5-HIAA in urine ranged from 1.2% to 3.9% and 3.2% to 7.6%, respectively. For urine samples from patients with known or suspected carcinoid tumors, results obtained by the automated method were highly correlated (r=0.988) with those from an established manual extraction method. For samples from autistic patients, urinary excretion of 5-HIAA was similar to that reported for healthy individuals. This SPE-HPLC method demonstrated lower imprecision and time per analysis than the manual solvent extraction method.