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

Abstract

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.