Received on April 3, 2005
Accepted on June 23, 2005

Automation and Analytical Techniques

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

¹ Child and Adolescent Psychiatry, University of Groningen, Groningen, The Netherlands
² Department of Pathology and Laboratory Medicine, University of Groningen, Groningen, The Netherlands
³ Child Study Center, Yale University School of Medicine, New Haven, CT
⁴ Department of Medical Oncology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

^* To whom correspondence should be addressed. E-mail: i.p.kema{at}lc.umcg.nl.

Background: 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.

Methods: 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.

Results: The internal standard (5-HICA) and 5-HIAA were recovered in high yield (87.2%-114%). Within- and between-series CVs for the measurement of 5-HIAA in urine ranged from 1.3% 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.

Conclusion: This SPE-HPLC method demonstrated lower imprecision and time per analysis than the manual solvent extraction method.