Online extraction of 5-hydroxyindole acetic acid from urine for analysis by liquid chromatography–tandem mass spectrometry

Ann Clin Biochem 2008;45:149-152
© 2008 Association for Clinical Biochemistry


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Original Article

Heather Perry  and
Brian Keevil

Department of Clinical Biochemistry, Wythenshawe Hospital, University Hospital of South Manchester, Manchester M23 9LT, UK

Corresponding author: Dr Heather Perry. Email: heather.perry{at}

Background: Urinary 5-hydroxyindole acetic acid (5-HIAA) is a useful markerfor the turnover of tryptophan metabolites in the diagnosisand monitoring of carcinoid tumours and the carcinoid syndrome.We have developed a simple and cost-effective assay for urinary5-HIAA using liquid chromatography–tandem mass spectrometry(LC–MS/MS) incorporating an online sample clean-up processto replace a liquid chromatography electrochemical (LC-EC) technique.

Methods: Acidified urine was serially diluted in ammonium acetate buffer followed by ammonium acetate buffer enriched with 5-hydroxyindole-3-acetic-2,2-D2 acid internal standard. A 2.1 x 10 mm C18 column was used for primary online clean-up and eluted with 100% methanolic mobile phase onto a second dC18 Atlantis 2.1 x 20 mm column. Analyteswere detected by mass spectrometry using transitions 192.1 >146.3 and 194.1 > 148.0 for 5-HIAA and deuterated analyte,respectively.

Results: Run time was 3 min with 5-HIAA eluting at 1.37 min. The inter and intra-assay imprecision and accuracies of the three levels of inhouse quality control (QC) (30, 300 and 600 µmol/L) were acceptable with coefficient of variations (CVs) and deviation from target values <12% (n = 15). The average recovery of 5-HIAA spiked into urine was 93.7% with no ion suppression observed. The limit of detection was 2.8 and lower limit of quantification 4.0 µmol/L. Passing–Bablok regression of LC-EC with LC–MS/MS results showed good agreement between the methods, the relationship described as LC–MS/MS = 1.01(LC–EC)–1.22. No systematic or proportional biases were observed over the working range of the method. The assay was linear to at least 2000 µmol/L.

Conclusions: We have developed a robust method offering a more than six-foldimprovement in linearity compared to the existing LC–ECmethod.

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