Annals of Clinical Biochemistry > Volume 45, Number 2 > Pp. 149-152

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Original Article

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

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}smuht.nwest.nhs.uk

Background: Urinary 5-hydroxyindole acetic acid (5-HIAA) is a useful marker for the turnover of tryptophan metabolites in the diagnosis and monitoring of carcinoid tumours and the carcinoid syndrome. We have developed a simple and cost-effective assay for urinary 5-HIAA using liquid chromatography–tandem mass spectrometry (LC–MS/MS) incorporating an online sample clean-up process to 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. Analytes were 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-fold improvement in linearity compared to the existing LC–EC method.

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