title: Online extraction of 5-hydroxyindole acetic acid from urine for analysis by liquid chromatography-tandem mass spectrometry.
abstract: 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 micromol/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 micromol/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 micromol/L.
  
  
  CONCLUSIONS
  We have developed a robust method offering a more than six-fold improvement in linearity compared to the existing LC-EC method.
authors.affiliation: Department of Clinical Biochemistry, Wythenshawe Hospital, University Hospital of South Manchester, Manchester M23 9LT, UK. heather.perry@smuht.nwest.nhs.uk
authors.forename: Heather
authors.initials: H
authors.lastname: Perry
authors.forename: Brian
authors.initials: B
authors.lastname: Keevil
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D006897:Hydroxyindoleacetic Acid / Q000302:isolation & purification* / Q000652:urine*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006897:Hydroxyindoleacetic Acid