title: Analytical and preanalytical validation of a new mass spectrometric serum 5-hydroxyindoleacetic acid assay as neuroendocrine tumor marker.
abstract: BACKGROUND
  Serum 5-hydroxyindoleacetic acid (5-HIAA) could replace the determination of 24-h urinary 5-HIAA for diagnosis and follow-up of neuroendocrine tumors (NETs). We developed and validated a straightforward liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for serum 5-HIAA.
  
  
  METHODS
  We used serum samples from healthy volunteers (n=136) and patients suspected or followed for NET (n=129). Samples were spiked with 5-HIAA-D2, extracted and quantified by LC-MS/MS. We studied the effects of sample storage, sample device, a meal and diurnal variation on serum 5-HIAA. Furthermore, we established a reference range for serum 5-HIAA and compared our assay with a urinary 5-HIAA HPLC assay and a commercial plasma chromogranin A (CgA) immunoassay.
  
  
  RESULTS
  Our LC-MS/MS assay is sensitive (LOQ 5 nmol/L), has a wide assay range (5-10,000 nmol/L) and short analysis time (7 min). 5-HIAA in serum is stable for several days in various temperatures and during five freeze-thaw cycles. We found no diurnal variation (p ≥ 0.20) and a meal had no effect on serum 5-HIAA (p=0.89). We suggest an upper reference limit of 123 nmol/L for serum 5-HIAA. The area under curve (AUC) in receiver operator characteristics (ROC) analysis was 0.83 for urinary 5-HIAA, 0.81 for serum 5-HIAA and 0.76 for CgA, respectively.
  
  
  CONCLUSIONS
  The LC-MS/MS assay for serum 5-HIAA discriminates between healthy individuals and patients with NET and is well suited for the diagnosis and follow-up of NETs.
authors.affiliation: Haartman Institute, University of Helsinki, Helsinki, Finland. Electronic address: niina.tohmola@helsinki.fi.
authors.forename: Niina
authors.initials: N
authors.lastname: Tohmola
authors.affiliation: HUSLAB, Helsinki University Central Hospital, Helsinki, Finland.
authors.forename: Outi
authors.initials: O
authors.lastname: Itkonen
authors.affiliation: Division of Endocrinology, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland.
authors.forename: Timo
authors.initials: T
authors.lastname: Sane
authors.affiliation: HUSLAB, Helsinki University Central Hospital, Helsinki, Finland.
authors.forename: Helene
authors.initials: H
authors.lastname: Markkanen
authors.affiliation: Haartman Institute, University of Helsinki, Helsinki, Finland.
authors.forename: Sakari
authors.initials: S
authors.lastname: Joenväärä
authors.affiliation: Haartman Institute, University of Helsinki, Helsinki, Finland; HUSLAB, Helsinki University Central Hospital, Helsinki, Finland.
authors.forename: Risto
authors.initials: R
authors.lastname: Renkonen
authors.affiliation: HUSLAB, Helsinki University Central Hospital, Helsinki, Finland.
authors.forename: Esa
authors.initials: E
authors.lastname: Hämäläinen
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000097:blood*; D005260:Female; D006801:Humans; D006897:Hydroxyindoleacetic Acid / Q000097:blood*; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D018358:Neuroendocrine Tumors / Q000097:blood* / Q000175:diagnosis; D055815:Young Adult
chemical_list: D014408:Biomarkers, Tumor; D006897:Hydroxyindoleacetic Acid
keywords: 5-HIAA; 5-Hydroxyindoleacetic acid; 5-hydroxyindoleacetic acid; CgA; Chromogranin A; LC-MS/MS; LOD; LOQ; NET; Neuroendocrine tumor; SPE; chromogranin A; limit of detection; limit of quantitation; liquid chromatography tandem mass spectrometry; neuroendocrine tumor; solid phase extraction