Analytical and preanalytical validation of a new mass spectrometric serum 5-hydroxyindoleacetic acid assay as neuroendocrine tumor marker

• Serum 5-HIAA LC-MS/MS assay is well suited for diagnosis and follow-up of NETs in a clinical laboratory.
• The assay is sensitive (LOQ 5 nmol/L) and has a wide assay range (5–10,000 nmol/L).
• The assay discriminates well between the healthy individuals and patients with serotonin-producing NET.
• Serum 5-HIAA was stable for at least seven days at + 4 °C and at room temperature.
• There was no diurnal variation in serum 5-HIAA.

BackgroundSerum 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.MethodsWe 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.ResultsOur 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.ConclusionsThe 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.