Validation of an LC-MS/MS method for urinary homovanillic and vanillylmandelic ACIDS and application to the diagnosis of neuroblastoma

• Validation of LC-MS/MS method for HVA/VMA with derivatization and online SPE. • Progress towards harmonization of catecholamine measurements across SIOPEN labs. • Inter-laboratory comparison of 36 samples demonstrated result interchangeability. • New method facilitates harmonization in neuroblastoma biochemical diagnostics. Urinary catecholamine metabolites are well-established biomarkers for neuroblastoma (NB). Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are the most frequently measured metabolites within SIOPEN – Catecholamine Working Group laboratories. Here, we evaluated the performance of a new LC-MS/MS in vitro diagnostic (IVD) kit for HVA and VMA to facilitate inter-laboratory harmonization. HVA and VMA and their deuterated internal standards were analyzed with a commercial method, on a ThermoFisher Quantiva LC-MS/MS. Validation was performed first using internal quality control and external quality assessment (IQC and EQA) samples. Next by clinical validation on 120 samples, previously tested by HPLC-ECD. Finally, 36 samples were exchanged between SIOPEN reference laboratories and analyzed by three methods. Using QCs and EQA the method was validated in a wide calibration range (4.61–830 µmol/L for HVA and 4.44–800 µmol/L for VMA). Intra-day CVs ( n = 5) were 7 and 8 % for HVA and 5 and 6 % for VMA for QC low and QC high, respectively; Inter-day CV% were 7 and 3 % for HVA and 2 and 7 % for VMA at QC low and QC high, respectively. Its application to 120 clinical samples confirmed a high diagnostic accuracy. The inter-laboratory quality control assessment showed interchangeable results ( p = 0,73 and p = 0.15 for HVA and VMA, respectively). The LC-MS/MS IVD method could be considered a useful tool for clinical laboratories involved in the measurement of catecholamines, contributing to harmonization efforts.