Establishment and clinical application of A Candidate Reference Measurement Procedure for Quantification of Urinary Vanillylmandelic acid and Homovanillic Acid using ID-LC-MS/MS method

Neuroblastoma (NB), an embryonic tumor of the autonomous nervous system, poses a significant threat to the health and lives of children. Accurate measurement of vanillylmandelic acid (VMA) and homovanillic acid (HVA) in human urine is crucial for screening and diagnosis of NB. Although various laboratories have developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect VMA and HVA, the comparability between the results obtained from different laboratories and methods was poor. The absence of reference method for VMA and HVA hinders the standardization of their measurements. Therefore, a candidate reference measurement procedure (cRMP) based on isotope dilution LC-MS/MS (ID-LC-MS/MS) for the detection of VMA and HVA in human urine was established. Urine samples were spiked with VMA-d3 and HVA-d5 as internal standards and extracted using a protein precipitation method. The cRMP exhibited desirable precision with the total imprecision below 5%. The accuracy of this cRMP was demonstrated by the high analytical recovery (98.64% - 102.22% and 98.41% - 100.97% for VMA and HVA, respectively), and comparability between different reference systems. The limit of detection for HVA and VMA were 15.625ng/mL and 3.906ng/mL, respectively; the quantification limits were 62.5ng/mL and 7.813ng/mL, respectively, which can meet the clinical detection requirements. The linear range was from 78.125ng/mL to 20μg/mL. Specificity evaluations showed no corresponding interference from structurally similar analogues. In conclusion, we have established a cRMP based on ID-LC-MS/MS for the measurement of VMA and HVA in urine samples, demonstrating well-defined method performance including accuracy, precision, and specificity. This newly established cRMP is suitable for routine assay standardization and evaluation of clinical samples. Furthermore, this method has the potential to significantly enhance the diagnostic accuracy for neuroblastoma.