Simple dilute-and-shoot method for urinary vanillylmandelic acid and homovanillic acid by liquid chromatography tandem mass spectrometry

Abstract

BackgroundNeuroblastomas are pediatric tumors characterized by overproduction of catecholamines. The catecholamine metabolites, vanillylmandelic acid (VMA) and homovanillic acid (HVA), are used in clinical evaluation of neuroblastoma. Tandem mass spectrometry (LC-MS/MS) is an effective analytical method for measurement of VMA and HVA in urine. MethodsDilute-and-shoot sample preparation was performed in a 96-well format using a liquid handler. Chromatographic separation was achieved using a reverse phase column; detection was accomplished by triple quadrupole mass spectrometry with electrospray ionization in positive mode. Data were acquired by multiple reaction monitoring. Two transitions, quantifier and qualifier, were monitored for each analyte and its stable isotope-labeled internal standard. Analytical specificity studies were performed. ResultsInjection-to-injection time was 4min. The method was validated for linearity, limit of quantification, imprecision, accuracy, and interference. Linearity was 0.5–100mg/l for both analytes. Within-run, between-day, and total imprecision were 1.0–4.1% for VMA and 0.8–3.8% for HVA. The method correlated well with our established HPLC method. Interferences precluding quantitation of VMA in 3% of specimens were reduced significantly (to 0.1% of specimens) using a modified LC gradient to reanalyze affected samples. ConclusionsA simple, robust, economical, fast LC-MS/MS method was developed and validated for measurement of urinary VMA and HVA.