Neuroblastoma screening: labeling of HVA and VMA for stable isotope dilution gas chromatography-mass spectrometry

Neuroblastoma is a curable tumor if detected early enough. Therefore, a screening test is performed in Austrian infants by analyzing urine samples collected and dried on filter strips. Screening is performed by measuring homovanillic acid (HVA) and vanillylmandelic acid (VMA) using the enzymatic immunoassay chemistry (EIA) method and reexamining the elevated values by high-performance liquid chromatography. Both methods, however, give a relatively high number of false-positive results. Therefore, the positives are reexamined by stable isotope dilution (SID) gas chromatography-mass spectrometry (GC-MS), which is the most sensitive and precise method in clinical chemistry. The labeled substance is added as an internal standard to the sample; it has nearly identical chemical and physical properties as the natural analogue, undergoes the entire preparation step, and is detected simultaneously with the natural analogue by the same detector, namely the mass spectrometry, given by a difference in molecular weight. All loss is compensated in this way by the final calculation. At present, HVA but not VMA can be purchased labeled with deuterium, so we started with our method using deuterated HVA as the internal standard for both HVA and VMA. Because the coefficient of variation is not sufficient for VMA, we try to label VMA via different labeling procedures; for example, 1) exchange of the aromatic H against D by incubation with DCI or in alkaline R-OD or LiOD or 2) exchange of phenolic 16OH against 18OH by reaction with H2(18)O in an acidic environment and at high temperatures. Carboxylic 16O can be exchanged against 18O.