Background: Tyrosinemia type I (TYR 1) is a disorder causing early death if left untreated. Newborn screening (NBS) for this condition is problematic because determination of the diagnostic marker, succinylacetone (SUAC), requires a separate first-tier or only partially effective second-tier analysis based on tyrosine concentration. To overcome these problems, we developed a new assay that simultaneously determines acylcarnitines (AC), amino acids (AA), and SUAC in dried blood spots (DBS) by flow injection tandem mass spectrometry (MS/MS).
Methods: We extracted 3/16-inch DBS punches with 300 microL methanol containing AA and AC stable isotope-labeled internal standards. This extract was derivatized with butanol-HCl. In parallel, we extracted SUAC from the residual filter paper with 100 microL of a 15 mmol/L hydrazine solution containing the internal standard 13C5-SUAC. We combined the derivatized aliquots in acetonitrile for MS/MS analysis of AC and AA with additional SRM experiments for SUAC (m/z 155-137) and 13C5-SUAC (m/z 160-142). Analysis time was 1.2 min.
Results: SUAC was increased in retrospectively analyzed NBS samples of 11 TYR 1 patients (length of storage, 52 months to 1 week; SUAC range, 13-81 micromol/L), with Tyr concentrations ranging from 65 to 293 micromol/L in the original NBS analysis. The mean concentration of SUAC in 13 521 control DBS was 1.25 micromol/L.
Conclusion: The inclusion of SUAC analysis into routine analysis of AC and AA allows for rapid and cost-effective screening for TYR 1 with no tangible risk of false-negative results.