Newborn screening for hepatorenal tyrosinemia by tandem mass spectrometry: analysis of succinylacetone extracted from dried blood spots

Objectives To develop a method for the determination of succinylacetone (SA) in dried blood spots (DBS) using tandem mass spectrometry (MS/MS). Methods SA was extracted from DBS with an acetonitrile and water solution (80:20 by volume) containing formic acid and hydrazine hydrate (both at 0.1% by volume), and analyzed by MS/MS with a total run time per sample under 2 min. The reference range for SA in newborns was determined by analyzing a control group of 3199 DBS. SA was also measured in stored newborn specimens from three patients diagnosed clinically with hepatorenal tyrosinemia (HT). Results The within-run precision was ≤7.4% and total precision was ≤12.2% on blood spots fortified with SA at 2, 10, and 50 μmol/L. The limit of quantitation was 1 μmol/L and the calibration was linear from 1 to 50 μmol/L. A comparison of SA analysis of this MS/MS method with an established enzyme assay indirectly quantitating SA by inhibition of δ-aminolevulinic acid dehydratase demonstrated a strong correlation. The reference range in the control group of unaffected newborn was determined to be <2 μmol/L, while SA in retrieved DBS from HT patients was significantly elevated, measuring 46.7, 36.5, and 23.2 μmol/L. Conclusions We report a simple method that can be used for screening HT in newborns. Based on our limited experience, a 2 μmol/L cutoff could result in up to 100% sensitivity and specificity.