Quantitative determination of succinylacetone in dried blood spots for newborn screening of tyrosinemia type I

Tyrosinemia type I (TYR 1) is a severe disorder causing early death if left untreated. While tyrosine can be determined in dried blood spots (DBS), it is not a specific marker for TYR 1 and most often associated with benign transient tyrosinemia of the newborn. Succinylacetone (SUAC) is a specific marker for TYR 1 but not detectable by routine newborn screening. We developed a new assay that determines SUAC in DBS by liquid-chromatography tandem mass spectrometry (LC–MS/MS). Whole blood is eluted from a 3/16-in. DBS by an aqueous solution containing deuterium labeled SUAC as internal standard (IS). SUAC and IS are oximated, then extracted, butylated, and analyzed by LC–MS/MS. Quantitation is from SUAC spiked calibrator DBS over the range 0–200 μM using selected reaction monitoring of transitions m/ z 212 to 156 and m/ z 214 to 140 for SUAC and IS, respectively. Analysis time is 5 min. To assess the effectiveness of a two-tier screening approach for TYR 1 we applied this assay to our newborn screening program over the last 15 months. The intra-assay precision was determined for three different levels of SUAC (5, 20, and 50 μmol/L) and the CV calculated to be 4.7, 2.6, and 3.1%, respectively ( n = 5). Inter-assay precision CVs were 12.7, 8.2, and 7.8%, respectively on the same samples. SUAC levels in DBS from 10 confirmed TYR 1 cases not treated with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) were clearly abnormal (16–150 μmol/L; mean: 61 μmol/L; controls: