title: Quantitative determination of succinylacetone in dried blood spots for newborn screening of tyrosinemia type I.
abstract: BACKGROUND
  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).
  
  
  METHODS
  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 microM 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.
  
  
  RESULTS
  The intra-assay precision was determined for three different levels of SUAC (5, 20, and 50 micromol/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 micromol/L; mean: 61 micromol/L; controls: <5 micromol/L). Over a 15-month period, SUAC was determined in newborn screening samples with elevated tyrosine concentrations when applying different cut off values until it was settled at 150 micromol/L. No case of TYR 1 was detected in 124,780 newborns tested.
  
  
  CONCLUSIONS
  We have developed a new LC-MS/MS based method for the determination of SUAC in DBS. This assay has the potential to significantly reduce the number of false positive results in newborn screening for TYR 1 and can also be used for the laboratory follow up of patients treated for TYR 1.
authors.affiliation: Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Magera
authors.forename: Nishantha D
authors.initials: ND
authors.lastname: Gunawardena
authors.forename: Si Houn
authors.initials: SH
authors.lastname: Hahn
authors.forename: Silvia
authors.initials: S
authors.lastname: Tortorelli
authors.forename: Grant A
authors.initials: GA
authors.lastname: Mitchell
authors.forename: Stephen I
authors.initials: SI
authors.lastname: Goodman
authors.forename: Piero
authors.initials: P
authors.lastname: Rinaldo
authors.forename: Dietrich
authors.initials: D
authors.lastname: Matern
mesh_terms: D000465:Algorithms; D002853:Chromatography, Liquid / Q000379:methods; D006537:Heptanoates / Q000097:blood*; D006801:Humans; D007231:Infant, Newborn; D015997:Neonatal Screening / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D020176:Tyrosinemias / Q000175:diagnosis* / Q000453:epidemiology; D014481:United States / Q000453:epidemiology
chemical_list: D006537:Heptanoates; C020804:succinylacetone