Quantitative determination of succinylacetone in dried blood spots for newborn screening of tyrosinemia type I.
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.
Magera
Mark J
MJ
Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Gunawardena
Nishantha D
ND
Hahn
Si Houn
SH
Tortorelli
Silvia
S
Mitchell
Grant A
GA
Goodman
Stephen I
SI
Rinaldo
Piero
P
Matern
Dietrich
D
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
D006537:Heptanoates; C020804:succinylacetone