Nonstandard abbreviations: NBS, newborn screening; AFP, [alpha]-fetoprotein; TYR1, tyrosinemia type I; SUAC
, succinylacetone; NTBC, 2-(2-nitro-4-trifluoromethylbenzoyl)1,3-cyclohexanedione.
In 2008, NSQAP began development of a SUAC QA program to support laboratories that perform NBS tests for tyrosinemia type I.
In this document, we report results from 2 distributions of the SUAC pilot blood spot materials and the first 2 SUAC proficiency testing (PT) challenge panels.
The SUAC (4,6-dioxoheptanoic acid, 99.5% purity) used to prepare stock solutions for enriching whole blood was purchased from Sigma-Aldrich.
We prepared a set of pilot SUAC DBS calibrators by dividing 1 unit of hematocrit-adjusted blood into 7 portions for SUAC enrichment at 0, 1.5, 3.0, 5.0, 10.0, 20.0, and 50.0 j[micro]mol/L and validated the SUAC concentrations by performing duplicate MS/MS analyses (11) of every calibrator in each of 5 runs (Table 1).
(15) requires no substantive new technology or instruments for a laboratory to implement analysis of SUAC, although the laboratory must be set up to deal with hydrazine, a suspected carcinogen.
First, systems must be put in place to be sure there is no mix-up in the sample extracts during recombination of SUAC and amino acid/ acylcarnitine extracts.
The combined result of SUAC and tyrosine appears to be definitive for Tyr-I.
We prepared DBS for SUAC calibration, recovery, stability, and imprecision studies as follows: aliquots of pooled whole blood were spiked with SUAC to achieve final concentrations of 0, 5, 10, 20, 50, and 100 [micro]mol/L, then spotted on filter paper (Whatman ProteinSaver 903) and dried overnight at room temperature.
Sample preparation entails a parallel work-up of eluates from the same DBS that contain either amino acids and acylcarnitines or SUAC. The analytes are subsequently recombined for ESI-MS/MS analysis as outlined in Fig.
Wetransferred the methanol eluates to another round-bottom 96-well plate, leaving the residual filter paper discs for subsequent elution of SUAC for 30 min at 65 [degrees]C by addition of 100 [micro]L acetonitrile/water/formic acid solution (80:20:0.1, vol:vol:vol), which also contains 0.1% hydrazine monohydrate (15 mmol/L) and the internal standard 13C5-SUAC (0.25 [micro]mol/L).
After extraction of SUAC from the leftover dried filter paper spots, the eluates were transferred to another round-bottom 96-well plate and dried under heated (40 [degrees]C) nitrogen for approximately 7 min.