Podium Presentation in Room 2 on Wednesday at 16:15 (Chair: Amy Caudy)
Authors: Joshua A. Dubland (1,2,3), Bojana Rakić (1,2,3), Hilary Vallance (1,2,3), Graham Sinclair (1,2,3)
INTRODUCTION: Methylmalonic acidemias, propionic acidemias, and combined re-methylation disorders are included in newborn screening programs. The presence of one of these heterogeneous disorders is initially suggested by elevated propionylcarnitine (C3) and/or methionine in the flow-through tandem mass spectrometry (MS/MS) first-tier newborn screen of a dried blood spot (DBS) card. Unfortunately, analysis of C3 and methionine concentrations alone lack sensitivity and disease specificity. Conservative C3 and methionine cutoff values along with acylcarnitine ratios and the methionine/phenylalanine ratio can increase screening sensitivity, but second-tier testing is required to increase analytical specificity and confirmation of disorder. Currently, positive DBS screens for C3 and/or methionine are recalled for a urine organic acids profile typically by GC/MS or for plasma analysis of homocysteine by tandem MS to differentiate between disorders. Analysis of the same neonatal DBS card from the first-tier analysis can greatly decrease both clinical diagnostic turn-around time and parental anxiety. Here we describe a tandem MS approach for the analysis of homocysteine, methylmalonic acid, and methylcitric acid in DBS to support newborn screening programs.
METHODS: Standard curves were prepared by spiking stock solutions of homocysteine, methylmalonic acid, and methylcitric acid (0-50 µM) into whole blood and spotting 100 µL of blood onto screening cards. Two discs 3-mm in diameter (6.2 µL of blood) were punched for standards, QCs, and patient samples. As methylcitric acid suffer from poor ionization we derivatized using 4-[2-(N,N-dimethylamino) ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE) to increase sensitivity. Homocysteine and methylmalonic acid were converted to butyl-esters using n-butanol-3N HCl following disulfide cleavage using dithiothreitol. Analytes were separated from interferences on a reverse-phase UPLC column and analyzed by tandem MS in positive ESI mode.
RESULTS: Excellent sensitivity for methylcitric acid and sufficient reverse-phase retention of homocysteine was obtained with our derivatization approach. Analytical results had good correlation with external proficiency samples for all analytes. QCs had CVs<15% across levels and calibration curves had an r2>0.99. Our method separated methylmalonic acid from the isomeric compound succinic acid. Quantifier-to-qualifier ion ratios were incorporated in the screening approach as a confirmatory measure. Matrix effects were determined to be minimal by post-column infusion analysis. Run times were 7 minutes which facilitated rapid second-tier screening. No carryover was observed.
CONCLUSION: DBS metabolite analysis can be inherently challenging due to limited sample volume. We report a tandem MS approach to determine DBS methylcitric acid, methylmalonic acid, and homocysteine levels for the determination of associated inborn errors of metabolism.
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