US EPA

1461410 

Journal Article 

Determination of Total Homocysteine, Methylmalonic Acid, and 2-Methylcitric Acid in Dried Blood Spots by Tandem Mass Spectrometry 

Turgeon, CT; Magera, MJ; Cuthbert, CD; Loken, PR; Gavrilov, DK; Tortorelli, S; Raymond, KM; Oglesbee, D; Rinaldo, P; Matern, D 

2010 

Yes 

Clinical Chemistry
ISSN: 0009-9147
EISSN: 1530-8561 

56 

11 

1686-1695 

English 

20807894 

10.1373/clinchem.2010.148957 

WOS:000283581800011 

BACKGROUND: Newborn screening (NBS) for inborn errors of propionate, methionine, and cobalamin metabolism relies on finding abnormal concentrations of methionine and propionylcarnitine. These analytes are not specific for these conditions and lead to frequent false-positive results. More specific markers are total homocysteine (tHCY), methylmalonic acid (MMA), and methylcitric acid (MCA), but these markers are not detected by current NBS methods. To improve this situation, we developed a method for the detection of tHCY, MMA, and MCA in dried blood spots (DBSs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

METHODS: The analytes were extracted from a single 4.8-mm DBS punch with acetonitrile:water:formic acid (59:41:0.42) containing dithiothreitol and isotopically labeled standards (d(3)-MMA, d(3)-MCA, d(8)-homocystine). The extract was dried and treated with 3 N HCl in n-butanol to form butylesters. After evaporation of the butanol, the residue was reconstituted and centrifuged and the supernatant was subjected to LC-MS/MS analysis. Algorithms were developed to apply this method as an efficient and effective second-tier assay on samples with abnormal results by primary screening.

RESULTS: The 99th percentiles determined from the analysis of 200 control DBSs for MMA, MCA, and HCY were 1.5, 0.5, and 9.8 μmol/L, respectively. Since 2005, prospective application of this second-tier analysis to 2.3% of all NBS samples led to the identification of 13 affected infants.

CONCLUSIONS: Application of this assay reduced the false-positive rate and improved the positive predictive value of NBS for conditions associated with abnormal propionylcarnitine and methionine concentrations.