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

olism relies on finding abnormal concentrations ofme-thionine 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 NBSmethods. To improve this situation, we developed a method for the detection of tHCY,MMA, andMCA 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 iso-topically labeled standards (d3-MMA, d3-MCA, d8-homocystine). The extract was dried and treated with 3 N HCl in n-butanol to form butylesters. After evapo-ration of the butanol, the residuewas 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 controlDBSs forMMA,MCA, andHCY were 1.5, 0.5, and 9.8mol/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. © 2010 American Association for Clinical Chemistry Several inborn errors of propionic acid, methionine (Met)3, and cobalamin (Cbl) metabolism are included in the panel of conditions for which every newborn should be screened, as recommended by the USHealth and Services Administration and the American College of Medical Genetics, and now adopted by all newborn screening (NBS) programs in the US (Table 1