title: Determination of total homocysteine, methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass spectrometry.
abstract: 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.
authors.affiliation: Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
authors.forename: Coleman T
authors.initials: CT
authors.lastname: Turgeon
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Magera
authors.forename: Carla D
authors.initials: CD
authors.lastname: Cuthbert
authors.forename: Perry R
authors.initials: PR
authors.lastname: Loken
authors.forename: Dimitar K
authors.initials: DK
authors.lastname: Gavrilov
authors.forename: Silvia
authors.initials: S
authors.lastname: Tortorelli
authors.forename: Kimiyo M
authors.initials: KM
authors.lastname: Raymond
authors.forename: Devin
authors.initials: D
authors.lastname: Oglesbee
authors.forename: Piero
authors.initials: P
authors.lastname: Rinaldo
authors.forename: Dietrich
authors.initials: D
authors.lastname: Matern
mesh_terms: D001800:Blood Specimen Collection; D002853:Chromatography, Liquid; D002951:Citrates / Q000097:blood*; D005189:False Positive Reactions; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D007231:Infant, Newborn; D057230:Limit of Detection; D008764:Methylmalonic Acid / Q000097:blood*; D015997:Neonatal Screening; D011237:Predictive Value of Tests; D012016:Reference Values; D053719:Tandem Mass Spectrometry
chemical_list: D002951:Citrates; D006710:Homocysteine; C031605:2-methylcitric acid; D008764:Methylmalonic Acid