Determination of total homocysteine, methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass spectrometry.
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.
Turgeon
Coleman T
CT
Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Magera
Mark J
MJ
Cuthbert
Carla D
CD
Loken
Perry R
PR
Gavrilov
Dimitar K
DK
Tortorelli
Silvia
S
Raymond
Kimiyo M
KM
Oglesbee
Devin
D
Rinaldo
Piero
P
Matern
Dietrich
D
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
D002951:Citrates; D006710:Homocysteine; C031605:2-methylcitric acid; D008764:Methylmalonic Acid