Received on February 19, 2007
Accepted on April 24, 2007

Pediatric Clinical Chemistry

Rapid 2nd-Tier Test for Measurement of 3-OH-Propionic and Methylmalonic Acids on Dried Blood Spots: Reducing the False-Positive Rate for Propionylcarnitine during Expanded Newborn Screening by Liquid Chromatography-Tandem Mass Spectrometry

¹ Metabolic Unit, Department of Paediatrics, Meyer Children's Hospital
² Department of Pharmaceuticals Department, University of Florence, Florence, Italy

^* To whom correspondence should be addressed. E-mail: g.lamarca{at}meyer.it.

Background: The expansion of newborn screening programs has increased the number of newborns diagnosed with inborn errors of metabolism in the presymptomatic phase, but it has also increased the number of costly, stress-producing false-positive results. Because propionylcarnitine (C3) is one of the analytes most frequently responsible for false-positive results, we aimed to develop a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to identify free methylmalonic and 3-OH propionic acids (3-OH-PAs) in blood spots.

Methods: We studied newborn screening spots from 250 healthy controls; 124 from infants with abnormal C3, of whom only 5 (4%) were truly affected; 124 from infants with altered isolated methylmalonylcarnitine; and 4 from clinically diagnosed patients. Whole blood was eluted from a 3.2-mm dried blood spot by a CH₃CN/H₂O 7:3 and 5 mL/L formic. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray without derivatization. Total analysis time was 5 min per sample.

Results: The assays were linear up to 3300 nmol/L for both metabolites. Intra- and interassay imprecision data were 3.6%-8% and 3.1%-%, respectively, for methylmalonic acid (MMA) and 5.2%-20% and 3.6%-17% for 3OH-PA. Limit of detection and limit of quantitation were 1.95 and 4.2 µmol/L, respectively, for MMA and 8 and 10 µmol/L for 3OH-PA. The recoveries were 92.9%-106.1%. No deterioration was noted on the columns after 500 chromatographic runs. If the new method had been used as a 2nd-tier test for the 124 samples, only the 5 true positives would have been recalled for additional samples, and the positive predictive value would have been 100%.

Conclusions: This method has the potential to markedly reduce false-positive results and the associated costs and anxiety. It may also be suitable for diagnosing and routinely monitoring blood spots for methylmalonic aciduria and propionic acidemia.