Diagnosis of Methylmalonic Acidemia from Dried Blood Spots by HPLC and Intramolecular-Excimer Fluorescence Derivatization

Abstract

Methylmalonic acidemias, a group of heterogeneous disorders, are characterized by accumulation of methylmalonic acid (MMA) and its byproducts in biological fluids (1)(2). Methylmalonic acidemia is now included in all tandem mass spectrometry (MS/MS)-based newborn screening programs (3)(4)(5). Detection is based on the finding of increased propionylcarnitine and/or increased propionylcarnitine-to-acetylcarnitine ratio in dried blood spots (DBS) by MS/MS. These markers, however, are not specific because they are increased in propionic acidemia and, possibly, in multiple carboxylase deficiency (3). In most programs, newborns or patients with initial positive results are recalled for a second blood spot, and a urine sample is collected for organic acid analysis to differentiate among the three disorders. In the present study, we used the intramolecular-excimer fluorescence derivatization approach of Nohta and coworkers (6)(7) to form a fluorescent derivative of MMA. This would allow the detection of MMA in DBS samples from affected neonates, leading to a conclusive diagnosis with the remains of the DBS within a short time, often the same working day. From a DBS, four 3.2-mm discs were punched and extracted into 250 μL of methanol containing 20 μmol/L malonic acid (MA) as internal calibrator by vortex-mixing for 30 s and standing at room temperature for 1 h. After evaporation and reconstitution of the residue …