Analysis of dicarboxylic acids by tandem mass spectrometry. High-throughput quantitative measurement of methylmalonic acid in serum, plasma, and urine

Background: Methylmalonic acid (MMA) is a dicarboxylic acid whose concentra tion can be increased in blood and urine in patients with an inborn error o f metabolism or vitamin B,, deficiency. We developed a method for the selec tive analysis of dicarboxylic acids that exploits the high specificity of t andem mass spectrometry (MS/MS) and the substantial difference in fragmenta tion patterns of the isomers methylmalonic (MMA) and succinic acid (SA). Methods: Dicarboxylic acids were extracted from samples with methyl-tert-bu tyl ether and derivatized with butanolic HCl to form dibutyl esters. The de rivative was injected into the liquid chromatography (LC)-MS/ MS system usi ng TurbolonSpray (TM) (nebulizer-assisted electrospray) ionization and quan tified by the multiple reaction monitoring mode of MS/MS. Results: The assay for MMA was linear up to 150 mu mol/L. The total impreci sion was less than or equal to7.5% at both low and high concentrations. The limits of quantification and detection were 0.1 and 0.05 mu mol/L, respect ively. The degree of interference from SA could be predicted from the branc hing ratios of the major product ions. Conclusions. The method is specific for dicarboxylic acids. The LC-MS/MS an alysis for MMA requires minimal chromatographic separation and takes < 60 s per sample. The entire analysis, including sample preparation, for a batch of 100 specimens can be performed in <4 h. (C) 2001 American Association f or Clinical Chemistry.