Analysis of Dicarboxylic Acids by Tandem Mass Spectrometry. High-Throughput Quantitative Measurement of Methylmalonic Acid in Serum, Plasma, and Urine

Abstract

BACKGROUND: Methylmalonic acid (MMA) is a dicarboxylic acid whose concentration can be increased in blood and urine in patients with an inborn error of metabolism or vitamin B(12) deficiency. We developed a method for the selective analysis of dicarboxylic acids that exploits the high specificity of tandem mass spectrometry (MS/MS) and the substantial difference in fragmentation patterns of the isomers methylmalonic (MMA) and succinic acid (SA). METHODS: Dicarboxylic acids were extracted from samples with methyl-tert-butyl ether and derivatized with butanolic HCl to form dibutyl esters. The derivative was injected into the liquid chromatography (LC)-MS/MS system using TurboIonSpray (nebulizer-assisted electrospray) ionization and quantified by the multiple reaction monitoring mode of MS/MS. RESULTS: The assay for MMA was linear up to 150 micromol/L. The total imprecision was < or =7.5% at both low and high concentrations. The limits of quantification and detection were 0.1 and 0.05 micromol/L, respectively. The degree of interference from SA could be predicted from the branching ratios of the major product ions. CONCLUSIONS: The method is specific for dicarboxylic acids. The LC-MS/MS analysis 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.