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 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.
Kushnir
M M
MM
ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA. kushnmm@aruplab.com
Komaromy-Hiller
G
G
Shushan
B
B
Urry
F M
FM
Roberts
W L
WL
D002853:Chromatography, Liquid; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008764:Methylmalonic Acid / Q000097:blood* / Q000652:urine*; D010949:Plasma / Q000737:chemistry; D012680:Sensitivity and Specificity; D019802:Succinic Acid / Q000097:blood* / Q000652:urine*
D008764:Methylmalonic Acid; D019802:Succinic Acid