A simple high-throughput method for the determination of plasma methylmalonic acid by liquid chromatography-tandem...

Cobalamin (Cbl) deficiency is a common clinical phenomenon, in particular among the elderly and possibly also among infants. Methylmalonic acid (MMA) is the most sensitive and specific marker of intracellular Cbl status, but its application is hindered by limited methods available for accurate and high-throughput MMA determination.

We developed a non-laborious method for determination of MMA without the need for prior derivatization using HPLC combined with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Stable isotope-labeled methyl-d(3)-malonic acid (MMA-d(3)) was added to 100 microL of plasma as an internal standard. After deproteinization by ultrafiltration, an acidified aliquot of the eluate was injected into the HPLC system and analyzed by LC-ESI-MS/MS monitoring of the carbonyl loss of MMA and MMA-d(3).

Calibrations between 0.1 and 1.0 microM exhibited consistent linearity and reproducibility. The lower limit of detection for plasma MMA was 0.1 microM (signal-to-noise ratio > or = 10). The intra- and inter-assay CVs of ten determinations of a plasma sample were 1.5% and 6.7%, respectively, at a mean concentration of 0.29 microM. Inter-assay CVs for 25 determinations of low, medium and high concentrations (0.22, 0.45 and 0.94 microM MMA) were 8.3%, 5.9% and 4.6%, respectively. The mean recovery of MMA added to plasma was 100%.

By avoiding derivatization, we developed a new, non-laborious, simple and reliable high-throughput method for the determination of MMA that is suitable for automation.