Analysis of Methylmalonic Acid in Plasma by Liquid Chromatography-tandem Mass Spectrometry

Background: Methylmalonic acid (MMA) is a biochemical marker for cobalamin deficiency, particularly in cases where the cobalamin concentration is moderately decreased or in the low-normal range. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization is a rapid, robust method that has been used in MMA analysis. We developed a simple method combining solid-phase extraction (SPE) and derivatization to prepare serum or plasma for LC-MS/MS analysis of MMA.

Methods: Deuterated internal standard d(3)-MMA was added to serum or plasma before SPE on strong anion-exchange (SAX) columns. After elution with HCl-butanol (10:90 by volume) and addition of 1 g/L formic acid, the samples were simultaneously derivatized and evaporated by heating to 70 degrees C for 15 min followed by 54 degrees C overnight in uncapped vials. Acetonitrile and 1 g/L formic acid were added to the samples before injection into the LC-MS/MS system. MMA and d(3)-MMA were quantified in the multiple-reaction monitoring mode. Calibrators were prepared in serum by the standard addition method.

Results: The MMA assay was linear up to 200 micromol/L. Interassay CVs were 6.7%, 5.0%, and 5.0% for mean concentrations of 0.15, 0.36, and 0.65 micromol/L, respectively.

Conclusions: Our simplified sample preparation and derivatization method is suitable for use in MMA analyses. MMA elutes with the derivatization reagent, and derivatization and evaporation are performed simply by leaving the uncapped vials in a heating block overnight. The method shows good linearity and precision.