Received on December 20, 2007
Accepted on September 11, 2008

General Clinical Chemistry

Quantification of Methylmalonic Acid in Human Plasma with Hydrophilic Interaction Liquid Chromatography Separation and Mass Spectrometric Detection

¹ Department of Clinical Pharmacology, University of Umeå, Umeå, Sweden
² SeQuant AB, Umeå, Sweden

^* To whom correspondence should be addressed. E-mail: jorn.schneede{at}medbio.umu.se.

BACKGROUND: Measurement of methylmalonic acid (MMA) in serum or plasma is useful for diagnosing cobalamin deficiency. We developed a method for quantifying MMA in plasma based on hydrophilic interaction liquid chromatography (HILIC) and single-stage negative electrospray ionization (ESI) mass spectrometry.

METHODS: We deproteinized plasma samples (200 µL) with 800 µL acidified acetonitrile containing 0.17 µmol/L deuterated MMA (D₃-MMA) internal standard, centrifuged the samples, and injected 4 µL of the supernatant into the LC-MS instrument. Separation was achieved within 3 min on a Merck SeQuant ZIC®-HILIC column with a mobile phase consisting of 4 volumes acetonitrile plus 1 volume 100 mmol/L ammonium acetate buffer, pH 4.5, at a flow rate of 400 µL/min. Subsequent column washing and reconditioning contributed to a total run time of 10 min. MMA and D₃-MMA were quantified by single-ion monitoring (m/z 117.2 and 120.2, respectively) in negative ESI mode at a drying-gas flow rate of 10 L/min, 300 °C, and a capillary voltage of 3.0 kV.

RESULTS: The estimated limits of MMA quantification and detection were 0.09 µmol/L and 0.03 µmol/L, respectively, in plasma. The assay was linear to 200 µmol/L. Interassay and intraassay CVs were 5% at all tested concentrations. Recoveries were 90%–93%.

CONCLUSIONS: This robust assay allows analysis of MMA in human plasma without derivatization. Sample preparation is simple and suitable for automation.