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

Abstract

Abstract 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.