Quantification of Methylmalonic acid in human plasma with hydrophilic interaction liquid chromatography separation and mass spectrometric detection

(MMA) in serum or plasma is useful for diagnosing co-balamin deficiency. We developed a method for quanti-fying MMA in plasma based on hydrophilic interaction liquid chromatography (HILIC) and single-stage nega-tive electrospray ionization (ESI) mass spectrometry. METHODS: We deproteinized plasma samples (200 L) with 800Lacidified acetonitrile containing 0.17mol/L deuterated MMA (D3-MMA) internal standard, cen-trifuged the samples, and injected 4 L of the super-natant into the LC-MS instrument. Separation was achieved within 3 min on a Merck SeQuant ZIC-HILIC columnwith amobile phase consisting of 4 vol-umes acetonitrile plus 1 volume 100 mmol/L ammo-nium acetate buffer, pH 4.5, at a flow rate of 400 L/ min. Subsequent column washing and reconditionin