Quantification of methylmalonic acid in human plasma with hydrophilic interaction liquid chromatography separation and mass spectrometric detection
2008 (English)In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 54, no 12, 2028-2035 p.Article in journal (Refereed) Published
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 microL) with 800 microL acidified acetonitrile containing 0.17 micromol/L deuterated MMA (D(3)-MMA) internal standard, centrifuged the samples, and injected 4 microL 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 microL/min. Subsequent column washing and reconditioning contributed to a total run time of 10 min. MMA and D(3)-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 degrees C, and a capillary voltage of 3.0 kV.
Results: The estimated limits of MMA quantification and detection were 0.09 micromol/L and 0.03 micromol/L, respectively, in plasma. The assay was linear to 200 micromol/L. Interassay and intraassay CVs were < or = 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.
Place, publisher, year, edition, pages
Washington, DC: American Association for Clinical Chemistry , 2008. Vol. 54, no 12, 2028-2035 p.
IdentifiersURN: urn:nbn:se:umu:diva-25991DOI: 10.1373/clinchem.2007.101253PubMedID: 18845771OAI: oai:DiVA.org:umu-25991DiVA: diva2:235658