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Quantification of methylmalonic acid in human plasma with hydrophilic interaction liquid chromatography separation and mass spectrometric detection
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Pharmacology.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
2008 (English)In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 54, no 12, 2028-2035 p.Article in journal (Refereed) Published
Abstract [en]

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.
Identifiers
URN: urn:nbn:se:umu:diva-25991DOI: 10.1373/clinchem.2007.101253PubMedID: 18845771OAI: oai:DiVA.org:umu-25991DiVA: diva2:235658
Available from: 2009-09-17 Created: 2009-09-17 Last updated: 2011-09-02Bibliographically approved

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CiteExportLink to record
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