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

Lakso, Hans-Ake (author)
Umeå universitet,Klinisk farmakologi
Appelblad, Patrik (author)
Schneede, Jörn (author)
Umeå universitet,Klinisk kemi
 (creator_code:org_t)
2008-12-01
2008
English.
In: Clinical Chemistry. - Washington, DC : American Association for Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 54:12, s. 2028-2035
  • Journal article (peer-reviewed)
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  • 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.

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