Semi-automated quantification of methylmalonic acid in human serum by LC-MS/MS

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Abstract

Background. Methylmalonic acid (MMA), a sensitive biomarker of functional vitamin B12 deficiency, is commonly determined by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods using manual extraction and derivatization of MMA to reduce polarity prior to separation. Methods. In the present study we introduce a semi-automated extraction on a strong anion exchanger, HPLC separation on a BEH-amide column to separate serum MMA from its abundant isoform, succinic acid, followed by MS/MS detection and quantification. Results. The extraction of MMA plus internal standard provides full recovery and the method is linear between 0.03 mu mol/L and 20.0 mu mol/L (r(2) = 1.0) with intra-and inter-assay imprecision of 2.2%. Agreement with other laboratories has been demonstrated in external proficiency testing. Compared to both conventional GC-MS and LC-MS/MS methods, the correlation is r(2) > 0.99. Conclusions. The use of robotic pipetting, elimination of derivatization and improved separation by the BEH-amide column combined with HILIC chromatographic conditions significantly improve sample throughput compared to conventional methods. Using a single pipetting robot and LC-MS/MS instrument, this method is currently performing 180 analyses per day from 10 regional hospitals and several additional distant sites.

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Subject classification (UKÄ) – MANDATORY

  • Medicinal Chemistry
  • Pharmacology and Toxicology

Keywords

  • Chromatography, high performance liquid, tandem mass spectrometry, gas, chromatography-mass spectrometry, methylmalonic acid, vitamin B12
Original languageEnglish
Pages (from-to)441-446
JournalScandinavian Journal of Clinical & Laboratory Investigation
Volume72
Issue number6
Publication statusPublished - 2012
Publication categoryResearch
Peer-reviewedYes

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