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Automation and Analytical Techniques |
1 LOCUS for Homocysteine and Related Vitamins, and Section for Pharmacology, Institute of Medicine, University of Bergen, Bergen, Norway.
aAddress correspondence to this author at: LOCUS for Homocysteine and Related Vitamins, Section for Pharmacology, Institute of Medicine, University of Bergen, Armauer Hansens Hus, N-5021 Bergen, Norway. Fax 47-55-97-4605; e-mail amrei.windelberg{at}farm.uib.no.
Background: The combined measurement of methylmalonic acid (MMA) and total homocysteine (tHcy) in serum or plasma is useful in diagnosing and distinguishing between cobalamin and folate deficiencies. We developed and validated an isotope-dilution gas chromatography–mass spectrometry (GC-MS) method with automated sample workup for the determination of MMA, tHcy, and the related amino acids Met, total cysteine (tCys), Ser, and Gly in serum or plasma.
Methods: Serum or plasma samples (100 µL) were treated with a reductant (dithioerythritol), deproteinized with ethanol, and derivatized and extracted in a single step by the addition of methylchloroformate and toluene. All liquid handling was performed in 96-well (1 mL) microtiter plates by a robotic workstation. The N(S)-methoxycarbonyl ethyl ester derivatives were analyzed by GC-MS in the selected-ion monitoring mode.
Results: Detection limits (signal-to-noise ratio, 5:1) were between 0.03 µmol/L (MMA) and 10 µmol/L (Ser, tCys). The assay was linear to 100 µmol/L for MMA and tHcy and to 1000 µmol/L for Met, tCys, Ser, and Gly. The within-day CVs ranged from 0.7% to 3.6% (n = 20), and the between-day CVs from 2.1% to 8.1% (n = 20). The recovery was between 79% and 99% for the different analytes.
Conclusion: This assay combines a simple and automated sample preparation with selective and sensitive GC-MS analysis and is well suited for the combined measurement of MMA, tHcy, and the related amino acids.
The following articles in journals at HighWire Press have cited this article:
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