Automated assay for the determination of methylmalonic acid, total homocysteine, and related amino acids in human serum or plasma by means of methylchloroformate derivatization and gas chromatography-mass spectrometry.
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 microL) 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 micromol/L (MMA) and 10 micromol/L (Ser, tCys). The assay was linear to 100 micromol/L for MMA and tHcy and to 1000 micromol/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.


CONCLUSIONS
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.
Windelberg
Amrei
A
LOCUS for Homocysteine and Related Vitamins, and Section for Pharmacology, Institute of Medicine, University of Bergen, Norway. amrei.windelberg@farm.uib.no
Arseth
Ove
O
Kvalheim
Gry
G
Ueland
Per Magne
PM
D000596:Amino Acids / Q000097:blood*; D001322:Autoanalysis; D005561:Formates; D008401:Gas Chromatography-Mass Spectrometry; D005998:Glycine / Q000097:blood; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D007202:Indicators and Reagents; D008715:Methionine / Q000097:blood; D008764:Methylmalonic Acid / Q000097:blood*; D010949:Plasma; D012016:Reference Values; D012680:Sensitivity and Specificity; D012694:Serine / Q000097:blood; D044967:Serum
D000596:Amino Acids; D005561:Formates; D007202:Indicators and Reagents; D006710:Homocysteine; D012694:Serine; D008764:Methylmalonic Acid; D008715:Methionine; C014667:methyl chloroformate; D005998:Glycine