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
| Autor: | Gry Kvalheim, Amrei Windelberg, Per Magne Ueland, Ove Årseth |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Serum
Homocysteine Formates Clinical Biochemistry Methylmalonic acid Glycine Sensitivity and Specificity Gas Chromatography-Mass Spectrometry chemistry.chemical_compound Plasma Methionine Reference Values Serine Humans Sample preparation Amino Acids Derivatization Detection limit chemistry.chemical_classification Chromatography Autoanalysis Chemistry Biochemistry (medical) Thiol Indicators and Reagents Gas chromatography Gas chromatography–mass spectrometry Methylmalonic Acid |
| Zdroj: | Clinical chemistry. 51(11) |
| ISSN: | 0009-9147 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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