Homocysteine and other thiols determined in plasma by HPLC and thiol-specific postcolumn derivatization
- 1 August 1993
- journal article
- research article
- Published by Oxford University Press (OUP) in Clinical Chemistry
- Vol. 39 (8) , 1590-1597
- https://doi.org/10.1093/clinchem/39.8.1590
Abstract
We describe a versatile high-performance liquid-chromatographic method for determining homocysteine and other plasma sulfhydryls. Using three different procedures for preparation of plasma, we determined total, free (non-protein-bound), and reduced forms of homocysteine, cysteine, glutathione, cysteinylglycine, and gamma-glutamylcysteine in human plasma. Sample preparation involves disulfide reduction with dithiothreitol and protein precipitation with sulfosalicylic acid. The assay utilizes isocratic reversed-phase ion-pair liquid chromatography at pH 2.4, postcolumn derivatization with 4,4'-dithiodipyridine, and colorimetric detection at 324 nm. The intra-assay precision (CV) of the method for total homocysteine is 1.5%; the interassay precision over 2.5 months is 2.5%. The detection limit for homocysteine is < 50 nmol/L plasma.Keywords
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