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Clinical Chemistry 39: 1590-1597, 1993;
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Clinical Chemistry, Vol 39, 1590-1597, Copyright © 1993 by American Association for Clinical Chemistry

Homocysteine and other thiols determined in plasma by HPLC and thiol- specific postcolumn derivatization

A Andersson, A Isaksson, L Brattstrom and B Hultberg
Department of Clinical Chemistry, University Hospital, Lund, Sweden.

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.


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