Homocysteine and other thiols determined in plasma by HPLC and thiol-specific postcolumn derivatization.
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.
Andersson
A
A
Department of Clinical Chemistry, University Hospital, Lund, Sweden.
Isaksson
A
A
Brattström
L
L
Hultberg
B
B
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003124:Colorimetry; D003545:Cysteine / Q000097:blood; D004151:Dipeptides / Q000097:blood; D004220:Disulfides; D005978:Glutathione / Q000097:blood; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D011725:Pyridines; D013438:Sulfhydryl Compounds / Q000097:blood*
D004151:Dipeptides; D004220:Disulfides; D011725:Pyridines; D013438:Sulfhydryl Compounds; D006710:Homocysteine; C007542:4,4'-dipyridyl disulfide; C028505:cysteinylglycine; D005978:Glutathione; D003545:Cysteine; C017341:gamma-glutamylcysteine