title: Simultaneous determination of albumin and low-molecular-mass thiols in plasma by HPLC with UV detection.
abstract: In this paper, we describe a simple and robust HPLC based method for determination of total low- and high-molecular-mass thiols, protein S-linked thiols and reduced albumin in plasma. The method is based on derivatization of analytes with 2-chloro-1-methylquinolinium tetrafluoroborate, separation and quantification by reversed-phase liquid chromatography followed by UV detection. Disulfides were converted to their thiol counterparts by reductive cleavage with tris(2-carboxyethyl)phosphine. Linearity in detector response for total thiols was observed over the range of 1-40 μmol L(-1) for Hcy and glutathione (GSH), 5-100 μmol L(-1) for Cys-Gly, 20-300 μmol L(-1) for Cys and 3.1-37.5 μmol L(-1) (0.2-2.4gL(-1)) for human serum albumin (HSA). For the protein S-bound forms these values were as follows: 0.5-30 μmol L(-1) for Hcy and GSH, 2.5-60 μmol L(-1) for Cys-Gly and 5-200 μmol L(-1) for Cys. The LOQs for total HSA, Cys, Hcy, Cys-Gly and GSH were 0.5, 0.2, 0.4, 0.3 and 0.4 μmol L(-1), respectively. The estimated validation parameters for all analytes are more than sufficient to allow the analytical method to be used for monitoring of the total and protein bound thiols as well as redox status of HSA in plasma.
authors.affiliation: University of Łódź, Faculty of Chemistry, Department of Environmental Chemistry, 163 Pomorska Str., 90-236 Łódź, Poland.
authors.forename: Kamila
authors.initials: K
authors.lastname: Borowczyk
authors.affiliation: University of Łódź, Faculty of Chemistry, Department of Environmental Chemistry, 163 Pomorska Str., 90-236 Łódź, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Wyszczelska-Rokiel
authors.affiliation: University of Łódź, Faculty of Chemistry, Department of Environmental Chemistry, 163 Pomorska Str., 90-236 Łódź, Poland.
authors.forename: Paweł
authors.initials: P
authors.lastname: Kubalczyk
authors.affiliation: University of Łódź, Faculty of Chemistry, Department of Environmental Chemistry, 163 Pomorska Str., 90-236 Łódź, Poland. Electronic address: glowar@uni.lodz.pl.
authors.forename: Rafał
authors.initials: R
authors.lastname: Głowacki
mesh_terms: D000418:Albumins / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013438:Sulfhydryl Compounds / Q000097:blood*
chemical_list: D000418:Albumins; D013438:Sulfhydryl Compounds
keywords: Albumin; Derivatization; Endogenic plasma thiols; High performance liquid chromatography; Ultraviolet detection