Simultaneous determination of albumin and low-molecular-mass thiols in plasma by HPLC with UV detection

• The paper describes HPLC-UV method for determination of HSA and main plasma thiols.
• The assay enables direct injection of not deproteinized sample.
• Compounds with different physic-chemical properties can be simultaneously assayed.
• The assay uses fewer steps in sample work-up speeding up the analytical procedure.

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 (0.2–2.4 g L−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.