Analysis of berry superfruits for reduced and total thiol content by LC-UV-based methods

The article presents the first methods for determining total and reduced glutathione (GSH), cysteine (Cys), cysteinyl-glycine (Cys-Gly), and homocysteine (Hcy) in berries using high-performance liquid chromatography with ultraviolet detection. The assay employs N-acetyl-cysteine as an internal standard. Sample preparation involves homogenization in phosphate buffer, tris(2-carboxyethyl)phosphine-mediated reduction of disulfide bonds (if total thiol content is determined), 2-chloro-1-methylquinolinium tetrafluoroborate derivatization, perchloric acid acidification, and filtration through a polyethersulfone membrane. Separation is achieved on a Zorbax SB-C18 column using gradient elution with trichloroacetic acid and acetonitrile. Both total and reduced thiol assays showed linearity at 0.5-50 μmol/L for GSH and 0.5-20 μmol/L for Cys and Cys-Gly. Applied to various Polish market berries (e.g., strawberry, raspberry, blueberry, blackberry, forest berry, haskap berry, wild strawberry, currant, gooseberry, cranberry, chokeberry, and elderberry) results identified GSH as the predominant thiol. Cys appeared at lower levels, Cys-Gly was found in traces, and Hcy was undetected.