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Hydrophilic interaction liquid chromatographic (HILIC) separation of biologically important thiols derivatized with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) was performed for the first time. After optimization of mobile phase conditions, including acetonitrile content, pH, and buffer concentration, eight ABD-thiols (cysteamine, cysteine, N-acetylcysteine, glutathione, homocysteine, cysteinylglycine, -glutamylcysteine, and N-(2-mercaptopropionyl)glycine as an internal standard) were successfully separated in 16 min on ZIC-HILIC column with sulfoalkylbetaine groups. The optimum mobile phase was acetonitrile/50 mM ammonium formate buffer, pH 2.5 (82/18, v/v). The limits of quantitation for ABD-thiols, when signal-to-noise ratio was 10, ranged from 1–120 nM, lower than previous reports that used reversed-phase conditions. The increased sensitivity was partly due to enhancement of fluorescence intensity of ABD-thiols in the acetonitrile-rich mobile phase used in HILIC conditions.

chromatography

Efficient Separation and Sensitive Detection of Biothiols by Hydrophilic Interaction Liquid Chromatography with Fluorescence Detection after Derivatization with 4-Aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole