Fast and simultaneous analysis of biothiols by high-performance liquid chromatography with fluorescence detection under hydrophilic interaction chromatography conditions

Abstract

A method for analyzing biothiols based on high-performance liquid chromatography (HPLC)-fluorescence detection under hydrophilic interaction chromatography (HILIC) conditions has been developed. Thiols were derivatized with nonfluorescent ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), which selectively reacts with the thiol groups to furnish the corresponding fluorescent SBD–thiols. Among the six different kinds of HILIC columns examined, the ZIC-HILIC column with sulfobetaine groups in the stationary phase proved to be the best for the separation of SBD–thiols. Eight thiols—N-acetylcysteine, cysteamine, homocysteine, cysteine, cysteinylglycine, glutathione, γ-glutamylcysteine, and internal standard N-(2-mercaptopropionyl)glycine—were baseline-separated within 10 min. The detection sensitivity was improved partly due to the increase in the SBD–thiol fluorescence owing to the acetonitrile-rich mobile phase used. The detection limits at a signal-to-noise ratio of 3 were 0.02–3.4 nmol l⁻¹. The method could successfully quantify six thiols in a human plasma sample, while cysteamine could not be detected. Both the intra- and interday precisions were below 4% for homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine except for N-acetylcysteine. This method should be a useful tool for investigating the relationship between sulfur metabolism and related diseases, since a multicomponent system consisting of different thiol compounds could be analyzed simultaneously with high sensitivity within a short time.