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.
Highlights
Low-molecular-weight thiols such as cysteine (Cys), homocysteine (Hcy), N-acetylcysteine (NAC), glutathione (GSH), cysteinylglycine (CysGly), g-glutamylcysteine (g-GluCys), and cysteamine (CA) shown in Fig. 1A are metabolites of the sulfur cycle and play important roles in biological processes
We aim to develop a method for the simultaneous analysis of different thiols derivatized with SBD-F reagent under Hydrophilic interaction chromatography (HILIC) conditions
TSKgel NH2-100 was excluded because the SBD–thiols were so strongly retained on TSKgel
Summary
Low-molecular-weight thiols such as cysteine (Cys), homocysteine (Hcy), N-acetylcysteine (NAC), glutathione (GSH), cysteinylglycine (CysGly), g-glutamylcysteine (g-GluCys), and cysteamine (CA) shown in Fig. 1A are metabolites of the sulfur cycle and play important roles in biological processes. Biothiols in uence the extra- or intracellular redox state, help in eliminating free radicals, including reactive oxygen species, and remove toxic compounds.[1] In addition, the sulfur metabolism is coupled with the one-carbon metabolism, where both DNA/RNA methylation and nucleotide/thymidylate synthesis take place.[2] An imbalance in the sulfur metabolism causes many diseases such as vascular disease, Alzheimer's disease, human immunode ciency virus infection, and cancer.[3] genetic defects in the enzymes regulating sulfur pools lead to homocystinuria, cystinuria, homocysteinemia, cysteinemia, and neural tube defects.[3] simultaneous detection and quanti cation of different biothiols is important.[4,5,6,7,8,9] Among the Hydrophilic interaction chromatography (HILIC), which was rst introduced in 1990 by A. Alpert,[14] is advantageous for the retention and separation of polar, ionizable analytes.[15,16,17] In HILIC, an organic-rich mobile phase, as in RPLC, and a polar stationary phase, as in normal-phase liquid chromatography, are used for the strong retention of polar compounds by hydrogen-bonding, ion exchange, and hydrophilic partitioning
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