title: 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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
authors.forename: Muneki
authors.initials: M
authors.lastname: Isokawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Funatsu
authors.forename: Makoto
authors.initials: M
authors.lastname: Tsunoda
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005464:Fluorobenzenes / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000097:blood / Q000737:chemistry*; D013997:Time Factors
chemical_list: D005464:Fluorobenzenes; D013438:Sulfhydryl Compounds; C037802:7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate