title: Determination and characterization of total thiols in mouse serum samples using hydrophilic interaction liquid chromatography with fluorescence detection and mass spectrometry.
abstract: Biothiols such as homocysteine, cysteine, and glutathione play many biologically important roles, especially in reduction-oxidation homeostasis and resistance to oxidative stress, and the measurement of their concentrations in model animal fluids is important in clarifying the pathology of thiol-related diseases. In this study, an analytical method for total biothiols in mouse serum using hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection was developed. Mouse serum samples were derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), after reduction by tris(2-carboxyethyl)phosphine. Five biothiols (homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine) in the mouse sera were separated within 16 min on an amide-type HILIC column. The method possessed good linearity, good reproducibility with an intra-day variance of less than 3%, and low detection limits of 0.2-4 nM. Concentrations of homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine in the mouse serum samples were calculated as 6.7 ± 0.3, 227.7 ± 16.9, 1.2 ± 0.4, 77.5 ± 29.2, and 8.2 ± 0.9 μM, respectively (mean ± S.D., n = 4). Furthermore, HILIC-negative electrospray ionization-mass spectrometry (MS) analysis using a high-resolution mass spectrometer was conducted to determine the exact masses of two unknown peaks, which were found in the mouse serum samples with high signal intensity and were not detected in human plasma samples. The exact masses of the unknown compounds were determined as 1184.519 and 800.281 (as SBD-derivatized negative ions), which possessed a product ion common to SBD-thiols (m/z 230.954, as [SBD-SH](-)) upon tandem MS spectrometric analysis.
authors.affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Muneki
authors.initials: M
authors.lastname: Isokawa
authors.affiliation: Graduate School of Medicine, University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Shimosawa
authors.affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Funatsu
authors.affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address: makotot@mol.f.u-tokyo.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Tsunoda
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D051379:Mice; D013438:Sulfhydryl Compounds / Q000097:blood* / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D013438:Sulfhydryl Compounds
keywords: Amide column; Oxidative stress; SBD-F; Thiol metabolism