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Optimization of tris(2-carboxyethyl) phosphine reduction conditions for fast analysis of total biothiols in mouse
Chunfang Chang1, Muneki Isokawa1, Takashi Funatsu1
1Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Abstract:
In this study, we investigated suitable conditions for the reduction of disulfides in mouse serum samples by tris(2-carboxyethyl) phosphine (TCEP) for fast analysis of total biothiols. Disulfides were reduced with TCEP, and then, thiols were derivatized with the fluorogenic reagent, ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Interference peaks on chromatograms of mouse serum samples disappeared when the TCEP reaction was conducted on ice instead of at room temperature, which is used classically. Low-molecular-weight disulfides, such as cystine and glutathione disulfide, were nearly completely reduced by TCEP on ice. Six SBD-biothiols (homocysteine, cysteine, cysteinylglycine, glutathione, γ-glutamylcysteine, and N-acetylcysteine) were separated within 7.5 min on a sulfoalkylbetain-type column (ZIC-HILIC: 150 × 2.1 mm i.d., 3.5 μm), without interference peaks. The developed method showed good linearity and reproducibility, with inter- and intra-day precisions of less than 3%.
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