Optimization of tris(2-carboxyethyl) phosphine reduction conditions for fast analysis of total biothiols in mouse serum samples

• Main Authors: Chunfang Chang, Muneki Isokawa, Takashi Funatsu, Makoto Tsunoda
• Format: Article
• Language: English
• Published: Elsevier 2019-05-01
• Series: Heliyon
• Subjects: Pharmaceutical chemistry; Analytical chemistry
• Online Access: http://www.sciencedirect.com/science/article/pii/S2405844018391722

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%.