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

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%.

Analysis of Total Thiols in the Urine of a Cystathionine β-Synthase-Deficient Mouse Model of Homocystinuria Using Hydrophilic Interaction Chromatography

“…Pretreatment of mouse urine samples and standard solutions was based on methodology from a previous report [18]. In brief, the samples were incubated with tris(2-carboxyethyl)phosphine (TCEP) to reduce disulfide bonds into thiols.…”

A New and Rapid LC-MS/MS Method for the Determination of Cysteamine Plasma Levels in Cystinosis Patients

“…The determination of free cysteamine in plasma requires reducing disulfides prior to the analysis. Several reducing agents have been proposed, including dithiothreitol (DTT) and Tris(2-carboxyethyl) phosphine (TCEP) [14,30,31]. In the present study, in order to selectively and quantitatively reduce disulfide bonds, we used TCEP as previously described in [31].…”

“…In the present study, in order to selectively and quantitatively reduce disulfide bonds, we used TCEP as previously described in [31]. Optimal TCEP-reducing conditions for measuring total biothiols in mouse serum samples have been previously described in [30,32,33]. These studies showed that TCEP reduces disulfide bonds as effectively as dithiothreitol (DTT) but offers addi-tional advantages because it is non-volatile, odorless, and, unlike most other reducing agents, resistant to air oxidation.…”