Determination of Thiols and Free Thiol Content in a Protein with Liquid Chromatography Coupled to Amperometric Detection at a Silver Based Carbon Paste Electrode

Abstract

Liquid chromatography (LC) coupled to an electrochemical detector was applied for the determination of small thiols such as cysteine (Cys), glutathione (GSH) and dithiothreitol (DDT) and the free thiol content in a protein. The thio‐compounds were first derivatized by 2,2′‐dipyridyldisulfide (DPDS) at pH 4.5 to yield the corresponding disulfide and 2‐thiopyridone (2‐TP). The reaction mixture was separated isocratically on a reversed‐phase C18 column with amperometric detection (AD) at a carbon paste electrode modified with silver microparticles and polarized at 0.080 V versus Ag/AgCl. The LC‐AD response corresponding to the generated 2‐TP allowed for the indirect determination of the studied thiol derivatives in the 1×10−5–1×10−4 M range for Cys and GSH and 5×10−6–5×10−5 M range for DTT. The LOD and LOQ values were in the range of 9×10−7–4×10−6 M and 3×10−6−‐5.5×10−6 M, respectively for the three studied compounds. Application to the determination of the free thiol content of human and bovine serum albumin as model proteins was realized. The LC‐AD results were validated with respect to a developed spectrophotometric method performed under identical experimental derivatization conditions.

Voltammetric Electrochemical Behavior of Carbon Paste Electrode Containing Intrinsic Silver for Determination of Cysteine

“…Phase composition of the synthesized material was determined by x-ray powder diffraction (XRD), as previously reported [11,40]. The pattern for the material clearly indicates the existence of the metallic silver and silver-copper sulfides (Ag1.2Cu0.8S and CuAgS).…”

“…Electrode material comprising metallic silver and jalpaite showed the best electrochemical behavior towards cysteine [10]. A number of studies have shown that silver is a good substrate for electrodes for the selective determination of thio compounds, in the range of submicromolar concentrations, at low applied potential [11].…”

“…Accordingly, many chemical and instrumental methods have been reported for the determination of cysteine. These include chromatography separation [11,13,14], spectrophotometry [2,5,7,15] and electrochemical techniques [15][16][17][18]. Compared to other methods, electroanalytical approaches have inherent advantages such as simplicity, high sensitivity, good stability and relatively low price.…”

Recent Advances in Electrochemical Sensors for Sulfur-Containing Antioxidants

“…The slow kinetics of sulfur-containing antioxidants oxidation at bare electrodes significantly decreases the sensitivity and requires the application of high positive working potentials. A promising approach to solve this problem is the application of chemically modified electrodes based on Au nanoparticles [ 177 ], Ag microparticles [ 178 ] or carbon nanomaterials [ 179 ].…”