| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1165208 | Analytica Chimica Acta | 2013 | 6 Pages |
In general, the reduction of disulfide bonds with tris(2-carboxyethyl)phosphine (TCEP) is performed using off-line operation, which is not only time-consuming but also vulnerable to the spontaneous re-oxidation of thiols during sample preparation and subsequent analysis procedures. To the best of our knowledge, there has been not any case on the on-line reduction for biological disulfides coupled with high performance liquid chromatography (HPLC). In this study, these obstacles are overcome by packing Zn(II)-TCEP complexes into a home-made column. The as-synthesized Zn(II)-TCEP complexes enable efficient reduction of disulfide bonds at pH 3.0. This acidic pH value was compatible with that of the mobile phase for HPLC separation of thiols and disulfides. Therefore, using fluorosurfactant-prepared triangular gold nanoparticles as HPLC postcolumn specific chemiluminescence (CL) reagents for thiols, the feasibility of the established on-line reduction column has been confirmed for the direct identification of both thiols and disulfides by incorporating this reduction column into a single chromatographic separation. Detection limits for these analytes range from 8.3 to 25.4 nM and the linear range in a log–log plot can comprise three orders of magnitude. Finally, the utility of this automated on-line reduction of disulfides-HPLC-CL system has been demonstrated for the reliable determination of thiols and disulfides in human urine and plasma samples.
Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► An on-line Zn(II)-TCEP reduction column for disulfides is fabricated. ► This reduction column is coupled with HPLC-CL system for assay of thiols and disulfides. ► The optimum pH for disulfides reduction is compatible with that of the HPLC mobile phase. ► The proposed method has been applied to analyze real samples.
