Automated high performance liquid chromatography with on-line reduction of disulfides and chemiluminescence detection for determination of thiols and disulfides in biological fluids.

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.

The new approach for captopril detection employing triangular gold nanoparticles-catalyzed luminol chemiluminescence.

In this work, we utilize the triangular gold nanoparticles (AuNPs) prepared by trisodium citrate reduction of HAuCl(4) in presence of nonionic fluorosurfactant (FSN) as a novel chemiluminescence (CL) probe for the determination of captopril. Captopril can induce a sharp decrease in CL intensity from the triangular AuNPs-catalyzed luminol system. Under the selected experimental conditions, a linear relationship was obtained between the logarithm of CL intensity and the logarithm of concentration of captopril in the range of 23.0-920 nM, and the detection limit at a signal-to-noise ratio of 3 for captopril was 4.6 nM. The as-prepared triangular AuNPs were easier to synthesize, stable at a wider pH range and high ionic strength, and exhibited a high selectivity and an excellent sensitivity toward captopril. The applicability of the proposed method has been validated by determining captopril in commercial pharmaceutical formulations and human urine samples with satisfactory results. The recoveries for captopril in spiked samples were found to be between 95.0% and 103.5%. The method shows promise for routine control analysis of pharmaceutical preparations and human urine samples. Moreover, based on the CL spectra, UV-vis spectra and transmission electron microscope (TEM) measurements, a possible CL mechanism was proposed. The mechanism of high selectivity toward captopril is supposed to originate from the tight binding of the sulphydryl groups of captopril to the active site of the as-prepared triangular AuNPs, leading to oxygen-related radicals cannot easily be generated from H(2)O(2) on the surface of triangular AuNPs.