An on-line post-column detection system for the detection of reactive-oxygen-species-producing compounds and antioxidants in mixtures

Abstract

Reactive oxygen species (ROS) can damage proteins, cause lipid peroxidation, and react with DNA, ultimately resulting in harmful effects. Antioxidants constitute one of the defense systems used to neutralize pro-oxidants. Since pro-oxidants and antioxidants are found ubiquitously in nature, pro-and antioxidant effects of individual compounds and of mixtures receive much attention in scientific research. A major bottleneck in these studies, however, is the identification of the individual pro-oxidants and antioxidants in mixtures. Here, we describe the development and validation of an on-line post-column biochemical detection system for ROS-producing compounds and antioxidants in mixtures. Inclusion of cytochrome P450s and cytochrome P450 reductase also permitted the screening of compounds that need bioactivation to exert their ROS-producing properties. This pro-oxidant and antioxidant detection system was integrated on-line with gradient HPLC. The resulting highresolution screening technology was able to separate mixtures of ROS-producing compounds and antioxidants, allowing each species to be characterized rapidly and sensitively.

Development of On-Line High Performance Liquid Chromatography (HPLC)-Biochemical Detection Methods as Tools in the Identification of Bioactives

“…The simplest configuration, used for most on-line antioxidant assays, has no split in the HPLC effluent and only a single reaction coil ( Figure 2a ) [ 5 ]. Most EAD and RAD assays, as well as some on-line antioxidant assays, make use of flow-splitters to divide the HPLC effluent [ 6 – 9 ]. Part of the flow is directed to the BCD assay, while the rest can be linked to additional detectors used for compound identification or sent to waste.…”

Miniaturized Bioaffinity Assessment Coupled to Mass Spectrometry for Guided Purification of Bioactives from Toad and Cone Snail

“…New screening approaches for natural extracts, e.g., affinity-selection MS (AS-MS) [ 26 ], ultrafiltration MS [ 27 ] and high-resolution screening (HRS) [ 28 , 29 , 30 , 31 , 32 ], implement integrated liquid chromatographic (LC) separation and LC-MS. AS-MS techniques measure binding between a protein and its potential ligands by separating the protein-ligand complexes from the non-binding mixture components. Identification of the ligands is done subsequently with MS or MS-MS, whereas HRS techniques apply biochemical assays coupled on-line to LC-MS.…”

The Optimisation of Analytical Parameters for Routine Profiling of Antioxidants in Complex Mixtures by HPLC Coupled Post‐column Derivatisation

“…Accordingly, many reviews and monographs have been published on the evaluation of antioxidant assays (Huang et al , ; Moon and Shibamoto ). More recently, some of the principles of bulk spectrophotometric or electrochemical assays detecting substances with antioxidative properties have been modified for on‐line, post‐column coupling with high‐performance liquid chromatography (HPLC; Dapkevicius et al , ; Exarchou et al , ; Bartasiute et al , ; Kool et al , ; Milasiene et al , ; Niederländer et al , ). The aim of such on‐line approaches is not only the rapid measurement of total activity, but also the profiling of antioxidants in complex mixtures following their chromatographic separation from the matrix.…”