On-line screening method for antioxidants by liquid chromatography with chemiluminescence detection

Abstract A novel screening method for individual antioxidants in complex matrices was developed. The determination of antioxidants is based on the decrease of chemiluminescence (CL) intensity derived from luminol and superoxide anion radical (O −⋅ 2 ) which is generated from the enzyme reaction of xanthine oxidase with hypoxanthine. The constant CL intensity, that is recorded as background (baseline) is required for an accurate and precise detection before injection of sample solutions containing antioxidants. Each of the antioxidant separated by the liquid chromatography (LC) column was observed as a negative peak corresponding to its antioxidative potential. Some authentic antioxidants, i.e. ascorbic acid, isoascorbic acid, glutathione, (+)-catechin, (−)-epicatechin, and (−)-epigallocatechin gallate, seemed to possess scavenging activity for O −⋅ 2 . However, the potential was different for each antioxidant and relatively stronger activity was found in the flavonoids. The detection limits (signal-to-noise ratio of two) of (+)-catechin and (−)-epicatechin with on-line LC-CL were 4.4 and 4.3 pmol, respectively. The proposed method was applied to the detection of antioxidants in extracts of green tea leaves ( Thea sinensis L.). Judging from the observation of negative peaks, (−)-epicatechin was the main component of antioxidants among the various intrinsic substances in green tea extracts. Detection of (−)-epicatechin in the extracts diluted 2000 times was possible, and the sensitivity was higher than that of UV detection at 280 nm. The structure of (−)-epicatechin was also identified with LC-electron spray ionization/mass spectrometry.