Rapid screening and characterization of natural antioxidants in Polygonum viviparum by an on-line system integrating the pressurised liquid micro-extraction, HPLC-DAD-QTOF-MS/MS analysis and antioxidant assay.

Rapid discovery of active ingredients from complex matrices is one of great challenges for modern drug development. Traditional methods often require many sample treatment steps, including an extraction step with exclusively dedicated solvents followed by repeated separation and activities assessment. This present work described an integrated analytical setup for natural antioxidants discovery in which the online extraction (OLE) of a solid sample is directly coupled to its analysis by high performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry and 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) antioxidant assay (OLE-HPLC-DAD-QTOF-MS/MS-ABTS). This developed approach makes sample extraction, chromatographic separation and chemical detection, and antioxidant assay integrated into a single HPLC injection and was successfully applied for the rapid discovery of natural antioxidant bioactives from Polygonum viviparum. A total of 21 secondary metabolites were characterized according to their retention times, ultraviolet (UV) spectra, exact mass and fragmentation ions in MS/MS spectra, and 18 of them displayed antioxidant activity (response as negative peaks in antioxidant assay). This work describes a simple, green and efficient approach to minimize the sample consumption (only 0.4 mg was required) and eliminate complex sample treatment procedures. The developed OLE-HPLC-DAD-QTOF-MS/MS-ABTS system offers new perspectives for rapid chemical profiling of natural products and their antioxidants discovery.