Development of a triple hyphenated HPLC-radical scavenging detection-DAD-SPE-NMR system for the rapid identification of antioxidants in complex plant extracts.

A rapid method for the simultaneous detection and identification of radical scavenging compounds in plant extracts was developed by combining an HPLC with on-line radical scavenging using DPPH* as a model radical and an HPLC-DAD-SPE-NMR system. Using this method a commercial rosemary extract was investigated. All major compounds present in the extract were collected on SPE cartridges after their separation. Advantages of on-line SPE peak trapping are the possibility to perform HPLC with non-deuterated solvents, a concentration effect and being able to record NMR spectra in pure 100% deuterated solvents. After comparing DAD and DPPH scavenging chromatograms, 1H NMR spectra of compounds having radical scavenging activities were recorded. Afterwards all compounds were collected and infused into an ESI-MS. The five main active compounds - carnosol, carnosic acid carnosaldehyde, 12-methoxycarnosic acid and epiisorosmanol could be identified from the combined UV, NMR and mass spectral data without actually isolating them. It was possible to record on-line an HMBC spectrum of carnosic acid. Also one compound was tentatively identified as epirosmanol methyl ether.