Coupling HPLC-SPE-NMR with a microplate-based high-resolution antioxidant assay for efficient analysis of antioxidants in food – Validation and proof-of-concept study with caper buds

This work describes the coupling of a microplate-based antioxidant assay with a hyphenated system consisting of high-performance liquid chromatography–solid-phase extraction–nuclear magnetic resonance spectroscopy, i.e., HPLC-SPE-NMR/high-resolution antioxidant assay, for the analysis of complex food extracts. The applicability of the microplate-based antioxidant assay for high-resolution screening of common food phenolics as well as parameters related to their trapping efficiency, elution behavior, and recovery on/from SPE cartridges are described. It was found that the microplate-based high-resolution antioxidant assay is an attractive and easy implementable alternative to direct on-line screening methods. Furthermore, it was shown that Resin SH and Resin GP SPE material are superior to RP C18HD for trapping of phenolic compounds. Proof-of-concept study was performed with caper bud extract, revealing the most important antioxidants to be quercetin, kaempferol, rutin, kaempferol-3-O-β-rutinoside and N1,N5,N10-triphenylpropenoyl spermidine amides. Targeted isolation of the latter, and comprehensive NMR experiments showed them to be N1,N10-di-(E)-caffeoyl-N5-p-(E)-coumaroyl spermidine, N1-(E)-caffeoyl-N5,N10-di-p-(E)-coumaroyl spermidine, N10-(E)-caffeoyl-N1,N5-di-p-(E)-coumaroyl spermidine, and N1,N5,N10-tri-p-(E)-coumaroyl spermidine amides.