Online coupling solid-phase ligand-fishing with high-performance liquid chromatography-diode array detector-tandem mass spectrometry for rapid screening and identification of xanthine oxidase inhibitors in natural products.

Screening and analysis of bioactive compounds from natural products is challenging work due to their complexity. This study presents the first report on hyphenation of solid-phase ligand-fishing using immobilized xanthine oxidase microcolumn (IXOM) and high-performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS) for screening and identification of XO inhibitors from complex mixtures. Solid-phase ligand-fishing system was hyphenated with the HPLC system via four-port switching valve and a six-port injection valve as an interface for transferring effluents from IXOM to HPLC, and collecting chromatograms from LFMC (ligand-fishing microextraction column) and C18 column in a run by only one DAD. Mixtures containing allopurinol (positive control) and tryptophane (negative control) were analyzed in order to verify the specificity and reproducibility of the approach. Subsequently, the newly developed system was applied to screening and identification of XO inhibitors from L. macranthoides and its human microsomal metabolites. Six prototype compounds (3-caffeoylquinic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid) and three metabolites (3-caffeoyl-epi-quinic acid, 5-caffeoyl-epi-quinic acid, 4-caffeoyl-epi-quinic acid) with XO binding affinities were identified. The XO inhibition activities of six prototype compounds were evaluated and confirmed using in vitro enzymatic assay. With the online system developed here, we present a feasible, selective, and effective strategy for rapid screening and identification of enzyme inhibitors from complex mixtures.