In recent years, hyphenation of analytical-scale high-performance liquid chromatography and high-resolution mass spectrometry with solid-phase extraction and nuclear magnetic resonance spectroscopy, i.e., HPLC-HRMS-SPE-NMR, has proven successful for dereplication as well as full structure elucidation directly from crude extracts without any prepurification steps [1 – 3]. However, the HPLC-HRMS-SPE-NMR technology is exclusively a chemical analysis tool and does not give any information about the bioactivity of individual constituents in the crude extract. Thus, the recent development of microplate-based high-resolution bioassays [4], ligand fishing [5] and bioactivity-correlated metabolomics has allowed targeting subsequent HPLC-HRMS-SPE-NMR experiments towards the bioactive constituents only – as indicated in the figure below.
In this talk, the principles of ligand-fishing technologies and bioactivity-correlated metabolomics in combination with HPLC-HRMS-SPE-NMR analysis will be outlined. Thus, ligand-fishing using α-glucosidase immobilized on magnetic beads were used to effectively fish out myricetin 3-O-α-L-rhamnopyranoside, myricetin, quercetin, and kaempferol as α-glucosidase inhibitors in crude extract of Eugenia catharinae [5], and bioactivity-labelling of 1H NMR spectra of crude extracts of Radix astragalii were used for facilitating visual interpretation of scores in principal component analysis. Both methods were combined with HPLC-HRMS-SPE-NMR for structural elucidation of analytes.
Acknowledgements: Arife Önder is acknowledged for technical assistance.
Keywords: High-resolution bioassay, ligand-fishing, metabolomics, HPLC-HRMS-SPE-NMR.
References:
[1] Vinther JM, Wubshet SG, Staerk D. NMR-based metabolomics and hyphenated NMR techniques – a perfect match in natural products research. In: Heinrich M, Jäger AK, editors. Ethnopharmacology – a Reader. Chichester: Wiley, 2015; 63 – 74
[2] Wubshet SG, Nyberg NT, Tejesvi MV, Pirttilä AM, Kajula M, Mattila S, Staerk D. Targeting high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance analysis with high-resolution radical scavenging profiles – bioactive secondary metabolites from the endophytic fungus Penicillium namyslowskii. J Chromatogr A 2013; 1302: 34 – 39
[3] Kongstad KT, Wubshet SG, Nyberg NT. HPLC-NMR revisited: Using time-slice HPLC-SPE-NMR with database assisted dereplication. Anal Chem 2013; 85: 3183 – 3189
[4] Tahtah Y, Kongstad KT, Wubshet SG, Nyberg NT, Jønsson LH, Jäger AK, Qinglei S, Staerk D. Triple aldose reductase/α-glucosidase/radical scavenging high-resolution profiling combined with high-performance liquid chromatography – high-resolution mass spectrometry – solid-phase extraction – nuclear magnetic resonance spectroscopy for identification of antidiabetic constituents in crude extract of Radix Scutellariae. J Chromatogr A 2015; 1408: 125 – 132
[5] Wubshet SG, Brighente IMC, Moaddel R, Staerk D. Magnetic ligand fishing as a targeting tool for HPLC-HRMS-SPE-NMR: α-glucosidase inhibitory ligands and alkylresorcinol glycosides from Eugenia catharinae. J Nat Prod 2015; 78: 2657 – 2665
