Advancing HPLC-PDA-HRMS-SPE-NMR Analysis of Coumarins in Coleonema album by Use of Orthogonal Reversed-Phase C₁₈ and Pentafluorophenyl Separations

Abstract

A hyphenated procedure involving high-performance liquid chromatography, photodiode array detection, high-resolution mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy, i.e., HPLC-PDA-HRMS-SPE-NMR, has proven an effective technique for the identification of compounds in complex matrices. Most HPLC-PDA-HRMS-SPE-NMR investigations reported so far have relied on analytical-scale reversed-phase C columns for separation. Herein is reported the use of an analytical-scale pentafluorophenyl column as an orthogonal separation method following fractionation of a crude ethyl acetate extract of leaves of Coleonema album on a preparative-scale C column. This setup allowed the HPLC-PDA-HRMS-SPE-NMR analysis of 23 coumarins, including six new compounds, 8-O-β-d-glucopyranosyloxy-6-(2,3-dihydroxy-3-methylbut-1-yl)-7-methoxycoumarin (4), (Z)-6-(4-β-d-glucopyranosyloxy-3-methylbut-2-en-1-yl)-7-hydroxycoumarin (6), 6-(4-β-d-glucopyranosyloxy-3-methylbut-1-yl)-7-hydroxycoumarin (8), (Z)-7-(4-β-d-glucopyranosyloxy-3-methylbut-2-en-1-yloxy)coumarin (13), (S)-8-(3-chloro-2-hydroxy-3-methylbut-1-yloxy)-7-methoxycoumarin (19), and 7-(3-chloro-2-hydroxy-3-methylbut-1-yloxy)coumarin (20). The use of the pentafluorophenyl column even allowed separation of several regioisomers that are usually difficult to separate using reversed-phase C columns. The phytochemical investigation described for C. album in this report demonstrates the potential and wide applicability of HPLC-PDA-HRMS-SPE-NMR for accelerated structural identification of natural products in complex mixtures.

Current status and contemporary approaches to the discovery of antitumor agents from higher plants

“…It is also possible to use two or more spectroscopic methods in combination, allowing for the separation and acquisition of all spectral data at once, particularly for samples available only in limited amounts (Wilson and Brinkman, 2007). In this regard, a widely used dereplication technique in drug discovery is LC-MS-NMR (Corcoran and Spraul, 2003), with many variants available such as LC-PDA-HRMS-SPE-NMR, where solid-phase extraction (SPE) is intended to resolve the technical issues associated with using in combination two detection methods (i.e., NMR and MS) with different degrees of sensitivity (Jaroszewski, 2005b;Lima et al, 2017). This procedure offers the ability of the investigator to elucidate the structure of individual natural products when present in mixtures (i.e., crude extracts of organisms and chromatographic fractions), thereby facilitating early dereplication without isolation, in addition to allowing preparative-scale isolation, and has been applied for the isolation of numerous secondary metabolites (Clarkson et al, 2006;Johansen et al, 2011;Johansen et al, 2013;Kesting et al, 2011;Staerk et al, 2009).…”

Metabolomics in the Context of Plant Natural Products Research: From Sample Preparation to Metabolite Analysis

“…The sample is split after the initial HPLC separation with a small portion being routed for PDA and MS analysis and the remainder to the SPE cartridges for collection [173]. HPLC-PDA-MS-SPE-NMR has been used for the dereplication of six novel quinolinone alkaloids, named haplacutine A-F from the Haplophyllum acutifolium crude extract [174] and 23 coumarins including six new compounds from the crude ethyl acetate extract of Coleonema album leaves [175] and even minor metabolites could also be detected and fully characterized using this advanced platform [176]. These developments thus suggest that NMR, despite its lower sensitivity will retain a role in role in drug discovery in the decades to come.…”

Metabolomics-Guided Elucidation of Plant Abiotic Stress Responses in the 4IR Era: An Overview

“…Additionally, HPLC-SPE-NMR has been enhanced with the integration of a photodiode array detector (PDA) and MS for authentication and the structural characterization of secondary metabolites from complex plant extracts [ 34 , 130 ]. For instance, HPLC-PDA-HRMS-SPE-NMR analysis of Coleonema album leaves enabled the structural identification of 23 coumarins [ 131 ]. An automated HPLC-MS-SPE-NMR approach was used to characterize flavonoid structures in crude tomato extracts [ 130 ].…”