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

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 C₁₈ 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 <i>Coleonema album</i> on a preparative-scale C₁₈ column. This setup allowed the HPLC-PDA-HRMS-SPE-NMR analysis of 23 coumarins, including six new compounds, 8-<i>O-</i>β-d-glucopyranosyloxy-6-(2,3-dihydroxy-3-methylbut-1-yl)-7-methoxycoumarin (<b>4</b>), (<i>Z</i>)-6-(4-β-d-glucopyranosyloxy-3-methylbut-2-en-1-yl)-7-hydroxycoumarin (<b>6</b>), 6-(4-β-d-glucopyranosyloxy-3-methylbut-1-yl)-7-hydroxycoumarin (<b>8</b>), (<i>Z</i>)-7-(4-β-d-glucopyranosyloxy-3-methylbut-2-en-1-yloxy)­coumarin (<b>13</b>), (<i>S</i>)-8-(3-chloro-2-hydroxy-3-methylbut-1-yloxy)-7-methoxycoumarin (<b>19</b>), and 7-(3-chloro-2-hydroxy-3-methylbut-1-yloxy)­coumarin (<b>20</b>). The use of the pentafluorophenyl column even allowed separation of several regioisomers that are usually difficult to separate using reversed-phase C₁₈ columns. The phytochemical investigation described for <i>C. album</i> 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