Combining HPLC-DAD-QTOF-MS and HPLC-SPE-NMR to Monitor In Vitro Vitetrifolin D Phase I and II Metabolism

By combining HPLC-DAD-QTOF-MS and HPLC-SPE-NMR, the in vitro metabolism of vitetrifolin D, a pharmacologically active key molecule from Vitex agnus-castus in liver cell fractions, was investigated. Twenty-seven phase I and phase II metabolites were tentatively identified from the culture broth by HPLC-DAD-QTOF-MS. The subsequent HPLC-SPE-NMR analysis allowed for the unequivocal structural characterization of nine phase I metabolites. Since the preparative isolation of the metabolites was avoided, the substance input was much lower than in conventional strategies. The study did prove that the use of hyphenated instrumental analysis methodologies allows for the successful performance of in vitro metabolism studies, even if the availability of substances is very limited.

Automated summary

The study investigated the in vitro metabolism of vitetrifolin D using HPLC-DAD-QTOF-MS and HPLC-SPE-NMR, identifying 27 phase I and phase II metabolites, with structural characterization of nine phase I metabolites. By avoiding preparative isolation of metabolites, the study demonstrated the successful performance of in vitro metabolism studies using hyphenated instrumental analysis methodologies even with limited substance availability.