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

ABSTRACT: 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.

SUBMITTER: Sturm S

PROVIDER: S-EPMC8400717 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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

Sturm Sonja S Högner Christina C Seger Christoph C Stuppner Hermann H

Metabolites 20210809 8

By combining HPLC-DAD-QTOF-MS and HPLC-SPE-NMR, the in vitro metabolism of vitetrifolin D, a pharmacologically active key molecule from <i>Vitex agnus-castus</i> 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 ...[more]

PMID: 34436470

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Project description:BackgroundDanmu preparations (Danmu Capsule and Danmu Syrup), which are made from Nauclea officinalis stem extracts, have good clinical efficacy in acute tonsillitis, acute pharyngitis and upper respiratory tract infection. However, there is currently no reliable and systematic method to control the quality of these two Danmu preparations.MethodsUsing high-performance liquid chromatography (HPLC) coupled with diode array detection (DAD), the fingerprints of the Danmu preparations were established at 250 nm to comprehensively investigate the stability of preparation process. The chemical constituents in the Danmu preparations were separated and identified by HPLC coupled with quadrupole-time-of-flight high-definition mass spectrometry (HPLC-Q-TOF-MS). And seven major components were simultaneously determined at dual wavelengths (250 nm, 326 nm).ResultsThe results of HPLC fingerprint similarity evaluation showed that the similarity values of 25 batches of Danmu preparations were more than 0.993. Twenty-three compounds, including 10 alkaloids, 6 phenolic acids, 2 iridoids, and 5 unknown compounds, were identified or tentatively characterized according to the retention times and MS/MS fragment patterns of compounds. The developed assay method of seven components was validated with acceptable linearity, precision, repeatability, stability and recovery. The contents of strictosamide belonging to alkaloids as the most abundant constituent in Danmu Capsule and Danmu Syrup were 43,681.20-99,652.49 μg/g and 1567.83-2427.25 μg/mL respectively. The contents of protocatechuic acid which were the highest in measured phenolic acids were 2633.01-7739.78 μg/g in Danmu Capsule and 192.05-448.71 μg/mL in Danmu Syrup, respectively. As an iridoid, the contents of sweroside in Danmu Capsule and Danmu Syrup were 1573.82-2789.81 μg/g and 70.32-182.81 μg/mL, respectively.ConclusionThe established qualitative analysis method of fingerprint can be used to attain standardization, uniformity and stability of the preparation process. Meanwhile, the quantitative analysis in this study can be used as an accurate assay method for preparations.

Project description:Commercial preparations of Ginkgo biloba are very complex mixtures prepared from raw leaf extracts by a series of extraction and prepurification steps. The pharmacological activity is attributed to a number of flavonoid glycosides and unique terpene trilactones (TTLs), with largely uncharacterized pharmacological profiles on targets involved in neurological disorders. It is therefore important to complement existing targeted analytical methods for analysis of Ginkgo biloba preparations with alternative technology platforms for their comprehensive and global characterization. In this work, (1)H NMR-based metabolomics and hyphenation of high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) were used for investigation of 16 commercially available preparations of Ginkgo biloba. The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that (1)H NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone-d(6). Unexpected or unwanted extract constituents were also easily identified in the (1)H NMR spectra, which contrasts traditional methods that depend on UV absorption or MS ionizability and usually require availability of reference standards. Automated integration of (1)H NMR spectral segments (buckets or bins of 0.02 ppm width) provides relative distribution plots of TTLs based on their H-12 resonances. The present study shows that (1)H NMR-based metabolomics is an attractive method for non-selective and comprehensive analysis of Ginkgo extracts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-009-0195-x) contains supplementary material, which is available to authorized users.

Dataset Information

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

Publications

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

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