Combining PARAFAC Analysis of HPLC-PDA Profiles and Structural Characterization Using HPLC-PDA-SPE-NMR-MS Experiments:  Commercial Preparations of St. John's Wort

Abstract

Herbal preparations represent very complex mixtures, potentially containing multiple pharmacologically active entities. Methods for global characterization of the composition of such mixtures are therefore of pertinent interest. In this work, chemometric analysis of high-performance liquid chromatography with photodiode-array detection (HPLC-PDA) data from extracts of commercial preparations of Hypericum perforatum (St. John's wort) that originate from several continents is described. The spectral HPLC profiles were aligned in the elution mode using correlation optimized warping in order to remove peak misalignment caused by retention time shifts due to matrix effects. Furthermore, the warping was assisted by HPLC-PDA-SPE-NMR-MS (SPE = solid-phase extraction) experiments that yielded 1H NMR and 13C NMR data (from 1H-detected heteronuclear correlations), as well as ESI-MS and HRMS data, which enabled the identification of all major mixture constituents. The preprocessed HPLC-PDA data were subjected to parallel factor analysis (PARAFAC), a chemometric method that is a generalization of principal component analysis (PCA) to multi-way data arrays. PCA of the peak areas obtained from the PARAFAC analysis was used to facilitate sample comparison and allowed straightforward interpretation of constituents responsible for the differences in composition between individual preparations. In addition, loadings from the PARAFAC analysis provided pure elution profiles and pure UV spectra even for coeluting peaks, thus enabling the identification of chromatographically unresolved components. In conclusion, PARAFAC analysis of the readily accessible HPLC-PDA data provides the means for unsupervised and unbiased assessment of the composition of herbal preparations, of interest for assessment of their pharmacological activity and clinical efficacy.

Polyphenol characterization and encapsulation in β-cyclodextrin of a flavonoid-rich Hypericum perforatum (St John's wort) extract

“…An ion at m/z 473, which was ascribed to Na þ adduct, was characteristic of its structure, assigned to astilbin (dehydroquercetin rhamnoside) (Makris, Boskou, Andrikopoulos, & Kefalas, 2008). Astilbin has been also detected in commercial St John's wort preparations (Schmidt, Jaroszewski, Bro, Witt, & Staerk, 2008), while the other quercetin glucosides detected were also present in St John's wort samples from Greece (Tatsis et al, 2007).…”

Magnetic Ligand Fishing as a Targeting Tool for HPLC-HRMS-SPE-NMR: α-Glucosidase Inhibitory Ligands and Alkylresorcinol Glycosides from Eugenia catharinae

“…The above challenges can be overcome by combining ligand fishing with a more powerful structural analysis tool, i.e., HPLC-HRMS-SPE-NMR, that already has proven to be a powerful technique for full structural identification of constituents directly from crude extracts at analytical-scale HPLC conditions. 12,13 …”

Metabolome Classification of CommercialHypericum perforatum(St. John's Wort) Preparations via UPLC-qTOF-MS and Chemometrics

“…One possible explanation is the much more acidic nature of the chosen mobile phase (pH 2.9) compared to ammonium acetate (pH 5) as an eluent, which might not be appropriate for the elution and further detection of these special constituents. Indeed, separation methods reported in the literature using formic acid in aqueous phase failed to detect hypericins in H. perforatum extracts [27,35]. UV and MS spectra were recorded for hypericin standard prepared in 0.1 % formic acid and injected directly without going through the column, ruling out a problem due to an ionization suppression effect by the eluent or possible degradation.…”

“…Exposure of the extract to light converts protohypericin into hypericin and also leads to the degradation of hyperforin, which is unstable and extremely sensitive to air oxidation [24]. Since the efficacy of St. Johnʼs wort medical preparations is based on a mixture of relevant metabolites (synergism), rather than the presence of a single constituent, the development of quick methods allowing for the analysis of such complex unstable extracts is of high relevance [25][26][27][28]. Standardization of commercial H. perforatum extracts is only based on the total content of hypericins, as required by the European Pharmacopoeia monograph, which implies that extracts may be variable with respect to other classes of metabolites [26].…”

“…A near infrared spectroscopic (NIRS) method was established as an alternative to liquid chromatography for quantitative determination of hypericins and phloroglucinols in St. Johnʼs wort extracts [29]. Nuclear magnetic resonance (NMR) based metabolomics used in conjunction with multivariate data analysis, such as principal component analysis (PCA) and parallel factor analysis (PARAFAC) have also been established for the analysis of St. Johnʼs wort products based on their global composition [11,[25][26][27]. However, NMR has the problem of its low sensitivity compared to MS and the overlap of 1 H-NMR signals that hinders robust metabolite identification.…”