Using UHPSFC to Determine Anthraquinone Derivatives in Herbal Medicine

Scientists from the University of Innsbruck in Austria used ultrahigh-performance supercritical fluid chromatography (UHPSFC) to facilitate the qualitative and quantitative determination of seven anthraquinone derivatives in alder buckthorn. Their findings were published in the Journal of Chromatography A (1).

Buckthorn (Rhamnus catharticus). Black berries on a branch with leaves, on thorn bush in the family Rhamnaceae | Image Credit: © Denny - stock.adobe.com

Alder buckthorn or Frangula alnus Mill., is a type of shrub native to most of Europe and parts of western Asia (2). Its components have been used in various ways, with dried bark and fruit being used for its laxative effects to treat acute constipation since the Middle Ages. The bark contains chemicals that, by stimulating the intenstines, allow for laxative effects (3). However, its metabolites are also associated with toxic side effects. Because of this reliable analytical techniques for determination are vital. The current edition of the European Pharmacopoeia monographs the bark with a minimum content of 7.0% glucofrangulins, which are determined by a photometric assay based on the modified Bornträger reaction (a type of reaction used for identifying anthraquinones in plant extracts and powders) (4).

In this study, the scientists presented an alternative approach using UHPSFC, to facilitate the qualitative and quantitative determination of anthraquinone derivates in the shrub. Using UHPSFC, the lipids are separated according to a headgroup’s polarity, which results in the coelution of all lipid species within one lipid class in one chromatographic peak; unlike typical SFC systems, UHPSFC systems offer stable and reproducible results, the scientists wrote (4,5). The overall process was done in under six minutes. With selectivity, linearity (R² ≥ 0.9995), precision, accuracy, and recovery rates between 96.6 and 103.3%, the results were in line with their respective ICH requirements.

The assay’s practical applicability was confirmed by analyzing different samples, including commercially available plant materials, phytopharmaceuticals that contained Frangula bark, and wild collected samples. With exception to the latter, glucofrangulin A was shown to be the dominating compound. However, overall anthraquinone content proved to be very variable, ranging from 2.18 to 51.32 mg.

With the presented method, analysis time was significantly shortened to 7 minutes (compared to 14 with the original method), with 3 additional compounds (emodin, emodin-8-O-glucoside and physcion-8-O-rutinoside) having capacity for assessment outside of the dominant derivatives frangulin A and B as well as glucofrangulin A and B. This method also allowed for simultaneous analysis of anthraquinone aglyca and glycosides. The validation parameters were all within acceptable limits and quantitative results for real samples, which were comparable to those already published.

Overall, the UHPSFC method presented in this study was shown to surpass currently used approaches for buckthorn bark analysis. This stems from significant advantages regarding from separation time and the number of analytes, as well as assays being fully validated and successfully applied to diverse types of real samples.

References

(1) Zwerger, M.; Deisl, A.; Hammerle, F.; Ganzera, M. Determination of Anthraquinones in Frangula alnus by Supercritical Fluid Chromatography. J. Chromatogr. A 2024, 1737, 465432. DOI: 10.1016/j.chroma.2024.465432

(2) Alder Buckthorn. Woodland Trust 2024. https://www.woodlandtrust.org.uk/trees-woods-and-wildlife/british-trees/a-z-of-british-trees/alder-buckthorn/ (accessed 2024-12-13)

(3) Alder Buckthorn. RxList 2020. https://www.rxlist.com/supplements/alder_buckthorn.htm (accessed 2025-1-7)

(4) Dihydroxyanthraquinone. ScienceDirect 2018. https://www.sciencedirect.com/topics/chemistry/dihydroxyanthraquinone (acccessed 2024-12-16)

(5) Lásko, Z.; Hájek, T.; Jirásko, R.; et al. Four-Dimensional Lipidomic Analysis Using Comprehensive Online UHPLC × UHPSFC/Tandem Mass Spectrometry. Anal. Chem. 2024, 96 (49), 19439–19446. DOI: 10.1021/acs.analchem.4c03946

(6) Botcherby, L. Lipidomic Analysis Using UHPSFC–MS. LCGC International 2022. https://www.chromatographyonline.com/view/lipodomic-analysis-using-uhpsfc-ms (accessed 2025-1-6)