What’s in the box? Authentication ofEchinaceaherbal products using DNA metabarcoding and HPTLC

Abstract Background Differences in regulatory policies between countries as well as a lack of appropriate standardized methods for authentication and quality control of herbal products lead to concerns over quality and safety. Echinacea products are among the top selling herbal products in Europe and the United States with indications for broad range of ailments. Purpose This study approached the need for a novel analytical strategy in authentication of herbal products. Methods A combination of high performance thin layer chromatography (HPTLC) and DNA metabarcoding was employed. Fifty-three Echinacea herbal products marketed across Europe were tested to evaluate the accuracy of these methods in plant identification and their potential in detecting substitutes, adulterants and other unreported plant constituents. Results HPTLC provides a high resolution in detecting Echinacea phytochemical target compounds, but does not offer information on the other species within the product. Alternatively, we showed that the limitation of HPTLC to detect non-targeted species can be overcome through the complementary use of DNA metabarcoding. Using DNA metabarcoding, Echinacea species were detected in 34 out of the 38 retained products (89 %), but with a lack of discriminatory resolution at the species level due to the low level of molecular divergence within the Echinacea genus. All herbal products showed considerable discrepancies between ingredients listed on the label and the ones detected using DNA metabarcoding registering an overall ingredient fidelity of 43 %. Conclusion The results confirm that DNA metabarcoding can be used to test for the presence of Echinacea and simultaneously to detect other species present in even highly processed and multi-ingredient herbal products. Abbreviations EMA European Medicines Agency BLAST basic local alignment search tool FTIR Fourier-transformed infrared spectroscopy IR infrared spectrometry HPTLC high performance thin layer chromatography mat K maturase K MS mass spectrometry MOTU molecular taxonomic unit nrITS nuclear ribosomal internal transcribed spacer Ph.Eur. European Pharmacopoeia rbc L ribulose bisphosphate carboxylase TLC thin-layer chromatography UV-VIS ultraviolet-visible