Quantitative Analysis and Simultaneous Characterization of Triterpenoids and Phenolics in Inonotus obliquus (Chaga) Using LC-PDA-ELSD and LC-DAD-QToF

Authors: AVULA, BHARATHI - University Of Mississippi; KATRAGUNTA, KUMAR - University Of Mississippi; TATAPUDI, KIRAN - University Of Mississippi; KHAN, IKHLAS - University Of Mississippi

Submitted to: Planta Medica
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/24/2025
Publication Date: 9/11/2025
Citation: Avula, B., Katragunta, K., Tatapudi, K.K., Khan, I.A. 2025. Quantitative Analysis and Simultaneous Characterization of Triterpenoids and Phenolics in Inonotus obliquus (Chaga) Using LC-PDA-ELSD and LC-DAD-QToF. Planta Medica. 2025 Sep 11. https://doi.org/10.1055/a-2689-8131.
DOI: https://doi.org/10.1055/a-2689-8131

Interpretive Summary: Inonotus obliquus (Ach. ex Pers.) Pilát, a member of the Hymenochaetaceae family within the Agaricomycetes class, is widely recognized as the chaga mushroom. Chaga contains various bioactive compounds, including polysaccharides, triterpenoids, polyphenols, and melanin. To address the characterization and simultaneous quantitative analysis of triterpenoids and phenolics in Inonotus obliquus (chaga), a multi-analytical approach has been developed. This integrated methodology enables comprehensive profiling and quantification of bioactive compounds, ensuring product authenticity and quality control in chaga-based supplements. These findings provide valuable insights for the quality assessment of dietary or food supplements marketed as containing chaga. Furthermore, these results contribute to dereplication studies on I. obliquus sclerotia, aiding in the identification and characterization of its bioactive constituents.

Technical Abstract: Inonotus obliquus (Ach. ex Pers.) Pilát, a member of the Hymenochaetaceae family within the Agaricomycetes class, is widely recognized as the chaga mushroom. Chaga contains various bioactive compounds, including polysaccharides, triterpenoids, polyphenols, and melanin. To address the characterization and simultaneous quantitative analysis of triterpenoids and phenolics in Inonotus obliquus (chaga), a multi-analytical approach combining LC-PDA-ELSD (Liquid Chromatography with Photodiode Array and Evaporative Light Scattering Detection) and LC-DAD-QToF (Liquid Chromatography-Diode Array Detection-Quadrupole Time-of-Flight Mass Spectrometry) has been developed. This integrated methodology enables comprehensive profiling and quantification of bioactive compounds, ensuring product authenticity and quality control in chaga-based supplements. These methods were designed to quantify 11 compounds, comprising seven triterpenoids and four fatty acids, using LC-PDA-ELSD, and four phenolics using the LC-DAD-QToF method. Calibration curves for these compounds demonstrated excellent linearity (r² = 0.99) within the tested range. The methods exhibited high precision, with intra- and inter-day relative standard deviations (% RSD) below 3% and recoveries ranging from 91% to 104%. The validated method was applied to analyze eleven I. obliquus sclerotia samples, one mycelium sample, three grain-based samples, and eighteen dietary supplements. Results revealed that eight of the eighteen supplements (44%) contained ground mycelium grown on grain biomass, which primarily shows the presence of fatty acids but lacks detectable levels of triterpenoid and phenolic markers characteristic of chaga. Triterpenoids and hispidin, identified as key bioactive compounds, were detected in eight (44%) of the eighteen supplements, but these products also showed the presence of fatty acids and/or betulin. Two (11%) of the 18 supplements showed the presence of phenolic compounds only; no triterpenoids were detected. Additionally, untargeted metabolomic screening using LC-DAD-QToF tentatively identified 73 compounds from diverse chemical groups, including eight reference compounds. These included twenty-seven styrylpyrones and eleven triterpenoids, constituting the majority of the other identified classes of compounds. These findings provide valuable insights for the quality assessment of dietary or food supplements marketed as containing chaga. Furthermore, these results contribute to dereplication studies on I. obliquus sclerotia, aiding in the identification and characterization of its bioactive constituents.