Supercritical fluid-based protocol for the identification and quantitation of triterpenoids in commercial Inonotus obliquus (chaga) samples

Inonotus obliquus is a fungal pathogen that parasitizes the trunks of broadleaved tree species. The sclerotia of this fungus, so-called chaga, show a broad range of traditional and medicinal uses. The increasing market for chaga supplements, promoted for their antioxidant, anti-inflammatory, and antidiabetic properties, highlights the need for standardized identity and quality control methodologies. In this study, the first supercritical fluid-based protocol for the extraction, isolation, identification, and quantitation of six characteristic chaga constituents [lanosterol (1), ergosterol (2), inotodiol (3), trametenolic acid B (4), betulin (5) and inonotsutriol A (6)] was developed and validated. Semi-preparative supercritical fluid chromatography was utilized, to obtain lanosterol (1), inotodiol (3) and inonotsutriol A (6). Ultra-high-performance supercritical fluid chromatography allowed for a separation of the standard compounds within 10 min. Good results regarding linearity (R² ≥ 0.99) were obtained for photodiode array detection, with a lowest detection limit of 0.424 μg/mL. A 20-minute exhaustive SFE method was applied to 14 commercial chaga samples, yielding extracts ranging from 0.17 % to 0.58 %. The qualitative composition of the extracts was consistent, with inotodiol (3), trametenolic acid B (4), and lanosterol (1) as the most abundant constituents. Notably, inonotsutriol A (6) was quantified in commercial chaga samples for the first time. The use of supercritical fluid technology enabled a fast, selective, and environmentally friendly extraction, isolation, and analysis of compounds. This protocol offers a sustainable alternative to traditional chromatographic methods and could serve as a valuable tool for the authenticity and quality control of chaga raw materials and supplements.