An Integrated Strategy Combining Shotgun Metabarcoding With Conventional Methods for Comprehensive Authentication of Food‐Medicine Homologous Species and Fungal Contamination Detection in Yangyin Qingfei Wan

ABSTRACT OTC medicines frequently incorporate species with “food‐medicine homology”, navigating regulatory gray areas, particularly concerning fungal pathogens and adulterated bioactive components that pose health risks to consumers. This research employed a hybrid approach combining shotgun metabarcoding with conventional methods (microscopic identification, thin‐layer chromatography [TLC] and high‐performance liquid chromatography [HPLC]) to authenticate ingredients and assess fungal contamination in Yangyin Qingfei Wan (YYQFW). Analysis encompassed two mock samples and 19 commercial YYQFW samples. Conventional methods confirmed adherence to Chinese Pharmacopeia standards: microscopic analysis identified essential tissue structures, TLC detected key compounds (paeonol, paeoniflorin), and HPLC quantified paeonol content (6.2–9.1 mg/pill), surpassing the standard (5.8 mg/pill). Shotgun metabarcoding retrieved sequences from four DNA barcoding regions (ITS2, psbA‐trnH , matK , and rbcL ), yielding 350, 58, 49, and 53 operational taxonomic units (OTUs), respectively. All labeled ingredients were authenticated via DNA barcodes matching reference databases. Importantly, ITS2 analysis identified 53 fungal OTUs (17 genera, predominantly Aspergillus , Fusarium , and Cladosporium ), indicating potential mycotoxin presence. This research underscores the efficacy of integrating methods to ensure the reliability of OTC medications containing food‐medicine homologous ingredients, which is crucial given their widespread clinical use. Furthermore, ITS2 emerges as the optimal barcode sequence in shotgun metabarcoding studies, verifying ingredient authenticity and detecting fungal contaminants effectively.