The critical role of trace constituents in differentiating easily confused crop species — A case study of six commercially cultivated Bupleurum varieties

Differentiating easily confounded herbal species is challenging, and the relative roles of high-abundance versus trace components in species discrimination remain poorly defined. This study aims to systematically evaluate their contributions using Bupleurum as a proof of concept. LC-MS chemical profiling of two authentic and four adulterant Bupleurum species that are widely cultivated as cash crops across China were analyzed, identifying 489 constituents, revealing compositional overlaps and authentication challenges. Quantitative LC-MRM-MS analysis of 14 high-abundance saikosaponins, corroborated by HPTLC, showed that these major compounds alone failed to discriminate the six species. This prompted a shift toward minor constituents. HPLC fingerprinting indicated that trace peaks played a decisive role, and LC-HRMS-based metabolomics targeting lower-abundance metabolites achieved complete species discrimination. Following this, a customized R-based pipeline combined with LC-QDa MS identified 13 robust trace markers, which were then applied to 68 commercial samples, yielding authentication rates of ~55% for raw materials and ~64% for formulations. Thus, this first systematic authentication of six Bupleurum species demonstrates that trace constituents possess superior discriminatory power over high-abundance components and provides a methodological framework for tackling easily confounded species