Core-Multiplex-Micro Component Profiling for Precision Discrimination of Multi-sources and Adulterated Arnebiae Radix, from Herbs to Formulations

Background The “one-to-multiple” phenomenon and counterfeiting are prevalent in herbal medicines. Accurately identifying these issues in both crude herbs and commercial formulated products is crucial for ensuring drug safety and efficacy. However, this task remains inherently challenging due to the complex nature of the matrices involved. Purpose This study aims to develop methodologies for the precise authentication of multi-sourced herbal medicines and their counterfeits, and to trace constitute species in commercial products. Methods This paper proposes an integrated strategy termed “Core-Multiplex-Micro (CMM) component profiling for precision discrimination amid multi-sources and adulterants”, using Arnebiae Radix as a case study. The strategy begins with the comparative analysis of primary component, shikonin derivatives, across two genuine species and three adulterants, emplying single standard to determine multi-components (SSDMC) approach. It then expands to include a broader range of mid- to low-abundance metabolites through fingerprinting-based classification. Subsequently, trace-level components are explored using LTQ-Orbitrap MS combined with metabolomics to authenticate the five species. Robust chemical markers are identified, validated, and translated to a cost-effective QDa MS platform for targeted detection. This tiered methodology was applied to authenticate 15 batches of commercially available raw materials and 10 batches of formulations; Results The study successfully distinguished between two authentic Arnebiae Radix species and three adulterants simultaneously for the first time. Through cross-validation, nine diagnostic chemical markers were identified and effectively transferred from expensive LTQ-Orbitrap MS platform to more cost-effective QDa MS system for practical use in commercial product traceability. The research revealed that 60% raw Arnebiae Radix materials were authentic, while 30% formulations matched their labeled species composition. Conclusion This study sets a precedent for precise traceability for “one-to-multiple” and counterfeit products: tracing them from raw herbs to final formulations.