Complementary Orthogonal Approaches for Metabolite Profiling of Chamaelirium luteum (False Unicorn) Root Preparations and Commercial Products

Abstract

Chamaelirium luteum (L.) A. Gray (Melanthiaceae), commonly known as false unicorn, is a North American medicinal herb traditionally used for women’s reproductive health. Its major bioactive constituents are open-chain steroidal saponins, a structurally distinctive compound subclass whose increased conformational flexibility may confer bioactivities distinct from common steroidal saponin scaffolds. Because C. luteum is slow-growing and primarily wild-harvested, it faces conservation concerns. Although individual metabolites, including chamaelirosides A (1) and B (2), have been characterized, commercial reference standards and extract-level authentication methods are lacking. To address this need, we developed complementary orthogonal analytical approaches integrating high-performance thin-layer chromatography (HPTLC) chemometric profiling with untargeted liquid chromatography−mass spectrometry (LC-MS) metabolomics and molecular networking. HPTLC band extraction coupled with LC-MS and nuclear magnetic resonance (NMR) enabled rapid structural verification of marker compounds while reducing analysis time and resource requirements. The workflow established robust phytochemical profiles for authenticated C. luteum materials and was subsequently applied to purchased commercial formulations. The majority of products lacked characteristic C. luteum profiles, highlighting species substitution and adulteration. The generalizable workflow and reference data presented here provide a practical framework for phytochemical characterization of botanicals containing open-chain steroidal saponins and, more broadly, for other taxa lacking authenticated reference materials.