Database-matching method enables advanced LC-MS technologies to identify 51 “unreported” components and quantify 8 toxins in Yunnan Baiyao powder

Yunnan Baiyao powder (YNBYp), a traditional chinese medicine (TCM) formulation with a rich historical legacy, serves as an indispensable medicinal preparation within the majority of Chinese households to date. However, its bioactive, toxic, and potentially hazardous components remain poorly characterized. In this study, a database-matching methodology was developed to facilitate the application of UHPLC-Q-Orbitrap-MS/MS technology, enabling the comprehensive identification of diverse components in YNBYp. A total of 62 components were putatively identified, including 51 “previously unreported” components in YNBYp, representing the first such identification in this formulation. These novel components predominantly comprise 8 spirostene saponins (polyphyllin Ⅶ, saponin C from Liriope muscari , polyphyllin Ⅵ, polyphyllin I, deltonin, dioscin, prosapogenin A, and ophiopogonin D), 6 toxic alkaloids (matrine, karacoline, 12- epi -napelline, fuziline, benzoylaconitine, and yunaconitine), and two potentially hazardous components (β-ecdysterone and diosgenin). Subsequently, all detected toxic components were quantified utilizing a complementary advanced LC-MS (liquid chromatography-mass spectrometry) platform, specifically database-matching ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-Q-TOF-MS/MS). Key findings included: (1) the enrichment of spirostene saponins, ginsenosides, and aconitine-type diterpenoid alkaloids constitutes a defining chemical signature of YNBYp; (2) toxic alkaloids and steroidal compounds were present in quantifiable concentrations (2.3–129.8 μg/g) within YNBYp. Collectively, these observations provide not only critical insights into the chemical basis of YNBYp but also empirical evidence to inform risk assessment and control strategies. Furthermore, this study establishes a methodological framework for the comprehensive characterization of the bioactive, toxic, and potentially hazardous components contained within other complex TCM formulations.