A standard-free strategy for the differentiation of isomers and deep profiling of chemicalome in Chinese medicinal formulas.

The precise differentiation of isomers is crucial for comprehensive mass spectrometry-based analysis of complex Chinese medicinal formulas (CMFs). To address this challenge, a standard-free strategy was proposed by integrating chromatographic behavior, mass spectrometric behavior, and quantum chemical calculations. Firstly, structural isomers and certain stereoisomers were differentiated based on characteristic fragment ions and calculated partition coefficient (clogP). For remaining indistinguishable isomers, fragment ions with significant intensity differences were identified, and 3D molecular structures of the precursor ions, product ions, and neutral loss fragments were constructed. The bond dissociation enthalpies (BDE) for critical fragmentation pathways were computed using density functional theory (DFT) and correlated with relative fragment ion abundances to achieve differentiation, following the principle that higher cleavage energies correspond to reduced fragmentation efficiency and lower product ion intensities. The methodology was validated using limited standards and the optimal collision energy (OCE) fitting. Using Qiangli Pipa syrup as a case, this integrated approach enabled the identification of 203 compounds, including 40 isomer groups comprising 27 structural isomers and 13 stereoisomers. This strategy overcomes the limitations of conventional methods that are typically restricted to specific structural classes, demonstrating robust capability for systematic isomer differentiation in CMFs.