Optimized normalized collision energy and characteristic ion fragments for LC-MS/MS identification of caffeoylquinic...

The rapid discrimination of caffeoylquinic acid (CQA) positional isomers remains challenging in complex matrices due to highly similar tandem mass spectra and frequent chromatographic co-elution. An energy-resolved LC-MS/MS strategy is presented that exploits the sensitivity of low-energy fragmentation to acyl substitution positions. By systematically screening normalized collision energies (NCEs) and quantitatively evaluating MS/MS spectral differences using cosine similarity, optimized normalized collision energies (ONCEs) and characteristic ion fragments (CIFs) were objectively determined for mono- and di-caffeoylquinic acids. Low-NCE regimes were found to maximize isomer-specific differences in fragment ion abundances, enabling reliable discrimination of difficult isomeric pairs, including 1-CQA and 3-CQA. The resulting hierarchical identification workflow was validated across multiple chromatographic systems and successfully applied to the analysis of CQA isomers in Blumea aromatica and Blumea repanda, including cases of severe chromatographic co-elution. This strategy provides a robust, standard-independent approach for high-confidence identification of positional isomers and establishes a generalizable framework for NCE-resolved MS/MS analysis of structurally similar compounds. Therefore, further cross-platform validation screening criteria and evaluation in more complex co-elution scenarios are necessary in the future.