This study systematically evaluates the comparative performance of two mass spectrometry acquisition modes, data-dependent acquisition (DDA) and data-independent acquisition (DIA), coupled with ultra-high-performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) for comprehensive chemical profiling of complex traditional Chinese medicine (TCM) formulations. Huaihua Powder, a classical formulated prescription, was employed as a model system for empirical assessment. Optimized DDA and DIA acquisition methods were separately established: the DDA method incorporated a targeted precursor ion selection strategy with customized fragmentation parameters, while the DIA method employed a segmented variable window strategy to cover the target m/z range, performing unbiased fragmentation on all precursor ions. Compound identification was executed using Compound Discoverer software. The comparative evaluation specifically focused on the performance characteristics of the two acquisition modes, encompassing the number of identified compounds, reproducibility, MS/MS spectral quality, and detection sensitivity for low-abundance active constituents. Results demonstrated that the DDA mode yielded a higher total number of detected compounds, whereas the DIA mode generated a greater proportion of high-confidence identifications (10.63 % with spectral match scores >0.8). Notably, the DIA approach exhibited significantly superior reproducibility in retention time and peak area for six representative compounds, with rutin showing >3-fold difference in retention time RSD between the two acquisition modes. However, DDA produced cleaner MS/MS spectra with distinct fragment ions, whereas DIA spectra exhibited interference from contaminant ions. Concurrently, DIA effectively detected low-abundance active constituents whose ion chromatograms and MS/MS fragments could not be extracted in DDA mode. This study contributes critical experimental evidence and analytical datasets to inform the selection of high-resolution mass spectrometry acquisition modes for complex TCM formulation research. Subsequent researchers may integrate the complementary advantages of both approaches to achieve dual objectives in comprehensive compound characterization.

This study systematically evaluates the comparative performance of two mass spectrometry acquisition modes, data-dependent acquisition (DDA) and data-independent acquisition (DIA), coupled with ultra-high-performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) for comprehensive chemical profiling of complex traditional Chinese medicine (TCM) formulations. Huaihua Powder, a classical formulated prescription, was employed as a model system for empirical assessment. Optimized DDA and DIA acquisition methods were separately established: the DDA method incorporated a targeted precursor ion selection strategy with customized fragmentation parameters, while the DIA method employed a segmented variable window strategy to cover the target m/z range, performing unbiased fragmentation on all precursor ions. Compound identification was executed using Compound Discoverer software. The comparative evaluation specifically focused on the performance characteristics of the two acquisition modes, encompassing the number of identified compounds, reproducibility, MS/MS spectral quality, and detection sensitivity for low-abundance active constituents. Results demonstrated that the DDA mode yielded a higher total number of detected compounds, whereas the DIA mode generated a greater proportion of high-confidence identifications (10.63 % with spectral match scores >0.8). Notably, the DIA approach exhibited significantly superior reproducibility in retention time and peak area for six representative compounds, with rutin showing >3-fold difference in retention time RSD between the two acquisition modes. However, DDA produced cleaner MS/MS spectra with distinct fragment ions, whereas DIA spectra exhibited interference from contaminant ions. Concurrently, DIA effectively detected low-abundance active constituents whose ion chromatograms and MS/MS fragments could not be extracted in DDA mode. This study contributes critical experimental evidence and analytical datasets to inform the selection of high-resolution mass spectrometry acquisition modes for complex TCM formulation research. Subsequent researchers may integrate the complementary advantages of both approaches to achieve dual objectives in comprehensive compound characterization.