Comprehensive Chemical Profiling and Quality Control of Gegen–Danshen Herb Pair Using Advanced Online Two‐Dimensional Liquid Chromatography Coupled With Hybrid Linear Ion Trap Orbitrap Mass Spectrometry

The Gegen-Danshen (GD) herb pair is extensively employed in the treatment of cardiovascular diseases, attributed to its wide spectrum of bioactive constituents. This study presented a comprehensive chemical analysis of GD using an advanced analytical platform, which integrated online two-dimensional liquid chromatography (LC × LC) coupled with diode array detector (DAD) and hybrid linear ion trap orbitrap mass spectrometry (LTQ-orbitrap). In this setup, a Hypersil GOLD CN column (150 mm × 2.1 mm, 3 µm) was employed in the first dimension, while an Accucore PFP column (50 mm × 4.6 mm, 2.6 µm) was used in the second dimension. Under optimized conditions, the online LC × LC-DAD-ESI/HRMS/MSⁿ system exhibited high orthogonality (90.20%) and 102 compounds were identified or tentatively characterized based on high-resolution accurate mass and MSⁿ fragmentation data, acquired in both positive and negative ion modes. Furthermore, a quantitative analysis method was developed and validated using LC × LC-DAD contour plots, targeting 26 bioactive components in GD herb pair. This method demonstrated excellent linearity (R² ≥ 0.9970) and precision with relative standard deviation (RSD) values below 4.95%. The established analytical approach offers robust separation, reliable identification, and precise quantification of chemical constituents in GD herb pair, providing a valuable tool for the study of bioactive compounds in complex herbal matrices.