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

ABSTRACT 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/MS n system exhibited high orthogonality (90.20%) and 102 compounds were identified or tentatively characterized based on high‐resolution accurate mass and MS n 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 ( R 2 ≥ 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.