Online comprehensive two-dimensional hydrophilic interaction chromatography × reversed-phase liquid chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry for the analysis of phenolic acids in Salvia miltiorrhiza

In the present study, a system was developed for the analysis of phenolic acids in Salvia miltiorrhiza using online comprehensive two-dimensional hydrophilic interaction chromatography and reversed-phase liquid chromatography coupled to a DAD detector and hybrid linear ion trap-Orbitrap mass spectrometry (HILIC × RP-DAD-ESI/HRMS/MSn). The system was configured based on the combination of an XBridge Amide column (150 mm × 4.6 mm, 3.5 μm) and Accucore PFP column (50 mm × 4.6 mm, 2.6 μm) for the first and second dimensions, respectively. An additional LC pump was used to dilute the eluent from the first dimension to decrease its elution strength in the second dimension. A back-flush trap column was selected as an interface to make up for the loss of efficiency and resolution due to the online dilution. Under the optimized conditions, a total of 196 peaks of polar compounds were successfully separated and detected in Salvia miltiorrhiza using the developed online HILIC × RP system, which exhibited high orthogonality (73%). The online combination of HILIC and RP provides powerful separation capability for the analysis of polar compounds in samples with complex matrices.