Separation and analysis of phenolic acids from Salvia miltiorrhiza and its related preparations by off-line two-dimensional hydrophilic interaction chromatography × reversed-phase liquid chromatography coupled with ion trap time-of-flight mass spectrom

Salvia miltiorrhiza (SM) is one of the most widely used Traditional Chinese Medicine. Active constituents of SM mainly contain hydrophilic phenolic acids (PAs) and lipophilic tanshinones. However, due to the existing of multiple ester bonds and unsaturated bonds in the structures, PAs have numerous chemical conversion products. Many of them are so low-abundant that hard to be separated using conventional methods. In this study, an off-line two-dimensional liquid chromatography (2D-LC) method was developed to separate PAs in SM and its related preparations. In the first dimension, samples were fractionated by hydrophilic interaction chromatography (HILIC) (Acchrom × Amide, 4.6 × 250 mm, 5 μm) mainly based on the hydrogen bonding effects. The fractions were then separated on reversed-phase liquid chromatography (RP-LC) (Acquity HSS T3, 2.1 × 50 mm, 1.7 μm) according to hydrophobicity. For the selective identification of PAs, diode array detector (DAD) and electrospray ionization tandem ion trap time-of-flight mass spectrometry (ESI-IT-TOF-MS) were employed. Practical and effective peak capacities of all the samples were greater than 2046 and 1130, respectively, with the orthogonalities ranged from 69.7% to 92.8%, which indicated the high efficiency and versatility of this method. By utilizing the data post-processing techniques, including mass defect filter, neutral loss filter and product ion filter, a total of 265 compounds comprising 196 potentially new PAs were tentatively characterized. Twelve kinds of derivatives, mainly including glycosylated compounds, O-alkylated compounds, condensed compounds and hydrolyzed compounds, constituted the novelty of the newly identified PAs. The HILIC × RP-LC/TOF-MS system expanded our understanding on PAs of S. miltiorrhiza and its related preparations, which could also benefit the separation and characterization of polar constituents in complicated herbal extracts.