An integrative UHPLC-MS/MS untargeted metabonomics combined with quantitative analysis of the therapeutic mechanism of Si-Ni-San.

A UHPLC-MS/MS untargeted serum metabonomic method combined with quantitative analysis of five potential biomarkers in rat serum was developed and validated, to further understand the anti-liver injury effect of Si-Ni-San and its mechanism on liver injury rats in this study. The metabolites were separated and identified on BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using the ACQUITY UHPLC-MS system (Waters Corp., Milford, MA, USA). Principal component analysis (PCA) was used to identify potential biomarkers. Primary potential biomarkers including phenylalanine, tryptophan, Glycochenodeoxycholic acid (GCDCA) and hysophosphatidylcholine (LPC), which were related to amino acid metabolism, lipid metabolism, bile acid biosynthesis and oxidation-antioxidation balance, were found in the untargeted metabonomic research. Moreover, these targeted biomarkers were further separated and quantified in multiple-reaction monitoring (MRM) with positive ionization mode. The proposed method was linear for each analyte with correlation coefficients over 0.99. The intra- and inter-day precision values (relative standard deviation, RSD) were less than 13.1% and accuracy (relative error, RE) was from -9.5% to 10.3% at all quality control (QC) levels. The validated method was successfully applied to study the serum samples of control group, model group, positive control group (silymarin group) and Si-Ni-San group in rats.