Optimization of metabolomics pretreatment method of cholangiocarcinoma cells based on ultrahigh performance liquid chromatography coupled with mass spectrometry

Metabolomics intends to maximize the quantity of available metabolites for the global metabolome, which largely depends on sample pretreatment protocols. However, there are few studies that comprehensively examined the effects of extraction and reconstitution solvents on metabolome coverage of adherent mammalian cells. In this study, the human cholangiocarcinoma TFK-1 cells were chosen as a cell model, and eight extraction solvents and five reconstitution solvents were used for the pretreatment based on ultrahigh performance liquid chromatography coupled with mass spectrometry (UPLC/MS). The coverage, reproducibility, and stability of the data were norms to evaluate the effectiveness of different extraction solvents and reconstitution solvents. Based on the number of metabolites, the mean Euclidean distance (ED_MEAN) in the principal component analysis (PCA) 3D score plots and the relative standard deviation (RSD) distribution of metabolites, it was demonstrated that MeOH-CHCl₃-H₂O (8:1:1, v/v/v) was the optimal extraction solvent and MeOH-H₂O (1:1, v/v) or H₂O was superior to other reconstitution solvents for RP column analysis, and the extraction solvent MeOH-ACN-H₂O (2:2:1, v/v/v) and the reconstitution solvents ACN-H₂O (4:1, v/v) or MeOH-H₂O (1:1, v/v) provide the best performance for HILIC column analysis. The optimized pretreatment methods explored in this study expand the coverage of polar and non-polar metabolites and improve the reproducibility and stability of the metabolic data, which can be applied to UPLC/MS-based global metabolomics study on cholangiocarcinoma cells, potentially providing better extraction solvents and reconstitution solvents for other adherent mammalian cells with similar chemical and physical properties.