Cell Metabolomics Reveals the Hepatotoxic Mechanism of Copper in Normal Rat Liver Cells Using Reversed‐Phase and Hydrophilic Interaction Liquid Chromatography‐Quadrupole‐Time‐of‐Flight Mass Spectrometry

ABSTRACT Copper (Cu) is an essential trace element for maintaining normal cellular functions; however, excessive Cu accumulation has been confirmed to induce hepatotoxicity, while the metabolic mechanisms underlying Cu‐induced hepatotoxicity remain unclear. In this study, an innovative integrated separation strategy was established, combining hydrophilic interaction liquid chromatography (HILIC) and reversed‐phase liquid chromatography (RPLC), coupled with quadrupole‐time‐of‐flight mass spectrometry (Q‐TOF/MS), to systematically resolve metabolomic perturbations in CuCl 2 ‐exposed rat BRL‐3A hepatocytes. Based on their complementary separation mechanisms—HILIC enables efficient retention and separation of polar metabolites via hydrophilic interactions, while RPLC separates nonpolar/weakly polar lipid molecules based on hydrophobic interactions—this analytical strategy significantly expanded the coverage of detectable metabolites and improved the reliability of metabolite identification through cross‐validation between the two chromatographic platforms. The results showed that a total of 25 metabolites with significant changes were identified when BRL‐3A cells were exposed to 50 µM CuCl 2 (with a cell viability of 85%). These changes were mainly enriched in metabolic pathways such as glutathione metabolism (characterized by a significant decrease in the GSH/GSSG ratio, p < 0.01), arachidonic acid (AA) metabolism (a 42% reduction in AA, p < 0.05), and glycerophospholipid metabolism (a 1.8‐fold increase in the levels of lysophospholipids [LysoPCs/LysoPEs], p < 0.05). These findings reveal that oxidative stress, membrane structure damage, and energy metabolism imbalance are the core mechanisms of Cu‐induced hepatotoxicity. The integrated liquid chromatography‐mass spectrometry (LC‐MS) analytical framework established in this study not only provides a novel molecular perspective for elucidating the mechanisms of Cu‐induced hepatotoxicity but also demonstrates the application potential of advanced complementary separation technologies in the risk assessment of environmental pollutants.