Analysis of fatty acids in mouse cells using reversed-phase high-performance liquid chromatography

Summary A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to analyze various fatty acids in recombinant mouse L cells. These fatty acids were the metabolites of oleic acid. A process was developed to extract fatty acids from the cell samples before RP-HPLC analysis. The samples were first saponified with 0.5 M NaOH in 96% ethanol then extracted with acidified ethyl acetate. After extraction, the sample was dried and dissolved in HPLC-grade methanol. After centrifugation to remove insoluble impurities, the sample was applied to a C₁₈RP-HPLC column using a gradient of acetonitrile (ACN)-H₂O. The eluted fatty acids were monitored by ultraviolet (UV) absorption at 195 nm and identified by retention time and adsorption spectrum comparison. This method successfully resolved various fatty acids and provided a tool for the elucidation of the fatty acid metabolic pathway in the cells.