Optimization of untargeted lipidomics analysis of human plasma after Dangguijakyak-san administration

Untargeted lipidomics enables comprehensive characterization of lipid profiles; however, analytical variability in sample preparation, chromatographic conditions, and data processing often limits reproducibility. Thus, this study aimed to optimize untargeted lipidomics analysis by evaluating chromatographic columns, mobile-phase additives, and lipid extraction methods to maximize feature detection, reproducibility, and overall data quality. Chromatographic performance was compared between ethylene-bridged hybrid (BEH) and charged-surface hybrid columns. For mobile-phase additives, 10 mM ammonium formate (AmFa) with or without 0.1% formic acid (Fa) was evaluated in positive ionization mode, whereas 10 mM ammonium acetate (AmAc) with or without 0.1% acetic acid was evaluated in negative ionization mode. Lipid extraction was assessed using chloroform-, methyl tert-butyl ether-, or butanol-based methods, with or without re-extraction. The final optimized workflow consisted of a BEH column, 10 mM AmFa with 0.1% Fa in positive mode, 10 mM AmAc in negative mode, and the Folch extraction method. The optimal analytics applied to plasma samples collected from women with primary dysmenorrhea, before and after Dangguijakyak-San (DJS) administration, identifying 321 lipid species across major classes, including glycerophospholipids, glycerolipids, and sphingolipids. Principal component analysis of quality control samples demonstrated tight clustering, and 14 internal standards exhibited relative standard deviations below 30%, confirming analytical robustness. Four lipid species were significantly altered: three phosphatidylcholines decreased, and one ceramide phosphoinositol increased after DJS administration. These alterations suggest modulation of oxidative stress-related lipid pathways and sphingolipid metabolism. This optimized analytical workflow provides reproducible plasma lipidomics and has potential applications in studies of traditional herbal medicines.