박진주 (경북대학교)

윤영란 (경북대학교)

박진주 (경북대학교)

윤영란 (경북대학교)

Reproducible lipid analysis relies on the careful design of analytics, including chromatographic columns, mobile phase additives, and extraction methods. This study aims to optimize analytics for consistent untargeted lipidomics analysis. Human plasma samples were analyzed using BEH and CSH columns. In positive ionization mode, we tested a mixture of 0.1% formic acid and 10 mM ammonium formate and 10 mM ammonium formate alone. In negative mode, we evaluated a mixture of 0.1% acetic acid and 10 mM ammonium acetate and 10 mM ammonium acetate alone. Lipid extraction methods included: chloroform-based (with/without re-extraction), MTBE-based (with/without re-extraction), and butanol-based. Each analysis was conducted in quadruplicate using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Optimal conditions were established based on detected features, PCA, lipid annotation, and RSD values. The BEH column detected more features than the CSH column, particularly in negative ionization mode. Use of 0.1% formic acid and 10 mM ammonium formate in positive mode, and 10 mM ammonium acetate in negative mode, improved precision. Chloroform and MTBE-based extraction methods showed distinct clustering in the PCA plot. While chloroform extraction produced fewer features than MTBE, the similar annotations suggest that chloroform is more selective and efficient. This study compares key factors in lipidomics analysis and presents optimal analytics.​