Abstract

Lipidomics is the study of lipids in biological matrices. Challenges in the field include the use of toxic solvents and data harmonization. Ionic liquids (ILs), organic salts that can extract organic compounds and biomolecules, offer potential solutions, has not been extensively studied for lipidomics. Two ILs, 1-hexyl-3-methylimidazolium hexafluorophosphate and trihexyltetradecylphosphonium hexafluorophosphate, were assessed for lipid extraction using IL dispersive liquid-liquid microextraction with methanol as a disperser. Both ILs showed selectivity towards lipids of intermediate polarity, with limited extraction efficiency towards more polar lipids. However, slight solubility of ILs in the aqueous layer led to significant ion suppression for lipids tested when analyzed using liquid chromatography-mass spectrometry. Overall, the tested ILs were unsuitable for untargeted lipidomics of plasma.
Internal standards are commonly used for semi-quantification in untargeted lipidomics. However, electrospray ionization (ESI) differences between internal standards and endogenous lipids can impact quantification accuracy. The adduct formation for phospholipids, glycerolipids, sphingolipids, and sterol lipids was studied in human plasma, murine plasma, murine spinal cord, and murine liver tissue. Within-class adduct formation was consistent for most lipid classes, except for diglyceride and cholesteryl ester lipid subclasses, necessitating multiple internal standards. Mismatch between internal standard and endogenous lipid adduct formation was observed for phosphocholine, ceramide, triglyceride, diglycerol, and cholesteryl ester lipid subclasses in at least one of the four matrices. Inter-batch variability in sodium concentrations significantly influenced sodium adduct formation and could not be controlled with usage of ammonium volatile salts. Finally, monoglycerides and diglycerides were detected to form acetate adducts in ESI-.