Comparison of sample preparation approaches for phospholipids profiling in human serum by liquid chromatography–tandem mass spectrometry

Fractionation of biological fluids for proper analysis by LC–MS of low-abundance metabolites in the presence of high-abundant endogenous metabolites is of interest, particularly when the latter cause undesirable phenomena such as ionization suppression, as is the case with phospholipids (PLs). An exhaustive study and comparison of different protocols for sample preparation based on deproteinization, liquid–liquid extraction (LLE) and solid-phase extraction (SPE) has been carried out with the main aim of implementing metabolomics analysis of PLs within a global metabolite profiling of serum. After selecting LLE and SPE as the best strategies for sample preparation, the fractions from each approach were analyzed by LC–TOF/MS in positive and negative ionization modes, and the highest number of molecular features was found by the latter strategy (2667 versus 1911 in the positive ionization mode). SPE also allows obtaining a clean elution of PLs which made possible tentative identification of these metabolites in both ionization modes by matching to the Human Metabolome Database. Confirmatory analysis to assess PLs identity was supported on MS/MS experiments at different collision energies.

► Analysis of phospholipids within a global metabolite profiling of serum.
► Comparison of sample preparation based on deproteinization, LLE and SPE were done.
► Fractions collected from each approach were analyzed by LC–TOF/MS.
► Identification of metabolites using Human Metabolome Database has been carried out.
► Confirmatory analysis to assess phospholipids identity was supported on MS/MS.