Glycerophospholipid profiling by high‐performance liquid chromatography/mass spectrometry using exact mass measurements and multi‐stage mass spectrometric fragmentation experiments in parallel

Abstract

A profiling method for glycerophospholipids (GPs) in biological samples was developed using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry (LIT-FTICRMS) with electrospray ionization (ESI) in the negative ionization mode. The method allowed qualitative (identification and structure elucidation) and relative quantitative determination of various classes of GPs including phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines, phosphatidic acids, phosphatidylglycerols, and cardiolipins in a single experiment. Chromatographic separation was optimized by the examination of different buffer systems and special emphasis was paid on the detection by ESI-MS. The hybrid LIT-FTICRMS system was operated in the data-dependent mode, switching automatically between FTICRMS survey scans and LIT-MS/MS experiments. Thereby, exact masses for elemental composition determination and fragmentation data for identification and assignment of fatty acid residues are provided at the same time. The low absolute instrumental limits of detection (0.05 pmol for phosphatidylglycerol to 1 pmol for phosphatidic acid) complemented by a linear dynamic range of 1.5 to 2.5 orders of magnitude facilitated the relative quantification of GP species in a lipid extract from Saccharomyces cerevisiae. The developed method is a valuable tool for in-depth GP profiling of biological systems.