A liquid chromatography-mass spectrometry method for profiling global phospholipid methylation via Headgroup analysis...

Global analysis of phospholipid methylation is crucial for understanding the biological roles of phospholipids in mammalian membranes. This study developed and validated a robust liquid chromatography-mass spectrometry (LC-MS) method for the simultaneous determination of ethanolamine (EA) and choline (CL) released from the hydrolysis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), respectively. The extraction, hydrolysis, and detection procedures were optimized. The compounds were separated on an HILIC column with isocratic elution and detected using a mass spectrometer with electrospray ionization in positive ion mode. The selected ions monitored were m/z 104.1 for CL, m/z 113.1 for d9-CL (internal standard for CL), m/z 62.1 for EA, and m/z 66.1 for d4-EA (internal standard for EA). The method showed satisfactory linearity (r² > 0.999), precision (intra- and inter-day RSDs ≤6.3 %), and accuracy (-2.1-6.8 %). The limits of detection (LOD) were 0.30 μM for EA and 0.02 μM for CL. This method was applied to human erythrocytes and several tumor cell lines (Hela, EC109, Saos-2), revealing significant differences in the EA/CL ratio (reflecting the PE/PC ratio) among different cell types. This study indicated that the global phospholipid methylation status is cell-type specific and established the approach as a valuable tool for investigating membrane lipid composition in health and disease.