High-resolution sphingolipid analysis in vitro using LC-qToF MS

The complexity of the sphingolipidome, characterized by variations in chain length, saturation, and headgroup composition, makes it essential to develop analytical strategies capable of high sensitivity and structural precision. Traditional biochemical methods lack the resolution to discriminate among closely related species, underscoring the transformative role of liquid chromatography-mass spectrometry (LC-MS) in sphingolipidomics. LC-MS provides unparalleled capabilities for sphingolipid analysis, combining chromatographic separation with high-resolution mass detection to achieve both qualitative and quantitative accuracy. In particular, liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-qToF MS) has emerged as a powerful platform, offering mass accuracy, broad dynamic range, and rapid acquisition rates. These features enable confident identification of isobaric and structurally related sphingolipids, which is essential for understanding their roles in cellular physiology and pathology. This chapter focuses on an optimized LC-qToF MS method tailored for sphingolipid profiling in cultured mammalian cells. By focusing on the analytical strengths of LC-MS, the approach provides a robust foundation for dissecting sphingolipid metabolism and its dysregulation in cellular processes.