Accurate Sphingolipid Quantification Reducing Fragmentation Bias by Nonlinear Models

[Image: see text] Quantitative sphingolipid analysis is crucial for understanding the roles of these bioactive molecules in various physiological and pathological contexts. Molecular sphingolipid species are typically quantified using sphingoid base-derived fragments relative to a class-specific internal standard. However, the commonly employed “one standard per class” strategy fails to account for fragmentation differences presented by the structural diversity of sphingolipids. To address this limitation, we developed a novel approach for quantitative sphingolipid analysis. This approach utilizes fragmentation models to correct for structural differences and thus overcomes the limitations associated with using a limited number of standards for quantification. Importantly, our method is independent of the internal standard, instrumental setup, and collision energy. Furthermore, we integrated this method into a user-friendly KNIME workflow. The validation results illustrate the effectiveness of our approach in accurately quantifying ceramide subclasses from various biological matrices. This breakthrough opens up new avenues for exploring sphingolipid metabolism and gaining insights into its implications.