A sensitive LC‑MS/MS method for the simultaneous quantification of hexosylceramides and hexosylsphingosines, their precursors and metabolites in cells, plasma, and tissue homogenates.

Hexosylceramides (HexCers) and hexosylsphingosines (HexSphs) are bioactive glycosphingolipids implicated in neurodegenerative diseases such as Parkinson's disease (PD), Gaucher disease, and Krabbe disease. Despite their biological relevance, no existing method enables the simultaneous quantification of both HexCers and HexSphs across multiple biological matrices. In this study, we report the development, validation, and application of a novel LC-MS/MS method capable of quantifying 11 lipid species, including glucosylceramides, galactosylceramides, glucosylsphingosine, galactosylsphingosine, ceramide, lactosylceramide, and sphingosine, in a single chromatographic run. The method integrates optimized liquid-liquid extraction and solid-phase extraction protocols, achieving high recovery and selectivity. Validation was performed according to internal and external (e.g., ICH and FDA guidelines) international guidelines, demonstrating excellent sensitivity, linearity (R2 > 0.99), precision (cv < 15%), and stability across cells, mouse plasma, and brain tissue homogenates. Proof-of-concept applications included wild-type (WT) SKMEL28 cells treated and non-treated with the glucocerebrosidase inhibitor CBE, as well as WT mouse plasma and brain regions (cortex, hippocampus, midbrain, cerebellum), establishing baseline levels for future pharmacokinetic studies. This highly comprehensive method offers a robust analytical platform for sphingolipid profiling in neurodegenerative disease research.