A Parallel Reaction Monitoring-based approach for ceramide quantification. (a) MS2 of d18:1/16:0 ceramide standard in positive ionisation mode. Fragment structures are based on published assignments43,44. (b) Schematic of Parallel Reaction Monitoring. (c) Example PRM chromatograms for C41:1 ceramide from 48 hpf zebrafish embryos, demonstrating presence of multiple LCB-acyl chain isomers with the same m/z. RT: retention time. (d) MS2 spectrum of C41:1 ceramide from 48 hpf zebrafish embryos, the four major LCB fragments are labelled. (d,e) m/z 200-m/z 350, the four major LCB fragments and additional LCB-derived minor fragments are labelled. Unlabelled minor fragments: m/z 224.2371 (d16:1 LCB), m/z 238.2525 (d17:1 LCB).
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