High-throughput quantitation of serological ceramides/dihydroceramides by LC/MS/MS: Pregnancy baseline biomarkers and potential metabolic messengers

•A LC/MS/MS assay quantifying 16 ceramides and 10 dihydroceramides in human serum.•The assay provides excellent sensitivity and specificity in high-throughput fashion.•The assay identifies unusual serum ceramides and dihydroceramides at low abundances.•Gestation baselines of ceramides and dihydroceramides are established in pregnancy. Ceramides and dihydroceramides are sphingolipids that present in abundance at the cellular membrane of eukaryotes. Although their metabolic dysregulation has been implicated in many diseases, our knowledge about circulating ceramide changes during the pregnancy remains limited. In this study, we present the development and validation of a high-throughput liquid chromatography-tandem mass spectrometric method for simultaneous quantification of 16 ceramides and 10 dihydroceramides in human serum within 5 min. by using stable isotope-labeled ceramides as internal standards. This method employs a protein precipitation method for high throughput sample preparation, reverse phase isocratic elusion for chromatographic separation, and Multiple Reaction Monitoring for mass spectrometric detection. To qualify for clinical applications, our assay has been validated against the FDA guidelines for Lower Limit of Quantitation (1 nM), linearity (R2>0.99), precision (imprecision85 %), and carryover (