Quantitative LCMS for ceramides/dihydroceramides: pregnancy baseline biomarkers and potential metabolic messengers

Abstract

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 (LC/MS/MS) method for simultaneous quantification of 16 ceramides and 10 dihydroceramides in human serum within 5 mins by using stable isotope-labeled ceramides as internal standards (ISs). This method employs a protein precipitation method for high throughput sample preparation, reverse phase isocratic elusion for chromatographic separation, and Multiple Reaction Monitoring (MRM) for mass spectrometric detection. To qualify for clinical applications, our assay was validated against the FDA guidelines: the Lower Limit of Quantitation (LLOQ as low as 1 nM), linearity (R2>0.99), precision (Coefficient of Variation90%), stability (>85%), and carryover (<0.1%). With enhanced sensitivity and specificity from this method, we have, for the first time, determined the serological levels of ceramides and dihydroceramides to reveal unique temporal gestational patterns. Our approach could have value in providing insights into disorders of pregnancy.