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 highthroughput 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 (R 2 >0.99), precision (Coefficient of Variation<15%), accuracy (Percent Error<15%), extraction recovery (>90%), 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.

Case finding of early pregnancies at risk of preeclampsia using maternal blood leptin/ceramide ratio: multi-omics discovery and validation from a longitudinal study

“…Serum concentrations of 16 Cers and 10 DHCers were measured by liquid chromatography-tandem mass spectrometric assay. The measurements were implemented for sera by the analytical methodology as previously described (55).…”

“…The copyright holder for this this version posted December 19, 2020. ; https://doi.org/10.1101/2020.12.17.20248418 doi: medRxiv preprint LC/MS/MS analysis Serum concentrations of 16 Cers and 10 DHCers were measured by liquid chromatography-tandem mass spectrometric assay. The measurements were implemented for sera by the analytical methodology as previously described (Huang et al, 2020a). In brief, 10 µL of serum was extracted with methanol containing deuterated Cers to remove the proteins.…”

Blood-Derived Lipid and Metabolite Biomarkers in Cardiovascular Research from Clinical Studies: A Recent Update

“…Given the critical role of ceramides in the development of CVD, several analytical methods have been developed and validated to quantify ceramides in human plasma and serum [104][105][106] samples.…”