High-throughput quantitation of serological ceramides/dihydroceramides by LC/MS/MS: Pregnancy baseline biomarkers and potential metabolic messengers
Qianyang Huang, Shiying Hao, Xiaoming Yao, Jin You, Xiao Li, Donghai Lai, Chunle Han, James Schilling, Kuo‐Yuan Hwa, Sheeno Thyparambil, John C. Whitin, Harvey J. Cohen, Henry Chubb, Scott R. Ceresnak, Doff B. McElhinney, Ronald J. Wong, Gary M. Shaw, David K. Stevenson, Karl G. Sylvester, Xuefeng B. Ling
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 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 (imprecision<15 %), accuracy (inaccuracy<15 %), extraction recovery (>90 %), stability (>85 %), and carryover (<0.01 %). 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.