Ultrafast Measurement of Circulating Ceramides in Human Cohorts

Ceramides (Cer) are sphingolipids that can accumulate in human blood plasma during metabolic disorders, insulin resistance, and diabetes. Monitoring levels of four circulating plasma ceramides─Cer 18:1;O2/16:0, Cer 18:1;O2/18:0, Cer 18:1;O2/24:0, and Cer 18:1;O2/24:1─can predict the risk for cardiovascular disease (CVD) events and death. Here, using a RapidFire instrument, we present an online Solid Phase Extraction-Tandem Mass Spectrometry (SPE–MS/MS) methodology for ultrafast (∼15 s/sample) measurements of these ceramides in human plasma. The addition of authentic deuterated standards enables absolute quantitation of each ceramide species, and linear calibration lines were obtained for all analytes in the biological and clinical range. The aim of this study was also to determine whether the results produced with the RapidFire-MS/MS platform are in agreement with an existing validated LC-MS/MS method currently used in the clinic. The applicability of the novel methodology was demonstrated by determining ceramide concentrations using both analytical methods in various biological samples, including mouse plasma, NIST reference materials, a human cohort of 99 individuals, and by performing a multicenter comparison. In all of these cases, the RapidFire-MS/MS method yielded results that were in agreement with the validated LC-MS/MS determination, addressing the need for increased speed of lipid analyses and facilitating the measurement of ceramides in large human cohorts for research and clinical applications.