Simplified Mass Spectrometric Analysis of Ceramides using a Common Collision Energy

Ceramides (CER) are biologically active sphingolipid precursors that are mechanistically linked to several pathogenic states including cancer, insulin resistance, and neurodegeneration. CER are commonly quantified through mass spectrometry-based methods founded upon a product ion scan (PIS) in positive mode to produce a characteristic m/z 264 ion. The ionization efficiency (IE) of CER species decreases with an increase in CER mass, thus quantitation of CER typically involves application of mass-dependent response factors (RF) for each CER species. In this work, we observed that the RF were systematically dependent on the number of fatty acid acyl carbons and the collision energy (CE) used to generate the m/z 264 ion. Using these complimentary trends, we determined an "isosbestic" CE where the RF for all CER species were equivalent, allowing for CER quantitation without postcollection correction factors. A comparison of this common CE/common RF method to the multiple RF method demonstrated good agreement between the two methods. Use of the common CE/common RF method will reduce data processing and reduce the use of multiple CER species standards.