Comprehensive Quantitation Using Two Stable Isotopically Labeled Species and Direct Detection of <Emphasis Type="Italic">N</Emphasis>-Acyl Moiety of Sphingomyelin

Sphingomyelin (ceramide‐phosphocholine, CerPCho) is a common sphingolipid in mammalian cells and is composed of phosphorylcholine and ceramide as polar and hydrophobic components, respectively. In this study, a qualitative liquid chromatography‐electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS/MS) analysis is proposed in which CerPCho structures were assigned based on product ion spectra corresponding to sphingosylphosphorylcholine and N‐acyl moieties. From MS/MS/MS analysis of CerPCho, we observed product ion spectra of the N‐acyl fatty acids as RCO₂]^? ions as well as sphingosylphosphorylcholine. A calibration curve for CerPCho was constructed using two stable isotopically labeled CerPCho species and then used to quantify the CerPCho species in HeLa cells as a proof‐of‐principle study. The present study proposes an accurate method for quantifying and assigning structures to each CerPCho species in crude biologic samples by LC–ESI–MS/MS/MS analysis.