Fourier Transform Mass Spectrometry and Nuclear Magnetic Resonance Analysis for the Rapid and Accurate Characterization of Hexacosanoylceramide.

Ceramides are a central unit of all sphingolipids which have been identified as sites of biological recognition on cellular membranes mediating cell growth and differentiation. Several glycosphingolipids have been isolated, displaying immunomodulatory and anti-tumor activities. These molecules have generated considerable interest as potential vaccine adjuvants in humans. Accurate analyses of these and related sphingosine analogues are important for the characterization of structure, biological function, and metabolism. We report the complementary use of direct laser desorption ionization (DLDI), sheath flow electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) and high-field nuclear magnetic resonance (NMR) analysis for the rapid, accurate identification of hexacosanoylceramide and starting materials. DLDI does not require stringent sample preparation and yields representative ions. Sheath-flow ESI yields ions of the product and byproducts and was significantly better than monospray ESI due to improved compound solubility. Negative ion sheath flow ESI provided data of starting materials and products all in one acquisition as hexacosanoic acid does not ionize efficiently when ceramides are present. NMR provided characterization of these lipid molecules complementing the results obtained from MS analyses. NMR data was able to differentiate straight chain versus branched chain alkyl groups not easily obtained from mass spectrometry.

A novel coaxial electrospray ionization method for characterizing hexacosanoylceramides by Fourier transform ion cyclotron resonance mass spectrometry.

Because of poor aqueous solubility and lack of UV chromophores, the characterization of long-chain hydrocarbons and ceramides by conventional UV and mass spectrometric methods has not been successful. Therefore, a novel coaxial electrospray ionization method was developed for characterizing reaction products of phytosphingosine and hexacosanoic acid in toluene and tetrahydrofuran (THF), by high resolving power Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). Simultaneous spraying of a solution of apolar analytes and polar reagents into the gas phase readily enabled protonation and/or sodiation of analyte with enhanced signal-to-noise (S/N). Sample introduction was by direct infusion such that the sprayers were arranged either along the instrument line-of-sight (for monospray and coaxial spray modes) or in a 45 degrees configuration for dual-spray mode. For dual-spray and coaxial spraying, p-toluenesulphonic acid was used as a reagent and sprayed simultaneously with the analyte dissolved in toluene or THF. Compounds were characterized by accurate mass measurement of the protonated and/or sodiated molecules.