Electrospray ionization with higher-energy collision dissociation tandem mass spectrometry toward characterization of ceramides as [M þ Li] þ ions: Mechanisms of fragmentation and structural identification Fong-Fu Hsu Mass Spectrometry Resource, Division of Endocrinology, Diabetes, Metabolism, and Lipid Research, Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63130, USA highlights graphical abstract  High resolution HCD tandem mass spectrometry permits complete structural characterization of ceram- ides as [M þ Li] þ ions.  Multiple sets of fragment ions define the long-chain base and fatty acyl chain, and minor isomer can be identified.  High resolution MS precisely assign the formula of molecular and frag- ment ions, and facilitates fragmenta- tion mechanism recognition.  The fragmentation pathways of ceramides in 12 subclasses are proposed. article info Article history: Received 19 July 2020 Received in revised form 14 September 2020 Accepted 25 September 2020 Available online 5 October 2020 Keywords: Tandem mass spectrometry High resolution mass spectrometry Electrospray ionization abstract Ceramide is a huge lipid family consisting of diversified structures in which various modifications are seen in the fatty acyl chain and the long chain base (LCB). In this contribution, a higher collision energy (HCD) linear ion-trap mass spectrometric method (LIT MS n ) was applied to study the mechanisms un- derlying the fragmentation processes of ceramide molecules in 12 subclasses, which were desorbed by ESI as the [M þ Li] þ ions. Multiple sets of fragment ions reflecting the fatty acyl chain and LCB were observed in the HCD MS 2 spectra for all the ceramide classes, resulting in unambiguous definition of the ceramide structures, including the chain length and the modification (a-hydroxy-, b-hydroxy-, u-hy- droxy-FA) of the fatty acyl moiety, and the types of LCB (sphingosine, phytosphigosine, 6-hydroxy- sphingosine). Thereby, this approach permits differentiation of isomeric structures and ceramide species in the biological specimen can be unveiled in detail. By application of sequential MS 3 , the double bond Abbreviations: ESI-MS, electrospray ionization-MS; HRMS, high resolution mass spectrometry; LIT, linear ion-trap; HCD, higher-energy collision dissociation; Cer, ceramide; LCB, long-chain base; FA, fatty acid; nFA, non-hydroxy fatty acid; hFA, hydroxy fatty acid; ahFA, a-hydroxy fatty acid; bhFA, b-hydroxy fatty acid; uhFA, u- hydroxy fatty acid; LCB/nFA-Cer, ceramide class consisting of nonhydroxy FA; LCB/ hFA, ceramide class consisting of hydroxy FA; d18:1-LCB, sphigosine long-chain base; d20:1, C20:1 sphingosine long-chain base; t18:1, 6-hydroxy sphingosine; t18:0, phytosphingosine. E-mail address: fhsu@im.wustl.edu. Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca https://doi.org/10.1016/j.aca.2020.09.056 0003-2670/© 2020 Elsevier B.V. All rights reserved. Analytica Chimica Acta 1142 (2021) 221e234