Ceramide lipids in alive and thermally stressed mussels: an investigation by hydrophilic interaction liquid chromatography-electrospray ionization Fourier transform mass spectrometry Laura Facchini, a Ilario Losito, a,b * Tommaso R.I. Cataldi a,b and Francesco Palmisano a,b Hydrophilic interaction liquid chromatography coupled to electrospray ionization-Fourier transform mass spectrometry was employed to study ceramide lipids occurring in mussels of sp. Mytilus galloprovincialis. Lipid extracts from alive mussels and mus- sels deliberately subjected to specific thermal treatments were analyzed. In particular, single and tandem MS measurements were performed on a hybrid quadrupole-Orbitrap mass spectrometer and then complemented by MS n analyses (n = 2, 3) achieved by a linear ion trap mass spectrometer. This approach enabled the characterization of 66 ceramide lipids, encompassing ceramide phosphoethanolamines (CPE), ceramide aminoethylphosphonates (CAEP) and N-monomethylated CAEP. The sphingoid and acyl chains of each ceramide lipid could be distinctly recognized in terms of numbers of carbon atoms and C=C bonds, and indications on the possible location of the latter on the sphingoid chain could be often inferred from fragmentation patterns. The occurrence of several species hydroxylated on the α carbon of the acyl chain was also discovered. On the other hand, the sphingoid chain of ceramide lipids was never found to be involved in oxidation processes, unless forced exposure of the mussel lipid extracts to at- mospheric oxygen was performed. CPE(d19:3/16:0) and its hydroxylated form, CPE(d19:3/2-OH-16:0), were found to be the pre- vailing species among CPE, whereas CAEP(d18:2/16:0), CAEP(d19:3/16:0) and CAEP(d19:3/2-OH-16:0) were the most abundant CAEP. Finally, ceramide lipids showed a remarkably higher stability, compared with glycerophospholipids, in mussels subjected to different thermal treatments. This finding opens interesting perspectives on the role of ceramide-based lipids in the adaptation of aquatic organisms to thermal stresses. Copyright © 2016 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: mussels; Mytilus galloprovincialis; ceramides; lipidomics; hydrophilic interaction liquid chromatography; Fourier transform mass spectrometry Introduction Mussels are lamellibranch bivalve molluscs belonging to the family of Mytilidae, living attached to solid surfaces by means of keratin fil- aments produced by the byssus gland. They are widely distributed in temperate waters of both hemispheres, where they are produced through intensive aquaculture, for human nutrition purposes. [1–3] Beside their interest as a seafood containing nutraceuticals, such as omega-3 fatty acids, [4–9] mussels are also considered potential bioindicators of abiotic stresses, due to their ability to accumulate and tolerate many organic and inorganic pollutants. [10–13] The lipid fraction characterization, so far mainly focused on fatty acids, [14–16] phospholipids and triacylglycerols, [17–19] represents an important aspect of studies on mussels, both as seafood and as biomarkers. Recently, polar phospholipids of Mytilus galloprovincialis, the mussel species prevailing in the Mediterranean Sea, have been the object of an extended characterization in our laboratory. In particular, the possible effects of thermal treatments (i.e. refrigeration and freezing) commonly performed on mussels for transportation and storage purposes [20] have been evaluated. As a result, the lyso forms of phosphatidylcholines (LPCs) and phosphatidylethanolamines (LPEs), arising from the hydrolytic cleavage of one of the acyl chains of such phospholipids, have been found to be significantly in- creased after long-term refrigeration or freezing. A similar effect has been observed upon storage at room temperature for prolonged times. [20] Other polar lipids occurring in mussels, such as sphingolipids, in which the glycerol backbone of major phospholipids is replaced by sphingosine (i.e. an amino alcohol bearing a long aliphatic side chain), have received much less consideration. Sphingolipids are di- vided into different subclasses, depending on the polar head linked to sphingosine, which can be a H atom (ceramides), a sugar mole- cule (glycosphingolipids) or a phosphorus-containing moiety, like phosphocholine and phosphoethanolamine. [21] As pointed out by Pruett et al., [22] sphingosines exhibit a significant biodiversity, * Correspondence to: Ilario Losito, Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Via E. Orabona 4, 70126 Bari, Italy. E-mail: ilario.losito@uniba.it a Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Via E. Orabona 4, 70126, Bari, Italy b Centro Interdipartimentale SMART, Università degli Studi di Bari Aldo Moro, Via E. Orabona 4, 70126, Bari, Italy J. Mass Spectrom. 2016, 51, 768–781 Copyright © 2016 John Wiley & Sons, Ltd. Research article Journal of MASS SPECTROMETRY Received: 2 May 2016 Revised: 25 July 2016 Accepted: 28 July 2016 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jms.3832 768