**1. Introduction**

Lipid hydroperoxides are labile compounds derived from lipids containing carbon-carbon double bonds. The formation of these primary peroxidation products occurs in enzymatic and non-enzymatic (autooxidation, photo-oxidation) reactions [1]. Lipid hydroperoxides, formed in biological systems, have not only multiple damaging effects on cellular macromolecules, but are also important regulators of many cellular processes [2]. In some pathological situations, lipid hydroperoxides are generated at higher than normal rates. Such overproduction is implicated in several human diseases and exposures including atherosclerosis, cancer, diabetes, acute lung injury, chronic alcohol exposure, and neurodegenerative disorders. The complex nature of lipid peroxidation and its potential biological significance have attracted the attention of scientists across many disciplinary fields, ranging from chemistry and biochemistry to biology and clinical science (see review [3] and references cited herein).

Recent progress in the characterization and quantification of oxygenated fatty acids (FAs) has facilitated a better understanding of lipid oxidation, but the methods currently available still suffer from unresolved sensitivity and selectivity problems [4]. The main reason for these problems is the complexity of the product profile formed by oxidation from a single lipid molecular species. In particular, peroxidation of any unsaturated fatty acyl group may generate a mixture of allylic hydroperoxides with different double bond positions and/or configurations. These hydroperoxides are easily decomposed or go through further reactions to form different secondary peroxidation products including epoxides, allylic alcohols, α,β-unsaturated ketones (enones), and chain-cleavage products [1]. The constituents of these mixtures are difficult to separate, isolate, and identify. Additionally, natural extracts are characterized, as a rule, by extremely low levels of oxidized lipids, causing further difficulties in their isolation and analysis [3,4]. Undoubtedly, structural elucidation of primary and secondary peroxidation products, formed from complex lipids, remains a challenge for lipid analysts.

Monoenes are much less prone to undergo oxidation than polyenes. Respectively, direct addition of peroxyl radicals to monounsaturated lipids is not generally observed with the exceptions of cholesterol [3] and related sterols. In particular, sphingolipids, containing mainly saturated or monounsaturated hydrocarbon chains, are poor substrates for peroxidation. We found only one report on the isolation of peroxidized sphingolipids from a natural source, but these compounds formed due to oxidation of unique sphingoid base moiety with conjugated double bonds. Namely, some stereoisomeric glycosphingolipids, containing endoperoxide and allylic hydroperoxide functions in their dienoic sphingoid base moieties, were isolated from the extract of the sponge *Axinella corrugate* [5]. The locations of the double bonds and peroxide-containing groups were determined by 1H,1H-COSY (proton-proton correlation spectroscopy), НМBC (heteronuclear multiple-bond correlation spectroscopy), and ROESY (rotating-frame nuclear Overhauser effect spectroscopy) experiments due to the proximity of the previously mentioned functionalities to the polar portion of the sphingolipids. Methanolysis (MeOH/HCl) of the peroxidized glycosphingolipids was used to release methyl esters of saturated FAs, but oxidized dienoic sphingoid bases were not obtained, apparently, due to rapid decomposition under acidic conditions.

In mass spectrometric (MS) studies of oxidative stress markers (disease biomarkers), oxidation of some standard glycosphingolipids, containing monoenoic FAs, was induced by a Fenton reaction (H2O2/FeCl2) or UVA (ultraviolet A) irradiation [6,7]. In particular, Couto et al. obtained galactosylceramides with allylic hydroperoxy, hydroxy, or keto groups in FA chains. These cerebrosides were characterized using ESI-MS (electrospray ionization mass spectrometry) and HPLC-MS (high-performance liquid chromatography-mass spectrometry) methods (including tandem mass spectrometry (MS/MS)) [6]. In our continued study on lipids from far eastern marine sponges [8], similar oxidized cerebrosides (Figure 1), presumably derived from glucosylceramides with monounsaturated fatty acyl groups, were found in the extract of a deep-sea glass sponge, *Aulosaccus* sp. As seen in Figure 1, the depicted compounds contain an allylic hydroperoxy, hydroxy, or keto group in each acyl chain, but their monoenoic sphingoid base moieties are not oxidized. A fraction of these minor cerebrosides, along with fractions of non-oxidized cerebrosides [8], was isolated from the sponge extract using high-performance liquid chromatography on reversed-phase column (RP-HPLC). The present study presents structural elucidation of rare oxidized glycosphingolipids using mass spectrometry (ESI-MS and gas-chromatography-mass spectrometry (GC-MS)), <sup>1</sup>Н-, 13C-NMR (nuclear magnetic resonance) spectroscopy, GC, chemical transformations, and optical rotation measurement.

A classical approach to the analysis of any complex lipid includes methanolysis or hydrolysis, which is followed by analyses of liberated derivatized simple lipids and sugar. However, prolonged high temperatures and treatments with acidic or alkaline solutions, required for solvolysis of *N*-acyl lipids, presents significant problems with respect to the potentially labile allylic oxygen-containing groups of the acyl chains. To solve these problems, we used catalytic hydrogenation to fix the starting positions of the oxygenated groups prior to chemical degradation of the oxidized glycosphingolipids. In addition, attempts were made to fix the double bonds of allylic substructures by reacting with

dimethyl disulfide (DMDS) because, in our preliminary research, the DMDS adduct of methyl oleate did not lose *S*-methyl groups during hydrolysis with HCl in MeCN-H2O. To minimize possible allylic rearrangements (1,3-isomerizations) and other alterations, we avoided elevated temperatures and strong acid/base conditions in the derivatization reactions before hydrolysis. Thus, our attention was mainly focused on procedures suitable for an initial detailed structural analysis of a complex mixture of glycosphingolipids, containing an allylic hydroperoxy, hydroxy, or keto group in the monoenoic acyl chain.

**Figure 1.** Oxidized cerebrosides from the extract of a sponge *Aulosaccus* sp.
