**1. Introduction**

The densely packed content of carbohydrates, lipids, proteins, and other micronutrients in the endosperm of nuts and seeds is the storage of nutrients for the development and early growth of the seed into a new plantlet [1,2]. This characteristic makes them a complete and healthy food for animals and humans. Therefore, due to their traditional importance as a food staple in many countries and as a highly prized nutraceutical complement of health-promoting diets and of value-added traditional foods, the study of their composition is a topic of grea<sup>t</sup> importance [3]. Composition studies are especially important to relate the contents of micronutrients to health status [4,5] and to confirm the presence of specific components in complex preparations [6].

In particular, the ceramides present in the fatty component of nuts display a degree of chemical diversity in the sphingoid bases, fatty acid substituents, and C-1 appendages [7]. Limited information is available on occurrence and levels of ceramides in nuts. Miraliakbari and Shahidi et al. [8] reported

that almond and pistachio oils contain 240 and 330 mg/100 g of total sphingolipids, respectively, measured utilising TLC-FID as a non-specific method of quantification. Using 750 Da as a mean representative value for sphingolipid molecular masses, this value corresponds to approximately 500 nmol/100 g. By using LC-MS/MS, we found 200 pmol/g = 20 nmol/100 g of ceramide species with the "standard" (i.e., mammalian) sphingoid base, 1,3-dihydroxy-D4-C18 sphingosine [9]. Even accounting for ine fficient extraction and unspecific measurement, this discrepancy suggests that there is a major pool of sphingolipids unaccounted for in nuts.

The measured "standard" ceramides are not the only derivatives of sphingoid bases that are currently known to occur in nuts. Sang et al. [10] used high-resolution 1D and 2D NMR data to identify in almonds a monoglucocerebroside with a modified sphingoid base, sphinga-4,8-dienine. Fang et al. [6] employed liquid chromatography coupled to electrospray mass spectrometry with in-source fragmentation to identify further compounds in several plants, among which were almonds. Among those identified in almonds, there are ceramides and cerebrosides with trihydroxy bases with zero or one double bond, mainly 4-hydroxy-8-sphingenine (t18:1), amide-linked to very long chain fatty acids, with or without an α-hydroxy group. In this study, cerebrosides, expressed as d18:2-C16:0H-GLU, were measured at 68 μg/g (approximately 100 nanomoles/g, or 10 μmoles/100 g); thus, at a level that is at least two orders of magnitude higher than sphingosine d18:1 ceramides [6].

To systematically search for unexpected ceramides in lipid extracts, we propose an innovative use of the triple quadrupole tandem mass spectrometry based on a precursor ion scan with iso-energetic collision activated dissociation, aimed at systematic discovery and preliminary characterization of the main sphingosine components of almond and pistachio. Specifically, we searched for components with di fferent C18 long chain bases containing di fferent structural motifs (d18:0, d18:1, d18:2, t18:0, t18:1). The presence of hexoside unit was screened with a specific neutral loss experiment under iso-energetic collision activated dissociation conditions. Connectivity confirmation was achieved by fragment ions analysis with accurate mass measurements.
