**5. Conclusions**

This report furthers our previous one on the targeted analysis of ceramides (d18:0, d18:1) in almond and pistachio [9]. This technique exploits the performance of the triple quadrupole tandem mass spectrometer well beyond its traditional use by analytical chemists.

Success with this approach will prompt a complete investigation of the structural details of the observed phytochemicals that cannot be resolved with the analytical and spectroscopic techniques employed in this preliminary survey. Semi-preparative chromatographic separation of crude lipid preparations driven by the selective scan will enable to obtain enriched fractions of the unexpected components. The use of high-energy collision induced dissociation will characterize the connectivity of the ceramide and fatty acid subunits [11], and deglycosidation will enable to separately identify the linked hexose.

Availability of a fast and comprehensive method to screen lipid extracts for trace sphingosides with chemically decorated long chain bases will be applied to analyze several more seeds of a nutraceutical interest that are the base of typical foods of the Italian lore.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/9/2/110/s1. Figure S1: High-resolution mass spectrometry analysis for the confirmation of the unexpected ceramide species in the extract of a pistachio cultivar. Table S1: High-resolution measurement of fragment ion spectra of the four main ceramide species. Figure S2: High-resolution molecular (a, c, e) and fragment ion spectra (b, d, f) of triglycerides. Figure S3: Chromatographic traces of the Par263 and Par264 scans for a representative extract. Figure S4: Formation of protonated diglyceride and acylium fatty acid fragments from triglycerides. Figure S5: Plot of the relationship of chromatographic retention vs. molecular size for ceramides.

**Author Contributions:** Conceptualization, F.M.R.; methodology, F.M.R., M.D.C.; investigation, F.M.R., M.D.C., M.B.; resources, M.I., R.P.; data curation, F.M.R., M.D.C., M.B.; writing—original draft preparation, F.M.R., M.D.C, R.P.; writing—review and editing, F.M.R., M.D.C, R.P, R.G, M.I.; visualization, F.M.R., M.D.C.; supervision, R.P.; project administration, R.P., M.I.; funding acquisition, R.P., M.I., R.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** MDC receives support from the PhD program in Molecular and Translational Medicine of the Università degli Studi di Milano, Italy. Part of this work was carried out in OMICs, an advanced mass spectrometry platform established by the Università degli Studi di Milano.

**Conflicts of Interest:** The authors declare no conflict of interest.
