**3. Discussion**

Research on the characterization of oils and solid fats (butter) derived from nuts is extensive [17], due to the long traditional use of these products and of increasing consumption as alternatives to dairy sources. Most analyses focus on the composition of major components, such as the presence of fatty acids [18,19]), of triglycerides and of some minor components with nutraceutical properties, such as vitamins and antioxidants [8,20,21].

Sphingolipid research in plants has highlighted the presence of modified long chain bases, mainly t18:1 and d18:2, linked to commonly occurring and more rare odd-carbon and hydroxylated fatty acids [6,10,12,22,23].

Observation of ceramides with a modified long chain base and with specific fatty acids may increase the variety of chemical indicators that can be employed to confirm the presence of specific high-value nuts, such as pistachios and almonds, in food preparations, and to highlight contamination or adulteration with extraneous materials [2,24,25]. Several techniques are currently used for this task, including direct Near-Infra-Red [26] and Raman [27] spectroscopy, mineral element pattern [28], and chemometric evaluation of untargeted LC-HRMS analysis [29].

It is of note that selective scans in the Precursor Ion and Neutral Loss modes are unique of the tandem triple quadrupole (and, earlier, of some magnetic) mass spectrometer configuration. The sole limitation of the use of triple quadrupole is the unit resolution of the mass filters. As highlighted in our case, spurious signals can be generated from the unexpected contribution of isotopic components of bulk constituents. This approach allowed observing and understanding for the first time the interference of a

high load of triglycerides in the selective discovery search for trace level nitrogen-containing lipophilic secondary metabolites by triple quadrupole tandem mass spectrometry methods. In addition, the comparatively lower sensitivity of the iso-energetic Precursor Ion approach is inherent to the use of a scan mode, rather than MRM, and is an unavoidable trade-off of the demonstrated higher coverage of different chemical species [30,31].

Complete characterization of the discovered ceramide hexosides entails recognition of the hexose stereochemistry and confirmation of the position and stereochemistry of the structural modifications of the fatty acid and of the sphingosine. These structural details cannot be easily differentiated by simple, *on the fly* recording of tandem mass spectra but need isolation of more substantial amounts of purified materials by mass spectrometry guided preparative methods.

#### **4. Materials and Methods**

#### *4.1. Reagents, Chemicals, and Standards*

Sphingolipid standards, including ceramides with d18:1D4 sphingosine long-chain base (LCB) and saturated/singly unsaturated straight-chain even-carbon fatty acids (12–24 FA) and C12:0 cerebroside were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Methanol, ethanol, acetonitrile, ammonium formate, and formic acid (all analytical grade) were supplied from Merck (Darmstadt, Germany). Water was MilliQ-grade (Millipore, Milford, MA, USA).
