2.3.2. Extraction and Derivatization of Non-Polar Metabolites

For the extraction process, 1000 mg of frozen pericarp powder was mixed with chloroform (1250 μL), methanol (2500 μL), and n-tridecane (800 μg.mL−1, internal standard; 20 μL), followed by vortexing for 10 s and incubation on ice for 30 min. Then, 1.5% sodium sulfate (1250 μL) and chloroform (1250 μL) were added to the mixture, incubated on ice for 5 min and centrifuged at 4 ◦C for 1000× *g* and 15 min. The upper polar phase was collected and dried under nitrogen gas. The sample was redissolved in hexane (1000 μL), toluene (200 μL), methanol (1500 μL), and 8% chloridric acid (300 μL), mixed for 10 s, and incubated for 1.5 h at 100 ◦C. Subsequently, hexane (1000 μL) and Milli-Q water were added to the sample and mixed [16–18]. The hexane phase was separated and dried under nitrogen gas. The sample was redissolved in hexane (80 μL) and pyridine (20 μL) and derivatized with MSTFA (40 μL). Finally, the derivatized samples were moved into glass vials and analyzed by GC-MS. A pool of fatty acid methyl ester (FAME) external standards (Sigma-Aldrich) was applied to certify the identified metabolites by mass spectral comparison: methyl laurate (C12:0, 0.8 mg.mL−1); methyl tetradecanoate (C14:0, 0.8 mg.mL−1); methyl palmitate (C16:0, 0.8 mg.mL<sup>−</sup>1); methyl octadecanoate (C18:0, 0.4 mg.mL−1); methyl arachidate (C20:0, 0.4 mg.mL<sup>−</sup>1); methyl docosanoate (C22:0, 0.4 mg.mL−1); methyl lignocerate (C24:0, 0.4 mg.mL−1); methyl linoleate (C 18:2, 0.4 mg.mL−1); (Z)-9-oleyl methyl ester (C 18:1, 0.4 mg.mL−1); methyl linolenate (C 18:3, 0.4 mg.mL−1); methyl palmitoleate (C 16:1, 0.8 mg.mL<sup>−</sup>1) [15].
