2.3.1. Extraction and Derivatization of Polar Metabolites

The extraction and derivatization of polar metabolites were conducted as described in [14]. For the extraction process, 100 mg of frozen pericarp powder was mixed with 100% distilled methanol at −<sup>20</sup> ◦C (1400 <sup>μ</sup>L) and ribitol (200 <sup>μ</sup>g.mL−1, internal standard; 60 μL). The mixture was vortexed, incubated in a thermomixer at 950 rpm for 10 min at 70 ◦C, centrifuged at 11,000× *g* for 10 min, and the supernatant was collected. To the upper phase was added chloroform at −20 ◦C (750 μL) and Milli-Q water (1500 μL), followed by mixing and centrifugation at 2200× *g* for 15 min. The upper hydrophilic phase (150 μL) was collected and dried under nitrogen gas. Sample derivatization comprised adding 20 mg.mL−<sup>1</sup> methoxyamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA; 40 μL) and pyridine with subsequent incubation in an orbital shaker at 1000 rpm and 37 ◦C for 2 h. Consecutively, N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA; 70 μL) was added to the sample, followed by incubation in an orbital shaker at 1000 g and 37 ◦C for 30 min. Finally, the derivatized samples were moved into glass vials and analyzed by GC-MS. A pool of polar metabolite external standards (1 mg.mL−1, Sigma-Aldrich) was applied to certify the identified metabolites by mass spectral comparison: D-glucose; D-fructose; maltose; sucrose; D-galactose; myo-inositol; citric acid; L-alanine; L-serine; L-proline; L-aspartate; L-glutamate [15].
