Methyl linolenate

The chemical reagents used for VOO characterization were of GC or HPLC grade. Methanol, acetonitrile, o-phosphoric acid and the Folin-Ciocateu (F-C) reagent were provided from Sigma-Aldrich (Steinheim, Germany), n-hexane from Hipersolv Chromanorm (Pennsylvania, USA), Na₂CO₃ anhydrous from Acros organics (New Jersey, USA) and KOH from Fisher Scientific (Loughborough, UK). Regarding the commercial standards, methyl palmitate, methyl palmitoleate, methyl stearate, methyl oleate, methyl linoleate, methyl linolenate, hydroxytyrosol, tyrosol, p-coumaric acid, luteolin, and apigenin were purchased from Sigma-Aldrich (Steinheim, Germany) and Fluka (Steinheim, Germany), vanillic and syringic acid standards were obtained from Acros organics (New Jersey, USA), gallic acid from Fisher Scientific (Loughborough, UK), and oleacein and oleocanthal were supplied by Prof. Prokopios Magiatis (Athens, Greece). Finally, deionized water (18 MΩ cm) from a Milli-Q water purification system (Millipore, Bedford, MA, USA) was used to prepare the mobile phases for HPLC analysis.

Methyl linolenate (methyl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate) and deuterochloroform (CDCl₃, 99.8 d-%,) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Millet was purchased from the Jianjun agricultural and agricultural products processing plant (Shijiazhuang, China). High-temperature-resistant amylase was purchased from Novozyme (Denmark). Anhydrous glucose was purchased from Sigma (China). Methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, and methyl linolenate were obtained from Sigma (China). Calcium, zinc, manganese, copper, iron, and magnesium standard stock solutions (10 mg/mL) were purchased from the national center of analysis and testing for nonferrous metals and electronic materials. Amino acids were purchased from Hitachi (Japan). Boron trifluoride-methanol solution (14% BF₃-methanol) was purchased from CNW Technologies (Dusseldorf, Germany). N-hexane (chromatographic purity) was purchased from Fisher Chemical.

Five hundred milliliters of the reaction mixture were mixed with 1.0 mL isooctane for two min. Following centrifugal separation, the upper organic layer was collected and washed twice with distilled water and dried over anhydrous Na₂SO₄. The solvent was dried under N₂ steam and dissolved in 0.25 mL of CH₂Cl₂. The previous GC condition of WCO analysis was applied. The prepared FAMEs were then analyzed using particular fatty acid methyl ester standards (methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, and methyl linolenate; Sigma-Aldrich).

0.5 mL of reaction mixture was mixed with 1.0 mL isooctane for 2 min. Following centrifugal separation, the organic upper layer was collected and washed twice with distilled water and dried over anhydrous Na₂SO₄. The solvent was dried under N₂ steam and dissolved in 0.25 mL of CH₂Cl₂. The previous GC condition of soybean oil analysis was applied. FAMEs formations were analyzed by means of authentic fatty acid methyl ester standards (methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, and methyl linolenate; Sigma-Aldrich).