2.6.4. Phytosterols Concentration

Extraction and quantification of phytosterols were done according to Domínguez-Avila et al. and Nair et al., respectively [28,29]. HCl and KOH solutions were made using EtOH as solvent. Oil was mixed with 6 M HCl (1:5 *w*/*v*) and incubated (1 h, 80 ◦C). The mixture was cooled in a water bath and 5 mL of 1.3% KOH was added and left to react (30 min, 80 ◦C). To phytosterols extraction, 2 mL of distilled water and 5 mL of hexane were included to the mixture, vortexed for 1 min, and centrifuged (3750 rpm, 15 min, 20 ◦C). The addition of distilled water and hexane was performed twice. Hexane was evaporated from pooled supernatants using a vacuum evaporator (2.5 h, 45 ◦C). Extracts were reconstituted in 0.5 mL of hexane for chromatographic analysis. A HPLC-ELSD system (Agilent 1200, Agilent Technologies, Santa Clara, CA, USA) equipped with a 5 μm, 4.6 mm × 500 mm Luna C8 column (Torrance, CA, USA) was employed to identify and

quantify phytosterols from pecan nut oil. Column and ELSD temperature were maintained at 40 ◦C and 50 ◦C, respectively. Aliquots (10 μL) were analyzed employing a mobile phase consisted of MeOH:H2O (95:5 *v*/*v*) at a flow rate of 1 mL/min and the detector was set at a gain of 16. Standard curves of β-sitosterol (0.2–1.2 mM), stigmasterol (0.2–1.2 mM), and campesterol (0.05–0.25 mM) were prepared to quantification. Concentrations were expressed as mg per kg of pecan nut oil (mg/kg).
