*4.3. Extraction and Quantification of Policosanols from Barley and Wheat Seedlings*

The preparation of barley and wheat crude extracts, policosanol standards, GC/MS parameters, and the quantification method of GC/MS as described elsewhere [17,36], was used in this study. Briefly, 1 g of freeze-dried and chopped samples collected from all treatments was extracted separately into 10 mL of hexane on a shaker for 24 h at room temperature. The supernatant of the mixture was collected by centrifugation at 3000 g for 3 min, filtered through a syringe filter with a pore size of 0.45 µm (Whatman Inc., Maidstone, UK), and kept under vacuum conditions until the hexane was completely removed. To the final extract, 250 µL of N-Methyl-N-trimethylsilyfluoroacetamide (MSTFA) and 0.5 mL of chloroform was added and stirred for 15 min at 60 ◦C. Chloroform was added to make up one ml of sample and, then, one µL was injected into the gas chromatograph using an auto sampler with a split ratio of 1:5. The GC was equipped with an HP-5MS UI (diphenyl 5%-dimethylsiloxane 95% co-polymer) capillary column (30 m by 0.25 µm by 0.25 µm film thickness) and a 5977A series mass spectroscopy (Agilent Technologies, Palo Alto, CA). The policosanol content was quantified according to the methods of Ra et al. [17]. The policosanol standards—Eicosanol (C20), heneicosanol (C21), docosanol (C22), tricosanol (C23), tetracosanol (C24), hexacosanol (C26), heptacosanol (C27), octacosanol (C28), and triacontanol (C30)—hexane, and chloroform solutions were purchased from Sigma (Sigma-Aldrich, St. Louis, MO).
