α tocopherol standard

α-Tocopherol content in co-products and silage samples were analyzed according to Larsson and Undeland^{26 (link)} with modifications as described by Sajib and Undeland¹¹ . Briefly, 8 ml chloroform extract from a chloroform:methanol extraction (as described earlier) was dried under N₂, followed by dilution in 1 ml methanol, centrifugation at 2000 × g for 3 min, whereafter the supernatant was analyzed using HPLC with fluorescence detection. Quantification was done using α-tocopherol standard (Sigma-Aldrich, USA).

An aliquot of this extract in n-hexane was filtered through a 0.45 μm filter, and 0.4 mL was taken and diluted with 1 mL of ethanol/hexane (70:30 v/v). The sample was then injected into a Waters 2690 HPLC high-performance liquid chromatography (HPLC) instrument equipped with a Waters 2475 fluorescence detector (Waters Corporation, Milford, MA, USA). A Nucleodur 100-5 C18 ec, 4.6 × 250 mm column (Macherey-Nagel, Dueren, Germany) was used for the separation. The mobile phase consisted of methanol (A) and ethanol (B), and an isocratic flow rate of 0.8 mL min^-1 (80A/20B) was used. The column temperature was set at 30 °C, and the total chromatogram time was 6 min. Peaks were identified by comparing the retention times with those of an α-tocopherol standard (Sigma-Aldrich Chemical Co., St. Louis, MO, USA). The concentration of α-tocopherol was expressed as μg per g FW.