2.5 Extraction and Determination of Vitamin E (Tocopherol)
A known amount (0.05g) of sample (PFAD and HNPFAD) was dissolved in 2 ml of n-hexane and 5\(\mu l\) of the mixture was injected into a HPLC equipment. HPLC analyses for all samples were performed at the Multi-Purpose Laboratory, located in the department of Chemistry of the Ahmadu Bello University (ABU), Zaria, Kaduna State, Nigeria. Prepared samples were analyzed using the HPLC (Agilent Technologies) at reverse phase mode. The HPLC (Agilent 1100 series) operated at Agilent isocratic condition with a stack configuration of: solvent cabinet, vacuum degasser, HP-1100 pump system, an autosampler, column compartment and fluorescence detector (G1321 A FLD). The column was a Hypersil ODS, 125 mm × 4 mm internal diameter with 5\(\mu\)m particles; a capillary length of 150 mm and 0.17 mm i.d. The mobile phase was a mixture of methanol and water (95:5, v/v) and eluted at a flow rate of 1.0ml/min. The analytical column was set at 290nm excitation wavelength and 325nm emission wavelength. The total separation time was 10mins. The tocopherols were identified by comparison of retention times standards of the \(\alpha,\ \gamma\ \)and\(\delta\)- tocopherols. Peak area was used for quantification.\(\alpha\)-, \(\gamma\)- and \(\delta\)- tocotrienols standards were unavailable and because of this, only tocopherol standards were used for analysis. The sum of concentrations of \(\alpha,\ \gamma\ \)and\(\delta\)- tocopherols was used as the total tocopherol content (vitamin E) of the FAD.