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.