Non-aqueous Reversed-phase Liquid-chromatography based Quantification of Tocopherols in Groundnut Oil

A convenient non-aqueous reverse-phase liquid-chromatographic method was developed, validated and applied for measuring vitamin E isomeric forms (α-, β + γ- and δ-tocopherol) in groundnut oil. Analytes were separated on 5 µm Polar Advantage II, 120 Å, C-18 column, with dimensions of 4.6 × 150 mm, maintained at 25°C. The separation of vitamin E homologues was optimized in terms of sample diluents, mobile-phase composition and chromatographic conditions on the basis of the efficiency, resolution and run time. Isocratic non-aqueous reversed-phase separation starts at 5.5 min with absolute methanol as mobile phase with flow rate of 1.0 mL/min. Detection is accomplished with in 10 min only using PDA-UV detector at the wavelength of 295 nm. The method was validated on the basis of precision, linearity, limit of detection (0.08–0.746 µg/mL) and quantification (0.241–2.26 µg/mL). The elution order of tocopherols in TG-51 genotype was δ-tocopherol, (β + γ)- tocopherol and α-tocopherol with retention time of 4.87, 6.01 and 6.43 min, respectively. The total tocopherol content in TG-51 was found to be 567 ppm with 49 ppm of δ-tocopherol, 109 ppm of β + γ-tocopherol and 409 ppm of α-tocopherol. Peaks of α-, β + γ- and δ-tocopherol in groundnut oil were identified by comparing their retention times with standards.