Fast gradient normal-phase high pressure liquid chromatography for rapid baseline separation of vitamin E forms in palm-derived tocotrienol rich fractions (TRFs)
A rapid, accurate and precise normal phase high pressure liquid chromatographic method was developed and validated for the simultaneous determination of nine vitamin E forms comprised of α-tocomonoenol, α-, β-, γ-, and δ-tocopherols and α-, β-, γ-, and δ-tocotrienols in tocotrienol rich fractions (TRFs) derived from palm oil. The chromatographic separation of vitamin E was performed on a silica HPLC column thermostated at 40 °C using n-heptane and ethyl acetate as the mobile phase in a gradient elution mode. The calibration standard curves were linear over the concentration range from 0.1 to 1.0 mg mL−1 for all vitamin E forms with overall R2 > 0.9980. The percent relative standard deviation of the method repeatability was less than 2.0% and the recovery ranged between 90 and 110%. The instrument limit of detection and quantitation ranged from 0.04 to 0.05 mg mL−1 and from 0.11 to 0.18 mg mL−1 respectively. The separation was completed in 15 minutes with an additional 2 minutes for re-equilibration. In conclusion, a method has been successfully developed and validated for the simultaneous analysis of nine vitamin E forms in palm oil which is suitable for routine quality control of all vitamin E forms in palm TRFs at different compositions.