Quantitative determination of tocopherols in single seeds of rapeseed (Brassica napus L.)

A high-performance liquid chromatography (HPLC) method was developed to determine tocopherols in single seeds of oilseed rape (Brassica napus L.). Single seeds were extracted with isooctane, centrifuged, and the overlayer extract injected into the HPLC instrument without further purification. Separation was performed on a diol-column (250 mm × 3 mm I.D.) using a mobile phase consisting of 94% isooctane and 6% tert-butyl methyl ether. Quantification of individual tocopherols was done by fluorescence detection and with β-tocopherol as internal standard. The standard error (SE) of the present method for total tocopherol content, as determined with 30 ground samples of Samourai (mean = 304 mg kg -1 dry seed) and 20 ground samples of Lirabon (mean = 350 mg kg -1 dry seed), was 9.5 mg tocopherol/kg dry seed. The SEs of the method for the determination of α- and γ-tocopherol percentages were 1.2% and 1.3%, respectively. The low SE values indicate that the method can be effectively used to detect small differences for tocopherol content and composition in studies requiring the analysis of reduced amounts of plant material. In particular, the comparison between the SE of the method and the SD of single seeds within individual plants of Samourai (36.8 mg kg -1 dry seed) and Lirabon (37.6 mg kg -1 dry seed) shows the potential of the present method for the detection of the tocopherol content within plant differences.