A small-scale HPLC method for determination of α-tocopherol in fresh plant tissue

This paper presents an high-performance liquid chromatographic (HPLC) method for the determining of alpha-tocopherol in small samples of fresh plant material. Ryegrass was used as the test material. The samples of 5-100 mg are saponified for 30 min at 60 degrees C. After evaporating the n-hexane extract of the unsaponifiables and dissolving the residue in n-hexane, the sample is ready for the HPLC analysis. The HPLC system involves an amino column, n-hexane-isopropanol (99:1) flowing at the rate of 2 ml/min as the mobile phase and a fluorescence detector. The recovery of alpha-tocopherol added to ryegrass samples was 110 % when the quantification was based on an alpha-tocopherol standard run through the whole sample preparation procedure. The variation in the alpha-tocopherol contents was 11 % (coefficient of variation CV %) when 18 samples ranging 5-100 mg were analysed within one day. The day-to-day variation was only 4.6 % when samples of 50-100 mg were analysed. The results confirm that this simple, rapid and sensitive method is suitable both for routine analysis of fresh plant tissues and for plant physiological studies where live plants are to be monitored by repeated sampling.