A small-scale sample preparation method with HPLC analysis for determination of tocopherols and tocotrienols in cereals

A small-scale sample preparation method was developed for reliable and economic analysis of tocopherols and tocotrienols in rye and other cereals. The saponification parameters were optimized using two experimental design protocols with statistical analyses. Three critical factors were optimized for hot saponification: time, temperature and amount of potassium hydroxide (KOH). The amount of KOH had the greatest effect. Two direct solvent extraction methods without saponification were tested and found to yield in comparable values than with saponification. Finally, three solvent mixtures for extracting tocopherols and tocotrienols after the optimized saponification step were studied in order to examine the effect of solvent polarity on their recovery. Adding a polar modifier to the solvent, tocopherol and tocotrienol values of rye were significantly increased. The optimized sample preparation method included saponification with 0.5 mL KOH at 100°C for 25 min followed by extraction with n-hexane:ethyl acetate (8:2). The repeatability of the recommended analytical procedure was good and the recoveries of added tocopherols from rye flour samples ranged from 90.3% to 94.3%. The procedure developed was applied to examine the amounts and distribution of tocopherols and tocotrienols in ten rye varieties. The average total tocopherol and tocotrienol content of rye grains was 48.8 μg/g with a 9.3% standard deviation between varieties.