Simultaneous determination of alpha-tocopheryl acetate and tocopherols in aquatic organisms and fish feed

In aquaculture, alpha-tocopheryl acetate (alpha-TA) is the main source of vitamin E used to fortify fish feed. alpha-TA in fish is often determined indirectly, i.e., by alkaline hydrolysis, followed by quantitation of "total alpha-tocopherol" (alpha-T) and subtraction of the natively present alpha-T. The aim of this study was to develop an HPLC method for the simultaneous quantitative determination of alpha-TA and free tocopherols in aquatic organisms and fish feed. The assay consists of a simple extraction with methanol containing butylhydroxytoluene (BHT) as an antioxidant, followed by reversed-phase chromatography with consecutive UV and fluorescence detection of alpha-TA and tocopherols, respectively. The peak of the internal standard tocol in the fluorescence trace was used for quantitation. Linearity was achieved over the range of 0.2 to 4.2 micrograms alpha-TA per ml extract of Artemia nauplii, which would correspond to 30.7 to 614.4 micrograms/g dry mass. The within-run coefficient of variation was 1.9% at a level of 310 micrograms/g dry mass. The recovery of alpha-TA ranged from 97.7 to 100.8% (concentration = 2.1 and 20.5 micrograms/ml, n = 6). The detection limit was about 7 ng and the quantification limit on spiked samples was 0.2 microgram/ml. This method was routinely applied to determine alpha-TA and alpha-, gamma- and delta-tocopherol (alpha-T, gamma-T, delta-T) simultaneously in Artemia, fish feed, shrimp eggs and various other aquatic organisms.