Specific determination of alpha-tocopherol in food and feed by fluorometry. Part 1. Manual method.

The proposed fluorometric method for determining alpha-tocopherol is highly specific and sensitive, yet requires low-cost equipment available in any laboratory. It is robust and fairly fast (4 determinations in 100 min, sample preparation not included). It has been tested in parallel with a conventional thin layer chromatographic method on foods and feeds. The only necessary cleanup is the usual saponification. The unsaponifiable fraction can be extracted with ethyl ether or, preferably, with Extrelut columns. Isooctane is used as a carrier solvent. Reagents and their solvents are added to the isooctane solution before each successive reaction and are then eliminated by partition with water. The alpha-tocopherol (alpha-T) derivative always remains in isooctane. The first step is nitrosation and elimination of tocopherols and tocotrienols other than alpha-isomers. alpha-T is then oxidized to alpha-tocored (alpha-TR) with a mixture of sulfuric acid, ferric chloride, and iodine bromide. alpha-TR is then condensed to a new reagent: 4,5-dimethyl-o-phenylenediamine. The phenazine formed is strongly fluorescent. Iodine and bromine add to the double bonds of alpha-tocotrienol present and quench the fluorescence of its phenazine. A procedure for blank assays specifically inhibits the conversion of alpha-T to alpha-TR.