Tocopherol color reactions

The color complex produced with ferric chloride-dipyridyl is equally sensitive (0.5 jag a-tocopherol detectable) and unspecific. The color reaction can be used, however, for quantitative determination of a-tocopherol in pharmaceutical forms and concentrates after localization of the spot in UV light and elution from the adsorbant (Bolliger, 1962). 

Tocotrienols differ from tocopherols by the presence of three isolated double bonds in the branched alkyl side chain. Oxidation of tocopherol leads to ring opening and the formation of tocoquinones that show an intense red color. This species is a significant contributor to color quaUty problems in oils that have been abused. Tocopherols function as natural antioxidants (qv). An important factor in their activity is their slow reaction rate with oxygen relative to combination with other free radicals (11). 

The selectivity of the trap towards hydroxyl radicals was demonstrated by several control experiments using different radicals, showing that the formation of the respective hydroxylation product, 5-hydroxy-6-0-zso-propyl-y-tocopherol (57), was caused exclusively by hydroxyl radicals, but not by hydroperoxyl, alkylperoxyl, alkoxyl, nitroxyl, or superoxide anion radicals. These radicals caused the formation of spin adducts from standard nitrone-and pyrroline-based spin traps, whereas a chemical change of spin trap 56 was only observed in the case of hydroxyl radicals. This result was independent of the use of monophasic, biphasic, or micellar reaction systems in all OH radical generating test systems, the trapping product 57 was found. For quantitation, compound 57 was extracted with petrol ether, separated by adsorption onto basic alumina and subsequently oxidized in a quantitative reaction to a-tocored, the deeply red-colored 5,6-tocopheryldione, which was subsequently determined by UV spectrophotometry (Scheme 23). 

On spraying with 2,6-dichloro-p-benzoquinone-4-chloroimine, ceric sulfate reagent (Seher, 1960, 1961) and especially with antimony penta-chloride (BoUiger, 1962), the tocopherols give different colors which vary also with the adsorbant, as shown in Table IX and Figs. 7 and 8. The reaction with antimony pentachloride (20% in chloroform) depends on the number and position of the methyl gi-oup on the benzene ring and on the nature of the side chain. These color complexes are not stable, however. 

Non-specific visualization procedures (e.g., charring) for tocopherols/tocotrienols are based on spraying with sulfuric acid, perchloric acid, nitric acid, or copper (II) sulfate phosphoric acid (54). More specific though still subject to many interferences are reactions which take advantage of the reducing properties of vitamin E derivatives. In the Emmerie-Engel reaction and its later modifications, ferric ions are reduced to ferrous ions, which form a red-colored complex with a,a -dipyridine ot bathophenanthroline (50,51). A comparable sensitivity is achieved by spraying with phospho-molybdic acid. The color can be stabilized by subsequent exposure of the plates to ammoniacal 2,7-dichlorofluorescein (51). 

Ali and Iqbal (2008) reported a modified spectrophotometric method for determination of plasma levels of a-tocopherol in normal adults. The method was based on the work of Fabianek et al. (1968). Iron(III) was reduced to iron(II) in the presence of reducing agent (a-tocopherol) and then coupled with a chelating agent 4,7-diphenyl-l,10-phen-anthroline to produce pink iron(II) colored complex. The absorbance of complex was monitored at 536 nm and was proportional to the concentration of a-tocopherol in the reaction over the range 0.5-4 pg/mL with a limit of detection 0.2 pg/mL. This modified assay involved extraction of a-tocopherol in -hexane (less hazardous and improved precision of the assay) rather than xylene. 

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