STANDARDS FOR VITAMIN E 819 



When the tocopherols are treated with nitric acid alone, a different result 

 obtains for 5-tocopherol, as compared with the other tocopherols. Both 

 natural and synthetic 5-tocopherol show a maximum absorption band at 

 373 m^ with extinction coefficients^^ of 59 and 48, respectively. The 

 divergencies are explained as being due to the fact that the oxidation 

 products were not purified. On the other hand, the a-, jS-, and 7-toco- 

 pherols yield the same results with nitric acid as with silver nitrate, namely, 

 o-quinones with maxima between 460 and 480 mju.'^ The intensity of the 

 color production at 373 m^t should furnish a quantitative method for the 

 determination of 5-tocopherol in the presence of the other tocopherols. 



There are several color reactions which are more or less specific for the 

 tocopherols. One of these is based upon the oxidation of vitamin E in 

 alcoholic solution by ferric chloride, ^^^ or by the quantitative reduction of 

 ferric to ferrous chloride by the tocopherols.^^* When tocopherol is mixed 

 with the ferric chloride-Q:,a:'-dipyridyl solution, a characteristic red color de- 

 velops due to the formation of ferrous dipyridyl."* Since the amount of 

 ferrous ions formed depends upon the amount of tocopherols present, the 

 measurement of the red color is an index of that factor. This is the basis of 

 the Emmerie-Engel method for the determination of tocopherol. '^*'"^ 

 While this procedure is quite satisfactory for a-, /3-, and 7-tocopherols, it 

 can be used only under controlled conditions with 5-tocopherol.'''^ If the 

 reaction is allowed to proceed for 10 minutes instead of for 2^/2 minutes, a 

 22% excess of color develops over that produced by the isomeric toco- 

 pherols. 



An especially satisfactory reaction for 7- and 5-tocopherol has been de- 

 scribed by Weisler, Robeson, and Baxter.®^ This involves the reaction be- 

 tween diazotized o-anisidine and 7- or 5-tocopherol. 



6. Standards for Vitamin E 



The international standard for vitamin E which has been adopted by the 

 League of Nations is synthetic racemic a-tocopherol acetate. One Inter- 

 national Unit is defined as the specific activity of 1 mg. of a standard 

 preparation. The international standard contains the 1 mg. of a-tocoph- 

 erol in 100 mg. of olive oil. This is the amount which, when admin- 

 istered orally, prevents resorption-gestation in rats deprived of vitamin 

 E.ii« 



The Rat Unit or "fertility dose" is more widely employed. This is the 

 smallest amount of vitamin E which, when given 'per os daily to resorption- 



113 J. Waddell and H. Steenbock, /. Biol. Chem., 80, 431^42 (1928). 

 '" A. Emmerie and C. Engel, Nature, 142, 873 (1938). 

 !"> A. Emmerie and C. Engel, Rec. trav. chim., 57, 1351-1355 (1938). 

 "« E. M. Hume, Nature, I48, 472-473 (1941). 



