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Hydrogenation versus Chromatography in the Estimation of Tocopherol in Calf Plasma¹

Storrs (Conn.) Agricultural Experiment Station

ABSTRACT

In the Emmerie-Engel reaction for the estimation of tocopherol in blood plasma, interference is due principally to carotenoids (1), and these apparently can be eliminated by either selective hydrogenation (1, 6–9) or chromatography (2, 9).

Data, with which to base a choice between these two procedures in estimating tocopherol concentration in calf plasma, were not found in the literature. In the course of experiments conducted during 1955–1956, plasma samples from 19 calves fed varying amounts of carotene and/or vitamin A, as well as D--tocopherylacetate, were analyzed by two procedures, (I) hydrogenation (7, 8), or (II) chromatography (2), to determine whether differences existed between the two methods.

Although saponification is not included in the method of Quaife and Harris (8), it was found necessary for calf plasma, in order to eliminate turbidity in the final ethanol solutions, as was the case whether (I) or (II) was employed.

In both (I) and (II) two five-ml. samples of calf plasma in 70-ml. glass stoppered centrifuge tubes were saponified (9), with ten ml. of two per cent ethanolic (aldehyde-free) potassium hydroxide containing 0.5% p-hydroxyacetanilide, by boiling gently for 20 minutes on a steam bath.

¹ This study was supported in part by funds from the Chas. H. Hood Dairy Foundation, the Chas. M. Cox Co., Boston, Mass., and the American Cyanamid Co., Pearl River, N.Y.