Determining Vitamins D₂ by Two Physical Chemical Methods

Abstract

Two methods for determining the ,itamin Dz content of oil solutions of irradiated ergosterol are described. In a spectrophotometric method the nonsaponifiable fraction of the oil is chromatographed, using a column of Superfiltrol and a solvent composed of a mixture of 30 parts of hexane, 10 parts of ether, and 1 part of alcohol. Interfering substances are adsorbed on the Superfiltrol while the vitamin D1 portion passes through the column.The vitamin Di content is evaluated by measuring its extinction at 263 mp. . i colorimetric method is proposed in which the nonsaponifiable fraction of .kRIOUS methods have been proposed for determining the vitamin D: content of irradiated provitamins D solutions and ah exten-ive review of these methods has been of fish liver oils. given ( 5 ) . Yield, Russell, and Zimmerli (7,12) found that by adding acetyl chloride to the antimony trichloride reagent of Brockmann and Chen ( 3 ) , its sensitivity could be increased and the presence of zinc, tin, or antimony had a tendency t o stabilize the color reaction. They also observed that sterols having double bonds in the side chain did not affect the reaction and those having double bonds in their ring structures gave the following absorption coefficients: El% 2 . 2 a t 500 mp El% 7 a t 515 i nM El% 1800 at 500 inlr 1 om.

Studies on the analysis of vitamins D. 1. The reaction of vitamins D and related substances with iodine trichloride

“…Experience has shown, however, that without the best attainable purification of vitamin D by removal of sterols, such methods do not give results agreeing with biological assays, when applied to widely varying products. This is so for the method of Ewing et al (1948).…”

“…The analysis of vitamins D in irradiation products appears at first sight to be easier and advances have been made, particularly by Ewing, Powell, Brown & Emmett (1948). It is often assumed that most of the sterols in an irradiation product of ergosterol or 7-dehydrocholesterol do not interfere with the analysis of vitamin D by the best known colorimetric methods.…”

“…Since the paper by Ewing et al (1943), the main advances have been due to Sobel, Mayer & Kramer (1945) with their discovery of the glycerol dichlorohydrin reaction, and De Witt & Sullivan (1946), who used a chromatographic separation on magnesia and diatomaceous earth and a modified antimony trichloride-acetyl chloride reagent in ethylene dichloride for determining vitamin D in several products. Ewing, Powell, Brown & Emmett (1948) developed two methods *for determining vitamin D in irradiated ergosterol products, one by direct estimation with the improved antimony tri-chloride reagent of Nield etal. (1940) and the other, after a chromatographic separation on 'Superfiltrol', by direct measurement of extinction at 265 mnu.…”

Spectrophotometric Determination of Vitamins D in Presence of Vitamin A

“…equivalent units of vitamin D per gram. This was on the basis of data from a number of samples of irradiated ergosterol in corn oil which had previously been assayed spectrophotometrically by the method of Ewing et al (6) and also bioassaved using the method and reference standard of U.S.P. XIII'.…”

“…FOR several years work in these laboratories has been directed toward the determination of vitamins D present in various oils using chromatographic techniques to separate the vitamin D from other nonsaponifiable oil constituents. Prior to this work two methods were proposed: the first by Ewing, Kingsley, Brown, and Emmett (5), and the second by Ewing, Powell, Brown, and Emmett (6).…”