An improved assay method of vitamin D using superfiltrol chromatography.

Abstract

1. In the determination of vitamin D in liver oils using Superfiltrol chromatography, the determination can be disturbed by the appearance of the degradation products of vitamin A in the vitamin D fraction, when a large quantity of vitamin A is present. The fraction eluted prior to the vitamin D fraction contains more amount of the non-vitamin D substance.2. The E values at 500 and 550mμ of the colored solution by antimony reaction of the non-vitamin D substance derived from vitamin A were about equal 120 seconds after mixing the reagents.3. In the antimony trichloride reaction of vitamin D the absorption maximum lies at 500mμ and the E value is 0 at 550mμ.4. The difference E550-E550 after 120 seconds corresponds to E500 of vitamin D, with no interference by non-vitamin D substances. Determination of vitamin D is not affected, if the amount of vitamin A is less than 4000μg for one Superfiltrol chromatography.5. Use of ethylene dichloride containing acetyl chloride in the antimony reaction makes the reagent more stable and the color reaction more intense than the use of chloroform. The E values are less affected by temperature. The optimal conditions for the determination have been described.6. The vitamin D contents of several liver oils, which are considered to produce large amounts of non-vitamin D interfering substances, were assayed. The values obtained by the present method were definitely lower than those calculated from E265, assuming that the absorbance was entirely due to vitamin D.7. The values of E500-E550 of solutions containing vitamin A fraction were negative. Therefore the values for vitamin D are lower than the true values when the solutions contain the vitamin A fraction.