Measuring Vitamins A and D in Milk

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Abstract
Quantitative estimations of vitamin A palmitate in nonfat milk and vitamin D in whole milk were made by reverse phase high performance liquid chromatography. [Milk fortification makes it necessary to be able to ascertain their levels in the milk]. The procedure developed for vitamin D analysis consisted of extraction of the lipid material, formation of methyl esters from triglycerides by alcoholysis, TLC to separate the vitamin D from the methyl esters and final analysis by reverse phase high performance liquid chromatography with a methanol:water, 97:3 (vol/vol), mobile phase. The mean corrected recovery of the vitamin D was 94% with coefficients of variation ranging from 2.5 to 5.3%. The analysis for vitamin A palmitate in nonfat milk utilized the same basic procedure except for elimination of TLC. Mean recovery of vitamin A palmitate was 99% with coefficients of variation 3.2 to 10.0%. The major advantage of this method over other methods is the use of alcoholysis instead of saponification. Alcoholysis saves time and subjects vitamins to milder conditions than does saponification. Another advantage lies in the use of TLC plates, which provides a good method of separation of vitamin D3 from other material and allows for instant visualization of vitamin D3. Finally, the reverse phase high performance liquid chromatography system gives good separation of vitamin D and vitamin A palmitate from other substances in their respective chromatograms plus virtual baseline separation of vitamins D2 and D3.