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A simplified and accurate method for determination of naturally occurring vitamin D3 in bovine milk was established by high-performance liquid chromatography (HPLC) using successively reversed-phase and straight-phase columns. Exactly 25.0 ml of a sample of bovine milk was taken and the lipid was extracted with a solvent mixture of petroleum ether and ethyl ether (1:1) with small amounts of ethanol and Triton X-100, present. The extracted lipid was subjected to the first preparative HPLC using a Nucleosil 5C18 column (reversed-phase type) with acetonitrile-methanol (1:1) as the mobile phase, and a fraction containing vitamin D3 was isolated. The fraction was subsequently subjected to the second analytical HPLC using a Zorbax SIL column (straight-phase type) with 0.4% isopropanol in n-hexane as the mobile phase. Vitamin D3 was assayed by estimating the peak height on the chromatogram. The overall recovery and CV values were 92.1 +/- 8.7% and 9.4%, respectively, which were satisfactory. The proposed method was applied to several kinds of colostrum, early and later bovine milk in pairs. The assayed values in the colostrum of the group with large amounts of vitamin D3 administered before delivery to prevent parturient paresis were higher than those in the group with no administration. However, the values of the former group generally decreased in the respective early and later milk in step and there were few differences among those in the later milk of the two groups. The assayed values in later milk were 30-80 IU/liter.

High-performance liquid chromatographic determination of vitamin D3 in bovine colostrum, early and later milk.