High-performance liquid chromatographic determination of vitamin D in foods, feeds and pharmaceuticals by successive use of reversed-phase and straight-phase columns.

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Abstract
A simplified and accurate method for the determination of vitamin D in foods, feeds and pharmaceuticals was established by high-performance liquid chromatography (HPLC) using successively reversed-phase and straight-phase columns. About 1-2 g of a sample was accurately weighed and directly saponified. The extracted unsaponifiable matter was subjected to preparative HPLC using a reversed-phase column and a vitamin D fraction was collected. The fraction was subsequently subjected to analytical HPLC using a straight-phase column and vitamin D was assayed by estimating the peak height. The proposed method was applied to various kinds of samples, e.g., fishery products, fish meals, mixed feeds for fish farming and chicken farming, egg yolk, milk products, cattle liver, Shiitake, fortified foods and multivitamin preparations. The proposed method was useful for the determination of vitamin D in such samples, because the peak of vitamin D in the profile of the second HPLC was always clearly separated from other concomitants and good recovery was obtained. The method is probably useful as a routine one for the determination of vitamin D in foods, feeds and pharmaceuticals.