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A simplified method for the determination of vitamin D2 in fortified dried milk was established by using two steps of high-performance liquid chromatography (HPLC). About 1 g of fortified dried milk was accurately weighed and directly saponified. The extracted unsaponifiable matter was first subjected to preparative HPLC using a Nucleosil 5C18 column (reversed-phase type) with acetonitrile-methanol (1:1) as a mobile phase and a fraction containing vitamin D2 was separated. The separated fraction was subsequently subjected to analytical HPLC using a Zorbax SIL column (straight-phase type) with 0.4% isopropanol in n-hexane as a mobile phase. Since the peak corresponding to vitamin D2 was clearly observed with separation from other concomitants on the chromatogram of the analytical HPLC, the vitamin was assayed by estimating the peak height. The overall recovery of vitamin D2 by the proposed method was 94.3 +/- 2.3% (mean +/- SD). Naturally occurring vitamin D3 derived from cow's milk was negligible in commercial fortified dried milk sold in Japan. When the proposed method was applied to 5 kinds of commercial fortified dried milk, satisfactory results were obtained.

Simplified assay of vitamin D2 in fortified dried milk by using two steps of high-performance liquid chromatography.