DETERMINATION OF VITAMIN-D IN FOOD BY MEANS OF HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY (HPLC) - RESULTS OF COLLABORATIVE STUDIES OF THE WORKING GROUP VITAMIN ANALYSIS ACCORDING TO SECTION 35 LMBG (GERMAN FOOD ACT)

A standardized method to determine the vitamin D content of food by means of HPLC is described. After the test material was homogenized and saponified with ethanolic aqueous potassium hydroxide solution, vitamin D was extracted using n-hexane. Using HPLC on a silica gel column, the fraction containing vitamin D is separated from the nonsaponifiable residue. After the fraction was reduced, the residue was dissolved in methanol and the vitamin D content determined after HPLC separation on an RP-C18 column. For evaluation, either a conventional external standard method using laboratory and matrix specific recovery rates as correction factors, or an internal standard method using vitamin D2, and D3, respectively as internal standards were employed. The method was developed and standardized by the working group "Vitamin Analysis" in accordance with sectional sign 35 LMBG (German Food Act). Repeatability and comparability of the results were checked in collaborative studies (14 laboratories) in milk powder and gruel that had been enriched with vitamin D3. Applicability of the method to other food (eggs, milk, fish, margarine) was checked separately. The statistical evaluation of the results of the collaborative studies has shown that the method is reliable enough to be included into the "Amtliche Sammlung" (official collection of analytical methods) according to sectional sign 35 LMBG. The present method may be used to determine the content of vitamin D2 and D3 in natural and vitaminized food. It is specific for the vitamins D2 and D3 released during saponification of food, but does not allow separate determination of the pre-vitamins D already present in the sample.