Patent ID: 7358092

Claim:
A process for the quantitative determination of 25-hydroxy-cholecalciferol in animal feed which comprises the steps of a) dispersing a feed sample in water to form an aqueous dispersion and adding to the sample a defined amount of an internal standard compound hydroxycholecalciferol which is a derivative of, an isomer of, or isotopically labeled 25-hydroxycholecalciferol to obtain an aqueous dispersion; b) extracting the aqueous dispersion with tert. butyl methyl ether; c) submitting the ether extract to semipreparative HPLC; d) collecting fractions containing 25-hydroxycholecalciferol and the internal standard compound; e) submitting the fractions collected in d) or an aliquot thereof to HPLC combined with mass speetrometry; f) determining MS peak areas of 25-hydroxycholecalciferol and of the internal standard compound added; and g) calculating the amount of 25-hydroxycholecalciferol by computing the MS peak areas measured.