Determination of 25-Hydroxycholecalciferol Content in Egg Yolk by HPLC

A high-performance liquid chromatographic (HPLC) procedure for determining 25-hydroxy-cholecalciferol contents in chicken egg yolk has been developed. The procedure consists of the following steps: saponification, extraction of the unsaponifiable fraction, purification using solid-phase extraction, clean-up on straight-phase HPLC, and quantification with reverse-phase HPLC using an internal standard (25-OH-D 2) method and diode array detection. The suitability of the method was tested on a pooled egg-yolk sample. The overall mean recovery for 25-OH-D 2 was 71 ± 5.4% and for 25-OH-D 3 was 71 ± 3.2%. The recovery of 25-OH-D 3, calculated on the basis of the internal standard, was 98 ± 4.6%. The detection and determination limits of the method were 2 and 8 ng/injection, respectively. The pooled egg yolk contained 0.98 ± 0.069 μg of 25-OH-D 3/100 g of egg yolk. A simultaneous determination of the vitamin D 3 contents in yolks is also possible with the method described.