Influence of the extraction technique in the assay of serum 25-hydroxyvitamin D

Various extraction procedures for the assay of serum 25-hydroxyvitamin D were compared in order to investigate their specificity and accuracy. Extraction with chloroform/methanol followed by chromatography on silicic acid columns was chosen as the reference method. The dried extracts and eluates from column chromatography were used in a competitive protein binding assay. The values obtained using the different extraction procedures without chromatography did not differ significantly, but were constantly higher than the values obtained using the reference method. These results confirm the necessity of a chromatographic step after extraction of samples in the assay of serum 25OHD,, since all the techniques tested 25OHD together with other substances which interfere with the competitive binding assay. Ethanol extraction gave the best recoveries; furthermore this technique is also very simple, therefore it could be considered the best extraction procedure in the assay of 25OHD.