[The quantitative determination of vitamin D3 and its metabolites in plasma]

A method is described which enables determination of vitamin D3 and its physiologically most important metabolites, i.e. 25-OHD3, 24,25-(OH)2D3, 25,26-(OH)2D3 and 1,25-(OH)2D3 in a plasma sample of about 2 to 4 ml. The whole procedure involves two preparative and one analytical steps: Extraction with methanol/methylene chloride (2:1), chromatographic separation on Lipidex 5000 using a stepwise gradient of n-hexane and chloroform and finally HPLC separation on Zorbax-Sil columns with n-hexane isopropanol mixtures and subsequently reversed phase separation on RP 18-columns and mixtures of methanol and water. Except for 1,25-(OH)2D3 all D compounds were quantified by UV-detection with 1.4 ng of substance being the lowest detectable amount. 1,25-(OH)2D3 was measured by radioimmunoassay. Prior to HPLC analysis the extract was separated into three fractions on Lipidex 5000 which contained 1) vitamin D3, 2) 25-OHD3 and 3) the dihydroxy metabolites. The three fractions were separated by HPLC using different mixtures of isopropanol/n-hexane and methanol/water, respectively. Retention times of the individual D-components longer than 10 min appeared to be essential to separate these compounds from accompanying material. Overall recoveries of the individual metabolites were for vitamin D3 48.9%, for 25-OHD3 54.2%, for 24,25-(OH)2D3 50.9% and for 1,25-(OH)2D3 52.5%. Application of the methods to plasma samples from pigs with pseudovitamin D deficiency rickets, typ I, revealed a reduced concentration of 1,25-(OH)2D3 and 24,25-(OH)2D3 and an elevated level of 25-OHD3 in these animals. The results obtained by this method contributed substantially to a better understanding of the aetiological factors associated with this disease.