Determination of 25-hydroxyvitamin D3 in serum by high performance liquid chromatography and isotope dilution-mass spectrometry.

Four different HPLC methods for analysis of 25-hydroxyvitamin D3 in serum were evaluated with a method based on isotope dilution-mass spectrometry (ID-MS). Method I utilized Sephadex LH-20 chromatography as the only prepurification step. No correlation with the ID-MS method was obtained. Method II utilized Sephadex LH-20 chromatography and a subsequent reversed phase HPLC step as prepurification. The correlation coefficient was 0.99 (regression coefficient 1.2 and intercept - 3.9 micrograms/l). Method III included open silicic acid chromatography and straight phase HPLC as prepurification. The correlation when compared with the ID-MS method was 0.94 (regression coefficient 1.2 and intercept - 0.4 micrograms/l). In method IV Sep-pak C18 chromatography and open silicic acid chromatography were used as prepurification. The correlation coefficient when compared with the ID-MS method was 0.97 (regression coefficient 0.8 and intercept 0.1 microgram/l). It is concluded that a single Sephadex LH-20 step is not sufficient as prepurification and that method IV had an accuracy sufficient for its intended use to analyse 25-hydroxyvitamin D3 in serum from cattle.