Determination of 25‐hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation

Interest in the metabolism and physiological action of vitamin D is increased exponentially. The most important metabolites of vitamin D are 25-hydroxyvitamin and 1,25-dihydroxyvitamin D(3). The aim of the study was to develop a rapid and simple HPLC method for the measurement of 25-hydroxyvitamin D(3) in human plasma. A method for the measurement of 25-hydroxyvitamin D(3) using HPLC with UV detection and investigation into the extraction techniques with regard to stability and recovery are described. For the separation, RP column LiChroCart 125-4, Purospher RP-18e, 5 microm, was used. The mixture of methanol and deionized water (95:5 v/v) was used as mobile phase. The analytical performance of this method is satisfactory: the intra- and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma samples were between 92.0-103.2%. The LOD was 10 nmol/L. The preliminary reference range of 25-hydroxyvitamin D(3) in a group of blood donors is 62 +/- 26 nmol/L.