Determination of 25‐hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation |
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Authors: | Roman Kand'ár Pavla Žáková |
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Affiliation: | Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic. Fax: +420‐466037068 |
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Abstract: | 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 D3. The aim of the study was to develop a rapid and simple HPLC method for the measurement of 25‐hydroxyvitamin D3 in human plasma. A method for the measurement of 25‐hydroxyvitamin D3 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 μm, 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 D3 in a group of blood donors is 62 ± 26 nmol/L. |
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Keywords: | HPLC with ultraviolet detection 25‐hydroxyvitamin D3 Retinyl acetate and solid‐phase extraction |
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