title: Determination of 25-hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation.
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 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.
authors.affiliation: Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic. roman.kandar@upce.cz
authors.forename: Roman
authors.initials: R
authors.lastname: Kand'ár
authors.forename: Pavla
authors.initials: P
authors.lastname: Záková
mesh_terms: D000328:Adult; D002112:Calcifediol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D012016:Reference Values; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods*; D014466:Ultraviolet Rays
chemical_list: D002112:Calcifediol