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

• Published in: Journal of separation science 2009-09, Vol.32 (17), p.2953-2957
• Main Authors: Kand'ar, Roman, Zakova, Pavla
• Format: Article
• Language: English
• Subjects: 25-hydroxyvitamin D3; Adult; Analysis; Biological and medical sciences; Calcifediol - blood; Chromatography, High Pressure Liquid - instrumentation; Chromatography, High Pressure Liquid - methods; Female; General pharmacology; HPLC with ultraviolet detection; Humans; Male; Medical sciences; Pharmacology. Drug treatments; Reference Values; Retinyl acetate and solid-phase extraction; Sensitivity and Specificity; Solid Phase Extraction - methods; Ultraviolet Rays
• ISSN: 1615-9306; 1615-9314
• DOI: 10.1002/jssc.200900213

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.