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

✍ Scribed by Roman Kand'ár; Pavla Žáková

• Publisher: John Wiley and Sons
• Year: 2009
• Tongue: English
• Weight: 243 KB
• Volume: 32
• Category: Article
• ISSN: 1615-9306
• DOI: 10.1002/jssc.200900213

✦ Synopsis

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 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 D~3~ in a group of blood donors is 62 ± 26 nmol/L.