An improved method for routine determination of vitamin D and its hydroxylated metabolites in serum from children and adults

Clin Chim Acta. 1981 Aug 10;114(2-3):233-47. doi: 10.1016/0009-8981(81)90396-x.


A method for routine determination of vitamin D and its major metabolites 25-hydroxyvitamin D (25(OH)D), 24,25-dihydroxyvitamin D (24,25(OH)2D) and 1,25-dihydroxy-vitamin D (1,25(OH)2D) in serum samples from normal children and adults has been developed. Methodological improvements enable a rapid and accurate analysis of 25(OH)D and also the microscale screening of other metabolites present in large concentrations in serum. Vitamin D and its metabolites are extracted from serum samples using hexane/propan-2-ol, which allows a convenient separation of the water soluble and lipid soluble fractions from each other and also from proteins. Preparative silicic acid chromatography was used to separate vitamin D from its metabolites and also from the major portion of co-eluting lipid contaminants. An automated LC solvent delivery and sample introduction system was used to achieve the rapid separation of metabolites. Vitamin D was further purified using adsorption high-performance liquid chromatography and assayed using reverse phase high-performance liquid chromatography connected with UV detection. The 25(OH)D fraction from the preparative chromatography was measured by a competitive protein-binding assay along with 24,25(OH)2D, which was purified by reverse phase high-performance liquid chromatography along with 1,25(OH)2D. A diluted human serum from a pregnant woman (3rd trimester of pregnancy) was used as source of the binding protein for 25(OH)D and 24,25(OH)2D. 1,25(OH)2D was determined by a competitive protein-binding assay using a diluted cytoplasmic 1,25(OH)2D receptor protein isolated from the intestinal mucosa of rachitic chicks. Vitamin D and its metabolite levels were assayed in serum samples from normal children and adults.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 24,25-Dihydroxyvitamin D 3
  • Adolescent
  • Adult
  • Aged
  • Binding, Competitive
  • Calcifediol
  • Calcitriol / blood
  • Carrier Proteins
  • Child
  • Child, Preschool
  • Chromatography, High Pressure Liquid / methods
  • Dihydroxycholecalciferols / blood
  • Ergocalciferols / blood
  • Humans
  • Hydroxycholecalciferols / blood
  • Infant
  • Infant, Newborn
  • Middle Aged
  • Vitamin D / blood*
  • Vitamin D-Binding Protein


  • Carrier Proteins
  • Dihydroxycholecalciferols
  • Ergocalciferols
  • Hydroxycholecalciferols
  • Vitamin D-Binding Protein
  • Vitamin D
  • 24,25-Dihydroxyvitamin D 3
  • Calcitriol
  • Calcifediol