Elsevier

Clinica Chimica Acta

Volume 114, Issues 2–3, 10 August 1981, Pages 233-247
Clinica Chimica Acta

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

https://doi.org/10.1016/0009-8981(81)90396-XGet rights and content

Abstract

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.

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