Application of High Pressure Liquid Chromatography for Assay of Vitamin D Metabolites

A method for the quantitative estimation of dihydrotachysterol-2 is described using high performance liquid chromatography coupled to an UV monitor. Since radioactive labelled DHT2 is not available, recovery is based on the assumption that dihydrotachysterol-2 and cholecalciferol behave identically during chromatic procedures. Evidence is presented to support this assumption. In patients treated with DHT2 the serum concentrations of DHT2 rose in proportion to the administered dose.

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An improved competitive protein-binding assay for 25-hydroxy vitamin D

Aksnes

1978

Scandinavian Journal of Clinical and Laboratory Investigation

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A competitive protein binding assay for 25-hydroxy vitamin D in serum is described using serum from a pregnant woman as source of binding protein. Introduction of a polyvinyl alcohol to the reaction buffer enhanced the solubility of 25-hydroxy vitamin D in aqueous solutions and nearly eliminated loss of material to the walls of reaction tubes. Addition of a protein fraction obtained by gel filtration of human serum, shown to bind only vitamin D, enhanced the specificity and reproducibility of the assay without interfering with the binding properties of 25-hydroxy vitamin D. Serum samples were extracted by chloroform-methanol. Comparison of column chromatography on silicic acid and Sephadex LH-20 showed that both systems gave good separation of vitamin D and 25-hydroxy vitamin D and with a similar recovery of about 80%. The silicic acid columns were less time consuming, either to handle and cheaper in use, and was therefore preferred as the routine separation method. The assay has a detection limit of 0.4 nmol/l in serum, and an intra- and inter-assay coefficient of variation of 6.8 and 8.1%, respectively.

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Application of High Pressure Liquid Chromatography for Assay of Vitamin D Metabolites

Cited by 3 publications

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Recent Advances in the Quantitation of Vitamin D and Vitamin D Metabolites

Recent Advances in the Quantitation of Vitamin D and Vitamin D Metabolites

Serum Concentrations of Dihydrotachysterol‐2 in the Treatment of Osteoporosis and Hypoparathyroidism

An improved competitive protein-binding assay for 25-hydroxy vitamin D

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