Quantitation of vitamin D and its metabolites and their plasma concentrations in five species of animals |
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Authors: | R L Horst E T Littledike J L Riley J L Napoli |
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Affiliation: | 1. National Animal Disease Center, Agricultural Research, Science and Education Administration, U. S. Department of Agriculture, Ames, Iowa 50010 USA;2. Department of Biochemistry, The University of Texas Health Science Center, Dallas, Texas 75235 USA |
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Abstract: | Chromatographic methods suitable for the resolution of 24,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D2, 25-hydroxyvitamin D3-26,23 lactone, and 25,26-dihydroxyvitamin D2 are described. These four metabolites comigrated in high-pressure liquid chromatography on silicic acid columns developed in 11:89 isopropanol:hexane. Adequate resolution was achieved by subjecting the four-metabolite complex to high-pressure liquid chromatography column developed in 2:98 isopropanol:methylene chloride. This additional chromatographic step, coupled with modifications of assay procedures previously described, allowed for the estimation of plasma concentrations of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D2, 24,25-dihydroxyvitamin D3, 25,26 dihydroxyvitamin D2, 25,26-dihydroxyvitamin D3, 25-hydroxyvitamin D3-26,23 lactone, and 1,25-dihydroxyvitamin D (1,25-dihydroxyvitamin D2 plus 1,25-dihydroxyvitamin D3). The samples automatically were introduced onto the high-pressure liquid chromatography columns with a Waters 710A “intelligent” processor. The metabolites were automatically collected with the aid of a programmable timer that advanced a fraction collector at predetermined intervals. The assays were used to determine the plasma vitamin D and vitamin D metabolite concentrations in five species of adult farm animals. |
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Keywords: | To whom correspondence should be addressed. |
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