The Estimation of Vitamin D2, Vitamin D3, and Their 25-Hydroxy Metabolites in Animal Tissues and Foods (Hplc)
Travis, Benjamin David
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https://hdl.handle.net/2142/70096
Description
Title
The Estimation of Vitamin D2, Vitamin D3, and Their 25-Hydroxy Metabolites in Animal Tissues and Foods (Hplc)
Author(s)
Travis, Benjamin David
Issue Date
1987
Department of Study
Food Science
Discipline
Food Science
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Agriculture, Food Science and Technology
Abstract
Nutritional and compositional studies of vitamin D have been hampered by the lack of a quick, sensitive, direct method of measuring vitamin D in tissues and foods. Past studies have relied on biological assays which are time consuming, expensive, equivocate total antirachitic activity with vitamin D levels, and are subjective. Recent physicochemical methods have been time consuming or insensitive. A fast, sensitive, objective, and direct assay is desirable.
A method was developed for the determination of vitamins D(,2) and D(,3) and their 25-hydroxy metabolites that sould be applicable to a variety of tissues in foods. Tritiated vitamin D(,3) and 25-hydroxyvitamin D(,3) were used to monitor vitamin losses through the assay procedure. The first step used a saponification and extraction to free the vitamins D from their matrix and reduce the lipids. Most samples were saponified using the AOAC method, while a new procedure was developed for high fat samples. This utilized greater extraction volumes, a more polar extraction solvent, and three salt washes.
Three different types of chromatography were then used to enable quantitation. Mini-columns of phenyl sulfonic acid-bonded silica were argentated using 0.5 M silver nitrate and used to remove contaminants, and separate vitamin D from 25-hydroxyvitamin D. The vitamin D and 25-hydroxyvitamin D fractions were further purified by HPLC on cyanopropyl-bonded silica. Quantitation was achieved by reverse phase HPLC on two C18-bonded silica columns in series, and the measurement of peak areas following absorption at 254 nm. Peak areas were related to standard curves. Peak purity and identity was established, and monitored.
The method was reproducible and accurate for the estimation of vitamin D(,3) and 25-hydroxyvitamin D(,3), but not as accurate for determining vitamin D(,2) and 25-hydroxyvitamin D(,2) due to a lack of radioisotope standards of these compounds. The method was applied to a number of fortified and nonfortified foods and animal tissues, and to an examination of the effect of heat on the vitamin D content of milk. Most fortified foods were found to be moderately overfortified, while other results agreed with available literature values. Vitamin D in milk was found to be stable to heat treatment.
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