VITAMINS

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Procedures for the qualitative and quantitative analysis of vitamin D and related compounds in foods, skin, plasma and other biological materials were established and the following results were obtained : (1) The formation of previtamin D_3, vitamin D_3 and tachysterol_3 in the skin of vitamin D-deficient rats irradiated with ultraviolet (UV) light was physicochemically confirmed by gas-liquid chromatography/mass spectrometry (GC-MS) and/or high-performance liquid chromatography (HPLC). The experimental results showed that the endogenous 7-dehydrocholesterol in the skin was photo-chemically and thermally isomerized to vitamin D_3 via previtamin D_3 in the same manner as in a test tube. (2) The major circulating metabolite of vitamin D in human plasma is 25-hydroxyvitamin D (25-OH-D) and its concentration directly reflects the repletion status of vitamin D nutrition. We assayed the concentrations of exogenous 25-OH-D_2 and endogenous 25-OH-D_3 in the plasma samples obtained from about 1,000 subjects including adults, mothers, infants and children, and very interesting results were obtained. (3) We established a HPLC method for micro-determination of vitamin D in foods, feeds and pharmaceuticals using reversed-phase and straight-phase columns. The proposed method was confirmed to be useful for many kinds of samples.

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To determine total vitamin C (AsA plus DAsA) in foodstuffs we developed a high-performance liquid chromatography method with precolumn derivatization using 2,4-dinitrophenylhydrazine (DNPH). A bishydrazone derivative of ascorbic acid was separated on a straight-phase system from other components such as saccharides and reductones which may react with DNPH. Moreover erythorbic acid were separated from ascorbic acid. Thus, the selectavility in analysis of total vitamin C was greatly improved. The separation was achieved on a Nucleosil (silica 3 /μm) column using a mixture of n-hexane ・ ethylacetate ・ acetic acid ・ n-propanol (4:3:0.1:0.2 v/v) as the mobile phase with・detection at 495 nm. Total vitamin C can be determined in concentrations as low as 0.1mg/100g.

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As a method for determination of ubiquinones (UQ) in biological materials, ubiquinols (UQH_2) were oxidized to UQ with ferric chloride as oxidation reagent, and total contents of ubiquinones (UQH_2 + UQ) were determined by high-performance liquid chromatography (HPLC). To human plasma and each homogenate of the liver, heart, kidney and brain of rat, ferric chloride ethanolic solution was added, and UQH_2 was oxidized to UQ. The UQ was extracted with n-hexane, which was then re-moved by evaporation. The residue was dissolved in isopropyl alcohol and subjectecl to HPLC. This method is simpler in procedure and permits to determine more samples at the same time than the conventional alkali saponification method.

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