Vitamins D2 and

Nomenclature. The Vitamin D compounds ate steroidal materials and thus ate named according to the lUPAC-IUB rules for nomenclature (1) (Table 1). Vitamin [520-91 -2] is a mixture of vitamin D2 and lumisterol. 

In 1981, the lUPAC-IUB Joint Commission on Biochemical Nomenclature proposed that there be a set of trivial names for the important vitamin D compounds, including calciol [67-97-0] for vitaminD, calcidiol [19356-17-3] ion 25-hydroxy-vitaminD, and calcitriol [32222-06-3] ion 1 a,25-dihydroxy-vitamin D. This nomenclature has met with varying degrees of acceptance, as has the proposal to use calcine [69662-75-5] (deoxy-vitamin D2) and ercalcine [68323-40-0] (deoxy-vitamin D ) to name the triene hydrocarbon stmcture for 9,10-j (9-cholesta-5,7,10(19)-ttiene and... 

Physical Properties. The physical properties of the provitamins and vitamins D2 and are Hsted ia Table 6. The values are Hsted for the pure substances. The D vitamins are fat-soluble and, as such, are hydrophobic. 

Physical Methods. Vitamins D2 and D exhibit uv absorption curves that have a maximum at 264 nm and an (absorbance) of 450—490 at 1% concentration (Table 8). The various isomers of vitamin D exhibit characteristically different uv absorption curves. Mixtures of the isomers are difficult to distinguish. However, when chromatographicaHy separated by hplc, the peaks can be identified by stop-flow techniques based on uv absorption scanning or by photodiodearray spectroscopy. The combination of elution time and characteristic uv absorption curves can be used to identify the isomers present in a sample of vitamin D. 

Infrared and nmr spectroscopy have been used to help distinguish between vitamins D2 and D (87—89). X-ray crystallographic techniques are used ... 

Vitamin D is available ia a variety of forms. Cod Hver oil and percomorph Hver oil historically were good sources of vitamin D. Recent cost iacreases of these materials have caused a decline ia their market position. Cod Hver oil sold for 0.40—0.45/L ia 1970 and as high as 1.45/L ia 1979 and 3.43/L ia 1996. The prices of the cod Hver oils and of vitamin D2 and vitamin D from 1955 to 1995 are shown ia Table 12. 

Vitamin D is often combined in varying amounts with calcium salts. A multiple vitamin is another good source of vitamin D. Most multivitamins contain 400 IU per tablet. Vitamin D is also available as a single entity. Doses above 2000 IU/day should be avoided owing to the risk of hypercalciuria and hypercalcemia. Ergocalciferol (vitamin D2) and... 

The active vitamins are produced by conversion of provitamins by ultraviolet light. Ergosterol, a yeast sterol, is converted to its active form, ergocalciferol (vitamin D2), and 7-dehydrocholesterol, which is found in many natural foods and is also synthesized in man, is converted to cholecalciferol (vitamin D3). Fish liver oils are virtually the only source of vitamin D3 in nature. The most active form of vitamin D3 is 1,25-dihydroxycholecalciferol and this is produced by the hydroxylation of cholecalciferol at position 25 in the liver and then at position 1 in the kidney. 

Note In recovery experiments, the spike levels were 150mg for vitamin C, 2-20mg for the other WSVs, 5.0mg of vitamin A acetate, 0.5mg of vitamin D2 and 25mg of vitamin E acetate. 

In solution, vitamin D (both D2 and D3) isomerizes to previtamin D and forms a temperature-dependent equilibrium mixture [520], which leads to quantification problems. Previtamin D is difficult to quantify because of interference from co-eluted contaminants. The reversibility of the isomerization is very slow, therefore the percentage of previtamin can be considered constant during the entire analysis. The quantification of the potential vitamin D can be performed using an external standard that has undergone saponification procedure as the sample [521]. Vitamin D2 and D3 can be used as an internal standard to quantify the other one. Indeed, the isomerization rates of vitamins D2 and D3 are virtually the same thereby the previtamin D/vitamin D ratio will be the same for both vitamers at any temperature. The isomerization problem can be resolved by... 

NP chromatography is unable to separate vitamin D2 from vitamin Dj. So it is usually used as semipreparative chromatography [527-531]. Instead, RP chromatography is able to resolve vitamin D2 and Dj, thus it is widely applied as aualy tical chromatography. Mattila et al. [532] describe a two dimensioual LC procedure. The sample is saponified and an NP semipreparative column is used before the quantification in a tandem column set (Zorbax ODS x Vydac 201 TP54 CIS). 

Vitamin D2 and D3 exhibit identical UV absorption spectra and they do not possess fluorescence. Electrochemical detection is limited and only few methods are applied in food analysis [530,533], MS detection has been applied achieving satisfactory detection limit (10 mol/mL) [534,535],... 

Figure 2.8 Ergocalciferol (vitamin D2) and the calculated absorption maximum...

Formation of 1,25-diOH D3 Vitamins D2 and D3 are not biologically active, but are converted in vivo to the active form of the D vitamin by two sequential hydroxylation reactions (Figure 28.23). The first hydroxylation occurs at the 25-position, and is catalyzed by a specific hydroxylase in the liver. The product of the reaction, 25-hydroxycholecalciferol (25-OH D3), is the predominant form of vitamin D in the plasma and the major storage form of the vitamin. 25-OH D3 is further hydroxylated at the one position by a specific 25-hydroxycholecalciferol 1 -hydroxylase found primarily in the kidney, resulting in the formation of 1,25-dihydroxycholecalciferol j (1,25-diOH D3). [Note This hydroxylase, as well as the iver 25-hydroxylase, employ cytochrome P450, molecular oxygen, and NADPH.]... 

However, it was not until 1924, when Steenbock and Hess showed that irradiation of certain foods generated protective activity against the disease, that vitamin D (calciferol) was recognized as a second lipid-soluble vitamin. Vitamin D is a family of compounds formed by the irradiation of A5/7-unsaturated sterols such as ergosterol and 7-dehydrocholesterol. The former yields ergocalciferol (vitamin D2) and the latter cholecalciferol (vitamin D3). 

Members of the vitamin D family are extremely difficult to isolate and identify in pure form from any source. Fish liver oils are rich sources, and vitamins D2 and D3 have been isolated from them. Most ordinary foods are such poor sources in terms of amounts present, that the presence of D vitamins in them has not been demonstrated. Sterols that can be converted into some form of vitamin D by ultraviolet light are, however, widespread, and it may be inferred tlial D vitamins are often present even when their presence has never been demonstrated. 

Vitamin D3 (C27H440, MW = 384.62) and vitamin D2 (C28H440), MW = 396.63) differ structurally only in the C-17 side chain, which in vitamin D2 has a double bond and an additional methyl group (Fig. 4). Both compounds occur naturally with 5,6 double bond in the cis configuration. The biological potencies of vitamins D2 and D3 in humans are essentially equal. 

In solution, vitamins D2 and D3 exhibit reversible thermal isomerization to their corresponding previtamins, forming an equilibrium mixture. Equations and calculations have been published to determine the ratio of previtamin D to vitamin D as a function of temperature and reaction time (39). When equilibrated at 20°C, the ratio of previtamin D to vitamin D is 7 93. The isomerization rates of vitamins D2 and D3 are virtually equal (40) and are not affected by solvent, light, or catalysis (41). 

Solid-phase extraction effectively separates vitamin D from its more polar 25-hydroxy metabolite. In the analysis of human milk (64), the dried lipid fraction of milk was dissolved in 35% dichloromethane in hexane and then applied to a preconditioned silica cartridge. The sample was fractionated using the following elution sequence 9 ml hexane (discard), 3 ml 7% ethyl acetate in hexane (discard), 15 ml 7% ethyl acetate in hexane (vitamins D2 and D3), 25 ml 15% ethyl acetate in hexane (discard), and 9.5 ml 3% 2-propanol in hexane (25-hydroxyvitamin D2 and 25-hydroxy vitamin D3). 

Vitamins D2 and D, exhibit identical UV absorption spectra, with Amax at 265 nm and an e value of 18,000 in ethanol or hexane. The e value is less than that predicted for a conjugated m-triene structure because the degree of conjugation is reduced by the C-19 methylene group being above the plane of the other two double bonds. Reported on-column detection limits range from 1 to 10 ng (68,124,125). 

Add vitamin D2 and 25(OH)D2 to homogenized sample as internal standards. Saponify (ambient), extract unsaponifiables with diethyl ether/petroleum ether, 1 1. 

Normal-phase HPLC, using either silica or polar-bonded stationary phases, separates, isocrati-cally, vitamin D2 or D3 from their respective previtamins and inactive isomers (207). Vitamin D (D2 + D3), 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 can be separated from one another and from other hydroxylated metabolites (215), but vitamins D2 and D3 cannot be resolved from one another. The inability to resolve vitamins D2 and D3 means that one vitamer cannot be used as an internal standard for the other. 

Normal-phase/reversed-phase chromatography is the ideal combination for semipreparative and quantitative separations in two-dimensional HPLC. Vitamins D2 and D3 coelute during the semipreparative stage, allowing a narrow retention window to be collected for analysis using internal standardization. By this means, Johnsson et al. (201) obtained a vitamin D3 detection limit of 0.1 yug/kg for milk and milk products. 

Indyk and Woollard (195) demonstrated that the removal of cholesterol from the un-saponifiable fraction of vitamin D-supplemented whole milk powder by methanolic precipitation and filtration was an adequate cleanup procedure, making semipreparative HPLC unnecessary. This simplified procedure was made possible by connecting two analytical columns in series. The tandem columns adequately separated vitamins D2 and D3 from one another and from vitamins A and E. The analysis of infant formulas (100) required cleanup by silica solid-phase extraction to remove the minor tocopherols and tocotrienols, which constituted potential sources of interference. 

Mattila et al. (205) described a two-dimensional HPLC procedure for determining vitamin D3 and 25-hydroxyvitamin D3 in meat and milk products. Samples were saponified in the presence of vitamin D2 and 25-hydroxyvitamin D2 as internal standards, and the extracted un-saponifiable matter was subjected to normal-phase semipreparative HPLC to obtain a fraction containing 25-hydroxyvitamin D2 + 25-hydroxyvitamin D3 and a fraction containing vitamin D2 + vitamin D3. The collected fractions were evaporated and purified by reversed-phase HPLC. Fractions were again collected, after which vitamin D3 was quantified by tandem-column reversed-phase HPLC and 25-hydroxyvitamin D3 by tandem-column normal-phase HPLC. Analytical chromatograms of a purified extract of chicken are shown in Fig. 12. 

JF Muniz, CT Wehr, HM Wehr. Reverse phase liquid chromatographic determination of vitamins D2 and D, in milk. J Assoc Off Anal Chem 65 791-797, 1982. 

G Jones, HF DeLuca. High-pressure liquid chromatography separation of the metabolites of vitamins D2 and D3 on small-particle silica columns. J Lipid Res 16 448-453, 1975. 

Radial-PAK cartridge containing Vitamins D2 and D, 195 either Resolve Cl8 or Nova-PAK Cl8 5 pm (two cartridges connected in series)... 

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