Vitamin Tocopherols, formulas

Currently, high-performance liquid chromatography (HPLC) methods have been widely used in the analysis of tocopherols and tocotrienols in food and nutrition areas. Each form of tocopherol and tocotrienol can be separated and quantified individually using HPLC with either a UV or fluorescence detector. The interferences are largely reduced after separation by HPLC. Therefore, the sensitivity and specificity of HPLC methods are much higher than those obtained with the colorimetric, polarimetric, and GC methods. Also, sample preparation in the HPLC methods is simpler and more efficiently duplicated than in the older methods. Many HPLC methods for the quantification of tocopherols and tocotrienols in various foods and biological samples have been reported. Method number 992.03 of the AOAC International Official Methods of Analysis provides an HPLC method to determine vitamin E in milk-based infant formula. It could probably be said that HPLC methods have become dominant in the analysis of tocopherols and tocotrienols. Therefore, the analytical protocols for tocopherols and tocotrienols in this unit are focused on HPLC methods. Normal and reversed-phase HPLC methods are discussed in the separation and quantification of tocopherols and tocotrienols (see Basic Protocol). Sample... 

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. 

For the determination of supplemental vitamin E in infant formulas, Woollard and Blott (222) employed a radially compressed Radial-PAK cartridge. This enabled lipid material to be rapidly cleared by stepping up the mobile-phase flow rate from 2 ml/min to 10 ml/min after elution of the a-tocopheryl acetate. Fluorescence detection, using a filter-type fluorometer, allowed the indigenous a-tocopherol to be conveniently estimated, while UV absorbance detection was used to quantify the a-tocopheryl acetate. Supplemental retinyl acetate could be assayed simultaneously with either added or indigenous vitamin E using the appropriate detection mode. With the aid of a dual-monochromator spectrofluorometer, a-tocopheryl acetate and a-tocopherol could be determined simultaneously with wavelengths of 280 nm (excitation) and 335 nm (emission), but the increased selectivity eliminated detection of the vitamin A esters (233). 

AOAC official method 992.03. Vitamin E activity (all-rac-a-tocopherol) in milk-based infant formula. Liquid chromatographic method. In MP Bueno, ed. Official Methods of Analysis of AOAC International. 16th ed. Arlington, VA AOAC International, 1995, p. 50-4-50-5. 

With the exception of the diol 9, that was obtained from the corresponding aldehyde in up to 35% yield, most of the chiral diols mentioned above were isolated in yields of only 20-25%. The formation of the acyloin-type condensation products is in competition with the much more efficient reduction of the carbonyl carbon and saturation of the double bond of the unsaturated aldehydes that were used as substrates. We became interested in the mode of reduction of particular aldehydes such as 54-56 (Scheme 8) in a study of the total synthesis of natural a-tocopherol (vitamin E) (23). We expected to obtain chiral alcohols that would be useful for conversion into natural isoprenoids from the reduction of the a-double bond of the above aldehydes. Indeed, 54-56 afforded up to 75% yield of the saturated carbinols 57-59 by treatment with yeast. Whereas the ee of 57 and 58 was ca 85%-90%, that of 59 is 99%, as shown by NMR experiments on the (-)-MTPA derivative (24). The synthetic significance of carbinol 59 was based on the structural unit present in natural isoprenoids (see brackets in structural formulas). This protected synthon can be unmasked by ozonolysis, as indicated by the high yield conversion of 59 into (S)-(-) -3-methyl-y-butyrolactone 60 (Scheme 9). Product 59 is a bifunctional chiral intermediate which does not need protective manipulation in that... 

Any of the group of related substances (a-, P-, y, -d tocopherols) constitutes vitamin E which is a natural antioxidant. The a-form is the most potent having the following structural formula and properties [38] ... 

Erhard Fernholz was the first to demonstrate the relationship of vitamin E to duro-hydroquinone by isolating that substance from the pyrolysis products of a-tocopherol. In a masterly degradation study (1937,1938), he proposed that a-tocopherol could be represented by the structural formula we now know it to possess. In the untimely death of this young genius, science lost a chemist of outstanding ability. 

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Vitamin E activity is shown by several closely related substances, called tocopherols. The most potent is a-tocopherol, with formula... 

The others (3, y, 5, e, 0 have slightly different formulas. The tocopherols are yellow oils. Vitamin E is required for reproduction and lactation in rats, and probably is required for good health by man. One lU is equal to 1 mg of D,L-a-tocopheryl acetate (0.74 mg of the d acetate, 1.36 mg of the L acetate). Vitamin E is the principal fat-soluble antioxidant it protects the tissues, especially the unsaturated fatty acids, against damage by oxidation. Animals deficient in vitamin E develop muscular dystrophy and heart disease. 

Vitamin E in Foods, Feeds, and Infant Formulas (3). The determination of tocopherols and tocotrienols in foods and feeds serves a dual purpose, namely nutritional assessment and quality control. To establish the vitamin E nutritional value of foods such as cereals, oils, margarine, and vegetables as weU as infant formulas, both the naturally present tocopherols and the esters used for fortification should be quantitated. 

Tables 7 and 8, pertaining to the analysis of oils, fats, foods, and feeds, show a different picture in that the assessment of the nutritional value of these materials also requires the determination of tocopherols other than the a-form, as well as the tocotrienols. Accordingly, normal-phase HPLC, GC, and SEC occupy a more important position in this context than reversed-phase HPLC. In foods, notably infant formulas (67,125,139), in feeds (158), and in pharmaceutical preparations (44,92,103,108,164), a-tocopheryl acetate is also frequently included as an analyte, whereas in dairy products vitamin E may be determined concurrently with other fat-soluble vitamins, particularly A and D (72,109,161), sometimes P-carotene (161), and vitamin K (109).

Contrary to the predictions in 1989 (27), LC-MS has not experienced a breakthrough as an analytical technique in the vitamin E area. One research group reported two studies on the determination of liposoluble vitamins in foods and infant formulas by LC-particle beam MS (44,50). Caimi and Brenna coupled HPLC with combustion isotope ratio mass spectrometry for the analysis of mixtures of liposoluble vitamins, including a- and y-tocopherol (171). 

Vitamin E tocopherol) is related to the quinones with the isoprenoid side chain and can easily be oxidized to a quinone (tocoquinone). The formula and synthesis have been discussed in Chapt. XIV-9. Various homologs of tocopherol occur naturally they differ only in the number of methyl groups. Not enough is known about the biochemical mechanism of action of this Adtamin. Several authors believe that it is involved in the respiratory chain (cf. Chapt. X—4). 

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