A,p,y and 5-Tocopherols

The term vitamin E describes a family of eight antioxidants, a-, P-, y-, and 5-tocopherol and a-, P-, y-, and 5-tocotrienol, which are synthesized only by plants. Tocopherols and tocotiienols are characterized by a substituted hydroxylated ring system (chromanol ring). However, tocotrienols have an unsaturated side chain, while tocopherols have a saturated side chain with three asymmetric carbon atoms (Figure 21.2). Out of eight possible tocopherol stereoisomers, only the AAA-stereoisomer occurs naturally. ... 

Tocopherols and tocotrienols comprise the group of eight chromanol homologs that possess vitamin E activity in the diet. They are natural monophenolic compounds with varying antioxidant activities. The a-, P-, y-, and 5-tocopherols are characterized by a saturated side chain consisting of three isoprenoid units, whereas their corresponding tocotrienols have double bonds at the 3, 7, and 11 position of the isoprenoid side chain (Figure 5.3). 

Kofler (1942, 1947) uses the fluorescence of a phenazine derivative, obtained by condensation of the tocopheryl-o-quinones with o-phenylene-diamine, to determine a-, p, y-, and 5-tocopherol. This method is specific, but time consuming and therefore unsuitable for routine analysis. 

Abidi and Mounts [338] studied a-, P-, y, and 5-tocopherol and 5,7-dimethyltocol retention on j5- and y-cyclodextrin columns (A = 298 nm, ex 345 nm, em) using both cyclohexane and hexane mobile phases modified with alcohols (ethanol, IPA, n-propyl alcohol, 1-butanol, and 2-methyl-2-propanol), ethers (dioxane, THE, diisopropyl ether, MrBE, or tetrahydropyran) or ethyl acetate. A k versus percent hexane and percent cyclohexane plot was shown for each modifier, Selected chromatograms and extensive tables of k and a values are presented. Most elutions were complete in less than 45 min. In general, peak shapes were excellent with the notable exception of when MtBE was used as the modifier. In this instance, very broad peaks were generated. Why this occurred for MrBE and not for diisopropyl ether is not explained, nor is it readily explainable. Also observed was a significant decrease in fluorescence intensity when ediyl acetate was the mobile phase modifier. The authors ascribed this result to the decreased solubility of the analytes in the solvent, since the effect was not observed with any other solvent system. Anotho-possibility is that ethyl acetate may effectively quench the fluorescence (it is the only carbonyl-containing solvent used in the study). 

Vitamin E is a collective term for tocopherols and tocotrienols. Their basic structural element is tocol or 2-methyl-2-(4, 8, 12 -trimethyltridecyl)-6-chromanol. The four naturally occurring tocopherols, a-, p-, y-, and 5-tocopherol (abbrevi-... 

Oilseed rape a-, P-, y-, and 5-Tocopherols Three modified sample preparation protocols used for extraction (i) evaporation of solvent after extraction without silylation, (ii) direct supernatant collection after overnight extraction with drying and silylation, and (iii) trimethylsilylation with BSTFE GC-FID 5Sil MS column, temperature from 180°C 280°C 100,150,200,250, and 300 pg/mL with r values of 0.995,0.995, 0.988, 0.994, and 0.995 forT-, a-, P, y-, and 8-tocopherols, respectively 0.35 pg/g sample Hussain etal. (2013)... 

Ergoniil, P. G. Koseo, 0. (2014). Changes in a-, P-, y- and 5-tocopherol contents of mostly consumed vegetable oils during refining process. CyTA J. Food, 12(2), 199-202. 

The all-rac forms of P-, y-, and 5-tocopherols can be synthesized using the same condensation reaction as used for i7//-n7iC-a-tocopherol. To synthesize i7//-n7iC-P-tocopherol, 2,5-dimethylhydroquinone instead of trimethyUiydroquinone is condensed with isophytol. For all-rac- - and 5-tocopherol, 2,3-dimethylhydroqiiinone and methyUiydroquinone are used, respectively. 

M. oleifera seed oil is high in the natural antioxidants - tocopherols (128) with homologues (a- P, y-, and 8-tocopherol) (42). Extraction methods influence the quantities of tocopherols extracted (2). a-Tocopherol is the primary vitamer with biological activity, while the other vitamers have been shown to have decreased activity, y-tocopherol has 10% of the activity of a-tocopherol, and 5-tocopherol has 1% of the activity of a-tocopherol (38,115). a-tocopherol content of M. oleifera leaves averages 90.0 mg/kg. However, a-, y- and 5-tocopherols are detected up to levels of 105.0, 39.5 and 77.6 mg/kg of oil, respectively as shown in Table VII (2,82). 

In the case of the tocopherols the three chiral centres result in the potential existence of eight stereoisomer in each of the a, p, y and 5 compounds while similarly in the case of the tocotrienols all four members have the E-configuration and each has a chiral centre at the 2-position. The eight... 

Vitamin E is a group of fat-soluble compounds including four tocopherols (designated as a, P, y, and 5) and four tocotrienols (designated as a, P, y, and 8). The food that we consume may contain different combinations and amounts of tocols. a-Tocopherol has become synonymous with vitamin E because... 

The retention behavior of retinol, retinal, ergocalciferol, cholecalciferol, a-, p-, y-, and (J-tocopherol, menadione, and phylloquinone was studied on a silica column (A = 254 nm or 292 nm) using a THF-modified hexane mobile phase [677]. A plot of versus percent THF in hexane (from 5% to 20%) is shown and is a good resource for method development work. THF provided superior selectivity for these solutes as compared with IPA. Conversely, IPA yielded sharper more symmetric peaks, especially for those solutes that have accessible hydroxyl functional groups. 

Three types of tocopherol are produced for sale (i) natural tocopherols isolated from soybean, sunflower or other vegetable oils which are mixtures of the a, p, Y and 5 compounds, (ii) natural mixtures which have been methylated to convert the P, y and 8 compounds to a tocopherol (the trimethyl compound) with higher vitamin E activity, and (iii) synthetic a-tocopherol made from trimethylhydroquinone and phytyl bromide. This is entirely the trimethyl compound but a mixture of eight stereoisomers. All three show antioxidant and vitamin E activity but only the last two can be sold as vitamin E. 

The combination of SEC with MS has been briefiy addressed in V.C.3. Mobile phases in SEC consist of CO2 and a polar modifier such as ethanol. Oils and fats are either extracted by SEE or dissolved in a nonpolar solvent prior to SEC. A thorough study dedicated to SEC of tocopherols is that of Yarita et al. (144). These investigators examined the retention behavior of a-, P-, y-, and 8-tocopherol on a silica ODS column eluted with mixtures of CO2 and methanol. At low modifier concentrations (0.5% of methanol), P- and y-tocopherol were baseline separated. Increasing the modifier content of the eluent improved the resolution of the other tocopherols but at the expense of that of the P and y pair. The four homologs elute according to the number of methyl groups, but the elution order of P- and y-tocopherol depends on the concentration of the modifier. 

Vitamin E has eight biologically active forms [1] four tocopherols (saturated isoprenoid side chain) and four tocotrienols (imsaturated iso-prenoid side chain), designed as a-, P-, y-, and 5-according to the number and position of methyl groups on the chromanol ring. Of these, a-tocopherol is the most important (plant sources) and active. 

Diaz et al. (2006) utilized fluorometric techniques and partial least squares (PLS-1) multivariate analysis for simultaneous determination of quaternary mixture of tocopher-ols (a-, P-, y-, and 5-T) in vegetable oils dissolved in hexane diethyl ether (70 30 v/v). In the proposed study, PLS-1 was applied to matrices made up of FL excitation and emission spectra (EEM) and with FL excitation, emission, and synchronous spectra (EESM) of tocopherols. When synthetic samples were analyzed, recoveries around 100% were obtained and detection limits were calculated using EEM and EESM. For the analysis of the oils, the samples, diluted in hexane, were cleaned in silica cartridges and tocopherols were eluted with hexaneidiethyl ether (90 10 v/v). The results were satisfactory for a-, P-, and y-tocopherol, but worse for 8-tocopherol. 

Gomas, P. (2015). Unique variability of tocopherol composition in various seed oils reeovered from by-products of apple industry Rapid and simple determination of all four homologues (a, p, y and 5) by RP-HPLC/FLD. Food Chem., 172, 129-134. 

The correlation between the TEARS assay and MDA dnring oxidation of edible oils may be complicated by the presence of tocopherols (e.g. Vitamin E, 21) . An evaluation was carried of MDA, determined by an independent method , and TEARS as indices for direct oxygen uptake of edible oils and unsatnrated fatty acids. The linear increase of MDA and TEARS with oxygen consumption of soybean oil, in a closed vessel at 170 °C, stops when the latter value reaches 500 p.molL, when both MDA and TEARS start to decrease on further O2 consumption. The same process carried out at 40 °C, using 2,2 -azobis(2,4-dimethylvaleronitrile) (171) as initiator, shows linearity up to 1500 p,molL O2 consumption . A similar behavior is observed for nnsatnrated fatty acids snch as oleic, linoleic and linolenic acids . On the other hand, depletion of Vitamin E (a-tocopherol, 21) and its analogs y- and 5-tocopherol (172, 173) present in the oil show a linear dependence on O2 consumption of the oil, np to 1800 p,molL . This points to the consumption of these antioxidants, and especially 21, as a good index for the O2 uptake in oils at high temperature. The determination of the tocopherols is carried ont by HPLC-FLD (Xex = 295 nm, Ah = 325 nm) . ... 

Tocopherols and tocotrienols, collectively known as tocols, are monophenolic and lipophilic compounds that are widely distributed in plant tissues (7). The main commercial source of natural tocopherols is the soybean oil. Tocotrienols, less common than tocopherols, are present in palm oil, rice bran oil, as well as cereals and legumes (11). Tocopherols and tocotrienols are classified into a-, (3-, y-, and 5-, depending on their chemical structures (Figure 7). In general, tocotrienols have a stronger antioxidant effect on lipid oxidation than tocopherols. The antioxidant activity of tocopherols is dependent on temperature and is in the order of 5- > y->P-> oc-tocopherol (7). Tocopherols (mixed natural concentrate) are a golden... 

The separation of a, / , y and 5-tocopherols on LH-20 in chloroform was reported [205], separation being related to structure of the to-copherols and independent of molecular size. The correlations between the differences in conformation and elution behaviour of 2, 5 - and 3, 5 -dinucleoside monophosphates on LH-20 has been studied [206]. Separation of the dinucleoside monophosphates was apparently related to conformation which affected their adsorption to the LH-20. The different degrees of interaction of biogenic amines, aromatic amino acids, and phenylphrine with Bio-Gel P-2 were used to separate these species [207]. 

Good sources of vitamin E are vegetable oils, such as com, soy, and peanut oil. Animal fats, such as butter and lard, contain lower amounts of the vitamin. The content of the most important form of vitamin E, a-tocopherol, in various foods is as follows. Com oil contains about 16 mg of a-tocopherol per 100 g simflower oil 50 mg/100 g wheat germ oil 120 mg/100 g and fish, eggs, and beef 0.5 to 2.0 mg/100 g. In plants, a-tocopherol resides in chloroplasts, while other forms of tocopherol (P-, y-, and 6-tocopherol) occur elsewhere in the plant cell. Tocotrienols, which also have vitamin E activity, are not foimd in the green parts of plants, but in the bran and germ of seeds. 

According to a suggestion by Scudi and Buhs (1942), tocopherols which are not methyl-substituted in ii-position could be coupled at this site with diazotized aromatic amines. Quaife (1944) found that y-tocopherol can l)e coupled within a pH range of 4.5-11 with diazotized p-nitroaniline to a red dye with an absorption maximum at 520 m , whereas tocopherol does not couple. These two tocopherols ( an thus be quantitatively determined by colorimetry in presence of each other. On the other hand, the a- and /3-fractions in a mixture of a-, fi-, and y-tocopherol cannot be measured individually. Weisler et al. (1947) coupled y- and 5-tocopherol with the more stable diazotized o-dianisidine. They found that the color intensity of the two coupling products was dependent on pH, and they based on this observation their method of determining y- and 5-toeopherol individually. Colorimetric measurements are carried out in the one case in soda alkaline and in the other in potassium hydroxide solution. 

Hara et al. [677] studied the effect of changing the mobile phase composition on the retention of 10 fat-soluble vitamins (e.g., Ini 5-retinol, retinal, ergocalciferol, cholecalciferol, menadione, phylloquinone). A silica column (2 —254nm or 292 nm) was used. The ethyl acetate level in hexane was systematically changed from 5% to 20% and the results were plotted in a Ink vs. percent ethyl acetate format. As expected, when the percent ethyl acetate increased, the overall retention of the analytes decreased. Interesting, however, was the fact that the changes in retention of both menadione and retinal were so different from the other analytes (e.g., a-, P-, y-, and -tocopherol) that reversals in retention order occurred. These data present an excellent basis from which to develop a new method for fat-soluble vitamins. 

Unesterified tocopherols are found in a variety of foods however, concentration and isomer distribution of tocopherols vary gready with source. Typically, meat, fish, and dairy contain <40 mg/100 g of total tocopherols. Almost all (>75%) of this is a-tocopherol for most sources in this group. The variation in the content of meat and dairy products can be related to the content of the food ingested by the animal. A strong seasonal variation can also be observed. Vegetable oils contain significant levels of y-, P-, and 5-tocopherol, along with a-tocopherol (Table 3). 

---