Tocopherols quantitative determination

K Abe, Y Yuguchi, G Katsui. Quantitative determination of tocopherols by high-speed liquid chromatography. J Nutr Sci Vitaminol 21 183-188, 1975. 

Kadioglu, Y. Demirkaya, F. Demirkaya, A.K. 2009. Quantitative determination of underivatized alpha-tocopherol in cow milk, vitamin and multivitamin drugs by GC-FID. Chromatographia 70 665-670. 

Melchert HU, Pabel E. Quantitative determination of alpha-, beta-, gamma- and delta-tocopherols in human serum by high-performance liquid chromatography and gas chromatography-mass spectrometry as trimethylsilyl derivatives with a two-step sample preparation. J Chromatogr A 2000 896(l-2) 209-15. 

Goffman, F.D., Velasco, L., and Thies, W. 1999. Quantitative Determination of Tocopherols in Single Seeds of Rapeseed (Brassica napus L.). Fett/Lipid. 101 142-145. 

Muller-Mulot, W. (1976) Rapid method for quantitative determination of individual tocopherols in oils and fats. J. Am. Oil Chem. Soc., 53, 732-736. 

Tocopherol carries six methyl groups three on the hydroquinone ring and three on the side chain. The three chiral centers are all of R configuration (2-R, 4 -R, 8 -R). The most significant chiral center at C2 is as stable to oxidative cleavage as the remote centers at C4 and C8, but it disappears, of course, upon oxidation to the quinone (see Scheme 7.2.1). The UV spectrum shows a maximum at 284 nm (e = 30,000) in petrol ether and at 292 nm in ethanol. Quantitative determination in food occurs by extraction with ether, HPLC, and reduction of Fe(III) ions to Fe(II). The iron(II) concentration is then determined colorimet-rically with 2,2 -dipyridyl = 520 nm) or 4,7-diphenyl-1,10-phenanthrolin. Solutions of free tocopherol fluoresce in the UV( ), whereas neither tocopherol acetate nor the quinone shows any fluorescence (Lang, 1974 Isler and Bmbacker, 1982). This phenomenon is not understood. 

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. 

The color complex produced with ferric chloride-dipyridyl is equally sensitive (0.5 jag a-tocopherol detectable) and unspecific. The color reaction can be used, however, for quantitative determination of a-tocopherol in pharmaceutical forms and concentrates after localization of the spot in UV light and elution from the adsorbant (Bolliger, 1962). 

Estimation of true vitamin E in foods requires quantitative determination of all its components since they vary in their biological potency. This vitamin consists of four tocopherols (a, jS, y, and 6) and four tocotrienols (a, jS, y, and d), but the three major constituents responsible for vitamin E activity are the a-, jS-, and y-tocopherols. While these compounds are fluorescent, their esters must be reduced to free alcohols for total tocopherol assays. Total vitamin E can be directly obtained through fluorimetry, but the determination of individual components is carried out using LC with fluorimetric detection. This procedure has been used to determine the composition of vitamin E in seed oils from maize, olives, soya beans, sesame, safflower, and sunflower by measuring the content of all the four tocopherols plus a-tocotrienol. The simultaneous determination of tocopherols, carotenes, and retinol in cheese has been carried out using LC with two programmable detectors coimected in series, a spectrophotometer and a fluorimeter. Carotenes have been determined photometrically, and fluorimetric measurements have been obtained for tocopherol and retinol. 

In a nice study by Ye et al. [704] the comparative performance of narrow-bore and analytical silica columns on the sqiaration of a-, fi-, y-, and -tocopherols was determined. Temperature was held at 29°C and peaks were detected at A = 298 run. A 99.2/0.8 hexane/IPA mobile phase generated baseline resolution and elution in 14 min. Linear ranges of 0.33-1 S.Sng injected was established and quantitation limits (analyte dependent) were 14-51 pg injected and 72-307 pg injected for narrow-bore and analytical, respectively. 

Strohecker [97] has worked out a procedure for quantitative determination of ascorbyl palmitate in oils and fats he oxidises with 2,6-dichlorophenol-indophenol and treats the reaction product with 2,4-dinitrophenylhydrazine. The 2,4-dinitroosazone of ascorbic acid is formed under these conditions and may be easily separated from other compounds containing phenolic groups such as tocopherols. He uses a silica gel H layer and chloroform-ethyl acetate (50 + 50) for TLC. After development, the brick red zone of the osazone is scraped off and determined colorimetrically in solution m sulphuric acid. Down to 0.001% of ascorbyl palmitate in antioxidant mixtures and in oils and fats can be determined with this procedure. 

Fig. e. Quantitative determination of a-tocopherol in water-soluble vitamin A + E concentrate, after hydrolysis [8,130]. Solvent cyclohexane-ether (80 + 20). a-T zone located in UV (254 nm) in part B, scraped ofT, eluted and submitted to colour reaction. 1 a-T standard 2 extract of the hydrolysed sample solution A Eefer-ence chromatogram, sprayed with A SbCls-reagent ... 

Prieto et al. (1999) reported another spectrophotometric method for the quantitative determination of antioxidant capacity based on the reduction of Mo(VI) to Mo(V) by vitamin E in acidic conditions with incubation at 95°C for 90 min. The subsequent green phosphate/Mo(V) complex, after cooling at room temperature was monitored at 695 nm with a calibration range of 0.2-2 x 10 M (r = 0.997) and a detection limit of 0.135 pmol vitamin E. The method was applied for measurement of total antioxidant capacity of plant extracts and to determine vitamin E in a variety of grains and seeds, including corn and soybean. The recovery of vitamin E from seeds was determined by supplementing the samples with the different vitamin E isomers or a-tocopherol acetate as internal standard and applying both the proposed method and a standard HPEC assay (Huo et al., 1996) and yielded a recovery of 93%-97% tocopherols. 

Abe, K., Yuguchi, Y. Katsui, G. (1975). Quantitative determination of tocopherols by highspeed liquid chromatography. J. Nutr. Sci. Vitaminol, 21, 183-188. 

Robledo, S. N., Zachetti, V. G. L., Zon, M. A. Fernandez, H. (2013). Quantitative determination of tocopherols in edible vegetable oils using electrochemical ultra-microsensors combined with chemometric tools. Talanta, 116, 964-971. 

Two-dimensional NMR analysis (COSY and heteronu-clear correlation spectroscopy [HETCOR]) was performed to make the complete H and C NMR assignment for a-, (3-, 7-, and 8-tocopherol, as well as for the acetate and hemisuccinate of a-tocopherol. C NMR was found to be especially useful for distinguishing between the various tocopherols and between natural D-isomer and the DL-racemic mixture. High-resolution NMR allows for the quantitative determination of a mixture of the four diastereomeric pairs in racemic a-tocopherol. ° ... 

The selectivity of the trap towards hydroxyl radicals was demonstrated by several control experiments using different radicals, showing that the formation of the respective hydroxylation product, 5-hydroxy-6-0-zso-propyl-y-tocopherol (57), was caused exclusively by hydroxyl radicals, but not by hydroperoxyl, alkylperoxyl, alkoxyl, nitroxyl, or superoxide anion radicals. These radicals caused the formation of spin adducts from standard nitrone-and pyrroline-based spin traps, whereas a chemical change of spin trap 56 was only observed in the case of hydroxyl radicals. This result was independent of the use of monophasic, biphasic, or micellar reaction systems in all OH radical generating test systems, the trapping product 57 was found. For quantitation, compound 57 was extracted with petrol ether, separated by adsorption onto basic alumina and subsequently oxidized in a quantitative reaction to a-tocored, the deeply red-colored 5,6-tocopheryldione, which was subsequently determined by UV spectrophotometry (Scheme 23). 

Vegetable oil modification involves not only the FA qualitative and quantitative manipulations, but also the manipulation of components such as tocopherols and phytosterols. To fully characterize oils from genetically modified plants, these oils must be analyzed for their stereochemical stracture, which determines several oil properties and the suitability of the oil for various purposes. 

Some work has recently been completed in our laboratory which further explores the absorption characteristics of the tocopherols in the chick. Two aspects were of interest, namely, to determine the rate of absorption of a-tocopherol and a-tocopheryl acetate after a single dose at physiological levels and to determine the effect on the quantitative relationships of intake versus plasma and liver tocopherols on increasingly higher levels of vitamin E intake. 

The method described is identical to the lUPAC method 2.403 (Paquot, 1979b). The AOCS method Ce 3-74 (AOCS, 1978) describes a method for the gas chromatographic determination of tocopherols and sterols in soya sludges and residues. In this method, the sample is saponified and the extracted unsaponifi-able matter is reacted with butyric anhydride. The butyrate esters of the tocopherols and the sterols present in the unsaponifiable matter are subjected to gas chromatographic analysis and determined quantitatively. Relative retention times for the various tocopherol and sterol butyrates are given. 

Rapid UPLC method for the determination of retinol and a-, y-, and 5-tocopherols in foods was accomplished by Shim and his coworkers [100]. The method was validated in terms of precision, accuracy, and linearity. The separation was performed on a reversed-phase Cjg column with 2 4m particle size, 2 mm i.d., and 75 mm length, followed by fluorescence detection. The recovery of retinol was more than 84.5% in all cases, while the detection and quantitation limits of the UPLC analysis were 0.015 and 0.045 mg/kg, respectively. The precision values were less than... 

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. 

Reversed-Phase. Reversed-phase chromatography continues to form the backbone of most assays of tocopherols and, rarely, tocotrienols in biological materials. Its popular status in the vitamin E area has been rationalized in ni.A.2. When the methods for the simultaneous determination of tocopherols and retinoids/carotenoids are also taken into account, reversed-phase systems outnumber their normal-phase counterparts by a factor 2. A survey of reversed-phase systems for the separation and quantitation of tocopherols, tocopheryl esters, tocotrienols, and a-tocopherolquinone is presented in Table 2. Methods specifically... 

The applications surveyed in Tables 4 through 9 illustrate the general principles of vitamin E assays outlined in I.E. 1. Specifically, each matrix puts a different emphasis on the E vitamers to be determined. Thus, in serum/plasma a-tocoph-erol is clearly the main compound of interest. Accordingly, leversed-phase chromatography with UV detection is the indicated technique for this purpose. In addition, as part of the assessment of the antioxidant status of humans, tocopher-ols are determined concurrently with retinoids (retinol, retinyl palmitate) and carotenoids (particularly p-carotene). a-Tocopherol is also the principal target compound in erythrocytes and platelets but here, predictably, the quantitation of a-tocopherolquinone may also be meaningful as an indicator of oxidative stress (7). The need to assay this minor constituent in turn justifies coulometric detection. The analysis of tissues is complementary to that of plasma and red blood cells and mainly concerns the determination of a-tocopherol as well as retinoids, carotenoids, and ubiquinones (Table 6). 

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