Tocopherol assay

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 the 1985 edition of this book, TLC received the credit of occupying an important position among analytical methods in the vitamin E field. Before the advent of modern liquid chromatography and given the inability of the older GC systems to separate P- and y-tocopherol, assays aiming at the differentiation of positional isomers nearly exclusively relied on TLC. Furthermore, TLC was frequently used as a lipid prefractionation step for complex biological samples prior to the quantitation of the E vitamers by colorimetry or GC. However, due to the poor resolving... 

Hossu et al. (2009) presented spectrofluorimetric methods for the determination of fat-soluble vitamin E in multivitamin pharmaceutical products. In method I, M-hexane was used as solvent for a-tocopherol assay, while in method II, ethanol was used as a carrier between the aqueous solution and the n-hexane fluorescent solution of a-tocopherol. At 290/306 nm excitation and emission wavelengths, method I was linear for a-tocopherol over the range 1-100 p,g/mL R = 0.97687), having a limit of detection 1 p,g/mL and a limit of quantification 2 p,g/mL, and method II was linear over the range 2-50 p,g/mL R = 0.9709) with a limit of detection 0.68 J.g/mL and a limit of quantification 2.27 j,g/mL. 

Basic procedure (ACL kit) Mix 2400 pL of ACL reagent 1 (diluter) with 100 pL of ACL reagent 2 (buffer) and 25 pL of photosensitizer reagent (luminol based). Start measurement after brief vortexing. Assayed solution (lipid extract) is added before addition of photosensitizer reagent. Volume of ACL reagent 1 is reduced by the volume of assayed solution. Standard substance a-tocopherol or Trolox. Duration of measurement 1 min. Measured parameter integral (area under the kinetic curve of PCL). 

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) . ... 

TOCO see Thiol-olefin co-oxygenation Tocopherols, TEARS assay, 668 Torsion angles, hydroperoxides, 690 Tosylhydrazones, superoxide reactions, 1036 Total hydroperoxides see Peroxide value Total oxidative capacity, titration methods, 674 Total polar phenols, colorimetry, 664 Toxicity... 

Recently, Maruta et al. [112] have found that methanol extracts of roots of burdock show a significant antioxidant activity in an in vitro lipid peroxidation assay, and have isolated five caffeoylquinic acid derivatives (CQAs) from the roots of burdock (Arctium lappa L ), an edible plant in Japan. Antioxidant activities of DCQAs and related compounds have been investigated by measuring the hydroperoxidation of methyl linolate via radical chain reaction. This study indicates that in this particular system caffeic acid and CQAs are more effective than a-tocopherol. These results approximately agree with our findings [38], Additionally, CQAs as the principle antioxidative substance in burdock root have been characterized. 

The nutritional evaluation of the vitamin E-rich vegetable oils and the products made from them using a nonbiological assay necessitates the determination of the individual tocopherols and to-cotrienols, for these vitamers vary widely in biological activity. High-performance LC is ideally suited for this purpose, and the overall vitamin E value of such foods can be estimated by applying appropriate factors based on relative biological activities. For the analysis of those animal products known to contain predominantly a-tocopherol, only this vitamer need be determined. In vitamin E-fortified foods it is usually sufficient to determine either the added a-tocopheryl acetate or the total a-tocopherol. 

Amperometric detection in the oxidative mode produced on-column detection limits of 0.07, 4.3, and 0.19 ng for retinol, vitamin D3, and a-tocopherol, respectively (143). A limitation of amperometric detection in vitamin E assays is that it cannot measure a-tocopheryl acetate, owing to the absence of the oxidizable hydroxyl group. 

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). 

CK Chow, HH Draper, A Saari Csallany. Method for the assay of free and esterified tocopherols. Anal Biochem 32 81-90, 1969. 

Association of Vitamin Chemists. Vitamin E (tocopherols). Methods of Vitamin Assay. 3rd ed. New York Interscience, 1966, pp 363-402. 

Procedure Chromatograph 2 to 5 xL of the Assay Preparation, and record the chromatogram as described under Calibration. Measure the areas under the major peaks occurring at relative retention times of approximately 0.51 (a-tocopherol) and 1.00 (hexadecyl hexadecanoate), and record the values as ajj and ah respectively. Calculate the weight, in milligrams, of all-rac-a-Tocopherol in the sample by the formula... 

Assay Not less than 40.0% of total tocopherols, of which not less than 95.0% consists of RRR-a-tocopherol (C29H50O2). Acidity Passes test. 

Assay, Acidity, and Optical (Specific) Rotation Determine as directed in the monograph for all-rac-a-Tocopherol. Lead Determine as directed in the Flame Atomic Absorption Spectrophotometric Method under Lead Limit Test, Appendix IIIB, using a 10-g sample. 

Assay High-Alpha Type Not less than 50.0% of total tocopherols, of which not less than 50.0% consists of RRR-a-tocopherol (C29H50O2) and not less than 20.0% consists of d-3- plus D-y- (C28H48O2) plus D-S-tocopherols (C27H46O2). Low-Alpha Type Not less than 50.0% of total tocopherols, of which not less than 80.0% consists of d-(3- plus D-y- plus D-S-tocopherols. 

Note In the Assay and Optical (Specific) Rotation tests, use low-actinic glassware for all solutions containing tocopherols. 

Optical (Specific) Rotation Transfer an accurately weighed amount of sample, equivalent to about 100 mg of total tocoph-erols, into a separator, and dissolve it in 50 mL of ether. Add 20 mL of a 10% solution of potassium ferricyanide in a 1 125 sodium hydroxide solution, and shake for 3 min. Wash the ether solution with four 50-mL portions of water, discard the washings, and dry over anhydrous sodium sulfate. Evaporate the dried ether solution on a water bath under reduced pressure or in an atmosphere of nitrogen until about 7 or 8 mL remains, and then complete the evaporation, removing the last traces of ether without the application of heat. Immediately dissolve the residue in 5.0 mL of isooctane, and determine the optical rotation. Calculate the optical rotation [see Optical (Specific) Rotation, Appendix HB], using as c the concentration expressed as the number of grams of total tocopherols, as determined in the Assay (above), in 100 mL of the solution. 

In the UVB study we also measured malondialdehyde, a marker of lipid peroxidation, but were unable to detect any increase in response to UV light, in contrast to other reports. There are a number of possible explanations. In our experiment, a-tocopherol levels remained high after irradiation, and it is known that lipid peroxidation does not begin in in vitro membrane systems until a-tocopherol is completely depleted [29], Several studies have shown a time lag of one to several hours after irradiation to occur before a measurable increase in cutaneous lipid peroxidation can be detected [26-28] since skin was processed immediately in our experiments, lipid peroxidation may not have reached detectable limits. Finally, the TBARS assay for malondialdehyde is notoriously fraught with artifact and has a relatively high background [30], and noise levels may simply have been too high to detect peroxidation. Results... 

Aguilar-Garcia, C. Gavino, G. Baragano-Mosqueda, M. Hevia, P. Gavino, V.C. 2007. Correlation of tocopherol, tocotrienol, y-oryzanol and total polyphenol content in rice hran with different antioxidant capacity assays. Food Chem. 102 1228-1232. Awad, A.B. Fink, C.S. 2000. Phytosterols as anticancer dietary components evidence and mechanism of action. J. Nutr. 130 2127-2130. 

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