Tocopherol and tocotrienol

In inner layers, changes are much the same as during boiling. Tocopherols and tocotrienols present in wheat and rye are partially destroyed during baking. In ordinary wheat bread, losses of a-tocopherol amount to about 25%, but in the case of rye bread, prepared by traditional technology, a loss of about 50% was reported (Piironen et al, 1987). Losses of natural antioxidants in coffee brews and tomato puree were also observed (Nicoli et al, 1997). 

Tocopherols and tocotrienols belong to the vitamin E family of compounds, which are potent antioxidants. The four isomers of tocotrienols (a-T3, P-T3, y-T3, 8-T3) are structurally related to their corresponding homologues of tocopherols (a-T, p-T, y-T, 8-T), but differ in their side-chain in that T3... 

The molecular target site of triketone herbicides is the enzyme -hydroxyphenylpyruvate dioxygenase (HPPD). Inhibition of this enzyme disrupts the biosynthesis of carotenoids and causes a bleaching (loss of chlorophyll) effect on the foliage similar to that observed with inhibitors ofphytoene desaturase (e.g. norflurazon). However, the mechanism of action of HPPD inhibitors is different. Inhibtion of HPPD stops the synthesis of homogen tisate (HGA), which is a key precursor of the 8 different tocochromanols (tocopherols and tocotrienols) and prenyl quinones. In the absence of prenylquinone plastoquinone, phytoene desaturase activity is interrupted. The bleaching of the green tissues ensues as if these compounds inhibited phytoene desaturase. 

The main problem in the vitamin E analysis is that it is easily oxidized, thereby an antioxidant, such as butyl hydroxy toluene (BHT) or ascorbic acid, is added to prevent degradation during the extraction step. The traditional method for extraction of tocopherols and tocotrienols in foods is solvent extraction (like soxhlet) and saponification with KOH [457,458]. Some authors have recently proved that saponification is not necessary [459-462], nevertheless, it has been widely applied until the present day. 

Determination of vitamin E isomers in seeds and nuts Determination of tocopherol and tocotrienol in rice bran Investigation of vitamin E content in olives Determination of tocopherol and tocotrienol in olive oils... 

Determination of tocopherols in vegetable oils Determination of tocopherols and tocotrienols in rice bran Determination of tocopherols in tomato... 

Vitamin E is a collective term for all of the tocopherols and tocotrienols. Of these, one of the most active is (-)-o-tocopherol 6. Lutz Tietze of the Universitiit Gottingen has reported (Angew. Chem. bit. Ed. 2005,44,257) that the Pd-catalyzed cascade cyclization of 4 to 5 proceeds with 96% . 

Basic Protocol HPLC Analysis of Tocopherols and Tocotrienols Dl.5.2... 

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... 

There are numerous papers that refer to the quantification of tocopherols and tocotrienols using HPLC methods. Normal-phase HPLC methods with a silica column as well as reversed-phase HPLC methods with a C18 column are commonly used. A silica normal-phase column is able to separate all eight tocopherols and tocotrienols in a typical chromatographic procedure. Because plant tissues possess most forms of tocopherol and tocotrienol, it is recommended that the normal-phase HPLC method be applied to food samples from plants. In the reversed-phase HPLC method, [3- and y-tocopherol and (3-and y-tocotrienol are not usually completely separated. This method can be used in animal tissues, which either lack or have reduced levels of [3- and y-tocopherol and (3- and y-tocotrienol. The resolution of the normal-phase HPLC method is higher than that of reversed-phase HPLC method however, the reversed-phase HPLC column is more long-lasting than the normal-phase HPLC column (see Critical Parameters and Troubleshooting). 

Prepare different concentrations of standard solution using mobile phase (generally, 0 to 10 fig/ml for a- and y-tocopherols and tocotrienols and 0 to 5 pg/ml for other tocopherols and tocotrienols). 

Compared to refined vegetable oils, the compositions of crude vegetable oils and oil and fat products are more complicated. These samples contain proteins, carbohydrates, and minerals that interfere with HPLC separation and reduce the lifetime of the HPLC column. These compounds need to be largely eliminated from the extract before HPLC analysis. Saponification and heating are used to weaken sample matrices to allow the solvent to fully access all tocopherols and tocotrienols of the sample. Liquid/liquid extraction is used to remove these polar compounds from the organic solvent layer that contains tocopherols and tocotrienols. The normal-phase HPLC method is usually used for crude vegetable oils and vegetable oil products reversed-phase HPLC can be used for animal fat products. 

As the levels of tocopherols and tocotrienols in meat samples are usually lower than in oil and fat samples, a larger sample size is needed in the sample preparation. The meat sample is homogenized to weaken the sample matrix. As in Basic Protocol 2, saponification, heating, and liquid/liquid extraction are used to increase the recovery and remove interference compounds. Satisfactory results can be achieved using a reversed-phase HPLC method. 

Cereals and nuts are blended to fine particles before sample preparation. Saponification, heating, and liquid/liquid extraction are employed in the sample preparation to weaken sample matrix and eliminate interference compounds. Most cereals and nuts contain various forms of tocopherol and tocotrienol. The normal-phase HPLC method is recommended for these types of samples. 

Tocopherols and tocotrienols are pale yellow and viscous oils at high concentration. They are readily soluble in nonpolar organic solvents, e.g., hexane and octane. These solvents are usually used to extract tocopherols... 

Column. Although the advantage of the normal-phase column is the high resolution in the separation of all tocopherols and tocotrienols, the silica packing material in a normal-phase column is very reactive to strong polar chemicals. Any high-polarity compounds in the sample extract and mobile phase will diminish the column performance and shorten the column lifetime. It is very important to ensure that the sample extract is free of water and metal ions, and it is also advisable to regenerate the column routinely. 

Detectors. Fluorescence and UV detectors are used in the HPLC analysis. The high sensitivity and specificity of fluorescence detection in tocopherols and tocotrienols make the fluorescence detector the first choice. The fluorescence detector is ten times more sensitive and has less background noise than the UV detector. Electrochemical detectors are also used in the analysis of tocopherols and tocotrienols (Murphy and Kehrer, 1987 Sanchez-Perez et al., 2000). As a high-polarity mobile phase is needed for the electrolytes when using an elec-... 

Saponification. Before solvent is added for extraction, saponification (alkaline hydrolysis) is a step used in most extractions of tocopherols and tocotrienols. It should be noted that acetate forms of tocopherols or tocotrienols in a sample are changed to free tocopherols and tocotrienols after saponification. This process breaks down the ester bonds of lipids and sample matrices as well. In most extraction procedures, a 60% to 80% (w/v) aqueous solution of KOH is used to perform the saponification. The volume of KOH required varies according to the amount of lipid contained in the sample. Also, ethanol is needed to stabilize the saponified solution and prevent the precipitation of soap material. Usually, the ratio of KOH, ethanol, and fat (in sample) during saponification is 3 (g) 15 (ml) 1 (g), respectively (Ball, 1988). The ratio may need to be adjusted based on the types of fats in the sample. Although ethanol concentration has no effect on the extraction of a-tocopherol by hexane, a concentration above 30% may cause lower recoveries of other tocopherols (Ueda and Igarashi, 1990). For most food samples, saponification for 30 min at70°C is sufficient. 

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