Analysis of Vitamin E in Vegetable Oil

Introduction

Vitamin E is a fat-soluble vitamin that is found in various foods, oils, and fats. Vitamin E is an antioxidant; therefore, it is helpful in preventing damage to the cells of the body. Besides, antioxidants may protect against serious illnesses such as heart disease and cancer. The vitamin also helps the body in making red blood cells and utilizing vitamin K. Individuals who are not able to absorb fat properly may suffer from vitamin E deficiency. The symptoms of vitamin E deficiency include vision problems, muscle weakness, loss of muscle mass, unsteady walking, and unusual eye movements (National Health Services, 2015).

Naturally occurring vitamin E is found in eight chemical forms including alpha, beta, gamma, as well as delta-tocopherol, and alpha, beta, gamma, and delta-tocotrienol with varying levels of activities. Alpha-tocopherol is the only form identified as meeting human requirements.

As an antioxidant, vitamin E protects cells from the destructive effects of free radicals, the molecules that contain an unshared electron (National Institutes of Health, 2016). Cells damaged by free radicals can contribute to the development of cancer and cardiovascular disease. Unshared electrons are usually very energetic and undergo rapid reactions with oxygen forming reactive oxygen species (ROS) (National Institutes of Health, 2016). Usually, the body forms reactive oxygen species endogenously when converting food to energy. The free radicals in the body result from environmental exposures including air pollution, cigarette smoke, and ultraviolet radiation that comes from the sun (National Institutes of Health, 2016). Reactive oxygen species form part of the signaling mechanisms among cells. Vitamin E is fat-soluble and stops the production of reactive oxygen species that are formed whenever fat is oxidized.

Besides its role as an antioxidant, vitamin E has an immune function in cell signaling and regulating gene expression as well as other metabolic processes. Notably, alpha-tocopherol inhibits protein kinase C activity (National Institutes of Health, 2016). This enzyme takes part in cell proliferation and differentiation that occurs in platelets, smooth muscle cells, and monocytes. Vitamin-E-full endothelial cells that line the inner surface of blood vessels are able to resist blood-cell components that stick to this surface (National Health Services, 2015). Importantly, vitamin E increases the expression of the enzymes, which suppresses the metabolism of arachidonic acid, therefore enhancing the release of prostacyclin from the epithelium to dilate blood vessels and inhibit the aggregation of platelets (National Institutes of Health, 2016).

Numerous foods act as sources of vitamin E, including seeds, nuts, and vegetable oils. These products are considered to be among the best sources of alpha-tocopherol, and significant amounts of the vitamin are also found in green leafy vegetables as well as fortified cereals. A further breakdown reveals that the important sources of alpha-tocopherol include wheat germ oil, sunflower seeds, almonds, hazelnuts, peanut butter, peanut, corn oil, spinach, broccoli, soybean oil, kiwi fruit, and mango (National Institutes of Health, 2016).

It is the liver that affects the level to which serum will be saturated with vitamin E (alpha-tocopherol) since the organ receives nutrients after intestinal absorption. Notably, the liver selectively re-secrets alpha-tocopherol only through the hepatic alpha-forms transfer protein while metabolizing and excreting other forms of vitamin E (National Institutes of Health, 2016). Consequently, the concentrations of other forms of vitamin E in the blood and cells are lower than those of tocopherol.

Long-term deficiency of vitamin E may result in kidney and liver problems. The digestive tract often requires fat to absorb vitamin E (National Health Services, 2015). Consequently, people having malabsorption disorders are likely to be more deficient of the vitamin than those without such disorders. Some of the deficiency symptoms are ataxia, peripheral neuropathy, retinopathy, skeletal myopathy, as well as impaired immune response.

Those with such conditions as cystic fibrosis, Crohn’s disease, and the inability to secrete bile from the liver into the digestive tract may suffer from chronic diarrhea or pass greasy stool. Therefore, at times, the individuals with such conditions may need the forms of vitamin E that are soluble in water, for instance, tocopheryl polyethylene succinate. However, individuals suffering from abetalipoproteinemia, which is associated with poor absorption of fat in diet, may require significant doses of supplemental vitamin E (National Institutes of Health, 2016). In sum, vitamin E supplementation may be preferred by people having digestive disorders such as chronic bowel disease, or in the case of individuals who have undergone gastrointestinal surgeries because their systems are less likely to absorb vitamins that are soluble in fat (National Health Services, 2015). The lack of vitamin E, which is secondary to abetalipoproteinemia, results in poor nerve impulse transmission, weakness of muscles, and retinal degeneration, a condition associated with blindness.

There have been claims regarding the potential of vitamin E in promoting health and treating as well as in preventing diseases. The strategies by which the vitamin might offer protection include acting as an antioxidant in anti-inflammatory processes, immune enhancement, and inhibiting platelet aggregation (National Institutes of Health, 2016).

Review of Literature on Vegetable Oil and Vitamin E

Vegetable oils remain the main sources of dietary vitamin E that decreases the risk of cancer and cardiovascular diseases. Cold-pressed vegetable oils are an excellent source of alpha-tocopherol in particular. Among the major sources of vegetable oils, there are seeds of sunflower, canola, soybean, and wheat germ. Similarly, aromatic almond, sesame, and apricot oils also have healthy vitamin E. According to Sarwar, Quadri, and Moghal (2013), oilseeds remain the leading supplier of superior quality and specialty vegetable oils to natural food, nutritional products, as well as premium snack food globally. The authors noted further that the important oil-producing crops include oat, cotton, corn, mustard, soybean, camelina, safflower, crambe, coconut, rapeseed, olives, and oil palm.

In most countries, oilseeds are produced for oil extraction. The content of oil varies from one crop to another, for instance, small grains of wheat contain only between 1 and 2% of oil, while it is 20% for soybean, and over 40% for canola and sunflower (Sarwar et al., 2013). The main sources of edible seed oils in the world include soybean, rapeseed, sunflower, peanut, and cotton. Indeed, Imoisi, Ilori, Agho, and Ekhator (2015) noted in their research that palm oil has the highest content of vitamin E among vegetable-oil-producing crops. The authors reported that palm oil usually contains between 500 and 800 parts per million of tocopherols, which act as antioxidants. Palm oil contains alpha, beta, and gamma forms. Tocotrienols found in vitamin E have anti-cancer and antioxidant activities (Imoisi et al., 2015). Therefore, such antioxidants as carotenes and tocotrienols are added to cosmetics and foods because of the perceived health benefits that they confer. The antioxidant property of gamma-tocotrienol can be helpful in preventing increased rates of blood pressure by reducing lipid peroxides while increasing antioxidant status. Besides, increased melanoma can be reduced using delta levels of tocotrienols (Imoisi et al., 2015).

In another research, Ejoh and Ketiku (2013) sought to determine the levels of vitamin E in traditionally processed and raw melon seeds and groundnut products. Among the products included in the study, there were roasted groundnut, groundnut oil, melon seed oil, and roasted melon seed. The outcomes of the study revealed that apart from the stated high protein and energy content, groundnut and melon seed are useful sources of vitamin E, particularly tocopherol from the oils of these products. Significantly, the investigators noted that due to high oil content and vitamin E, vegetable oils still remain among the major sources of vitamin E.

De Almeida, Sobrinho, Manzi, Lima, Endo, V, and Zeola (2014) conducted a study to assess the impacts of supplementation with sunflower seeds and vitamin E for fattening lambs on the cholesterol, chemical composition, level of vitamin E, fatty acid profile, as well as oxidation of meat from sheep. This study revealed that sunflower seed supplementation was able to increase the levels of different acids including linoleic acid, conjugated linoleic acid, and vaccenic acid (de Almeida et al., 2014). Importantly, vitamin E supplementation increased the concentration of this vitamin in the meat but decreased lipid oxidation. Therefore, the authors concluded that as the demand for healthy foods increases including sunflower seeds as well as vitamin E in the diets, it is a worthwhile consideration to be made (de Almeida et al., 2014).

Maarasyid, Muhamad, and Supriyanto (2011) also found out that vitamin E, which is essential for the human body, is found naturally in vegetable fats and oil as well as from their derivatives. Tocopherols and tocotrienols, which are the isomers of vitamin E, can be extracted using different methods. In their study, Maarasyid et al. (2011) identified several methods of extraction including solvent-based extraction, adsorption, chemical modification, enzymatic process, membrane technology, micro-wave assisted extraction, and molecular distillation. The other methods of extracting tocols from vegetable oils include high-performance liquid chromatography (HPLC) and pressurized liquid extraction (Lampi, 2011; Silva et al., 2011).

In their study, Pantsi, Bester, Esterhyse, and Aboua (2014) have identified different health benefits that people can derive from vegetables. First, the authors have noted that vegetable oils have monounsaturated fat in products such as canola oil, olive oil, and peanut oil. Monounsaturated fat plays an important role in the prevention of coronary heart disease. It is noteworthy that the main monounsaturated fat in the human diet is oleic acid. The intake of monounsaturated fat is said to provide a cardioprotective effect (Pantsi et al., 2014). The fat has a beneficial impact on serum lipid profile, thereby decreasing possible risks of cardiovascular disease. Red palm oil, which is a major source of vitamin E, is noted to have anti-arrhythmogenic effects that can minimize sudden death following ischemic incidents (Pantsi et al., 2014).

Extraction of Vitamin E from Vegetable Oil Using Different Methods

Tocotrienols and tocopherols (tocols) are lipid-soluble and amphipathic compounds that are oxidized with ease when exposed to heat, light, and alkaline conditions. Tocols have a ring in their polar chromanol as well as a chain on its hydrophobic carbon side attaching the ring through carbon-2 atom. Of note, tocopherols have saturated phytyl side chains as opposed to tocotrienols, which have isoprenyl side chains with three double bonds. They differ in their number as well as position taken by methyl groups within the ring. All tocols are 2R-stereoisomers, implying that the side chain is usually attached to the ring using the same stereochemistry. There are additional asymmetric centers that are found in the side chains at carbon four and eight, and they both are R-stereoisomers in natural isomers. Additionally, double bonds found in side chains at carbon number three and carbon number seven of tocotrienols have a trans configuration. This section offers a description of the various methods that are used for extracting vitamin E from vegetable oils.

Method 1: High-Performance Liquid Chromatography (HPLC) for Detecting Vitamin E from Vegetable Oils

There are several reverse-phase high-performance liquid chromatography (RP-HPLC) methods of determining tocopherols in oils. Unfortunately, the methods involve saponification, implying that multiple solvent extractions must be utilized, but this is disadvantageous because of the possibility of drying as well as because of many concentration steps. Since tocopherols tend to be slight and air-sensitive, the procedures that may require numerous manipulations are likely to cause a partial degradation of the antioxidants as well as significant quantification errors. Besides, alkaline conditions considerably decrease alpha-tocopherol. Consequently, sample preparation remains a major step of analysis (Pacheco et al., 2013). Even though direct analysis of diluting the oil in an organic matter has been performed, the reversed-phase against normal-phase strategy results in higher column stability, retention time reproducibility, shorter time of analysis, and quicker equilibrium. Furthermore, reversed-phase high-performance liquid chromatography solvent systems often help in the preservation of the environment compared with the normal high-performance liquid chromatography solvent systems. However, it has been reported that the procedure that involves using reversed-phase supports the separation of all the acceptable tocopherols. During the process, vegetable oil can be diluted in hexane before injection to an immiscible as well as incompatible mobile phase that allows shorter column life.

Once the samples have been prepared, the next course is to analyze the vegetable oil used for vitamin E extraction. Chromatographic analysis entails injection with an appropriate volume. Also, the mobile phase may be water- and ethanol-based, while elution is done at a lower flowrate. Furthermore, the analytical column would require a temperature of about 450C. It is always important to analyze working standard solutions with the samples to determine the compounds that are in these samples. Importantly, peak-ratio areas can be identified and used in calculations following a standard method. Detection is performed at an appropriate wavelength such as 290 nm, and each run has to be timed well (Varzakas & Kiokas, 2016).

There are a number of quality assurance strategies that need to be in place. Firstly, the analyst needs to conduct an initial demonstration in order to gauge the ability to achieve acceptable accuracy as well as precision with the method. Secondly, the analyses of spiked samples are necessary to determine the accuracy of the method. Thirdly, the analyses of duplicate samples are necessary to determine the precision of the method. Moreover, there is a need to conduct an analysis of a blank to ensure that the set-up is free from contamination. An ongoing calibration is also necessary for verification as well as analysis of the precision in order to ensure that the system of analysis is under control. Finally, it is important to have a record that defines the quality of data that is generated. Assessment of accuracy has to be done regularly (Varzakas & Kiokas, 2016).

Method 2: Using Solvent Extraction for Detecting Vitamin E from Vegetable Oils

The other method of extracting vitamin E from vegetable oils is solvent extraction. In fact, solvent-based extraction has always been the conventional method of extracting the natural product. In this method, organic solvents are used including hexane, chloroform, as well as short-chain alcohols. In industry, the short-chain alcohols that are often used include isopropanol and ethanol. The use of organic solvents brings a similarity between this method and HPLC. In fact, hexane and ethanol are used in both methods.

In the food industry, the use of short-chain alcohol is preferred over chloroform and hexane in solvent extraction because of the potential health hazards. Besides, ethanol and isopropanol extract more non-glyceride than hexane due to greater polarity. Similarly, HPLC and solvent extraction methods entail laborious procedures that usually require enhanced purity and higher costs. Besides, these two methods have procedures that require a long time. Nonetheless, solvent extraction can extract up to 83 percent of vitamin E from such products as soybean (Maarasyid et al., 2011).

Solvent extraction is a simple procedure of extracting tocotrienols and tocopherols from tissues and oilseeds. In this method, the extract is used either directly or it can be dissolved in the mobile phase followed by filtration. Hexane and a mixture of hexane and ethanol have been used to extract tocols from food samples, while a mixture of chloroform and methanol have been used to extract the vitamin from the seeds of pumpkin, and methanol from the grains of cereals. Ethanol treatment before solvent extraction helps in releasing tocols from the proteins that are in the wet tissues. Just as in HPLC, vortexing may be used to improve extraction. Solvent extractions are performed under mild conditions, and antioxidants have been added to them to protect tocols from being degraded at the time of extraction or during storage (Maarasyid et al., 2011).

Over the years, the solvent extraction method has been modified to other newer methods such as pressure liquid extraction. On the one hand, this method often uses organic solvents that are supplied at high pressure and temperatures which are above boiling points of extraction to aid in the process of extracting the analyte from the sample mediums. High pressure increases the contract between the extracting fluids with the sample. On the other hand, high temperatures are used in breaking the bonds between the analyte and the matrix. Likewise, increased temperatures also increase the extraction efficiency because of the rate of the enhanced diffusion and solubility of the analyte in the solvent. Higher concentrations of vitamin E have been obtained using pressurized liquid extraction. As the high yields are achieved, the pressure liquid extraction reduces the number of organic solvents used in the extraction process (Maarasyid et al., 2011). However, it is critical to be careful with the sake of one’s own safety while handling the pressure liquid extraction system because of the high temperatures and pressure. Similarly, there is a high risk of human consumption of organic solvents. It is necessary to note that organic solvents are inflammable, partially toxic, can cause an explosion, and are not friendly to the environment.

Method 3: Chemical Modification as a Method of Extracting Vitamin E from Vegetable Oil

Chemical modification entails the conversion of a substance component into various properties to enable easier separation. Due to the fact that sterols, free fatty acids, and vitamin E all have similar properties, there have been proposals for reactions that would facilitate the process of separation. The transformation of a molecule often involves the alteration of physical properties based on the principles that are used in the process of separation. The latter is achieved using two strategies, namely esterification, and saponification (Maarasyid et al., 2011).

Saponification is used to free vitamin E from the sample matrices and convert esterified products of the vitamin into their free forms as well as to minimize a load of extracted materials into the organic phase. Unlike HPLC and solvent extractions which are time-consuming, chemical modification requires less time. However, vitamin E yields are higher in HPLC and solvent extraction methods than in chemical modification, where it is reported that the yields can be between 15 and 25 percent (Maarasyid et al., 2011). Saponification is able to trigger emulsion whenever a sample’s acid content is highly fatty or in a case where there is no proper control of the conditions, which is always a problem in the process. Just like in pressure liquid extraction, the chemical modification method is characterized by high-temperature conditions during saponification. The high temperatures and alkaline conditions may cause degradation of unsaturated vitamin E.

Chemical modification may also occur through esterification. Importantly, the latter.is used to convert large fatty acids and triglyceride molecules into smaller molecules of methyl esters. Fatty acid esters are often easy to extract than their fatty acids and triglycerides having a similar corresponding molecular weight of carbon numbers. Therefore, the concentration of vitamin E is enhanced. Combining esterification with superfluid extraction improves the solubility of fatty acids and increase the quantity of vitamin E extracted (Maarasyid et al., 2011). It is noted that the contents of vitamin E in the esterification of soybean is increased with the use of superficial fluid extraction. However, the major problem with this method is that even though it increases recovery of vitamin E, the irreversible reactions that take place during chemical modification may change the properties of the components of oil to the level of making them non-edible.

The Choice of High-Performance Liquid Chromatography (HPLC) as the Best Method for Extracting Vitamin E from Vegetable Oil

In high-performance liquid chromatography (HPLC), the extraction of vegetable oil begins by sample preparation, which is also considered to be a very time-consuming process in the analysis. Besides, it is this step that is often the source of error in the process. Therefore, a simple workup is necessary to avoid tocol losses. HPLC is the most widely used technique in analyzing tocols. Both normal-phase (NP) and reversed-phase (RP) chromatography are used in the process of analyzing tocols (Lampi, 2011). Notably, tocols are always stable under high-performance liquid chromatography conditions, they can be easily dissolved in an appropriate solvent, and the detection of tocols is easy because of the availability of several detectors which combine with HPLC. Usually, fluorescence detection and ultraviolet detection are the commonest methods used in food analysis. However, gas chromatography could also be useful in the analysis of tocols, but this would require that analytes are derived first, which might come with the risk of decomposition because of high temperatures. However, where there is a need for all eight tocols to be extracted. Afterward, the normal-phase high-performance liquid chromatography can be utilized. Reversed-phase high-performance liquid chromatography can be used where there are mixtures of vitamins that are soluble in fat and free, and esterified tocols need to be separated.

During the normal-phase high-performance liquid chromatography, the isomers are dissolved in fairly non-polar organic solvents, and separation takes place through adsorption that is also considered as the most effective strategy for separating isomers. The numbers of methyl groups that are found in the chromanol ring usually influence the polarity of tocols. The steric effects of the methyl groups, as well as the increased polarity of unsaturated side chains of tocotrienols, influence the polarity of tocols to a lesser extent. It is usually difficult to separate beta and gamma tocols because their ring structure consists of three methyl groups (Maarasyid et al., 2011).

Silica columns have been used in the base-line separation of all eight tocols in the normal phase of HPLC. Using hexane with a moderately strong modifier of 1, 4-dioxine during the mobile phase compared with a weaker modifier such as methyl tert-butyl ester is helpful in achieving better selectivity. In the stationary phase, the silica columns include Alltima SI 5U, Inertsil Silica, Genesis, Kromasil Phenomenex, Partisil PAC, LiChrosorb Si-60, Supelcosil LC-SI, and Taxil PFD (Lampi, 2011).

Normal-phase HPLC fluorescence detection chromatograms may produce additional peaks, which represent other compounds other than the usual tocols. A notable example of the compounds is the plastochromanol-8, which is a homolog of gamma-tocotrienol with eight unsaturated isoprene units. The compound is found in the side chain and has a relative molecular mass of 750. It is also found in indifferent vegetable oils and may be wrongly identified as beta tocopherol or gamma tocotrinol. Similarly, alpha tocomonoenol has been identified in pumpkin seeds and palm oil.

Reversed-phase high-performance liquid chromatography has also been used in analyzing tocols. The advantage of RP-HPLC over NP-HPLC is less harmful mobile phases such as ethanol or methanol which may be used; nonetheless, sample preparation would require that acyl lipids are removed to prevent the reversed-phase from getting contaminated. It is possible to separate alpha-tocopherol as well as all tocotrienols in crude palm oil using methanol and C-30 bonded silica gel as the mobile phase. A reversed-phase column with pentafluorophenyisilica column has been used to extract tocols from plant seeds as well as tocopherols from sunflower oil. It is noted that pentafluorophenylsilica column has a mobile phase of silica, and water may be used to extract all eight tocols. It has been easy to separate and quantify tocopherols fast and efficiently using the reversed-phase technique because of the invention of an ultra-high pressure liquid chromatography (Lampi, 2011).

Tocols usually absorb ultraviolet light at the wavelength between 290 and 300 nm; however, maximal absorbances are often small. Hence, ultraviolet absorption can be used for detecting and quantifying tocols in tocol-containing samples such as vegetable oils. Higher selectivity and better sensitivity of HPLC is achieved using fluorescence detection, which is always the preferred method when using biological samples. The wavelength for excitation is between 290 and 296 nm, while the emission wavelength is between 325 and 330 nm (Lampi, 2011). Nonetheless, the linearity of calibration curves of tocols using fluorescence detection needs to be evaluated for each instrument and method due to significant differences that occur in linearity ranges. Negative ion atmospheric pressure chemical ionization (APCI) has been identified as a better choice among positive and negative ion electrospray as well as APCI ionization in detecting and quantifying tocopherols.

Reliable tocopherol identification and quantification standards are available in commercial forms; therefore, it is fairly easy to identify and characterize them from chromatograms using ultraviolet and fluorescence detection. The literature on elution orders for different tocopherols are available.

Measurements for tocopherols’ calibration curves also need to be done regularly. Due to the absence of pure tocotrienols commercially, their quantification is often performed using tocopherols. Luckily, tocotrienols have been known to have fluorescent responses that are similar to respective tocopherols. Consequently, tocotrienols can be quantified based on tocopherol standards. Quantification of tocols is done using an external method due to difficulties in finding a suitable compound that cannot interfere with their analysis (Lampi, 2011).

Validating the method for analyzing tocols found in biological samples has to be done before the introduction of a new method as well as the type of the sample to be introduced. Besides, it is important to evaluate and present the performance of chromatographic systems used in the separation and detection of tocols (Maarasyid et al., 2011). Validation of sample preparation is often difficult because of the lack of certified materials for making references to natural tocols. A few certified reference materials that are available can be fortified with alpha-tocopherol or its esters. Nonetheless, the reference materials are of less value in homogenous or complex samples. Therefore, to validate a sample preparation, an indirect strategy must be used. The indirect method is accomplished by studying recoveries as well as precisions of tocols, which often describes the retention of the added analytes during the sample workup as well as HPLC analysis (Maarasyid et al., 2011).

Conclusion

It can be concluded that vegetable oils are important sources of vitamin E. The fats and oils derived from these sources are important because they are unsaturated, therefore preventing cardiovascular diseases. Vegetable oils have tocols that make up vitamin E; however, vitamin E sources have to be extracted from vegetable oils. Consequently, there have been a number of methods of extracting vitamin E from vegetable oils as indicated in this study, including solvent extraction, chemical modification, and the use of high-performance liquid chromatography. Even though solvent extraction was traditionally the method of choice for extraction, some newer methods which seem to be replacing it have been invented. Importantly, there are notable resemblances between HPLC and solvent extraction, but HPLC seems to be currently the method of choice because of the quality control mechanisms that have been proposed for using the method.

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