Tocopherols and tocotrienols in plants and their products: A review on methods of extraction, chromatographic separation, and detection

Food Research International 82 (2016) 59–70

Contents lists available at ScienceDirect

Food Research International journal homepage: www.elsevier.com/locate/foodres

Review

Tocopherols and tocotrienols in plants and their products: A review on methods of extraction, chromatographic separation, and detection Ramesh Kumar Saini ⁎, Young-Soo Keum ⁎ Department of Bioresource and Food Science, College of Life and Environmental Sciences, Konkuk University, Seoul 143-701, Republic of Korea

a r t i c l e

i n f o

Article history: Received 29 December 2015 Received in revised form 24 January 2016 Accepted 24 January 2016 Available online 26 January 2016 Keywords: Tocochromanols Tocols Ultrasonic-assisted extraction (UAE) Supercritical fluid chromatography (SFC) Supercritical fluid extraction (SCFE) Mass spectrometry

a b s t r a c t Vitamin E consists of four tocopherols (α-, β-, γ-, and δ-tocopherol) and four tocotrienols (α-, β-, γ-, and δtocotrienols), collectively referred to as tocochromanols or tocols. Tocols are well-known for potent antioxidant, anticancer, anti-inflammatory, immuno-stimulatory and nephroprotective properties. For human nutrition, diet is the major source of tocols (vitamin E) in the body. Thus, there is a need to analyze the different forms of tocols in the diet for the recommendations and to monitor the intake in the body accurately. Several methods have been developed for effective extraction, selective chromatographic separation and sensitive detection of tocols in food. Major advancements also have been made in the field of mass spectrometry for high throughput analysis of primary and secondary metabolites in fruits, vegetables, and grains. This review discusses the theoretical aspects and modern developments in methods of extraction, chromatographic separation, and detection of tocols in plants and their products. Additionally, future research challenges in this perspective are also identified. © 2016 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemistry, biosynthesis, biological activities, and RDA of tocols . . . . . Methods of extraction . . . . . . . . . . . . . . . . . . . . . . . 3.1. Solvent extraction . . . . . . . . . . . . . . . . . . . . . . 3.2. Ultrasonic-assisted extraction (UAE) . . . . . . . . . . . . . 3.3. Extraction with matrix solid-phase dispersion (MSPD) . . . . . 3.4. Supercritical fluid extraction (SCFE) . . . . . . . . . . . . . . 3.5. Pressurized liquid extraction (PLE) . . . . . . . . . . . . . . 4. Methods of chromatographic separation . . . . . . . . . . . . . . . 4.1. High-performance liquid chromatography (HPLC) . . . . . . . 4.2. Supercritical fluid chromatography (SFC) . . . . . . . . . . . 4.3. Nano-LC, capillary LC and capillary electrochromatography (CEC) 5. Methods of detection . . . . . . . . . . . . . . . . . . . . . . . . 6. Direct determination of tocols by FT-IR and SFS techniques . . . . . . 7. Other methods of separation and detection . . . . . . . . . . . . . . 8. Summary and future perspective . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

59 60 61 62 62 62 63 64 65 65 65 65 66 67 67 67 68 68 68

1. Introduction ⁎ Corresponding authors. E-mail addresses: [email protected] (R.K. Saini), [email protected] (Y.-S. Keum).

http://dx.doi.org/10.1016/j.foodres.2016.01.025 0963-9969/© 2016 Elsevier Ltd. All rights reserved.

In nature, vitamin E consists of four tocopherols (α-, β-, γ-, and δtocopherol) and four tocotrienols (α-, β-, γ-, and δ-tocotrienols), determined by the numbers and position of methyl groups (−CH3) present

60

R.K. Saini, Y.-S. Keum / Food Research International 82 (2016) 59–70

on the chromanol ring. The tocopherols and tocotrienols are collectively referred to as tocochromanols or tocols. Vitamin E is the major bioactive constituent of human diet and is well-known for its potent antioxidant and anticancer activities. In addition, numerous studies have demonstrated the potential health benefits, since the discovery of vitamin E in 1922, which includes, hypolipidemic (Minhajuddin, Beg, & Iqbal, 2005), antiatherogenic (Kirmizis & Chatzidimitriou, 2009), antihypertensive (Kizhakekuttu & Widlansky, 2010), allergic dermatitis suppressive (Tsuduki, Kuriyama, Nakagawa, & Miyazawa, 2013), nephroprotective (Siddiqui, Ahsan, Khan, & Siddiqui, 2013), neuroprotective (Rashid Khan, Ahsan, Siddiqui, & Siddiqui, 2014) and anti-inflammatory (Mocchegiani et al., 2014) activities. Tocopherols and tocotrienols are also used to increase the shelf life and the stability of foods (Kamal-Eldin & Budilarto, 2015; Seppanen, Song, & Csallany, 2010). The relative antioxidant activity of tocopherols and tocotrienols generally depends on the food system. α-Tocopherols have shown superior antioxidant activity than γ-tocopherols in oils and fats (Seppanen et al., 2010). Vitamin E is also receiving growing attention in cosmetic and clinical dermatology because of its photoprotection and antioxidant properties (Thiele, Hsieh, & Ekanayake-Mudiyanselage, 2005). Unlike any other antioxidant molecule; evolutionary, genetic and biochemical evidence suggest cell signaling as the prime activity of α-tocopherol in a cellular system (Azzi, 2007). α-Tocopherol plays a significant role in maintaining the integrity of long-chain polyunsaturated fatty acids in the cell membranes. Thus, bioactive lipids such as tocopherol are the important signaling molecules that change their levels differentially in response to the external environment, depending on the extent and type of the stimulations. This phenomenon is the key to cellular events that are governed by α-tocopherol and responded to by cells (Traber & Atkinson, 2007). With this mechanism only, αtocopherol plays a vital role in plant stress tolerance. It is assumed that increased level of α-tocopherol contributes to stress tolerance while decreased levels favor oxidative damage in plants (MunnéBosch, 2005). In plants, tocopherol also plays an important role as singlet oxygen scavenger in photosystem II (Kruk, Holländer-Czytko, Oettmeier, & Trebst, 2005). Several separation techniques have been developed for the analysis of tocopherols in food. These include gas chromatography (GC), normal phase- or reverse phase-high performance liquid chromatography (NP/ RP-HPLC), capillary electrochromatography (CEC), nano-liquid chromatography (Nano-LC), capillary liquid chromatography (CLC), supercritical fluid chromatography (SFC), capillary liquid chromatography (CLC) and thin layer chromatography (TLC). Also, Fourier transform-infrared spectroscopy (FT-IR) and synchronous fluorescence spectroscopy (SFS) techniques have been developed and applied for the direct determination of α-tocopherol in various oils. NP/RP-HPLC with UV–visible, fluorescent and mass spectrometric detection is routinely used for analysis of tocopherols (Lanina, Toledo, Sampels, Kamal-Eldin, & Jastrebova, 2007). With the introduction of new stationary phases, such as long-chain alkyl-bonded C30-silica and solid-core Penta fluorophenyl (PFP) column, separation of different tocopherols isomers in RP–HPLC has become easier. This review discusses the theoretical aspects and modern developments in methods of extraction and analysis of tocopherols and tocotrienols. Additionally, future research challenges in this perspective are also identified. 2. Chemistry, biosynthesis, biological activities, and RDA of tocols Tocochromanols or tocols (vitamin E) are the group of four tocopherols (α-, β-, γ-, and δ-tocopherol) and four tocotrienols (α-, β-, γ- and δ-tocotrienols), determined by the numbers and position of methyl groups (− CH3) present at the 5- and 7-positions on the chromanol ring. All these tocols have a 16-carbon phytyl side chain attached to chromanol ring, in which tocopherols are saturated, and tocotrienols have three double bonds. Tocols are produced at different levels by photosynthetic organisms. However, their chemistry and function are

originally studied in animals due to the vitamin E activity in the diet. The outline of biosynthesis, general structure and antioxidant activity of tocols are shown in Fig. 1. The polar head group (chromanol ring) of tocols are derived from homogentisic acid (HGA), biosynthesized from aromatic amino-acid metabolism (shikimate pathway), whereas the hydrocarbon tail is derived from geranylgeranyl diphosphate (GGDP), biosynthesized from methylerythritol phosphate (MEP) pathway. Tocols interact with polyunsaturated acyl groups of membrane lipids and protect from peroxidation by scavenging reactive oxygen species (ROS) (Szarka, Tomasskovics, & Bánhegyi, 2012). During scavenging reaction with ROS, tocopherols are converted to the corresponding quinone (DellaPenna & Pogson, 2006). The presence of other antioxidant such as ascorbic acid (vitamin C) is required to regenerate the tocols (Jiang, 2014) (Fig. 1). The content and composition of tocols vary immensely among plant tissues, with photosynthetic tissues (green leafy vegetables) accumulating low levels of total tocols and a high proportion of α-tocopherol (Saini, Prashanth, Shetty, & Giridhar, 2014), whereas seeds accumulate 10–20 times higher amount of total tocols, with large proportion of γtocopherol (DellaPenna & Pogson, 2006). The summarized data on tocopherol content in selected food crops (in Table 1) shows that αtocopherol is predominantly found in wheat germ, hazelnut, sunflower, almond, rice bran, grapeseed oil, whereas γ-tocopherol is the major proportion of vitamin E in peanut, corn, canola and soybean oil. Surprisingly, the significant high content of α-tocopherol (53.3 mg/100 g FW) is recorded in black chokeberry (Aronia melanocarpa) leaves collected from greenhouse-grown in vitro plants (Sivanesan, Saini, & Kim, 2016). Among the natural sources of tocopherols, wheat germ oil is one of the most abundant, containing 149.40 mg of tocopherol in 100 g wheat germ (Table 1). Soybean and sunflower seeds and raspberries are the rich sources of δ-tocopherol. Tocotrienols are much less prevalent then tocopherols (≈ 1%), found commonly in paprika and chili spices, oat bran (raw), and coconut oil as listed in USDA, ARS, National nutrient database for standard (http://ndb.nal.usda.gov/ndb/ search, Release 28, Accessed on 7th November 2015). Due to the extensive use of soybean and corn oil in food preparation, γ-tocopherol represents ≈ 60–70% of the total vitamin E, in typical U.S. diet, whereas, α-tocopherol accounts only 20–30% (Jiang, 2014). In food, tocols are coexisting with fatty acids. Interestingly, the majority of γ-tocopherol is mainly associated with polyunsaturated fatty acids (PUFA), especially with omega-6 fatty acids (Ω − 6 or n − 6) whereas α-tocopherols are associated with monounsaturated fatty acids (MUFA). Thus, collectively γ-tocopherol and Ω − 6 fatty acids plays a vital role in disease prevention (Jiang, 2014). The antioxidant, non-antioxidant and radical scavenging functions of tocols are well studied. α-Tocopherol functions as a fat soluble chainbreaking (hydrogen atom donor) antioxidant, intercepting the chain-carrying peroxyl radicals during lipid oxidation (ROO•) in cellular membranes and low-density lipoproteins (LDLs) (Higdon, 2015) (Fig. 1). LDLs play a significant role in cholesterol transportation from the liver to the tissues of the body. The oxidized LDLs are associated with the development of cardiovascular disease, due to lipid deposition in the arterial wall (Trpkovic et al., 2015). The antioxidant function of α-tocopherol is also important in cell-mediated immunity by strengthening the immune synapse between CD4+ T cells and antigen-presenting cells (APC), and enhanced production of interleukin-2 (IL-2), resulting in provoked T cell activation and proliferation (Molano & Meydani, 2012). In contrast with αtocopherols, γ-tocopherols are capable of trap electrophiles and reactive nitrogen species (RNS) during inflammation (Constantinou, Papas, & Constantinou, 2008). Recent studies have demonstrated that tocotrienols have potent antioxidant and anti-inflammatory properties that are superior to tocopherols in prevention and treatment against major chronic diseases (Jiang, 2014). The unsaturated side chain of tocotrienols allows an efficient penetration into tissues with saturated fatty layers. Tocotrienols possess potent

R.K. Saini, Y.-S. Keum / Food Research International 82 (2016) 59–70

61

Fig. 1. The outline of biosynthesis, general structure and antioxidant activity of tocols. The polar head group of tocols are derived from homogentisic acid (HGA), biosynthesized from aromatic amino-acid metabolism (shikimate pathway), whereas the hydrocarbon tail is derived from geranylgeranyl diphosphate (GGDP), biosynthesized from methylerythritol phosphate (MEP) pathway. Tocols are biosynthesized from GGDP and HGA with the methylation and cyclization reactions. All four tocopherols (α-, β-, γ-, and δ-tocopherol) and four tocotrienols (α-, β-, γ- and δ-tocotrienols), are determined by the numbers and position of methyl groups (−CH3) present at the 5- and 7-positions on the chromanol ring. The tocols interact with polyunsaturated acyl groups of membrane lipids and protect from peroxidation by scavenging reactive oxygen species (ROS). During scavenging reaction with ROS, tocopherols are converted to the corresponding quinone. The presence of other antioxidant such as ascorbic acid (vitamin C) is required to regenerate the tocols.

anti-cancer, anti-diabetic, anti-inflammatory, immuno-stimulatory and nephroprotective properties (Ahsan, Ahad, Iqbal, & Siddiqui, 2014; Constantinou et al., 2008). The RDA (recommended dietary allowance) of vitamin E for adolescent (14–18 years) and adult men and non-lactating women (19 years and older) is 15 mg (22.5 IU) per day. For lactating (breastfeeding) women, it is 19 mg (28.5 IU) per day (Monsen, 2000). However, more than 90% of individuals in the US do not meet the RDA for vitamin E from food sources alone, due to the consumption of soybean and corn oil, which contains only 20–30% of α-tocopherol. Therefore, daily multivitamin/mineral (MVM) supplement of 30 IU of synthetic vitamin E is recommended (Higdon, 2015). For human nutrition, diet is the major source of tocols (vitamin E) in the body. Thus, there is a need to analyze the different forms of tocols in the diet (food), for the recommendations and to monitor the intake in the body accurately. For tocopherol analysis, in the first step, tocols are released from the food matrix with suitable extraction methods. In the second step, different forms of tocols are separated with chromatographic

techniques, and finally, these various forms are identified and quantified with detection technologies. Till date, several methods and techniques have been developed for the extraction, separation, and detection of tocols in plants and their products. In coming sections, we have discussed some of these methods/techniques. 3. Methods of extraction Wide varieties of methods have been developed for the extraction of tocols from various food samples. The extraction methods include solvent extraction (direct solvent extraction, soxhlet extraction and saponification), maceration (MAC), extraction with matrix solid-phase dispersion (MSPD), pressurized liquid extraction (PLE), supercritical fluid extraction (SCFE), and ultrasonic-assisted extraction (UAE) (Ramos, 2012; Wang & Weller, 2006). There are certain advantages and disadvantages to each of the extraction method (Table 2). So, the choice of an extraction method for the release of tocols depends on the physical and chemical characteristics of the sample, and also on the available

62

R.K. Saini, Y.-S. Keum / Food Research International 82 (2016) 59–70

Table 1 Tocopherol content in selected plants and their products. Source: USDA, ARS, National Nutrient Database for Standard (Reference: http:// ndb.nal.usda.gov/ndb/search, Release 28, assessed on 7th November 2015). Crop/food Oil

Wheat germ Hazelnut Sunflower Almond Rice bran Grapeseed Peanut Corn and canola Olive Soybean Spices Chili powder Paprika Oregano, dried Basil, dried Parsley, dried Turmeric, powder Peppers, sweet, green, freeze-dried Cumin seed Peppers, hot chili, sun-dried Mustard seed, ground Cinnamon, ground Poppy seed Nuts Almonds Pine nuts, dried Peanuts, all types, raw Nuts, pistachio nuts, raw Walnuts, black, dried Pecans Vegetables Carrot, dehydrated Dandelion greens, raw Turnip greens, raw Coriander (cilantro) leaves, raw Taro, raw Chicory greens, raw Collards, raw Fruits Avocados, raw, Florida Sapote, mamey, raw Avocados, raw, California Kiwifruit, green, raw Cranberries, raw Seeds pumpkin and squash, dried Flaxseed Other Seaweed, spirulina, dried

α-Tocopherol γ-Tocopherol δ-Tocopherol – – – – – – 15.95 35.37 0.83 64.26 3.41 3.54 24.42 0.77 1.53 0.72 –

– – – – – – 1.37 1.28 – 21.30 0.00 0.25 0.92 0.00 0.00 0.00 –

– –

– –

5.07 3.32 1.77 25.63 9.33 8.33 2.86

19.82 10.44 8.82 0.64 11.15 – 20.41

0.81 0.26 0.23 0.07 0.00 – 0.80

2.08 1.40 5.45 3.44 2.86 2.50

28.78 24.44 – – – –

1.51 0.47 – – – –

2.38 2.26 2.26 2.66 2.11 1.97

– – – – – –

– – – – – –

1.46 1.32 2.18

– – 35.10

– – 0.44

3.31 5.00

19.95 –

0.35 –

149.40 47.20 41.08 39.20 32.30 28.80 15.69 14.82 14.35 8.18 38.14 29.10 18.23 10.70 8.96 4.43 4.00 3.33 3.14

Values are mg/100 of food.

resources and instruments. In coming sections, we have described different extraction methods. 3.1. Solvent extraction Solvent extraction is the most widely used method for the extraction of tocols from grains, oilseeds, and biological tissues, due to its lipidsoluble (hydrophobic) nature. Alkaline hydrolysis is often used during saponification to extract the tocols from plants samples. This helps in the disintegration of carbohydrates and proteins, which are often associated with tocols in plants. The samples are properly grounded and homogenized by vortexing, sonication, and ultrasound to improve the extractability from solid food matrix. Several type of solvents and solvent mixtures have been used for the extraction of tocols from plant derived samples. The developments in the solvents and solvent mixtures used for the extraction of tocols from various plant-derived samples are summarized in Table 3. Extraction with ethanol followed by hot

saponification is the widely used method for the extraction of tocols from grain samples, oil (Mitei, Ngila, Yeboah, Wessjohann, & Schmidt, 2009) and bakery products (Mignogna, Fratianni, Niro, & Panfili, 2015). This procedure was originally developed by Ueda and Igarashi (1985), and modified by Panfili, Fratianni, and Irano (2003, 2004), Panfili, Manzi, and Pizzoferrato (1994), and Shammugasamy, Ramakrishnan, Ghazali, and Muhammad (2013). Important steps used in this procedure are illustrated in Fig. 2. Hidalgo, Brandolini, Pompei, and Piscozzi (2006) tested four extraction methods including, water-saturated 1-butanol, hot saponification, methanol and room temperature saponification to extract the tocols of einkorn wheat (Triticum monococcum), and suggested to use hot saponification for higher extractability. Tocols from biological samples (plasma and milk) is extracted after deproteinization with ethanol (Chauveau-Duriot, Doreau, Nozière, & Graulet, 2010; Kadioglu, Demirkaya, & Demirkaya, 2009), to avoid the interference of proteins. Annunziata et al. (2012) compared the efficiency of three extraction methods and solvent for the extraction of tocols from Brassica rapa (subsp. sylvestris) leaves. Authors recorded significantly high amount of tocols in a sample extracted with methanol, compared to chilled acetone, and ethanol with saponification. All the extraction procedures are performed in the presence of chemical antioxidants (stabilization) under subdued light and inert environment to prevent the oxidation of tocols (Moreau & Lampi, 2012). Knecht, Sandfuchs, Kulling, and Bunzel (2015) emphasized the importance of sample stabilization for vitamin E analysis in raw vegetables. Even if the samples were instantly extracted following the homogenization step, the degradation of tocols occurs very rapidly. Thus, stabilization of tocols is required during homogenization and extraction steps. Tocols can be stabilized by freeze-drying of fresh fruits and vegetables prior to initial homogenization. During extraction, tocols can be stabilized by addition of antioxidants, such as ascorbic acid, butylated hydroxytoluene (BHT) and pyrogallol (Valdivielso et al., 2015). In extraction, saponification is used for removal of chlorophyll and interfering lipids from leafy specimens. These impurities interfere during detection of tocols by mass spectrometric (MS) techniques. However, these impurities do not interfere in the ultraviolet (UV) and fluorescence detection methods (Saini, Shetty, Prakash, & Giridhar, 2014). 3.2. Ultrasonic-assisted extraction (UAE) Ultrasound- or ultrasonic-assisted extraction (UAE) is used for extraction of oil, protein and bioactives, including carotenoids, polyphenols, and gingerol from various plant samples. In UAE, ultrasound is used to disrupt the sample matrix that helps in improved penetration of the solvent into the cell and enhances mass transfer. UAE is employed in numerous extraction methods in order to achieve high extraction efficiencies in short times at low temperatures, with a small amount of sample and extraction solvents (Vilkhu, Mawson, Simons, & Bates, 2008). These extractions includes β-carotene from carrot, polyphenols from red grape marc, black tea and apple, gingerol from ginger, oil from almonds, and protein from soybean seeds. RSM (response surface methodology) coupled with the CSD (central composite design) were used to optimize the ultrasonic-assisted extraction of α-tocopherol from the fronds of oil palm (Elaeis guineensis Jacq) (Ofori-Boateng & Lee, 2013). In this study, extraction conditions of 40 °C extraction temperature, 1:20 g/mL sample/solvent ratio (hexane), and 50 min extraction time was found the optimum. The authors also compared the extraction efficiency of commonly used methods, and found the highest recovery of tocopherols with UAE, compared to Soxhlet extraction, saponification, and maceration. 3.3. Extraction with matrix solid-phase dispersion (MSPD) In the extraction with matrix solid-phase dispersion (MSPD), a sample, and suitable dispersion sorbent is manually blended and transferred into a column for sequential elution with solvents. The MSPD method requires a smaller amount of solvents (≈ 95%), and time (≈ 90%)

R.K. Saini, Y.-S. Keum / Food Research International 82 (2016) 59–70

63

Table 2 Advantages and disadvantages of commonly used extraction methods of tocols. Extraction methods

Advantages

Disadvantages

Solvent extraction

• Most simple extraction method. • Solvent extraction with saponification helps in eliminating the impurities and reduces interferences during chromatographic analysis. • Useful in deacidification and refining of edible oils under mild conditions of temperature and pressure. • Inexpensive, simple, fast and efficient alternative to conventional techniques. • Simultaneous extraction of a vast number of samples at once in an ultrasonic bath. • Ultrasound helps in improved penetration of the solvent into the cell and enhances mass transfer. • Low operating temperature helps for the extraction of thermolabile compounds. • Required small amount of sample and solvents. • Low cost of extraction. • Offers simultaneous disruption and extraction of solid, semisolid, and viscous samples. • Useful in selective elution of a single compound. • Requires smaller amount of solvents (≈95%), and time (≈90%). • Offers selective extractions and fractionations as physicochemical characteristics including density, viscosity, diffusivity and dielectric constant of supercritical fluid (CO2) can be manipulated by changing the pressure and temperature. • Higher diffusivity of supercritical fluid permits rapid mass transfer, resulting in increased extraction efficiency. • CO2 is non-corrosive in the presence of water, non-toxic, non-flammable, and can be obtained at low cost. • Extraction prevents the thermal damage to labile compounds. • High temperature (above their boiling point), and high pressure for enhanced solubility and mass transfer. • Reduce the use of harmful organic solvents in extraction.

• Only lipid-soluble (hydrophobic) compounds can be extracted. • Required long extraction time with a lot of toxic solvents consumption.

Ultrasonic-assisted extraction (UAE)

Extraction with matrix solid-phase dispersion (MSPD)

Supercritical fluid extraction (SCFE)

Pressurized liquid extraction (PLE)

(Tsochatzis & Tzimou-Tsitouridou, 2014). MSPD is an efficient method for isolation of several classes of substances, such as pesticides, drugs, pollutants, and bioactive compounds from a wide variety of plant samples (Capriotti et al., 2015). In MSPD extraction of tocols from barley grains, a sample/eluent solvent (methanol) ratio of 1:50 and sample/ dispersion sorbent (alumina) ratios of 1:5 was found optimum (Tsochatzis & Tzimou-Tsitouridou, 2014). 3.4. Supercritical fluid extraction (SCFE) Supercritical (SC) fluid extraction (SCFE) is the widely used method for the extraction of lipids, flavors, and bioactive compounds, removal of alcohol from wine and beer, and encapsulation of liquids for engineering solid products (Brunner, 2005). SCFE use carbon dioxide (CO2) as the solvent that can be recovered without damaging the substrate and extract. The use of CO2 is also having several other advantages as it is non-corrosive in the presence of water, non-toxic, non-flammable, and can be obtained at low cost (Brunner, 2005). SC extraction with CO2 operate near critical temperature and pressure (Tc = 304 K, Pc = 7.4 MPa). SC-CO2 extraction prevents the thermal damage to labile compounds, as

• The efficiency of extraction depends on nature of the plant matrix. • Low experimental reproducibility due to lack of uniformity of the distribution of ultrasound energy. • Cooling of the sonication vessel is required due to the large amount of heat generation.

• Laborious and time-consuming.

• Higher cost of the equipment and blockage in the systems as a result of the presence of water in the sample.

• Not suitable for extraction of thermolabile compounds and are found in low concentrations.

it operates near-environmental temperature. Imsanguan et al. (2008) comparatively studied the efficiency of SC-CO2, solvent and soxhlet extraction methods to extract α-tocopherol from rice bran, and found the highest extractability of α-tocopherol with SC-CO2 extraction. In SCCO2 extraction method, 48 MPa pressure, and 55 °C temperature was found optimum for maximum extractability of α-tocopherol from rice bran. The author also observed that neither ethanol nor hexane can extract α-tocopherol at atmospheric pressure, however, under soxhlet extraction, hexane was found better than ethanol. Thus, the choice of extraction method and extraction solvent mostly depends on the type of sample. Although the SCFE is the efficient method for extraction of thermally labile bioactive compounds, however, it is generally not suitable at industrial scale, as it allows processing of materials only in batch mode, due to high-pressure operation. To overcome this problem, simulation of an SCFE-CO2, using pseudo-continuously operated SFE process is introduced by adding three-six extractors vessels, to extract the tocopherols from plant samples (Moraes, Zabot, & Meireles, 2015; Núñez, Gelmi, & del Valle, 2011). Moraes et al. (2015) also proposed the incorporation of SCFE with low-pressure solvent extraction (LPSE) using a

Table 3 Commonly used solvents and solvent mixtures used for the extraction of tocols from various plant-derived samples. Solvent

Sample

Reference

Ethanol Hexane Methanol:hexane (1:1) Isopropanol:chloroform (3:1) Ethanol:hexane (4:3) Methanol Acetonitrile Hexane:ethyl acetate (9:1) Methanol:dichloromethane (1:2) Hexane:isopropanol (3:2) Acetone

Cereal grains, oil, bakery products Legumes Mushrooms Oil Tomato Brassica leaves Barley Coffee brews Green leafy vegetables Lentil Wheat

Panfili et al. (2003) Kalogeropoulos et al. (2010) Heleno, Barros, Sousa, Martins, and Ferreira (2010) da Costa, Ballus, Teixeira-Filho, and Godoy (2010) Van Meulebroek, Vanhaecke, De Swaef, Steppe, and De Brabander (2012) Annunziata et al. (2012) Tsochatzis, Bladenopoulos, and Papageorgiou (2012) Górnaś, Siger, Polewski, Pugajeva, and Waśkiewicz (2013) Cruz and Casal (2013) Zhang et al. (2014) Ziegler, Schweiggert, and Carle (2015)

64

R.K. Saini, Y.-S. Keum / Food Research International 82 (2016) 59–70

Fig. 2. The outline of most commonly used extraction methods of tocols from plants and their products.

mixture of water and ethanol to remove Bixin, an apocarotenoid biological pigment extracted from the defatted annatto seeds. LPSE conditions of 60 °C temperature with solvent/feed ratio (S/F) of 8 g solvent/g annatto seeds for ethanol, and 50 °C temperature with S/F of 8 g solvent/g annatto for water was optimum for the highest extractability of Bixin. 3.5. Pressurized liquid extraction (PLE) In pressurized liquid extraction (PLE) technique, a mixture of extraction solvents and samples are subjected to a high temperature (above their boiling point), and high pressure for enhanced solubility and mass transfer. PLE is successfully utilized for the extraction of phenolic compounds, lignans, carotenoids, essential oils and other nutraceuticals from foods and herbal plants (Mustafa & Turner, 2011). PLE is an environmentally friendly (green chemistry) technique and helps to reduce the use of harmful organic solvents in extraction. PLE offers several advantages over other traditional extraction methods, such as the low

solvent volume, and reduced extraction times. However, PLE is not suitable for extraction of thermolabile compounds and are found in low concentrations. PLE has been used for the determination of tocopherols and tocotrienols in cereals. The use of methanol as extraction solvent at a temperature of 50 °C and a pressure of 110 bar, with a static time of 5 min were optimized in order to maximize the extraction efficiency (Bustamante-Rangel, Delgado-Zamarreño, Sánchez-Pérez, & CarabiasMartínez, 2007). PLE was also employed for the extraction of αtocopherols from Brazilian grape seeds of wine industry waste (Dos Santos Freitas, Jacques, Richter, da Silva, & Caramão, 2008), cereals (Delgado-Zamarreño, Bustamante-Rangel, Sierra-Manzano, VerdugoJara, & Carabias-Martínez, 2009), fruits and vegetables (Viñas, BravoBravo, López-García, Pastor-Belda, & Hernández-Córdoba, 2014). Viñas et al. (2014) used the PLE of tocopherols from spinach, corn, cranberry, pomegranate and mango–apple juice, and also optimized the temperature of 50 °C, 1600 psi (110.3 bar) pressure, 3 g sample amount, 5 min static time, and single extraction cycle for maximum yield of tocopherols.

R.K. Saini, Y.-S. Keum / Food Research International 82 (2016) 59–70

4. Methods of chromatographic separation After extraction with suitable techniques, tocopherols are separated with the help of chromatographic techniques. Similar chemical structure and polarity of γ- and β-tocopherol and tocotrienol, differing only in the position of methyl groups on chromanol structure, make difficulties in separating these forms. Several separation techniques (methods) have been developed for the analysis of tocopherols in food. These include gas chromatography (GC), high-performance liquid chromatography (HPLC), capillary electrochromatography (CEC), nano-liquid chromatography (Nano-LC), supercritical fluid chromatography (SFC), capillary liquid chromatography (CLC) and thin layer chromatography (TLC). Also, Fourier transform- infrared spectroscopy (FT-IR) and synchronous fluorescence spectroscopy (SFS) techniques have been developed and applied for the direct determination of tocols in food without separating the different forms. 4.1. High-performance liquid chromatography (HPLC) Normal-phase (NP-) and reversed-phase (RP-) high-performance liquid chromatography (HPLC) are commonly used for the separation of tocols from food samples. The selectivity of NP-HPLC towards the sequential elution of tocols from column arises due to the affinity between the hydroxyl group of chromanol ring and the silanol groups on the silica gel surface. The polarity of tocols decreases with increasing number of methyl substituents on the chromanol ring, and also the steric hindrances due to the position of methyl groups on chromanol ring has an effect of the selectivity, leading to an elution order tocopherol (T) and tocotrienol (T3) in the following trends: α-T → α-T3 → βT → β-T3 → γ-T → γ-T3 → δ-T → δ-T3. The tocotrienols are eluted after tocopherols because of their higher affinity for the stationary phase (silica) caused by their slightly higher polarity. With two methyl groups on chromanol ring, β and γ forms of tocols differ only in the position, for example β forms of tocols possess methyl groups on 5′ and 8′ position, whereas, γ forms of tocols possess methyl groups on 7′ and 8′ position (Fig. 1). Thus, the steric hindrances due to the position of methyl groups on chromanol ring, selective elution of β and γ forms are achieved. In RP-HPLC, the elution order is reversed, and this technique is preferred due to shorter analysis time, use of less harmful solvents, with higher reproducibility, compared to NP-HPLC. In RP-HPLC, The separation of tocols is achieved on the basis of the difference in the polarity and degree of saturation of the side chain (Stöggl, Huck, Scherz, Popp, & Bonn, 2001). Before the year 2000, NP-HPLC methods were commonly used due to the better selectivity for the separation of βand γ-tocopherols and tocotrienols. Moreover, NP-systems allows direct analysis of oils, since nonpolar mobile phase can elute these compounds. However, with recent developments in column stationary phase (SP), now it is possible to separate the β- and γ-tocopherols and tocotrienols using RP-HPLC. Grebenstein and Frank (2012) successfully achieved the baseline separation of all eight tocopherols and tocotrienols by RP-HPLC with a solid-core Penta fluorophenyl (PFP) column. This new PFP stationary phase is a solid core and a porous shell that allows reduced diffusion of the analytes through the particles, helps in the better mass transfer, and finally results in better resolution at a lower backpressure than conventional stationary phases. This method is widely followed for the separation of the tocols in butter (Górnaś, Siger, Czubinski, Dwiecki, Segliņa, & Nogala-Kalucka, 2014), fresh fruits and vegetables (Viñas et al., 2014), coffee beans (Górnaś, Siger, Pugajeva, Czubinski, Waśkiewicz, & Polewski, 2014), rice bran and rice bran oil (Shammugasamy, Ramakrishnan, Manan, & Muhammad, 2014), and cooked vegetables (Knecht et al., 2015). In the studies of Górnaś, Siger, Czubinski et al. (2014) increasing the proportion of water in mobile phase (methanol–water) found beneficial for separation of β/γ-tocopherols and tocotrienols with an increase in the retention time, and the highest resolution was recorded with 8% of water in the mobile phase. Similarly, a reduction of column oven

65

temperature from 40 to 10 °C resulted in an improved separation between homologs of β/γ-tocopherols and tocotrienols, with the increase in the analysis time by 4 min. In the investigation of Shammugasamy et al. (2014), the baseline separation of all eight tocol isomers and the γ-oryzanols was achieved within a runtime of 20 min, at a flow rate of 1.0 mL min−1 using a gradient elution of mobile phase consisting of methanol and water at column oven temperature of 30 °C. In the study of Knecht et al. (2015), chromatographic conditions for baseline separation of all eight tocol isomers were optimized on both, a PFP and a C30 column. On the PFP column, the elution order of tocopherol (T) and tocotrienol (T3) was observed in following trends: δ-T3 → βTe3 → γTe3 → αTe3 → δαTerved in foll whereas on the C30 column, γ-isomers eluted before δ-isomers, with the elution order of δT3 → γT-3 → βT-3 → αT-3 → δT-isomers elut. The better resolution in a shorter run time of 25 min and column oven temperature of 24 °C was achieved using the PFP column, compared to C30 column (45 min, 18 °C), making it the preferred method. RP-HPLC with a triacontyl (C30) stationary phase is also successfully used for baseline separation of α-, β-, γ-, and δ-tocopherols (Saha, Walia, Kundu, & Pathak, 2013). Authors also compared the different mobile phases and found that methanol-tertbutyl methyl ether (TBME) in the ratio of 95:5 (v/v), at a flow rate of 0.75 mL min− 1 is best for the baseline separation of tocopherols, compared to methanol, methanol–acetonitrile–dichloromethane (50:44:6, v/v) and acetonitrile–tetrahydrofuran–water (70:20:10, v/v). 4.2. Supercritical fluid chromatography (SFC) In SFC, supercritical CO2 are mixed with a modifier, such as alcohol or acetonitrile, for eluting analyze from a column. Due to of low viscosity and high diffusivity of CO2 , analysis time can be significantly reduced. Moreover, SFC can be performed in the large varieties of stationary phases. There are several reports on analysis of tocols by SFC. Recently, Méjean, Brunelle, and Touboul (2015) reported the quantification of tocopherols and tocotrienols in soybean oil by SFC coupled to the atmospheric pressure photoionization (APPI) mass spectrometer. The authors also optimized the different analytical conditions, including the stationary phase (NH2), modifiers (ethanol), flow rate (1.5 ml min− 1), and the oven temperature (30 °C), for the finest separation of tocols in the possibly shortened time. In advancements of SFC, ultra-performance convergence chromatography (UPC2 ) is developed by integrating SCF and UFLC (ultra-fast liquid chromatography) techniques, and utilized for a rapid, reliable, and cost-effective separation and quantification tocopherols (Gee, Liew, Thong, & Gay, 2016; Gong, Qi, Wang, Li, & Lin, 2014). By using UPC2, Gee et al. (2016) reported a new trace vitamin E component as α-tocodienol, and constitutes nearly 0.2% of the total vitamin E in the tocotrienol soft gelatin capsule. All vitamin E components (tocols) were well-resolved using 0.5% methanol in supercritical CO2 with an isocratic separation in ethylene-bridged hybrid column (3 mm × 100 mm). 4.3. Nano-LC, capillary LC and capillary electrochromatography (CEC) Miniaturization is one of the recent trends in the field of analytical chemistry. Nano-liquid chromatography (nano-LC) and capillary liquid chromatography (CLC) are the miniaturization techniques, currently used for the separation of tocopherols. These techniques offer several advantages as better separation in shorter analysis time with higher sensitivity, lower sample and reagent consumption. In Nano-LC, separation is performed in a capillary column of id's in the range between 10 and 100 μm with flow rates of 50–800 nL min−1. While, making use of higher id columns of 100–500 μm, and higher flow rates of 1–100 μL min− 1, the method is termed as capillary LC (CLC) (Fanali, Dugo, Dugo, & Mondello, 2013; Hernández-Borges, Aturki, Rocco, & Fanali, 2007). Capillary electrochromatography (CEC) is also the

66

R.K. Saini, Y.-S. Keum / Food Research International 82 (2016) 59–70

miniaturized analytical technique; use the combination HPLC and capillary electrophoresis (CE). In the CEC, separation is achieved as a result of differential partitioning during electrophoretic migration of solutes driven by electroosmosis. In CEC and Nano-LC/CLC, analyze is separated in a capillary containing the selected stationary phase (SP). The SP can be a packed particles or a polymer (monolith) or bonded only to the wall that is called open tubular (OT) (Fanali et al., 2013). These miniaturized analytical techniques are widely used in pharmaceutical and food analysis. The CEC method was applied to separate α-, γ-, δtocopherols and α-tocopherol-acetate from different commercially available virgin olive, hazelnut, sunflower, and soybean oil, using fused-silica capillary (75 mm id), partially packed with ChromSpher C18 stationary phase (Aturki, D'Orazio, & Fanali, 2005). Recently, a reverse-phase monolithic stationary phase developed with copolymerization of 3-methylacryloyl-3-oxapropyl-3-(N,N-dioctadecyl carbamoyl)-propionate (AOD) and ethylene glycol dimethacrylate (EDMA), showed good long-term stability, reproducibility and exhibited good selectivity for β- and γ-tocopherol isomers with chromatographic resolution (Rs) of 1.1 (Duan et al., 2014). Nano-LC has been applied to the separation of tocopherols in several vegetable oils including avocado, corn, grapeseed, hazelnut, and red palm, using chromolith RP-C18 capillary column (Cerretani, LermaGarcía, Herrero-Martínez, Gallina-Toschi, & Simó-Alfonso, 2010). The monolithic column employed in this work was found efficient to separate different isomers of tocols, except β-T and γ-T isomers. This can be explained by reverse-phased characteristics of the column, which is unable to resolve the β-T and γ-T isomers. 5. Methods of detection Liquid chromatography (LC) detectors used in the analysis of tocols includes diode or photodiode array (DAD or PDA), ultraviolet (UV), fluorescence, evaporative light scattering (ELSD), electrochemical (ED) (pulsed amperometric and coulometric), flame ionization (FID), charged aerosol detectors (CAD) and mass spectrometer (MS). Every detector has its advantages and drawbacks. The absorption spectra of column eluted compounds can be easily obtained by DAD and UV detectors, which are used in identification and purity analysis. Conversely, both detectors suffer from lack of sensitivity and selectivity. Fluorescence detector (FD) is the most sensitive for the detection of tocols due to its native fluorophore properties (Moreau & Lampi, 2012). The ED is the preferred option for tocopherol analysis due to the sensitivity, selectivity, and easy handling. The electrochemical detector is also highly sensitive and selective. However, its drawbacks are related to the difficulties encountered in daily handling due to low intra-assay and intermediate precision, with higher relative standard deviation (RSD) values, compared to a fluorescence detector (Ruperez, Mach, & Barbas, 2004). In a comparative study among three detection systems, ELSD (at 40 °C evaporator temperature, 3 bar air pressure, and photomultiplier sensitivity of 4), a UV (at 295.0 nm) and a DAD connected in series with a fluorescence detector (at 290.0 nm excitation, and 330.0 nm emission wavelength), for quantification of tocols in Portuguese olive oils using NP-HPLC, the best results for linearity, detection limits and resolution were obtained with the fluorescence detector (Cunha, Amaral, Fernandes, & Oliveira, 2006). In another study, fluorescence detection was found the most sensitive, followed by PDA in terms of linearity, detection limits, and resolution of the tocopherols (Saha et al., 2013). Fluorescence detection resulted in fewer interfering peaks in the analysis of tocopherols from sesame, corn, and soybean. ELSD was the least sensitive that may be due to small size of tocopherol molecule. Thus, in general trends, on the basis of detection limit and sensitivity, various detectors appear to fall in following pattern: ELSD b UV b FL b EC. After HPLC, gas chromatography (GC) is the most common type of method used for separating and analyzing tocols. Before the 1990s, gas chromatography (GC) methods were widely used for the analysis of tocols in food, which have been reviewed by (Abidi, 2000). However,

the use of GC methods for tocols analysis is limited due to heat decomposition during derivatization and less-volatile nature of tocols. Flame ionization detectors (FID) are most commonly used in GC analysis of trimethylsilyl ether derivatives of tocols (Hussain et al., 2013; Kim, Ha, et al., 2012). In a comparative study of three extraction methods for GC-FID analysis of tocopherol in oilseed rape (Brassica napus L.), trimethylsilylation with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) was found best with highest recovery percentage, compared to other direct extraction methods performed without silylation (Hussain et al., 2013). Mass spectrometry (MS) is the most powerful method of qualitative analysis of tocols and other bioactive compounds, in terms of sensitivity and selectivity. Also, it can provide information regarding molecular weight (Mw), isotopic and fragmentation patterns, for precise identification of unknown metabolites. Several liquid chromatography–mass spectrometry (LC–MS), gas chromatography–mass spectrometry (GC– MS), capillary electrophoresis–mass spectrometry (CE–MS), and nuclear magnetic resonance (NMR) techniques are applied for detection of tocols. Among all LC–MS methods, LC-APCI-MS (atmospheric pressure chemical ionization) and LC-ESI–MS (electrospray ionization) in positive and negative ion modes using a single quadrupole, triple quadrupole or time-of-flight mass (TOF) spectrometers are commonly used. ESI and APCI in negative ion mode are more efficient (50-fold better response) as it only produces target deprotonated pseudo-molecular ions [M–H]−, without any fragmentation. In comparison between ESI and APCI, the APCI in negative ion mode showed a higher range of linearity, lower detection limits, and was less sensitive to the difference in chemical structures of tocopherols than negative ion ESI (Lanina et al., 2007). Authors recorded, at least, 50-fold better response of all tocopherols in negative APCI and 10–45-fold better response in negative ESI compared to positive ion mode using MeOH–H2O (95:5, v/v) as an eluent. In both APCI and ESI, the use of methanol–water (95:5, v/v) as mobile phase was found batter with a considerable gain in MS signal, compared to acetonitrile-water. Thus, the MS response of γ- and δ-tocopherols in various detectors was observed in the following trend: APCI (negative) N ESI (negative) N ESI (positive) N APCI (positive) (Lanina et al., 2007). LC-ESI–MS in negative and positive ions mode was also employed for analysis of vitamin E and γ-oryzanol components in rice bran and germ (Yu, Nehus, Badger, & Fang, 2007). ESI is the most commonly used ionization method in LC based mass spectrometers (MS). However, the lack of a site for protonation or deprotonation on non-polar substances, such as tocopherols, hinders their ionization. The addition of a metal salt, such as Ag+, to the mobile phase, enhances the ionization of tocols; this ionization technique is called coordinated ion spray (CIS). This CIS approach has been used for the detection and identification of tocols in animal tissue (Al-Talla & Tolley, 2005). Similarly, the addition of ammonia (6.0 mM) in the mobile phase (methanol–water; 97:3, v/v), was reported beneficial for enhancing the ionization and sensitivity of LC-ESI–MS for analyzing the vitamin E content of rye and wheat samples (Bustamante-Rangel et al., 2007). Authors also reported the better accuracy of TOF measurements than the quadrupolar mode. Supercritical fluid chromatography (SFC) with an atmospheric pressure photoionization (APPI) source for the detection of the mass spectrum of non-polar compounds with enhancements in signal to noise was reviewed nearly one decade ago (Bolaños et al., 2004). Recently, an SFC–APPI–MS method for the quantification of vitamin E congeners in soybean oil is described. Authors also evaluate the ionization efficiency of ESI, APCI, and APPI coupled to the SFC system and a high-mass resolution mass spectrometer. Results showed that, increasing the flow rate led to a reduction in sensitivity for ESI in both ionization modes, and APPI in the positive ion mode. At a low flow rate of 0.1– 0.2 mL min− 1, similar ionization efficiency of α-tocopherol was observed with use of methanol and ethanol as modifiers. The sensitivity and degree of ionization were recorded in the following trend: APCI (negative) b APCI (positive) b ESI (negative) b APPI (negative) b ESI

R.K. Saini, Y.-S. Keum / Food Research International 82 (2016) 59–70

(positive) b APPI (positive). Interestingly, these observations regarding the sensitivity of ESI in negative and positive mode was opposite to the findings of Lanina et al. (2007). Gas chromatography–mass spectrometry (GC–MS) is also widely used to determine the tocols in diverse plant tissues (Lytovchenko et al., 2009). GC with TOF (time-of-flight: GC–TOF-MS) is capable of separating co-eluted and overlapping peaks mathematically used in the high-throughput analysis in the field of metabolomics. Metabolomics approaches enable the simultaneous assessment of the broad range of metabolites (metabolome) in the biological sample (Fernie & Schauer, 2009). One-dimensional (1D) and twodimensional (GC × GC) gas chromatography–TOF-MS is a novel approach for enhancing the GC throughput, resolution, and sensitivity. This approach has been used for comprehensive metabolic phenotyping of natural variants in rice, including α-tocopherol, enabling the selection of nutritionally useful rice varieties for breeding program (Kusano et al., 2007). Similarly, GC–TOF-MS technique is used for the metabolic profile of Arabidopsis thaliana (Sun et al., 2010) and a fermented traditional Korean food made from soybeans, called Cheonggukjang (CGJ) (Kim, Choi, et al., 2012).

67

7. Other methods of separation and detection Thin layer chromatography (TLC) is also used for quantification of tocopherols in food (Kıvçak & Akay, 2005). In TLC analysis of tocols, silica gel plates with mobile phase of petroleum ether (60–90 °C)/ethyl acetate (95:5), or chloroform with cyclohexane (55:45 v/v) are commonly used. The quantification is performed with densitometry at 280–290 nm, that can give the detection limit of 1.2 μg mL−1 for αtocopherol (Hossu, Maria, Radulescu, Ilie, & Magearu, 2009). Molecular imprinting polymer (MIP) technique using CdSe/ZnS quantum dots (QDs) sensors were also employed for optosensing of tocopherol in vegetable oils and rice (Liu, Fang, Zhu, & Wang, 2014; Liu et al., 2012). MIP acts as a fluorescence nano-sensing material, and fluorescence intensity decreases with the increasing tocopherol concentration. MIP is capable of quantification of tocopherols with the detection limit of 5.80 × 10−8 mol L−1, with the precision of 2.17% (RSD; relative standard deviation). The MIP technique can be used without separation and derivatization process, thus, it is a cost-effective and rapid method for quantification of total tocopherol. 8. Summary and future perspective

6. Direct determination of tocols by FT-IR and SFS techniques Several spectrophotometric methods, such as UV-spectrophotometry (Kıvçak & Akay, 2005), Raman spectroscopy (Beattie et al., 2007), and Fourier transform-infrared spectroscopy (FT-IR) are developed and utilized for tocopherol analysis (Silva, Rosa, Ferreira, Boas, & Bronze, 2009). Raman spectroscopy evaluates the inelastic scattering of photons generated by the changes in the vibrational energy of tocopherol molecule after excitation with monochromatic light. Raman spectroscopy combined with optical microscopy, called as Raman microscopy is a powerful technique for identifying, mapping and localizing the tocopherols in biological samples (Beattie et al., 2007). Man, Ammawath, and Mirghani (2005) analyzed α-tocopherol in refined bleached and deodorized (RBD) palm olein by FT-IR using sodium chloride (NaCl) window utilization. The content of α-tocopherol measured by FT-IR was concordant with results of HPLC. In another study, tocopherols, tocotrienols were analyzed in seeds oil of canola, flax, soybean and sunflower by FT-IR using ZnSe attenuated total reflection (ATR) crystal surface at an angle of incidence of 45° (Ahmed, Daun, & Przybylski, 2005). With this method also, an excellent correlation was obtained between the FT-IR and HPLC results. Thus, the author concluded that FT-IR can be employed as a rapid screening tool for tocopherols and tocotrienols, without the use of large quantities of hazardous solvents. Similarly, αtocopherol was accurately quantified in 13 vegetable oils by FT-IR, using ZnSe ATR crystals surface at an angle of incidence of 45° (Silva et al., 2009). In the advancement of fluorescence spectroscopy, synchronous fluorescence spectroscopy (SFS) technique, allows simultaneous scanning of both excitation and emission wavelengths, keeping a specified constant difference of 10–80 nm (Sikorska, Khmelinskii, & Sikorski, 2012). This technique is widely used for the characterization of tocopherols in edible oils. Sikorska et al. (2008) used SFS method to monitor the content of tocopherols, chlorophylls, and phenolic compounds during storage of virgin olive oil to study the effects on these constituents. In another study, synchronous fluorescence spectra combined with principal component analysis (PCA) was found useful for monitoring olive oil deterioration under UV irradiation at 80 °C (Poulli, Mousdis, & Georgiou, 2009). The SFS technique was also proved to be useful for detecting the addition of olive oil to extra virgin olive oil due to significant differences in fluorescent intensities in the region in the wavelength range of 240–700 nm (Dankowska & Małecka, 2009). Recently, SFS technique is used for detecting the addition of plant fat to hard cheese, with lowest detection limits of adulteration of 3.0%, at the wavelength intervals of 60 nm (Dankowska, Małecka, & Kowalewski, 2015).

During the last decade, the primary research in tocol analysis has expanded to metabolomics for qualitative and quantitative determination of the metabolome in a biological sample with high precision, using mass spectrometry techniques. The supercritical fluid extraction (SCFE) is emerging as a potentially important technique for extraction of heat labile compounds, such as tocopherols, without using toxic solvents. However, the solvent extraction with saponification is the most commonly used method for the extraction of tocols from grains, oilseeds, and biological tissues. The degradation of tocols occurs very rapidly during the sample preparation, thus, the tocols must be stabilized with ascorbic acid, butylated hydroxytoluene (BHT) or pyrogallol. With the development of new long-chain alkyl-bonded C30-silica and solid-core Penta fluorophenyl (PFP) stationary phases, the separation of different tocopherols isomers in RP-HPLC has become easier. RPHPLC with fluorescence/electrochemical detection is the most convenient and sensitive method for the analysis of tocopherols and tocotrienols in the majority of complex food samples. Liquid chromatography (LC) with APCI and ESI in negative ionization is most sensitive for mass spectrum analysis of tocopherol; the sensitivity can be further improved by the addition of an ionizing agent (such as NH3) in the mobile phase. One-dimensional (1D) and two-dimensional (GC × GC) gas chromatography–TOF-MS is a novel approach for enhancing the GC throughput, resolution, and sensitivity in the field of metabolomics. Although the significant advancement has been achieved in the extraction of tocopherols, however, still the analysis of tocopherols from complex food matrix is time-consuming and requires a considerable amount of toxic solvents. Thus, simplified extraction protocols are required to be developed by using eco-compatible solvents. And also, the feasibility of supercritical fluid extraction (SCFE), matrix solidphase dispersion extraction (MSPDE) and other extraction methods should be studied with the samples of diverse physical and chemical characteristics. The metabolic profiling with metabolome approach is investigated to few food crops only. In the future, the metabolome can be studied in major breeding genotypes, that could enable its powerful applications in metabolomics-assisted breeding (MAB) for the development of nutritionally rich verities. The content and various forms of tocopherols are successfully utilized as authenticity markers in determining the quality and authenticity of bakery and milk products. The markers can be further developed and used in assessing the adulteration of dairy products with plant-derived fats to ensure the consumers' protection. δ-Tocopherol and γ-tocotrinols have shown the potent antioxidant and anti-inflammatory properties that are superior to αtocopherols, however, the data of their content is not available in the major food sources. Thus, the nutrient database on tocopherols and

68

R.K. Saini, Y.-S. Keum / Food Research International 82 (2016) 59–70

tocotrienols contents in major foods should be updated. And also, it is important to provide the recommended dietary allowances (RDAs) for humans not only limited to α-tocopherol. Recent reports recorded a new trace vitamin E component as α-tocodienol, constituting nearly 0.2% of the total vitamin E in the tocotrienol soft gelatin capsule. The detailed investigation can be made to understand its biological functions. And also, the presence and concentration of α-tocodienol in various foods can be examined. Conflict of interest The authors have declared that there is no conflict of interest. Acknowledgment This paper was supported by KU research professor program of Konkuk University, Seoul, Republic of Korea. References Abidi, S.L. (2000). Chromatographic analysis of tocol-derived lipid antioxidants. Journal of Chromatography A, 881(1–2), 197–216. http://dx.doi.org/10.1016/ S0021-9673(00)00131-X. Ahmed, M.K., Daun, J.K., & Przybylski, R. (2005). FT-IR based methodology for quantitation of total tocopherols, tocotrienols and plastochromanol-8 in vegetable oils. Journal of Food Composition and Analysis, 18(5), 359–364. Ahsan, H., Ahad, A., Iqbal, J., & Siddiqui, W.A. (2014). Pharmacological potential of tocotrienols: A review. Nutrition & Metabolism, 11. http://dx.doi.org/10.1186/17437075-11-52. Al-Talla, Z.A., & Tolley, L.T. (2005). Analysis of vitamin E derivatives in serum using coordinated ion spray mass spectrometry. Rapid Communications in Mass Spectrometry, 19(16), 2337–2342. Annunziata, M.G., Attico, A., Woodrow, P., Oliva, M.A., Fuggi, A., & Carillo, P. (2012). An improved fluorimetric HPLC method for quantifying tocopherols in Brassica rapa L. subsp. sylvestris after harvest. Journal of Food Composition and Analysis, 27(2), 145–150. http://dx.doi.org/10.1016/j.jfca.2012.05.006. Aturki, Z., D'Orazio, G., & Fanali, S. (2005). Rapid assay of vitamin E in vegetable oils by reversed-phase capillary electrochromatography. Electrophoresis, 26(4–5), 798–803. http://dx.doi.org/10.1002/elps.200410181. Azzi, A. (2007). Molecular mechanism of α-tocopherol action. Free Radical Biology and Medicine, 43(1), 16–21. http://dx.doi.org/10.1016/j.freeradbiomed.2007.03.013. Beattie, J.R., Maguire, C., Gilchrist, S., Barrett, L.J., Cross, C.E., Possmayer, F., & Schock, B.C. (2007). The use of Raman microscopy to determine and localize vitamin E in biological samples. The FASEB Journal, 21(3), 766–776. http://dx.doi.org/10.1096/fj.067028com. Bolaños, B., Greig, M., Ventura, M., Farrell, W., Aurigemma, C.M., Li, H., & Phillipson, D. (2004). SFC/MS in drug discovery at Pfizer, La Jolla. International Journal of Mass Spectrometry, 238(2), 85–97. http://dx.doi.org/10.1016/j.ijms.2003.11.021. Brunner, G. (2005). Supercritical fluids: Technology and application to food processing. Journal of Food Engineering, 67(1–2), 21–33. http://dx.doi.org/10.1016/j.jfoodeng. 2004.05.060. Bustamante-Rangel, M., Delgado-Zamarreño, M.M., Sánchez-Pérez, A., & CarabiasMartínez, R. (2007). Determination of tocopherols and tocotrienols in cereals by pressurized liquid extraction–liquid chromatography–mass spectrometry. Analytica Chimica Acta, 587(2), 216–221. http://dx.doi.org/10.1016/j.aca.2007.01.049. Capriotti, A.L., Cavaliere, C., Foglia, P., Samperi, R., Stampachiacchiere, S., Ventura, S., & Laganà, A. (2015). Recent advances and developments in matrix solid-phase dispersion. TrAC Trends in Analytical Chemistry, 71, 186–193. http://dx.doi.org/10.1016/j. trac.2015.03.012. Cerretani, L., Lerma-García, M.J., Herrero-Martínez, J.M., Gallina-Toschi, T., & SimóAlfonso, E.F. (2010). Determination of tocopherols and tocotrienols in vegetable oils by nanoliquid chromatography with ultraviolet–visible detection using a silica monolithic column. Journal of Agricultural and Food Chemistry, 58(2), 757–761. http://dx. doi.org/10.1021/jf9031537. Chauveau-Duriot, B., Doreau, M., Nozière, P., & Graulet, B. (2010). Simultaneous quantification of carotenoids, retinol, and tocopherols in forages, bovine plasma, and milk: Validation of a novel UPLC method. Analytical and Bioanalytical Chemistry, 397(2), 777–790. http://dx.doi.org/10.1007/s00216-010-3594-y. Constantinou, C., Papas, A., & Constantinou, A.I. (2008). Vitamin E and cancer: An insight into the anticancer activities of vitamin E isomers and analogs. International Journal of Cancer. Journal International Du Cancer, 123(4), 739–752. http://dx.doi.org/10.1002/ ijc.23689. da Costa, P.A., Ballus, C.A., Teixeira-Filho, J., & Godoy, H.T. (2010). Phytosterols and tocopherols content of pulps and nuts of Brazilian fruits. Food Research International, 43(6), 1603–1606. http://dx.doi.org/10.1016/j.foodres.2010.04.025. Cruz, R., & Casal, S. (2013). Validation of a fast and accurate chromatographic method for detailed quantification of vitamin E in green leafy vegetables. Food Chemistry, 141(2), 1175–1180. Cunha, S.C., Amaral, J.S., Fernandes, J.O., & Oliveira, M.B.P.P. (2006). Quantification of tocopherols and tocotrienols in Portuguese olive oils using HPLC with three different

detection systems. Journal of Agricultural and Food Chemistry, 54(9), 3351–3356. http://dx.doi.org/10.1021/jf053102n. Dankowska, A., & Małecka, M. (2009). Application of synchronous fluorescence spectroscopy for determination of extra virgin olive oil adulteration. European Journal of Lipid Science and Technology, 111(12), 1233–1239. http://dx.doi.org/10.1002/ejlt.200800295. Dankowska, A., Małecka, M., & Kowalewski, W. (2015). Detection of plant oil addition to cheese by synchronous fluorescence spectroscopy. Dairy Science & Technology, 95(4), 413–424. http://dx.doi.org/10.1007/s13594-015-0218-5. Delgado-Zamarreño, M., Bustamante-Rangel, M., Sierra-Manzano, S., Verdugo-Jara, M., & Carabias-Martínez, R. (2009). Simultaneous extraction of tocotrienols and tocopherols from cereals using pressurized liquid extraction prior to LC determination. Journal of Separation Science, 32(9), 1430–1436. http://dx.doi.org/10.1002/jssc. 200800707. DellaPenna, D., & Pogson, B.J. (2006). Vitamin synthesis in plants: Tocopherols and carotenoids. Annual Review of Plant Biology, 57, 711–738. Dos Santos Freitas, L., Jacques, R.A., Richter, M.F., da Silva, A.L., & Caramão, E.B. (2008). Pressurized liquid extraction of vitamin E from Brazilian grape seed oil. Journal of Chromatography A, 1200(1), 80–83. http://dx.doi.org/10.1016/j.chroma.2008.02.067. Duan, Q., Liu, C., Liu, Z., Zhou, Z., Chen, W., Wang, Q., & Jiang, Z. (2014). Preparation and evaluation of a novel monolithic column containing double octadecyl chains for reverse-phase micro high performance liquid chromatography. Journal of Chromatography A, 1345, 174–181. http://dx.doi.org/10.1016/j.chroma.2014.04.036. Fanali, C., Dugo, L., Dugo, P., & Mondello, L. (2013). Capillary-liquid chromatography (CLC) and nano-LC in food analysis. TrAC Trends in Analytical Chemistry, 52, 226–238. http:// dx.doi.org/10.1016/j.trac.2013.05.021. Fernie, A.R., & Schauer, N. (2009). Metabolomics-assisted breeding: A viable option for crop improvement? Trends in Genetics, 25(1), 39–48. http://dx.doi.org/10.1016/j.tig. 2008.10.010. Gee, P.T., Liew, C.Y., Thong, M.C., & Gay, M.C.L. (2016). Vitamin E analysis by ultraperformance convergence chromatography and structural elucidation of novel αtocodienol by high-resolution mass spectrometry. Food Chemistry, 196, 367–373. http://dx.doi.org/10.1016/j.foodchem.2015.09.073. Gong, X., Qi, N., Wang, X., Li, J., & Lin, L. (2014). A new method for determination of αtocopherol in tropical fruits by ultra performance convergence chromatography with diode array detector. Food Analytical Methods, 7(8), 1572–1576. http://dx.doi. org/10.1007/s12161-014-9789-7. Górnaś, P., Siger, A., Czubinski, J., Dwiecki, K., Segliņa, D., & Nogala-Kalucka, M. (2014). An alternative RP-HPLC method for the separation and determination of tocopherol and tocotrienol homologues as butter authenticity markers: A comparative study between two European countries. European Journal of Lipid Science and Technology, 116(7), 895–903. http://dx.doi.org/10.1002/ejlt.201300319. Górnaś, P., Siger, A., Polewski, K., Pugajeva, I., & Waśkiewicz, A. (2013). Factors affecting tocopherol contents in coffee brews: NP-HPLC/FLD, RP-UPLC-ESI/MSn and spectroscopic study. European Food Research and Technology, 238(2), 259–264. http://dx. doi.org/10.1007/s00217-013-2103-x. Górnaś, P., Siger, A., Pugajeva, I., Czubinski, J., Waśkiewicz, A., & Polewski, K. (2014). New insights regarding tocopherols in Arabica and Robusta species coffee beans: RP-UPLC-ESI/ MSn and NP-HPLC/FLD study. Journal of Food Composition and Analysis, 36(1), 117–123. Grebenstein, N., & Frank, J. (2012). Rapid baseline-separation of all eight tocopherols and tocotrienols by reversed-phase liquid-chromatography with a solid-core pentafluorophenyl column and their sensitive quantification in plasma and liver. Journal of Chromatography A, 1243, 39–46. Heleno, S.A., Barros, L., Sousa, M.J., Martins, A., & Ferreira, I.C.F.R. (2010). Tocopherols composition of Portuguese wild mushrooms with antioxidant capacity. Food Chemistry, 119(4), 1443–1450. http://dx.doi.org/10.1016/j.foodchem.2009.09.025. Hernández-Borges, J., Aturki, Z., Rocco, A., & Fanali, S. (2007). Recent applications in nanoliquid chromatography. Journal of Separation Science, 30(11), 1589–1610. http://dx.doi.org/10.1002/jssc.200700061. Hidalgo, A., Brandolini, A., Pompei, C., & Piscozzi, R. (2006). Carotenoids and tocols of einkorn wheat (Triticum monococcum ssp. monococcum L.). Journal of Cereal Science, 44(2), 182–193. Higdon, J. (2015). Vitamin E. Retrieved December 6, 2015, from http://lpi.oregonstate. edu/mic/vitamins/vitamin-E#antioxidant-activity Hossu, A. -M., Maria, M. -F., Radulescu, C., Ilie, M., Magearu, V., et al. (2009). TLC applications on separation and quantification of fat-soluble vitamins. Romanian Biotechnological Letters, 14(5), 4615–4619. Hussain, N., Jabeen, Z., Li, Y., Chen, M., Li, Z., Guo, W., & Jiang, L. (2013). Detection of tocopherol in oilseed rape (Brassica napus L.) using gas chromatography with flame ionization detector. Journal of Integrative Agriculture, 12(5), 803–814. http://dx.doi. org/10.1016/S2095-3119(13)60301-9. Imsanguan, P., Roaysubtawee, A., Borirak, R., Pongamphai, S., Douglas, S., & Douglas, P.L. (2008). Extraction of α-tocopherol and γ-oryzanol from rice bran. LWT- Food Science and Technology, 41(8), 1417–1424. Jiang, Q. (2014). Natural forms of vitamin E: Metabolism, antioxidant, and antiinflammatory activities and their role in disease prevention and therapy. Free Radical Biology and Medicine, 72, 76–90. Kadioglu, Y., Demirkaya, F., & Demirkaya, A.K. (2009). Quantitative determination of underivatized α-tocopherol in cow milk, vitamin and multivitamin drugs by GCFID. Chromatographia, 70(3–4), 665–670. Kalogeropoulos, N., Chiou, A., Ioannou, M., Karathanos, V.T., Hassapidou, M., & Andrikopoulos, N.K. (2010). Nutritional evaluation and bioactive microconstituents (phytosterols, tocopherols, polyphenols, triterpenic acids) in cooked dry legumes usually consumed in the Mediterranean countries. Food Chemistry, 121(3), 682–690. http://dx.doi.org/10.1016/j.foodchem.2010.01.005. Kamal-Eldin, A., & Budilarto, E. (2015). Tocopherols and tocotrienols as antioxidants for food preservation. Handbook of Antioxidants for Food Preservation, 141.

R.K. Saini, Y.-S. Keum / Food Research International 82 (2016) 59–70 Kim, J., Choi, J.N., John, K.M., Kusano, M., Oikawa, A., Saito, K., & Lee, C.H. (2012). GC–TOFMS- and CE–TOF-MS-based metabolic profiling of Cheonggukjang (fast-fermented bean paste) during fermentation and its correlation with metabolic pathways. Journal of Agricultural and Food Chemistry, 60(38), 9746–9753. Kim, J.K., Ha, S. -H., Park, S. -Y., Lee, S.M., Kim, H.J., Lim, S.H., & Cho, H.S. (2012). Determination of lipophilic compounds in genetically modified rice using gas chromatography–time-of-flight mass spectrometry. Journal of Food Composition and Analysis, 25(1), 31–38. Kirmizis, D., & Chatzidimitriou, D. (2009). Antiatherogenic effects of vitamin E: The search for the Holy Grail. Vascular Health and Risk Management, 5, 767–774. Kıvçak, B., & Akay, S. (2005). Quantitative determination of α-tocopherol in Pistacia lentiscus, Pistacia lentiscus var. chia, and Pistacia terebinthus by TLC-densitometry and colorimetry. Fitoterapia, 76(1), 62–66. Kizhakekuttu, T.J., & Widlansky, M.E. (2010). Natural antioxidants and hypertension: promise and challenges. Cardiovascular Therapeutics, 28(4), e20–e32. http://dx.doi. org/10.1111/j.1755-5922.2010.00137.x. Knecht, K., Sandfuchs, K., Kulling, S.E., & Bunzel, D. (2015). Tocopherol and tocotrienol analysis in raw and cooked vegetables: A validated method with emphasis on sample preparation. Food Chemistry, 169, 20–27. http://dx.doi.org/10.1016/j.foodchem.2014. 07.099. Kruk, J., Holländer-Czytko, H., Oettmeier, W., & Trebst, A. (2005). Tocopherol as singlet oxygen scavenger in photosystem II. Journal of Plant Physiology, 162(7), 749–757. http:// dx.doi.org/10.1016/j.jplph.2005.04.020. Kusano, M., Fukushima, A., Kobayashi, M., Hayashi, N., Jonsson, P., Moritz, T., & Saito, K. (2007). Application of a metabolomic method combining one-dimensional and two-dimensional gas chromatography-time-of-flight/mass spectrometry to metabolic phenotyping of natural variants in rice. Journal of Chromatography B, 855(1), 71–79. http://dx.doi.org/10.1016/j.jchromb.2007.05.002. Lanina, S.A., Toledo, P., Sampels, S., Kamal-Eldin, A., & Jastrebova, J.A. (2007). Comparison of reversed-phase liquid chromatography–mass spectrometry with electrospray and atmospheric pressure chemical ionization for analysis of dietary tocopherols. Journal of Chromatography A, 1157(1–2), 159–170. http://dx.doi.org/10.1016/j.chroma.2007. 04.058. Liu, H., Fang, G., Li, C., Pan, M., Liu, C., Fan, C., & Wang, S. (2012). Molecularly imprinted polymer on ionic liquid-modified CdSe/ZnS quantum dots for the highly selective and sensitive optosensing of tocopherol. Journal of Materials Chemistry, 22(37), 19882–19887. Liu, H., Fang, G., Zhu, H., & Wang, S. (2014). Application of molecularly imprinted polymer appended onto CdSe/ZnS quantum dots for optosensing of tocopherol in rice. Food Analytical Methods, 7(7), 1443–1450. Lytovchenko, A., Beleggia, R., Schauer, N., Isaacson, T., Leuendorf, J.E., Hellmann, H., & Fernie, A.R. (2009). Application of GC–MS for the detection of lipophilic compounds in diverse plant tissues. Plant Methods, 5(4), 1–11. Man, Y.C., Ammawath, W., & Mirghani, M.E.S. (2005). Determining α-tocopherol in refined bleached and deodorized palm olein by Fourier transform infrared spectroscopy. Food Chemistry, 90(1), 323–327. Méjean, M., Brunelle, A., & Touboul, D. (2015). Quantification of tocopherols and tocotrienols in soybean oil by supercritical-fluid chromatography coupled to highresolution mass spectrometry. Analytical and Bioanalytical Chemistry, 407(17), 5133–5142. http://dx.doi.org/10.1007/s00216-015-8604-7. Mignogna, R., Fratianni, A., Niro, S., & Panfili, G. (2015). Tocopherol and tocotrienol analysis as a tool to discriminate different fat ingredients in bakery products. Food Control, 54, 31–38. Minhajuddin, M., Beg, Z.H., & Iqbal, J. (2005). Hypolipidemic and antioxidant properties of tocotrienol rich fraction isolated from rice bran oil in experimentally induced hyperlipidemic rats. Food and Chemical Toxicology, 43(5), 747–753. Mitei, Y.C., Ngila, J.C., Yeboah, S.O., Wessjohann, L., & Schmidt, J. (2009). Profiling of phytosterols, tocopherols and tocotrienols in selected seed oils from Botswana by GC–MS and HPLC. Journal of the American Oil Chemists' Society, 86(7), 617–625. Mocchegiani, E., Costarelli, L., Giacconi, R., Malavolta, M., Basso, A., Piacenza, F., et al. (2014). Vitamin E–gene interactions in aging and inflammatory age-related diseases: Implications for treatment. A systematic review. Ageing Research Reviews, 14, 81–101. Molano, A., & Meydani, S.N. (2012). Vitamin E, signalosomes and gene expression in T cells. Molecular Aspects of Medicine, 33(1), 55–62. http://dx.doi.org/10.1016/j.mam. 2011.11.002. Monsen, E.R. (2000). Dietary reference intakes for the antioxidant nutrients: Vitamin C, vitamin E, selenium, and carotenoids. Journal of the American Dietetic Association, 100(6), 637–640. Moraes, M.N., Zabot, G.L., & Meireles, M.A.A. (2015). Extraction of tocotrienols from annatto seeds by a pseudo continuously operated SFE process integrated with lowpressure solvent extraction for bixin production. The Journal of Supercritical Fluids, 96, 262–271. http://dx.doi.org/10.1016/j.supflu.2014.09.007. Moreau, R.A., & Lampi, A. -M. (2012). Analysis methods for tocopherols and tocotrienols. Analysis of Antioxidant-Rich Phytochemicals, 353. Munné-Bosch, S. (2005). The role of -tocopherol in plant stress tolerance. Journal of Plant Physiology, 162(7), 743–748. http://dx.doi.org/10.1016/j.jplph.2005.04.022. Mustafa, A., & Turner, C. (2011). Pressurized liquid extraction as a green approach in food and herbal plants extraction: A review. Analytica Chimica Acta, 703(1), 8–18. http:// dx.doi.org/10.1016/j.aca.2011.07.018. Núñez, G.A., Gelmi, C.A., & del Valle, J.M. (2011). Simulation of a supercritical carbon dioxide extraction plant with three extraction vessels. Computers & Chemical Engineering, 35(12), 2687–2695. http://dx.doi.org/10.1016/j.compchemeng.2011.04.002. Ofori-Boateng, C., & Lee, K.T. (2013). Ultrasonic-assisted extraction of α-tocopherol antioxidants from the fronds of Elaeis guineensis Jacq.: Optimization, kinetics, and thermodynamic studies. Food Analytical Methods, 7(2), 257–267. http://dx.doi.org/10. 1007/s12161-013-9619-3.

69

Panfili, G., Fratianni, A., & Irano, M. (2003). Normal phase high-performance liquid chromatography method for the determination of tocopherols and tocotrienols in cereals. Journal of Agricultural and Food Chemistry, 51(14), 3940–3944. Panfili, G., Fratianni, A., & Irano, M. (2004). Improved normal-phase high-performance liquid chromatography procedure for the determination of carotenoids in cereals. Journal of Agricultural and Food Chemistry, 52(21), 6373–6377. Panfili, G., Manzi, P., & Pizzoferrato, L. (1994). High-performance liquid chromatographic method for the simultaneous determination of tocopherols, carotenes, and retinol and its geometric isomers in Italian cheeses. Analyst, 119(6), 1161–1165. Poulli, K.I., Mousdis, G.A., & Georgiou, C.A. (2009). Monitoring olive oil oxidation under thermal and UV stress through synchronous fluorescence spectroscopy and classical assays. Food Chemistry, 117(3), 499–503. http://dx.doi.org/10.1016/j.foodchem.2009. 04.024. Ramos, L. (2012). Critical overview of selected contemporary sample preparation techniques. Journal of Chromatography A, 1221, 84–98. http://dx.doi.org/10.1016/j. chroma.2011.11.011. Rashid Khan, M., Ahsan, H., Siddiqui, S., & Siddiqui, W.A. (2014). Tocotrienols have a nephroprotective action against lipid-induced chronic renal dysfunction in rats. Renal Failure, 37(1), 136–143. Ruperez, F.J., Mach, M., & Barbas, C. (2004). Direct liquid chromatography method for retinol, α-and γ-tocopherols in rat plasma. Journal of Chromatography B, 800(1), 225–230. Saha, S., Walia, S., Kundu, A., & Pathak, N. (2013). Effect of mobile phase on resolution of the isomers and homologues of tocopherols on a triacontyl stationary phase. Analytical and Bioanalytical Chemistry, 405(28), 9285–9295. http://dx.doi.org/10. 1007/s00216-013-7336-9. Saini, R.K., Prashanth, K.V.H., Shetty, N.P., & Giridhar, P. (2014). Elicitors, SA and MJ enhance carotenoids and tocopherol biosynthesis and expression of antioxidant related genes in Moringa oleifera Lam. leaves. Acta Physiologiae Plantarum, 36(10), 2695–2704. http://dx.doi.org/10.1007/s11738-014-1640-7. Saini, R.K., Shetty, N.P., Prakash, M., & Giridhar, P. (2014). Effect of dehydration methods on retention of carotenoids, tocopherols, ascorbic acid and antioxidant activity in Moringa oleifera leaves and preparation of a RTE product. Journal of Food Science and Technology, 51(9), 2176–2182. Seppanen, C.M., Song, Q., & Csallany, A.S. (2010). The antioxidant functions of tocopherol and tocotrienol homologues in oils, fats, and food systems. Journal of the American Oil Chemists' Society, 87(5), 469–481. Shammugasamy, B., Ramakrishnan, Y., Ghazali, H.M., & Muhammad, K. (2013). Combination of saponification and dispersive liquid–liquid microextraction for the determination of tocopherols and tocotrienols in cereals by reversed-phase high-performance liquid chromatography. Journal of Chromatography A, 1300, 31–37. Shammugasamy, B., Ramakrishnan, Y., Manan, F., & Muhammad, K. (2014). Rapid reversed-phase chromatographic method for determination of eight vitamin E isomers and γ-oryzanols in rice bran and rice bran oil. Food Analytical Methods, 8(3), 649–655. Siddiqui, S., Ahsan, H., Khan, M.R., & Siddiqui, W.A. (2013). Protective effects of tocotrienols against lipid-induced nephropathy in experimental type-2 diabetic rats by modulation in TGF-β expression. Toxicology and Applied Pharmacology, 273(2), 314–324. http://dx.doi.org/10.1016/j.taap.2013.09.004. Sikorska, E., Khmelinskii, I., & Sikorski, M. (2012). Analysis of Olive Oils by Fluorescence Spectroscopy: Methods and Applications. INTECH Open Access Publisher (Retrieved from http://cdn.intechopen.com/pdfs/27027/InTech-Analysis_of_olive_oils_by_ fluorescence_spectroscopy_methods_and_applications.pdf). Sikorska, E., Khmelinskii, I.V., Sikorski, M., Caponio, F., Bilancia, M.T., Pasqualone, A., & Gomes, T. (2008). Fluorescence spectroscopy in monitoring of extra virgin olive oil during storage. International Journal of Food Science & Technology, 43(1), 52–61. http://dx.doi.org/10.1111/j.1365-2621.2006.01384.x. Silva, S.D., Rosa, N.F., Ferreira, A.E., Boas, L.V., & Bronze, M.R. (2009). Rapid determination of α-tocopherol in vegetable oils by fourier transform infrared spectroscopy. Food Analytical Methods, 2(2), 120–127. Sivanesan, I., Saini, R.K., & Kim, D.H. (2016). Bioactive compounds in hyperhydric and normal micropropagated shoots of Aronia melanocarpa (michx.) Elliott. Industrial Crops and Products, 83, 31–38. http://dx.doi.org/10.1016/j.indcrop.2015.12.042. Stöggl, W.M., Huck, C.W., Scherz, H., Popp, M., & Bonn, G.K. (2001). Analysis of vitamin E in food and phytopharmaceutical preparations by HPLC and HPLC-APCI–MS–MS. Chromatographia, 54(3–4), 179–185. http://dx.doi.org/10.1007/BF02492241. Sun, X., Zhang, J., Zhang, H., Ni, Y., Zhang, Q., Chen, J., & Guan, Y. (2010). The responses of Arabidopsis thaliana to cadmium exposure explored via metabolite profiling. Chemosphere, 78(7), 840–845. Szarka, A., Tomasskovics, B., & Bánhegyi, G. (2012). The ascorbate-glutathione-αtocopherol triad in abiotic stress response. International Journal of Molecular Sciences, 13(4), 4458–4483. http://dx.doi.org/10.3390/ijms13044458. Thiele, J.J., Hsieh, S.N., & Ekanayake-Mudiyanselage, S. (2005). Vitamin E: Critical review of its current use in cosmetic and clinical dermatology. Dermatologic Surgery, 31, 805–813. http://dx.doi.org/10.1111/j.1524-4725.2005.31724. Traber, M.G., & Atkinson, J. (2007). Vitamin E, antioxidant and nothing more. Free Radical Biology and Medicine, 43(1), 4–15. http://dx.doi.org/10.1016/j.freeradbiomed.2007. 03.024. Trpkovic, A., Resanovic, I., Stanimirovic, J., Radak, D., Mousa, S.A., Cenic-Milosevic, D., & Isenovic, E.R. (2015). Oxidized low-density lipoprotein as a biomarker of cardiovascular diseases. Critical Reviews in Clinical Laboratory Sciences, 52(2), 70–85. http:// dx.doi.org/10.3109/10408363.2014.992063. Tsochatzis, E.D., & Tzimou-Tsitouridou, R. (2014). Validated RP-HPLC method for simultaneous determination of tocopherols and tocotrienols in whole grain barley using matrix solid-phase dispersion. Food Analytical Methods, 8(2), 392–400. http://dx.doi.org/ 10.1007/s12161-014-9904-9.

70

R.K. Saini, Y.-S. Keum / Food Research International 82 (2016) 59–70

Tsochatzis, E.D., Bladenopoulos, K., & Papageorgiou, M. (2012). Determination of tocopherol and tocotrienol content of Greek barley varieties under conventional and organic cultivation techniques using validated reverse phase high-performance liquid chromatography method. Journal of the Science of Food and Agriculture, 92(8), 1732–1739. Tsuduki, T., Kuriyama, K., Nakagawa, K., & Miyazawa, T. (2013). Tocotrienol (unsaturated vitamin E) suppresses degranulation of mast cells and reduces allergic dermatitis in mice. Journal of Oleo Science, 62(10), 825–834. http://dx.doi.org/10.5650/jos.62.825. Ueda, T., & Igarashi, O. (1985). Evaluation of the electrochemical detector for the determination of tocopherols in feeds by high-performance liquid-chromatography. Journal of Micronutrient Analysis, 1(1), 31–38. Valdivielso, I., Bustamante, M.Á., Ruiz de Gordoa, J.C., Nájera, A.I., de Renobales, M., & Barron, L.J.R. (2015). Simultaneous analysis of carotenoids and tocopherols in botanical species using one step solid–liquid extraction followed by high performance liquid chromatography. Food Chemistry, 173, 709–717. http://dx.doi.org/10.1016/j. foodchem.2014.10.090. Van Meulebroek, L., Vanhaecke, L., De Swaef, T., Steppe, K., & De Brabander, H. (2012). UHPLC–MS/MS to quantify liposoluble antioxidants in red-ripe tomatoes, grown under different salt stress levels. Journal of Agricultural and Food Chemistry, 60(2), 566–573. Vilkhu, K., Mawson, R., Simons, L., & Bates, D. (2008). Applications and opportunities for ultrasound assisted extraction in the food industry — A review. Innovative Food Science & Emerging Technologies, 9(2), 161–169. http://dx.doi.org/10.1016/j.ifset. 2007.04.014.

Viñas, P., Bravo-Bravo, M., López-García, I., Pastor-Belda, M., & Hernández-Córdoba, M. (2014). Pressurized liquid extraction and dispersive liquid–liquid microextraction for determination of tocopherols and tocotrienols in plant foods by liquid chromatography with fluorescence and atmospheric pressure chemical ionization-mass spectrometry detection. Talanta, 119, 98–104. http://dx.doi.org/10.1016/j.talanta.2013. 10.053. Wang, L., & Weller, C.L. (2006). Recent advances in extraction of nutraceuticals from plants. Trends in Food Science & Technology, 17(6), 300–312. Yu, S., Nehus, Z.T., Badger, T.M., & Fang, N. (2007). Quantification of vitamin E and γoryzanol components in rice germ and bran. Journal of Agricultural and Food Chemistry, 55(18), 7308–7313. Zhang, B., Deng, Z., Tang, Y., Chen, P., Liu, R., Ramdath, D.D., ... Tsao, R. (2014). Fatty acid, carotenoid and tocopherol compositions of 20 Canadian lentil cultivars and synergistic contribution to antioxidant activities. Food Chemistry, 161, 296–304. http://dx.doi. org/10.1016/j.foodchem.2014.04.014. Ziegler, J.U., Schweiggert, R.M., & Carle, R. (2015). A method for the simultaneous extraction and quantitation of lipophilic antioxidants in Triticum sp. by HPLC-DAD/FLDMSn. Journal of Food Composition and Analysis, 39, 94–102. http://dx.doi.org/10. 1016/j.jfca.2014.11.011.