Analysis of Tocopherols and Tocotrienols

Currently, high-performance liquid chromatography (HPLC) methods have been widely used in the analysis of tocopherols and tocotrienols in food and nutrition areas. Each form of tocopherol and tocotrienol can be separated and quantified individually using HPLC with either a UV or fluorescence detector. The interferences are largely reduced after separation by HPLC. Therefore, the sensitivity and specificity of HPLC methods are much higher than those obtained with the colorimetric, polarimetric, and GC methods. Also, sample preparation in the HPLC methods is simpler and more efficiently duplicated than in the older methods. Many HPLC methods for the quantification of tocopherols and tocotrienols in various foods and biological samples have been reported. Method number 992.03 of the AOAC International Official Methods of Analysis provides an HPLC method to determine vitamin E in milk-based infant formula. It could probably be said that HPLC methods have become dominant in the analysis of tocopherols and tocotrienols. Therefore, the analytical protocols for tocopherols and tocotrienols in this unit are focused on HPLC methods. Normal and reversed-phase HPLC methods are discussed in the separation and quantification of tocopherols and tocotrienols (see Basic Protocol). Sample 

AOAC 971.30 a-Tocopherol and a-Tocopheryl Acetate in Foods and Feeds, Colorimetric Method (First Action 1971 and Final Action 1972) 

AOAC 975.43 Identification of RRR- or all-rac-alpha-Tocophcm in Drugs and Food or Feed Supplements, Polarimetric Method (First Action 1975 and Final Action 1980) 

AOAC 969.40 Vitamin E in Drugs, Gas Chromatographic Method (First Action 1969 and Final Action 1974) 

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

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

Table D1.5.3 Analysis of Tocopherols and Tocotrienols of Foods Using HPLC Methods... 

Gertz, C. and Hermann, K. (1982) Analysis of tocopherols and tocotrienols in foods. Z Lebensm. Unters. Forsch., 174, 390-394. 

This previous review provides a comprehensive overview of the major chromatographic methods for the analysis of tocopherols and tocotrie-nols developed until about 2000. The current chapter is not a comprehensive overview, but instead is a summary of the major advances in the field that have occurred since 2000. Two other valuable resources that review methods for the analysis of tocopherols and tocotrienols are the websites AOCS Lipid Library (2011) and Cyberlipid (2011). 

In recent years several normal-phase HPLC methods have been reported for the quantitative analysis of tocopherols and tocotrienols (Table 11.5). The best of these methods have been able to achieve baseline separation of all four tocopherols and all four tocotrienols, as shown in Figures 11.2 and 11.3. Kamal-Eldin et al. (2000) reported the optimal baseline separation of all eight common tocols using a Diol-bonded phase column and an isocratic mobile phase of hexane/methyl tert-butyl ether (MTBE), 96 4, v/v (Figure 11.2). Similar separations were reported by Moreau et al. (2007) using the same type of column and mobile phase. Schwartz et al. (2008) reported that, with a normal-phase silica column, plastochromanol-8 in rapeseed oil eluted between y-tocopherol and 5-tocopherol. 

Several reversed-phase HPLC methods have also been reported for the quantitative analysis of tocopherols and tocotrienols (Table 11.6). To be able to separate all eight tocols a pentafluorophenylsilica column (Abidi, 2003) was used. A C30-bonded phase silica column separated the three tocotrienols, a-tocopherol, and a-tocomonoenol in palm oil (Ng et al., 2004). C30-bonded silica columns have also been used to simultaneously analyze tocopherols, other fat-soluble vitamins, and carotenoids (Gentili and Caretti, 2011). 

Several capillary electrochromatography (CEC) methods have also been reported for the quantitative analysis of tocopherols and tocotrienols (Table 11.7). Three of these report baseline separation of all four tocopherols, but none has investigated the separation of tocotrienols. 

Although tocopherols and tocotrienols can be detected by UV absorbance at 280 nm, fluorescence detection (excitation 294 nm and emission 326 nm), as shown in Figure 11.3, has proven to be a much more sensitive method. Electrochemical detection such as pulsed amperometric and coulometric (Uspitasari-Nienaber, 2002) has also proven to be sensitive and potentially valuable for the quantitative analysis of tocopherols and Tocotrienols (Abidi, 2000), especially for tocol analysis in blood and serum samples. HPLC mass detectors such as flame-ionization detectors, evaporative light-scattering detectors, and charged aerosol detectors have proven to be valuable for the quantitative analysis of many types of lipids, but because tocols have... 

As noted above in our experience, normal-phase HPLC with fluorescence detection is the most convenient and most sensitive method for the analysis of tocopherols and tocotrienols in the majority of samples. 

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