CAPÍTULO 1: FUNDAMENTACIÓN TEÓRICA
1.2 Fundamentación de la Tecnología
1.2.8 Herramientas
Both normal-phase and reversed-phase HPLC have been applied in vitamin E analysis.
Reversed-phase HPLC is unable to completely separate all tocopherols and toco-trienols. Because β- and γ-vitamers have very similar structures, their separation cannot be obtained with reversed-phase HPLC. It is, however, applicable when only tocopherols or α-tocopheryl esters are analyzed (Gimeno et al., 2000; Iwase, 2000).
There are reversed-phase methods to analyze tocopherols together with other lipid constituents from biological and food samples such as carotenoids (Epler et al., 1993; Salo-Väänänen et al., 2000), ubiquinols and ubiquinones (Podda et al., 1996) or sterols (Warner and Mounts, 1990).
There are many applications of normal-phase chromatography to separate all eight vitamers using silica or bonded silica phases such as diol or amino (Table 1.5;
Figure 1.4). The separation of β- and γ-tocopherols and tocotrienols is the most difficult task, but it can be achieved with normal-phase HPLC. In addition to its
TABLE 1.5
Normal-Phase HPLC Conditions Used to Separate Eight Tocopherol and Tocotrienol Isomers from Food and Feed Samples
Column Mobile Phase Reference
Kramer et al., 1997, 1999
Silica
Hexane:dioxane, 96:4 and 95:5 (v/v) 1.5–2 ml/min
Kamal-Eldin et al., 2000
Tocopherols and Tocotrienols from Oil and Cereal Grains 27
higher separation ability, it is superior to reversed-phase chromatography because neutral lipids and other nonpolar compounds do not interfere with the analysis. The reproducibility of normal-phase columns, which used to reduce the use of these columns, has recently improved (Kramer et al., 1999; Kamal-Eldin et al., 2000).
An advanced HPLC system has been developed to separate cis/trans isomers of tocotrienols using a chiral permethylated β-cyclodextrin column and an acetonitrile/
water eluent mixture (Drotleff and Ternes, 1999).
Detection of tocopherols and tocotrienols after HPLC separation is based on their ability to absorb ultraviolet light and create fluorescence. Tocopherols and tocotrienols show typical UV spectra with maximum absorption at 290–300 nm (Table 1.6). If the samples contain sufficient amounts of analytes, e.g., vegetable oils and supplemented products, a UV detector is sensitive enough. When higher sensitivity and better selectivity is needed, a fluorescence detector is the commonly used detector. With a fluorescence detector, it is possible to analyze tocopherols FIGURE 1.4 Normal-phase HPLC chromatogram of tocopherols (T) and tocotrienols (T3) of oat extract. Column: Genesis Silica (Jones Chromatography; 250 × 4.6 mm, dp = 4 µm);
eluent: n-hexane:1,4-dioxane (96:4, v/v) 1.5 ml/min at 30°C; fluorescence detection; λex = 294 nm, λem = 326 nm.
TABLE 1.6
Characterizing Tocopherols and Tocotrienols by Ultraviolet Light
Vitamer
λmax (nm),
in EtOH E1%, 1 cm
α-tocopherol 292 75.8
β-tocopherol 296 89.4
γ-tocopherol 298 91.4
δ-tocopherol 298 87.3
α-tocotrienol 292 91.0
β-tocotrienol 295 87.5
γ-tocotrienol 298 103.0
δ-tocotrienol 292 83.0
Data modified from Podda et al. (1996).
Fluorescence response 0 5 10 15 20
Retention time (min) α -T
α -T3
β -T β -T3
γ -T3 δ -T
δ -T3 TX69027-ch01-Frame Page 27 Wednesday, January 16, 2002 7:05 AM
from as little as single-seed samples (3–7 mg) of rapeseed (Goffman et al., 1999).
Usually, the excitation wavelength is set at 292 nm (285–297 nm) and the emission wavelength at 320 nm (310–324 nm) (Eitenmiller and Landen, 1999). Different tocopherols and tocotrienols have different fluorescence responses (Chase et al., 1994; Kramer et al., 1997), which means that calibration curves should be prepared individually. However, all tocopherols and their respective tocotrienols have similar fluorescence response, e.g., α-tocopherol and α-tocotrienol. Thus, tocotrienols are quantitated with their respective tocopherols (Piironen et al., 1984; AOCS, 1990;
Kramer et al., 1999).
Electrochemical detection has also been applied to detect tocopherols and toco-trienols (Ueda and Igarashi, 1987a; Podda et al., 1996). It has the advantage of about 20-fold greater sensitivity relative to fluorescence detection (Nelis et al., 2000).
1.8.3 QUALITY CONTROLOF TOCOPHEROL AND
TOCOTRIENOL ANALYSIS
For identification of tocopherols and tocotrienols after chromatographic separation, pure tocopherols and tocotrienol mixtures are commercially available. Their purity can be checked by both HPLC and GC and the concentration of stock solutions by UV spectroscopy (Table 1.6). It is common to use lipid extracts from cereals and rice bran and their mixtures with pure compounds and vegetable oils to tentatively identify the vitamers and to study the performance of the HPLC separation.
Quantitation of tocopherols and tocotrienols is based on either internal or external standard calibration procedures. When analyzing oils and biological samples, it is easy to find a suitable internal standard among the other tocopherols that are not present in the sample, e.g., δ-tocopherol. However, with complex materials, it is more difficult to find a relevant internal standard that would be similar enough to pass the sample preparation step similar to the vitamers, and would be different enough to be separated by HPLC. Compounds used as internal standards include 2,2,5,7,8-pentamethyl-6-chromanol (Ueda and Igarashi, 1987b), 3-octadecycloxy-1,2-propanediol (De Greyt et al., 1998) and 5,8-dimethyl tocol (Epler et al., 1993;
Chase et al., 1994; Lechner et al., 1999).
1.8.4 OFFICIAL METHODSAND REFERENCE MATERIALS
Determination of four tocopherols and four tocotrienols in vegetable oils and fats by the official American Oil Chemists’ Society method is based on separation by normal-phase HPLC and fluorescence detection (AOCS, 1990). Oil samples are dissolved in hexane, whereas margarines and other fats containing vitamer esters need a cold saponification step to liberate the vitamers. The American Association of Cereal Chemists has a method to analyze vitamin E in various foods. This method (AACC, 1997) is applicable to a vitamin E range of 1 × 10–4 – 100%, and it includes hot saponification and separation by reversed-phase HPLC. Results are calculated as α-tocopherol acetate. The Royal Society of Chemistry has approved a method to analyze vitamin E in animal feedstuffs by normal-phase HPLC after the vitamers have been liberated by hot saponification (Analytical Methods Committee, 1990).
Tocopherols and Tocotrienols from Oil and Cereal Grains 29
The National Institute of Standards and Technology (NIST) and the Community Bureau of Reference (BCR) have developed several food matrix standard reference materials for vitamin E analysis (Sharpless et al., 2000). Most of the materials contain either tocopherol acetate or α-tocopherol as the only certified constituent, but some materials contain all tocopherols (Table 1.7). It has been proposed to add tocopherols in baking chocolate for reference material (Sharpless et al., 2000).
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TABLE 1.7
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