Tocopherols detection

The color complex produced with ferric chloride-dipyridyl is equally sensitive (0.5 jag a-tocopherol detectable) and unspecific. The color reaction can be used, however, for quantitative determination of a-tocopherol in pharmaceutical forms and concentrates after localization of the spot in UV light and elution from the adsorbant (Bolliger, 1962). 

The total tocopherol content of sesame seed oil is approximately 400 mg/kg for white varieties and 540 mg/kg for dark varieties (Mohamed and Awatif, 1998 Yoshida et al, 2001). Others (Kamal-Eldin and Appelqvist, 1994 Yoshida and Kajimoto, 1994 Shahidi et al, 1997) have reported tocopherol levels in the range of 330-680 mg/kg oil, with y-tocopherol being the only tocopherol detected. Yoshida et al (2001) reported that y-tocopherol accounted for 96% of the tocopherols, with the 5 compound being the only other tocopherol present. Interestingly, wild species (Sesamum radiatum and S. angustifolium) had higher y-tocopherol contents (750 mg/kg oil and 800 mg/ kg oil, respectively) than cultivated varieties. 

Numerous high pressure Hquid chromatographic techniques have been reported for specific sample forms vegetable oHs (55,56), animal feeds (57,58), seta (59,60), plasma (61,62), foods (63,64), and tissues (63). Some of the methods requite a saponification step to remove fats, to release tocopherols from ceHs, and/or to free tocopherols from their esters. AH requite an extraction step to remove the tocopherols from the sample matrix. The methods include both normal and reverse-phase hplc with either uv absorbance or fluorescence detection. AppHcation of supercritical fluid (qv) chromatography has been reported for analysis of tocopherols in marine oHs (65). 

Detection and result The chromatogram was freed from mobile phase in a stream of warm air, immersed in the freshly prepared reagent mixture for 1 s and then dried in a stream of cold air. After a short time a-tocopherol yielded a red chromatogram zone on a colorless background at hRf 35—40. The visual detection limit on the HPTLC layer was 20 — 25 ng per chromatogram zone. 

The reaction of eq. 16.9 will regenerate the antioxidant Arj-OH at the expense of the antioxidant At2-OH. Despite the fact that such regeneration reactions are not simple electron transfer reactions, the rate of reactions like that of eq. 16.9 has been correlated with the E values for the respective Ar-0. Thermodynamic and kinetic effects have not been clearly separated for such hierarchies, but for a number of flavonoids the following pecking order was established in dimethyl formamid (DMF) by a combination of electrolysis for generating the a-tocopherol and the flavonoid phenoxyl radicals and electron spin resonance (ESR) spectroscopy for detection of these radicals (Jorgensen et al, 1999) ... 

CARDENOSA R, MOHAMED R, PINEDA M and AGUILAR M (2002) On-line HPLC detection of tocopherols and other antioxidants through the formation of a phosphomolybdemun complex, /dgricihod Chem, 50, 3390-5. 

Rentel, C. et al.. Silver-plated vitamins a method of detecting tocopherols and carotenoids in LC/ESI-MS coupling. Ana/. Chem., 70, 4394, 1998. 

Oxidation of the fatty acids in an LDL particle shares many of the characteristics associated with lipid peroxidation in other biological or chemical systems. Once initiated peroxyl radicals are formed and this results in the oxidation of a-tocopherol to give the a-tocopheroyl radical (Kalyanaraman etal., 1990). This can be demonstrated by e.s.r. techniques that allow the direct observation of stable radicals such as the a-tocopheroyl radical. After the a-tocopheryl radical is consumed, lipid-derived peroxyl radicals can be detected after reaction with spin traps (Kalyanaraman etal., 1990, 1991). 

By a combination of synthetic approaches, isotopic labeling, using tocopherols with 13C-labeling at C-5a and C-7a, EPR spectroscopy, and high-level DFT computations, it was shown that there is no radical formation at either C-5a or C-7a and that chromanol methide radical 10 does not occur in tocopherol.11 EPR failed to detect... 

Mortensen, A. and L. H. Skibsted. 1997b. Real time detection of reactions between radicals of lycopene and tocopherol homologues. Free Rad. Res. 27 229-234. 

Various hydroxyl and amino derivatives of aromatic compounds are oxidized by peroxidases in the presence of hydrogen peroxide, yielding neutral or cation free radicals. Thus the phenacetin metabolites p-phenetidine (4-ethoxyaniline) and acetaminophen (TV-acetyl-p-aminophenol) were oxidized by LPO or HRP into the 4-ethoxyaniline cation radical and neutral V-acetyl-4-aminophenoxyl radical, respectively [198,199]. In both cases free radicals were detected by using fast-flow ESR spectroscopy. Catechols, Dopa methyl ester (dihydrox-yphenylalanine methyl ester), and 6-hydroxy-Dopa (trihydroxyphenylalanine) were oxidized by LPO mainly to o-semiquinone free radicals [200]. Another catechol derivative adrenaline (epinephrine) was oxidized into adrenochrome in the reaction catalyzed by HRP [201], This reaction can proceed in the absence of hydrogen peroxide and accompanied by oxygen consumption. It was proposed that the oxidation of adrenaline was mediated by superoxide. HRP and LPO catalyzed the oxidation of Trolox C (an analog of a-tocopherol) into phenoxyl radical [202]. The formation of phenoxyl radicals was monitored by ESR spectroscopy, and the rate constants for the reaction of Compounds II with Trolox C were determined (Table 22.1). 

Romeu-Nadal, M., Morera-Pons, S., Castellote, A. 1., and Lopez-Sabater, M. C. (2006). Determination of gamma- and alpha-tocopherols in human milk by a direct high-performance liquid chromatographic method with UV-vis detection and comparison with evaporative Ught scattering detection. /. Chromatogr. A 1114,132-137. 

Although the majority of analytes do not possess natural fluorescence, the fluorescence detector has gained popularity due to its high sensitivity. The development of derivatization procedures used to label the separated analytes with a fluorescent compound has facilitated the broad application of fluorescence detection. These labeling reactions can be performed either pre- or post-separation, and a variety of these derivatization techniques have been recently reviewed by Fukushima et al. [18]. The usefulness of fluorescence detectors has recently been further demonstrated by the Wainer group, who developed a simple HPLC technique for the determination of all-trani-retinol and tocopherols in human plasma using variable wavelength fluorescence detection [19]. 

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-... 

Recovery of extraction. The recovery of extraction can be monitored using internal standard and spiking methods. For meat products, 8-tocopherol is used as an internal standard because it is usually not detectable in these samples. Ueda and Igarashi (1987) also introduced 2,2,5,7,8-pentamethyl-6-chromanol as an internal standard for the recovery examination. As uneven recoveries occurred in the different forms of tocopherols and tocotrienols during extraction, spiking, which is adding the standards into the sample prior to saponification and extraction, is a reliable method to obtain the recoveries of the tocopherols and tocotrienols. 

Figure D1.5.3 Chromatogram of tocopherols and tocotrienols of rice bran oil in normal-phase HPLC (see Basic Protocol). p-Tocopherol was below the level of detection.

Chase, G.W. Jr., Akoh, C.C., and Eitenimiller, R.R. 1994. Analysis of tocopherols in vegetable oils by high-pressure liquid chromatography Comparison of fluorescence and evaporative lightscattering detection. J. Am. Oil. Chem. Soc. 71 877-880. 

Murphy, M.E. and Kehrer, J.P. 1987. Simultaneous measurment of tocopherols and tocopheryl quinines in tissue fractions using high-performance liquid chromatography with redox-cycling electrochemical detection../. Chromatogr. 421 71-82. 

MacCrehan, W.A. and Schonberger, E. 1987. Determination of retinol, a-tocopherol, and a-caro-tene in serum by liquid chromatography with absorbance and electrochemical detection. Clin. Chem. 33 1585-1592. 

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