Fluorescence tocopherol

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... 

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). 

Fluorimetric methods of analysis make use of the natural fluorescence of the analyte, the formation of a fluorescent derivative or the quenching of the fluorescence of a suitable compound by the analyte. Fluorescence cannot occur unless there is light absorption, so that all fluorescent molecules absorb, but the reverse is not true only a small fraction of all absorbing compounds exhibits fluorescence. The types of molecule most likely to show useful fluorescence are those with delocalised ji-orbital systems. Often, the more rigid the molecule the stronger the fluorescence intensity. Naturally fluorescent compounds include Vitamin A, E (tocopherol). 

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]. 

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... 

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-... 

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. 

Tocopherols can be measured simultaneously by using a diode array detector, a second UV detector set at 280 to 300 nm, or a fluorescence detector set at 296 nm excitation and 336 nm emission. 

For tocopherols (optional) Place fluorescence detector after UV-Vis detector and monitor excitation at 296 nm and emission at 336 nm. 

Retinol and its esters and unesterified tocopherols and tocotrienols possess strong native fluorescence, but neither vitamin D nor vitamin K fluoresce. The carotenoids commonly associated with foods do not fluoresce to any significant extent, except notably phytofluene, which is found in considerable amounts in tomatoes (22) and in smaller amounts in carrots (130) and which fluoresces six times more intensely than retinyl acetate (131). 

Thompson and Hatina (135) showed that the sensitivity of a fluorescence detector toward unesterified vitamin E compounds under normal-phase conditions was at least 10 times greater than that of a variable-wavelength absorbance detector. The relative fluorescence responses of the tocopherols at 290 nm (excitation) and 330 nm (emission), as measured by HPLC peak area, were a-T, 100 /3-T, 129 y-T, 110 and 5-T, 122. The fluorescence responses of the corresponding to-cotrienols were very similar to those of the tocopherols, and therefore tocotrienol standards were not needed for calibration purposes. The fluorescence detector also allows the simultaneous monitoring of ubiquinone derivatives for example ubiquinone-10 has been detected in tomato (136). 

For the determination of supplemental vitamin E in infant formulas, Woollard and Blott (222) employed a radially compressed Radial-PAK cartridge. This enabled lipid material to be rapidly cleared by stepping up the mobile-phase flow rate from 2 ml/min to 10 ml/min after elution of the a-tocopheryl acetate. Fluorescence detection, using a filter-type fluorometer, allowed the indigenous a-tocopherol to be conveniently estimated, while UV absorbance detection was used to quantify the a-tocopheryl acetate. Supplemental retinyl acetate could be assayed simultaneously with either added or indigenous vitamin E using the appropriate detection mode. With the aid of a dual-monochromator spectrofluorometer, a-tocopheryl acetate and a-tocopherol could be determined simultaneously with wavelengths of 280 nm (excitation) and 335 nm (emission), but the increased selectivity eliminated detection of the vitamin A esters (233). 

Reversed-phase HPLC with fluorescence detection is the preferred system for the routine determination of total a-tocopherol in vitamin E-supplemented foods after saponification. The use of NARP chromatography with a predominantly hexane mobile phase allows aliquots of hexane extracts of the unsaponifiable matter to be injected directly onto the column, thus avoiding the evaporation step necessary when a semiaqueous mobile phase is used (234). 

Fig. 13 HPLC of vitamin E. (A) Standards of vitamin E vitamers. Column, 5-p.m Supelcosil LC-Si (250 X 4.6-mm ID) mobile phase, isooctane/ethyl acetate (97.5 2.5), 1.6 ml/min fluorescence detection, excitation 290 nm, emission 330 nm. Peaks (1) a-tocopherol (2) a-tocotrienol (3) /3-tocopherol (4) y-tocopherol (5) /3-tocotrienol (6) y-tocotrienol (7) 5-tocopherol (8) 5-tocotrienol. (B) Saponified rice bran sample. Chromatographic conditions as in (A) except for mobile phase isooctane/ethyl acetate/2,2-dimethoxypropane (98.15 0.9 0.85 0.1). (From Ref. 228. AOCS Press.)...

Butter, whole milk Saponify (hot), extract un- LiChrosorb Si-60 Hexane containing a -Tocopherol, all-trans- UV and fluorescence detectors 250... 

MK Balz, E Schulte, H-P Thier. Simultaneous determination of a-tocopheryl acetate, tocopherols and tocotrienols by HPLC with fluorescence detection in foods. Fat Sci Techno] 95 215-220, 1993. 

CJ Hogarty, C Ang, RR Eitenmiller. Tocopherol content of selected foods by HPLC/fluorescence quantitation. J Food Comp Anal 2 200-209, 1989. 

Tocopherols can be analysed readily in vegetable oils by normal phase HPLC with UV or fluorescence detection. A standard method is described by AOCS (1998). 

FIGURE 5-52. Examples of normal phase separations, (a) Corn-oil tocopherols. Sample 10 /xL of corn oil in 100 /xL of mobile phase. Column Nova Pak Silica (4 jam), 3.9 mm ID x 150 mm. Mobile phase 0.3% isopropyl alcohol in isooctane. Flow rate 1.0 mL/min. Detection fluorescence 290 nm excitation and 335 nm emission. (b) Separation of vitamin E from vitamin A. Mobile phase 0.5% isopropyl alcohol in isooctane. Other conditions are the same as those in a with the exception that retinol was detected with 365 nm excitation and 510 nm emission, (c) Structures of compounds. 

Fig. 6.1. HPLC separation of a-tocopherol (peak 1), y-tocopherol (peak 2) and tocol (peak 3). (a) Using hexane and methyl-t-butyl ether (88 12), flow rate 2.0 ml/min, ambient temp., fluorescence detector, Lichrosorb Si60 column, (b) Using hexane and methyl-t-butyl ether (92 8), flowrate 2.0 ml/min, ambient temp., fluorescence detector, Lichrosorb Si60 column, (c) Using hexane and methyl-/-butyl ether (95 5), flowrate 2.0 ml/ min, ambient temp., fluorescence detector, Lichrosorb Si60 column.

Figure 11.3 Normal-phase HPLC separation of tocopherols (Ts) and tocotrienols (T3s) from barley kernels. A. Detection via fluorescence, ex 294 nm, em 326 nm, B. Detection via a charged aerosol detector. For FIPLC parameters see Moreau et al. (2006).

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. 

Sikorska, W. Gliszczynska-Swiglo, A. Khmelinskii, L Sikorski, M. 2005. Synchronous fluorescence spectroscopy of edible vegetable oils. Quantification of tocopherols. J. Agric. Food Chem. 53 6988-6994. 

Subsequent studies in our iaboratory (not yet pubiished) have revealed that these exceptionally high y-tocopherol values (obtained via UV-280 nm detection) were not accurate. When we repeated the HPLC analyses using fluorescence detection, the levels of y-tocopherol in corn fiber oil (extracted before and after heat-preatment) were similar to above reported levels in corn germ oil and corn kernel oil (< 500 mg/kg oil). 

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