HPLC tocopherol

Antioxidants (qv) have a positive effect on oils when present in the proper concentration. Sterols and tocopherols, which are natural antioxidants, may be analy2ed by gas-Hquid chromatography (glc), high performance Hquid chromatography (hplc), or thin-layer chromatography (tic). Synthetic antioxidants maybe added by processors to improve the performance or shelf life of products. These compounds include butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), / fZ-butyUiydroquinone (TBHQ), and propyl gallate. These materials may likewise be analy2ed by glc, hplc, or tic. Citric acid (qv), which functions as a metal chelator, may also be deterrnined by glc. 

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). 

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. 

BHA, BHT, PG, TBHQ and tocopherols) a variety of stationary phases, mobile phases and detectors can be used [711]. Common antibacterials such as carba-dox, thiamphenicol, furazolidone, oxolinic acid, sul-fadimethoxine, sulfaquinoxaline, nalidixic and piromidic acid can be analysed by GE-RPLC-UV (at 254 nm). Collaborative studies have been reported for the HPLC determination of the antimicrobial sodium benzoate in aqueous solutions [712], Plastics devices used for field collection of water samples may contain polymer additives (such as resorcinol monobenzoate, 2,4-dihydroxybenzophenone or bisphenol A) or cyanobac-terial microcystins [713],... 

Krucker, M., Lienau, A., Putzbach, K., Grynbaum, M. D., Schuler, P., and Albert, K. 2004. Hyphenation of capillary HPLC to microcoil 11 NMR spectroscopy for the determination of tocopherol homologues. Anal. Chem. 76 2623-2628. 

Figure 8 Effect of LDL supplementation with a-tocopherol in vitro determined by HPLC and PCL techniques. The surplus in comparison to the initial value is represented for each parameter. (From Ref. 28.)...

Figure 9 Antiradical capacity in the lipid phase of blood plasma (ACL) determined with the PCL method versus vitamin E (VE) as a sum of a- and y-tocopherols determined with HPLC. (From Ref. 28.)...

Figure 17 Time course of the antiradical parameters ACL0 and ACW of LDL measured by the PCL method its a-tocopherol content (AT) measured by the HPLC technique and conjugated dienes (LDL-abs. at 234 nm) during Cu2+-initiated oxidation in vitro. (From Ref. 36.)...

The effect of vitamin E supplementation on a-tocopherol and /(-carotene concentrations in tissues from pasture- and grain-fed cattle was also elucidated with HPLC analysis. The investigation was motivated by the fact that a-tocopherol influences beneficially meat colour and stability [53], and the presence of /(-carotene can modify the amount of a-tocopherol in tissues [54], Samples were extracted with hexane and the concentration of /(-carotene was assessed by HPLC. Some data are listed in Table 2.20. It was concluded from the data that... 

GC analyses of the pupal secretion of E. borealis have indicated the presence of vitamin E acetate and other tocopherol derivatives [49,50]. However, in tests with ants, these compounds proved to be essentially inactive, whereas the secretion itself was potently deterrent. To find and identify the active components in the pupal Epilachna borealis secretion, NMR spectroscopic studies on the fresh secretion were carried out. One and two-dimensional NMR experiments revealed that the tocopheryl acetates account for only a relatively small percentage of the beetles5 total secretion (20%), whereas the major components represented a group of previously undetected compounds. By analysis of the COSY, HSQC and HMBC spectra of the mixture, these components were shown to be esters and amides derived from three (co-l)-(2-hydroxyethylamino)alka-noic acids 44-46. HPLC analyses coupled to a mass spectrometric detector revealed that the secretion contain a highly diverse mixture of macrocyclic polyamines, the polyazamacrolides (PAMLs) 47-52 (Fig. 8). 

Strohschein, S., Pursch, M., Lubda, D., and Albert, K., Shape selectivity of C-30 phases for RP-HPLC separation of tocopherol isomers and correlation with MAS NMR data from suspended stationary phases. Anal. Chem., 70, 13, 1998. 

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]. 

HPLC has been applied to lipid analysis mainly in consideration of the necessity to avoid high temperatures, so at the very beginning, its applications dealt with thermally unstable molecules (e.g., tocopherols, phenolics, oxidation products) and often it was used as an ancillary technique, as a preparative step prior to MS analysis. The limits were in the high volume of the HPLC band that strongly limited the possibility to transfer it to a GC or to a MS. Only in the last 20 years or somewhat less, this kind of hyphenation has become commercially available. 

The development of HPLC techniqne led to an increase in the number of scientific papers dealing with phenolic evalnation in foods and, in the meantime, it also improved the nnmber and type of foods in which phenolic snbstances were evalnated. Despite the high number of scientific papers, HPLC analysis of phenolic snbstances, except for wine and tocopherols in fats and oils, has never became an official method of analysis, so the nnmber of scientific papers has ever more increased. 

The correlation between the TEARS assay and MDA dnring oxidation of edible oils may be complicated by the presence of tocopherols (e.g. Vitamin E, 21) . An evaluation was carried of MDA, determined by an independent method , and TEARS as indices for direct oxygen uptake of edible oils and unsatnrated fatty acids. The linear increase of MDA and TEARS with oxygen consumption of soybean oil, in a closed vessel at 170 °C, stops when the latter value reaches 500 p.molL, when both MDA and TEARS start to decrease on further O2 consumption. The same process carried out at 40 °C, using 2,2 -azobis(2,4-dimethylvaleronitrile) (171) as initiator, shows linearity up to 1500 p,molL O2 consumption . A similar behavior is observed for nnsatnrated fatty acids snch as oleic, linoleic and linolenic acids . On the other hand, depletion of Vitamin E (a-tocopherol, 21) and its analogs y- and 5-tocopherol (172, 173) present in the oil show a linear dependence on O2 consumption of the oil, np to 1800 p,molL . This points to the consumption of these antioxidants, and especially 21, as a good index for the O2 uptake in oils at high temperature. The determination of the tocopherols is carried ont by HPLC-FLD (Xex = 295 nm, Ah = 325 nm) . ... 

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

Manzi, P., Panfili, G., and Pizzoferrato, L. 1996. Normal and reversed-phase HPLC for more complete evaluation of tocopherols, retinols, carotenes and sterols in dairy products. Chroma-tographia 43 89-93. 

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... 

There are numerous papers that refer to the quantification of tocopherols and tocotrienols using HPLC methods. Normal-phase HPLC methods with a silica column as well as reversed-phase HPLC methods with a C18 column are commonly used. A silica normal-phase column is able to separate all eight tocopherols and tocotrienols in a typical chromatographic procedure. Because plant tissues possess most forms of tocopherol and tocotrienol, it is recommended that the normal-phase HPLC method be applied to food samples from plants. In the reversed-phase HPLC method, [3- and y-tocopherol and (3-and y-tocotrienol are not usually completely separated. This method can be used in animal tissues, which either lack or have reduced levels of [3- and y-tocopherol and (3- and y-tocotrienol. The resolution of the normal-phase HPLC method is higher than that of reversed-phase HPLC method however, the reversed-phase HPLC column is more long-lasting than the normal-phase HPLC column (see Critical Parameters and Troubleshooting). 

Compared to refined vegetable oils, the compositions of crude vegetable oils and oil and fat products are more complicated. These samples contain proteins, carbohydrates, and minerals that interfere with HPLC separation and reduce the lifetime of the HPLC column. These compounds need to be largely eliminated from the extract before HPLC analysis. Saponification and heating are used to weaken sample matrices to allow the solvent to fully access all tocopherols and tocotrienols of the sample. Liquid/liquid extraction is used to remove these polar compounds from the organic solvent layer that contains tocopherols and tocotrienols. The normal-phase HPLC method is usually used for crude vegetable oils and vegetable oil products reversed-phase HPLC can be used for animal fat products. 

As the levels of tocopherols and tocotrienols in meat samples are usually lower than in oil and fat samples, a larger sample size is needed in the sample preparation. The meat sample is homogenized to weaken the sample matrix. As in Basic Protocol 2, saponification, heating, and liquid/liquid extraction are used to increase the recovery and remove interference compounds. Satisfactory results can be achieved using a reversed-phase HPLC method. 

Cereals and nuts are blended to fine particles before sample preparation. Saponification, heating, and liquid/liquid extraction are employed in the sample preparation to weaken sample matrix and eliminate interference compounds. Most cereals and nuts contain various forms of tocopherol and tocotrienol. The normal-phase HPLC method is recommended for these types of samples. 

In normal-phase HPLC on a silica column, separation is based on the number and position of methyl substituents on the chromanol ring. In reversed-phase HPLC on a Cl8 column, separation is based on the structure of the side chain and the number of methyl substituents. It is therefore difficult to completely separate [3-and y-tocopherol and (3- and y-tocotrienols by reversed-phase HPLC, because both have the same side-chain structure and number of methyl substituents on the chromanol ring. Only six peaks are usually found in the reversed-phase HPLC method. Thus, reversed-phase HPLC is recommended for samples from animal tissues, which contain little or no P and y vitamers. 

Mobile phase. Hexane is the major component of mobile phase in the normal-phase HPLC method. The percentage of hexane is up to 99% for silica normal-phase columns. Ethyl acetate, acetic acid, methanol, and isopropanol are used as modifier components (Shin and Godber, 1994). The flow rate of mobile phase is usually controlled at 1 to 1.5 ml/min to completely separate all eight tocopherols and... 

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-... 

Evaporation and redissolving. The solvent of the combined upper layer is evaporated under nitrogen flow or low-temperature vacuum distillation. An oily material appears after it is dried. A precisely measured aliquot of mobile phase is normally used to redissolve theextract. These procedures are intended to not only increase the concentration of tocopherols and tocotrienols to the measurable level of the detector, but also to avoid uncertain volume change of organic layer during extraction, which results in inaccurate results. The redissolved sample is transferred to a vial for HPLC analysis. 

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.

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