Tocopherols, HPLC analysis

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... 

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. 

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

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. 

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. 

Thirty minutes is sufficient for sample preparation beginning with refined vegetable oils. However, the other sample preparations take up to 120 min because of the incubation, re-extraction, and evaporation steps. Usually, a batch of samples is prepared at the same time to reduce the preparation time per sample. The running time for HPLC analysis is approximately 18 min for normal phase or 14 min for reversed phase in order to quantify all tocols. The running time for normal phase could be cut to 12 min for samples without 5-tocopherol and tocotrienol. 

Rustan et al. (123) used an isocratic HPLC method for the determination of alpha-, gamma-, and delta-tocopherol, BHT, BHA, PG, OG, DG, NDGA, TBHQ, ascorbyl palmitate, and beta-carotene in foods. An RP18 column was used in experiments, and seven mobile phases based on various combinations of acetonitrile, methanol, water, and tetrahydrofuran were tested. Trials with carrot juice, dried milk formula for infants, and aperitif cakes showed that all 12 antioxidants could be determined by a single isocratic HPLC analysis. The optimum mobile phase... 

NK Andrikopoulos, H Brueschweiler, H Felber, C Taeschler. HPLC analysis of phenolic antioxidants, tocopherols and triglycerides. J Am Oil Chem Soc 68 359-364, 1991. 

Lietz, G. Henry, C.J.K. 1997. A modified method to minimise losses of carotenoids and tocopherols during HPLC analysis of red palm oil. Food Chem. 60 109-117. 

Ryynanen, M. Lampi, A.M. Salo-Vaananen, P. Olliainen, V. Piironen, V. 2004. A small-seale sample preparation method with HPLC analysis for determination of tocopherols and tocotrienols in eereals. J. Food Comp. Anal. 17 749-765. 

Katsanidis, E. and Addis, P.B. 1999. Novel HPLC Analysis of Tocopherols, Tocotrienols, and Cholesterol in Tissue. Free Radic. Biol. Med. 27 1137-1140. 

Figure 7.2. HPLC analysis of tocopherols and sterols using normal-phase chromatography. Reprinted with permission from reference 9.

Carotenoid pigments were extracted by chloroform-acetone-isopropyl alcohol /2 l l/ after acetone extraction as described earlier. Lipids of different parts were extracted and their fatty acid composition were analysed by GLC. Tocopherols of different parts of the fruits were extracted, saponified and prepared for HPLC analysis according to Speek and co-workers. Organic acids were prepared by a method described previously. Following preparation the samples were redissolved in a minimal volume of the HPLC eluent. 

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). 

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). 

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. 

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... 

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