Tocopherol, analysis

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). 

Selected applications of coupled SEE-SEC consider the analysis of tocopherols in plants and oil by-products (65) or the analysis of lipid-soluble vitamins (66) by using a dynamic on-line SEE-SEC coupling, integrated in the SE chromatograph, based on the use of micropacked columns. 

E. Ibanez, J. Palacios and G. Reglero, Analysis of tocopherols by on-line coupling supercritical fluid extraction-superaitical fluid chromatography , ]. Microcolumn Sep. 11 605-611 (1999). 

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). 

McClure, TD and Liebler, DC, 1995. Electron capture negative chemical ionization mass spectrometry and tandem mass-spectrometry analysis of beta-carotene, alpha-tocopherol and their oxidation products. J Mass Spectrom 30, 1480-1488. 

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). 

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 main problem in the vitamin E analysis is that it is easily oxidized, thereby an antioxidant, such as butyl hydroxy toluene (BHT) or ascorbic acid, is added to prevent degradation during the extraction step. The traditional method for extraction of tocopherols and tocotrienols in foods is solvent extraction (like soxhlet) and saponification with KOH [457,458]. Some authors have recently proved that saponification is not necessary [459-462], nevertheless, it has been widely applied until the present day. 

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

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... 

Table D1.5.1 Conventional Analysis Methods of a-Tocopherol and a-Tocopheryl Acetate in the AOAC International Official Methods of Analysis...

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. 

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