Sterol ester, analysis

The analysis of sterols, sterols esters, erythrodiol and uvaol, and other minor components of oils and fats, is usually carried out by normal-phase HPLC-HRGC by using a loop-type interface and the concurrent eluent evaporation technique, as reported in the papers cited by Mondello et al. (48) (up to 1995) and in more recent papers (49, 50). More recently, reversed-phase LC-GC methods have been... 

Louter, A.J.H., Bauer-Plank, C.G., Duchateau, S.M.J.E. 2002. Analysis of plant sterol esters as functional food ingredients. Lipid Technol. 14, 87-90. 

Alcoholysis. Methanolysis of triacylglycerols is used to prepare methyl esters for fatty acid analysis, a process frequently referred to as transesterification. This can be acid-or base-catalyzed, the method being chosen to avoid modifying acid-or base-sensitive fatty acids and to minimize reaction times. Sterol esters of fatty acids react more slowly than triacylglycerols, and samples containing them require more vigorous reaction conditions. The preparation of methyl esters from oils and fats for GC and GC-MS analysis has been extensively reviewed (19, 22, 23). 

The analysis of sterols and sterol esters has been proposed as one way to identify oils in blends (311, 312). Johansson and Croon (313) discussed the use of 4-des-methyl-, 4-monomethyl-, and 4,4-dimethylsterols in characterizing different vegetable oils, and the results are summarized in Table (8). The levels of total sterols and sterol classes as well as the relative distribution of the individual sterol members vary between oils. The presence of steradienes can also be used as a marker for the presence of refined oils (314, 315). High oleic acid oils can easily be used to adulterate olive oil. The presence of rapeseed oil in other oils can be detected by the analysis of brassicasterol and its dehydration product, campestatriene. The presence... 

When sterol analysis is performed after saponification of ester bonds, a total value is obtained for the level of each sterol by GC analysis. Free sterols can be separated from sterol esters before saponification by means of thin-layer chromatography (326) or solid phase extraction (327-330) using hexane to elute the sterol esters and diethyl ether or a mixture of diethyl ether and hexane to elute the sterols. Once separated, sterol esters need be saponified to release sterols before gas... 

Fatty acids are GC analysed after their conversion to non-polar derivatives such as methyl or silyl esters (Evershed, 1992a). Often, esters are prepared as derivatives of the fatty acids liberated from wax esters or sterol esters by hydrolysis (Summchen et al., 1995 Szafranek Synak, 2006). The details and other important considerations of fatty add analysis have been well summarized by Christie (1994). Generally speaking, fatty add methyl esters (FAME) can be GC analysed employing non-polar stationary phases like other cuticular wax components. Unsaturated compoimds should be analysed on polar stationary phases (e.g. Carbowax). The determination of FAME often requires high resolution for the separation of positional and configurational isomers. The elution order is as follows (Evershed, 1992a) ... 

Analysis of the components of other fractions of simple lipids like free sterols, sterol esters, and wax esters by means of HPLC has some drawbacks, and it is usually performed using high-temperature GC. However, the most remarkable field of application of HPLC is the analysis of oxidized sterol and their esters, of biological interest, that used to be performed earlier using RP-LC with postcolumn fluorometric detection and is nowadays be carried out using LC coupled to MS, providing structural information on these complex derivatives. 

The simplest methylation method uses KOH (2 M in methanol). It is recommended, for instance, in the analysis of vegetable oUs or virgin ohve oil with reduced acidity (small amounts of free acids) by several European standards. The reaction is rapid and occurs at room temperature. It must be pointed out that sterol esters and waxes, as well as free FAs, do not react under these conditions, as mentioned earher. 

The liver (hepatopancreas or digestive gland) of freshwater snails contains mainly free sterols, triacylglycerols, and sterol esters (Figure 15.3, lane 2). Additionally, diacylglycerols may also be detected. The study described here is based on unpublished observations in our laboratory on the TLC analysis of the common viviparid snail, Campeloma decisum, found in freshwater ponds in the United States. Similar experiments can be done with the hepatopancreas of other available marine or freshwater snails (see Chapter 4). For this experiment, obtain 100... 

The potential for the preservation of lipids is relatively high since by definition they are hydrophobic and not susceptible to hydrolysis by water, unlike most amino acids and DNA. A wide range of fatty acids, sterols, acylglycerols, and wax esters have been identified in visible surface debris on pottery fragments or as residues absorbed into the permeable ceramic matrix. Isolation of lipids from these matrices is achieved by solvent extraction of powdered samples and analysis is often by the powerful and sensitive technique of combined gas chromatography-mass spectrometry (GC-MS see Section 8.4). This approach has been successfully used for the identification of ancient lipid residues, contributing to the study of artifact... 

The choice of an appropriate derivatisation of BAs is a crucial point for a good GC analysis [15]. Unconjugated BAs are commonly analysed as methyl ester-trimethyl-silyl (TMS) ether derivatives, although interference from plasma sterols may occur at the LCA retention time. Use of -butyl ester-TMS ether derivatives allows a better chromatographic separation between BAs, which elute later, and sterols. The advantage of this derivative procedure is the simplification of plasma pre-treatment. 

This specific derivatisation procedure allows a better separation between -butyl ester-TMS ether of LCA and TMS ether of cholesterol and /1-sitosterol (Fig. 5.4.4). Therefore, even if samples are not completely purified in the pre-analytic phase, sterols do not interfere in the quantitative analysis of plasma BAs, being well resolved on GC. Besides, the -butyl ester-TMS ether of nor-CA is eluted later than the sterols and can be used safely as internal recovery standard. 

The major limitation of GC is the requirement for heat stability and volatility of the sample. Obviously, compounds that decompose at elevated temperatures (below 250°C) cannot normally be subjected to GC analysis. Many compounds of biochemical interest are not volatile in the useful temperature range of GC (up to about 200-250°C). Such compounds can often be converted to volatile derivatives. Hydroxyl groups in alcohols, carbohydrates, and sterols are converted to derivatives by trimethylsilylation or acetylation. Amino groups can also be converted to volatile derivatives by acetylation and silylation. Fatty acids are transformed to methyl esters for GC analysis, as described in Experiment 6. 

Global lipid profiling methods typically cover molecular lipid species from the major classes of lipids such as cholesteryl esters, ceramides, mono-(MG), di- (DG), and TGs, and membrane PLs, for example, sphingomyelins (SMs), PCs, phosphatidylethanolamines (PEs), PSs, and lysophospholipids. In targeted lipid analysis, specific lipid classes that are poorly covered by the global profiling methods are usually analyzed. These lipids include steroids, sterols, bile acids, fatty acids, signaling lipids such as eicosanoids, and ceramides, as well as polar lipids and inositol lipids. 

The analysis of lanolin has concentrated on the lanolin alcohols (the unsaponifiable fraction of lanolin) and lanolin acids produced by hydrolysis rather than the esters in lanolin itself.20 Lanolin alcohols belong to three major groups (1) 69 aliphatic alcohols from C12 to C36, (2) sterols (cholesterol and dihydrocholesterol), and (3) trimethyl sterols (lanesterol, dihydrolanesterol, agnosterol, and dihydroagenosterol).21 The latter have been incorrectly termed triterpenoids. The relative proportion of each group is 22% (w/w) aliphatic alcohols, 35% (w/w) sterols, and 38% (w/w) trimethyl sterols.8... 

Much of the sterol component of shea butter is present as esters of cinnamic acid, which are less readily saponified than esters with glycerol (Peers, 1977) published data for this fat might therefore underestimate the hue value. Surprisingly measurement of cinnamic acid has not been used to test for the presence of shea butter in mixtures. Triterpene alcohols similar to those found in shea also occur in sal fat. Homberg and Bielefeld (1982) showed the presence of triterpene alcohols in illipe and sal fats and in commercial CBEs, and their analysis was proposed as a qualitative measure to detect cocoa butter adulteration. 

Standard methods of analysis of total sterol content of oils involve saponification of the oil, followed by extraction and isolation of total sterols from the unsaponihable fraction by thin layer chromatography (TLC) (AOCS, 1998). Quantification of individual sterols involves silylation of the sterol fraction and analysis by gas chromatography (GC). Sterols and steryl esters in oils and fats can be analysed by LC-GC after silylation or acylation of the free sterols (Artho et al., 1993). An alternative approach to the analysis of intact steryl esters involves separation of sterols and steryl esters by solid phase... 

To etherify sterols, it is recommended that the reaction is carried out in diethyl ether in the presence of potassium f rf.-butanolate. On the micro-scale the yields range from 78 to 90% [61]. Derivatives for the trace analysis of hydroxy compounds are prepared in a similar manner to esters (see p. 61) by reaction with a-bromopentafluorotoluene catalysed with potassium carbonate [62] (Scheme 4.12). The phenolic substrate is dissolved... 

Analytical methods for plant sterol analysis are commonly based on procedures used for cholesterol analysis. However, a significant shortcoming of these methods is the fact that cholesterol occurs only as free cholesterol and fatty acid esters. Therefore, the analytical methods optimized for cholesterol analysis are not suitable, or only suitable with some restrictions, for the analysis of conjugates found only in plants (SFs, SGs, and ASGs). Further, the methods described below only give the total amount of plant sterols and no information of the different sterol species found in the samples. However, if detailed information about the sterol composition is not required, and the amount of sterols to be analyzed is sufficiently high for these less sensitive but simpler methods, they provide a less laborious alternative for the analysis. 

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