Sterols, HPLC analysis

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

TLC is often used as a preparative technique to isolate individual lipid fractions for subsequent studies by a variety of analytical procedures. Preparative isolation of the sterol (Chitwood et al., 1985 Shetty et al., 1990) and triacylglyc-erol (Horutz et al., 1993) fractions have been reported. Preparative isolation of sterols may allow these lipids to be analyzed further by argentation TLC (Ditullio et al., 1965 Morris, 1966) or by GLC (Shetty et al., 1990). Moreover, preparative isolation of triacylglycerols will allow for TLC analysis of glyceryl ethers (Snyder, 1971) and HPLC analysis of specific triacylglycerols (Horutz et al., 1993). Experiment 7 is concerned with a preparative procedure for the analysis of sterols by argentation TLC. 

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... 

Not only fatty acids but every unsaturated lipid molecule can undergo oxidation. So sterol oxidation products have also been studied in this case, HPLC is used both as a preparative step and for analytical purposes [33,34], HPLC as a preparative step was proposed by several researchers to improve the speed of analysis in the case of sterols, stigmastadienes, and waxes [35,36], Stigmastadiene, which is used as a marker in the refining process applied to vegetable oils, is determined by capillary GC however, the International Organization for Standardization (ISO) method (ISO 15788-2 2003) [37] uses HPLC as a rapid screening technique. 

There are cases where HPLC separation is performed not in order to quantify the alcohols but as a technique for the purification of the analytes to be subjected to further instrumental analysis. This is the case, for example, with the identification and determination of the structure of an abscisic acid in starfruit extract (Averrhoa carambola L.). The separation and purification of the analytes was carried out also with HPLC using a mobile phase of diethyl ether, whereas the structure was elucidated by H and UC-NMR (6). In a similar way, to separate the sterols and alkanols from the unsaponifiable matter from olive oils on a silica column, a gradient composed of hexane/diethyl ether was chosen in an offline system (7), whereas an online HPLC-HRGC system uses as its mobile phase hexane/isopropanol (8). 

Recently Amelio et al. (7) described a method which may find routine applications and which makes use of SPE for the separation and clean-up of the unsaponifiable from olive oil, from which the aliphatic alcohols are separated by means of HPLC (besides sterols and the two triterpenic dialcohols erythrodiol and uvaol). The alkanols are then derivatized and analyzed by means of HRGC. The use of an autosampler and a fraction collector for use with HPLC permits a considerable automatization of the analysis. (Reprinted from Ref. 1, p. 581, by courtesy of Marcel Dekker Inc.)... 

Sterol analysis, fatty acid analysis of the whole fat and of the acids at the 2-position, and triglycerides by GC, can be very useful in determining adulteration of and by animal fats. In the future HPLC of triglycerides is likely to provide an even better method for some purposes, but much more data need to be collected before this can be evaluated. 

RP-HPLC The most commonly used methods for the analysis of SFs are reverse-phase liquid chromatography with UV-detection, which allow the separation of SFs with different sterol moieties. The number of components separated is naturally dependent on the type of column used, but most reverse-phase columns provide a full separation of at least five to six components of y-oryzanol. Cl 8-columns are most abundantly applied for the separation of SFs, but other reverse-phase... 

To separate free sterols and other non polar soluble component of the plant biomass from sapogenin steroid extraction was done in the following method. 1 gram (accurately weight) of dry biomass was extracted 3 times using a vortex-mixer with 5 ml CHCh for each. The residue was hydrolyzed with 2 N HC1 (100°C, 2 hours), cooled, neutralized with 10N NaOH, extracted 3 times with 5 mi CHCh- The chloroform phase was collected and evaporated in N2 to dryness. The hydrolysate extract was dissolved in 1 ml CHCL3 (accurately) before analysis (for TLC) or in methanol (for HPLC). 

Both normal-phase and reversed-phase HPLC have been applied in vitamin E analysis. Reversed-phase HPLC is unable to completely separate all tocopherols and toco-trienols. Because (1- and y-vitamers have very similar structures, their separation cannot be obtained with reversed-phase HPLC. It is, however, applicable when only tocopherols or a-tocopheryl esters are analyzed (Gimeno et al., 2000 Iwase, 2000). There are reversed-phase methods to analyze tocopherols together with other lipid constituents from biological and food samples such as carotenoids (Epler et al., 1993 Salo-Vaananen et al., 2000), ubiquinols and ubiquinones (Podda et al., 1996) or sterols (Warner and Mounts, 1990). 

Sterols of marine invertebrates have been found to comprise most complex mixtures more than 120 sterols have been isolated and their structures determined [1]. Analysis of these sterols has been performed largely by GC, GC/MS and HPLC. Stereochemical details were determined by NMR or by synthetic work. Fig. 1 demonstrates the effectivity of a capillary colunrn a and b showing the GC analysis of a sterol fraction obtained from the sponge, Hymeniacidon perleve using a packed and a capillary column, respectively [2]. 

Capillary GC analysis of the sterols of the Caribbean Xestospongia species, containing predominantly xestosterol (27), indicated a new sterol, xestospongesterol (31) which was separated by reversed-phase HPLC [22]. The structure was determined by mass spectrometry and NMR as a hitherto unprecedented quadruply biomethylated sterol. From the Indopacific sponge Strongylophora durissima, which has been reported to contain over 90% of strongylosterol, a new sterol named... 

The sterols include that group of compounds which contain an alcoholic hydroxyl group at C3 and a branched aliphatic chain of at least eight carbon atoms at C17. These compounds, which include cholesterol and its derivatives, can occur as free alcohols or as long-chain fatty acid esters. A number of HPLC techniques have been used in the analysis of the sterols including reversed phase, non-aque-ous reversed phase, normal phase, argentation and combinations of the above. 

Olive oil is often illegally adulterated with other less expensive vegetable oils. Oils widely used for this purpose include olive pomace oil, corn oil, peanut oil, cottonseed oil, sunflower oil, soybean oil, and poppy seed oil. °° Among the varions chemical and physical methods employed toward the detection of the adulteration of olive oil by low-grade olive oils and seed oils are (a) Sterol analysis (presence of stigmasterol and 3-sitosterol), (b) alkane analysis (C27, C29, and C31), (c) wax and aliphatic alcohol analysis, (d) fatty acids/(with HPLC) trans fatty acid, and (e) Triacylglycerol. 

The main area of application of HPLC in lipid analysis is the determination of larger molecules like triglycerides and phospholipids, while the analysis of smaller lipid classes like sterols and fatty acids is usually carried out using gas chromatography (GC), though it is possible to analyze these molecules using LC too. The subject can be divided in two topics separation of simple lipid classes from a lipid fraction and separation of the individual components, like the different sterols of a specific fraction. 

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. 

Segura-Carretero, A., Carrasco-Pancorbo, A., Cortacero, S., Gori, A., Cerretani, L., and Femdndez-Gutifeez, A. 2008. A simphfied method for HPLC-MS analysis of sterols in vegetable oil. Eur J. Lipid Sci. Technol. 110 1142-1149. 

One aspect of LC-MS which is rapidly being developed is the HPLC-MS and has great promise for the analysis of many compounds. The primary obstacle to easy coupling of HPLC to MS arises from the fact that the flow for conventional HPLC columns is approximately an order of magnitude greater than can be accommodated by the commonly used ion sources and pumping systems. The requirement for a transfer of a maximal quantity of sample and a minimal amount of solvent into the ion source of the mass spectrometer has necessitated the development of different interfaces. The need for introduction of smaller amounts of HPLC effluents into a mass spectrometer has stimulated the development of narrow bore columns. The application of HPLC-MS with a direct liquid introduction has been applied to the identification of marine sterol peroxides by Djerassi and Sugnaux in 1982. An ultrasphere ODS column of i.d. 5 ixm and methanol-water (99 1, v/v) solvent system were used. 

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