Sterols, determination

The sterols present in cocoa butter make up about 0.3 to 0.4% of the oil and, as in other oils, they exist as free sterols, esters with fatty acids, and as glucosides and acylated sterol glucosides. Sterol determinations are normally carried out on the saponified material after isolation of the sterol fraction by TLC, although recovery from the TLC plate is lower than can be achieved by the use of HPLC, which also gives improved separation of the desmethylsterols and triterpene alcohols. Gas chromatography can be performed on the non-derivatized sterols provided the column is in good condition but acetylation (which can precede TLC) or silylation gives more consistently reliable resolution and peak shape. 

There are difficulties in the lack of an ideal internal standard for sterol determinations. Most used for this purpose are a-cholestanol (dihydrocholestanol), cholestane and betulin. Cholestanol is perhaps the most suitable where its resolution from cholesterol can be demonstrated. Cholestane and betulin, having none and two hydroxyl groups, respectively, do not migrate closely with the sterols on TLC and clearly their derivatization requirements do not match those of the analytes. 

Total Sterol Content. The gas liquid chromatographic method for sterol determination using an internal standard (cholestanol) is used to calculate the absolute (total) sterol content of an oil (68, 69). Gravimetric, enzymatic, colorimetric, and liquid chromatographic methods have also been reported (69). Limits (mg/100 g) are as follows (12) virgin olive oil, refined olive oil, and olive oil (mixture of refined and virgin) >100 crude olive-pomace oil >250 and refined ohve-pomace oil, olive oil and olive-pomace oil (mixture) >180. 

A.- ng deduction. This is an irreversible reaction which is a foremost determinant of the secretion rate of cortisol (double bonds and C-3 carbonyl). Catalyzed predominantiy by cortisone P-reductase and 3a-hydroxysteroid dehydrogenases, SP sterols result, although 5a sterols are more prevalent in the case of other glucocorticoids. Urocortisol and urocortisone result from the metabohsm of cortisol and cortisone, respectively. Compounds can be complexed to glucuronic acid at this point. 

For the determination of sterols and their esters the chromatogram is immersed in a 10% aqueous copper(II) sulfate solution and then heated to 105°C for 30 min. In this case green-yellow fluorescent chromatogram zones are visible in long-wavelength UV light (X = 365 nm) [6]. 

Several applications involve the removal of large amounts of triglicerides, including the determination of wax esters in olive oil (39), sterols and other minor components in oils and fats (40, 41), PCBs in fish (42), lactones in food products (43, 44), pesticides (45), and mineral oil products in food (46,47). Grob et al. (47) studied the capacity of silica gel HPLC columns for retaining fats, and concluded that the capacity of such columns is proportional to their size, although the fractions of the volumes that are then transferred to the GC system grow proportionally with the column capacity. For these reasons, 2-3 mm i.d. LC columns are to be preferred for LC-GC applications. 

K. Grob, M. Eanfranchi and C. Mariani, Determination of free and esterified sterols and of wax esters in oils and fats by coupled liquid chromatography-gas chr omatography , 7. Chromatogr. 471 397-405 (1989). 

R Lanuzza, G. Micali and G. Calabro, On-line HPLC-HRGC coupling and simultaneous transfer of two different LC fractions determination of aliphatic alcohols and sterols in olive oil , 7. High Resolut. Chromatogr. 19 444-448 (1996). 

Ethanol and choline glycerolipids were isolated from calf brain and beef heart lipids by PTLC using silica gel H plates. Pure ethanol amine and choline plasmalogens were obtained with a yield of 80% [74]. Four phosphohpid components in the purple membrane (Bacteriorhodopsin) of Halobacterium halobium were isolated and identified by PTLC. Separated phosphohpids were add-hydrolyzed and further analyzed by GC. Silica gel G pates were used to fractionate alkylglycerol according to the number of carbon atoms in the aliphatic moiety [24]. Sterol esters, wax esters, free sterols, and polar lipids in dogskin hpids were separated by PTLC. The fatty acid composition of each group was determined by GC. 

Using PTLC six major fractions of lipids (phospholipids, free sterols, free fatty acids, triacylglycerols, methyl esters, and sterol esters) were separated from the skin lipids of chicken to smdy the penetration responses of Schistosoma cercaria and Austrobilharzia variglandis [79a]. To determine the structure of nontoxic lipids in lipopolysaccharides of Salmonella typhimurium, monophosphoryl lipids were separated from these lipids using PTLC. The separated fractions were used in FAB-MS to determine [3-hydroxymyristic acid, lauric acid, and 3-hydroxymyristic acids [79b]. 

Unfortunately, not all products that are used in clinical trials are available in the United States. In a randomized, double-blind, multicenter European study, 1069 men with moderate benign prostatic hyperplasia were randomized to receive saw palmetto (Permixon" )1 160 mg twice daily (90% free and 7% esterified fatty acids) or finasteride 5 mg once daily for 6 months [32]. As determined by patients and physicians, Permixon offered similar improvement in symptoms related to benign prostatic hyperplasia compared to finasteride. Since Permixon is not available in the United States, it should be recommended to patients to use a product that is similar to Permixon that contains a standardized extract of saw palmetto containing 85-95% sterols and fatty acids [18]. 

No class reaction has been proposed for the measurement of total sterols. Instead, various fractionation methods, usually derived from the biochemical litreature, have been adapted to the concentrated materials collected from seawater. Certain of the more important sterols, particularly those used in the evaluation of water quality, have been determined by the use of a compound-specific reaction, after concentration from solution. Thus Wann et al. [405,... 

Most often the sterols have been collected by liquid-liquid extraction using petroleum ether and ethyl acetate [408], chloroform and methanol [409], -hexane [410,411] or chloroform [412,413]. After concentration, gas chromatography was generally used for the final separation and determination, although thin-layer chromatography has also been employed. The extra sensitivity of the electron capture detector could be used by reacting the concentrated sterols with bromomethyldimethylchlorosilane (BMDS) before separation and measurement [414],... 

While most of the interest in sterols has been in the materials in solution, Kanazawa and Teshima [417] have investigated the compounds present in suspension. The suspended matter was fractionated by filtration through a graded series of filters, the sterols removed by extraction with an organic solvent, and the final separation and determination was made by flame ionisation gas chromatography. 

Dreier et al. [44] determined sterols in lacustrine sediments. Samples of wet lacustrine sediments were heated under anoxic conditions at 150, 175, 200 and 250°C for five days at 175°C for five days with influx of potassium hydroxide and methanol to remove sterols and at 175°C for 12, 18, 24 and 48h, after which extraction was performed. Heating the sediment increased the amounts of extractable sterols provided that the temperature did not exceed 200°C, because degradation became rapid above that temperature. The behaviour of sterol ketones was similar, but the temperature limit was slightly higher. The various levels of the sterols extracted are tabulated 4-methylsterols had a high stability towards thermal degradation under the conditions used. 

In many tissues cholesterol and other sterols exist as a mixture of the free alchohol and its long chain fatty acid ester (esterified at position 3 of the steroid nucleus). The determination of the cholesterol content of a sample may involve the measurement of either of these two fractions individually or the total cholesterol. It is possible to precipitate free cholesterol by adding an equal volume of digitonin (1 gl-1 in 95% ethanol), a naturally occurring glu-coside, to form a complex that is insoluble in most solvents, including water. 

Gagosian, R.B., J.K. Volkman, and G.E. Nigrelli. 1983. The use of sediment traps to determine sterol sources in coastal sediments off Peru. Pp. 369-379 in Advances in Organic Geochemistry 1981, M. Bjoroy et al., eds., New York John Wiley and Sons. 

Krahn et al. [479] developed a similar multi-residue scheme for the determination of organochlorines and PAHs in sediments. In this scheme, the preparation is semi-automated with GPC to separate the biogenic material and the sulfur from both the PAHs and organochlorines in the samples. The sterols were separated and purified with an amino-cyano HPLC column prior to derivatiza-tion with bis(trimethylsilyl)trifluoroacetamide (BSTFA). 

The success of the Potts-Guy equation led many authors to advocate a single mechanism as the rate determining step for permeation through the skin barrier for all or at least a wide range of solutes diffusion was assumed to occur primarily via the interkeratinocyte lipids of the stratum corneum, a mixture of ceramides, fatty acids, and sterols. While from a macroscopic point of view these lipids may be modeled as a bulk solvent, on a microscopic scale they... 

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. 

Crist, B.V. Li, X. Bergquist, P.R. Djerassi, C. (1983) Isolation, partial synthesis, and determination of absolute configuration of pulchrasterol. The first example of double bioalkylation of the sterol side chain at position 26. J. Org. Chem., 48,4472-9. 

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