Chemical databases Zinc Database. Curated database of small molecules.
The phytosterols found in the highest abundance in most plants are sitosterol, campesterol, and stigmasterol, which are all classified as 4-desmethylsterols Fig. However, there are reportedly over different sterol structures that have been discovered in various plant species .
Plant sterols can exist in plants in their free form, as esters with fatty acids, ferulic acid, or p-coumaric acid, or as glycosides and acylated steryl glycosides. See also web pages on plant sterols and oxidized plant sterols elsewhere on this site.
Structures of some common phytosterols and phytostanols Phytosterols are well-known for their ability to lower blood cholesterol by competing with absorption of cholesterol from the diet and reabsorption of bile cholesterol . In the yearthe FDA approved a health claim relating phytosterol ester or phytostanol ester consumption to reduced risk for coronary heart disease, in foods such as margarines and spreads, salad dressings, snack bars, and dietary supplements .
However, the FDA has recently proposed to extend the health claim to include free phytosterols and an increased number of conventional foods . The European Union EU has also approved similar health claims, and has established labeling guidelines for these foods .
Labeling requirements by both the FDA and the EU include a declaration of the content of plant sterols or sterol esters, and include specifications for phytosterol or phytostanol composition of the esters.
Thus, there is increased interest in qualitative and quantitative analysis of phytosterols in food products.
Gas chromatography GC is the most common method for analyzing phytosterol content and composition . There are HPLC methods available for separating and quantifying the various forms of phytosterols, such as free sterols, steryl fatty acid esters, steryl glycosides, acylated steryl glycosides, and hydroxycinnamic acid esters of sterols [1,10].
However, with over different structures, GC, often combined with mass spectrometry, is the best and most widely used tool for the chromatographic separation, identification, and quantification of phytosterols.
Most standardized methods are somewhat tedious in terms of the amount of glassware, sample, and solvent required and in the number of steps in the procedure, which make it difficult to analyze multiple samples at the same time.
These procedures have been modified by users to require smaller sample sizes, less solvent, and to eliminate some of the time-consuming cleanup steps .
However, for the most part, the latest techniques employ the same basic steps that the traditional methods use: The intent of this chapter is to describe these steps in both an informative and practical manner. Sample Preparation The first and most important step in the analysis of phytosterols is sample preparation.
The type of sample preparation needed depends on the sample matrix whole plant tissue or extracts, food product, supplement, vegetable oil, blood serum, etc. In some samples, such as blood serum, where phytosterols are present in very low concentrations, lipid extraction to concentrate the phytosterol-containing lipid fraction away from proteins and other compounds may be a necessary first step .
In other cases, the analyst may want to quantify other lipids in the sample, or the various forms of phytosterols, i. However, for many matrices, direct alkaline or acid hydrolysis has been used successfully [15,17].
If free sterols and sterol esters are the only sterol forms anticipated in the sample, such as in a refined oil or fat sample, then alkaline hydrolysis saponificationfollowed by extraction of the unsaponifiable material, is sufficient sample preparation for analysis of total phytosterols.
However, in samples such as cereal products, the phytosterols may be trapped within the carbohydrate matrix, or may have bonds that are not hydrolyzed by saponification, such as the glycosidic bonds in steryl glycosides.
In this case, acid hydrolysis prior to saponification is necessary . Sample Amount The sample amount is dependent on the concentration of phytosterols in the sample, the ability to completely hydrolyze the sample and liberate all of the phytosterols, and the quantification limits of the GC.
Therefore, sample weight needs to be optimized for each product. An estimate of the phytosterol concentration in a sample can usually be determined based on a survey of the literature for the same or similar sample types.
Laakso  suggests a sample size containing 1 mg phytosterols, but that method is targeted for foods that are supplemented with high concentrations of phytosterols. Internal Standard For phytosterol analysis, the addition of an internal standard IS is necessary to obtain accurate qualitative and quantitative results by GC.
Since retention times often shift from run to run, the main function of the IS is to aid in the identification of phytosterols in unknown samples, based on the relative retention time RRT of the phytosterol compared to the internal standard. In addition, when using a split injection, which is typically the case in phytosterol analysis, the amount of sample entering the column will vary slightly from injection to injection, so the IS is used to compensate for this variation.
Finally, large variations in peak response area for the IS is a good indicator that there is a problem with the extraction protocol or with the GC injection. The IS should be a compound that is commercially available in pure form, and that is similar in structure to phytosterols so that it will be extracted quantitatively with the sample phytosterols, and elute from the GC column within the time frame and with a similar detector response to other phytosterols, without interfering with the retention time of sample peaks.
The IS is typically added to the sample prior to alkaline or acidic hydrolysis, so that it undergoes the same extraction conditions as the sample phytosterols. However, the IS may also be added after extraction but prior to derivatization. Structure of sterols and other compounds commonly used as internal standards.
However, if there is a problem with the silylation reaction where samples are incompletely silylated, cholestane will not be affected and therefore will not serve as an indicator of the problem. Dihydrocholesterol is a good choice, but it is a hydrogenation product of cholesterol, so it could interfere with this peak in samples that contain cholesterol and have been subjected to hydrogenation.
Epicoprostanol does not occur naturally in plants, elutes early, and thus does not usually interfere with phytosterol peaks .
After solvent removal by rotary evaporation, saponification of the lipid sample proceeds as described below.The “compound” which is set to become the drug molecule undergoes safety tests and a series of experiments to prove that it is absorbed in the blood stream, distributed to proper site of action in the body, metabolized sufficiently and demonstrates its non-toxicity thus, can .
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Explore and learn more about Conference Series LLC . Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at .