Unsaponifiable analysis

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.)... 

Saponification of the sample simplifies the analysis by converting the vitamin A esters to retinol. The unsaponifiable material is extracted with hexane, or a predominantly hexane solvent mixture, which is compatible with the nonpolar mobile phase (146,153,156). In vitamin A-fortified foods there is no need to concentrate the unsaponifiable extract—an aliquot can be injected directly into the chromatograph (153). 

The removal of triglycerides from the food sample by saponification provides the opportunity to utilize reversed-phase chromatography. The unsaponifiable matter is conventionally extracted into a solvent [e.g., diethyl ether/petroleum ether (50 50) or hexane] that is incompatible with a semiaqueous mobile phase. It then becomes necessary to evaporate the unsaponifiable extract to dryness and to dissolve the residue in a small volume of methanol (if methanol is the organic component of the mobile phase). For the analysis of breakfast cereals, margarine, and butter, Egberg et al. (153) avoided the time-consuming extraction of the unsaponifiable matter and the evaporation step by acidifying the unsaponifiable matter with acetic acid in acetonitrile to precipitate the soaps. An aliquot of the filtered extract could then be injected, after dilution with water, onto an ODS column eluted with a compatible mobile phase (65% acetonitrile in water). 

Semipreparative HPLC has been employed to obtain a vitamin D-rich fraction of the unsaponifiable matter for subsequent quantitative HPLC. Combinations of chromatographic modes used for offline semipreparative and quantitative analysis have included polar bonded-phase/adsorption (211,212), reversed-phase/adsorption (194,213), and adsorption/reversed-phase (70,125). An online two-dimensional HPLC technique using two polar bonded-phase columns has also been described (214). 

Sterols, which are very abundant in some fruits, can be removed by precipitating them in different solvents. The unsaponifiable matter is dissolved in a minimum volume of methanol, petroleum ether, or acetone. The precipitation is completed overnight at — 20°C following centrifugation, the sterol-free supernatant is used for analysis (5). 

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... 

The official method of analysis of steradienes in olive oil (Commission regulation (EC) No. 656/95) involves saponification of the oil with an internal standard of cholesta-3,5-diene, followed by extraction of the unsaponifiable fraction into hexane. The steradiene fraction is then separated from other hydrocarbons, such as alkanes and squalene isomers, by column chromatography on silica gel. Quantitative analysis is then performed by GC. 

Most oils contain low levels of saturated and unsaturated hydrocarbons. In olive oil, the unsaturated hydrocarbon squalene can constitute up to 40% of the unsaponifiable fraction (Boskou, 1996). Other hydrocarbons commonly present in olive oil are straight chain alkanes and alkenes with 13 to 35 carbon atoms, along with very low amounts of branched chain hydrocarbons. Variations are found between different olive varieties but the main hydrocarbons are those with 23, 25, 27 and 29 carbon atoms (Guinda et al., 1996). Olive oil can clearly be differentiated from other vegetable oils on the basis of hydrocarbon components, and levels of 2.6% crude rapeseed oil or crude sunflower oil can be detected by hydrocarbon analysis (Webster et al., 1999). Terpenes have been identified in the volatile fraction of crude sunflower oil (Bocci and Frega, 1996). 

Most manufacturers use soapstock to spray on meal for animal feed, or ship the material to acidulators. Some seed oil producers treat soapstock on site with sulfuric acid at a temperature of 90-95 °C to produce acidulated soapstock (Dijkstra and Segers, 2007). Acidulated soapstock is very dark in color with a strong, rancid, burned odor from the free fatty acids and neutral oils. Free fatty acid content varies and can be in excess of 90%. Moisture content as well as unsaponifiables can be substantial and the pH (based on samples provided to Stepan Company) may vary from 3 to 4.5. An example of a typical analysis of an acid oil sample is listed below (Table 6.2). 

Coupled HPLC-GC methods are used for the detection of adulteration of oils and fats. The identification is done by analysis of unsaponifi-able minor constituents specific to a particular oil or fat as shown below ... 

Analysis of the sterol fraction isolated from the unsaponifiable fraction is very important, as will be seen later, for determining the authenticity of the oil. The triterpenes and sterols are present both as free alcohols and as fatty acid esters (46, 47). 

Sterol Composition. Sterol analysis involves preparation of the unsaponifiable fraction, fractionation by thin-layer chromatography (TLC), and gas chromatographic analysis of the TMS derivatives (66). The following limits apply to aU types of olive oil (12) ... 

Italian and Spanish ohve oil from the 1991-1992 crop year contained a very high level of 9,19-cyclolanosterol (>400 mg/kg), which was not found with the standard method for sterol analysis. Two isomers of this sterol were identified by GC/MS of the unsaponifiable fraction, and their levels were found to be inversely proportional to the levels of p-sitosterol in the oils. GC/MS of the unsaponifiable fraction with high-resolution GC capillary columns provides a relatively rapid means of checking product purity and the identity of individual components. Thus, triterpene diols were identifiable at m/z 203, ot-tocopherol at m/z 165, squalene at m/z 69, cholesterol at m/z 386, and brassicasterol, characteristic of canola oil and other Brassica oils, at m/z 398. 

Table III gives the physical and chemical properties of the M. oleifera oil. Some of the properties of the oil depend on the extraction medium. The M oleifera oil is liquid at room temperature and pale-yellow in colour. Electronic nose analysis shows that it has a flavor similar to that of peanut oil. The melting point estimated by differential scanning calorimetry is 19°C (15). The chemical properties of the oil depicted in Table III below are amongst the most important properties that determines the present condition of the oil. Free fatty acid content is a valuable measure of oil quality. The iodine value is the measure of the degree of unsaturation of the oil. The unsaponifiable matter represents other lipid- associated substances like, sterols, fat soluble vitamins, hydrocarbons and pigments. The density, iodine value, viscosity, smoke point and the colour of Moringa oil depends on the method of extraction, while the refractive index does not. Varietal differences are significant in all physical characteristics apart from refractive index and density (2). The heating profile of the M. oleifera seed oil using the differential scanning calorimetry (DSC) conventional scan rate shows that there is one major peak B and, two small shoulder peaks A and C...

Methods of analysis (ASTM D-128, IP 37) are available for the measurement of excessive acidity derived from oxidation. These methods cover conventional grease that consists essentially of petroleum oil and soap. Thus these test methods are applicable to many types but not all grease. The constituents covered by the test series are soap, unsaponifiable matter (base oil), water, free alkalinity, free fatty acid, fat, glycerin, and insoluble. A supplementary test method is also provided and is intended for application to grease that contains thickeners that are essentially insoluble in n-hexane and to grease that cannot be analyzed by conventional methods because of the presence of such constituents as nonpetroleum fluids or nonsoap-type thickeners, or both. These methods may not be applicable to grease analysis when lead, zinc, or aluminum soaps are present or in the presence of some additives such as sodium nitrite. 

Determination of unsaponifiable matter According to the lUPAC (1964) diethyl ether method (lUPAC) (Standard Methods for the Analysis of Oils, Fats and Soaps, 5th edition, 1966, II.D.5.1 and II.D.5.3). Results are expressed as grams unsaponifiable matter per kilogram oil. 

Saponification of the oil or fat, then separation of the unsaponifiable matter. Isolation of the sterols from the unsaponifiable matter by TLC. Analysis by GLC of the sterol fraction so isolated (or of the trimethyl-silyl ethers prepared from the sterol fraction) and interpretation of the chromatograms. 

The method described is identical to the lUPAC method 2.403 (Paquot, 1979b). The AOCS method Ce 3-74 (AOCS, 1978) describes a method for the gas chromatographic determination of tocopherols and sterols in soya sludges and residues. In this method, the sample is saponified and the extracted unsaponifi-able matter is reacted with butyric anhydride. The butyrate esters of the tocopherols and the sterols present in the unsaponifiable matter are subjected to gas chromatographic analysis and determined quantitatively. Relative retention times for the various tocopherol and sterol butyrates are given. 

Quality evaluation of cocoa bean and cocoa powder is by visual inspection for contamination, moldiness, and by aroma/flavor and tasting. Physical analysis of cocoa bean and cocoa powder includes analysis for total moisture (< 8%) and fat (<55%). Additionally, the quality of cocoa is characterized by the iodine number (degree of unsaturation of the fatty acid components), unsaponifiable matter, and GC analysis (for volatile and aroma components). 

Saponification of the extracts is generally desirable to remove unwanted lipid materials. However, this step is omitted in the isolation of carotenol esters, since these are hydrolyzed by this procedure. It is also omitted in the isolation of carotenoids such as fucoxanthin and peridinin, which are alkali-labile. If acetone has been used in the initial extraction, it is essential that all traces be removed before saponification. The general procedure used involves dissolving the total lipid fraction in an alcoholic (ethanol or methanol) solution of potassium hydroxide. The mixture is then either heated for a short period of time while kept in the dark, or left in the dark at room temperature for 12-16 h. There has been considerable discussion of the merits of these two procedures. Which method is used is dependent on the nature of the samples being analyzed and the requirements of the analysis (Davies, 1976 Liaaen-Jensen, 1971). After saponification, water is added, and neutral lipids (the unsaponifiable fraction) are extracted with diethyl ether or hexane. Acidic carotenoids remain in the alkaline phase and are extracted with diethyl ether or hexane after acidification with acetic acid. The unsaponifiable fraction usually contains sterols as well as carotenoids. If desired, sterol contaminants can be removed by precipitation from cold (- 10°C) petroleum ether or by precipitation of these compounds as their digitonides. 

Zarrouk, W., Carrasco-Pancorbo, A., Zarrouk, M., Segura-Carretero, A. and Fernandez-Gutierrez, A. (2009) Multi-component analysis (sterols, tocopherols and triterpenic dialcohols) of the unsaponifiable fraction of vegetable oils by liqnid chromatography-atmospheric pressure chemical ionization-ion trap mass spectrometry. Talanta 80, 924-934. 

Some fats which can not be unequivocally distinguished by their fatty acid or triacylglyceride composition may be identified by analysis of the unsaponifiable minor constituents. Examples are given in Table 14.26. 

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