Unsaponifiable Matter

Heiduschka, A. Unsaponifiable matter of sesame oil. Orig Com 8th Intern Congr Appl Chem 1912 11 13. 

Maxwell, R. J. and Schwartz, D. P. 1979. A rapid, quantitative procedure for measuring the unsaponifiable matter from animal, marine, and plant oils. J. Am. Oil chem. Soc. 56, 634-636,... 

Add 60 ml water to dilute the alcohol, and subsequently extract the unsaponifiable matter three times with 20 ml n-hexane. 

When the amount of water added is small and the alcohol concentration in the saponified solution is high, fatty acid potassium salts may transfer into the ether layer along with unsaponifiable matter, including cholesterol. For quantitative recovery of cholesterol, as well as the complete separation of the fatty acid potassium salts from the cholesterol fraction, lowering the alcohol concentration by adding excess water prior to ether extraction is recommended. When the alcohol concentration is sufficiently low, the saponified solution forms easily. 

Dry the unsaponifiable matter under a nitrogen stream and add the following ... 

A capillary gas chromatographic method is described for determination of major phytosterols and cholesterol in edible oils and fats. To extract the unsaponifiable matter and for sample cleanup, solid-phase extraction with C18 absorbent was used. 

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). 

A mixture of petroleum ether/diethyl ether (1 + 1) is suitable for extracting vitamin D from the unsaponifiable material and allows vitamins A and D to be coextracted. For the determination of vitamin D alone in fortified milks, margarine, and infant formulas, Thompson et al. (70) extracted the unsaponifiable matter three times with hexane in the presence of a 6 4 ratio of water to ethanol. The combined hexane layers were then washed with 55% aqueous ethanol, after the initial 5% aqueous KOH and water washes, to remove material, including 25-hydroxyvitamin D, that was more polar than vitamin D. This extraction process was based on partition studies that showed that insignificant amounts of vitamin D were extracted from hexane by aqueous ethanol when the ratio of ethanol to water was less than 6 4. 

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). 

Reversed-phase HPLC with fluorescence detection is the preferred system for the routine determination of total a-tocopherol in vitamin E-supplemented foods after saponification. The use of NARP chromatography with a predominantly hexane mobile phase allows aliquots of hexane extracts of the unsaponifiable matter to be injected directly onto the column, thus avoiding the evaporation step necessary when a semiaqueous mobile phase is used (234). 

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). 

Other extraction methods used in the lipid extraction include supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE). With SEE, good extraction yields have been obtained for nonpolar lipids including ester-ified fatty acids, acylglycerols, and unsaponifiable matter. However, complex polar lipids are only sparingly soluble in supercritical carbon dioxide alone and polar modifiers, such as methanol, ethanol, or even water is required to improve the extraction of polar lipids (10). SFE has been used for the extraction of lipids especially from various food matrices, such as different nuts, edible oils, and seeds (11). The recoveries of lipids in SFE were on the same levels than with conventional solvent extraction methods (12,13), no significant differences between the fatty acids extracted were observed. PLE has also been used in lipid extraction, although only in very few applications (14). The elevated temperatures used in PLE can cause alteration of the lipid composition. 

Joseph, D. and Neeman, I. (1982) Characterisation of avocado oil by polyalcoholic compounds in the unsaponifiable matter. Riv. Ital. Sost. Grasse, 59, 279-284. 

Frega, N., Bocci, E, Giovannoni, G. and Lercker, G. (1993) High resolution GC of unsaponifiable matter and sterol fraction in vegetable oils. Chromatographia, 36, 215-217. 

Eisner, J., Iverson, J.L. and Firestone, D. (1966) Gas chromatography of unsaponifiable matter. 4. Aliphatic alcohols, tocopherols and triterpenoid alcohols in butter and vegetable oils. J. Assoc. Off. Anal. Chem., 49, 580-590. 

Unsaponifiable Matter in Fats and Oils, Including Marine Oils (Ca bb-5, 3) determines substances dissolved in fats and oils that cannot be saponified (turned into sodium salts) by the usual caustic treatment, including higher aliphatic alcohols, sterols, pigments, and hydrocarbons. This method is not suitable for marine oils or feed grade fats. 

Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII. 

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