E. N. Frankel

Analysis of autoxidized fats by gas chromatography-mass spectrometry: VII. Volatile thermal decomposition products of pure hydroperoxides from autoxidized and photosensitized oxidized methyl oleate, linoleate and linolenate

To clarify the sources of undesirable flavors, pure hydroperoxides from autoxidized and photosensitized oxidized fatty esters were thermally decomposed in the injector port of a gas chromatograph-mass spectrometer system. Major volatile products were identified from the hydroperoxides of methyl oleate, linoleate and linolenate. Although the hydroperoxides from autoxidized esters are isomerically different in position and concentration than those from photosensitized oxidized esters, the same major volatile products were formed but in different relative amounts. Distinguishing volatiles were, however, produced from each type of hydroperoxide. The 9- and 10-hydroperoxides of photosensitized oxidized methyl oleate were thermally isomerized in the injector port into a mixture of 8-, 9-, 10- and 11-hydroperoxides similar to that of autoxidized methyl oleate. Under the same conditions, the hydroperoxides from autoxidized linoleate and linolenate did not undergo significant interconversion with those from the corresponding photosensitized oxidized esters. The compositions of the major volatile decomposition products are explained by the classical scheme involving carboncarbon scission on either side of alkoxy radical intermediates. Secondary reactions of hydroperoxides are also postulated, and the hydroperoxy cyclic peroxides from methyl linoleate (photosensitized oxidized) and methyl linolenate (both autoxidized and photosensitized oxidized) are suggested as important precursors of volatiles.

Formation of malonaldehyde from lipid oxidation products

Abstract An acetalation-acid decomposition procedure was developed to investigate the formation of malonaldehyde from a wide assortment of primary and secondary lipid oxidation products. The formation of malonaldehyde as the tetramethyl acetal derivative was determined quantitatively by gas chromatography with an internal standard technique and identified by gas chromatography-mass spectrometry. Five-membered hydroperoxy epidioxides and 1, 3-dihydroperoxides were found to be the most important precursors of malonaldehyde. 1, 4-Dihydroperoxides were less important, and monohydroperoxides were the least significant sources of malonaldehyde. No malonaldehyde was produced from either a six-membered hydroperoxy epidioxide or 1,7-and 1,8-dihydroperoxides. No correlation was found between the thiobarbituric acid values of the lipid oxidation products and analyses of malonaldehyde by the acetalation-acid decomposition procedure. By the specific methodology presented in this paper, the potential of lipid oxidation products to form malonaldehyde and its biological effect due to crosslinking can better be evaluated than the extensively used thiobarbituric acid test, which is not specific for most of the oxidation lipid products examined in this study.

Analysis of autoxidized fats by gas chromatography-mass spectrometry: V. Photosensitized oxidation

The role of singlet oxygen in oxidation was studied by analyzing hydroperoxide isomers in unsaturated fats and esters by gas chromatography-mass spectrometry (GC-MS). On oxidation photosensitized with methylene blue at 0 C, methyl oleate produced a 50–50% mixture of 9- and 10-hydroperoxides, linoleate a mixture of 66% conjugated (9+13) and 34% unconjugated (10+12) hydroperoxides, and linolenate a mixture of 75% conjugated (9+12+13+16) and 25% unconjugated (10+15) hydroperoxides. Cottonseed, safflower, and corn oil esters showed, as in soybean esters, the presence of varying amounts of 12-hydroxy esters derived from the corresponding hydroperoxide at low peroxide values. Since these oils do not contain linolenic acid, a likely source of the 12-hydroperoxide is linoleic acid by photosensitized oxidation. Several lines of evidence support the conclusion that singlet oxygen may contribute to the unique hydroperoxide composition of vegetable oil esters at low levels of oxidation. In the presence of photosensitizers such as methylene blue and chlorophyll, the unique hydroperoxide composition (high levels of 10- and 12-hydroperoxides) obtained in soybean esters was similar to that produced by oxidation at low peroxide values. In contrast, a normal hydroperoxide composition was produced, as expected from the fatty acid composition of soybean oil esters, when singlet oxygen quenchers such as β-carotene and α-tocopherol were used and when the esters were treated with carbon black to remove natural photosensitizers. GC-MS analyses of the derived unsaturated alcohols provided indirect evidence for 12-hydroperoxy-9,13-diene in soybean esters as expected by photosensitized oxidation of linoleate.

Secondary products of lipid oxidation

In the last decade, a multitude of secondary products have been identified from the radical and photosensitized oxidations of polyunsaturated lipids. These secondary products consist of oxygenated monomeric materials including epoxy-hydroperoxides, oxo-hydroperoxides, hydroperoxy epidioxides, dihydroperoxides, hydroperoxy bis-epidioxides, and hydroperoxy bicycloendoperoxides. More recently, higher molecular weight dimeric compounds have been identified from autoxidized methyl linoleate and linolenate. Decomposition of these oxidation products form a wide range of carbonyl compounds, hydrocarbons, furans, and other materials that contribute to the flavor deterioration of foods and that are implicated in biological oxidation. The interaction of some of these degradation products with DNA may be involved in cell-damaging reactions.

Analysis of autoxidized fats by gas chromatography-mass spectrometry: II. Methyl linoleate

The gas chromatography-mass spectrometry (GC-MS) approach developed in the preceding paper was applied for qualitative and quantitative investigations of autoxidation products of methyl linoleate. A GC-MS computer summation method was standardized with synthetic 9- and 13-hydroxyoctadecanoate. Equal amounts of 9- and 13-hydroperoxides were found in all samples of linoleate autoxidized at different temperatures and peroxide levels. The results are consistent with the classical free radical mechanism of autoxidation involving a pentadiene intermediate having equivalent sites for oxygen attack at carbon-9 and carbon-13. Minor oxygenated products of autoxidation indicated by GC-MS include keto dienes, epoxyhydroxy monoenes di- and tri-hydroxy monoenes. These hydroxy compounds are presumed to be present in the form of hydroperoxides. The quantitative GC-MS method was found suitable for the analysis of autoxidized mixtures of oleate and linoleate. By this method, it is possible to determine the origin of the hydroperoxides formed in mixtures of these fatty esters.

Analysis of autoxidized fats by gas chromatography-mass spectrometry: I. Methyl oleate

A structural investigation of autoxidation products of methyl oleate was carried out by gas chromatography-mass spectrometry (GC-MS) of trimethylsilyl (TMS) ether derivatives. GC-MS using computer plots of selected masses afforded structural assignments of GC peaks due to incompletely resolved mixtures. This method provided evidence of epoxy and keto esters which are not completely separated from the main components consisting of the TMS derivatives of the allylic hydroxy esters. Use of an MS-computer system also showed that the hydroxyoctadecanoate TMS ethers were partially separated by GC. The use of synthetic hydroxyoctadecanoates for the first time enabled us to demonstrate the quantitative reliability of a GC-MS computer summation approach to analyze the isomeric composition of oleate hydroperoxides (as the saturated TMS ether derivatives). Consistently higher concentrations were found of the 8- and 11-hydroperoxides than of the 9- and 10-hydroperoxides. Minor products of autoxidation identified by GC-MS include allylic enones, isomeric epoxyoctadecanoates, dihydroxyctadecenoates, and dihydroxyoctadecanoates.

Autoxidation of polyunsaturated triacylglycerols. I. Trilinoleoylglycerol

The hydroperoxides and secondary products formed from trilinoleoylglycerol autoxidized at 40°C were isolated and characterized to clarify their contribution to oxidative deterioration of vegetable oils. The products were purified by high performance liquid chromatography (HPLC) and identified, as intact triacylglycerols, by ultraviolet, infrared,1H NMR and13C NMR analyses, and after derivatization by lipolysis, gas chromatography, and gas chromatography-mass spectrometry. The main, primary products included mono-,bis- and tris-9-hydroperoxy-trans-10,cit-12-; 9-hydroperoxy-trans-10,trans-12; 13-hydroperoxy-cis-9,trans-11; and 13-hydroperoxy-trans-9,trans-11-linolenoyl glycerols. The structures of the minor secondary products analyzed after derivatization were consistent with known oxidative degradation products of linoleate hydroperoxides. HPLC analyses showed that thebis- and tris-hydroperoxides were formed from the mono-hydroperoxides during autoxidation at peroxide values above 18 and 28 meq/kg. Studies on the further oxidation of the mono-hydroperoxides support a mechanism for the consucutive formation ofbis- and tris-hydroperoxides from the monohydroperoxides. HPLC analyses showed that no preferential oxidation occurred between the 1(3)- and 2-triglyceride positions. Hydroperoxides of linoleate triacylglycerols may be important precursors of volatile compounds contributing to off-flavors of vegetable oils.

High pressure liquid chromatography of autoxidized lipids: II. Hydroperoxy-cyclic peroxides and other secondary products from methyl linolenate

A previous study of autoxidation products by high pressure liquid chromatography (HPLC) of methyl oleate and linoleate was extended to methyl linolenate. Autoxidized methyl linolenate was fractionated by HPLC either after reduction to allylic alcohols on a reverse phase system, or directly on a micro silica column. Isolated oxidation products were characterized by thin layer and gas liquid chromatography and by ultraviolet, infrared, nuclear magnetic resonance and mass spectrometry. Secondary products from the autoxidation mixtures (containing 3.5–8.5% monohydroperoxides) included epoxy unsaturated compounds (0.2–0.3%), hydroxy or hydroperoxy-cyclic peroxides (3.8–7.7%), epoxy-hydroxy dienes (<0.1%), dihydroxy or dihydroperoxides with conjugated diene-triene and conjugated triene systems (0.9–2.9%). Cyclization of the 12- and 13-hydroperoxides of linolenate would account for their lower relative concentration than the 9- and 16-hydroperoxides. Dihydroperoxides may be derived from the 9- and 16-linolenate hydroperoxides. Cyclic peroxides and dihydroperoxides are suggested as important flavor precursors in oxidized fats.

Photosensitized oxidation of methyl linoleate: Secondary and volatile thermal decomposition products

Studies of photosensitized oxidation of methyl linoleate show that the greater relative concentration of 9- and 13-hydroperoxides than 10- and 12-hydroperoxides is characteristic of singlet oxygenation and not due to either simultaneous autoxidation or type 1 photosensitized oxidation. Cyclization of the internal 10- and 12-hydroperoxides accounts for their lower relative concentrations. Secondary products separated by silicic acid and high pressure liquid chromatography were characterized spectrally (IR, UV,1H-NMR,13C-NMR, GC-MS). Major secondary products included diastereomeric pairs of 13-hydroperoxy-10,12-epidioxy-trans-8-octadecenote (I and III) and 9-hydroperoxy-10,12-epidioxy-trans-13-octadecenoate (II and IV); minor secondary products included hydroperoxy oxy genated and epoxy esters. Thermal decomposition of the hydroperoxy cyclic peroxides produced hexanal and methyl 10-oxo-8-decenoate as major volatiles from I and III and methyl 9-oxo-nonanoate and 2-heptenal from II and IV. Hydroperoxy cyclic peroxides may be important sources of volatile decomposition products of photooxidized fats.

Analysis of autoxidized fats by gas chromatography-mass spectrometry: III. Methyl linolenate

The gas chromatography-mass spectrometry (GC-MS) method developed in the preceding papers was extended to the analysis of autoxidation products of methyl linolenate. Four isomeric hydroxy allylic trienes with a conjugated diene system were identified after reduction of the linolenate hydroperoxides. All eight geometrictrans,cis- andtrans, trans-conjugated diene isomers of these hydroxy allylic compounds were identified and partially separated by GC of the trimethylsilyl (TMS) ether derivatives. The proportion found of 9- and 16-hydroperoxides was significantly higher (75–81%) than the 12- and 13-hydroperoxides (18–25%). The tendency of the 12- and 13-hydroperoxides to form cyclic peroxides, cyclic peroxidehydroperoxides, and prostaglandin-like endoperoxides was supported by indirect evidence for the presence of 9,10,12- and 13,15,16-trihydroxyoctadecanoate in hydrogenated derivatives of the highly oxygenated products. The quantitative GC-MS method was used to determine the relative contribution of linolenate, linoleate, and oleate in mixtures to the formation of hydroperoxides.