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. Presented at the AOCS Meeting, Chicago, September 1976.     

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. Presented at the AOCS Meeting, San Francisco, California, April 29–May 3, 1979. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

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 linolenate. Secondary products

Previous studies of secondary oxidation products by high-pressure liquid chromatography (HPLC) of autoxidized methyl oleate, linoleate and linolenate and photosensitized-oxidized linoleate are extended to photosensitized-oxidized linolenate. Photosensitized-oxidized linolenate was fractionated by silicic acid chromatography with diethyl ether/hexane mixtures. Selected silicic acid chromatographic fractions were separated by polar phase HPLC and characterized by thin layer and gas liquid chromatography and by ultraviolet, infrared, nuclear magnetic resonance and mass spectrometry. Secondary products from the photosensitized oxidation mixtures (containing 8.2 to 29.0% monohydroperoxides) included keto- and epoxy-dienes (0.4–1.6%), hydroperoxy epidioxides (0.8–4.9%), hydroperoxy bicyclic monoenes (0.1–0.3%), dihydroperoxides (1.0–5.6%), and hydroperoxy bisepidioxides (0.7–1.6%). Some of these secondary products are new and unique to photosensitized oxidation. Cyclization of the 10-, 12-, 13- and 15-hydroperoxides of linolenate would account for their lower relative concentration than that found for the 9- and 16-hydroperoxides. Dihydroperoxides may be derived from monohydroperoxides by singlet oxygenation or free radical oxidation. The hydroperoxy bis-epidioxides may be formed by further serial cyclization of the hydroperoxy epidioxides from 10- and 15-monohydroperoxides. Dihydroperoxides, hydroperoxy epidioxides and hydroperoxy bis-epidioxides are suggested as important flavor precursors in oxidized fats. The mention of firm names or trade products does not imply that they are endorsed by the US Department of Agriculture over other firms or similar products not mentioned.     

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. Presented at the AOCS Meeting, New York, May 1977.     

Analysis of autoxidized fats by gas chromatography-mass spectrometry: X. Volatile thermal decomposition products of methyl linolenate dimers

High-molecular weight compounds previously were found to be important secondary products from autoxidation of polyunsaturated fatty esters. The contribution of dimers to oxidative deterioration was investigated by analyzing their volatile thermal decomposition products by capillary gas chromatography-mass spectrometry. Dimers were isolated by gel permeation chromatography from autoxidized linolenate and from the corresponding monohydroperoxides, cyclic peroxides and dihydroperoxides. Major volatile decomposition products identified from these oxidative dimers were similar to those formed from the corresponding monomeric hydroperoxides. However, dimers from linolenate hydroperoxides produced more propanal and methyl 9-oxononanoate than the corresponding monomers but less methyl octanoate and much less or no 2,4-heptadienal and 2,4,7-decatrienal. Significant differences in minor volatile products also were observed between dimeric and monomeric products of methyl linolenate oxidation compounds. Mechanisms are suggested for the formation of volatile decomposition products from different dimeric structures. These dimers are believed to be important sources of volatile compounds contributing to flavor and oxidative deterioration of fats.     

Effect of α-tocopherol on the volatile thermal decomposition products of methyl linoleate hydroperoxides

α-Tocopherol and 1,4-cyclohexadiene were tested for their effect on the thermal decomposition of methyl linoleate hydroperoxide isomers. The volatiles generated by thermolysis in the injector port of a gas chromatograph at 180°C were analyzed by capillary gas chromatography. In the presence of either α-tocopherol or 1,4-cyclohexadiene, which are effective donors of hydrogen by radical abstraction, volatile formation decreased in all tests, and significant shifts were observed in the relative distribution of products in certain hydroperoxide samples. When an isomeric mixture of methyl linoleate hydroperoxides (cis, trans andtrans, trans 9- and 13-hydroperoxides) was decomposed by heat, the presence of α-tocopherol and 1,4-cyclohexadiene caused the relative amounts of pentane and methyl octanoate to decrease and hexanal and methyl 9-oxononanoate to increase. A similar effect of α-tocopherol was observed on the distribution of volatiles formed from a mixture of thetrans,trans 9- and 13-hydroperoxides. This effect of α-tocopherol was, however, insignificant with purecis,trans 13-hydroperoxide of methyl linoleate. The decrease in total volatiles with the hydrogen donor compounds, α-tocopherol and 1,4-cyclohexadiene, indicates a suppression of homolytic β-scission of the hydroperoxides, resulting in a change in relative distribution of volatiles. The increase in hexanal and methyl 9-oxononanoate at the expense of pentane and methyl octanoate in the presence of hydrogen donor compounds supports the presence of a heat-catalyzed heterolytic cleavage (also known as Hock cleavage), which seems to mainly affect thetrans,trans isomers of linoleate hydroperoxides.     

Analysis of autoxidized fats by gas chromatography-mass spectrometry. IV. Soybean oil methyl esters

An unusual isomeric distribution of hydroperoxides has been found in soybean oil esters oxidized at low levels (peroxide values below 50). The unexpectedly high concentration of the 12-hydroperoxide isomer is in marked contrast to the isomeric composition of oxidized pure linolenate. The different isomeric hydroperoxides observed at low levels of oxidation may contribute through their decomposition to the unique flavor deterioration of soybean oil. Quantitative gas chromatographymass spectrometry (GC-MS) used in this study provides for the first time an answer to the basic question of which hydroperoxides contribute to the state of oxidation of soybean oil. Results of GC-MS were confirmed by capillary gas chromatography. Analyses of highly oxidized soybean esters (peroxide values 468 and 2352) reveal the same main compounds as those found in oxidized pure linoleate, together with small amounts of oleate and linolenate hydroperoxides. Presented at AOCS Meeting, St. Louis, Missouri, May 14–18, 1978. Mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Analysis of oleate,linoleate and linolenate hydroperoxides in oxidized ester mixtures

The hydroperoxides in oxidized mixtures of methyl oleate, linoleate and linolenate were analyzed by reducing the hydroperoxides to the corresponding hydroxyesters and separating the hydroxyesters from the unoxidized esters by thin layer chromatography (TLC). The hydroxyesters from linolenate were separated from the other hydroxyesters by TLC on silver ion plates. The hydroxyesters were converted to TMS-hydroxy derivatives. The TMS-hydroxyoleate and TMS-hydroxylinoleate were separated by gas chromatography (GC), and all the TMS-derivatives were quantified by GC. The relative rates of oxidation of methyl oleate, linoleate and linolenate in mixtures were ca. 1∶10.3∶21.6. The hydroperoxides formed in the oxidation of soybean and olive oils were similar before and after randomization and similar to corresponding methyl ester mixtures. Journal Paper No. J-9657 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Project 2143.     

Analysis of autoxidized fats by gas chromatography-mass spectrometry: VIII. Volatile thermal decomposition products of hydroperoxy cyclic peroxides

Secondary oxidation products are important sources of volatiles because of their susceptibility to further decomposition. Volatiles from the thermal decomposition of hydroperoxy cyclic peroxides have been identified by capillary gas chromatography followed by mass spectrometry (GC-MS). By using a saturated hydroperoxy cyclic peroxide as a synthetic model, the thermal decomposition pathways have been elucidated. Main cleavage occurs between the peroxide ring and the carbon-bearing hydroperoxide group. Volatiles produced were generally similar to those from corresponding monohydroperoxides. New volatiles identified included methyl furan octanoate, methyl ketones, and conjugated diunsaturated aldehyde esters. The general fragmentation observed between the peroxide ring and the hydroperoxide-bearing carbons is sufficiently predictable that it can be used as a tool for the structural characterization of hydroperoxy cyclic peroxides. Hydroperoxy cyclic peroxides from autoxidized and photosensitized oxidized methyl linolenate are suggested as important precursors of volatiles that may affect flavor quality of lipid-containing foods.     

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. Presented at 72nd Annual Meeting of the American Oil Chemists Society, New Orleans, LA, May 1981.     

Products formed by photosensitized oxidation of unsaturated fatty acid esters

Photosensitized oxidation of unsaturated fatty acid methyl ester was carried out using methylene blue as a sensitizer. Oxidation products, monohydro-peroxides, were identified as trimethylsilyl derivatives. Methyl oleate gave the 9- and 10-isomers; methyl linoleate, the 9-, 10-, 12-, and 13-isomers; and methyl linolenate, the 9-, 10-, 12-, 13-, 15-, and 16-isomers, respectively. The double bond to which the hydroperoxide group attached was shifted to the adjacent position in each isomer. Thus, both conjugated and nonconjugated isomers were present in methyl linoleate monohydroperoxides and methyl linolenate monohydroperoxides. By the inhibition experiment, it was ascertained that the above reaction proceeded via singlet oxygen. The relative rates of methyl oleate, methyl linoleate, and methyl linolenate were 1.0∶1.7∶2.3, respectively. These results obtained from the methyl esters were applied to the photosensitized oxidation of triglycerides purified from vegetable oils, and the reaction mechanism on triglycerides was proposed.     

Carbonyls in oxidizing fat. V. The composition of neutral volatile monocarbonyl compounds from autoxidized oleate,linoleate, linolenate esters,and fats

The steam volatile monocarbonyl compounds in mildly autoxidized esters of oleic, linoleic, linolenic acids, and animal and vegetable fats were quantitatively estimated. The major aldehydes in oleate and linoleate were those that might be expected from the scission of reported monomeric hydroperoxide isomers. The predominance of hept-2,4-dienal and propanal in linolenate suggested that the major monomeric hydroperoxides were 12-and 16-hydroperoxy conjugated dienoic isomers. The number of minor aldehydes increased with degree of unsaturation of the fatty acid. The amounts of monocarbonyl compounds in the fats examined generally agreed with their average fatty acid composition. Appreciable amounts of heptanal in lamb and beef fat and heptanal and decanal in butterfat, under the conditions of oxidation, could not have come from the three unsaturated acids. Heating at 165°C. in all samples increased the proportions of the most unsaturated major aldehydes.     

Analyses of lipids and oxidation products by partition chromatography. Fatty acid hydroperoxides

A liquid partition chromatographic method was developed to isolated and determine hydroperoxides in autoxidized fatty acids or their methyl esters. By the use of benzene containing 2 to 4% methanol as the mobile solvent, the hydroperoxides were separated from unoxidized fatty acids or methyl esters and from secondary and polymeric decomposition products. In the analyses of oxidized fatty acids, diethyl ether was necessary to elute the secondary decomposition products. Saponification of autoxidized fatty esters destroyed the peroxides as determined iodometrically, but the resulting acids contained a fraction which was eluted in the same position as hydroperoxide acids. Evidence showed that this fraction is a monomeric hydroxy fatty acid containing conjugated cis-traux and trans-trans unsaturation. Fatty ester hydroperoxides were isolated chromatographically in yields and purity comparable to those reported in the literature by countercurrent distribution. The concentrations of methyl linoleate hydroperoxide determined chromatographically were smaller than indicated by the peroxide value and diene conjugation of the autoxidized methyl linoleate. This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.     

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. Presented at the AOCS Meeting, Chicago, September 1976.     

Rapid headspace gas chromatography of hexanal as a measure of lipid peroxidation in biological samples

A rapid, sensitive and convenient capillary gas chromatographic-headspace method was developed to determine hexanal as an important volatile decomposition product of hydroperoxides formed from n−6 polyunsaturated fatty acids in rat liver samples. Total volatiles were also determined as a measure of overall lipid peroxidation. Samples of headspace taken from sealed serum bottles incubated at 37°C were injected into a gas chromatograph. It was possible to make 15 determinations per hour. This method is convenient because no special sample manipulations are necessary. The addition of 0.5 mM ascorbic acid prior to gas chromatographic, analysis significantly increased hexanal production. The applicability of the method was demonstrated in studies of the effect of iron in the presence or absence of hydroperoxides of methyl linoleate and methyl linolenate andtert-butyl hydroperoxide on rat liver homogenates, slices and microsomes. A rapid silica cartridge chromatographic procedure was used to purify hydroperoxides from autoxidized methyl linoleate and methyl linolenate, and hydroperoxy epidioxides (cyclic peroxides) from autoxidized methyl linolenate in 20–40 mg quantities. The hydroperoxides and hydroperoxy epidioxides of methyl linolenate were, effective inducers n−6 polyunsaturated fatty acid peroxidation in liver homogenates. Hexanal and thiobarbituric acid-reacting substances were signficantly correlated in liver homogenates and microsomes but not in slices. This specific method for hexanal, a known product of peroxidation of n−6 polyunsaturated fatty acids, can be used as a good measure of lipid peroxidation. Presented at the joint meeting of the Amerian Society for Cell Biology and the American Society for Biochemistry and Molecular Biology, San Francisco, CA January 29–February 2, 1989.     

Quantitative analyses of hydroxystearate isomers from hydroperoxides by high pressure liquid chromatography of autoxidized and photosensitized-oxidized fatty esters

A high pressure liquid chromatography (HPLC) method is described for the determination of the isomeric hydroxysterates from hydroperoxides of oxidized fatty esters. The samples are hydrogenated and the mixtures of hydroxystearates are concentrated by partial removal of unoxidized esters and complete removal of polar materials. Isomeric hydroxystearates are then separated on a porous microparticulate adsorption (10 μ) column and elution with 0.25% isopropyl alcohol inn-hexane is monitored at 212 nm. The 8-OH, 9-OH, 10-OH, 11-OH and 16-OH isomers were completely separated, but the 12-OH, 13-OH and 15-OH were only partly resolved by HPLC. The relative percentages of isomeric hydroxy esters were analyzed quantitatively by a computer integration method. Accuracy of the method was checked with known mixtures of synthetic hydroxystearates. The isomeric hydroperoxide composition of oxidized methyl oleate, linoleate, linolenate and soybean methyl esters determined by HPLC were in good agreement with previous analyses by gas chromatography-mass spectrometry. Presented at ISF-OACS Meeting, New York, NY, April 27–May 1, 1980.     

Analysis of oleate and linoleate hydroperoxides in oxidized ester mixtures

The hydroperoxides in oxidized mixtures of methyl oleate and linoleate were reduced to the corresponding hydroxyesters, which were separated from unoxidized esters by thin layer chromatography on silica gel. The hydroxyesters from oleate and linoleate were converted to trimethylsilyl ethers and separated by gas chromatography on OV 225. The results suggest that methyl linoleate oxidizes about ten times faster than methyl oleate, but oleate hydroperoxides are formed in appreciable amounts, even in mixtures containing 87% methyl linoleate. Journal Paper No. J-8658 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Project No. 2143.     

Monocarbonyl compounds from catalytic decomposition of autoxidized unsaturated fatty acid esters

Monocarbonyl compounds formed by the decomposition of autoxidized triolein, methyl linolenate, and methyl arachidonate were converted into their 2,4-dinitrophenylhydrazone derivatives and analyzed by thin layer and paper chromatographies. From decomposition of autoxidized triolein with acid-washed Fuller’s earth alkanals were the only monocarbonyl products found, whereas, with metal catalysts or heat, 2-alkenals were the primary products. Autoxidized methyl linolenate and methyl arachidonate decomposed with metal catalysts or heat yielded 23–55% of 2,4-alkadienals but minor amounts with acid-washed Fuller’s earth. The differences in distribution of monocarbonyl products were attributed to a selective course of scission of the hydroperoxides that depended upon the conditions of decomposition.     

The oxidized-metallic and grassy flavor components of autoxidized milk fat

OCt-1-en-3-one accounts for the metallic flavor of autoxidized milk fat. In combination with small amounts of aldehydes it gives an oxidized flavor to milk. The grassy flavor component observed in autoxidized milk fat is shown to betrans- cis-2,6-nonadienal. Mechanisms are proposed for the production of oct-l-en-3-one from linoleate and 2,6-nonadienal from linolenate.