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

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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.     

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.     

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: 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.     

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 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.     

Thermal decomposition of methyl oleate hydroperoxides and identification of volatile components by gas chromatography-mass spectrometry

The role of methyl oleate hydroperoxides as precursors of volatile compounds was investigated by thermal decomposition in the injector port of a gas chromatograph attached to a computerized mass spectrometer. The major volatile compounds identified correspond to those formed from triolein heated in air at 192 C.     

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 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.     

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.     

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.     

Chemical lonization-mass spectrometry of secondary oxidation products from methyl linoleate and linolenate

Chemical ionization-mass spectrometry (CI-MS) with a direct exposure probe was used to analyze a series of hydroperoxy cyclic peroxides and dihydroperoxides obtained from methyl linoleate and linolenate by either autoxidation or photosensitized oxidation. The mass spectra obtained with isobutane and ammonia as reacting gases showed fragmentation patterns similar to those ditions of gas chromatography (GC)-electron impact (EI)-MS. Because the fragmentation patterns obtained under either CI-MS with a direct exposure probe or GC-(EI)-MS conditions are sufficiently predictable, these techniques are powerful analytical tools for the structural characterization of lipid oxidation products. These techniques are also useful to elucidate the cleavage pathways to volatile lipid oxidation products of flavor and biological significance.     

Autoxidation of polyunsaturated triacylglycerols. IV. Volatile decomposition products from triacylglycerols containing linoleate and linolenate

Trilinoleoylglycerol (LLL), trilinolenoylglycerol (LnLnLn) and four synthetic triacylglycerols were autoxidized and the volatile products were investigated to determine the effect of fatty acid glyceride position on the mechanism of hydroperoxide decomposition. Capillary gas chromatography provided a sensitive method to follow the volatile oxidation products of mixtures of LLL and LnLnLn and of synthetic triacylglycerols containing linoleate and linolenate in different known positions. The relative amount of linoleate oxidation was determined by analyzing for hexanal, 2-heptenal and 2,4-decadienal, and the relative amount of linolenate oxidation by analyzing for 2,4-heptadienal and 2,4,7-decatrienal. The volatiles from pure monohydroperoxides of LLL and LnLnLn were compared with those of the corresponding triacylglycerols by capillary gas chromatography. Significant differences in the distribution of volatile products were observed depending on the triacylglycerols precursor. A 1∶1 mixture of LLL and LnLnLn autoxidized at 40°C showed an equal contribution of linolenate and linoleate volatiles at a peroxide value of 34. The synthetic triacylglycerols LLnL and LLLn (L, linoleic; Ln, linolenic acid) formed initially about the same total volatiles, whereas LnLnL formed more volatiles than LnLLn. The ratio Ln to L volatile products was the same for the diL triacylglycerols, and higher for LnLnL than for LnLLn. This new information should permit us to better understand the influence of triacylglycerol structure on the relative oxidative stability of unsaturated triacylglycerols. Presented in part at the 80th Annual American Oil Chemists' Meeting, Cincinnati, OH, May 3–6, 1989.     

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. IX. Homolytic vs. Heterolytic cleavage of primary and secondary oxidation products

To elucidate the genesis of volatile lipid oxidation products, thermal homolytic and acid heterolytic decomposition processes were compared. Secondary oxidation products were decomposed thermally (200 C), and the volatiles formed were identified by capillary gas chromatography-mass spectrometry (GC-MS). Oxidation products also were decomposed in the presence of HCl-methanol, and the resulting dimethyl acetals were identified by GC-MS. The volatile thermal decomposition products were those expected by homolytic β-scission on both sides of the hydroperoxide group. No dialdehydes were identified under our thermal decomposition conditions. In contrast, the acetals formed by acid decomposition were those expected by selective heterolytic scission between the hydroperoxide group and the allylic double bond. Dialdehydes identified from acid decomposition of cyclic peroxides and dihydroperoxides included malonaldehyde and 2,4-hexadienedial.     

Analysis of autoxidized fats by gas chromatography-mass spectrometry: VI. Methyl 9,15- and 12,15-octadecadienoate

The gas chromatography-mass spectrometry (GC-MS) method developed in preceding papers was extended to the structural analysis of autoxidation products of methylcis-9,cis-15-octadecadienoate and of an 87% concentrate ofcis-12,cis-15-octadecadienoate. Eight isomeric hydroxydienes with one allylic and one isolated double bond were identified from oxidized 9,15-diene and 2 conjugated hydroxydienes from oxidized 12,15-diene, after reduction of the hydroperoxides. The proportions of 16-(15%) and 17-hydroperoxides (22%) from 9,15-diene were significantly higher than that of the other isomers (8–12% each: 8-, 9-, 10-, 11-, 14- and 15-OOH). Similarly, the amount of 16-hydroperoxide from 12,15-diene was larger (42%) than the 12-hydroperoxide (31%). Substantial amounts of dihydroxy esters with one OH substituent on carbons-8,-9,-10 or-11 and the other OH on carbons-14,-16 or-17, were identified after hydrogenation in highly oxidized 9,15-diene. The implications of these hydroperoxide analyses are discussed in relationship to the precursors of flavor deterioration of oils and partially hydrogenated oils containing an ω-3 double bond.     

Nutritive value of methyl linoleate and its thermal decomposition products

Methyl linoleate was heated for 10 hrs. at 300°C, in the absence of air and fractionated by alembic distillation and urea adduct-formation. Intestinal absorptions of the urea adduct-forming monomeric nonadduct-forming monomeric, and dimeric fractions were determined. It was found that dimers were half as well absorbed as the monomers. When fed to rats, dimers were better accepted and exhibited some toxicity symptoms different from the non-adduct-forming monomers. The dimers caused diarrhea, irritability, and loss of hair during the early period of administration. The nonadduct-forming monomers were lethal and produced an increase in liver weight. Both fractions depressed growth. With the technical assistance of Oscar giacomantone and Perla Mordujovich.