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.     

Quantification of carbonyls produced by the decomposition of hydroperoxides

Carbonyls produced by the decomposition of cyclohexene hydroperoxide and various hydroperoxides of linoleic and linolenic acids and their methyl esters were determined by gas chromatography of the 2,4,6-trichlorophenylhydrazones. The effect of temperature, iron and copper ions, ethanol and several antioxidants on the rate of decomposition, the nature of the products and their yield was observed. The hydroperoxides of methyl esters decomposed more slowly than those of free fatty acids. Ethanol slowed, and metal ions accelerated the rates of decomposition. Metal ions, especially copper, increased the yield and complexity of the carbonyls formed, but ethanol decreased carbonyl yields. Antioxidants and decomposition temperatures changed the relative yields of carbonyls produced. The 9- and 13-hydroperoxides of linoleic acid gave similar carbonyls, but those of linolenic acid did not. The carbonyl mixtures produced from autoxidized fatty acid methyl esters were more complex than those produced from lipoxygenase-treated fatty acids.     

Effects of β‐carotene and retinal on the formation of methyl linoleate hydroperoxides

In order to clarify the prooxidative role of carotenoids on the oxidation of unsaturated lipids this study examined the effects of β‐carotene and its oxidative breakdown product, retinal, on primary oxidation products of linoleic acid methyl ester. Formation as well as isomer distribution of methyl linoleate hydroperoxides were followed by highperformance liquid chromatography. Oxidation of methyl linoleate without or with added β‐carotene (5, 20, 200 μg/g) or retinal (7, 18, 180, 360 μg/g) was carried out in the dark under air at 40 °C. Both β‐carotene and retinal promoted the formation of hydroperoxides and thus acted as prooxidants in a concentration‐dependent way. Moreover, carotenoids also had an effect on the isomeric distribution of primary oxidation products as high contents of retinal increased the portion (%) of trans,trans‐hydroperoxides. Being thermodynamically more stable isomers than cis,trans‐isomers of hydroperoxides they are known to accumulate during later phases of oxidation or during hydroperoxide decomposition. The results showed that β‐carotene and retinal were not effective hydrogen donors. These findings raise the question that carotenoids and their oxidative breakdown products enhance the decomposition of lipid hydroperoxides and this effect partially explains the prooxidative effect of carotenoids.     

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.     

Ascorbic acid and ascorbyl palmitate have only minor effect on the formation and decomposition of methyl linoleate hydroperoxides

Effects of ascorbic acid (AA) and ascorbyl palmitate (AP) on lipid hydroperoxides were evaluated during the formation and decomposition of methyl linoleate hydroperoxides (ML‐OOH). AA and AP at 1 and 10 mM levels had no effect on the formation of ML‐OOH during the autoxidation of methyl linoleate at 40 °C. However, depending on the reaction medium, AA and AP at 0.2 and 2 mM either slightly inhibited or accelerated the decomposition of 40 mM cis, trans ML‐OOH in hexadecane or in hexadecane‐inwater emulsion. The increased decomposition rate of ML‐OOH, when compared to a control sample, was apparently due to the reductive activity of AA and AP on metal ions present in the system, as the addition of EDTA improved the stability of ML‐OOH. The more detailed analysis of the decomposition reactions of ML‐OOH suggests that under favorable reaction conditions AA and AP were, to some extent, capable of acting as hydrogen atom donors to peroxyl radicals and reducers of hydroperoxides to more stable hydroxy compounds. However, since all these effects of AA and AP on lipid hydroperoxides were relatively small, it is assumed that the antioxidative activity of AA and AP as well as their effect on the stability and reactions of lipid hydroperoxides in biological systems and in foods is mainly related to their synergistic interactions with other antioxidative compounds such as tocopherols.     

Analyses of lipids and oxidation products by partition chromatography: Hydroxy fatty acids and esters

A liquid-partition chromatographic procedure was used to separate hydroxy fatty acids, their methyl esters, and reduced fatty ester hydroperoxides. Mixtures of methyl stearate, mono- and dihydroxystearate, and mixtures of the corresponding free fatty acids were easily separated. Chromatographic determinations for ricinoleate in castor oils compared favorably with the chemical and infrared analyses. The chromatographic procedure was used to separate hydroxy fatty acids inDimorphotheca andStrophanthus seed oils. The methyl ester of dimorphecolic acid, the principal hydroxy fatty ester ofDimorphotheca oil, behaved like reduced methyl linoleate hydroperoxide and showed a polarity intermediate between methyl 12-hydroxystearate and methyl 9,10-dihydroxystearate. The 9-hydroxy-12-octadecenoic ester ofStrophanthus oil had a larger retention volume than methyl ous hydroxy fatty esters isolated chromatographically. The diene content of the reduced hydroperoxides agrees well with values reported in the literature (1,5,16). The diene content of the chromatographed methyl dimorphecolate is higher than reported by Smithet al. (20) for their preparations but agrees well with the value reported by Chipault and Hawkins (6) for puretrans-trans conjugated methyl linoleate. The extinction coefficient of methyl 12-hydroxystearate at 2.8 μ is higher than that reported for ricinoleate and the absorption band is much sharper. Because of these two conditions no association of the hydroxyl groups is indicated. These results also confirm the purity of the hydroxy fatty esters obtained by LPC. This method has been a valuable adjunct to the study of various oxygen-containing fatty acid and esters and was used to characterize the hydroxy esters obtained from the hydrogenation of methyl linolenate hydroperoxides (9). This work offers a basis for the development of analytical methods to determine the hydroxy and other polar acid content of fatty glycerides and their derivatives.     

Autoxidation ofAcholeplasma laidlawii membranes

Autoxidation ofAcholeplasma laidlawii membranes (with equimolar ratio of palmitic and linoleic acid) lacks an obvious induction period, and the overall rate of disappearance of substrate does not follow closely that of typical autocatalytic kinetics. Throughout the course of autoxidation, the major oxygenated products isolated were hydroperoxides (as hydroxy esters) and compounds that gave rise to trihydroxy esters. The yield of trihydroxy esters was appreciable even at the early stage of the oxidation and eventually grew to surpass that of hydroperoxides. The positions of the three hydroxyl groups in the trihydroxy esters were determined to be mostly of the 1,2,5-type rather than 1,2,3-type arrangement. To a lesser extent, some degraded products, including dimethyl nonanedioate, methyl myristate, methyl pentadecanoate, methyl hexadecadienoate and methyl heptadecadienoate also were obtained. Dimethyl nonanedioate was a previously known degradation product from 9-hydroperoxide. The shorter chain esters presumably arise from the cleavage of α-hydroperoxides of palmitate and linoleate moieties.     

Degradation of monocarbonyls from autoxidizing lipids

In an attempt to account for carbonyls found in oxidized lipid systems, but not theoretically predicted from the decomposition of lipid hydroperoxides, a member from each of the monocarbonyl classes commonly observed in oxidizing lipids was oxidized at 45C in a Warburg apparatus and the carbonyl products studied. The carbonyl compounds used weren-nonanal,n-non-2-enal,n-hepta-2,4-dienal andn-oct-1-en-3-one. Nonanal was relatively stable to oxidation and was oxidized to nonanoic acid. Oct-1-en-3-one did not absorb oxygen during a 52-hr period; however, the unsaturated aldehydes oxidized at faster rates than methyl linoleate or linolenate. Non-2-enal upon absorption of 0.5 mole of oxygen was oxidized almost quantitatively to non-2-enoic acid. Hepta-2,4-dienal was polymerized at 0.5 mole of oxygen uptake. In addition both of the unsaturated aldehydes produced shorter chain mono- and dicarbonyls as oxidative degradation products. The identification of these compounds helps to explain the presence of carbonyls in oxidizing lipids and model systems that are not accountable through the decomposition of theoretically predictable isomeric hydroperoxide esters. The relatively large yield of malonaldehyde from the oxidized dienal suggests that these carbonyls may serve as a major source of malonaldehyde in oxidizing diene esters. Significant quantities of malonaldehyde are not observed in methyl linoleate until late stages of oxidation, and the dienals formed through degradation of primary hydroperoxides may in turn oxidize to give malonaldehyde. Technical Paper No. 1804, Oregon Agricultural Experiment Station. Submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy.     

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.     

Participation of free fatty acids in the oxidation of purified soybean oil during microwave heating

Effects of 0 to 1.0% levels of caprylic, capric, lauric, myristic, palmitic or stearic acid on the oxidative stability of purified soybean oil were investigated under microwave heating conditions. A prooxidative effect of the fatty acids introduced into the systems was established. The extent of this effect depended on the acyl chin and levels of added fatty acids. During microwave heating, the oxidative rate of purified soybean oil by the fatty acids was rapid compared to the addition of their corresponding hydrocarbons; the shorter the chainlength and the higher the levels of fatty acids, the more accelerated was the thermal oxidation in the oil. The results are explained on the basis of the catalytic effect of the carboxylic group on the formation of free radicals by the decomposition of hydroperoxides. Therefore, particular attention should be paid to the free fatty acid content, which affects the oxidative stability of purified soybean oil.     

Inhibition of Hydroperoxy-, Keto- and Hydroxy-FAME by Alpha- and Delta-Tocopherol at Rancimat Conditions

The effects of α‐ and δ‐tocopherol on inhibition of hydroperoxides, keto and hydroxy compounds under Rancimat conditions, i.e. 100 °C and air bubbling, were studied in samples of fatty acid methyl esters (FAME) obtained from high linoleic (HL) and high oleic (HO) sunflower oils. Primary hydroperoxides from methyl linoleate and methyl oleate and secondary keto and hydroxy compounds derived from methyl linoleate hydroperoxides were analyzed by HPLC–UV‐ELS. Different tocopherol concentrations, namely, 10, 50, 100, 500 and 1000 mg/kg, were tested. Irrespective of the lipid substrate and the initial concentration of tocopherol, results showed that the content of hydroperoxides accumulated during the induction period was remarkably higher in the samples containing δ‐tocopherol. The relative concentrations of oleate hydroperoxides in the HO samples were also higher in the presence of δ‐tocopherol. α‐Tocopherol was more effective in inhibiting hydroperoxides at low levels, with 100 mg/kg as optimal concentration, while δ‐tocopherol displayed optimal protection at 1000 mg/kg. Under the oxidation conditions applied, neither α‐ nor δ‐tocopherol showed a protective effect on hydroperoxide decomposition at any level assayed. Formation of keto‐ and hydroxy‐dienes was more related to the concentration of their hydroperoxide precursors. Furthermore, both tocopherols gave rise to increased concentrations of ketodienes at 500 and 1000 mg/kg compared to the controls. Such an effect was more pronounced for α‐tocopherol and in the HL samples.     

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.     

The rates of oxidation of unsaturated fatty acids and esters

The rates of autoxidation of oleic acid, ethyl oleate, linoleic acid, 10,12-linoleic acid, ethyl linoleate, trilinolein, pentaerythritol linoleate, dipentaerythritol linoleate, elaidolinolenic acid, linolenic acid, ethyl linolenate, trilinolenin, and methyl arachidonate have been studied by oxygen uptake in a Warburg respirometer and the results are compared with the rates of enzymatic oxidation of lipoxidase substrates. The increase in the number of double bonds in a fatty acid by one increases the rate of oxidation of the fatty acid or its esters by at least a factor or two. Earlier findings that acids oxidize more rapidly than their esters have been confirmed. The initial rates of lipoxidase oxidation of ethyl linoleate, ethyl linolenate, and methyl arachidonate were found to be essentially the same.     

On the kinetics of the autoxidation of fats. II. Monounsaturated substrates

The autoxidation of methyl oleate and oleic acid shows some differences as compared to the autoxidation of linoleate,e.g., the formation of water at an early stage. Linearization of experimental data on the autoxidation to high oxidation degrees of methyl oleate and other monounsaturated substrates shows that the rate equations previously derived for methyl linoleate in the range of 1–25% oxidation are valid, provided the correct expression for the remaining unreacted substrate is used. With monounsaturated substrates, part of the oxygen is consumed by a secondary oxidation reaction almost from the beginning, and only a certain constant fraction α of the total O₂ consumption is consumed in hydroperoxide formation. The fraction α is different for methyl oleate, oleyl alcohol, oleic acid andcis 9-octadecene, but the rate constant for the hydroperoxide formation is the same for all of them when experimental conditions are the same. The main difference between oleate and linoleate autoxidation is the much faster decomposition of the oleate hydroperoxides relative to their slow formation.     

Oxygen absorption of methyl esters of fat acids,and the effect of antioxidants

Summary The oxygen absorption of methyl linolenate, methyl linoleate, methyl oleate, methyl stearate, the distilled methyl esters of lard, and various mixtures of the four individual methyl esters were measured at 100° C. in the Barcroft-Warburg apparatus. Mixtures of methyl esters absorbed oxygen at a rate which could be approximately predicted from the rate of oxygen absorption of each component and the percentage of each present. The antioxidants nordihydroguaiaretic acid (NDGA), propyl gallate, benzylhydroquinone, α-tocopherol, and their synergistic combinations with citric acid, d-isoascorbyl palmitate, and lecithin were tested with the substrates methyl linoleate, methyl oleate, methyl stearate, and the distilled methyl esters of lard. Citric acid showed marked synergism with each antioxidant. The two most effective were the combinations of citric acid with nordihydroguaiaretic acid and with propyl gallate. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, United States Department of Agriculture.     

Detection and measurement of hydroperoxides by near infrared spectrophotometry

Summary Near-infrared spectra have been measured on a group of hydroperoxides of fatty acid esters and related substances. Only those substances having an −OOH group were found to absorb at 1.46 and 2.07 μ. Dialkyl peroxides and ozonized unsaturated substances had no such maxima in their near infrared spectra although they had high iodometric peroxide values. In a study of the thermal decomposition of methyl oleate hydroperoxide and a study of the autoxidation of methyl linoleate, the intensity of absorption at 1.46 and 2.07 μ paralleled the iodometric peroxide value. This work was aided by grants-in-aid from the Hormel Foundation, the Atomic Energy Commission (Contract AT-11-1-108), the Office of Naval Research (Contract N8 onr66218), the National Live Stock and Meat Board, and the National Diary Council. Hormel Institute Publication No. 170.     

The reaction of mercaptoacetic acid with methyl linoleate and linoleic acid

The reaction of mercaptoacetic acid with methyl linoleate and with linoleic acid was investigated. The reaction proceeded at low and erratic rates, with and without catalysts, such as peroxides at various temperatures, but could be accelerated by use of a large excess of mercaptoacetic acid. Addition of 1 mole of mercaptoacetic acid to 1 mole of methyl linoleate resulted in a product containing about 40% of mono-adduct. Ozonolysis of the purified mono-adduct yielded approximately equimolar quantities of caproic and azelaic acids, indicating that addition occurred about equally at the 9,10- and 12,13-ethylenic bonds. The dicarboxylic acid and the dimethyl ester of the mono-adduct and the tricarboxylic acid and trimethyl ester of the di-adduct of linoleic acid and mercaptoacetic acid were prepared, and the infrared spectra and some physical and chemical characteristics of these products were determined. The infrared spectra of the reaction products were obtained and correlated with functional groups which give rise to them. Bands at about 7.8 and 8.8 μ, commonly observed in long chain acids and esters and ascribed to C−O vibrations, are intensified in the sulfur-containing reaction products, suggesting characteristic absorption of C−S compounds at almost identical wavelengths. The formation of adducts was accompanied by a high degree of isomerization of the unreacted ethylenic bonds from thecis to thetrans form both in the mono-adduct and in unreacted methyl linoleate. The methyl linoleate recovered contained about 12% diene conjugation, but catalytic quantities of mercaptoacetic acid were not effective in inducing conjugation.     

A study of fatty acid methyl esters with epoxy or alkyne functionalities

Described are the physical and chemical properties of the methyl esters of two uncommon fatty acids: vernolic acid, containing an epoxy group, and crepenynic acid, containing a triple bond. The incorporation of an epoxy or alkyne group into the fatty acid structure is shown to greatly affect the properties compared to conventional unsaturated fatty acids. The methyl esters have been characterized and compared with ordinary fatty acid methyl esters (i.e., methyl oleate and linoleate) with respect to spectroscopic characterization [¹H nuclear magnetic resonance (NMR)], ¹³C NMR, and Fourier transform infrared), rheological properties, and oxidative reactivity (using chemiluminescence). Both methyl vernoleate and methyl crepenynate could successfully be produced by transesterification under basic conditions without reaction of the epoxy or alkyne groups. Rheological measurements showed that the methyl esters had a significantly lower viscosity compared to their triglyceride analogs. Smaller differences were seen when comparing the different methyl esters where methyl vernoleate had the highest viscosity due to the presence of the more polar oxirane group. Very large differences were found with respect to the oxidation rate of the different methyl esters. Methyl crepenynate was shown to oxidize extremely rapidly, whereas methyl vernoleate was very stable toward oxidation.     

Kinetic investigation into glucose-, fructose-, and sucrose-activated autoxidation of methyl linoleate emulsion

Autoxidation of methyl linoleate emulsions in aqueous phosphate buffer solutions in the presence of glucose, fructose, and sucrose has been studied by the rate of oxygen uptake. Oxidation rates increased with increasing concentration of sugars in the system. At comparable molar ratios of sugar to methyl linoleate the rate of oxidation in the presence of fructose was greater than with glucose which, in turn, was greater than with sucrose. Oxidation rates increased with pH until a maximum rate was reached at pH 8.00. There was less conjugation and more CO₂ with fructose than with glucose at a comparable level of oxygen uptake and pH value. This suggested concurrent oxidation of methyl linoleate and sugars; fructose is the most readily oxidized of those studied. Sugars seemed to be effective only in combination with the resulting methyl linoleate hydroperoxide. The effect of sugars rests in an activation of the decomposition of the linoleate hydroperoxide, and on the acceleration of the autocatalysis. The activation energy values for the autoxidation of methyl linoleate emulsions in the presence of sucrose, glucose, and fructose are 14.9, 10.6, and 10.6 K. Cal./mol. at pH 5.50; 16.0, 10.8, and 10.4 K. Cal./mol. at pH 7.00; and 14.4, 10.2, and 8.8 K. Cal. at pH 8.00, respectively. Addition of ascorbic acid to the system at zero time or after 2 hrs. increased oxygen absorption. It appeared that oxidized methyl linoleate caused co-oxidation of the ascorbic acid. Additions of nordihydroguaiaretic acid, propyl gallate, and hydroquinone to the system were ineffective in stopping oxidation when they were added after oxidation had commenced. They reduced effectively the rate of oxidation when added at zero time. Presented at the 52nd annual meeting, American Oil Chemists' Society, St. Louis, Mo., May 1–3, 1961. American Meat Institute Foundation Journal Paper No. 215.     

Reactions of biological antioxidants: I. Fe(III)-catalyzed reactions of lipid hydroperoxides with α-tocopherol

Rates of α-tocopherol oxidation were measured for free-radical reactions produced by Fe(III)-catalyzed dissociations of hydroperoxides. The kinetics were treated as first-order in α-tocopherol. The hydroperoxides were preformed from methyl linoleate, methyl linolenate, ethyl arachidonate, methyl eicosapentaenoate, and a fraction of polyunsaturated fatty esters of menhaden oil. The degree of unsaturation of the lipid hydroperoxides had little effect on the rates of α-tocopherol oxidation. The rates of oxidation decrease with the concentration of water and increase with the acidity of the media. The pH data suggest a transition from one predominant mechanism to another, which may involve principally acid catalysis. A mechanism for α-tocopherol oxidation is suggested.