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: 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.     

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.     

Methods of analysis of mixtures of oleic,linoleic and saturated esters and their application to highly purified methyl oleate and methyl linoleate

Summary Iodine numbers by the Hanus and Wijs methods and thiocyanogen numbers using various absorption periods, were determined on methyl oleate and methyl linoleate and on mixtures of these esters. It is concluded that iodine numbers by the Wijs method and three-hour thiocyanogen numbers are more satisfactory for methyl linoleate and for mixtures containing large amounts of this ester. Small amounts of higher saturated acids were determined with a precision of about 0.1 unit-percent by means of the Bertram procedure. A purified specimen of methyl oleate was found to contain about 0.2 percent of saturated ester by this method. Food Research Division Contribution No. 428.     

Autoxidation of methyl linoleate. Separation and analysis of isomeric mixtures of methyl linoleate hydroperoxides and methyl hydroxylinoleates

The mixture of conjugated diene hydroperoxide isomers obtained from autoxidation of methyl linoleate was separated by high performance liquid chromatography (HPLC). Four major isomers were obtained from adsorption chromatography and identified as the 9 and 13 positional isomers having thetrans-trans andcis-trans configurations. The latter geometrical isomers have thetrans double bond adjacent to the hydroperoxide group. The hydroxy compounds (methyl hydroxylinoleates) obtained from the hydroperoxides by NaBH₄ reduction were similarly separated but with improved resolution. This is the first instance of the complete separation of these compounds and provides a rapid method for their analysis. Unlike adsorption chromatography, reversed-phase chromatography separates the mixtures only according to the geometrical isomerism of the double bonds and not according to the position of the hydroxy or hydroperoxide function.     

Structure of monohydroperoxides formed by chlorophyll photo-sensitized oxidation of methyl linoleate

The structures of the hydroperoxides prepared by photochemical oxidation of methyl linoleate in the presence of chlorophyll were determined by gas liquid chromatography-mass spectrometry (GLC-MS) of the trimethylsilyl (TMS) derivatives of the hydroxyesters. The position of the double bonds were deduced via hydroxylation with OsO₄, followed by GLC-MS analysis of the TMS derivatives. Structures of the original hydroperoxides were elucidated from these data. One of 28 papers presented at the Symposium “Metal-Catalyzed Lipid Oxidation,” ISF-AOCS World Congress, Chicago, September 1970.     

Hydrogen sulfide adducts of methyl oleate and linoleate

Sulfur compounds derived from photochemical addition of hydrogen sulfide to methyl oleate and linoleate were separated by preparative gas chromatography. The major compounds were investigated by NMR, mass and IR spectroscopy and by elemental analysis. The primary product of the methyl oleate reaction was methyl 9(10)-mercaptostearate. Gas chromatograms of the product from methyl linoleate showed four principal peaks. From mass spectra and NMR data, we identified methyl 9-(2-pentyl-1-thiolan-5-yl)nonanoate, methyl 8-(2-hexyl-1-thiolan-5-yl)octanoate and methyl 9-(3-hexyl-1,2-dithiolan-5-yl)nonanoate. Evidence for the formation of methyl mercapto-octadecenoates and methyl dimercaptostearates was also obtained. ARS, USDA.     

Singlet oxygen oxidation of methyl linoleate: Isolation and characterization of the NaBH4-reduced products

The mixture of diene hydroperoxides from methylene blue-sensitized oxidation of methyl linoleate was reduced with NaBH₄ and the resulting alcohols were separated by high pressure liquid chromatography (HPLC). Four diene alcohols were isolated in approximately equal yields from adsorption and reversed phase HPLC; the isomers were identified as methyl esters of 9-hydroxy-10,12-, 10-hydroxy-8,12-, 12-hydroxy-9,13- and 13-hydroxy-9,11-octadecadienoate. Formation of equal yields of both conjugated and nonconjugated diene alcohols from methyl linoleate is characteristic of singlet oxygen oxidations. The detection of the easily separated nonconjugated isomer methyl 10-hydroxy-trans-8,cis-12-octadecadienoate from methyl linoleate is proposed as a test to probe the involvement of singlet oxygen in biological oxidations. A preliminary report of these results was presented at the 177th meeting of the American Chemical Society, Honolulu, HI, April 1–6, 1979; see abstracts of papers, paper No. ORGN-375.     

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.     

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.     

Differences in Oxidation Kinetics Between Conjugated and Non-Conjugated Methyl Linoleate

The oxidation kinetics of conjugated methyl linoleate was compared with that of non-conjugated methyl linoleate under mild oxidation conditions (30 °C in the dark). Samples of methyl 9-cis,11-trans-linoleate, methyl 10-trans,12-cis linoleate and methyl 9-cis,12-cis linoleate were assayed separately and in mixtures. For comparative purposes, methyl α-linolenate and methyl oleate were also used. Two complementary analytical approaches were selected to monitor the progress of oxidation, (1) the traditional follow-up of residual substrate by gas liquid chromatography, and (2) an analytical procedure by high-performance size-exclusion chromatography (HPSEC) for direct measurement of the oxidation compounds formed. The HPSEC method enabled us to quantitate oxidized monomers, dimers and polymers concomitantly in a rapid and direct analysis. Results showed that conjugated methyl linoleate samples oxidized later than their non-conjugated counterparts, and showed a very different oxidation pattern. Thus, formation of oxidized monomers was negligible and the first and major compounds formed were polymerization products. Also, under the conditions used, non-conjugated and conjugated methyl linoleate samples in 1:1 mixtures led to decreased oxidation rate of non-conjugated methyl linoleate and increased oxidation rate of conjugated methyl linoleate. This study supports the view that oxidation kinetics of conjugated dienes differ substantially from that of methylene-interrupted dienes.     

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.     

Photochemical oxidation of fatty acid esters with and without chlorophyll

1. It has been confirmed that the principal products formed in the oxidation of methyl oleate by oxygen under a variety of conditions are predominantlytrans hydroperoxides. However no inversion of the double bond occurs in unoxidized oleate. Hence the conversion ofcis totrans double bonds and peroxide formation occur together in the same molecules.
2. The autoxidation of methyl linoleate at low temperature yields predominantlycis,trans conjugated hydroperoxides. Autoxidation at 25°C., oxidation catalyzed by visible light, or ultraviolet light and copper soap catalyzed oxidation at temperatures appreciably above 0°C., lead to the formation primarily oftrans,trans conjugated hydroperoxides. The inversion of the second double bond in this case appears to be independent of the peroxide-forming reactions.
3. The photochlorophyll oxidation of methyl linoleate leads to the formation of some unconjugated hydroperoxides, some of which containtrans double bonds.
4. Under all of the conditions employed in the present investigation, the oxidation of methyl oleate and linoleate led primarily to the formation of monomeric peroxides which retained most of the unsaturation of the parent compound.

     

Peroxide value determination—Comparison of some methods

A comparison between the determination of the peroxide value by the methods of Wheeler and Sully (iodometric titration) and that of Stine, et al. (ferric thiocyanate method) was made. Some oxidized vegetable oils, H₂O₂, t-butyl hydroperoxide, cumene hydroperoxide, methyl oleate hydroperoxides, and methyl linoleate hydroperoxides were used as substrates. One-hundred percent of the methyl linoleate hydroperoxides were recovered by the Wheeler reduction, 85% by the Sully method. The Wheeler method was used to reduce the methyl linoleate hydroperoxides to the corresponding hydroxy acids. In the Sully procedure, the hydroxy acids are only intermediates which are dehydrated to octadecatrienoic acids. One equivalent methyl linoleate hydroperoxide oxidized two equivalents of I⁻ (Wheeler) and four equivalents of Fe²⁺ (Stine, et al.). By way of contrast, H₂O₂ needs only two equivalents I⁻ or Fe²⁺ for reduction. The excess consumption of reduction equivalents in the ferric thiocyanate method probably is caused by secondary reactions of the methyl linoleate hydroperoxide acyl residue.     

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.     

Limitations of the method using peroxidase activity of hemoglobin for detecting lipid hydroperoxides

The method using peroxidase activity of hemoglobin (Hb) for the determination of lipid peroxides was examined by using pure methyl linoleate hydroperoxides, trilinoleoylglycerol hydroperoxides and egg yolk phosphatidylcholine hydroperoxides as substrates and tetramethyl benzidine as electron donor for the peroxidase reaction of Hb. The reactivities of these substrates were quite varied. Furthermore, some electron donors were tested for peroxidase activity of Hb, but none showed a complete reduction of methyl linoleate hydroperoxides. From these results, it seems the Hb method needs to be carefully applied to biological materials that contain mixtures of different types of lipid classes.     

Dimers formed in oxygenated methyl linoleate hydroperoxides

The formation of dimers was demonstrated in methyl linoleate hydroperoxides decomposed by bubbling with dry air at 30 C. The dimer fraction isolated by gel permeation chromatography was further fractionated by successive silicic acid column and high performance liquid chromatography. Major components were analyzed after derivatizations by gas chromatography-mass spectrometry with several ionization methods, i.e., electron impact, chemical ionization and field desorption. After aeration for 90 min, 1.4% of the hydroperoxides were decomposed. However, almost all of the secondary products were dimers, while polar monomeric and low molecular products were negligible. After aeration for 390 min, both polar monomeric (5.0%) and low molecular (0.8%) compounds formed, but dimers and polymers (18.1%) were still the major products. These results show the importance of polymerization in the aerobic breakdown of hydroperoxides. The dimers isolated from hydroperoxides aerated 90 min could be separated into two fractions according to their polarities. The dimers identified usually were composed of octadecadienoate and oxygenated octadecenoate moieties crosslinked through either ether or peroxy linkages across C-9 or C-13 positions. The oxygen-containing functional groups found in the dimers include hydroperoxy, hydroxy and oxo groups. The polar dimers had two of these groups per molecule, while the less polar dimers had one. The main constituents of dimers were linked through peroxy bridges and found to be similar to the dimers previously identified in autoxidized methyl linoleate. These dimers are suggested as important intermediates in linoleate oxidation and as precursors of flavor deterioration.     

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.     

Metal-requiring and non-metal - requiring catalysts in the autoxidation of methyl linoleate

During the autoxidation of methyl linoleate, peroxide-containing substances are formed which, when added to unoxidized methyl linoleate, will catalyze oxygen uptake. Materials active only in the presence of added metal ions (MCs) were not inactivated during aerobic thin-layer chromatography on silicic acid or alumina but were selectively inactivated by treatment with triphenylphosphine. Catalysts not requiring added metal ions for activity (NCs) are not affected by triphenylphosphine, but the catalytic activity is lost during aerobic thin-layer chromatography. Autoxidized methyl linoleate was separated into four peroxide-containing fractions by elution from a silicic acid column with hexane-diethyl ether mixtures. Each fraction was found to contain both MCs and NCs. Presented at the 4th International Symposium on Metal Catalyzed Lipid Oxidation, London, April 1975.     

Nucleophilic addition of hydrogen sulfide to methyl oleate,methyl linoleate,and soybean oil

Hydrogen sulfide was added to methyl oleate, methyl linoleate, and soybean oil at −70 and 25 C in the presence of boron trifluoride. Major reaction compounds were identified by gas liquid chromatography and mass spectrometry. At −70 C with a 200 molar ratio of hydrogen sulfide to ester, the reactions were complete in 4 hr. Primary reaction product from methyl oleate was methyl 9(10)-mercaptostearate. Methyl linoleate gave ca. equal amounts of methyl 9-(2-pentyl-1-thiolan-5-yl) nonanoate and methyl 8-(2-hexyl-1-thiolan-5-yl) octanoate. At 25 C, the reaction of methyl oleate and linoleate with hydrogen sulfide was less complete, and more side reactions were noted. When equimolar amounts of methyl oleate and methyl 9(10)-mercaptostearate were reacted in the presence of boron trifluoride at 25 C, a new compound was formed, bis(methyl n-octadecanoate 9[10]-yl) sulfide. The addition of liquid hydrogen sulfide to soybean oil at −70 C in the presence of boron trifluoride yields a product which, upon saponification, acidification, and methylation analyzes by gas liquid chromatography as ca. 52% thiolan, 27% mercaptostearate, 10% palmitate, 6% stearate, and 5% unidentified compounds.