Analysis of triacylglycerol positional isomers in food products as brominated derivatives by high-performance liquid chromatography coupled with a flame ionization detection.

Reversed-phase HPLC resolution and HPLC-flame ionization detection quantitation of model triacylglycerol positional isomer pairs (important in the study of food formulation lipids) after facile conversion to brominated derivatives is reported. The positional isomers in the triacylglycerol pairs were at least 98% resolved from each other during reversed-phase HPLC. Triacylglycerol quantitation obtained by HPLC-flame ionization detector was checked against standard positional isomer pairs known by mass. The flame ionization detection area percent gave absolute error range of 0.3-1.6% per triacylglycerol.

Effect of oleic and linoleic acids on the production of deep-fried odor in heated triolein and trilinolein.

To determine sources of desirable deep-fried flavor in frying oils, degradation products from heated triolein and trilinolein with 5-31% polar compounds representing low to high deterioration were evaluated by purge-trap gas chromatography-mass spectrometry-olfactometry. (E,E)-2,4-Decadienal, 2-heptenal, 2-octenal, 2,4-nonadienal, and 2,4-octadienal produced deep-fried odor at moderate-strong intensities in heated trilinolein. However, unexpected aldehydes-2,4-decadienal, 2,4-undecadienal, 2,4-nonadienal, and 2-octenal (all <15 ppm)-were produced in triolein heated for 6 h. These dienals possibly were produced by hydroperoxidation and/or hydroxylation followed by dehydration of 2-alkenals. The 2-alkenals were produced from thermal decomposition of hydroperoxides, epoxides, and keto and dimeric compounds produced during the heating of triolein. These aldehydes produced low intensities of deep-fried odor in triolein. This information helps to explain sources of the deep-fried flavor that is characteristic of high linoleic frying oils but which is only at low intensity levels in high oleic frying oils.

Autoxidation products of normal and genetically modified canola oil varieties determined using liquid chromatography with mass spectrometric detection.

Normal, high stearic acid and high lauric acid canola oil varieties were heated in the presence of air to allow autoxidation to occur. After the reaction, the oils were analyzed using a non-aqueous reversed-phase high-performance liquid chromatographic separation followed by detection using atmospheric pressure chemical ionization mass spectrometry. Oxidized products were separated and identified. The major autoxidation products which remained intact were epoxides and hydroperoxides. Two classes of epoxy triacylglycerols (TAGs) were formed. One class with the epoxy group replacing a site of unsaturation and one class adjacent to a site of unsaturation, as was previously reported for model TAGs. Intact oxidation products resulted mostly from oxidation of oleic acid, while oxidation products of linoleic and linolenic acid chains decomposed to yield chain-shortened species. Both neutral and polar chain-shortened products were observed. Polar chain-shortened decomposition products eluted at very short retention times and required a different chromatographic gradient to separate the molecules. This class of molecules was tentatively identified as core aldehydes. The high stearic acid canola oil yielded more intact oxidation products containing stearic acid, as expected. The high lauric acid oil produced intact oxidation products which contained lauric acid.

Triacylglycerol analysis of potential margarine base stocks by high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry and flame ionization detection.

Several margarine base stock candidates have previously been prepared for the purpose of finding better, more oxidatively stable food components: high-saturate vegetable oils, randomized vegetable oils, vegetable oil-hard stock blends, and interesterified vegetable oil-hard stock blends. Here are reported the triacylglycerol compositions of these products, determined using reverse-phase high-performance liquid chromatography (HPLC) coupled with a flame ionization detector or a quadrupole mass spectrometer with an atmospheric pressure chemical ionization source. Triacylglycerol percent composition results for samples of known composition (randomized and interesterified samples) exhibited less average error by HPLC coupled with a quadrupole mass spectrometer with an atmospheric pressure chemical ionization source, after application of response factors, than the results by HPLC coupled with a flame ionization detector. The fatty acid compositions calculated from the mass spectrometric data exhibited less average error than the fatty acid compositions resulting from the flame ionization detector data. The average error of the fatty acid compositions by the mass spectrometer was lowest for interesterified blend samples, next lowest for randomized samples, then followed by high-saturated fatty acid oils, normal oils, and blends. Analysis of the vegetable oil-hard stock blends by mass spectrometer required special treatment for calculation of response factors.

Non-volatile products of triolein produced at frying temperatures characterized using liquid chromatography with online mass spectrometric detection.

Oxidation products from triolein under model heated frying conditions have been analyzed using liquid chromatography with an evaporative light scattering detector and atmospheric pressure chemical ionization (APCI) mass spectrometric detection. Triolein was heated at 190 degrees C with 2% water added each hour, to simulate the moisture of a frozen product, until polar components reached approximately 30%. The samples were separated using reversed-phase high-performance liquid chromatography with APCI-MS detection. Triolein oxidation products included hydroperoxides, epoxides and a ketone. Other products were formed by shortening of an acyl chain on the intact triolein. Normal and oxygen-containing products formed by the dimerization of triolein were also observed. Other products included chain addition products formed by addition of acyl chain subunits to intact triolein to form higher molecular weight products.

Quantitative analysis of triglycerides using atmospheric pressure chemical ionization-mass spectrometry.

Atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) was used for quantitative analysis of triglycerides (TG) separated by reverse-phase high-performance liquid chromatography. APCI-MS was used for analysis of mono-acid TG standards containing deuterated internal standard, of a synthetic mixture of heterogeneous TG, of randomized and normal soybean oils and of randomized and normal lard samples. Quantitation of the TG by four approaches based on APCI-MS were compared, and these were compared to quantitation obtained using liquid chromatography (LC) with flame-ionization detection (FID). The APCI-MS methods were based on (i) calibration curves from data for mono-acid TG standards, (ii) response factors obtained from a synthetic mixture of TG, (iii) response factors calculated from comparison of randomized samples to their statistically expected compositions, and (iv) response factors calculated from comparison of fatty acid (FA) compositions calculated from TG compositions to FA compositions obtained by calibrated gas chromatography (GC) with FID. Response factors derived from a synthetic mixture were not widely applicable to samples of disparate composition. The TG compositions obtained using APCI-MS data without application of response factors had average relative errors very similar to those obtained using LC-FID. Numerous TG species were identified using LC/APCI-MS which were undetected using LC-FID. Two quantitation methods, based on response factors calculated from randomized samples or on response factors calculated from FA compositions, both gave similar results for all samples. The TG compositions obtained using response factors calculated from FA compositions showed less average relative error than was obtained from LC-FID data, and were in good agreement with predicted compositions for the synthetic mixture and for randomized soybean oil and lard samples.

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.

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.

Fluorescence formation from the interaction of DNA with lipid oxidation degradation products.

To clarify the mechanism of fluorescence formation between DNA and lipid degradation products in the presence of ferric chloride and ascorbic acid, a number of carbonyl compounds and decomposition products of pure methyl linolenate hydroperoxides were examined. Keto derivatives of methyl ricinoleate, linoleate, and oleate, alkanals and 2-alkenals produced little or no fluorescence with DNA in the presence of ferric chloride-ascorbic acid. 2,4-Alkadienals were more active and 2,4,7-decatrienal was the most active. Mixtures of volatile aldehydes prepared from linolenate hydroperoxide decomposed either thermally or with iron and ascorbate had the same activity as 2,4,7-decatrienal. Higher molecular-weight products from the decomposition of methyl linolenate hydroperoxides showed relatively low activity. beta-Carotene, alpha-tocopherol and other antioxidants effectively reduced the amount of fluorescence formed by linolenate hydroperoxides. The results suggest that, in addition to hydroperoxide decomposition products, singlet oxygen and/or free radical species contribute significantly to the fluorescence formed from the interaction of methyl linolenate hydroperoxides with DNA in the presence of ferric chloride and ascorbic acid.

Mutagenicity tests of lipid oxidation products in Salmonella typhimurium: monohydroperoxides and secondary oxidation products of methyl linoleate and methyl linolenate.

Nine hydroperoxy and hydroperoxy-epidioxy oxidation products derived from either autoxidation (AO) or photosensitized oxidation (PO) of methyl linoleate (MLo) or methyl linolenate (MLn) were tested for mutagenic activity by the Salmonella typhimurium his+ reversion assay using strains TA100, TA98, TA102, TA97 and TA1537. All nine oxidation products, monohydroperoxides from AO-MLn (I) or from PO-MLn (II), dihydroperoxides from PO-MLo (III), AO-MLn (IV) or PO-MLn (V), hydroperoxy epidioxides from PO-MLo (VI), AO-MLn (VII) or PO-MLn (VIII) and hydroperoxy bis-epidioxides from PO-MLn (IX), were weakly mutagenic in strains TA97 and/or TA100. The hydroperoxy epidioxides (VI-IX) exhibited significantly greater activity in strain TA97 than did the monohydroperoxides (I, II) or the dihydroperoxides (III-V). In strain TA100, all of the oxidation products tested exhibited similar activity. No major differences between products derived from autoxidized and photooxidized MLn (I v. II, IV v. V, VII v. VIII) were obtained. Rat-liver S-9 reduced the toxicity of all oxidation products to the tester strains. The greatest mutant yields were usually obtained in the presence of S-9, but mutagenic potency was sometimes greater without S-9. The structural feature common to all of the mutagenic oxidation products was the presence of a hydroperoxy group, suggesting that this characteristic is responsible for the observed mutagenicity, either directly or through a common degradative pathway to reactive products of lower molecular weight.

The reaction of DNA with lipid oxidation products, metals and reducing agents.

The interaction of lipid hydroperoxides and secondary oxidation products with DNA was investigated by evaluating the fluorescence formed in the presence of metals and reducing agents. We also investigated the effect of malonaldehyde, because it has been generally considered responsible for the formation of fluorescence with DNA. However, malonaldehyde usually has been estimated by the notoriously unspecific thiobarbituric acid test. At low concentration of oxidation products (1 mM), fluorescence formation required the presence of metals and ascorbic acid. In contrast, a positive thiobarbituric acid reaction was obtained with many lipid oxidation products without metals or ascorbic acid. Monohydroperoxides from autoxidized methyl linoleate and linolenate produced the highest level of fluorescence. Hydroperoxy epidioxides of linolenate and dihydroperoxides of linoleate and linolenate were among the most active secondary products in forming fluorescence with DNA. In contrast, malonaldehyde produced very little fluorescence under our conditions. The thiobarbituric acid values did not correlate with fluorescence formation. This study showed that, in our model reaction system, DNA forms fluorescent products by the breakdown of lipid oxidation products in the presence of metals and ascorbic acid into reactive materials other than malonaldehyde. Therefore, the importance of malonaldehyde in its crosslinking properties with DNA may have been exaggerated in the literature.

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,(1)H-NMR,(13)C-NMR, GC-MS). Major secondary products included diastereomeric pairs of 13-hydroperoxy-10,12-epidioxy-trans-8-octadecenote (I and III) and 9-hydroperoxy-10,12-epidioxy-trans-13-octadecenoate (II and IV); minor secondary products included hydroperoxy oxy genated and epoxy esters. Thermal decomposition of the hydroperoxy cyclic peroxides produced hexanal and methyl 10-oxo-8-decenoate as major volatiles from I and III and methyl 9-oxo-nonanoate and 2-heptenal from II and IV. Hydroperoxy cyclic peroxides may be important sources of volatile decomposition products of photooxidized fats.

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

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, dihydroxyoctadecenoates, and dihydroxyoctadecanoates.

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, epoxyhydroxymonoenes, 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.