cis andtrans Analysis of fatty esters by gas chromatography: Octadecenoate and octadecadienoate isomers

A gas chromatographic method has been developed for quantitative determination of thecis andtrans percentages in octadecenoate and octadecodienoate esters. To separatecis- andtrans-monoene and diene isomers on a packed GC column, the fatty esters were stereospecifically epoxipized with peracetic acid. A simple and quantitative epoxidation procedure allows thecis- andtrans-epoxyoctadecanoates to be analyzed without prior isolation from the reaction mixture. No positional or geometric isomerization of the double bond occurred during epoxidation. Synthetic mixtures containingcis- andtrans-6,-9 and-12 octadecenoate isomers were completely separated intocis andtrans fractionstrans-15-Octadecenoate was the only isomer investigated that partially interfered in the analysis. Diene mixtures containingrans,trans-, cis,trans- andcis,-cis-9,12-octadecadienoates were also successfully analyzed by gas liquid chromatography (GLC) after epoxidation with peracetic acid. Each diene isomer formed two pairs of diepoxy diastereomers, some of which could be separated. Onecis,cis-diepoxyoctadecanoate diastereomer peak overlapped thecis,trans-diepoxyoctadecanoate peaks. The totalcis,-cis-diepoxyoctadecanoate percentages were calculated by using the ratio of the twocis,cis-diepoxyoctadecanoate diastereomers. Other positional octadecadienoate isomers were also epoxidized and analyzed by GLC. The large number of possible octadecadienoate isomers requires that some preeiminary fractionation be made before GC analysis is practical for diene isomers. Presented at the AOCS Meeting, Atlantic, City, October 1971 N. Market. Nutr. Res. Div., ARS, USDA.     

Occurrence of octadecenoic fatty acid isomers from hydrogenated fats in human tissue lipid classes

The level oftrans-18∶1 isomers in several isolated lipid classes of human liver, heart, red blood cells and plasma was determined. Phospholipids contained substantially fewertrans-18∶1 isomers than triglycerides. The double bond distribution of thecis andtrans octadecenoate fraction of triglycerides and phosphatidylcholines from human liver and heart was determined. Whereas the double bond distribution of the triglycerides correlated closely with the pattern found in dietary hydrogenated vegetable oils, the phosphatidylcholine fraction showed evidence of selective incorporation or metabolism of specifictrans positional isomers. In general, isomers with double bonds near the methyl terminus were present at levels higher than expected from their relative abundance in the diet. Refinements in methodology needed to analyze octadecenoate double bond configuration and location in human tissues are presented.     

Isomers of monoethylenic fatty acids in some partially hydrogenated marine oils

An analytical study of the monoethylenic isomers in commercial samples of partially hydrogenated herring, whale and seal oils is presented. The results show that with hydrogenated herring oil there is a slight decline in monoenetrans content from 37% in C₁₆ through to 32% in C₂₂. With both whale and seal oils, monoenetrans contents were constant at 54% and 59%, respectively, throughout all chain lengths. In general thecis andtrans positional isomers from hydrogenated whale and seal oils were more scattered than those from hydrogenated herring oil; however in each oil the majorcis isomers of each chainlength were indicative of originalcis fatty acid isomers in the raw oils.     

The application of packed column gas chromatographic analysis to the determination oftrans unsaturation

A study of the separation of synthetic mixtures ofcis andtrans methyl octadecenoate with packed column gas chromatography indicated its usefulness for such analyses. Several margarines were analyzed by this method and the results compared with those obtained by the infrared spectroscopic method. Reasonable agreement between both methods was obtained, but the gas chromatographic determination yielded lower values (average 3.8%). The presence oftrans unsaturation was also shown in selected samples of human milk and blood lipids.     

Analysis of C18:1 cis and trans fatty acid isomers by the combination of gas-liquid chromatography of 4,4-dimethyloxazoline derivatives and methyl esters

Trans fatty acids in foods are usually analyzed by gas-liquid chromatography (GLC) of fatty acid methyl esters (FAME). However, this method may produce erroneously low values because of insufficient separation between cis and trans isomers. Separation can be optimized by preceding silver-ion thin-layer chromatography (Ag-TLC), but this is laborious. We have developed an efficient method for the separation of 18-carbon trans fatty acid isomers by combining GLC of FAME with GLC of fatty acid 4,4-dimethyloxazoline (DMOX) derivatives. We validated this method against conventional GLC of FAME, with and without preceding Ag-TLC. Fatty acid isomers were identified by comparison with standards, based on retention times and mass spectrometry. Analysis of DMOX derivatives allowed the 13t, 14t, and 15t isomers to be separated from the cis isomers. The combination of the GLC analyses of FAME and DMOX derivatives gave results comparable with those obtained by GLC of FAME after preceding Ag-TLC, while saving about 100 h of manpower per 25 samples. It allowed the identification and quantitation of 11 trans and 8 cis isomers and resulted in 25% higher values for total C_18:1 trans, compared with the analysis of FAME alone. The combination of DMOX and FAME analyses, as applied to the analysis of 14 foods that contained ruminant fat and partially hydrogenated vegetable and fish oils, indicated that the most common isomers were 11t in ruminant fats, 9t in partially hydrogenated fish fats, and either 9t or 10t in partially hydrogenated vegetable fats. The combination of GLC analyses of FAME and DMOX derivatives of fatty acids improves the quantitation of 18-carbon fatty acid isomers and may replace the laborious and time-consuming Ag-TLC.     

High performance liquid chromatography and glass capillary gas chromatography of geometric and positional isomers of long chain monounsaturated fatty acids

Positional and geometrical isomers of monounsaturated long chain fatty acids were analyzed by the combination of high performance liquid chromatography (HPLC) and glass capillary gas chromatography (GC). A preparative group separation ofcis andtrans isomers of the monounsaturated fatty acid methyl esters was achieved according to chain length by reversed-phase HPLC, and using a highly sensitive interference refractive index detector. After collection of the different fractions containingcis andtrans forms of the monounsaturated fatty acid methyl esters, the fractions were analyzed for their content of positional isomers using glass capillary GC with Silar-5 CP as stationary phase. The preparative step in the HPLC was also used analytically for the determination of the ratio between thecis andtrans monounsaturated fatty acids. A comparison was made between the results obtained with the HPLC technique and the results of a GLC technique with a packed OV-275 column. There was a good correlation between the 2 techniques with a tendency to highertrans values with the HPLC technique (4%). It was shown with reference substances that 18∶1ω6-cis to ω11-cis and 18∶1ω5-trans to ω12-trans, the most common monounsaturated fatty acid isomers in partially hydrogenated vegetable oils, could be almost quantitatively recovered in the HPLC step. Most of the individual positional isomers of monounsaturated fatty acids of varying chain length could be separated and determined in the glass capillary GC step with the exception of those isomers containing the double bond in a relatively high ω-position. The relative standard deviation of the technique as determined with reference substances was better than 4%. The described technique was applied to the analysis of the isomeric monounsaturated fatty acid content in partially hydrogenated vegetable and marine oils, and about 5 samples a day could be executed. Part of this work has been presented at the ISF/AOCS World Congress, New York (1980)JAOCS 58, (4), 1981, abstr. no. 184.     

Positional and geometrical isomers of linoleic acid in partially hydrogenated oils

The geometrical and positional isomers of linoleic acid of a partially hydrogenated canola oil-based spread were isolated and identified. Through partial hydrazine reduction and mass spectral studies,cis-9,trans-13 octadecadienoic acid was identified as the major isomer. Other quantitatively important isomers characterized werecis-9,trans-12;trans-9,cis-12 andcis-9,cis-15. These four were also the major isomers in margarine based on common vegetable oils. A number of minor isomers were detected and some structures identified weretrans-9,trans-12;trans-8,cis-12;trans-8,cis-13;cis-8,cis-13;trans-9,cis-15;trans-10,cis-15 andcis-9,cis-13. The proportions of the various isomers are given for some margarines in the Canadian retail market. The amounts oftrans-9,trans-12 isomer in Canadian margarines were generally below 0.5% of the total fatty acids.     

Linolenic acid artifacts from the deodorization of oils

Gas liquid chromatography on polar open tubular columns of the methyl esters of fatty acids from vegetable oils shows that the linolenic acid in deodorized oils is accompanied by two major artifacts identified as cis-9,cis-12,trans-15 and trans-9,cis-12,cis-15 isomers. Physicochemical studies, isolation, and partial degradation steps showed two additional isomers with trans-9,cis-12,trans-15 and cis-9,trans-12,cis-15 structures. Gas liquid chromatography also showed that linoleic acid was accompanied by the trans-9,cis-12 isomer. These artifacts were not present in unrefined oils or bleached oils but could be induced by deodorization in the laboratory. Proportions of the two major artifacts in total 18:3ω3 are given for some vegetable oils from the retail market. Presented in part at the AOCS Spring Meeting, New Orleans.     

Measurement oftrans and other isomeric unsaturated fatty acids in butterand margarine

A procedure is described for gas liquid chromatographic determination of cis andtrans isomers of unsaturated fatty acids after fractionation of the saturated, monenoic, dienoic, and polyenoic fatty acid methyl esters by argentation thin layer chromatography. To test its reliability, the procedure was used for quantitative measurement of transisomers of unsaturated fatty acids in a known mixture of simple triglycerides containing saturated fatty acids from 4:0 to 24:0 andcis andtrans isomers of 14:1. 16:1, 18:1, and 18:2. Results of the analyses of five margarine and five butter samples are presented, together with results of infrared spectrophotometric analyses fortrans fatty acid concentrations, ultraviolet spectrophotometric analyses for conjugated fatty acid concentrations, and enzymatic analyses forcis-cis-methylene interrupted fatty acid concentrations. The combined argentation thin layer and gas Chromatographic procedure is suitable for determination of the principal fatty acids in complex food lipids such as milk fat.     

Monoethylenic fatty acids of a partially hydrogenated herring oil

A Canadian Atlantic herring oil hydrogenated for margarine use to an iodine value of 76 and melting point of 32.5 C was found to have 30% saturated acids and 66% monounsaturated fatty acids. The monounsaturated fatty acids could be analytically determined ascis andtrans isomers by open tubular gas liquid chromatography.Trans acids were 33% of the C₁₆ and C₁₈ monounsaturated acids, and 32 and 28%, respectively, of the C₂₀ and C₂₂ monounsaturated acids. After separation of geometric isomers by Florisil-silver nitrate chromatography the positional isomers in each class were determined by oxidative fission. The double bond positions of the originalcis fatty acids were largely retained in bothcis andtrans isomers, but additional isomers were observed, especially in thetrans fatty acids.     

Raman spectroscopic analysis of thecis/trans isomer composition of edible vegetable oils

A new method is presented for determining thecis/trans isomer content of edible vegetable oils. The intensities of Raman lines near 1656 and 1670 cm⁻¹ are associated with thecis andtrans configuration, respectively. A precision of ca. 1% can be obtained in thecis/trans isomer analysis of binary mixtures of methyl esters and triglycerides of monoenes and dienes and of hydrogenated vegetable oils. The spectroscopic data also provide the iodine value of vegetable oils or isolated fractions with precision for a single determination of ca. 1%. Presented at the AOCS meeting, Houston, May 1971. W. Market. Nutr. Res. Div., ARS, USDA.     

Linoleic acid isomers in heat treated sunflower oils

Heat treatment of sunflower oil resulted in the formation of linoleic geometrical and positional isomers. These isomers were isolated using a combination of column chromatography, urea fractionation, high performance liquid chromatography (HPLC) on a C18 reverse phase column and silver nitrate thin layer chromatography (TLC). Each component was submitted to hydrazine reduction and the resulting monoenes to AgNO₃-TLC. The resultingcis andtrans fractions were submitted to ozonolysis in BF₃-MeOH in order to determine the position of the ethylenic bonds. The major isomers were thecis, trans andtrans, cis 18∶2 Δ9, 12, thetrans, trans 18∶2 Δ9, 12 and somecis, trans, trans, cis andtrans, trans 18∶2 conjugated dienes. Thecis, trans andtrans, cis conjugated dienes were the Δ9, 11, Δ10, 12, Δ11, 13 and Δ12, 14 while thetrans, trans isomers were the Δ9, 11, Δ10, 12 and Δ11, 13. These C18∶2 isomers also were detected in oils collected from restaurants and market vendors. Presented in part at the AOCS/JOCS annual meeting in Honolulu, Hawaii, in May 1986.     

Essential fatty acid-deficient rats: II. On the fatty acid composition of partially hydrogenated arachis,soybean and herring oils and of normal,refined arachis oil

The fatty acid composition of partially hydrogenated arachis (HAO), partially hydrogenated soybean (HSO) and partially hydrogenated herring (HHO) oils and of a normal, refined arachis oil (AO) was studied in detail by means of direct gas liquid chromatography, ultraviolet and infrared spectrophotometry and by thin layer chromatography fractionation on silver nitrate-silica gel plates followed by gas liquid chromatography. It was shown that the partially hydrogenated oils all contained fatty acids withtrans double bonds. In the plant oils, thetrans acids were present mainly as elaidic acid. The HHO showed an almost equal distribution betweentrans 18∶1 ω9,trans 20∶1 ω>9 andtrans 22∶1 ω>9. Sometrans configuration was also found in the C₂₀-and C₂₂-dienes and trienes of the HHO. In all the oils, conjugated fatty acids were present in minor amounts only (<0.5%). Special attention was given to the ω-acids known to be of specific nutritional value. The HSO contained about 32% linoleic acid, whereas the content ofcis, trans+trans, cis andtrans, trans octadecadienoic isomers was 1.7% and 0.5%, respectively. The amount of linoleic acid in the HSO was even higher than that of AO (29%). The HAO contained only 0.8% 18∶2 ω6 (linoleic acid). Further, two 18∶2 fatty acids with ω>6, acis, cis and atrans, trans isomer, were present in small amounts. The HHO contained 0.5% 18∶2 ω6 (linoleic acid). Isomers of 18∶2 ω>6 were also found in the HHO. They may be hydrogenation products of higher unsaturated C₁₈-acids orginally present. All the C₂₀- and C₂₂-dienes and trienes were shown to have an ω-chain greater than 6. Fatty acids with ω6-structure were not formed during partial hydrogenation of the oils studied.     

Incorporation of dietarycis andtrans octadecenoate isomers in the lipid classes of various rat tissues

The percentage distribution of the geometrical and positional isomers in the hexadecenoates and octadecenoates isolated from triglycerides, phosphatidylcholines, and phosphatidylethanolamines of brain, heart, kidney, liver, lung, muscle, spleen, and adipose tissues from rats maintained four weeks on a semipurified diet supplemented with 15% partially hydrogenated safflower fatty acids, has been determined. Except for brain, octadecenoate percentages were increased in each of the lipid classes of all the tissues by the dietary fat. Although the diet did not contain detectable hexadecenoates, the 16∶1 fraction from the lipid classes of all the tissues was composed of 10–70% of thetrans isomers, indicating chain shortening of the dietary octadecenotes. Distribution ofcis andtrans positional isomers in triglyceride hexadecenoates was approximately the same in all tissues. Relatively high percentages of the Δ9, Δ10, and Δ11 isomers were observed, but the Δ8 was the predominatingtrans hexadecenoate isomer, indicating preferential chain shortening of thetrans δ10 octadecenoate.Trans octadecenoates were found in all tissues, but concentrations were dependent on tissue and lipid class. The distribution of thecis andtrans octadecenoate isomers was similar in all the tissue triglycerides, with the distribution of thetrans isomers resembling the diet. In contrast, the percentage distribution of thetrans octadecenoates in the phospholipid classes differed dramatically from the diet, and the distribution was dependent on both the tissue and lipid class. The Δ12, Δ13, and Δ14trans octadiet, suggesting an accumulation of these isomers. Although thecis Δ10 octadecenoate was a significant dietary component, this isomer was not incorporated significantly into any lipid class of any tissue. The metabolic fate of this isomer remains unknown.     

Quantitative analysis of long-chain trans-monoenes originating from hydrogenated marine oil

Gas chromatography (GC) is used for the analysis of trans-fatty acids in partially hydrogenated vegetable oils. Although trans-isomers of C₁₈ carbon length predominate in partially hydrogenated vegetable oils, trans-isomers of C₂₀ and C₂₂ carbon length occur in partially hydrogenated fish oil. We report a simple silver ion chromatographic combined with capillary GC technique for quantitative analysis of trans-monoenes derived from partially hydrogenated fish oil. Silver nitrate thinlayer chromatographic (TLC) plates are developed in toluene/hexane (50∶50, vol/vol). Fatty acid methyl esters are separated into saturates (R _f 0.79), trans-monoenes (R _f 0.49), cis-monoenes (R _f, 0.27), dienes (R _f, 0.10), and polyunsaturated fatty acids with three or more double bonds remaining at the origin. The isolated trans-monoenes are quantitatively analyzed by capillary GC. The technique of argentation TLC with GC analysis of isolated methyl esters is highly reproducible with 4.8% variation (i.e., coefficient of variation, CV%) in R _f values and 4.3 and 6.9% CV% in quantification within batch and between batch, respectively. Furthermore, the combined technique revealed that direct GC analysis underestimated the trans-content of margarines by at least 30%. In this study, C₂₀ and C₂₂ trans-monoenes were found in relatively large quantities; 13.9% (range 10.3–19.6%) and 7.5% (range 5.3–11.5%), respectively, in margarine purchased in 1995, but these C₂₀ and C₂₂ trans-monoenes were much reduced (0.1%) in a fresh selection of margarine purchased in 1998. Compositional data from labels underestimated the trans-content of margarines, especially those dervied from hydrogenated marine oil. Low levels of C₂₀ trans-monoenes (range 0.1–0.3%) and C₂₂ trans-monoenes (range 0.0–0.1%) were identified in adipose tissue obtained from healthy volunteers in 1995, presumably indicating consumption of partially hydrogenated fish oil.     

Analysis of the monoenoic fatty acid distribution in hydrogenated vegetable oils by silver-ion high-performance liquid chromatography

A silver-ion high-performance liquid chromatography column (hexane/acetonitrile as solvent, ultraviolet detection) was used to analyze the fatty acid distribution (as fatty acid methyl esters) of a representative sample of hydrogenated oil. Fractions containingcis- andtrans-18:1 isomers were readily separated. The positional fatty acid isomers were separated by rechromatographing these fractions. The elution order and percent compositions were compared with results obtained by gas chromatography. Of the Δ8 to Δ14trans-18:1 isomers, only the Δ8 and Δ9 pair could not be separated. The Δ8 and Δ9cis-18:1 pair also could not be separated, and the Δ10 isomer was poorly separated from this pair. Area percents were comparable to results obtained by gas chromatography.     

The reaction of picric acid with epoxides. II. The detection of epoxides in heated oils

A colorimetric method, based upon the reaction of the oxirane group with picric acid, was used to determine the epoxide content of heated vegetable oils. The picration method is particularly suitable for measuring small quantities of epoxide because it is much more sensitive than the common titrimetric methods, and it is not subject to interference from cyclopropene, conjugated dieneols, or α, β-unsaturated carbonyls. Thin-layer chromatography was used to separate mixtures of picrated, epoxidized methyl esters. Separation ofcis- andtrans-methyl epoxystearate, methyl epoxyoleate, and methyl diepoxystearate in a mixture of these four esters was achieved in this manner. The presence of saturatedcis- andtrans-epoxystearates and unsaturated epoxides was demonstrated in heated vegetable oils. Presented, in part, at the AOCS Meeting in Cincinnati, October 1965.     

Chromatographic separation ofcis andtrans fatty esters by argentation with a macroreticular exchange resin

Methyl oleate and methyl elaidate, as well as other monoenecis andtrans isomers of fatty esters, can be separated quickly and conveniently by a preparative chromatographic procedure in which a silver-saturated ion-exchange resin is used. Separations are based on differences in stabilites of the silver-olefin complexes. Recoveries of better than 95% were made, and puretrans andcis monoene fractions were collected. This method can be used to separate saturates fromcis andtrans monoenes. Thecis,trans,cis,cis, andtrans,trans-9-12-octadecadienoates were separated. Whilecis,trans andtrans,trans dienes were cluted separately, thecis,cis diene isomer remained on the column. Presented at AOCS Meeting, in Minneapolis, 1963. A laboratory of the No. Utiliz. Res. and Dev. Div., ARS, USDA.     

The composition of hydrogenated fats by high-resolution13C nuclear magnetic resonance spectroscopy

The high-resolution¹³C nuclear magnetic resonance spectra of twelve hydrogenated fats have been examined. Each spectrum contains 50–100 signals and reveals much about the nature of the acyl chains of both double-bond position and configuration. The signals for the ω1, ω2 and ω3 carbon atoms give information on thecis andtrans isomers of the Δ15, Δ14, Δ13 and Δ12 18:1 esters, respectively. Allylic signals distinguish betweencis andtrans esters, and the proportion of totalcis to totaltrans isomers can be obtained from these. Olefinic signals are the most informative, and most of these have been assigned. This leads to a semi-quantitative estimate of the various 18:1 isomers present. Assignments are based mainly on information already in the literature, but some were confirmed after urea fractionation of the acids from a hydrogenated oil in whichcis andtrans monoene acids were separately concentrated.     

Methyl esters from a partially hydrogenated vegetable oil is a better infrared external standard than methyl elaidate for the measurement of totaltrans content

An infrared spectrophotometric procedure, based on the fatty acid methyl ester mixture derived from a partially hydrogenated vegetable oil as the calibration standard, has been developed for accurate analysis of the totaltrans content of hydrogenated fats. This procedure produces more accurate results than the current official methods of Association of Official Analytical Chemists and American Oil Chemists’ Society, both of which use methyl elaidate as the external standard. The results obtained with this procedure were in close agreement to those determined by the combined procedure of silver-nitrate thin-layer chromatography and capillary gas-liquid chromatography. The improved results, obtained with the partially hydrogenated vegetable oil methyl esters as the calibration standard, may be attributable to its assortment oftrans isomers, which may have different absorptivities relative to methyl elaidate.