High-performance liquid chromatography of the triacylglycerols ofVernonia galamensis andCrepis alpina seed oils

The triacylglycerols ofVernonia galamensis andCrepis alpina seed oils were characterized because these oils have high concentrations of vernolic (cis-12,13-epoxy-cis-9-octadecenoic) and crepenynic (cis-9-octadecen-12-ynoic) acids, respectively. The triacylglycerols were separated from other components of crude oils by solid-phase extraction, followed by resolution and quantitation of the individual triacylglycerols by reversed-phase high-performance liquid chromatography with an acetonitrile/methylene chloride gradient and flame-ionization detection. Isolated triacylglycerols were characterized by proton and carbon nuclear magnetic resonance and by capillary gas chromatography of their fatty acid methyl esters. The locations of the fatty acids on the glycerol moieties in the oils were obtained by lipolysis. TheVernonia galamensis oil contained 50% trivernoloyl and 21% divernoloyllinoleoyl glycerols along with 20% triacylglycerols with one vernolic and two other fatty acids. TheCrepis alpina oil contained 36% tricrepenynoyl and 33% dicrepenynoyllinoleoyl glycerols, 17% triacylglycerols with two crepenynic and one other fatty acid and 7% triacylglycerols with one crepenynic acid and two other fatty acids. Vernolic acid was found at both the 1(3)- and 2-glycerol carbons but was more abundant at the 1,(3)-position in theVernonia galamensis oil. Crepenynic acid was found at both glycerol carbon positions but was more abundant at the 2-position in theCrepis alpina oil. Visiting scientist from Technical Research Institute, Snow Brand Milk Company, Ltd., Saitana, Japan.     

Soybean oil triacylglycerol analysis by reversed-phase high-performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

Soybean oil triacylglycerols from genetically modified soybean lines were conclusively identified by reversed-phase high-performance liquid chromatography coupled with mass spectrometry with atmospheric pressure chemical ionization. Atmospheric pressure chemical ionization is a soft ionization technique which gives simple spectra for triacylglycerols. Spectral identification of the triacylglycerols was based on the molecular [M+1]⁺ ion and the 1(2)-, 2(3)- and 1(3)-diacylglycerol fragments. Triacylglycerols identified in high-stearic and high-palmitic soybean varieties were quantitated by reversed-phase high-performance liquid chromatography with flame-ionization detection. There was excellent agreement between the fatty acid composition calculated from the triacylglycerol composition and the fatty acid composition obtained by gas chromatography of the transmethylated oils. The oils of the modified soybean varieties, compared to typical soybean oil, contained increased content of triacylglycerols known to be more oxidatively stable, such as linoleoyloleoylstearoyl, linoleoylpalmitoylstearoyl, and linoleoyldipalmitoyl glycerols, and less triacylglycerols like trilinoleoylglycerol, known to decrease oxidative stability. This study showed that the atmospheric pressure chemical ionization technique is suitable for mass spectral identification of neutral molecules, such as triacylglycerols, which do not contain a chargeable functional group.     

Characterization of triacylglycerols in the seeds ofAquilegia vulgaris by chromatographic and mass spectrometric methods

A combination of analytical techniques is generally necessary to properly characterize complex lipid materials. Chromatographic separation in conjunction with spectroscopic characterization was utilized for the analysis of the triacylglycerols in the seeds ofAquilegia vulgaris. Reversed-phase high-performance liquid chromatography (HPLC), micropacked argentation supercritical fluid chromatography (SFC), and combinations of the two techniques were used. The fatty acid profile was determined by gas chromatography/mass spectrometry of the picolinyl esters and by gas chromatography/flame-ionization detection of the methyl esters. The major components were also identified by direct inlet mass spectrometry. The excellent selectivity of packed fused silica argentation SFC for the separation of triacylglycerols was demonstrated.     

The fatty acid composition of the seeds ofGinkgo biloba

The fatty acid composition of seeds ofGinkgo biloba has been examined by a combination of capillary gas chromatography, silver ion high-performance liquid chromatography and gas chromatography/mass spectrometry. Some of the fatty acids identified are unusual in plants and were rather different from those reported earlier. These include ananteiso-methyl branched fatty acid, 14-methylhexadecanoic acid, 5,9-octadecadienoic acid, and 5,9,12-octadecatrienoic acid. Fourier-transform infrared spectroscopy confirmed that all of the double bonds were of thecis-configuration.     

Triacylglycerols of winter butterfat containing configurational isomers of monoenoic fatty acyl residues. II. Saturated dimonoenoic triacylglycerols

Triacylglycerols of Finnish winter butterfat containing one saturated and two monoenoic fatty acyl residues were studied. With silver ion high-performance liquid chromatography (HPLC), molecules were separated according to the difference in the configuration of one fatty acyl moiety. The distribution of the saturatedcis,trans-dimonoenoic and saturatedcis,cis-dimonoenoic triacylglycerols according to their acyl carbon numbers was compared by means of reversed-phase HPLC and tandem mass spectrometry. Furthermore, two examples of the fatty acid composition of a specified molecular weight species were shown. The fatty acid compositions of corresponding saturatedcis,trans-dimonoenoic and saturatedcis,cis-dimonoenoic triacylglycerols were similar; however, there may be differences in the proportions of different fatty acid combinations or in the distribution of fatty acids between primary and secondary glycerol positions.     

Large-scale preparation of linoleic acid-d2-enriched triglycerides fromCrepis alpina seed oil

Catalytic deuteration ofCrepis alpina seed oil provided a convenient one-step method for the direct synthesis of large quantities of triglycerides enriched with deuteriumlabelled linoleic acid.Crepis alpina seed (19 kg) was crushed, and the oil [74% crepenynic acid (cis-9-octadecen-12-ynoic acid)] was extracted with hexane. After purification by column chromatography (silica gel), > 170-g batches of oil were deuterated with Lindlar catalyst and deuterium (D₂) gas. Purification (silica gel) resulted in > 150-g samples of triglyceride containing 74%cis-9,cis-12-octadecadienoic acid-12,13-d₂ (18:2-d₂) and 14% unlabelled linoleic acid. Pure (> 99%) tricrepenynin was recovered by further fractionation of theCrepis alpina triglycerides on silica gel. Deuteration of this sample produced deuteriumlabelled trilinolein containing linoleic acid-d₂ of > 98% isotopic purity.     

Triacylglycerols of winter butterfat containing configurational isomers of monoenoic fatty acyl residues. I. Disaturated monoenoic triacylglycerols

The triacylglycerols of winter butterfat were fractionated according to the type and degree of unsaturation into six fractions by silver ion high-performance liquid chromatography (Ag-HPLC). The acyl carbon number distribution of the triacylglycerols in each fraction was elucidated by reversed-phase HPLC and mass spectrometry (MS). The MS analysis of each fraction gave deprotonated triacylglycerol [M - H]⁻ ions which were produced by chemical ionization with ammonia. The daughter spectrum of each of the [M - H]⁻ ions provided information on its fatty acid constituents. Successful fractionation of triacylglycerols differing in the configuration of one fatty acyl residue by Ag-HPLC was important because geometrical isomers could not be distinguished by the MS system used. In addition to the fatty acid compositions, reversed-phase HPLC analysis demonstrated the purity of the collected fractions: molecules having acis-trans difference were separated nearly to the baseline. Triacylglycerols differing in the configuration of one fatty acyl residue were not equally distributed in relation to their acyl carbon numbers. This indicates that during the biosynthesis of triacylglycerols,cis- andtrans-fatty acids are processed differently. Although the fatty acid compositions of the corresponding molecular weight species of disaturatedtrans- and disaturatedcis-monoenoic triacylglycerols were similar, there may be differences in the amounts of different fatty acid combinations or in the distribution of fatty acids between the primary and secondary glycerol positions. In addition to the main components, it was possible to analyze minor triacylglycerols, such as molecules containing one odd-chain fatty acid, by the MS system used.     

Determination of vernolic acid content in the oil ofEuphorbia lagascae by gas and supercritical fluid chromatography

Determination of the content of vernolic acid (12,13-epoxy-9c-octadecenoic) in the oil ofEuphorbia lagascae has been performed by gas chromatography of the fatty acid methyl ester derivatives of the triacylglycerols in the oil and by supercritical fluid chromatography (SFC) of the raw oil and the fatty acid derivatives of the oil. The content of vernolic acid was found to be 55 wt%. The three methods were compared, and SFC analysis of the fatty acid derivatives was found to be the most accurate method.     

Variation in lipid composition of niger seed (Guizotia abyssinica Cass.) Samples collected from different regions in ethiopia

Niger seed samples were collected from different regions in Ethiopia for determination of oil content, and of fatty acid, tocopherol and sterol composition in the seed oil by gas-liquid chromatography and high-performance liquid chromatography methods. There was a large variation in oil content, ranging from 29 to 39%. More than 70% of the fatty acids was linoleic acid (18∶2) in all samples analyzed. The other predominant fatty acids were palmitic (16∶0), stearic (18∶0) and oleic (19∶1) at a range of 6 to 11% each. Total polar lipids recovered after preparative thin-layer chromatography comprised a small fraction of the total lipids. They had higher 16∶0 and lower 18∶2 contents than the triacylglycerols.α-Tocopherol was the predominant tocopherol in all samples, 94–96% of the total amounting to 630–800 μg/g oil. More than 40% of the total sterols wasβ-sitosterol,ca. 2000μg/g oil. The other major sterols were campesterol and stigmasterol, ranging from 11 to 14%. The Δ5- and Δ7-avenasterols were in the range of 4 to 7%. From the samples studied, no conclusion could be drawn regarding the influence of altitude or location on oil content, tocopherol and/or sterol contents. The results of the present study on niger seed oil are discussed in comparison with known data for common oils from Compositae,viz, safflower and sunflower.     

Comparison between two procedures for stereospecific analysis of triacylglycerols from vegetable oils—I: Olive oil

Two methods for stereospecific analysis of triacylglycerols are compared. Procedure A, based on stereospecific phosphorylation ofsn-1,2-diacylglycerols to phosphatidic acids, and procedure B, based on separation of the diastereomeric 1,2(2,3)-diacylglycerol urethane derivatives by high-performance liquid chromatography on silica, were applied to olive oil triacyl-sn-glycerols. Statistical evaluation of the results showed good reproducibility, and Student'st-test indicates no statistical differences between the two considered procedures, although some small differences were observed and discussed. Fifteen samples of extra-virgin olive oil, produced in the same region (Umbria, Italy), were analyzed with the two considered procedures.     

Lipase-catalyzed synthesis of structured low-calorie triacylglycerols

Because of their unique fatty acid specificities and regioselectivities, lipases have been found to be effective catalysts for the synthesis of structured lipids that have a predetermined composition and distribution of fatty acyl groups on the glycerol backbone. The prospective plant-derived lipase found in the exudate of Carica papaya is known for its shortchain acyl group specificity, 1,3-glycerol regioselectivity, and sn-3 stereoselectivity. Carica papaya latex (CPL) was therefore examined for its potential ability to synthesize structured lowcalorie short- and long-chain triacylglycerols (SLCT). In this paper, we describe the utility of CPL in the lipase-catalyzed interesterification reaction of triacetin and hydrogenated soybean oil. Normal-phase high-performance liquid chromatography, combined with mass spectrometry, was used to distinguish the structured SLCT synthesized using the lipase from the corresponding SLCT produced by chemical synthesis.     

Gradients of n-heptane and acetonitrile in silver-ion high-performance liquid chromatography analyses of cis and trans bonds in lipids

Cis and trans isomers of fatty acid methyl esters, fatty alcohols, and triacylglycerols were analyzed with a silverion high-performance liquid chromatography system. Gradients of n-heptane and acetonitrile were used to elute molecules with up to nine cis double bonds. The analyses were as fast and reliable and had a resolution similar to that of the best published analyses. However, published analyses were performed with chlorinated solvents, and these solvents are carcinogenic and mutagenic. The solvents we used, heptane and acetonitrile, are less dangerous to the analyst.     

Analysis of north atlantic and baltic fish oil triacylglycerols by high-performance liquid chromatography with a silver ion column

Triacylglycerols from Atlantic herring (Clupea harengus), sandeel (Ammodytes sp.) and Baltic herring (Clupea harengus membras) have been fractionated by silver ion high-performance liquid chromatography. An ion exchange column loaded with silver ions was the stationary phase, and a gradient in the mobile phase from 1,2-dichloroethane/dichloromethane (1∶1, v/v) to acetone and then to acetone/acetonitrile (2∶1, v/v) was used to effect the separation with light-scattering (i.e., mass) detection. Fractions were collected via a streamsplitter, and fatty acid methyl esters were prepared by transesterification in the presence of an internal standard for identification and quantification by gas liquid chromatography. Triacylglycerols were separated according to the number of double bonds in the fatty acyl residues. Resolution was excellent at first, when the least unsaturated molecules eluted (trisaturated to dimonoene-monodiene fractions). Base-line resolution could no longer be achieved when molecules containing trienoic or more highly-unsaturated fatty acids began to elute because of overlapping components. Nonetheless, some valuable separations of species containing two saturated and/or monoenoic fatty acids and one polyenoic fatty acid were achieved. Double bond indices (average number of double bonds in each triacylglycerol molecule) were calculated to estimate the separations possible. Fractions containing at least 11–14 double bonds per molecule were obtained.     

Positional analysis and determination of triacylglycerol structure ofArgania spinosa seed oil

The distribution of fatty acids between the sn-1, sn-2 and sn-3 positions of triacylglycerols fromArgania spinosa seed oil of Morocco has been determined. Saturated fatty acids showed a preference for external positions. The sn-1 position contained slightly more palmitic acid than the sn-3 position, whereas stearic acid was preferentially esterified at the sn-3 position. Linoleic acid occurred predominantly in the sn-2 position with lesser amount evenly distributed between the sn-1 and the sn-3 positions, as generally found in vegetable oils. Oleic acid was distributed with a slight preference shown for the internal position, whereas the distribution between the external positions revealed a slight preference for the sn-1 position. The distribution of the triacylglycerols determined from high-performance liquid chromatography (HPLC) is at variance with that calculated from the 1-random 2-random 3-random distribution theory. This is particularly true for trioleoyl and trilinoleoylglycerols. In contrast, the agreement between theory and experiment is good for triacylglycerols containing two oleoyl and one linoleoyl chains, one oleoyl, one linoleoyl and one palmitoyl chains or one oleoyl, one palmitoyl, and one stearoyl chains.     

Cyclopropenoid fatty acids in seed oils ofSida acuta andSida rhombifolia (malvaceae)

Seed oils ofSida acuta andSida rhombifolia were found to contain sterculic (11.0, 10.8%) and malvalic (1.7, 2.0%) acids respectively, in addition to the normal fatty acids. Co-occurrence of these acids was established by gas liquid chromatography of the silver nitrate-methanol-treated methyl esters usingSterculia foetida esters as a reference standard. This gas liquid chromatography technique of quantitation was found most suitable to estimate these acids in low level cyclopropenoid acid-containing seed oils.     

Characterization of sucrose polyesters-triacylglycerols mixtures

High-performance size-exclusion chromatography (HPSEC) and thin-layer chromatography/flame-ionization detection (TLC/FID) have been used for the characterization of mixtures of either monoacid sucrose octaesters with triacylglycerols (TAG) or sucrose polyesters (SPE) prepared from oils with natural oils. In mixtures of monoacid sucrose octaesters/TAG, no significant differences were found between the values obtained by either HPSEC or TLC/FID and the actual component proportions. Additionally, components could be separated by TLC, which was confirmed by fatty acid composition data of each fraction. Analysis of SPE/oil mixtures was attainable by HPSEC, but alternative quantitation by TLC/FID required previous silylation. Likewise, fatty acid composition could be determined only in the total mixture and in the sucrose octaester fraction. A formula derived for calculation of oil fatty acid composition, based on analytical data, showed the validity of the approach used in this study to determine component proportions in functional SPE/oil mixtures.     

Oxidative stability of purified canola oil triacylglycerols with altered fatty acid compositions as affected by triacylglycerol composition and structure

Canola oil triacylglycerols from genetically modified canola lines (InterMountain Canola Co., Cinnaminson, NJ) have been evaluated for their photooxidative and autoxidative stabilities, as influenced by their fatty acid compositions and their triacylglycerol compositions and structures. Purified canola oil triacylglycerols were oxidized in duplicate in fluorescent light at 25°C and in the dark at 60°C under oxygen, and their oxidative deterioration with time was monitored by determining colorimetric peroxide values. Also monitored with time, oxidation products were determined by reversed-phase high-performance liquid chromatography with ultraviolet absorbance detection. Total volatiles, generated by thermal decomposition of the oxidized triacylglycerols, were quantitated by static-head-space gas chromatography. These experimental parameters were statistically correlated with predicted oxidizability, fatty acid composition, position of fatty acids on glycerol carbons and triacylglycerol composition. Oxidative deterioration of canola triacylglycerols correlated negatively with oleic acid composition, with oleic acid content at carbon 2 and with trioleoylglycerol content of the oil. Deterioration was positively correlated with the amount of linolenic acid on nonspecific locations on glycerol carbons 1,2 and 3, the amount of linoleic acid on glycerol carbon 2 and withsn-oleoyllinoleoyllinolenoyl glycerol content. Differences in character or quantity of volatile product and triacylglycerol hydroperoxides were low, whether generated during autoxidation or photooxidation of the canola triacylglycerols. Presented at the joint meeting of the American and Japan Oil Chemists' Societies, April 25–28, 1993, Anaheim, California.     

Analysis of seed oils containing cyclopentenyl fatty acids by combined chromatographic procedures

The fatty acids of seed oils of the Flacourtiaceae,Hydnocarpus anthelmintica, Caloncoba echinata andTaraktogenus kurzii, have been examined by a combination of capillary gas chromatography, silver ion high performance liquid chromatography and gas chromatography-mass spectrometry. In addition to the common range of cyclopentenyl fatty acids found in such oils, 13-cyclopent-2-enyltridec-4-enoic acid was a major component ofH. anthelmintica and was identified by mass spectrometry as its picolinyl ester and dimethyldisulphide adduct. It has not previously been found in nature. In the other seed oils, the isolated double bond in the corresponding fatty acid was in position 6, as expected. Similarly,cis-4-hexadecenoic acid and C₁₆ and C₁₈ cyclopentyl fatty acids were identified for the first time inH. anthelmintica. Iso- andanteiso-methylbranched fatty acids were present in trace amounts.     

Two-step enzymatic synthesis of docosahexaenoic acid-rich symmetrically structured triacylglycerols via 2-monoacylglycerols

Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically form bonito oil in a two-step process: (i) ethanolysis of bonito oil TG to 2-monoacylglycerols (2-MG) and fatty acid ethyl esters, and (ii) reesterification of 2-MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2-MG with 43.5% DHA content in 2 h. The 2-MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricaprylolglycerol (weight percent). The amount of TG with two CA residues and one C₂₂ residue (22∶6=DHA, 22∶5, and 22∶4) was 51 wt%. The 1,3-dicapryloyl-2-docosahexaenoylglycerol to 1,2(2,3)-dicapryloyl-3 (1)-docosahexaenoylglycerol ratio (based on high-performance liquid chromatography peak area percentages) was greater than 50∶1. The recovery of TG as structured lipids after silica gel column purification was approximately 71%. Ethyl esters and 2-MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the intial TG owing to the high 1,3-regiospecificity of Novozym 435 and the reduced acyl migration in the system.     

The fatty acids of the spongeDysidea fragilis from the black sea

The fatty acid composition of the sponge,Dysidea fragilis, from the Black Sea has been determined by analytical gas chromatography, silver ion high-performance liquid chromatography and gas chromatography/mass spectrometry. More than a hundred different fatty acids were identified, of which many were similar to those in sponges from tropical seas. On the other hand, some of the fatty acids identified have not been found previously in sponges or other marine sources and perhaps are new to science. These include 13-methyl-tetradec-4-enoic and 14-methyl-hexadec-6-enoic acids, together with demospongic acids,i.e. 5,9,17-tetracosatrienoic, 5,9,17-pentacosatrienoic and 5,9,19-pentacosatrienoic acids. From the elution behavior on silver nitrate chromatography, all the double bonds were of thecis-configuration.