Determination of positional distribution of short-chain fatty acids in bovine milk fat on chiral columns

The positional distribution of acetic and butyric acids in bovine milk fat triacylglycerols was determined by chiral-phase high-performance liquid chromatography (HPLC) of the derived diacylglycerols. Enriched fractions of acetic and butyric acid-containing triacylglycerols were isolated by normal-phase thin-layer chromatography (TLC) from a molecular distillate of butter oil, and they were fully hydrogenated. Mixedsn-1,2(2,3)- andX-1,3-diacylglycerols of short- and long-chainlength, which were generated by partial Grignard degradation of the hydrogenated triacylglycerols, were isolated by borate-TLC. The enantiomericsn-1,2-andsn-2,3-diacylglycerols and theX-1,3-diacylglycerols as their 3,5-dinitrophenylurethanes were resolved by HPLC on chiral columns. Both acetic and butyric acids were exclusively associated with thesn- 2,3- andX-1,3-diacylglycerols of short and long chainlength. These results establish the presence of acetic and butyric acids in thesn-3-position of bovine milk fat triacylglycerols. Other short-and medium-chainlength acids were found in progressively increasing proportions also in thesn-1- andsn-2-positions.     

Stereospecific analysis of triacylglycerols rich in long-chain polyunsaturated fatty acids

Six oils of marine, algal, and microbial origin were analyzed for stereospecific distribution of component fatty acids. The general procedure involved preparation ofsn-1,2-(2,3)-diacylglycerols by partial deacylation with ethylmagnesium bromide or pancreatic lipase, separation of X-1,3- andsn-1,2(2,3)-diacylglycerols by borate thin-layer chromatography, resolution of thesn-1,2- andsn-2,3-enantiomers by chiral phase high-performance liquid chromatography following preparation of dinitrophenylurethane derivatives, and determination of the fatty acid composition by gas chromatography. Unexpected complications arose during a stereospecific analysis of triacylglycerols containing over 33% of either 20∶4 or 22∶6 fatty acids. Thesn-1,2(2,3)-diacylglycerols made up of two long-chain polyunsaturated acids migrated with the X-1,3-diacylglycerols and required separate chiral phase resolution. Furthermore, the enzymatic method yieldedsn-1,2(2,3)-diacylglycerols, overrepresenting the polyenoic species due to their relative resistance to lipolysis, but prolonged digestion yielded correct composition for the 2-monoacylglycerols. The final positional distribution of the fatty acids was established by pooling and normalizing the data from subfractions obtained by norman- and chiral-phase separation of diacylglycerols. The molecular species of X-1,3-,sn-1,2- andsn-2,3-diacylglycerol dinitrophenylurethanes were identified by chiral-phase liquid chromatography/mass spectrometry with electrospray ionization, which demonstrated a preferential association of the paired long-chain acids with thesn-1,2- andsn-2,3-diacylglycerol isomers.     

Identification of natural diacylglycerols as the 3,5-dinitrophenylurethanes by chiral phase liquid chromatography with mass spectrometry

This study reports a facile identification of the molecular species of enantiomeric diacylglycerols by combining chiral phase high-performance liquid chromatography with positive chemical ionization mass spectrometry. For this purpose the 3,5-dinitrophenylurethane (DNPU) derivatives ofsn-1,2(2,3)-diacylglycerols are separated on an (R+)-naphthylethylamine polymer column (25 cm × 0.46 cm ID) using an isocratic solvent system made up of hexane/dichloroethane/acetonitrile (85∶10∶5, by vol) or isooctane/tert-butyl methyl ether/acetonitrile/isopropanol (80∶10∶5∶5, by vol). About 1% of the column effluent (1 mL/min) was admitted to a quadrupole mass spectrometer (Hewlett-Packard, Palo Alto, CA)via direct liquid inlet interface, and positive chemical ionization spectra were recorded over the range of 200–900 mass units. The DNPU derivatives of diacylglycerols yield characteristic [M-DNPU]⁺ and [RCO+74]⁺ ions for each diacylglycerol species from which the molecular weight and exact pairing of fatty acids can be unequivocally obtained. The characteristic ions appear to be generated in nearly correct mass proportions as indicated by preliminary quantitative comparisons. The abbreviations 14∶0, 16∶1, 18∶2, etc. represent normal chain fatty acids of 14, 16, 18, etc. acyl carbons and 0, 1, 2, etc. double bonds, respectively; 16∶0–18∶1, etc. represent diacylglycerols containing 16∶0 and 18∶1 fatty acids of unspecified positional distribution;sn indicates stereospecific numbering of glycerol carbons;sn-1,2-diacylglycerols andsn-2,3-diacylglycerols are enantiomeric diacylglycerols of unspecified fatty acid composition;rac-1,2-diacylglycerols are racemic diacylglycerols representing equal amounts ofsn-1,2-andsn-2,3-enantiomers;sn-1,2(2,3)-diacylglycerols are a mixture ofsn-1,2-andsn-2,3-diacylglycerols of unspecified proportion of enantiomers and unspecified fatty acid compisition and positional distribution; X-1,3-diacylglycerols are diacylglycerols of unspecified fatty acid composition and reverse isomer content.     

Acylglycerol structure of genetic varieties of peanut oils of varying atherogenic potential

Detailed investigation was made of the triacylglycerol structure of three varieties of peanut oils of varying atherogenic activity. By means of chromatographic and stereospecific analyses, it was shown that all the oils had markedly nonrandom enantiomeric structures with the long chain saturated fatty acids (C₂₀−C₂₄) confined exclusively to thesn-3-position, whereas the palmitic and oleic acids were distributed about equally between thesn-1-andsn-3-positions, with the linoleic acid being found preferentially in thesn-2-position. On the basis of detailed studies of the molecular species of the separatesn-1,2-,sn-2,3- andsn-1,3-diacylglycerol moieties, it was concluded that the fatty acids in the three positions of the glycerol molecule are combined with each other solely on the basis of their relative molar concentrations. As a result, it was possible to calculate the composition of the molecular species of the peanut oil triacylglycerols (including the content of the enantiomers and the reverse isomers) by means of the 1-random 2-random 3-random distribution. In general, the three peanut oils possessed triacylglycerol structures which where closely similar to that derived earlier for a commercial peanut oil of North American origin. Since their oil has exhibited a high degree of atherogenic potential, it was anticipated that the present oils would likewise be atherogenic, which has been confirmed by biological testing. However, there are certain differences in the triacylglycerol structures among these oils, which can be correlated with the variations in their atherogenic activity. The major differences reside in the linoleic/oleic acid ratios in the triacylglycerols, especially in thesn-2-position, and in the proportions in which these acids are combined with the long chain fatty acids. On the basis of the characteristic structures identified in the earlier analyzed atherogenic peanut oil, the peanut oil of South American origin would be judged to possess the greatest atherogenic potential and this has been borne out by biological testing.     

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.     

The triacylglycerol structure of olive oil determined by silver ion high-performance liquid chromatography in combination with stereospecific analysis

The compositions of positionssn-1,sn-2 andsn-3 of triacylglycerols from “extra-virgin” olive oil (Olea europaea) were determined. The procedure involved preparation of diacyl-rac-glycerols by partial hydrolysis with ethyl magnesium bromide; 1,3-, 1,2- and 2,3-diacyl-sn-glycerols as (S)-(+)-1-(1-naphthyl)ethyl urethanes were isolated by highperformance liquid chromatography (HPLC) on silica, and their fatty acid compositions were determined. The same procedure was also carried out on the five main triacylglycerol fractions of olive oil after separation according to the degree of unsaturation by HPLC in the silver ion mode. Although stereospecific analysis of the intact triacyl-sn-glycerols indicated that the compositions of positionssn-1 andsn-3 were similar, the analyses of the molecular species demonstrated marked asymmetry. The data indicate that the “1-random, 2-random, 3-random” distribution theory is not always applicable to vegetable oils.     

Identification of triacylglycerols in the enzymatic transesterification of rapeseed and butter oil

A blend of rapeseed and butter oil was transesterified using immobilized Thermomyces lanuginosus lipase (Lipozyme® TL IM) as catalyst. The reaction was followed by reversed-phase HPLC and the triacylglycerol peaks were tentatively identified from their elution times by calculating equivalent carbon numbers. Further identification was made using HPLC-electrospray tandem mass spectrometry. A few of the triacylglycerols detected were typical combinations of fatty acids originating from rapeseed oil, such as α-linolenic acid, and short-chain fatty acids from butter oil. Due to the regioselectivity of the lipase, the transesterification reaction involved mainly fatty acids in the sn-1 and sn-3 positions. However, significant changes in the fatty acid composition in the sn-2 position were detected after 6 h.     

Studies of triacyglycerol structure of very low density lipoproteins of normolipemic subjects and patients with type III and type IV hyperlipoproteinemia

The triacylglycerols of very low density lipoproteins (VLDL-TG) were analyzed in samples from normal subjects and patients with Frederickson’s Type III and Type IV hyperlipoproteinemia. VLDL were obtained by conventional ultracentrifugation, and the triacylglycerols were isolated by thin-layer chromatography (TLC). Representative sn-1,2(2,3)- and sn-1,3-diacylglycerols were generated by Grignard degradation of the triacylglycerols, and were resolved by TLC on borate-treated silica gel. The molecular association of the fatty acids in the diacylglycerol moieties was determined by gasliquid chromatography with mass spectrometry (GC/MS) of the tertiary-butyldimethylsilyl ethers. The positional distribution of the fatty acids was established by the Brockerhoff stereospecific analysis. The results showed a marked asymmetry in the distribution of the fatty acids in all samples, with the saturated acids predominantly in the sn-1-position and the unsaturated fatty acids distributed about equally between the sn-2- and sn-3-positions. In all instances, the molecular species composition of the sn-1,2-, sn-2,3- and sn-1,3-diacylglycerols was found to be similar to that calculated for 1-random 2-random 3-random distribution of triacylglycerols. There were marked differences in the quantitative composition of the molecular species of the VLDL-TG between normal subjects and patients, but these discrepancies were attributed to differences in the fatty acid composition of the samples.     

Chromatographic resolution of chiral diacylglycerol derivatives: Potential in the stereospecific analysis of triacyl-sn-glycerols

Diacylglycerols have been separated as their (S)-(+)-or (R)-(−)-1-(1-naphthyl)ethyl urethanes by high performance liquid chromatography (HPLC) on a column of silica gel with 0.5% 2-propanol in hexane as the mobile phase. The elution order of components derivatized with the (S)-form of the reagent was 1,3-, followed by 1,2-, and finally 2,3-diacyl-sn-glycerols. The elution order of 1,2- and 2,3-diastereomers was reversed when the (R)-form of 1-(1-naphthyl)ethyl isocyanate was used for derivatization. Single-acid 1,2- and 2,3-diastereomers were separated to the baseline with a resolution factor from 5.2–5.7, and the resolution factor between 1,3- and 1,2- or 2,3-diacyl-sn-glycerol derivatives was more than 23. Molecular species of single-acid diacylglycerol derivatives were separated in the sequence 18∶1<18∶0<18∶2<16.0. In order to assess this methodology as part of a procedure for the stereospecific analysis of triacyl-sn-glycerols, we prepared diacyl-rac-glycerols from maize oil, evening primrose oil and egg yolk triacylglycerols by partial hydrolysis with ethyl magnesium bromide. The 1,3-, 1,2- and 2,3-diacyl-sn-glycerols as (S)-(+)-1-(1-naphthyl)ethyl urethanes were isolated and their fatty acid compositions were determined. Although this only permitted an indirect determination of the compositions of positionssn-1,-2 and-3, it was sufficient to indicate the potential of the methodology because results comparable to those published earlier were achieved.     

Stereospecific analysis of TAG from sunflower seed oil

Stereospecific analysis of TAG from a sunflower seed oil of Tunisian origin was performed. The TAG were first fractionated according to chain length and degree of unsaturation by RP-HPLC. The four major diacid- and triacid-TAG fractions were palmitoyldilinoleoyl-glycerol, dioleoyllinoleoylglycerol, oleoyldilinoleoylglycerol, and palmitoyloleoyl-linoleoyl-glycerol, amounting to 7.2, 16.6, 29.5, and 12 mol%, respectively. The TAG of the four fractions were individually submitted to stereospecific analysis, using a Grignard-based partial deacylation, separation of sn-1,2(2,3)-DAG from sn-1,3-DAG by boric acid-impregnated silica gel TLC plates, conversion of the sn-1,2(2,3)-DAG to their 3,5-dinitrophenylurethane (DNPU) derivatives, fractionation of DNPU derivatives by RP-HPLC, resolution of the DNPU-DAG by HPLC on a chiral column, transmethylation of each sn-DNPU-DAG fraction, and analysis of the resulting FAME by GC. The data obtained were used to determine the triacyl-sn-glycerol composition of the main TAG of the oil. Fifteen triacyl-sn-glycerols were identified and quantified, representing, along with the monoacid-TAG, trilinoleoylglycerol and trioleoylglycerol, more than 90% of the total oil TAG. The two major triacyl-sn-glycerols were trilinoleoyl-glycerol and 1-linoleoyl-2-linoleoyl-3-oleoyl-glycerol (18.6 and 18.5% of the total, respectively). Results clearly identified linoleic acid as the major FA at the sn-2 position, whereas oleic and palmitic acids were the major FA at the sn-3 position. The sn-1 position was occupied to nearly the same extent by linoleic and oleic acids, and to a greater extent by palmitic acid, which was practically absent at the sn-2 position.     

Regioselective analysis of triacylglycerols by lipase hydrolysis

A modified procedure for the regiospecific analysis of triacylglycerols (TAG) with a 1,3-specific lipase is described. After partial lipase hydrolysis of the triacylglycerol, the released free fatty acids (FFA) and 1,2(2,3)-diacylglycerols (DAG) were isolated by thin-layer chromatography (TLC) and converted to fatty acid methyl esters (FAME). The FAME were analyzed by gas-liquid chromatography (GLC). The 1,3-specific lipases used in this study included supported preparations from strains ofMucor miehei andRhizopus oryzae. The method also was applied to the regiospecific analyses of tung nut and Chinese melon seed oil triacyglycerols, both of which contain high proportions of α-elaeostearic acid. The TAG composition of the oils was substantiated in parallel analysis of the oils by highperformance liquid chromatography with chemical ionization mass spectrometric detection of intact TAG.     

Formation of trierucoylglycerol (trierucin) from 1,2-Dierucoylglycerol by a homogenate of microspore-derived embryos ofBrassica napus L

Homogenates of microspore-derived embryos of rape (Brassica napus L.) incubated with [1-¹⁴C]erucoyl-CoA and 1,2-dierucoylglycerol are able to assemble trierucoyl-glycerol (trierucin). In addition, radioactive triacylglycerols are formed by transferring [1-¹⁴C]-erucoyl moieties to endogenous lipid precursors. Stereospecific analysis of radioactive triacylglycerols revealed that labeled erucoyl moieties had been incorporated exclusively into thesn-1,3 positions with more than 95% of radioactivity in thesn-3 position. No incorporation of labeled erucic acid into thesn-2 position has been observed. All data agree with the involvement of 1,2-diacylglycerol acyltransferase (E.C. 2.3.1.20), which utilized 1,2-dierucoylglycerol as well as endogenous 1,2-diacylglycerols as acceptors of erucoyl moieties. This result is of particular interest for the genetic modification of rape and other Cruciferae for producing trierucin in their seed oils. NRCC No. 33513.     

Stereospecific analysis of triacyl-sn-glycerolsvia resolution of diastereomeric diacylglycerol derivatives by high-performance liquid chromatography on silica

The compositions of positionssn-1, 2 and 3 of triacylglycerols can be determined by partial hydrolysis with ethyl magnesium bromide, derivatization of the total products with (S)-(+)-1-(1-naphthyl)ethyl isocyanate and isolation of the diacyl-sn-glycerol urethane derivatives by chromatography on solid-phase extraction columns containing an octadecylsilyl phase. The diastereomericsn-1,2-and 2,3-diacylglycerol derivatives are separated by high-performance liquid chromatography on silica for determination of their fatty acids by gas chromatography. Each step in the process has been evaluated rigorously. The compositions of all three positions can be calculated with good accuracy from the analyses of these compounds and that of the total triacylglycerols. Although the 1,3-sn-diacylglycerol derivatives can also be isolated easily, they do not give reliable results for the composition of positionsn-2 because acyl migration occurs during their generation. The stereospecific analysis procedure has been applied to some plant and animal triacyl-sn-glycerols of commercial and scientific interest, containing predominantly C₁₆ and C₁₈ fatty acids,i.e. safflower, sunflower, olive and palm oils, tallow, egg and rat adipose tissue. The method is not at present suited to the analysis of more complex triacylglycerols, such as milk fat or fish oils, and problems associated with these are discussed.     

Determination of stereochemical configuration of the glycerol moieties in glycoglycerolipids by chiral phase high-performance liquid chromatography

This study reports a simple and sensitive method for determining the absolute configuration of the glycerol moieties in glycoglycerolipids. The method is based on chiral phase high-performance liquid chromatography (HPLC) separations of enantiomeric di- and monoacylglycerols released from glycosyldi- and monoacylglycerols, respectively, by periodate oxidation followed by hydrazinolysis. The released di- and monoacylglycerols were chromatographed as their 3,5-dinitrophenylurethane (3,5-DNPU), and bis(3,5-DNPU) derivatives, respectively. The derivatives were separated on two chiral phases of opposite configuration, (R)-and (S)-1-(1-naphthyl)ethylamine polymers for diacylglycerols and N-(R)-1-(1-naphthyl)ethylaminocarbonyl-(S)-valine and N-(S)-1-(1-naphyl)ethylamino-carbonyl-(R)-valine for monoacylglycerols. Clear enantiomer separations, which permit the assignment of the glycerol configuration, were achieved for sn-1,2(2,3)-dicyl- and sn-1(3)-monoacylglycerols generated from linseed oil triacylglycerols by partial Grignard degradation on all the chiral stationary phases employed. Using the method, we have determined the glycerol configuration in the glycosyl-diacylglycerols (monogalactosyl-, digalactosyl-, and sulfquinovo-syldiacylglycerols) and glycosylmonoacylglycerols (monogalactosyl-, digalactosyl-, and sulfoquinovosylmonoacylglycerols) isolated from spinach leaves and the coralline red alga Corallina pilulifera. The results clearly showed that the glycerol moieties in all the glycoglycerolipids examined have S-configuration sn-1,2-diacyl- and sn-1-monoacylglycerols). The new method demonstrates that chiral phase HPLC provides unambiguous information on the configuration of the glycerol backbone in natural glycosyldi- and monoacylglycerols, and that the two-step liberation of the free acylglycerols does not compromise glycerol chirality.     

Stereochemical course of diacylglycerol formation in rat intestine

The biosynthesis of diacylglycerols from 2-monoacylglycerols and free fatty acids was examined in evert sacs of rat intestinal mucosa. By means of alternate labeling of the monoacylglycerols and fatty acids, and conventional stereo-specific analysis, it was shown that the main products of synthesis were thesn-1,2-diacylglycerols (53–63%), butsn-2,3-diacylglycerols (37–47%) were also formed in significant amounts. The total yield and proportions of the isomeric diacylglycerols recovered appeared to vary with the nature of the monoacylglycerol and the complexity of the free fatty acid mixture supplied.     

Analysis of diastereomeric DAG naphthylethylurethanes by normal-phase HPLC with on-line electrospray MS

Normal-phase HPLC resolution of sn-1,2(2,3)- and x-1,3-DAG generated by partial Grignard degradation from natural TAG was carried out with both (R)-(−) and (S)-(+)-1-(1-naphthyl)ethylurethane derivatives. The diastereomeric sn-1,2- and sn-2,3-DAG derivatives were resolved using two Supelcosil LC-Si (5 μm, 25 cm × 4.6 mm i.d.) columns in series and an isocratic elution with 0.37% isopropanol in hexane at a flow rate of 0.7 mL/min. The DAG were detected by UV absorption at 280 nm and were identified by electrospray ionization MS in the positive ion mode following postcolumn addition of chloroform/methanol/30% ammonium hydroxide (75∶24.5∶0.5, by vol) at 0.6 mL/min. Application of the method to a stereospecific analysis of the molecular species of TAG of rat VLDL showed that the TAG composition of VLDL circulating under basal conditions differs markedly from that of VLDL secreted by the liver during inhibition of serum lipases. The inhibition of serum lipases resulted in a significant proportional decrease in 16∶0 and PUFA and an increase in 18∶0 and oligoenoic FA in the sn-1-position, whereas the FA compositions in the sn-2- and sn-3-positions were much less affected.     

In Vitro hydrolysis of fungal oils: Distribution of arachidonic acid-containing triacylglycerol molecular species

Four commercially prepared arachidonic acidrich oils from the fungus Mortierella alpina were analyzed by high-performance liquid chromatography and gas chromatography. The levels of arachidonic acid and the distribution of triacylglycerol (TG) molecular species varied significantly among these oils. The major arachidonate-containing TG species were AAA, LAA, DAA, OAA, PAA, SAA, OLA, PGA, PLA, POA, and SOA where the abbreviations A, D, G, L, O, P, and S represent arachidonic (20:4n-6), dihomo-γ-linolenic (20:3n-6), γ-linolenic (18:3n-6), linoleic (18:2n-6), oleic (18:1n-9), palmitic (16:0), and stearic (18:0) acids, respectively. In vitro incubation of the TG fractions, purified from these oils with porcine pancreatic lipase for 5 min, yields a mixture of intermediate products, such as 1,2- and 2,3-diacylglycerols (1,2- and 2,3-DG), 2-monoacylglycerol (2-MG) and free fatty acids (FFA), as well as residual TG. The degrees of hydrolysis varied significantly among the four oil preparations, ranging from 35 to 57%. The levels of arachidonic acid in the residual TG and 1,2(2,3)-DG were significantly higher than those in the original TG, whereas those in the FFA fraction were significantly lower than those in 1,2(2,3)-DG and 2-MG. Results from this study suggest that the bioavailability of arachidonic acid differs among fungal oils prepared by different suppliers. These differences could be attributed to the arachidonic acid content of the oil as well as to the association of arachidonic acid with other fatty acids in the same TG molecule.     

Rapid separations of diacyl- and dialkylglycerol enantiomers by high performance liquid chromatography on a chiral stationary phase

Rapid and practical separations of 1,2-and 2,3-diacyl-and dialkyl-sn-glycerol enantimers as their 3,5-dinitrophenylurethane derivatives were carried out by normal-phase high performance liquid chromatography on a chiral stationary phase, N-(R)-1-(α-naphthyl)ethylaminocarbonyl-(S)-valine chemically bonded to γ-aminopropyl silanized silica. Complete separations of the racemates into enantiomers were achieved for both of the diacyl-and dialkylglycerols within 10 min using a stainless steel column (25 cm long) packed with the 5-μ particles, an isocratic elution with a mixture of hexane/ethylene dichloride/ethanol as a mobile phase and a UV detector. Thesn-1,2-enantiomers were eluted ahead of the correspondingsn-2,3-enantiomers. Satisfactory separation of thesn-1,3-diacylglycerols from the corresponding enantiomers and the separation of the homologues differing in acyl and alkyl groups were also observed. The formations of hydrogen bonding and charge transfer complex between the urethane derivatives and the stationary phase may contribute to the enantiomer separations.     

Enzymatic Synthesis of Structured Triacylglycerols Containing CLA Isomers Starting from <Emphasis Type="Italic">sn</Emphasis>-1,3-Diacylglycerols

The synthesis of structured triacylglycerols (TAG) by the enzymatic reaction between sn-1,3-diacylglycerols (sn-1,3-DAG) and conjugated linoleic acid (CLA) isomers was studied. Both the substrates of the reaction were produced from vegetable oils, the sn-1,3-DAG from extra virgin olive oil and the CLA isomers from sunflower oil. The enzymatic reactions between these substrates were catalyzed for 96 h by an immobilized lipase from Rhizomucor miehei (Lipozyme IM) and the reactions carried out in solvent were monitored every 24 h by using high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The enzymatic reactions were carried out in different reaction media (hexane, isooctane and solvent free) and with different CLA/sn-1,3-DAG ratios. Total % acidic composition and structural analysis data were evaluated to verify the presence of CLA isomers in sn-2- position of synthesized TAG. The results showed good levels of CLA incorporation in sn-1,3-DAG, from 19.2% of TAG synthesized in solvent free conditions with a 0.5:1 substrate ratio, to 47.5% of TAG synthesized in isooctane with a 2:1 substrate ratio. It was observed that for all the reaction media, the best sn-2- acylic specificity was obtained with a 0.5:1 substrate ratio.     

Characterization of α- and γ-linolenic acid oils by reversed-phase high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

Triacylglycerols of oils rich in α- and/or γ-linolenic acids were analyzed by reversed-phase high-performance liquid chromatography (HPLC) coupled with atmospheric pressure chemical ionization mass spectrometry [(APCI)MS]. The (APCI)MS spectra of most triacylglycerols exhibited [M + H]⁺ and [M - RCOO]⁺ ions, which defined the molecular weight and the molecular association of fatty acyl residues, respectively. Reversed-phase HPLC resulted in, at least, partial separation of triacylglycerols containing α- and/or γ-linolenic acid moieties. Molecules containing α-linolenic acid residues exhibited a relatively weaker retention by the stationary phase than the corresponding molecules containing γ-linolenic acid residues. Good separation of triacylglycerols of cloudberry seed oil, evening primrose oil, borage oil, and black-currant seed oil was achieved. The molecular species identification of separated components was based on the (APCI)MS data as well as on the elution properties of triacylglycerols on reversed-phase HPLC. This study demonstrated the efficiency of HPLC-(APCI)MS in determining the molecular species compositions of triacylglycerols in complex natural mixtures. Good quality mass spectra could be extracted even from minor chromatographic peaks representing 0.2% or less of the total triacylglycerols.