Stereospecific analysis of triacyl-sn-glycerols by chiral high-performance liquid chromatography

A method for the stereospecific analysis of triacyl-sn-glycerols by high-performance liquid chromatography (HPLC) on a chiral column is described. Triacyl-sn-glycerols were partially degraded with ethyl magnesium bromide, and the monoacylglycerols produced were separated as 1- and 2-monoacylglycerol fractions by thin-layer chromatography on boric acid-impregnated silica gel plates. The 1-monoacylglycerols were resolved intosn-1 andsn-3 monoacylglycerol fractions by HPLC on a chiral column (Sumichiral OA-4100) after derivatization with 3,5-dinitrophenyl isocyanate. Fatty acid methyl esters obtained from the original triacyl-sn-glycerols, 1- and 2-monoacylglycerol fractions, andsn-1 andsn-3 monoacylglycerol fractions were analyzed by gas chromatography on open-tubular columns. Stereospecific acyl distributions in triacyl-sn-glycerols were calculated from the data. The acyl distributions of several oils were obtained. The method is rapid, simple and gave reproducible results.     

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.     

Positional distribution of 24∶6(n−3) in triacyl-sn-glycerols from flathead flounder liver and flesh

This paper presents the positional distribution of very long-chain fatty acids, 24∶6(n−3), in triacyl-sn-glycerols (TG) of flathead flounder (Hippoglossoides dubius). Each of the liver and flesh TGs was subjected to the stereospecific analysis. The liver TGs contained 24∶6(n−3) at concentrations of 1.5, 1.2 and 1.7 mole % in thesn-1,sn-2 andsn-3 positions, respectively, and the flesh TGs had 9.0, 7.8 and 7.1 mole % in thesn-1,sn-2 andsn-3 positions, respectively. This fatty acid was distributed almost evenly among the three positions of the TGs. No preference for thesn-2 position was observed in contrast to the general tendency for the distribution of longer-chain polyunsaturated fatty acids, such as 22∶6(n−3), 22∶5(n−3) and 20∶5(n−3). There was essentially no difference in the positional distributions of the liver and flesh TGs. The results obtained in this study give new fundamental information to the investigation of very long-chain fatty acids.     

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.     

Stereospecific analyses of seed triacylglycerols from high-erucic acid brassicaceae: Detection of erucic acid at thesn-2 position inBrassica oleracea L. Genotypes

Stereospecific analyses of triacylglycerols from selected high-erucic acid breeding lines or cultivars ofBrassica napus L. andB. oleracea L. have been performed. Initial lipase screening revealed that while allB. napus lines contained little or no erucic acid at thesn-2 position, several of theB. oleracea lines had significant proportions of erucic acid at this position. Detailed stereospecific analyses were performed on the triacylglycerols from these lines by using a Grignard-based deacylation, conversion of thesn-1,sn-2 andsn-3 monoacylglycerols to their di-dinitrophenyl urethane (DNPU) derivatives, resolution of the di-DNPU-monoacylglycerols (MAGs) by high-performance liquid chromatography on a chiral column, transmethylation of eachsn-di-DNPU MAG fraction and analysis of the resulting fatty acid methyl esters by gas chromatography. The findings unequivocally demonstrate for the first time that, within the Brassicaceae, there existsB. oleracea germplasm containing seed oils with substantial erucic acid (30–35 mol%) at thesn-2 position. This has important implications for biotechnology and breeding efforts designed to increase the levels of erucic acid in rapeseed beyond 66 mol% to supply strategic industrial feedstocks. In the first instance, the germplasm will be of direct use in retrieving a gene encoding aBrassica lyso-phosphatidic acid acyltransferase with an affinity for erucoyl-CoA. In a breeding program, the germplasm offers promise for the introduction of this trait intoB. napus by interspecific hybridization and embryo rescue.     

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.     

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.     

Stereospecific analysis of monounsaturated triacylglycerols in cocoa butter

The enantiomeric composition of the monounsaturated triacylglycerols (TG) from cocoa butter was estimated. The monounsaturated TG were separated into three fractions by reversed-phase high-performance liquid chromatography (HPLC), and each fraction was subjected to the stereospecific analysis with chiral-phase HPLC. The results indicated that the major TG consisted of equal amounts of 1-stearoyl-2-oleoyl-3-palmitoyl-sn-glycerol (SOP-sn-TG) and POS-sn-TG (47 mol%), 1,3-distearoyl-2-oleoyl-glycerol (SOS-TG) (33 mol%), and POP-TG (19 mol%). The contents of SOP-sn-TG and POS-sn-TG are 1.30 times that of the POP-TG content, and the SOS-TG content is 1.30² times that of the POP-TG content. The term “priority factor” is proposed for the ratio of the stearoyl group/palmitoyl group, 1:30 at thesn-1 andsn-3 or 1(3)-position. It shows a distinct specificity for particular fatty acids or their Coenzyme A esters in random esterification at each position of the glycerol moiety in the biosynthesis of cocoa butter TG.     

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.     

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 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.     

Structure Identification of Triacylglycerols in the Seed Oil of Momordica Charantia L. Var. Abbreviata Ser.

Triacylglycerols (TGs) in the seed oil of Momordica charantia L. var. abbreviata Ser. (MCV) were separated by non-aqueous reversed-phase (NARP)-HPLC. Many of the TGs contain two different fatty acyl chains, such as palmitic (P), stearic (S), oleic (O), linoleic (L), and conjugated linolenic acid (CLn). Seven pairs of AAB/ABA-type TGs might present in the seed oil of MCV, namely CLnCLnP/CLnPCLn, CLnCLnS/CLnSCLn, CLnCLnO/CLnOCLn, CLnCLnL/CLnLCLn, SSCLn/SCLnS, OOCLn/OCLnO and LLCLn/LCLnL. The positional isomers of a AAB/ABA-type TGs pair yielded mass spectra showing a significant difference in relative abundance ratios of the fragment ions [AA]⁺ to [AB]⁺, which were produced by preferred losses of the fatty acid from the 1/3-position compared to the 2-position of the glycerol backbone. The precise stereospecific structures of the predominant regioisomers of TGs in AAB/ABA pairs were identified by atmospheric pressure chemical ionization mass spectrometry (APCI-MS) according to the special relative abundance ratios of the fragment ions [AA]⁺ to [AB]⁺. TGs with CLn occupying the sn-2 position in seven pairs of AAB/ABA might be major constituents of the oil, such as CLnCLnS, LCLnL, CLnCLnP, and so on. Some of the TGs which were isolated and collected as fractions from the seed oil of MCV by NARP-HPLC were further analyzed by ¹³C-NMR. ¹³C-NMR data of type-AAA TGs containing α-eleostearic acyl have been complemented.     

Reinvestigation of positional distribution of fatty acids in docosahexaenoic acid-rich fish oil triacyl-sn-glycerols

Positional distribution of fatty acids in triacyl-sn-glycerols of docosahexaenoic acid (DHA)-rich tuna orbital and bonito head oils has been reanalyzed by a method based on chromatographic separation of isomeric and enantiomeric monoacyl-sn-glycerol (MAG) derivatives. When boric acid thinlayer chromatography (TLC) was used for separation of 1(3)- and 2-MAG analytical intermediates, the stereospecific analysis showed the preferential association of DHA to the sn-2 position followed by the sn-3 position. This distribution pattern differed from that obtained by silicic acid LTC of their bis-3,5-dinitrophenylurethane (DNPU) derivatives. Reversed-phase high-performance liquid chromatography elution profiles of 1(3)- and 2-MAG intermediates revealed that 1(3)- and 2-MAG made up of both short- and long-chain lengths cannot be clearly resolved by TLC after preparation of the DNPU derivatives. The 1(3)- and 2-MAG must be resolved by boric acid TLC prior to derivatization.     

Regioselective analysis of the fatty acid composition of triacylglycerols with conventional high-performance liquid chromatography

A new method for regioselective analysis of triacyglycerols via conventional high-performance liquid chromatography (HPLC) has been developed. The method is simple and avoids the time-consuming purification processes normally characteristics of regioselective analyses. The procedure utilizes an sn-1,3-specific lipase from Rhizopus arrhizus to deacylate the fatty acid residues located at the sn-1 and sn-3 positions of triacylglycerols. The fatty acid residues esterified at the sn-2 position are determined by subtraction of the results of the sn-1,3 analysis from an overall composition analysis based on complete saponification of the original sample. The fatty acid mixtures are converted to p-bromophenacyl esters and analyzed using conventional HPLC techniques. The analytical procedure has been verified using a standard structured triacylglycerol. The analytical results for three edible vegetable oils are compared with those obtained via the method proposed by P.J. Williams and co-workers.     

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.     

Fractionation of the triacylglycerols of lipase-modified butter oil

Triacylglycerols (TGs) of lipase-modified butter oil were separated into saturated, monoene, diene and triene fractions on ap-propylbenzene sulfonic acid solid-phase extraction column loaded with silver ions. Fatty acid analysis of the fractions showed that the amounts of saturated TGs (98.4 mol%) and monoene TGs (26.0 mol%) in the saturated and monoene fractions, respectively, were close to the theoretical amounts of TGs in pure fractions. The column method provides a useful alternative to AgNO₃-thin-layer chromatography as a means of separating the TGs of butterfat and producing relatively pure TG fractions for further analysis by gas chromatography (GC) or GC-mass spectrometry.     

The positional distributions of fatty acids in the triacylglycerols and phosphatidylcholines of the intestinal and popliteal lymph and plasma of sheep

As part of a study of the contribution of the intestinal lymph lipoproteins and their lipid constituents to the plasma lipids in sheep, the positional distributions of the fatty acids in the triacyl-sn-glycerols and phosphatidylcholines in very low density/low density lipoprotein and high-density lipoprotein fractions were determined by stereospecific analysis procedures. The triacyl-sn-glycerols of these lipoprotein fractions in intestinal lymph did not differ appreciably in structure and resembled the plasma triacyl-sn-glycerols in the composition of positionsn-2 especially. However, there were appreciable amounts of the essential fatty acid, linoleic acid, in positionssn-1 andsn-3 of the triacylglycerols in lymph but not in plasma. This result is discussed in terms of the metabolism of the triacylglycerols of lymph after they enter the plasma as part of a mechanism for the conservation of essential fatty acids in ruminants. No differences of metabolic note were observed in the structures of the phosphatidylcholines between lipoprotein fractions and among tissues.     

Changes in olive oil composition due to microwave heating

The effects of microwave heating on some components of extra-virgin olive oil were studied. Traditional parameters, including free acidity, peroxide value and ultraviolet absorbance values at 232 and 268 nm, were determined in six extra-virgin olive oil samples before and after the microwave treatment. Significant differences (P<0.01) were detected for free acidity, peroxide, and ultraviolet absorbance at 268 nm; also, the absorbances at 232 nm showed significant differences (P<0.05) between treated and untreated samples. The glycerolic fractions, triacylglycerols (TAG), diacylglycerols (DAG), and monoacylglycerols (MAG), were isolated by thin-layer chromatography. The respective percentage fatty acid (FA) composition and percentage amount were obtained by high-resolution gas chromatography with an internal standard. For the most abundant TAG fraction, the stereospecific analysis was carried out to obtain the FA percentage compositions of the three sn-positions. Small but significant modifications were observed regarding the decrease in the TAG percentage and increases in the DAG and MAG percentage amounts. No significant changes were observed for the FA compositions of TAG, DAG, and MAG fractions before and after the treatment. Nevertheless, the results of TAG stereospecific analysis showed losses of unsaturated FA in all sn-positions. Higher percentage changes in the sn-1- than in sn-2-position of TAG were observed. Regarding the volatile fraction, different profiles were obtained after the treatment.     

Determination of molecular species of oil triacylglycerols by reversed-phase and chiral-phase high-performance liquid chromatography

A method is described for the determination of molecular species of oil triacylglycerols. The method is based on the analytical separation of the enantiomericsn-1,2-andsn-2,3-diacylglycerols, derived from triacylglycerols, by high-performance liquid-chromatography (HPLC) on a chiral column containing N-(R)-1-(α-naphthyl)ethylaminocarbonyl-(S)-valinecarbonyl-(S)-valine as stationary phase. Model triacyl-glycerol molecules comprising three known fatty acids were isolated from peanut oil and cottonseed oil by a combination of argentation-TLC and reversed-phase HPLC and submitted to partial chemical deacylation. The derivedsn-1,2(2,3)-diacylglycerols were analyzed and fractionated as 3,5-dinitrophenyl urethane derivatives by reversed-phase HPLC according to chainlength and unsaturation. From thesn-1,2(2,3)-diacylglycerol composition and the diacylglycerolsn-1,2-andsn-2,3-enantiomer composition, the individual molecular species of four peanut oil triacylglycerols and one cottonseed oil triacylglycerol were identified and quantitated. The method can be applied to triacylglycerols of any other oil or fat.     

Synthesis of Urethane Derivatives of Mono- and Diacylglycerols for Use as HPLC Standards in the Enantiomeric Separation

This paper presents a convenient method for the preparation of reference standards for high-performance liquid chromatography (HPLC) used in stereospecific analysis of triacyl-sn-glycerols via monoacylglycerol or diacylglycerol intermediates. In the analysis, these partial acylglycerols are separated into their respective positional and enantiomeric isomer classes by chiral HPLC as their 3,5-dinitrophenylurethane derivatives or by silicic acid HPLC as their (S)- or (R)-1-(1-naphthyl)ethyl urethane derivatives. In this study, these urethane derivative standards were synthesized by the following novel procedure: first, partial urethane derivatives of glycerol were prepared by carbamoylation of glycerol with isocyanates; secondly, the products were separated into positional isomer classes by silicic acid HPLC, and; finally, a fatty acid was added to the partial urethanes using N,N′-dicyclohexylcarbodiimide. The identities of the resulting urethane derivatives of glycerol were verified by mass spectrometry and HPLC. This new procedure is advantageous in that standard urethane derivatives of partial acylglycerols can be synthesized from no more than 50 μg of fatty acids. This benefit is especially important in the case of rare and expensive fatty acids, such as very long chain polyunsaturated fatty acids, tetracosahexaenoic acid, and hexacosaheptaenoic acid, found in marine lipids.