High-performance liquid chromatography with light-scattering detection and desorption chemical-ionization tandem mass spectrometry of milk fat triacylglycerols

The utility of reverse-phase high-performance liquid chromatography (HPLC), desorption chemical-ionization mass spectrometry (DCI-MS) and tandem mass spectrometry (MS/MS) for the characterization of triacylglycerols in complex mixtures has been further explored. Triacylglycerols of anhydrous bovine milk fat were separated by using two reversephase C₁₈ HPLC columns, and eluents were monitored with an evaporative light-scattering detector. Fifty-eight fractions were resolved and analyzed by positive ion isobutane DCI-MS. The formation of protonated molecules and of major fragments corresponding to the random loss of any one of the constituent fatty acids readily identified acyl carbon numbers and the number of double bonds within each fatty acid. MS/MS was only required when the original mass spectra indicated the presence of more than one triacylglycerol or of impurities in a fraction. Protonated molecules produced by DCI were fragmented using high energy collisional activation, and the resulting ions were detected by MS/MS. Odd-chain triacylglycerols were also readily distinguished using this methodology. The positive ion DCI and MS/MS techniques described here demonstrate the usefulness of this approach for the characterization of triacylglycerols in complex mixtures.     

Quantitative analysis of short chain fatty acids using gas liquid chromatography

The short-chain fatty acids, propionic to pelargonic, have been analyzed by gas liquid chromatography, as their butyl, phenacyl, and decyl esters to overcome losses due to their volatility. The three methods are compared and the decyl ester procedure offers the most advantages. Presented in part at the AOCS meeting in Los Angeles, Calif., 1959. Issued as N.R.C. No. 7261. National Research Council of Canada Postdoctorate Fellow 1957–1959.     

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.     

Distribution of the major fatty acids of human milk betweensn-2 andsn-1,3 positions of triacylglycerols

Human milk triacylgycerols (TAG) were analyzed by tandem mass spectrometry. The SIMPLEX method and a simple linear model were used to interpret the distribution of fatty acids between thesn-2 andsn-1,3 positions in 24 major molecular weight groups of TAG. The number of regio-isomeric pairs of TAG varied between 3 and 18 in each of these groups. Hexadecanoic (16∶0), tetradecanoic (14∶0) and dodecanoic acids (12∶0) typically occupied thesn-2 position in TAG containing less than 54 acyl carbons, whereas long-chain C18 and C20 acids were predominantly located at the primary positions. The positions of the three fatty acids within a TAG molecule were shown to depend on the fatty acid combination. The maximum of 12∶0 in thesn-2 position appeared at acyl carbon number (ACN) 48, the maxima of 14∶0 were at ACN 44 and ACN 50, and for 16∶0 at ACN 46 and 52.     

Rapid determination of free fatty acids in vegetable oils by gas liquid chromatography

Determination of the free fatty acids in small quantities of vegetable oil is accomplished by gas liquid chromatography. The free fatty acids are isolated from a hexane solution of the vegetable oil into an aqueous solution of trimethylphenylammonium hydroxide (TMPH). Due to the alkalinity of TMPH, the free fatty acids readily partition into this aqueous phase. Injection of the free fatty acid-TMPH salts into a gas chromatograph results in pyrolytic methylation of the free fatty acid salts—yielding the methyl esters. Excellent results were obtained when this new procedure was used on neutral lipid oils containing known amounts of free fatty acids and compared with the results obtained by a modified BF₃/MeOH esterification procedure. When compared to the AOCS titration procedure, this new procedure gave comparable results. This new procedure has advantages over the AOCS procedure: it is more sensitive and gives quantitative results for individual free fatty acids. This new procedure also has several advantages over the modified BF₃/MeOH esterification procedure: it is easily and more rapidly performed, there is no deposition of glyceride on the column when the sample is injected, and because there is quantitative recovery, the new procedure is more sensitive and can be used on oils with a low weight percentage of free fatty acids.     

The composition of new zealand milk fat triacylglycerols by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography (HPLC) method was developed for the purpose of analyzing milk fat triacylglycerols by evaporative light scattering detection. With a binary solvent system, comprising dichloromethane and acetonitrile, the method allowed a separation of 61 distinct peaks, on the basis of chainlength and number of double bonds. Triacylglycerols of differing partition numbers were clearly resolved, and the resolution between peaks of the same partition number was high. An argentation thin-layer chromatography method, separating triacylglycerols on the basis of chainlength and degree of unsaturation, provided nine band extracts that were analyzed by HPLC. By using existing fatty acid methyl ester data of these bands, an identity for each HPLC peak has been proposed.     

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.     

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.     

Characterization of branched-chain fatty acids from fallow deer perinephric triacylglycerols by gas chromatography-mass spectrometry

Branched-chain fatty acids of perinephric triacylglycerols of semi-feral fallow deer (Dama dama dama) were analyzed by high resolution gas chromatography-mass spectrometry. Of the total fatty acids, 15.50% were Branched-chain components including 8.96% iso acids, mostly 14-methylpentacanoic acid, 2.85% anteiso acids and 1.73% of other monomethyl-substituted acids; dimethyl-branched acids with an iso structure (1.05%) and with an anteiso structure (0.18%) were also present. Whereas the predominant iso acids and methyl-substituted iso acids had chain lengths of 13 and 15 carbon atoms, the anteiso acids and methyl-substituted anteiso acids had chain lengths of 14 and 16 carbon atoms. Methyl substitution occurred on the even numbered carbon atoms relative to the carboxyl group. The general composition is also given of the fatty acids comprising the triacylglycerols of subcutaneous (rump area) and perinephric adipose tissue.     

Separation of milk fat triacylglycerols by argentation thin-layer chromatography

Argentation thin-layer chromatography was investigated as a method of obtaining detailed compositional information about milk fat. A modified argentation thin-layer chromatography procedure, developed to optimize the separation of the complex mixture of total milk fat triacylglycerols, provided nine different groups of triacylglycerols, based on both the degree of unsaturation and the total length of fatty acid groups. Fatty acid methyl ester (FAME) analysis was performed to determine the composition of each band. Separation on the basis of chainlength was most pronounced among the fully saturated triacylglycerol groups, as evidenced by the high level of C_4:0 and C_6:0 in bands 7 and 8, respectively. For the cis-monoenoic triacylglycerols, the separation of C_4:0 and C_6:0 was less distinct. The cis,cis dienes and other dienoic, trienoic, or tetraenoic species were principally observed in two bands of retention factor <0.08 on the chromatography plate. Minimal cross-contamination of bands was observed, with the exception of the lowest of the trisaturate bands, band 7, in which trans-monoenes were found to be present. Three samples from different points of the New Zealand dairy season were separated by argentation thin-layer chromatography, and their FAME distributions were measured. In addition to differences in the masses of band extracts from these samples, levels of C_10:0 and C_12:0 in all bands, and levels of monounsaturates in the dienoic and trienoic bands, were found to differ. These changes were generally consistent with a pattern of decreasing fat hardness over the November to March period of a typical dairy season.     

The regiospecific position of 18∶1 cis and trans monoenoic fatty acids in milk fat triacylglycerols

TAG of butterfat were fractionated according to the type and degree of unsaturation into six fractions by silver-ion HPLC. The fractions containing TAG with either cis-or trans-monoenoic FA were collected and fractionated further by reversed-phase HPLC to obtain fractions containing cis TAG of ACN:DB (acyl carbon number:double bonds) 48∶1, 50∶1, and 52∶1 as well as trans 48∶1, 50∶1, and 52∶1. The FA compositions of these fractions were elucidated by GC. The MW distribution of each fraction was determined by ammonia negative-ion CI-MS. Each of the [M-H]⁻ parent ions was fractionated further by collision-induced dissociation with argon, which gave information on the location of cis-and trans-FA between the primary and secondary positions of TAG. The results suggest that the sn-positions of the monoenoic cis-and trans-FA depend on the two other FA present in the molecule. With 14∶0 FA in the TAG molecule, the 18∶1 FA in the sn-2 position are mostly present as cis-isomers. When there is no 14∶0 in the TAG molecule, the trans-18∶1 isomers seem to be more common in the sn-2 position. Also when other long-chain FA are present, the trans-isomers are more likely to be located in the secondary (sn-2) position.     

Quantitative analysis of food fatty acids by capillary gas chromatography

The superior efficiency of capillary columns is desirable for the gas chromatographic analysis of complex mixtures of fatty acids, but there have been some reservations regarding quantitation and reproducibility. This paper discusses the use of wall-coated glass capillary columns in a semiautomated system for the determination of food fatty acids. Glass columns coated with SP2340 were used for extended periods at temperatures up to 200 C without appreciable deterioration. Up to 1900 samples were analyzed on a single column over an 11-month period, with only minor changes in retention ratios, response factors and column efficiency. Quantitative precision of results, calculated either as normalized weight percentage or as absolute amounts, based on the use of an internal standard, were typically within 2% relative deviation. Difficulties encountered in obtaining acceptable chromatograms and reproducible data are discussed, and typical analyses of the fatty acids from foods presented.     

Analysis of α-branched fatty acids by gas chromatography and mass spectrometry

Gas liquid chromatography and mass spectrometry were utilized in combination to identify isomeric α-branched chain fatty acid methyl esters. In a given isomeric series equivalent chain length, values decreased with increase in the number and size of α-alkyl substituents. Mass spectra of the α-monoalkyl derivatives are characterized by prominent McLafferty rearrangement ion peaks, whereas those of the α,α-dialkyl isomers contain the former ions plus an α-cleavage ion. Presented at the AOCS Meeting, Chicago, September 1973.     

Determination of milk fat in chocolates by gas-liquid chromatography of triglycerides and fatty acids

The combination of two routine methods is proposed to determine the content of milk fat (MF) in chocolates, which is applicable even in the presence of lauric fats or others. The content of MF is obtained from the sum of C₄₀, C₄₂, and C₄₄ medium-chain triglycerides, determined by capillary gas-liquid chromatography (GLC). A new method, based on methyl esters of lauric acid and on minor acids situated between myristic and palmitic, is proposed. It enables detection and estimation of potential lauric fats, as well as the determination of the actual content of MF. The influence of other vegetable and animal fats is discussed. We analyzed 45 MF samples extracted from industrial milk powders and from pure or fractionated MF for chocolate manufacturing or pastry by GLC of triglycerides. We also analyzed by capillary GLC the methyl esters from 22 of those fats. Mixtures of these 22 MF samples with a cocoa butter also were used for chromatographic analyses of methyl esters and triglyceride. Results from the various analytical methods have been presented.     

Analysis of isomeric monoethylenic fatty acids in a partially hydrogenated herring oil by glass capillary gas liquid chromatography

The use of a glass capillary column coated with Silar 10 CP for gas liquid chromatographic analysis of geometric and positional isomers of monoethylenic fatty acids of a partially hydrogenated herring oil is reported. The results are in agreement with previous studies performed by different methods and demonstrate the usefulness of this technique in detecting and determining many types of isomeric fatty acids.     

On the syntheses of triacylglycerols from branched saturated fatty acids

A number of triacylglycerols with branched acyl groups were prepared via 1,2-isopropylidene glycerol for the purpose of studying three different physical properties: gel point, refractive index, and density. The monoacid triacylglycerols were prepared either via the corresponding acids or the acyl chlorides.     

Stereospecific distribution of fatty acids in triacylglycerols of olive oils

The positional distribution of fatty acids (FA) in triacylglycerols (TAG) of 47 virgin olive oils from diverse cultivars grown in distinct areas of North‐Eastern Italy was studied. Few data were previously available on oils from these geographical areas. The effects of climatic and geographical conditions on the stereospecific distribution of TAG in olive oil were confirmed. Moreover, the results of the stereospecific analysis were used to evaluate the preferential esterification position of each FA on the basis of the degree of unsaturation and the chain length. The data of the stereospecific analysis of olive oil TAG can contribute to the determination of the selectivity of olive fruit acyltransferases for distinct FA.     

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.