Location of double bonds in fatty acids of fish oil and rat testis lipids. Gas chromatography-mass spectrometry of the oxazoline derivatives

A new approach to the derivatization and analysis of long chain polyunsaturated fatty acids is described. The method is based on the formation of 2-alkenyl-4,4-dimethyloxazolines by condensation of the starting material with 2-amino-2-methylpropanol. The derivatization method is rapid, efficient and specific with respect to the chain feature of the parent acids. Volatility, comparable with that of the corresponding simple esters, and improved gas chromatographic separation are achieved without difficulty. The derivatives exhibit clear and regular fragmentation patterns that allow easy discrimination of positional isomers and assignment of double bond location in the chain. Chemical Modification in Mass Spectrometry, Part 8. For preceding paper, see reference 1.     

Location of double bonds in long chain esters by mass spectroscopy of methoxyhalogeno derivatives prepared from methoxymercuriacetate adducts

Methyl esters of mono-, di-, and triunsaturated long chain fatty acids react quantitatively with mercuric acetate in methanol to produce methoxyacetoxy-mercuri derivatives. Demercuration of these products with solutions of bromine or iodine in methanol yields methoxybromo or methoxyiodo derivatives readily isolable by thin layer chromatography. Mass spectra of the methoxyhalogeno derivatives of monounsaturated esters are characterized by intense peaks due to loss of halogen and cleavage adjacent to methoxy functions; the latter fragmentation allows the position of the original double bond to be established. The mass spectra of the methoxyhalogeno derivatives of polyunsaturated esters are more complex.     

Fatty acids: part 27. Olefin inversion involving unsaturated fatty esters

Unsaturated long chain fatty esters gave the corresponding bromochloro ester derivatives when treated with N-bromosuccinimide and hydrogen chloride. Treatment of the intermediate halo esters with sodium iodide furnished the same positional unsaturated fatty ester isomers, but of opposite geometric configuration. This olefin isomerization method was found to be high yielding, stereospecific and to cause no double bond migration.     

Isomeric monoethylenic fatty acids in herring oil

Monoethylenic fatty acids from herring oil were concentrated by chromatography by chromatography on silver nitratesilicic acid columns. Examination of consecutive fractions by open tubular gas chromatography confirmed the preferential elution of longer chain length esters and of esters within one chain length with the double bond closer to the terminal methyl group. Isomeric monoethylenic fatty acids with double bonds in the positions closer to the carboxyl group than the approximate midpoint of the even-numbered fatty acid chains could not be adequately separated by gas chromatography and were determined by ozonolysis. The isomers observed are consistent with primary formation from saturated acids through the action of an enzyme specifically removing hydrogen atoms in positions Δ⁹ and Δ¹⁰ relative to the carboxyl group. Chain extension of particular monoethylenic isomers by two carbon atoms in the C₂₀ and longer chain lengths is apparently influenced by the position of the double bond. This work was carried out in partial fulfillment of MSc requirements at Dalhousie University.     

Double bond location in polyenoic fatty esters through partial oxymercuration

A rapid micro-procedure has been developed to locate double bonds in fatty acid methyl esters containing from one to four double bonds. Reaction of the ester with an equal molar amount of mercuric acetate in methanol and reduction with sodium borohydride, followed by hydrogenation, produce a mixture of monomethoxy alkanoates. The mass spectrum of this mixture is simpler and more definitive than that from the completely methoxylated polyenoate. Only one methoxyl group is present per molecule, and the mass spectrum of the mixture is indicative of all olefinic positions. Four intense ions are observed for all double bonds examined, except Δ3, where the double bond is represented, by only two ions. Hydrogenation in a gas chromatograph reduces total analysis time to 1 hr. Presented at the AOCS Meeting, Cincinnati, September 28–October 1, 1975.     

Characterization of unsaturated fatty acids by gas-liquid chromatography

The equivalent chain length (ECL) has been determined on 79 methyl esters of unsaturated fatty acids and on 7 ethyl esters by gas chromatography. Ethylene glycol succinate (EGS), diethylene glycol succinate, β-cyclodextrin acetate and Apiezon L were chosen as the liquid phases to be used. For methyl esters of mono- and polyenoic acids, the differences between ECL on EGS and ECL on Apiezon L approximate 0.84 per double bond. For positional isomers, the ECL on both EGS and Apiezon L are usually greater for the isomer having the longer proximal end of the molecule (smallest ω value). In these terms a triple bond is approximately equal to three double bonds. Esters of nonconjugated dienoic and trienoic acids of the same chain length are not separable on Apiezon L if their proximal structures are the same. This also applies to tetraenoic and pentaenoic acids of the same chain length and the same proximal structure. Conjugation of double bonds, either with the ester carbonyl group or with themselves, yields ECL values on Apiezon L greater than the number of carbon atoms in the acid. Monounsaturated and nonconjugated polyunsaturated esters have ECL values on Apiezon L lower than the number of carbon atoms of the acid. The ECL values of ethyl esters of 18 and 20 carbon acids are greater than the corresponding methyl esters on Apiezon L. Presented at the Chicago meeting of the American Oil Chemists’ Society, Oct. 13, 1964.     

Identification of 3, 6, 9, 12, 15-octadecapentaenoic acid in laboratory-cultured photosynthetic dinoflagellates

Polar and nonpolar chromatograms of fatty acid methyl esters derived from 11 species of photosynthetic, marine dinoflagellates cultured in modified Erd-Schrieber medium contained a component (4-23%) not identifiable by conventional graphic or arithmetic methods. Hydrogenation followed by gas liquid chromatography of the product showed the unknown component to be a straight chained 18 carbon fatty acid methyl ester. Chemical (ch4) ionization mass spectrometry of the isolated ester gave a spectrum characteristic of methyl esters and a mol wt of 288, indicating an 18 carbon molecule with 5 double bonds, or equivalent unsaturation. The IR spectrum showed that the double bonds are nonconjugated, and all are cis in geometry. Electron impact mass spectrometry of the pyrorolidide derivative provided evidence that double bonds are located in the 3, 9, 15 positions and probably also in the 6 and 12 positions of the molecule. these double bond positions were confirmed by NMR spectrometry. Data obtained by quantitation of the algal methyl esters suggest the possibility that these dinoflagellates synthesize 18:5omega3 (shorthand notation for chain length: number of double bond counted from the terminal methyl group) by a 2 carbon chain shortening of 20:5omega3, rather than by the insertion of a delta3 bond into 18:4omega3.     

Monoenoic fatty acids in human brain lipids: Isomer identification and distribution

The carbon chain length distribution and the double bond positional isomer composition of the monoenoic fatty acids of the lipids of total human brain tissue have been determined using gas chromatography and gas chromatography/mass spectrometry of the fatty acid methyl and picolinyl esters. The even chain length monoenoic C₁₆ to C₂₈ fatty acids contain predominantly two positional isomer series, the n−7 and n−9cis homologues, whose relative proportion varies significantly with chain length. The odd chain length long-chain fatty acids consist of n−8 and n−10 isomers, whereas the odd chain length very long-chain (more than 22 carbon) fatty acids are n−7 and n−9 isomers.     

Gas chromatographic separation of cholesteryl esters of fatty acids of different degrees of unsaturation

Cholesteryl esters prepared from the fatty acid methyl esters of linseed oil, pig liver lipids, and Japanese anchovy oil have been separated on the basis of their chain lengths and number of double bonds by gas lipid chromatography on a cyanosiloxane SILAR 10C column. The equivalent chain lengths of cholesteryl esters having acyl groups with 14–22 carbons and 0–6 double bonds are presented. A significant influence of the column temperature on the equivalent chain lengths of the polyenoic fatty acid cholesteryl esters has been found. Separation of the cholestanyl and epicholestanyl esters of linseed oil fatty acids, respectively, under the same conditions is also described.     

Localization of fatty acid double bonds by gas chromatography of intermediate aldehydes as 1,3-dioxane

A semi-micro quantitative method has been developed for the determination of double bond positions in unsaturated fatty acid methyl esters differing in position, number of double bonds and chain length. This method involves ozonolysis in methylene chloride in the presence of 1,3-propanediol, reduction of the hydroperoxides with dimethyl sulfide and conversion of aldehyde and aldester fragments to stable dioxanes, followed by gas chromatographic analysis under programmed temperature. Propanediol acts as a proton donor for the generation of hydroperoxides as well as a reactant for the formation of dioxanes. All the steps are carried out in a culture tube without any transfers. The absence of unoxidized ester and unreduced hydroperoxide or ozonide has been shown by thin layer chromatography. The method has been tested with pure esters and their binary mixtures. Experimental values for mole percentage composition in the mixtures agreed with the actual values. This method enabled us to isolate and estimate malondialdehyde for the first time as a stable bis-dioxane derivative.     

Melting points of synthetic wax esters

Saturated, monoenoic and dienoic wax esters, C₂₆−C₄₀, have been synthesized from even-numbered fatty alcohols and acids. In homologous series of saturated esters, the increments of melting points follow a regular trend except for those esters which have an acid moiety two carbon atoms shorter than the alcohol moiety. These wax esters have melting points higher than interpolation would predict. Monoenoic wax esters with the double bond in the alcohol chain have melting points about 10 C higher than their isomers with the double bond in the acid chain.     

Mass spectrometric location of double bonds in unsaturated fatty acids including conjugated acids as their methoxybromo derivatives

Methoxybromo derivatives of unsaturated fatty acids including conjugated acids yield simple mass spectra and can be used to locate the position of double bonds in these acids. The derivatives are prepared under mild conditions by bromination of the unsaturated fatty acids in methanol. The method is illustrated with the methoxybromo derivatives from methyl esters of oleic, petroselenic, erucic, undecenoic, linoleic, linolenic, the conjugated diene acids from dehydrated castor oil, α-eleostearic, punicic and parinaric acids. Unlike other methods using methoxy derivatives, the methoxybromo derivatives yield fewer ions, the diagnostic peaks forming the most intense ions of the spectra. While unambiguous double bond location is possible with monoenoic acids and conjugated fatty acids, only the end carbon atoms of the unsaturation system in nonconjugated polyenoic acids is located. But the characteristic appearance of fragments corresponding to [CH₃(CH₂)_nCH(OMe)CH(Br)CH₂−2H]⁺ and fragments 24 mass units higher than those which locate the end carbons in such fatty acids indicate a methylene-interrupted system of double bonds.     

The use of nicotinates and sulfoquinovosyl monoacylglycerols in the analysis of monounsaturated n-3 fatty acids by mass spectrometry

Fatty acyl groups (16∶1 and 16∶0) liberated from purified sulfoquinovosyl diacylglycerols produced by the unicellular marine microalga,Heterosigma carterae (formerlyH. akashiwo), were converted to either the corresponding alcohols or methyl esters. Nicotinate derivatives of the alcohols were examined by combined gas chromatography/mass spectrometry, and the methyl esters were examined by nuclear magnetic resonance (NMR) spectroscopy after separation by high-performance liquid chromatography. Three different hexadecenoyl fatty acyl groups were identified, one of which wascis 13-hexadecenoyl (16∶1n−3). Both the configuration and the n−3 position of the double bond in thecis 13-hexadecenoyl moiety were unequivocally established by NMR analysis of the corresponding methyl ester. The nicotinate derived from the alcohol of the 16∶1n−3 fatty acyl moiety gave a characteristic fragmentation series in the electron impact msss spectrum which, by careful interpretation, was consistent with, but not unambiguous for, the assigned location of the double bond. Tandem mass spectrometry experiments on a sulfoquinovosyl monoacylglycerol containing thecis 13-hexadecenoyl group in thesn-2 position, using negative-ion liquid secondary ion mass spectrometry, also gave a fragmentation pattern which was consistent with the positional assignment of the double bond.     

Separation of the Fatty Acids in Menhaden Oil as Methyl Esters with a Highly Polar Ionic Liquid Gas Chromatographic Column and Identification by Time of Flight Mass spectrometry

The fatty acids contained in marine oils or products are traditionally analyzed by gas chromatography using capillary columns coated with polyethylene glycol phases. Recent reports indicate that 100 % cyanopropyl siloxane phases should also be used when the analyzed samples contain trans fatty acids. We investigated the separation of the fatty acid methyl esters prepared from menhaden oil using the more polar SLB-IL111 (200 m × 0.25 mm) ionic liquid capillary column and the chromatographic conditions previously optimized for the separation of the complex mixture of fatty acid methyl esters prepared from milk fat. Identifications of fatty acids were achieved by applying Ag⁺-HPLC fractionation and GC-TOF/MS analysis in CI⁺ mode with isobutane as the ionization reagent. Calculation of equivalent chain lengths confirmed the assignment of double bond positions. This methodology allowed the identification of 125 fatty acids in menhaden oil, including isoprenoid and furanoid fatty acids, and the novel 7-methyl-6-hexadecenoic and 7-methyl-6-octadecenoic fatty acids. The chromatographic conditions applied in this study showed the potential of separating in a single 90-min analysis, among others, the short chain and trans fatty acids contained in dairy products, and the polyunsaturated fatty acids contained in marine products.     

Column types for the chromatographic analysis of oleochemicals

Chromatography has developed into one of the principle methods of analysis of oleochemicals. Gas chromatography has been used extensively for the analysis of long-chain fatty acids as well as for the analysis of triglycerides and plant sterols. In recent years, high pressure liquid chromatography (HPLC) has been used for the analysis of triglycerides as well as for other related materials. Specialized gas chromatography columns have been developed for the separation of long-chain fatty acids such as the methyl esters. These columns have generally used high polarity stationary phases which separate fatty acids by degree of unsaturation. A specialized use of these high polarity stationary phases is separation ofcis-trans isomers as well ascis-cis andtrans-trans isomers. In this paper, packed and capillary columns are compared for the separation of thecis-trans isomers of fatty acid methyl esters prepared from a hydrogenated vegetable oil. For HPLC separations, the presence of a double bond is approximately equivalent chromatographically to shortening the alkyl chain by two carbons. The long-chain polyenic acids or ethyl esters thus elute near but are resolved from the short-chain saturated fatty acids or esters. HPLC is the method of choice for relatively complex, high molecular weight, or labile esters, such as those of retinyl or cholesterol. Glyceryl esters are particularly well resolved by HPLC in terms of both total chain length and degree of unsaturation. This technique is also useful for lipid class separations and for the analysis of modified fatty acid products, such as prostaglandins and related materials. In general, these analyses are conducted with octadecyl bonded phase column packings.     

The monounsaturated acyl- and alkyl-moieties of wax esters and their distribution in commercial orange roughy (Hoplostethus atlanticus) oil

Wax esters were isolated from commercial orange roughy (Hoplostethus atlanticus) oil by column chromatography and fractionated by argentation thin layer chromatography. Following transesterification, the resultant fatty acid methyl esters and fatty alcohols were analyzed by gas chromatography. both acyl- and alkyl-moieties were mainly of the monoene structure within the 16∶1–22∶1 range. After derivatization, the positions of the double bonds of even numbered fatty acid and fatty alcohol isomers were located by chromatography-mass spectrometry and compared. Results of these positional analyses indicate that the primary desaturation reactions takes place in the Δ9 position of pre-existing (C₁₄ to C₂₄) acyl chains. It is proposed that acyl components from 18∶1 are subjected to chain elongation to form a mixture of 24∶1 isomers as the final product. Apart from the 24∶1 acyl moiety of the wax esters, in which the double bond was almost exclusively in the Δ15 position, de novo biosynthetic reactions on acids and alcohols appear to yield related acyl- and alkyl-moieties of resynthesized wax esters.     

Location of methyl branchings in fatty acids: Fatty acids in uropygial secretion of Shanghai Duck by GC-MS of 4,4-dimethyloxazoline derivatives

2-Substituted 4,4-dimethyloxazolines (DMOX) have been found to be a useful alternative to the commonly used methyl esters for the localization of unsaturated bounds and other substituents in the fatty chain by mass spectrometry. The powerful directed fragmentation coupled with good gas chromatographic ability enables the structure elucidation of modified fatty acids in complex mixtures. Continuing our previous study, 76 out of a total of 86 fatty acids obtained from the preen gland wax of shanghai duck now have been identified by gas chromatography-mass spectrometry (GC-MS) of their oxazoline derivatives. The identification was based on the interpretation of the mass spectra and comparison with the spectra and equivalent chain lengths (ECL) of the corresponding methyl esters. Main components of this lipid mixture are straight chain fatty acids (8.22%), and 2-, 4-or 6-monomethyl branched acids (53.69%), amounting to 61.91% of the total acid fraction. In addition, a large number of dimethyl-substituted fatty acids (31.4%) also have been found. Typical mass spectra, which are easily recognizable and highly specific for fatty acids substituted at various positions, are presented and classified according to the structural feature of the chain. Chemical Modification in Mass Spectrometry 6. For preceding paper in this series, see Ref. 17.     

On-line hydrogenation in GC-MS analysis of cyclic fatty acid monomers isolated from heated linseed oil

A simple on-line hydrogenation method in GC-MS analysis of unsaturated fatty acid esters is described. Using hydrogen as carrier gas, hydrogenation takes place in a capillary reactor connected to the outlet of the analytical column in the oven of the gas chromatograph. The reactor is a fused silica tube (60 cm×0.32 mm i.d.) coated with palladium acetylacetonate. Selective hydrogenation of olefinic bonds is achieved after a normal chromatographic run. Structural information (carbon-skeleton, double bond equivalents) can thus be deduced, and structural correlations between the saturated and unsaturated components can be obtained. Structures of cyclic fatty acid esters isolated from heated linseed oil were elucidated using this simple method which was found very useful for structural investigations on unsaturated compounds by GC-MS. Presented in part at the AOCS meeting in May 1988, in Phoenix, AZ. INRA—Station de Recherches sur la Qualite des Aliments de l'Homme     

Comparison of pyrrolidides with other amides for mass spectral determination of structure of unsaturated fatty acids

Amides of unsaturated fatty acids give mass spectra which indicate the locations of the double bonds. A survey of several amides of oleic acid was made to evaluate which amide might be most suitable for routine use in elucidation of structure. Methods of preparation of several amides of oleic acid are presented with mass spectral and gas chromatographic data. Tertiary amides have most easily interpretable mass psectra which indicate the position of the double bond in the fatty acid chain. The pyrrolidide is advantageous for fatty acid gas liquid chromatography and mass spectrometry analyses because it can be prepared easily in high yield on a microscale, it is volatile enough for gas liquid chromatographic separations, and it has a simple and easily interpretable mass spectrum which indicates the structure.     

Cis-5-polyenoic acids inLarix leptolepis seed oil

cis-5,cis-9-Octadecadienoic acid (2.7%) andcis-5,cis-9,cis-12-octadecatrienoic acid (24.9%) are present in the seed oil ofLarix leptolepis. The double bond positions were identified by ozonolysis and by gas chromatography-mass spectrometry of methoxy derivatives. Small quantities of branched chain acids of various chain lengths were indicated by gas chromatography. The presence of C₁₇ and C₁₉ branched methyl esters was confirmed by gas chromatography-mass spectrometry.