Formation of N- and O-methyl derivatives of lipids containing amino,amide or hydroxy groups by the pyrolytic reaction with trimethylsulfonium hydroxide

Base-catalyzed transesterification of acyl lipids with methanol in the presence of trimethylsulfonium hydroxide (TMSH) is an easy and convenient method for the preparation of fatty acid methyl esters for GC analyses. However, lipids containing functional groups such as amino, amide and hydroxy groups are converted in varying degrees to the corresponding N- and O-methyl derivatives by the pyrolytic reaction of TMSH occurring in the injector of the gas chromatograph. For example, lipids containing amino or amide groups are converted into the corresponding N-methyl and N,N-dimethyl derivatives, fatty acid ethanolamides to the corresponding N-methyl, O-methyl and N,O-dimethyl derivatives, whereas alkyl methyl ethers are formed from long-chain alcohols. Furthermore, 2-O- and 3-O-monomethyl ethers as well as 2,3-di-O-methyl ethers are formed from 1-O-alkylglycerols, methoxy fatty acid methyl esters from hydroxy fatty acids as well as steryl 3β-O-methyl ethers from cholesterol and other sterols. Since some of the mentioned artefacts may interfere with fatty acid methyl esters in GC separations the TMSH derivatization method is recommended only with caution for lipids containing amino, amide and hydroxy groups. The methylation reactions, which finally lead to the corresponding N-methyl, O-methyl, N,N-dimethyl or N,O-dimethyl derivatives of fatty acids and lipids may, however, be of some diagnostic value for the structural analysis of such lipids by GC/MS.     

Reaction of lipids containing hydroxy groups with trimethylsulfonium hydroxide: Formation ofO-methyl derivatives

Base-catalyzed transesterification of acyl lipids with trimethylsulfonium hydroxide (TMSH) is an easy and convenient method for the preparation of fatty acid methyl esters (FAME) for gas chromatography (GC) analyses. We have found, however, that lipids containing hydroxy groups are partially converted to the correspondingO-methyl ether derivatives which may interfere with FAME in GC separations. For example, long-chain alcohols are found to be converted to alkyl methyl ethers,rac-1-O-alkylglycerols to the corresponding 2-O-and 3-O-monomethyl ethers, as well as 2,3-di-O-methyl ethers, hydroxy fatty acids to methoxy FAME, and cholesterol to cholesteryl 3β-methyl ether. From our results, it is obvious that TMSH derivatization method is not recommended without limitation for lipids containing hydroxy groups; it may be, however, of some diagnostic value for the analysis of such lipids by GC/mass spectrometry.     

Saturated keto esters from lesquerolic,dimorphecolic, and densipolic acids

Summary The naturally occurring, unsaturated, hydroxy fatty esters, methyl lesquerolate (methyl 14-hydroxy-cis-11-eicosenoate), methyl dimorphecolate (methyl 9-hydroxy-trans, trans-10,12-octadecadienoate), and methyl densipolate (methyl 12-hydroxy-cis,cis-9,15-octadecadienoate) have been converted to the corresponding saturated keto esters by tow routes. The unsaturated esters were subjected to a hydrogenation-dehydrogenation reaction in the presence of Raney nickel or their saturated derivatives were dehydrogenated by copper chromite catalysis. Yields of the keto esters are 65–82% in the nickel-catalyzed reactions, and 71–94% by copper chromite-catalyzed dehydrogenation. In the hydrogenation-dehydrogenation system the order of reactivity is: methyl lesquerolate>methyl dimorphecolate>methyl densipolate. Relationships between structure and reactivity of these compounds, methyl 12-hydroxystearate, and methyl ricinoleate are discussed. W. Utiliz. Res. Dev. Div., ARS, USDA.     

Mild Method for Preparation of 4,4-Dimethyloxazoline Derivatives of Polyunsaturated Fatty Acids for GC–MS

A mild and convenient method has been developed for preparing 4,4-dimethyloxazoline (DMOX) derivatives of fatty acids for GC–MS analysis. First, fatty acid methyl esters are converted to corresponding amides by incubation overnight at room temperature with 2-amino-2-methyl-1-propanol and a catalytic amount of sodium methoxide. The resulting 2-(methylpropanol) amides were isolated by partition between hexane–diethyl ether and water, and then converted to 4,4-dimethyloxazoline derivatives by treatment with trifluoroacetic anhydride under mild conditions (50 °C for 45 min). Structures of 2-methylpropanol amide and a DMOX derivative of oleic acid were confirmed by GC–MS. This method was applied to different FAME prepared from animal, plant or microbial lipids. The suggested method is most suitable for structure analysis of polyunsaturated fatty acids (PUFA) and for acids with double bonds in close to terminal positions. Application of the method is illustrated with spectra of the DMOX derivatives of 16:1(n-13), 24:5(n-6) and 24:6(n-3) acids.     

Nonvolatile α-branched chain fatty acid derivatives: III. Addition of acid chlorides,anhydrides and amides to terminal olefins

At-butyl peroxide initiated free radical reaction was employed for the preparation of α-branched fatty acid chlorides, which were then converted in situ to methyl esters. Similarly prepared were an α-branched fatty acid amide and an α-branched acid anhydride. The latter was converted to the methyl ester. The use of the acid chloride and acid anhydride permitted reduction in the molar ratio of reactants to half or less than that used in the addition of esters to terminal olefins without affecting the yield. The resulting increase of α-branched product concentration in the reaction mixture also made isolation of the product easier. The direct addition of a variety of stearic acid derivatives to 1-decene under the same conditions (20:4:1.2 molar ratio of reactants at 160 C) gave the following olefin based yield order: stearoyl chloride > stearic anhydride > stearamide and methyl stearate > stearic acid. Presented at the AOCS Meeting, Chicago, October 1967. E. Utiliz. Res. Dev. Div., ARS, USDA.     

Analysis of oleate,linoleate and linolenate hydroperoxides in oxidized ester mixtures

The hydroperoxides in oxidized mixtures of methyl oleate, linoleate and linolenate were analyzed by reducing the hydroperoxides to the corresponding hydroxyesters and separating the hydroxyesters from the unoxidized esters by thin layer chromatography (TLC). The hydroxyesters from linolenate were separated from the other hydroxyesters by TLC on silver ion plates. The hydroxyesters were converted to TMS-hydroxy derivatives. The TMS-hydroxyoleate and TMS-hydroxylinoleate were separated by gas chromatography (GC), and all the TMS-derivatives were quantified by GC. The relative rates of oxidation of methyl oleate, linoleate and linolenate in mixtures were ca. 1∶10.3∶21.6. The hydroperoxides formed in the oxidation of soybean and olive oils were similar before and after randomization and similar to corresponding methyl ester mixtures. Journal Paper No. J-9657 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Project 2143.     

Fatty Acid Methyl Esters with Two Vicinal Alkylthio Side Chains and Their NMR Characterization

The addition reaction of dimethyl disulfide (DMDS) to double bonds in alkenes and monounsaturated fatty acid esters in the presence of iodine or other catalysts to give bis(methylthio) derivatives has largely served analytical purposes in mass spectrometry with scattered reports on the addition of other disulfides to alkenes also existing. In this work, this iodine-catalyzed reaction was expanded to include the addition of other dialkyl disulfides (RSSR; R=ethyl, propyl, butyl, iso-propyl) besides DMDS to the double bonds in monounsaturated fatty acid methyl esters with 16, 18, 20, and 22 carbon atoms in the fatty acid chain to give the corresponding methyl 1,2-bis(alkylthio)alkanoates. The products are obtained in high to moderate yield after a facile purification procedure and are analytically characterized not only by mass spectrometry but also ¹H and ¹³C NMR. The threo and erythro diastereomers obtained from (Z) and (E) fatty acid methyl esters, respectively, can be easily distinguished by the NMR shifts of the protons and carbons in and close to the 1,2-bis(alkylthio) moiety. Various other effects in the NMR spectra are discussed. The 1,2-bis(alkylthio) derivatives of a symmetrical alkene, 7(E)-tetradecene, serve to confirm the NMR assignments besides NMR techniques such as 2D correlations and DEPT. The compounds may show properties of interest for various applications.     

Elaidic acid in rat liver identified by gas chromatography and nuclear magnetic resonance spectroscopy

Fatty acid composition has been commonly determined by gas chromatography (GC). In the most commonly used methods, the lipids must be converted into methyl esters or other derivatives of the fatty acids before being analyzed by GC. Nuclear magnetic resonance (NMR) spectroscopy has become one of the most promising methods to determine organic structures, providing useful data for analyzing the fatty acid composition of edible vegetable oils. The major advantage of NMR is that identification of each fatty acid is carried out by evaluation of particular signals. We report in this work the fatty acid profile of rat liver after consumption of diets containing different concentrations of partially hydrogenated vegetable fat. The fatty acid composition was identified by GC, and trans fatty acids were also identified by ¹³C NMR spectroscopy. The results obtained by the ¹³C NMR method were close enough to those obtained by conventional GC.     

Soap-based detergent formulations: XXII. Sulfobetaine derivatives ofN-alkylglutaramides and adipamides

The acid chlorides of methyl hydrogen glutarate and methyl hydrogen adipate were allowed to react with various primary fatty amines. The resulting amido esters were converted into the corresponding amino diamides by sodium catalyzed reaction with N,N-dimethylaminoalkylamines. The desired sulfobetaines were obtained by reaction of the resulting N-alkyl-N′-(1,1-dimethylaminoalkyl) glutaramides and adipamides with 1,3-propanesultone. The compouds possess the same excellent lime soap dispersing ability, water solubility, calcium ion stability, and detergency properties as the analogous succinamide derivatives previous reported. This indicates that the spacing between the two amido groups or that between the quaternary nitrogen atom and the nearest amido group does not affect surface active properties.     

Improved Methods for the Fatty Acid Analysis of Blood Lipid Classes

Two improved methods have been developed for preparation of fatty acid methyl esters (FAME) from major O-ester lipid classes in blood, i.e., cholesterol ester, triacylglycerol, and glycerophospholipids. The methods involve simple operations, and use neither harmful solvents such as chloroform or benzene nor highly reactive volatile reagents such as acetyl chloride. The FAME synthesis reaction proceeds under mild temperature conditions. The methods include (1) extraction of lipids from 0.2 ml of blood with 0.2 ml of tert-butyl methyl ether and 0.1 ml of methanol, (2) separation of the total lipids into lipid classes using a solid-phase extraction column or thin-layer chromatography, and (3) methanolysis of each lipid class at room temperature or at 45 °C. In all the operations, solvent concentration is performed only once prior to gas–liquid chromatography (GC). No noticeable differences in composition determined by GC have been found between FAME prepared by the present methods and those prepared by a conventional method involving lipid extraction with chloroform/methanol. The mild reaction and simplified procedures of the present methods enabled safe and reproducible analysis of the fatty acid compositions of the major ester-lipid classes in blood.     

X-ray diffraction powder data of some thiol esters of long chain fatty acids

Summary X-ray diffraction powder data were obtained for 14 thiol esters of long chain fatty acids including benzyl, β-naphthyl,n-amyl, andn-hexyl thiol myristates; thiol palmitates; thiol stearates; and in some cases thiol laurates. All the individual compounds can be readily distinguished and identified by the diffraction data. Long spacings increase regularly with increase in hydrocarbon chain length. The benzyl and β-naphthyl thiol esters crystallize in tilted bimolecular layers while then-amyl andn-hexyl thiol derivatives crystallize in tilted monomolecular layers. One of the laboratories of the Eastern Utilization Research Branch, Agricultural Research Service, U. S. Department of Agriculture.     

Reduction of fatty ester Δ2-isoxazoline heterocycles. Preparation of fatty esters containing the β-hydroxy ketone moietyheterocycles. Preparation of fatty esters containing the β-hydroxy ketone moiety

Fatty ester compounds containing the β-hydroxy ketone moiety were prepared in good yields from their corresponding fatty Δ²-isoxazoline heterocyclic precursors by a reductive hydrogenolysis-hydrolysis procedure using Raney nickel as catalyst. By this methodology, C-17, C-18, and C-19 straight-chain fatty methyl esters containing the 10-hydroxy 12-keto moieties were prepared in 73, 83, and 92%, respectively, from their corresponding isoxazoline fatty ester compounds. Two other 10-hydroxy 12-keto C-12 and C-14 fatty ester compounds were prepared in 84 and 92% yield, respectively. The C-12 β-hydroxy ketone contains a phenyl ring at C-12, whereas the C-14 β-hydroxy ketone compound has two methyl substituents at C-13. GC-MS using electron impact ionization was used to determine the hydroxyl and ketone positions after conversion of the hydroxyl group into its corresponding trimethylsilyl ether. The precursor fatty ester Δ²-isoxazolines used in this study are readily available in one step from a 1,3-dipolar cycloaddition reaction between nitrile oxides and methyl 10-undecenoate. This overall two-step sequence, 1,3-dipolar cycloaddition followed by reductive ring opening, represent a convenient method to construct fatty ester compounds in good yields containing the β-hydroxy ketone functionality, an outcome not easily accessible by other methods.     

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.     

Fluorination of Hydroxyesters with N,N-Diethyl-1,1,2,3,3,3-Hexafluoro Propaneamine

The reactions of N,N-diethyl-1,1,2,3,3,3-hexafluoro propaneamine (PPDA) with Β-hydroxyl esters gave their corresponding fluorides. For example, methyl (S)-(+)-3-fluorobutyrate was obtained from the reaction of methyl (R)-(-)-3-hydroxybutyrate with PPDA. The reaction ofa-hydroxy esters with PPDA gave a mixture of their corresponding fluorides and 2,3,3,3-tetrafluoropropionate esters. However, γ- and δ-hydroxyesters did not give their fluorinated compounds. Hydrolysis of those racemic monofluoroesters with Lipase MY gave optical active monofluorocarboxylic acids and esters. To whom correspondence should be addressed.     

Autoxidation of fatty acid esters in the presence of a heavy metal catalyst “Salcomine” (Co[II]-bissalicylalethylenedi-imine): II. Formation of a reaction product from a peroxy radical and salcomine

The formation of ionic Co(II)-bis-salicylalaethylenedi-imine complexes of peroxy radicals of methyl esters of fatty acids was followed by means of UV spectra. Typical compounds were isolated and purified, and analyzed for Co content. IR spectra were determined. The mechanism of the autoxidation of fatty acid methyl esters in the presence of “Salcomine” (Co[II]-bissalicylalethylene-di-imine) has been outlined, and its effect is explained as an oxidative addition reaction, resulting from coordinative unsaturation.     

Simple, high-efficiency synthesis of fatty acid methyl esters from soapstock

We report a simple method that efficiently esterifies the fatty acids in soapstock, an inexpensive, lipid-rich by-product of edible oil production. The process involves (i) alkaline hydrolysis of all lipid-linked fatty acid ester bonds and (ii) acid-catalyzed esterification of the resulting fatty acid sodium salts. Step (i) completely saponified all glycerides and phosphoglycerides in the soapstock. Following water removal, the resulting free fatty acid sodium salts were rapidly and quantitatively converted to fatty acid methyl esters (FAME) by incubation with methanol and sulfuric acid at 35°C and ambient pressure. Minimum molar reactant ratios for full esterification were fatty acids/methanol/sulfuric acid of 1∶30∶5. The esterification reaction was substantially complete within 10 min and was not inhibited by residual water contents up to ca. 10% in the saponified soapstock. The product FAME contained >99% fatty acid esters, 0% triglycerides, <0.05% diglycerides, <0.1% monoglycerides, and <0.8% free fatty acids. Free fatty acid levels were further reduced by washing with dilute sodium hydroxide. Free and total glycerol were <0.01 and <0.015%, respectively. The water content was <0.04%. These values meet the current specifications for biodiesel, a renewable substitute for petroleum-derived diesel fuel. The identities and proportions of fatty acid esters in the FAME reflected the fatty acid content of soybean lipids. Solids formed during the reaction contained 69.1% ash and 0.8% protein. Their sodium content indicated that sodium sulfate was the prime inorganic component. Carbohydrate was the predominant organic constituent of the solid.     

Chemoenzymatic epoxidation of unsaturated fatty acid esters and plant oils

In the presence of an immobilized lipase fromCandida antacrtica (Novozym 435^R) fatty acids are converted to peroxy acids by the reaction with hydrogen peroxide. In a similar reaction, fatty acid esters are perhydrolyzed to peroxy acids. Unsaturated fatty acid esters subsequently epoxidize themselves, and in this way epoxidized plant oils can be prepared with good yields (rapeseed oil 91%, sunflower oil 88%, linseed oil 80%). The hydrolysis of the plant oil to mono- and diglycerides can be suppressed by the addition of a small amount of free fatty acids. Rapeseed oil methyl ester can also be epoxidized; the conversion of C=C-bonds is 95%, and the composition of the epoxy fatty acid methyl esters corresponds to the composition of the unsaturated methyl esters in the substrate. Based partly on a lecture at the 86th AOCS Annual Meeting & Expo, San Antonio, Texas, May 7–11, 1995.     

Reactions of conjugated fatty acids. V. Preparation and properties of diels-alder adducts and their esters fromtrans,trans-conjugated fatty acids derived from soybean oil

Summary Soybean fatty acids were conjugated with alkali, and the contained, conjugated dienoic acids were isomerized with iodine to thetrans,trans configuration. Adducts were prepared from thesetrans,trans-conjugated acids by condensation with maleic anhydride and acrylic acid. The adducts were isolated, purified, and converted to esters by using a variety of alcohols, including methyl, ethyl,n-propyl,n-butyl, and allyl alcohols. Esters made from saturated alcohols were converted into the corresponding epoxy derivatives. All of the esters (except allyl) and all of the epoxy esters were compatible with an equal weight of polyvinyl chloride and appeared to be primary plasticizers for this plastic. The epoxy esters were effective in inhibiting heat deterioration of polyvinyl chloride. Presented at fall meeting. American Oil Chemists' Society, September 23–26, 1956, Chicago, Ill.     

Determination of Polyunsaturated Fatty Esters (PUFA) in Biodiesel by GC/GC–MS and <Superscript>1</Superscript>H-NMR Techniques

In the present work, methods based on Gas Chromatography (GC), Gas Chromatography–Mass Spectrometry (GC–MS) and ¹H-NMR techniques was developed for the characterization and estimation of Polyunsaturated Fatty Esters (PUFA) in biodiesel. The GC method enables the separation, identification and estimation of these polyunsaturated fatty acid methyl esters to be carried out using a highly polar capillary column (100% cyanopropyl silicon). GC–MS was utilized for unambiguous identification and estimation of esters and isomers present in biodiesel. The estimation of PUFA content is important because PUFA content is a part of the EN 14214 specifications of biodiesel. There is no existing standard method for the estimation of total PUFA content in biodiesel. The developed GC method also quantifies EPA and DHA (C20:5 and C22:6) fatty acid methyl esters which can be used as markers for the estimation of fish oil biodiesel. The presence of EPA and DHA indicates the contamination of fish oil in biodiesel. Further, the ¹H-NMR technique has also been employed to identify and estimate PUFA containing ≥3 double bonds. The PUFA content was estimated from the integral intensities of the chemical shift region corresponding to all type of double bonds in the ¹H-NMR spectra. Based on the developed method, GC fingerprinting of various biodiesel samples was carried out to estimate a PUFA content as low as 1,000 ppm.     

Lipase-catalyzed reactions involving thia fatty acids and ester derivatives

Enzymatic hydrolysis of synthetic methyl 5-, 9-, and 12-thiastearates in aqueous media withCandida cyclindracea or porcine pancreatic lipases gave the corresponding fatty acids in 70–100% yield. Hydrolysis of the 3- and 4-positional isomers gave only 15–25% of the free thia fatty acids, suggesting discrimination against these isomers by lipases. No lipolysis was achieved with methyl 2-thialaurate under a range of reaction conditions. Esterification of the 3-, 4-, 5-, 9-, and 12-thiastearic acids withn-butanol inn-hexane using Lipozyme (immobilizedRhizomucor miehei) as the biocatalyst gave the corresponding butyl esters in 80–95% yield. Interesterification (acyl exchange) of triolein with methyl 9-thiastearate in the presence of Lipozyme showed the incorporation of 9-thiastearoyl chain at only one of the α-positions of triolein. In the case of methyl 2-thialaurate, no lipase-catalyzed acyl exchange reaction was possible. This study showed that the position of the sulfur atom in thia fatty esters affects the lipase-catalyzed hydrolysis and interesterification reactions.