Transesterification of phospholipids or triglycerides to fatty acid benzyl esters with simultaneous methylation of free fatty acids for gas-liquid chromatographic analysis

A novel method is presented for transesterification of fatty acid esters in phospholipids and triglycerides to benzyl esters while simultaneously recovering free fatty acids as methyl esters. Transesterification is catalyzed by 0.2 M (m-trifluoromethyl phenyl)trimethyl ammonium hydroxide in methylene chloride, 10% (v/v) benzyl alcohol, and 1% (w/v) potassium tert-butoxide, and is complete in 30 min at room temperature. Methyl esters of all common fatty acids separate from the benzyl esters formed from phospholipids. This method has broad utility and is applicable to the formation of esters optimized for detection by absorbance or fluorescence (high performance liquid chromatography), electron capture (gas-liquid chromatography), or negative ion chemical ionization (gas-liquid chromatography-mass spectrometry).     

Hydrocarbon and multibranched ester waxes from the uropygial gland secretion of grebes (Podicipediformes).

The uropygial gland secretion of some grebes (Podicipediformes) had been shown to contain saturated and unsaturated aliphatic hydrocarbons and monoester waxes. While ester waxes are common constituents of preen gland secretions, nonisoprenoid hydrocarbons have not been detected hitherto. The wax constituents are very complex, belonging to several multibranched homologous series, including unusual acids with ethyl branches. The waxes were identified by gas-liquid chromatography-mass spectrometry and equivalent chain length comparisons. A method for the prediction of equivalent chain length values of unknown methyl esters is offered. The results are discussed from a chemotaxonomic viewpoint.     

Metabolism of cholestane-3 beta,5 alpha,6 beta-triol. II. Identification of two major neutral metabolites in the rat

Rats were given a single oral dose of cholestane-3beta,5alpha,6beta-triol-4-(14)C, and their feces were collected. The two major neutral metabolites were separated and isolated by use of solvent fractionation and chromatographic methods. The metabolites were identified as cholestane-3beta,5alpha-diol-6-one and a mixture of long-chain fatty acid esters of cholestane-3beta,5alpha,-6beta-triol. Cholestane-3beta,5alpha-diol-6-one was identified using thin-layer and gas-liquid chromatography, infrared spectroscopy, and the spectrum produced by reaction with 65% sulfuric acid. The mixed esters of cholestane-3beta,5alpha,6beta-triol were subjected to basic hydrolysis, and the steroid moiety was identified using the same techniques employed for cholestane-3beta,5alpha-diol-6-one. The fatty acids were analyzed by gas-liquid chromatography of their methyl esters.     

Bis(trimethylsilyl)acetamide in the silylation of lipolysis products for gas-liquid chromatography

When bis(trimethylsilyl)acetamide is used to silylate lipolysates for gas-liquid chromatography, the pyridine solution containing the sample and silylating agent can be injected directly onto the column. Conversion of free fatty acids to methyl esters before silylation is unnecessary, since silyl esters elute as sharp gas-liquid chromatographic peaks.     

Fatty Acids of Mycobacterium kansasii

The cellular fatty acids of 35 strains of Mycobacterium kansasii isolated from clinical material were analyzed to establish properties by which we could identify and characterize these acid-fast microorganisms. The fatty acids were extracted from cells grown in liquid synthetic media, and they were analyzed as methyl esters by gas-liquid chromatography. The fatty acid profiles of all strains were similar. They differed from fatty acid profiles of other mycobacteria by their content of a saturated fatty acid with a methyl group at C2.     

Crystallization and positional specificity of hydroperoxidation of Fusarium lipoxygenase.

A lipoxygenase obtained from the fungus Fusarium oxysporum was purified and crystallized. Using the purified enzyme, the positional specificity of linoleate peroxidation was studied. Linoleate hydroperoxides were converted into the corresponding trimethylsilyl derivative by reduction, catalytic hydrogenation and treatment with hexamethyldisilazane/trimethylchlorosilane/pyridine and then analyzed by combined gas-liquid chromatography-mass spectrometry. Fusarium lipoxygenase was found to produce 9- or 13-hydroperoxy-octadecadienoates from linoleate. The ratio of 9- to 13-hydroperoxides produced by the enzyme was also determined by high performance liquid chromatography of their methyl esters. When the enzymic reaction proceeded at pH 9.0 and 12.0, the ratio of 9- to 13-hydroperoxide isomers was 70 : 30 and 56 : 44, respectively. With the use of the heavy isotope of oxygen (18O2), atoms of oxygen introduced into hydroperoxides were found to be derived from the gaseous phase and not from the aqueous phase.     

Steric analysis of 3-, ω4-, ω3- and ω2-hydroxy acids and various alkanols by gas-liquid chromatography

D-Phenylpropionate (PP) derivatives of racemic hydroxy acid methyl esters and alkanols were prepared and the gas-liquid chromatographic separation of diastereoisomers was investigated using QF-1 as stationary phase. Good separations were obtained for the diasteroisomeric PP-derivatives of methyl 3-, 15-, 16-, and 17-hydroxyoctadecanoates. Less separation was observed for methyl 2- and 14-hydroxyoctadecanoates as PP-derivatives and there was no visible separation for PP-derivatives of 4-, 7-, or 13-hydroxyoctadecanoic acid methyl esters. The use of optically active 15-, 16- and 17-hydroxyoctadecanoic acids showed, that in these cases, the diastereoisomers containing L-hydroxy acids had shorter retention times than the ones containing D-hydroxy acids. On the other hand, the D-phenylpropionate derivative of methyl 3D-hydroxydecanoate had shorter retention time than the derivative of its L-enantiomer. PP-derivatives of 3-hexanol, 3-heptanol, 3-octanol, 2-octanol and 2-eicosanol could be resolved by gas-liquid chromatography.     

Occurrence of fatty acid chlorohydrins in jellyfish lipids.

Fatty acid chlorohydrins are characterized as lipid components of an edible jellyfish. The four isomers 9-chloro-10-hydroxypalmitic acid, 10-chloro-9-hydroxypalmitic acid, 9-chloro-10-hydroxystearic acid, and 10-chloro-9-hydroxystearic acid were identified by gas chromatography-mass spectrometry comparison of the methyl esters and their trimethylsilyl derivatives with known synthetic samples. Two additional isomers, 11-chloro-12-hydroxystearic acid and 12-chloro-11-hydroxystearic acid, were also found in the lipid by the identification of the expected mass spectral fragments of the trimethylsilyl (Me3Si) derivative of their methyl esters. These six isomeric compounds represented approximately 1.4% of the total extractable jellyfish lipid and were released from the lipid as methyl esters by boron trifluoride-methanol treatment. These isomers account for only about 30% of the organic chlorine in the lipid. Evidence is given that the remaining organic chlorine is also present as fatty acid chlorohydrins containing more than one hydroxyl group.     

Partial methylation of methyl glycosides: a source of samples for analysis of methylation of carbohydrate-containing compounds.

Methyl ether mixtures were obtained by partial methylation of 15 methyl glycopyranosides using Purdie's procedure. Identification of ethers as acetates by gas-liquid chromatography-mass spectroscopy (glc-ms) techniques allowed us to assume them to be sample sources for methylation analysis.T-values were tabulated using a scale ranging from permethyl ether- (0) to peracetate methyl glycosides (100) for subsequent identification with the aid of glc in NPGS and QF-1 liquid phases.A scheme has been devised for isolating individual methyl ethers by preparative glc.     

Significance and taxonomic value of iso and anteiso monoenoic fatty acids and branded beta-hydroxy acids in Desulfovibrio desulfuricans.

The fatty acids obtained from extractable lipids of the anaerobic sulfate bacterium Desulfovibrio desulfuricans were identified. Saturated and monoenoic iso (C15-C19) and anteiso (C15, C17) fatty acids and saturated normal (C14-C18) and monoenoic normal (C16, C18) fatty acids were shown to be shown to be present by capillary gas chromatography-mass spectrometry. Iso and anteiso beta-hydroxy fatty acids were analyzed as trimethylsilyl ethers in the same way. The position of methyl branches in the monoenoic fatty acids was determined from characteristic fragment ions in the mass spectra of their methyl esters. Disilyloxy methyl esters, prepared by derivatization of the mono unsaturated methyl esters and analyzed by capillary gas chromatography-mass spectrometry, provided the position of double bonds. The monoenoic fatty acids identified in this way were normal (delta7-C16:1, delta9-C16:1, delta9-C18:1, delta11-C18:1), iso (delta7-C15:1, delta9-C16:1, delta9-C17:1, delta11-C18:1, delta11-C19:1), and anteiso (delta7-C15:1, delta9-C17:1). Iso delta9-C17:1 fatty acid is present as the major component. The occurrence of these monoenoic fatty acids in this bacterium is of taxonomical importance.     

The Collection and Elution of Radioactive Fatty Acid Methyl Esters During Quantitative Gas-Liquid Chromatography

Abstract

An improved procedure is described for the collection and elution of low levels of radioactive fatty acid methyl esters separated by gas-liquid chromatography. A gas chromatographic effluent splitter was employed to partition fatty acid methyl ester samples in the column effluent, Condensation of a portion of the eluted fatty acids was accomplished in borosilicate glass tubing collectors maintained at ?70°C, Quantitation of nanomolar levels of fatty acid methyl esters was accomplished by calibrating the gas chromatographic flame ionization detectors with the splitters opened or closed. The elution of condensed radioactive fatty acid methyl esters from the glass collectors was complete when benzene followed by a toluene based scintillation fluid were employed as solvents. The method described may be applicable to the analysis of cis-trans isomers of unsaturated fatty acids.     

Identification of 3 beta, 7 beta-dihydroxy-5 beta-cholan-24-oic acid in serum from patients treated with ursodeoxycholic acid

An unknown bile acid was found by gas-liquid chromatography in the serum of patients who were administered ursodeoxycholic acid for the treatment of cholesterol gallstones. Identification of the chemical structure of the unknown bile acid was performed by the use of gas-liquid chromatography-mass spectrometry. Mass spectrum analysis of the methyl ester trimethylsilyl ether of the bile acid showed explicitly that this is dihydroxy-5 beta-cholanoic acid, since peaks at m/e 460 and 370 characteristic of methyl ester trimethylsilyl ether of dihydroxy bile acid were clearly exhibited. Sites of the two hydroxyl groups on the steroid nucleus were determined to be at the 3- and 7-positions by conversion of the bile acid to the corresponding dioxo-cholanoic acid and by comparison of the gas-liquid chromatographic behavior with those of authentic dioxo bile acids. Four authentic 3,7-dihydroxy-5 beta-cholan-24-oic acids were chemically synthesized and retention times and mass spectra of their methyl ester trimethylsilyl ether derivatives compared precisely with that of the unknown bile acid. The results indicate that the unknown bile acid is 3 beta, 7 beta-dihydroxy-5 beta-cholan-24-oic acid. Preliminary experiments suggest that 3 beta, 7 beta-dihydroxy-5 beta-cholan-24-oic acid is absent as amino acid-conjugated forms in serum. It is also suggested that the bile acid is excreted into urine but not into bile.     

A reliable analysis of tissue free fatty acids by gas-liquid chromatography

A convenient and reliable gas-liquid chromatographic method for determining the free fatty acids in biological specimens is described. The free fatty acids were extracted with hexane in the presence of H3PO4 and then back-extracted from the hexane phase into a very small volume of trimethyl (alpha, alpha, alpha-trifluoro-m-tolyl)ammonium hydroxide solution. Direct injection of the resultant quaternary ammonium salts of the fatty acids into a gas-liquid chromatograph unit gave their methyl esters, with a high recovery. The presence of triglycerides, phospholipids, or cholesterol esters did not interfere with the determination of free fatty acids. This method was applied to determination of free fatty acids in the samples of serum or brain. The results were more precise and reliable than those reported with the conventional methods with TLC separation. This method should be a useful aid for providing precise information about the physiological or pathological roles of free fatty acids.     

Capillary gas-liquid chromatography of glycine-conjugated bile acids without prior hydrolysis

A method is described for the gas-liquid chromatographic (GLC) analysis of intact glycine conjugates of the major bile acids present in human plasma. It is, therefore, now possible to analyze glycine-conjugated and unconjugated bile acids together on a single GLC column without the necessity for a hydrolytic step. A large number of derivatives of bile acid glycine conjugates were examined, but only acetate- and silyl ether-derivatives of carboxylic acid methyl esters were found initially to be suitable. It was not possible to make acetates consistently, and trimethylsilyl ethers did not allow resolution of the glycine conjugates of cholic and chenodeoxycholic acids. Dimethylethylsilyl ether methyl ester derivatives were subsequently found to give the best results. Chromatographic conditions for successful analysis of these derivatives were examined and it was found to be necessary to use wall-coated capillary columns of thin film thickness (0.12 micron) and very high carrier gas flow rates (ca. 20 ml/min hydrogen). Using acetonitrile and Bond Elut extraction, fractionation on Sep-Pak SIL cartridges, and derivatization as dimethylethylsilyl ether methyl esters, the capillary gas-liquid chromatography of intact glycine-conjugated bile acids from human plasma was demonstrated for the first time.     

New method for the reductive ozonolysis of double bonds in monoenoic fatty acid methyl esters

Unsaturated methyl esters were cleaved to aldehydes and aldehydo-esters by ozonolysis followed by reduction with dimethyl sulfide after conversion to hydroperoxides. Cleavage products were identified by thin-layer chromatography and quantified by temperature programmed gas-liquid chromatography.     

A novel approach to structure proof of glyceryl ether-containing glycerophospholipids. Base-catalyzed methanolysis of platelet-activating factor (AGEPC) at 60 degrees C

A novel reaction was explored in which synthetic platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), upon treatment with 1 N NaOH in methanol at 60 degrees C for 20 min, sequentially released the acetyl group, then the choline moiety with concomitant formation of the monomethyl ester of 1-O-alkyl-glycero-phosphoric acid. A mechanism is proposed in which a transient cyclic phosphate intermediate is formed and then attacked by a CH3O moiety to yield a mixture of the sn-2 and sn-3 methyl esters. Proof of structure of the monomethyl ester derivative was achieved through the use of thin-layer chromatography, aluminum oxide chromatography, and examination of the trimethylsilyl derivative of the monomethyl ester by gas-liquid chromatography-mass spectrometry. Replacement of the acyl group on the 2 position with an ethyl or methyl residue completely prevented any attack by 1 N NaOH in methanol at 60 degrees C. Sphingomyelin was not attacked and only acetate removal was noted with 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine under similar conditions. The significance of these findings as they relate to the influence of substituents on the chemical and biological reactivity of AGEPC is discussed.     

Novel surface lipids of diapausing Manduca sexta pupae. Long chain oxoalcohol esters of acetoacetic, hydroxybutyric, and acetic acids

Ester components in the surface wax from diapausing tobacco hornworm pupae, Manduca sexta L., were separated by thin layer chromatography and gas-liquid chromatography, and characterized by infrared spectroscopy and gas-liquid chromatography-mass spectrometry. Three groups of esters were identified as natural derivatives of acetic acid, acetoacetic acid, and 3-hydroxybutyric acid. The major ester fraction was identified as a mixture of C26 (10%), C27 (5%), and C28 (85%) oxoalcohol esters of acetoacetic acid. The major homolog consisted of equal amounts of 11-oxooctacosanyl 3-oxobutanoate and 12-oxooctacosanyl 3-oxobutanoate. Lesser amounts of 11- and 12-oxooctacosanyl and n-octacosanyl esters of acetic and 3-hydroxybutyric acids were also identified. The chain length distributions of these C26, C27, and C28 oxoalcohol and n-primary alcohol ester moieties, as well as the isomeric ratios for the 11- and 12-oxoalcohol isomers, were similar to the oxoaldehydes and unesterified oxoalcohols previously identified by Buckner et al (Buckner, J. S., Nelson, D. R., Haak, H., and Pomonis, J. G. (1984) J. Biol. Chem. 259, 8452-8470) as lipid components of the surface wax of M. sexta pupae.     

Long chain oxoaldehydes and oxoalcohols from esters as major constituents of the surface lipids of Manduca sexta pupae in diapause

Lipid constitutents of diapausing pupae of the tobacco hornworm, Manduca sexta (L), were identified by thin layer and gas-liquid chromatography, IR spectroscopy, and gas-liquid chromatography-mass spectrometry. Surface wax was a mixture of long chain polar compounds: oxoalcohol esters, oxoaldehydes, primary alcohol esters, and oxoalcohols, as listed in descending order of abundance. The distribution of the alcohols and aldehydes was C28 (75-85%), C27 (5%), and C26 (10-15%). The C26 compounds were largely 11-oxo isomers, but the C28 compounds consisted of similar amounts of 11- and 12-oxo isomers. The identities of the oxoaldehydes were confirmed by selective and complete NaBH4 reductions to yield oxoalcohols and diols, respectively. Mass spectral interpretations were verified with mass spectra of the oxoaldehyde, oxoalcohol, and diol synthesized from 12-hydroxystearic acid. Reduction of the total lipids with NaBH4 and hydrolysis of the product with ethanolic KOH gave oxoalcohols (85%), primary alcohols (8%), and oxoacids (5%); 30-40% of the oxoalcohols were derived from NaBH4-reduced oxoaldehydes, 5-10% were from free oxoalcohols, and 50% were from esters. Primary alcohols only existed as esters. Large quantities of fatty oxoalcohols relative to fatty oxoacids in the saponified mixture suggested the presence of esters other than those composed of long chain acids and alcohols. Oxoacids appeared to be mainly oxidation products of the oxoaldehydes.     

Two-column gas-liquid chromatography of fatty acid methyl esters

Fatty acid methyl esters were separated into fractions according to chain length on a nonpolar gas-liquid chromatographic column. These fractions were collected and rechromatographed on a polar column. Temperature programming was used in both cases. Data are given for the accuracy of the double procedure applied to a synthetic mixture.     

A highly sensitive method for quantitative determination of abscisic Acid

An abscisic acid derivative was formed by reaction with pentafluorobenzyl bromide which allowed highly sensitive detection by gas-liquid chromatography with electron capture detection. In comparison to the methyl ester derivative, the pentafluorobenzyl derivative of abscisic acid was four times more sensitive to electron capture detection and was stable at room temperature in the presence of ultraviolet light. Derivatization was rapid and the molecular weight of the new compound was confirmed by gas-liquid chromatography-mass spectrometry.