Silver Ion High-performance Liquid Research Articles

Enhanced Resolution of Conjugated Linoleic Acid Isomers by Tandem-Column Silver-Ion High Performance Liquid Chromatography

Enhanced Resolution of Conjugated Linoleic Acid Isomers by Tandem-Column Silver-Ion High Performance Liquid Chromatography

Enhanced Resolution of Conjugated Linoleic Acid Isomers by Tandem-Column Silver-Ion High Performance Liquid Chromatography

A commercial mixture of conjugated linoleic acid (CLA) isomers, reportedly consisting of six components, was recently resolved into 12 peaks attributed to CLA isomers using silver-ion high performance liquid chromatography (Ag+-HPLC). In this study, the coupling of two analytical silver-ion high performance liquid chromatography columns (tandem-column Ag+-HPLC) in series led to the enhanced resolution of CLA isomers. Many CLA isomers were baseline resolved and the pair 18 : 2 8,10 c/t and 18 : 2 7,9 c/t found in cheese products, was resolved for the first time. In this work, a similar commercial CLA mixture was separated into 16 peaks, while CLA isomers from cheese also gave rise to 16 peaks. As expected, the CLA isomers were separated into three geometric groups in the order trans,trans, cis/trans, and cis,cis. Semi-preparative Ag+-HPLC, followed by gas chromatography–mass spectroscopy of the dimethyloxazoline derivatives, was used to confirm the identity of the newly resolved positional CLA isomers. The double bond configuration of CLA isomers was established by gas chromatography–Fourier transform infrared spectroscopy. Two minor t,t CLA isomers found in cheese, presumably 18 : 2 t6t8 and 18 : 2 t13t15, were also separated. The CLA isomeric composition of 16 commercial cheese products was determined.

Combined silver-ion and reversed-phase high-performance liquid chromatography for the separation and identification of C 20 metabolites of conjugated linoleic acid isomers in rat liver lipids

Reversed-phase high-performance liquid chromatography (RP-HPLC) and silver-ion high-performance liquid chromatography (Ag-HPLC) were successively combined for the separation of the longer-chain metabolites of conjugated linoleic acids (CLAs). Commercial silver nitrate-impregnated columns were used with an eluting solvent composed of a mixture of hexane–acetonitrile. Fatty acid methyl esters (FAMEs) from liver lipids of rats fed CLA were analysed. This method allowed separation both of the non-conjugated FAME, as C16:1, C18:2, C18:3, C20:4 and C22:5, but also the conjugated fatty acids like CLA, 8,12,14-20:3, 5,8,12,14-20:4 and 5,8,11,13-20:4. The presence of 8,11,13-20:3 is reported for the first time. This method is of interest for the isolation and identification of the C 20 conjugated metabolites that cannot be resolved by gas chromatography. Furthermore, it allows the isolation of FAME for further characterisation by GC–mass spectrometry (MS).

Monocyclic dienoic fatty acids formed from γ-linolenic acid in heated evening primrose oil

The cyclic fatty acids isolated from evening primrose oil that had been heated to 275°C were fractionated by silver ion high-performance liquid chromatography and analysed by gas chromatography-mass spectrometry as the dimethyloxazoline derivatives, before and after deuteration. Sixteen cyclic dienoic fatty acids derived from γ-linolenic acid were characterised. There were eight cyclopentenyl fatty acids with rings from either C-7 to C-11 or C-8 to C-12 of the original chain. One double bond was lost on cyclization and the remaining two were in their original positions (C-9 and C-12, and C-6 and C-9, respectively). There were eight cyclohexenyl fatty acids with rings from C-7 to C-12 and double bonds at either C-5 and C-9 or C-9 and C-13. All possible combinations of configurational isomers of both the straight chains about the ring and of the double bond in the straight chain were formed. The structures were analogous to those previously reported from α-linolenic acid in the sense that the trienoic unit reacted in a similar way irrespective of its position along the C 18 chain. The relative proportions of acids with different structural features were also similar for both trienoic acids. Mechanisms involving allylic radicals could account for all products, and the limited number of acids formed compared to possible products is discussed.

Identification of conjugated linoleic acid isomers in cheese by gas chromatography, silver ion high performance liquid chromatography and mass spectral reconstructed ion profiles. Comparison of chromatographic elution sequences

Commercial cheese products were analyzed for their composition and content of conjugated linoleic acid (CLA) isomers. The total lipids were extracted from cheese using petroleum ether/diethyl ether and methylated using NaOCH3. The fatty acid methyl esters (FAME) were separated by gas chromatography (GC), using a 100-m polar capillary column, into nine minor peaks besides that of the major rumenic acid, 9c,11t-octadecadienoic acid (18:2), and were attributed to 19 CLA isomers. By using silver ion-high performance liquid chromatography (Ag+ -HPLC), CLA isomers were resolved into seven trans,trans (5-9%), three cis/trans (10-13%), and five cis,cis (<1%) peaks, totaling 15, in addition to that of the 9c,11t-18:2 (78-84%). The FAME of total cheese lipids were fractionated by semipreparative Ag+ -HPLC and converted to their 4,4-dimethyloxazoline derivatives after hydrolysis to free fatty acids. The geometrical configuration of the CLA isomers was confirmed by GC-direct deposition-Fourier transform infrared, and their double bond positions were established by GC-electron ionization mass spectrometry. Reconstructed mass spectral ion profiles of the m + 2 allylic ion and the m + 3 ion (where m is the position of the second double bond in the parent conjugated fatty acid) were used to identify the minor CLA isomers in cheese. Cheese contained 7t,9c-18:2 and the previously unreported 11t,13c-18:2 and 12c,14t-18:2, and their trans,trans and cis,cis geometric isomers. Minor amounts of 8,10-, and 10,12-18:2 were also found. The predicted elution orders of the different CLA isomers on long polar capillary GC and Ag+ -HPLC columns are also presented.

Identification of cocoa butter equivalents added to cocoa butter

Separation of the six sub-fractions of triacylglycerols (TAG) from cocoa butter, three cocoa butter equivalents (CBEs: calvetta, choclin and coberine) and their mixtures was carried out by silver ion high-performance liquid chromatography. The percentage fatty acid composition of all these sub-fractions was then measured by high-resolution gas chromatographic analysis of the methyl esters of the constitutive fatty acids. In particular, the percentage quantities of all acids in the fully saturated sub-fraction of pure fats and some of their mixtures were evaluated as a function of the percentage CBEs added to cocoa butter in order to devise multiple regression models. The aim was to obtain statistical tools for the identification and/or quantification of added CBEs. The results obtained show the effectiveness of TAG sub-fraction percentage fatty acid composition data for the purposes of research, as well as the potential use of stereospecific analysis of the same sub-fractions. In fact, the stereospecific analysis of TAG fractions from cocoa butter and CBEs already carried out has enabled observation of the different distributions of various fatty acids in the glycerol backbone of TAG from cocoa butter and the CBEs under study, and this will certainly result in the development of more powerful and discriminating statistical models.

Distributions of conjugated linoleic acid (CLA) isomers in tissue lipid classes of pigs fed a commercial CLA mixture determined by gas chromatography and silver ion-high-performance liquid chromatography.

Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerization of sunflower oil, at 2% of the basal diet, from 61.5 to 106 kg live weight, and were compared to pigs fed the same basal diet with 2% added sunflower oil. The total lipids from liver, heart, inner back fat, and omental fat of pigs fed the CLA diet were analyzed for the incorporation of CLA isomers into all the tissue lipid classes. A total of 10 lipid classes were isolated by three-directional thin-layer chromatography and analyzed by gas chromatography (GC) on long capillary columns and by silver-ion high-performance liquid chromatography (Ag+-HPLC); cholesterol was determined spectrophotometrically. Only trace amounts (<0.1%; by GC) of the 9,11-18:2 cis/trans and trans,trans isomers were observed in pigs fed the control diet. Ten and twelve CLA isomers in the diet and in pig tissue lipids were separated by GC and Ag+- HPLC, respectively. The relative concentration of all the CLA isomers in the different lipid classes ranged from 1 to 6% of the total fatty acids. The four major cis/trans isomers (18.9% 11 cis,13 trans-18:2; 26.3% 10 trans,12 cis-18:2; 20.4% 9 cis,11 trans-18:2; and 16.1% 8 trans, 10 cis-18:2) constituted 82% of the total CLA isomers in the dietary CLA mixture, and smaller amounts of the corresponding cis,cis (7.4%) and trans,trans (10.1%) isomers were present. The distribution of CLA isomers in inner back fat and in omental fat of the pigs was similar to that found in the diet. The liver triacylglycerols (TAG), free fatty acids (FFA), and cholesteryl esters showed a similar pattern to that found in the diet. The major liver phospholipids showed a marked increase of 9 cis,11 trans-18:2, ranging from 36 to 54%, compared to that present in the diet. However, liver diphosphatidylglycerol (DPG) showed a high incorporation of the 11 cis,13 trans-18:2 isomer (43%). All heart lipid classes, except TAG, showed a high content of 11 cis,13 trans-18:2, which was in marked contrast to results in the liver. The relative proportion of 11 cis,13 trans-18:2 ranged from 30% in the FFA to 77% in DPG. The second major isomer in all heart lipids was 9 cis,11 trans-18:2. In both liver and heart lipids the relative proportions of both 10 trans,12 cis-18:2 and 8 trans, 10 cis-18:2 were significantly lower compared to that found in the diet. The FFA in liver and heart showed the highest content of trans,trans isomers (31 to 36%) among all the lipid classes. The preferential accumulation of the 11 cis,13 trans-18:2 into cardiac lipids, and in particular the major phospholipid in the inner mitochondrial membrane, DPG, in both heart and liver, appears unique and may be of concern. The levels of 11 cis,13 trans-18:2 naturally found in foods have not been established.

Fractionation of cis- and trans-oleic, linoleic, and conjugated linoleic fatty acid methyl esters by silver ion high-performance liquid chromatography

Silver ion high-performance liquid chromatography (Ag-HPLC) using two commercially available columns connected in series and a solvent of 0.5% acetonitrile in hexane (1.0 ml/min), was used to fractionate a mixture of conjugated 18:2 isomers (as methyl esters) and resulted in an elution order (10 μg sample size) of: 9- trans,11- trans-18:2; 9- cis,11- trans-18:2. A more complex mixture of cis and trans 9-18:1, 9,11-18:2 and 9,12-18:2 methyl esters could also be fractionated within 25 min. The elution order (10 μg sample size) was found to be: 9- trans-18:1; 9- trans,11- trans-18:2; 9- trans,12- trans-18:2; 9- trans,11- cis-18:2; 9- cis,11- trans-18:2; 9- cis-18:1; 9- trans,12- cis-18:2; 9- cis,12- trans-18:2; 9- cis,12- cis-18:2. The Ag-HPLC elution orders differed significantly from those obtained via gas chromatography on a polar stationary phase (30 m SP2380 capillary column; Elution order: 9- trans-18:1; 9- cis-18:1; 9- trans,12- trans-18:2; 9- cis,12- trans-18:2; 9- trans,12- cis-18:2; 9- cis,12- cis-18:2; 9- cis,11- trans-18:2; 9- trans,11- cis-18:2; 9- trans,11- trans-18:2). The Ag-HPLC system was also used in the semi-preparative separation of a 10 mg sample of 9- trans,11- trans-18:2 and 9- cis,11- trans-18:2 fatty acid methyl esters.

Silver ion high-performance liquid chromatography of isomeric cis- and trans-octadecenoic acids : Effect of the ester moiety and mobile phase composition

The effects of the ester moiety and of the mobile phase composition on the resolution of cis and trans positional isomers of octadecenoates have been studied by silver ion HPLC with UV detection. The efficiency of the separation increases in the order phenethyl<phenacyl< p-methoxyphenacyl esters. Retention and resolution are substantially affected by small changes in the proportion of acetonitrile in the mobile phase. Dichloromethane influences retention but has a small effect on resolution. Clear resolution of cis and trans positional isomers of octadecenoic acid in partially hydrogenated sunflower oil has been achieved after conversion into p-methoxyphenacyl esters on a silver ion column by isocratic elution with a mobile phase of hexane–dichloromethane–acetonitrile (60:40:0.2, v/v) in only 22 min.

Sterol synthesis. A timely look at the capabilities of conventional and silver ion high performance liquid chromatography for the separation of C27 sterols related to cholesterol biosynthesis

Sterol intermediates in the biosynthesis of cholesterol have recently assumed a very prominent position in a number of important problems in medicine and biology. In studies of these matters, the separation and identification of the sterol intermediates present formidable challenges, a situation which does not appear to be generally appreciated. High performance liquid chromatography (HPLC) is a simple and rapid approach for the separation of the concerned compounds. Reversed phase HPLC is very commonly used for this purpose. In the present studies, we have evaluated the capabilities of reversed phase, normal phase, and silver ion HPLC for the separation of sterols. Using an extensive collection of authentic sterols, our studies indicate very limited capabilities of reversed phase and normal phase HPLC for the separation of C27 sterols differing in the number and location of olefinic double bonds. In contrast, silver ion HPLC provided remarkable separations of the same compounds, either as the free sterols or their acetate derivatives. These findings, coupled with the results of recent studies of the properties of the same compounds by gas chromatography and by nuclear magnetic resonance and mass spectroscopy, have important implications regarding current application of methodologies for the separation, identification, and quantitation of sterol intermediates in cholesterol biosynthesis as critical portions of investigations on a number of current and emerging problems in biology and medicine.

Normal-phase separation effects with lipids on a silver ion high-performance liquid chromatography column

The separation of saturated fatty acids (as the methyl esters and as triacylglycerols) of differing chain lengths by silver ion chromatography (ChromSpher Lipids; containing silver ions; Chrompack, Middelburg, Netherlands) was studied. The retention characteristics (isocratic solvent system of acetonitrile in hexane) of saturated fatty acid methyl esters and homogeneous, saturated triacylglycerols were found to be inversely related to the number of carbon atoms in the fatty acid(s). Thus methyl stearate eluted first and methyl acetate eluted last; for triacylglycerols, tripalmitoylglycerol eluted first while triacetin was strongly retained (11 min vs. 70 min). While mixtures of fatty acid methyl esters and of triacylglycerols could be separated, resolution was found to improve at higher concentrations of acetonitrile, a characteristic of normal-phase separations. In the absence of carbon–carbon double or triple bonds, the effects of the more polar silanol groups of the column packing tended to override the silver ion contributions (interaction of silver ions with the unpaired electrons of the carbonyl oxygens), resulting in separations similar to those observed in silica thin layer and normal-phase high-performance liquid chromatography.

Novel di-, tri-, and tetraenoic fatty acids with bis-methylene-interrupted double-bond systems from the sponge Haliclona cinerea.

Unusual fatty acids, with up to 34 carbon atoms and containing one or two bis-methylene-interrupted double-bond systems, have been identified in the sponge Haliclona cinerea from the Black Sea. These include the dienes-5,9-16:2, 7,11-18:2, 9,13-20:2, 13,17-24:2, 15,19-26:2, 17,21-28:2, 19,23-30:2 and 21,25-32:2; trienes-5,9,23-30:3, 5,9,24-31:3, 5,9,25-32:3, and 5,9,27-34:3; and the tetraenes-5,9,19,23-30:4, 5,9,21,25-32:4, and 5,9,23,27-34:4. In addition, 5,9,13-eicosatrienoic acid was present. Many of these do not appear to have been described before, and only 5,9-16:2 and 5,9,23-30:3 are found often in sponges. They were identified by using silver-ion high-performance liquid chromatography to simplify the complex mixture of fatty acids for subsequent analysis by gas chromatography-mass spectrometry as picolinyl ester derivatives. Deuteration with Wilkinson's catalyst in homogeneous solution confirmed the structures. We speculate that the di- and tetraenoic fatty acids arise by chain elongation of 5,9-hexadecadienoic acid, also a major component of the lipids, followed by further insertion of double bonds in the 5 and 9 positions. The trienes may be formed from 9-hexadecenoic acid by similar mechanisms.

Analysis of triacylglycerols by silver-ion high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

Triacylglycerols of the seed oils rich in alpha- and/or gamma-linolenic acid moieties were separated by silver-ion high-performance liquid chromatography (HPLC) followed by on-line atmospheric pressure chemical ionization-mass spectrometric (APCI-MS) detection. Mass spectra of most triacylglycerols exhibited abundant [M + H]+ and [M - RCO2]+ ions, which defined the molecular weight and the molecular association of fatty acyl residues of a triacylglycerol, respectively. Silver ions formed weaker complexes with triacylglycerols containing gamma-linolenic acid than with those containing alpha-linolenic acid, i.e., the elution order of molecules was XYT gamma > XYT alpha, XT gamma T gamma > XT gamma T alpha > XT alpha T alpha, and T gamma T gamma T gamma > T gamma T gamma T alpha > T gamma T alpha T alpha > T alpha T alpha T alpha, where T alpha = alpha-linolenic acid, T gamma = gamma-linolenic acid, and X, Y = fatty acids different from linolenic acid. Furthermore, silver-ion HPLC resulted in partial separation within equally unsaturated triacylglycerols according to differences in the combined number of acyl carbons. Regioisomeric forms of triacylglycerols were not determined from the seed oil samples, although differences were measured with reference compounds in the relative abundances of [M - RCO2]+ ions formed by a loss of a fatty acyl residue from the sn-2 position and the sn-1/3 positions. Silver-ion HPLC/APCI-MS provided valuable information for structure elucidation of seed oil triacylglycerols: 43 molecular species were identified from cloudberry seed oil, 39 from evening primrose oil, 79 from borage oil, 44 from alpine currant, and 56 from black currant seed oils. The quantitation requires to be studied further, especially in those cases where several molecular weight species of triacylglycerols eluted in a single chromatographic peak.

Mechanistic aspects of fatty acid retention in silver ion chromatography

The retention properties of various benzyl, phenacyl, and normal- and branched-chain alkyl esters of 6–18:1, 9–18:1 and 11–18:1 fatty acids have been studied by silver ion high-performance liquid chromatography. The weak electron-donating effect of the alkyl substituents on the carbonyl oxygen in the aliphatic alkyl esters ensures baseline resolution of isopropyl and tert-butyl esters, but not of n-alkyl esters. Comparison between benzyl and phenacyl derivatives confirmed that the silver ion interacted simultaneously with the double bond of the fatty acid and the carbonyl oxygen of the phenacyl moiety. This dual interaction enabled the separation of positional isomers. Introduction of electron-withdrawing or electron-donating substituents in the p-position in the benzene ring decreased or increased retention, respectively. The substituents strongly affect the resolution, and p-methoxyphenacyl esters have not only the highest k′ values but also the best resolution of the three positionally isomeric fatty acids, established so far.

Analysis of fatty acid mono- and diacylglycerol positional isomers by silver ion high-performance liquid chromatography

Mono- and diacylglycerol positional isomer pairs were separated (as acetates) by silver ion high-performance liquid chromatography on a commercially available column using an isocratic solvent system of 1.2% (v/v) acetonitrile in hexane and flame ionization detection. The acetate derivative(s) from the 1- and 2-monoacylglycerols and 1,2- and 1,3-diacylglycerols were prepared by acetic anhydride-pyridine. Conversion of the thermodynamically less stable 2-mono- and 1,2- diacylglycerols to triacylglycerols containing acetate(s) in the 1-, 3- and 2-positions, respectively, resulted in <3% isomerization. Conversion of the thermodynamically more stable 1-mono- and 1,3-diacylglycerol analogues yielded <1% isomers. Less than 0.5% inter-esterification by-products were noted. The triacylglycerol, the diacylglycerol-monoacetate isomer pair, the monoacylglycerol-diacetate isomer pair and triacetin were completely separated for the 16:0 and 18:1 fatty acid series. The triacylglycerols eluted first and the triacetin eluted last. The 16:0 elution pattern is unusual, since silver ion chromatographic separations are generally ascribed to the interaction of silver ions with carbon-carbon double bond π-electrons, a condition absent in the 16:0 series.

Monocyclic saturated fatty acids formed from oleic acid in heated sunflower oils

Fractions of saturated cyclic fatty acids, formed from oleic acid in normal and high-oleate sunflower oils during small-scale frying operations, were isolated by silver ion high-performance liquid chromatography. By means of gas chromatography of the methyl esters and gas chromatography/mass spectrometry of the dimethyloxazoline derivatives, eight monocyclic saturated fatty acids, comprising four basic structures, were identified: cyclopentyl fatty acids with rings from C-5 to C-9 and C-10 to C-14, and cyclohexyl fatty acids with rings from C-4 to C-9 and C-10 to C-15 of the original fatty acid chain. Each structure was represented by two components corresponding to cis and trans ring isomers. The cyclopentyl fatty acids were twice as abundant as those with a cyclohexane ring. Possible mechanisms for formation of the cyclic fatty acids via radical intermediates are discussed.

The nature of cyclic fatty acids formed in heated vegetable oils

The nature of the complex mixtures of monocyclic fatty acids formed in heated sunflower oils and linseed oil are summarized. The analytical approach involved fractionation of total cyclic fatty acids by silver ion high-performance liquid chromatography and characterization by gas chromatography - mass spectrometry of the picolinyl esters and/or dimethyloxazoline derivatives and Fourier transform infrared spectroscopy of the methyl esters. The cyclic fatty acids derived from oleate were saturated, those from linoleate were monoenes and those from linolenate were dienes. They contained five- and six-membered rings and differed according to the positions and configurations of the double bonds and rings. Possible mechanisms for their formation via allylic radical intermediates are discussed.

Changes in molecular species of triacylglycerols during frying

The loss of specific molecular species of triacylglycerols from sunflower, high-oleate sunflower and palm oils has been investigated in commercial frying operations and simulated frying experiments. The non-oxidized triacylglycerols were isolated and molecular species separated by silver ion highperformance liquid chromatography. Linoleate-containing species were lost more rapidly than those containing oleate, as expected. However, all species were liable to oxidation and those containing oleate were lost more rapidly than might have been anticipated. It is suggested that oxidation of linoleate is the probable initiation step, but then the reaction can be propagated readily to all unsaturated species.

Triacylglycerol Analysis by High Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry: Crepis Alpina and Vernonia Galamensis Seed Oils

Abstract Unusual seed oils having significance for chemical synthesis, Crepis alpina, or with fatty acids which contain functional groups important in the preparation of plastics, Vernonia galamensis, were analyzed by a new reversed-phase high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry technique. Using this method, we have identified 16 triacylglycerols in the Crepis alpina oil and 18 triacylglycerols in Vernonia galamensis oil and showed greater sensitivity for detection of and improved identification of triacylglycerols compared to previous analyses using the techniques of reversed-phase and silver ion high performance liquid chromatography with a flame ionization detector. The most abundant Crepis alpina triacylglycerols were: linoleoyldicrepenynoylglycerol (33.0%), tricrepenynoyl (32.3%), palmitoyldicrepenynoyl (11.5%), dilinoleoylcrepenynoyl (6.7%) glycerols. The remaining triacylglycerols occurred at five or less mole percent abundance. The most abun...

Silver ion chromatography of lipids and fatty acids.

Silver ion chromatography as applied to the analysis of lipids is reviewed. Thin-layer, column, high-performance liquid and supercritical fluid chromatography in the silver ion mode are included. The lipid types covered are fatty acids, triacylglycerols and complex lipids. Separations are divided into those according to number, geometry and position of double bonds, as well as acyl positional isomers for triacylglycerols. The mechanism of silver ion chromatography is discussed in relation to recent studies using silver ion high-performance liquid chromatographic methodology.