Silver Ion High-performance Liquid Research Articles

Cyclohexanediol Fatty Acid Diesters as Model Compounds for Mechanistic Studies in Silver Ion High Performance Liquid Chromatography

Series of cis‐1,2‐ and cis/trans‐1,4‐cyclohexanediol diesters of saturated, oleic and linoleic fatty acids were employed as model compounds in a study of the effect of the unsaturation and the position of the acyl moieties on the retention of acylglycerol molecules in silver ion high‐performance liquid chromatography (HPLC). cis‐1,2‐Diunsaturated cyclohexanediol diesters were retained slightly more strongly than were the respective cis/trans‐1,4‐compounds, thus indicating that formation of chelate‐type complexes between silver ions and the double bonds of two adjacent unsaturated fatty acid residues during silver ion chromatography may occur to a limited extent.

Alternative pathways of sterol synthesis in yeast: Use of C 27 sterol tracers to study aberrant double-bond migrations and evaluate their relative importance

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the Δ 8–Δ 7 isomerase (Erg2p) or Δ 5 desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5α-cholest-8-en-3β-ol, 8-dehydrocholesterol (Δ 5,8 sterol), or isodehydrocholesterol (Δ 6,8 sterol), together with the corresponding 3 α- 3 H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven Δ 22E sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag +-HPLC) and identified by gas chromatography–mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag +-HPLC. When Δ 8–Δ 7 isomerization was blocked, exogenous Δ 8 sterol underwent desaturation to Δ 5,8, Δ 6,8, and Δ 8,14 sterols. Formation of Δ 5,8 sterol was strongly favored over Δ 6,8 sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The Δ 5,8 sterol was metabolically almost inert except for Δ 22 desaturation, whereas the Δ 6,8 sterol was readily converted to Δ 5,7, Δ 5,7,9(11), and Δ 7,9(11) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, Δ 5,7 sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of Δ 5,8 and Δ 5,7,9(11) sterols in wild-type yeast compared to Smith–Lemli–Opitz individuals.

Ternary gradient elution markedly improves silver-ion high performance liquid chromatography of unsaturated sterols

A wide variety of unsaturated sterols can accumulate in eukaryotic cells as a consequence of normal metabolism, genetic disorders, and actions of enzyme inhibitors. Resolving these sterol mixtures into individual components by conventional chromatographic methods is inefficient because unsaturated sterols differ little in polarity, hydrophobicity, and volatility. Although sterol mixtures are well-resolved by silver-ion high performance liquid chromatography (Ag +-HPLC), existing methods require derivatization to acetates for best results, and the isocratic mobile phases lead to long analysis times and low sensitivity for late-eluting sterols. We show that these problems can be overcome with ternary gradient elution using acetone, hexanes, and acetonitrile. Separation of a mixture of 20 underivatized sterols gave dramatically shortened analysis times, with good peak shapes for both early- and late-eluting components. In a similar separation of blood sterols from a patient with Smith–Lemli–Opitz syndrome, the band for 7-dehydrocholesterol was much narrower than with isocratic elution. Column re-equilibration was rapid, and the separations could be monitored with ultraviolet spectroscopy at 210 nm, which affords universal, non-destructive detection of unsaturated sterols. Also discussed are retention mechanisms and reproducibility of Ag +-HPLC separations. The overall results represent a major advance in chromatographic methods for resolving mixtures of unsaturated sterols differing in the number and position of olefinic bonds.

Chromatographic separation and identification of conjugated linoleic acid isomers

There are 56 possible geometric and positional isomers of conjugated octadecadienoic acids (18:2), better known as conjugated linoleic acid (CLA). Positive health benefits are ascribed to the consumption of the 9 c,11 t-18:2 and 10 t,12 c-18:2 isomers. The dietary significance of the other isomers is not known. Our understanding of the biological role of these acids relies on their proper identification and quantitation in complex biological extracts. Gas chromatography (GC) alone cannot completely separate the naturally occurring CLA isomers. The combination of silver ion high performance liquid chromatography (Ag + HPLC) and GC offers the best separation of these isomers with complementary identification by GC–mass spectrometry (GC–MS) and GC–Fourier transform infrared (FTIR) analyses.

SILVER ION HPLC OF p-METHOXYPHENACYL DERIVATIVES OF UNSATURATED FATTY ACIDS. III. MOBILE PHASE EFFECTS ON TRANS 6-, 9-, AND 11–18 : 1

The effect of the mobile phase composition on the retention and resolution of trans positionally isomeric 6-, 9-, and 11–18 : 1 fatty acids was studied on a model mixture by silver ion high performance liquid chromatography (Ag-HPLC). Prior to chromatography, the fatty acids were converted into p-methoxyphenacyl derivatives. Hexane-based and dichloromethane-based mobile phases modified with either acetonitrile or methanol, or isopropanol were examined. The modifiers were found to have a different effect on the resolution, depending on the main solvent component in the mobile phase. In case of hexane being the main mobile phase solvent, best resolution was achieved when using isopropanol as a modifier. In dichloromethane-based mobile phases, best results were achieved with acetonitrile or methanol. The three trans isomers were completely resolved in practically all mobile phases tested, the elution order being 11-, 9-, 6–18 : 1, irrespective of the mobile phase composition.

Analysis of conjugated linoleic acid-enriched triacylglycerol mixtures by isocratic silver-ion high-performance liquid chromatography

Silver-ion HPLC (Ag-HPLC) was applied to the fractionation of a triacylglycerol (TAG) sample enriched (>80%) with conjugated linoleic acid (CLA). After conversion of the TAGs to fatty acid methyl esters using sodium methoxide in methanol, Ag-HPLC (dual-column; isocratic solvent system of 0.1% acetonitrile in hexane; UV detection at 233 nm) was used to determine the CLA isomer distribution (50:50 mixture of 9c,11t- and 10t,12c-18:2). Three or four Ag-HPLC columns connected in series (0.6–1.0% acetonitrile in hexane as solvent; UV detection at 206 nm) were used to analyze the sample in TAG form. Elution times for CLA-enriched TAGs averaged 30 min or less. Isocratic solvent conditions were used to eliminate the solvent equilibration times (often 30 min or more) required between sample injections when solvent programming is used. The ratio of TAGs containing three vs. only two CLA molecules was found to be approximately 3 to 1. Ag-HPLC has thus been shown to be a useful method for rapidly analyzing not only CLA isomers as esters, but also in the TAG form.

SILVER ION HPLC OF p-METHOXYPHENACYL DERIVATIVES OF UNSATURATED FATTY ACIDS. III. MOBILE PHASE EFFECTS ON TRANS 6-, 9-, AND 11?18 : 1

The effect of the mobile phase composition on the retention and resolution of trans positionally isomeric 6-, 9-, and 11–18 : 1 fatty acids was studied on a model mixture by silver ion high performance liquid chromatography (Ag-HPLC). Prior to chromatography, the fatty acids were converted into p-methoxyphenacyl derivatives. Hexane-based and dichloromethane-based mobile phases modified with either acetonitrile or methanol, or isopropanol were examined. The modifiers were found to have a different effect on the resolution, depending on the main solvent component in the mobile phase. In case of hexane being the main mobile phase solvent, best resolution was achieved when using isopropanol as a modifier. In dichloromethane-based mobile phases, best results were achieved with acetonitrile or methanol. The three trans isomers were completely resolved in practically all mobile phases tested, the elution order being 11-, 9-, 6–18 : 1, irrespective of the mobile phase composition.

Separation of conjugated linoleic acid-containing mono-, di- and triacylglycerols by silver-ion high-performance liquid chromatography

L'acide linoleique conjugue, present dans les produits laitiers et le boeuf, pssede chez l'animal des proprietes anticarcinogenes, antiatherogenes, antidiabetiques, favorisant la croissance et augmentant la masse musculaire. La chromatographie Ag + -HPLC est une technique appropriee pour separer des melanges simples d'isomeres de triacylglycerols (TAG) ou de TAG contenant des acides gras de diverses longueurs de chaine mais cette technique est peu appliquee pour l'analyse de melanges complexes de TAG. Deux a quatre colonnes en serie devraient suffire pour analyser les TAG d'oleagineux contenant des acides gras conjugues.

Separation of conjugated linoleic acid-containing mono-, di- and triacylglycerols by silver-ion high-performance liquid chromatography

Separation of conjugated linoleic acid-containing mono-, di- and triacylglycerols by silver-ion high-performance liquid chromatography

Separation of glyceride positional isomers by silver ion chromatography

Separation of triglyceride and diglyceride positional isomers by silver ion high-performance liquid chromatography coupled with an evaporative light-scattering detector is described. The triglyceride isomers had a fatty acid composition of CLC and CCL, where C and L were caprylic acid and linoleic acid, respectively. Diglyceride isomers, 1,2(2,3)-diglyceride and 1,3-diglyceride, which contained caprylic acid were separated too. A solvent system based on n-hexane, 2-propanol, ethyl acetate, and acetonitrile with a flow-rate of 0.8 ml/min was developed. Calibration curves of CLC and CCL were achieved with triolein as internal standard. Using this method, the incorporation of linoleic acid onto specific a position of glycerol backbone can be monitored.

Changes in conjugated linoleic acid composition within samples obtained from a single source.

Conjugated linoleic acid (CLA; 9c,11t-18:2) and CLA isomers have been reported, in animals, to exhibit a variety of health-related benefits. Silver ion high-performance liquid chromatography (Ag-HPLC) was found to provide better resolution of the isomers than gas chromatography. Most commercially available samples of CLA, prepared by base-catalyzed isomerization of linoleic acid (9c,12c-18:2), are composed of mixtures of four major isomers. While these isomers have been characterized, we found significant changes in CLA isomer rations within samples obtained from the same producer/commercial supplier over a period of 1.5 yr. In the first sample, the four cis/trans isomers (8t,10c-18:2, 9c,11t-18:2, 10t,12c-18:2 and 11c,13t-18:2) were present in a ratio of approximately 1:2:2:1, while in the second sample they were present in almost equal proportions. If indeed certain daily levels of CLA intake are required to produce suggested health benefits in humans, changes in concentrations of specific CLA isomers could significantly impact these effects. Care must be taken to analyze the CLA used in human and animal studies.

Aberrant pathways in the late stages of cholesterol biosynthesis in the rat: origin and metabolic fate of unsaturated sterols relevant to the Smith-Lemli-Opitz syndrome

Minor aberrant pathways of cholesterol biosynthesis normally produce only trace levels of abnormal sterol metabolites but may assume major importance when an essential biosynthetic step is blocked. Cholesta-5,8-dien-3β-ol, its Δ5,7 isomer, and other noncholesterol sterols accumulate in subjects with the Smith-Lemli-Opitz syndrome (SLOS), a severe developmental disorder caused by a defective Δ7 sterol reductase gene. We have explored the formation and metabolism of unsaturated sterols relevant to SLOS by incubating tritium-labeled Δ5,8, Δ6,8, Δ6,8(14), Δ5,8(14), and Δ8 sterols with rat liver preparations. More than 60 different incubations were carried out with washed microsomes or the 10,000 g supernatant under aerobic or anaerobic conditions; some experiments included addition of cofactors, fenpropimorph (a Δ8–Δ7 isomerase inhibitor), and/or AY-9944 (a Δ7 reductase inhibitor). The tritium-labeled metabolites from each incubation were identified by silver ion high performance liquid chromatography on the basis of their coelution with unlabeled authentic standards, as free sterols and/or acetate derivatives. The Δ5,8 sterol was converted slowly to cholesterol via the Δ5,7 sterol, which also slowly isomerized back to the Δ5,8 sterol. The Δ6,8 sterol was metabolized rapidly to cholesterol by an oxygen-requiring pathway via the Δ7,9(11), Δ8, Δ7, and Δ5,7 sterols as well as by an oxygen-independent route involving initial isomerization to the Δ5,7 sterol. The Δ8 sterol was partially metabolized to Δ5,8, Δ6,8, Δ7,9(11), and Δ5,7,9(11) sterols when isomerization to Δ7 was blocked. The combined results were used to formulate a scheme of normal and aberrant biosynthetic pathways that illuminate the origin and metabolic fate of abnormal sterols observed in SLOS and chondrodysplasia punctata.—Ruan, B., J. Tsai, W. K. Wilson, and G. J. Schroepfer, Jr. Aberrant pathways in the late stages of cholesterol biosynthesis in the rat: origin and metabolic fate of unsaturated sterols relevant to the Smith-Lemli-Opitz syndrome. J. Lipid Res. 2000. 41: 1772–1782.

Quantitative determination of conjugated linoleic acid isomers in beef fat

The amounts of 14 conjugated linoleic acid (CLA) isomers (t12t14, t11t13, t10t12, t9t11, t8t10, t7t9, t6t8; 12,14 c/t, t11c13, c11t13, t10c12, 9,11 c/t, t8c10, t7c9-18:2) in 20 beef samples were determined by triple-column silver-ion high-performance liquid chromatography (Ag+-HPLC). Quantitation was performed using an external CLA reference standard consisting of cis9,trans11-18:2,trans9,trans11-18:2 and cis9,cis11-18: 2. Linearity was checked as being r > 0.9999 between 0.02 × 10-3 to 2 mg/ml. The determination limit (5-fold signal/noise ratio) of the CLA reference was estimated to be 0.25, 0.50, 1.0 ng/injection for the cis/trans, trans,trans and cis,cis isomers, respectively. As expected, cis9,trans11-18:2 was the predominant isomer (1.95 ± 0.54 mg/g fat) in beef, followed by trans7,cis9-18:2 (0.19 ± 0.04 mg/g fat); cis,cis isomers were below the determination limit in most beef samples. Total CLA amounts determined by Ag+-HPLC were compared to total CLAs determined by gas chromatography (GC, 100 m CPSilTM 88 column). The amounts obtained by GC were generally higher than those determined by Ag+ -HPLC due to co-eluting compounds.

SILVER ION HPLC OF p-METHOXYPHENACYL DERIVATIVES OF UNSATURATED FATTY ACIDS. I. MOBILE PHASE EFFECTS

The effect of the mobile phase composition on the retention and resolution of positionally isomeric octadecamonoenoic (18:1), eicosamonoenoic (20:1), octadecatrienoic (18:3), and eicosatrienoic (20:3) fatty acids in silver ion high performance liquid chromatography was studied on model fatty acid mixtures. Prior to chromatography, the fatty acids were converted into p-methoxyphenacyl derivatives. Hexane-based and dichloro-methane-based mobile phases that contained acetonitrile, methanol, and iso-propanol as modifiers were examined. All three modifiers ensured reliable resolution of the respective isomeric fatty acids and are of practical interest. In contrast to acetonitrile and methanol, iso-propanol was found to reverse the elution order of difficult to resolve components that gave adjacent chromatographic peaks. This finding indicates the active role of mobile phase in the chromatographic process.

Oxidative stability of conjugated linoleic acid isomers.

Conjugated linoleic acids (CLAs) have been shown to be a strong anticarcinogen in a number of animal models. Our previous study demonstrated that CLA as a whole was extremely unstable in air. The present study was undertaken further to examine the oxidative stability of individual CLA isomers using the combination of gas-liquid chromatography (GLC) and silver ion high-performance liquid chromatography (Ag-HPLC). It was found that CLA as a whole oxidized rapidly and more than 80% was degraded within 110 h in air at 50 degrees C. Four c,c-CLA isomers were most unstable followed by four c,t-CLA isomers. In contrast, four t,t-CLA isomers were relatively stable under the same experimental conditions. Both the oxygen consumption and the GLC analysis revealed that 200 ppm jasmine green tea catechins (GTCs) exhibited protection to CLA and were even stronger than 200 ppm butylated hydroxytoluene (BHT) when added to either CLA or canola oil containing 10% CLA. The present study emphasized that oxidative unstability of CLA should not be overlooked although CLA has many biological effects.

SILVER ION HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF POLYNUCLEAR AROMATIC DERIVATIVES OF POSITIONALLY ISOMERIC OCTADECENOIC FATTY ACIDS

The chromatographic properties of positionally isomeric octadecenoic fatty acids in silver ion high performance liquid chromatography with hexane-based and dichloromethane-based mobile phases were studied after conversion in 2-naphthacyl, 2-naphthylmethyl and 9-anthrylmethyl derivatives. The retention decreased in the order 2-naphthacyl, 9-anthrylmethyl, 2-naphthylmethyl derivatives. Dichloromethane-acetonitrile mobile phases provided better resolution and 2-naphthacyl and 9-anthrylmethyl derivatives of 6-, 9-, and 11–18:1 were fully resolved with dichloromethane-acetonitrile 100:0.025 (v/v). The behavior of the aromatic derivatives, examined so far by Ag-HPLC, under comparable experimental conditions, was compared and a clear tendency of improving the resolution was observed when the number of aromatic nuclei in the aromatic fragment increased. The effect was ascribed to the increased electron-donating effect of the aromatic rings in the ester moiety, which supposedly enhanced the formation of a three center complex between double bond, a carbonyl oxygen, and silver ion. The good resolution of 9-anthrylmethyl derivatives is of potential practical value since it allows for the determination of nanogram amounts of fatty acids by silver ion HPLC with fluorescent detection.

Silver Ion High-Performance Liquid Chromatographic Separation of Conjugated Linoleic Acid Isomers, and other Fatty Acids, after Conversion top-Methoxyphenacyl Derivatives

Silver Ion High-Performance Liquid Chromatographic Separation of Conjugated Linoleic Acid Isomers, and other Fatty Acids, after Conversion top-Methoxyphenacyl Derivatives

Synthesis of [3α- 3H]cholesta-5,8-dien-3β-ol and tritium-labeled forms of other sterols of potential importance in the Smith-Lemli-Optiz syndrome☆

Five unsaturated sterols relevant to the Smith-Lemli-Opitz syndrome have been prepared in high radiochemical purity with a tritium label at the 3α position. Swern oxidation of cholesta-5,8-dien-3β-ol and other unlabeled C 27 sterols afforded the corresponding 3-ketosteroids, and reduction with tritiated NaBH 4 gave the desired 3α- 3H sterols, with double bonds at the Δ 5,8, Δ 5,8(14), Δ 6,8, Δ 6,8(14), and Δ 8 positions. High radiochemical purity of the tritiated sterols was demonstrated by normal phase, reversed phase, and silver-ion (Ag +) high-performance liquid chromatography (HPLC). In the course of this work, we developed a medium-pressure variant of Ag +-HPLC for purifying radiolabeled samples, documented significant isotopic fractionation of the 3α-tritiated sterols and their acetates on Ag +-HPLC, and discovered unexpected effects of a Δ 8(14) bond on the conformation of 3-keto-Δ 5-steroids. The synthetic and analytical methodologies described herein should provide a sound basis for investigating the origin and metabolism of sterols involved in the Smith-Lemli-Opitz syndrome and in late stages of cholesterol biosynthesis.

Recent advances in conjugated linoleic acid research

New results on the physiological properties of conjugated linoleic acid have been published by several working groups, especially showing the effects of single conjugated linoleic acid isomers on carcinogenesis and body composition. Recently, other studies have shown that conjugated linoleic acid has an influence on diabetes mellitus, platelet aggregation and the immune system. Conjugated linoleic acid was found to modify prostaglandin metabolism and delta9-desaturase activity and influence apoptosis. Furthermore, improved analytical methods using 13C nuclear magnetic resonance and silver ion high performance liquid chromatography are available to investigate the composition of conjugated linoleic acid mixtures and the exact structure of separated isomers. Also, the synthesis of isolated isomers is described, as published by different authors, in order to determine further the effects of each single conjugated linoleic acid isomer. In addition, new data on the contents of conjugated linoleic acid in foods, human adipose tissue and fluids are given in this review. More data need to be obtained using isolated isomers, with particular emphasis on studies in humans.

An improved synthesis of (20 R,22 R)-cholest-5-ene-3β,20,22-triol, an intermediate in steroid hormone formation and an activator of nuclear orphan receptor LXRα

Asymmetric dihydroxylation of (20(22) E)-cholesta-5,20(22)-dien-3β-ol acetate ( 2a), prepared from pregnenolone, gave a 1:1 mixture (67% yield) of (20 R,22 R)-cholest-5-ene-3β,20,22-triol 3-acetate ( 3a) and its 20 S,22 S isomer 3b. Highly purified 3a and 3b were obtained by semipreparative silver ion high performance liquid chromatography. Saponification of 3a and 3b gave (20 R,22 R)-cholest-5-ene-3β,20,22-triol ( 4a) and its 20 S,22 S isomer 4b. This simple approach provided the natural isomer 4a more efficiently than previously described chemical or enzymatic syntheses. Full 1H and 13C nuclear magnetic resonance data were presented for triols 4a and 4b and their synthetic precursors. Side-chain conformations of 2a, its 20(22)Z isomer, 4a, and 4b were studied by molecular mechanics and nuclear Overhauser effect difference spectroscopy.