Analysis of free and esterified sterols in vegetable oils

In vegetable oils, phytosterols occur as free sterols or as steryl esters. Few analytical methods report the quantification of esterified and free sterols in vegetable oils. In this study, esterified and free sterols were separated by silica gel column chromatography upon elution with n-hexane/ethyl acetate (90∶10 vol/vol) followed by n-hexane/diethyl ether/ethanol (25∶25∶50 by vol). Both fractions were saponified separately and the phytosterol content was quantified by GC. The analytical method for the analysis of esterified and free sterols had a relative standard deviation of 1.16% and an accuracy of 93.6–94.1%, which was comparable to the reference method for the total sterol analysis. A large variation in the content and distribution of the sterol fraction between different vegetable oils can be observed. Corn and rapeseed oils were very rich in phytosterols, which mainly occurred as steryl esters (56–60%), whereas the majority of the other vegetable oils (soybean, sunflower, palm oil, etc.) contained a much lower esterified sterol content (25–40%). No difference in the relative proportion of the individual sterols among crude and refined vegetable oils was observed.     

Fractionation of oligomeric triacylglycerides and the relation to rejection limits for used frying oils

Precipitates enriched in oligomeric triacylglycerides were separated from thermally oxidized olive residue oil, conventional and high-oleic sunflower oils, and soybean oil by solvent fractionation in methanol/acetone at 4–5°C for 16 h. Different fractionation conditions were evaluated in an effort to isolate the oligomeric triacylglycerides (OTG). OTG, formed in frying oils upon heating at low concentations, were not detectable with conventional methods to determine polymeric compounds. The best conditions found from the different assays were the following: (i) weight of oil sample-to-solvent volume ratio of 1∶20; and (ii) solvent system methanol/acetone 10∶90 (vol/vol) for monounsaturated oils and 15∶85 (vol/vol) for polyunsaturated oils. Precipitates, enriched in oligomers, were formed when heated oils and used frying oils contained more than 27% polar compounds, a value which is widely accepted as the upper limit for use of frying oils.     

Comparison between extraction of lipids and fatty acids from microalgal biomass

Seven solvent mixtures have been used to extract the lipid fraction of lyophilized biomass ofIsochrysis galbana. Six of them were composed of biocompatible solvents. Each method was carried out under relaxed operating conditions (i.e., one hour at room temperature) with extraction in a nitrogen atmosphere to prevent autooxidation and degradation of polyunsaturated fatty acids (PUFAs). Apart from the well-established Bligh and Dyer method [Can. J. Biochem. Physiol. 37:911 (1959)] (Cl₃CH/MeOH/H₂O, 1∶2∶0.8, vol/vol/vol), which rendered the highest yield of lipids (93.8%), ethanol (96%) and hexane/ethanol (96%), 1∶2.5 vol/vol produced the best results (84.4 and 79.6%, respectively). To obtain free fatty acids, KOH was added to the solvent mixtures used to extract the total lipids, except for Cl₃CH/MeOH/H₂O, and direct saponification was carried out at 60°C for 1 h or at room temperature for 8 h. The highest yields obtained by direct saponicification were 81% with hexane/ethanol (96%), 1∶2.5, vol/vol and 79.8% with ethanol (96%). Partial yields of the mainn-3 PUFAs found inI. galbana, stearidonic acid (SA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were calculated for both extraction methods. For lipid extraction with ethanol (96%), yields of 91, 82 and 83% were obtained for SA, EPA and DHA, respectively. When direct saponification was used, hexane/ethanol (96%; 1∶2.5, vol/vol) produced the best yields of (91, 79 and 69% for SA, EPA and DHA, respectively).     

Estimation of polyunsaturated fatty acid content in lipids of aquatic organisms using thin-layer chromatography on a plain silica gel plate

We have designed a rapid method for the separation of polyunsaturated fatty acids (PUFA, ≥trienes) from non-PUFA, and for estimation of total amounts of PUFA in lipids of aquatic organisms. Lipids from thirty-one species, including marine and fresh water fishes, shell fishes, marine algae, and other aquatic animals, and from terrestrial organisms, were transesterified with sodium methoxide in methanol. The resulting fatty acid methyl esters were separated by thin-layer chromatography on commercially available plain silica gel plates with a developing solvent ofn-hexane/ethyl ether/acetic acid (95∶5∶1, by vol). All of the methyl esters from aquatic organisms tested separated into two spots, whereas those from terrestrial sources, except for linseed oil, showed a single unresolved spot. The upper and lower spots were scraped separately from the plate, and their fatty acid compositions were determined by gas-liquid chromatography. The lower spot was composed of PUFA having more than two double bonds, whereas components of the upper spot were saturated, monoenoic, and the greater part of the dienoic fatty acids. When the spots on the silica gel plate were stained with Coomassie brilliant blue, the amounts of PUFA in aquatic organisms could be estimated satisfactorily using a scanning densitometer. Presented in Japanese at the general Meeting of JSSF held in Mie University, Tsu-city, Japan, October 1994.     

Identification of natural diacylglycerols as the 3,5-dinitrophenylurethanes by chiral phase liquid chromatography with mass spectrometry

This study reports a facile identification of the molecular species of enantiomeric diacylglycerols by combining chiral phase high-performance liquid chromatography with positive chemical ionization mass spectrometry. For this purpose the 3,5-dinitrophenylurethane (DNPU) derivatives ofsn-1,2(2,3)-diacylglycerols are separated on an (R+)-naphthylethylamine polymer column (25 cm × 0.46 cm ID) using an isocratic solvent system made up of hexane/dichloroethane/acetonitrile (85∶10∶5, by vol) or isooctane/tert-butyl methyl ether/acetonitrile/isopropanol (80∶10∶5∶5, by vol). About 1% of the column effluent (1 mL/min) was admitted to a quadrupole mass spectrometer (Hewlett-Packard, Palo Alto, CA)via direct liquid inlet interface, and positive chemical ionization spectra were recorded over the range of 200–900 mass units. The DNPU derivatives of diacylglycerols yield characteristic [M-DNPU]⁺ and [RCO+74]⁺ ions for each diacylglycerol species from which the molecular weight and exact pairing of fatty acids can be unequivocally obtained. The characteristic ions appear to be generated in nearly correct mass proportions as indicated by preliminary quantitative comparisons. The abbreviations 14∶0, 16∶1, 18∶2, etc. represent normal chain fatty acids of 14, 16, 18, etc. acyl carbons and 0, 1, 2, etc. double bonds, respectively; 16∶0–18∶1, etc. represent diacylglycerols containing 16∶0 and 18∶1 fatty acids of unspecified positional distribution;sn indicates stereospecific numbering of glycerol carbons;sn-1,2-diacylglycerols andsn-2,3-diacylglycerols are enantiomeric diacylglycerols of unspecified fatty acid composition;rac-1,2-diacylglycerols are racemic diacylglycerols representing equal amounts ofsn-1,2-andsn-2,3-enantiomers;sn-1,2(2,3)-diacylglycerols are a mixture ofsn-1,2-andsn-2,3-diacylglycerols of unspecified proportion of enantiomers and unspecified fatty acid compisition and positional distribution; X-1,3-diacylglycerols are diacylglycerols of unspecified fatty acid composition and reverse isomer content.     

A multivariate optimization of triacylglycerol analysis by high-performance liquid chromatography

In the present study we have used statistical experimental design and multivariate optimization to formally optimize a reversed-phase high-performance liquid chromatography method for the analysis of triacylglycerol molecular species of natural oils. The optimal conditions found were, on an octadecylsilan-column, from acetonitrile/isooctane (90∶10, vol/vol) to acetonitrile/ethanol/isooctane (40∶35∶25, by vol), at a column temperature of 50°C and a flowrate of 1.5mL/min using a negative exponential gradient profile. Several examples of separations of natural seed and animal oils,i.e., soybean oil, rapeseed oil, palm oil, linseed oil, tallow and fish oil, are given. A version of the equivalent carbon number concept, utilizing the Snyder polarity index, was used to identity the molecular species.     

Separation and identification of isomeric conjugated fatty acids by high-performance liquid chromatography with photodiode array detection

A simple, high-performance liquid chromatographic method is described for the separation of tetraenoic, trienoic and dienoic conjugated fatty acids on a Zorbax ODS reversed-phase column using acetonitrile/tetrahydrofuran (95∶5, vol/vol) at a flow rate of 1.2 mL/min as mobile phase. Also described is the separation of the isomeric conjugated fatty acids with acetonitrile/water/tetrahydrofuran (90∶90∶1, by vol) as mobile phase. The simultaneous detection and identification of the separated geometrical isomers in the eluant was accomplished using photodiode array detection.     

Effect of extraction methods on lipid yield and fatty acid composition of lipid classes containing γ-linolenic acid extracted from fungi

The effect of extraction procedures on the lipid yield and fatty acid composition of total lipid and main lipid structures (phospholipids, diacylglycerols, triacylglycerols, free fatty acids, and sterol esters) of fungal biomass (Mucor mucedo CCF-1384) containing γ-linolenic acid (GLA) was investigated. Seventeen extraction methods, divided into three groups, were tested: six with chloroform/methanol, five with hexane/alcohols, and six with common solvents or mixtures. The chloroform/methanol procedure (2∶1) was selected as standard, where lipid yield (TL/DCW, total lipid per dry cell weight) was 17.8%, considered to be 100% of lipids present. All chloroform/methanol extractions yielded more than 83% recorvey of lipids. Use of hexane/isopropanol solvent systems led to a maximum of 75% recovery. The best lipid yield was achieved by a two-step extraction with ethanol and hexane (120%). Extraction efficiency of the other solvent systems reached a maximum of 73%. Triacylglycerols were the main structures of lipid isolated; only methanol-extracted lipid contained 58.5% phospholipids. The fatty acid content of total recovered lipid was variable and depended on both the lipid class composition and the solvent system. GLA concentrations in total lipids isolated by hexane/alcohol procedures (7.3–10.7%) are comparable with classical chloroform/methanol systems (6.5–10.0%). The maximal GLA yield was obtained with chloroform/methanol/n-butanol/water/0.1 M ethylenediaminetetraacetic acid (EDTA) (2∶1∶1∶1∶0.1, by vol) and after two-step extraction with ethanol and hexane (14.3 and 13.7 g GLA/kg DCW, respectively). The highest GLA content was analyzed in the phospholipid fraction (16.1%) after using chloroform/methanol/n-butanol/water/0.1 M EDTA (2∶1∶1∶1∶0.1, by vol). Remarkably low concentrations of polyunsaturated fatty acids were determined in the free fatty acid fraction.     

Separation and determination of glycolipids from edible plant sources by high-performance liquid chromatography and evaporative light-scattering detection

Glycolipids from edible plant sources were accurately quantified by silica-based, normal-phase high-performance liquid chromatography using an evaporative light-scattering detector. Five major glycolipid classes (acylated steryl glucoside, steryl glucoside, ceramide monohexoside, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol) were separated and determined with a binary gradient system consisting of chloroform and methanol/water (95∶5, vol/vol) without any interference from other lipid classes and pigments. The described method was applied to 48 edible plants available in Japan including cereals, legumes, vegetables, and fruits. Examined plant species contained glycolipids in wide concentration ranges, such as 5–645 mg/100 g tissue.     

Rapid separation of the major phospholipid classes on a single aminopropyl cartridge

A rapid method for the separation of the individual phospholipid classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) by a single solid-phase extraction was developed. PC, PE, PS and PI were sequentially eluted from aminopropyl bonded silica with acetonitrile/n-propanol (2∶1, vol/vol), methanol, isopropanol/methanolic HCl (4∶1, vol/vol) and methanol/methanolic HCl (9∶1, vol/vol). Standard recoveries were over 95% for PC and PE and over 85% for PS and PI with undistorted fatty acid composition. The separation of complex lipid mixtures on aminopropyl minicolumns can be refined to the level of individual phospholipid classes.     

Antioxidant activity of extracts of defatted seeds of niger (<Emphasis Type="Italic">Guizotia abyssinica</Emphasis>)

Niger (Guizotia abyssinica) seed was ground and then defatted with hexane. The meal remaining after oil extraction was tested as a source of antioxidants. Three solvent systems, A [80∶20 (vol/vol) ethanol/water], B [80∶20 (vol/vol) acetone/water], and C (water) were evaluated as extraction media. Crude extracts were examined for their antioxidant activity in a β-carotene-linoleate and a meat model system. Extract A exhibited superior antioxidant activity, compared to extracts B and C, and its composition was studied further by using column chromatography and HPLC. Four fractions (I–IV) were obtained, of which fractions III and IV showed activity in the β-carotene-linoleate model system. Fraction IV was also highly effective in scavenging the 2,2-diphenyl-1-picrylhydrazyl radical but was less active when used in a bulk oil model system. Preparative TLC showed fraction IV as consisting of two components. UV spectroscopy suggested that the major active component pressent was a chlorogenic acid-related compound. Furthermore, HPLC analysis established that chlorogenic acid was dominant in the free phenolics fraction (2.6 mg/g). Upon hydrolysis, however, a substantial amount of caffeic acid (42.8 mg/g) was released, presumably from esterified and glycosylated chlorogenic acid. Thus, niger extracts derive their antioxidant activity, at least in part, from the chlorogenic acid-related compounds.     

Quantitative determination of butylated hydroxyanisole, butylated hydroxytoluene, and tert-butyl hydroquinone in oils, foods, and biological fluids by high-performance liquid chromatography with fluorometric detection

Concentrations of synthetic antioxidants butylated hydroxyanisole, butylated hydroxytoluene, and tert-butyl hydroquinone were quantified using a high-performance liquid chromatograph with spectrofluorometric detector. The antioxidants were separated and eluted on a reversed-phase column by gradient of a mixture of H₂O/acetonitrile/acetic acid (66.5: 28.5∶5, by vol) and a mixture of acetonitrile/acetic acid (95∶5, vol/vol). The eluants were monitored at emission and excitation wavelengths of 310 and 280 nm, respectively. Calibration curves obtained using peak areas against concentration showed ligh coefficients of multiple determination (R ²>0.99) for all antioxidants. Known concentrations of added antioxidant standards were recoverable within 98–99% from oils and over 93% from mouse blood. This method requires minimum sample extraction and purification before analysis and provides a relatively high percentage recovery. The method has been applied successfully for the measurement of antioxidant concentrations in oils, dried foods, and biological fluids.     

Gangliosides from rat cerebellum: Demonstration of considerable heterogeneity using a new solvent for thin layer chromatography

Using a new solvent (methyl acetate/n-propanol/chloroform/methanol/0.25% aqueous KCl, 25∶20∶20∶20∶17, v/v) and high performance silica gel thin layer chromatographic plates, all common gangliosides found in brain can be easily separated with 1 hr. This system is reproducible and “tailing” is negligible compared with previous solvents. When such a system is applied to separate the gangliosides found during the development of the rat cerebellum, a considerable heterogeneity is observed. Data are presented (using rechromatography experiments, fractionation on DEAE-Sephadex, treatment with neuraminidase or alkaline medium and carbohydrate analysis) suggesting that the complex profiles obtained with this chromatographic system are not due to chromatographic artifacts but result from the high resolving power of the system. After separation by ion-exchange chromatography, 28 gangliosides can be detected.     

Analysis of tocopherols in vegetable oils by high-performance liquid chromatography: Comparison of fluorescence and evaporative light-scattering detection

A comparison of the responses of an evaporative light-scattering detector (ELSD) and a fluorescence detector for tocopherols in vegetable oils by high-performance liquid chromatography is presented. The tocopherols were separated from acylglycerols by gel-permeation chromatography (GPC). The tocopherol fraction was collected off a set of four GPC columns with a mobile phase of methylene chloride before separation on a normal-phase silica column with a mobile phase of hexane/isopropanol, 99.7∶0.3 (vol/vol). An internal standard of 5,7 dimethyltocol, which was detected by both the ELSD and fluorescence detector, was used to obtain quantitative data. The fluorescence detector was ten times more sensitive than the ELSD. γ-Tocopherol was the major tocopherol detected in the vegetable oils studied and ranged from 24.1–93.3 mg/100 g. The amounts of tocopherols found in the vegetable oils agreed favorably with the literature values.     

High performance reversed phase chromatography of cholesterol and cholesteryl esters of human plasma lipoproteins

Cholesterol and cholesteryl esters were separated according to their carbon number and number of double bonds by high performance reversed-phase chromatography (HPRC) using acetonitrile/chloroform/methanol (1∶1∶1, v/v) as a mobile phase. It was found that within the same equivalent carbon number (ECN) category, cholesterol esters with the highest number of double bonds eluted ahead of those with a lower number of double bonds, and with thecis isomers eluting ahead of theirtrans partners. Thus, cholesteryl oleate (C27-18∶1c) elutes ahead of cholesteryl palmitate (C27-16∶0) and ahead of cholesteryl elaidate (C27-18∶1t). Human lipoprotein, as well as rat liver cholesteryl esters, were separated using this technique.     

Recovery of eicosapentaenoic acid from fungal mycelia by solvent extraction

Utilization of lipids containing eicosapentaenoic acid (EPA) produced by microorganisms requires processes for their efficient recovery from microbial cells. Recovery of EPA from mycelia of the fungusPythium irregulare by solvent extraction with hexane-isopropanol (HIP) in a pilot-plant colloid mill was investigated. Extraction efficiencies of 96% for lipid and EPA were achieved with a 3∶2 (vol/vol) HIP mixture by milling wet, filtered mycelia for 5 min at a solvent/dry solids ratio of 100 L/kg. The process yielded a crude extract that contained up to 96% lipid and an EPA content as high as 24% (with no selectivity for EPA).     

Potential bile acid metabolites. 24. An efficient synthesis of carboxyl-linked glucosides and their chemical properties

A facile and efficient synthesis of the carboxyl-linked glucosides of bile acids is described. Direct esterification of unprotected bile acids with 2,3,4,6-tetra-O-benzyl-d-glucopyranose in pyridine in the presence of 2-chloro-1,3,5-trinitrobenzene as a coupling agent afforded a mixture of the α- and β-anomers (ca. 1∶3) of the 1-O-acyl-d-glucoside benzyl ethers of bile acids, which was separated effectively on a C₁₈ reversedphase chromatography column (isolated yields of α- and β-anomers are 4–9% and 12–19%, respectively). Subsequent hydrogenolysis of the α- and β-acyl glucoside benzyl ethers on a 10% Pd−C catalyst in acetic acid/methanol/EtOAc (1∶2∶2, by vol) at 35°C under atmospheric pressure gave the corresponding free esters in good yields (79–89%). Chemical specificities such as facile hydrolysis and transesterification of the acyl glucosides in various solvents were also discussed.     

Determination of squalene in olive oil using fractional crystallization for sample preparation

A simple, rapid method for the determination of squalene in virgin olive oil was developed using RP-HPLC with detection at 208 nm. Fractional crystallization from methanol/acetone (7∶3, vol/vol) was applied to obtain squalene in the liquid fraction of the oil prior to HPLC. Elution of squalene was then carried out isocratically with acetone/acetonitrile (40∶60 vol/vol) within 11 min. The detection limit was 23 mg/kg, and the limit of quantification 79 mg/kg. The precision of the crystallization procedure (CV%=3.76, n=7) and the mean recovery (92.5 and 81.5% for the 7,000 and 700 mg/kg levels of addition, respectively) were satisfactory. The method is easily applicable to fulfill future needs for nutrition labeling.     

Cottonseed extraction with mixtures of acetone and hexane

Cottonseed flakes were extracted with mixtures of n-hexane and acetone, with the concentration of acetone varying between 10 and 75%. Adding small amounts of acetone (≤25%) to n-hexane significantly increased the extraction of free and total gossypol from cottonseed flakes. Sensory testing detected no difference in the odor of cottonseed meals produced either by extraction with 100% n-hexane or by extraction with a 10∶90 (vol/vol) mixture of acetone/hexane. More than 80% of the free gossypol was removed by the 10∶90 mixture of acetone/hexane, whereas pure n-hexane extracted about 47% of the free gossypol from cottonseed flakes. A solvent mixture containing 25% acetone removed nearly 90% of the free gossypol that was removable by extraction with pure acetone; the residual meal had only a minimal increase in odor. In contrast, cottonseed meals produced by extraction with pure acetone had a much higher odor intensity. The composition of the cottonseed crude oil was insignificantly affected by the acetone concentration of the extraction solvent. The results indicate that mixtures of acetone and n-hexane can be used as extraction solvents to produce cottonseed crude oil without the concomitant development of odorous meals.     

Characterization of feline omentum lipids

Feline omental lipid extracts, previously reported to be angiogenic in the cornea of rabbits, were fractionated and the major lipid components characterized. Approximately 97% of the chloroform/methanol extract consisted of triglycerides containing primarily 16∶0, 18∶0, 18∶1 and 18∶2 fatty acids. Trace quantities of free fatty acids, cholesterol, di- and monoglycerides were also detected. The phospholipid fraction, obtained by solvent partition and Unisil column chromatography and characterized by high performance liquid chromatography (HPLC)-mass spectrometry, was found to consist of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and phosphatidylserine. The neutral glycolipids, isolated by solvent partition and Unisil column chromatography and identified by high performance thin layer chromatography and HPLC of their perbenzoylated derivatives, were found to consist of glucosyl- and galactosylceramides, galabiosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. The complex glycolipid fraction, obtained from Folch upper phase solvent partition, was found to consist primarily of Forssman glycolipid and gangliosides GM₃ and GD₃. Smaller amounts of GM₁ and other unidentified gangliosides were also present. The ganglioside nomenclature is according to the system of Svennerholm (J. Neurochem. 10, 613–623, 1963)