The triacylglycerol structure of olive oil determined by silver ion high-performance liquid chromatography in combination with stereospecific analysis

The compositions of positionssn-1,sn-2 andsn-3 of triacylglycerols from “extra-virgin” olive oil (Olea europaea) were determined. The procedure involved preparation of diacyl-rac-glycerols by partial hydrolysis with ethyl magnesium bromide; 1,3-, 1,2- and 2,3-diacyl-sn-glycerols as (S)-(+)-1-(1-naphthyl)ethyl urethanes were isolated by highperformance liquid chromatography (HPLC) on silica, and their fatty acid compositions were determined. The same procedure was also carried out on the five main triacylglycerol fractions of olive oil after separation according to the degree of unsaturation by HPLC in the silver ion mode. Although stereospecific analysis of the intact triacyl-sn-glycerols indicated that the compositions of positionssn-1 andsn-3 were similar, the analyses of the molecular species demonstrated marked asymmetry. The data indicate that the “1-random, 2-random, 3-random” distribution theory is not always applicable to vegetable oils.     

Stereospecific analysis of monounsaturated triacylglycerols in cocoa butter

The enantiomeric composition of the monounsaturated triacylglycerols (TG) from cocoa butter was estimated. The monounsaturated TG were separated into three fractions by reversed-phase high-performance liquid chromatography (HPLC), and each fraction was subjected to the stereospecific analysis with chiral-phase HPLC. The results indicated that the major TG consisted of equal amounts of 1-stearoyl-2-oleoyl-3-palmitoyl-sn-glycerol (SOP-sn-TG) and POS-sn-TG (47 mol%), 1,3-distearoyl-2-oleoyl-glycerol (SOS-TG) (33 mol%), and POP-TG (19 mol%). The contents of SOP-sn-TG and POS-sn-TG are 1.30 times that of the POP-TG content, and the SOS-TG content is 1.30² times that of the POP-TG content. The term “priority factor” is proposed for the ratio of the stearoyl group/palmitoyl group, 1:30 at thesn-1 andsn-3 or 1(3)-position. It shows a distinct specificity for particular fatty acids or their Coenzyme A esters in random esterification at each position of the glycerol moiety in the biosynthesis of cocoa butter TG.     

Micro method for stereospecific analysis of triacyl-sn-glycerols by chiral-phase high-performance liquid chromatography

A procedure for micro stereospecific analysis of triacyl-sn-glycerols (TGs) by high-performance liquid chromatography (HPLC) on a chiral column is presented. TGs were partially hydrolyzed with ethyl magnesium bromide, and total products were immediately converted to 3,5-dinitro-phenylurethane derivatives. Each of the 1- and 2-monoacylglycerol (MG) derivatives was isolated by HPLC on a silica column. The 1-MGs were resolved intosn-1 andsn-3 MG fractions by HPLC on a Sumichiral OA-4100 column (Sumitomo Chemical, Osaka, Japan). Fatty acid methyl esters obtained from thesn-1,sn-2 andsn-3 MG fractions were analyzed by gas-liquid chromatography on a capillary column. Analyses of standard TGs showed that, even with 1 mg of sample, accuracy was comparable to that obtained with 100-mg samples. Applying this procedure to the stereospecific analysis of 5 mg of jujube pulp, TGs revealed the positional distribution of the (n-5) series of monounsaturated fatty acids they contained. Honored Student Award Address presented at the 83rd AOCS Annual Meeting held in Toronto, Canada, May 10–14, 1992.     

Stereospecific analysis of fish oil triacyl-sn-glycerols

Stereospecific analysis of fish oil triacyl-sn-glycerols was carried out by novel high-performance liquid chromatography on a chiral stationary phase. The positional distributions of fatty acids were determined without accumulation of errors in a particular position and preferential hydrolysis for a particular fatty acid. High-resolution gas-liquid chromatography on an open tubular column detailed the distribution of unsaturated fatty acid isomers having olefinic bonds in different positions. The distribution of fatty acids was not independent of other fatty acids. The distribution of long-chain highly unsaturated fatty acids 22:6(n-3), 22:5(n-3) and 20:5(n-3) was governed by total amounts of 20:1 and 22:1 in triacyl-sn-glycerols. Long-chain monounsaturated acids 20:1 and 22:1 were influenced by the position of the olefinic bond in 20:1 occurring in triacyl-sn-glycerols. Shorter-chain C14-C18 fatty acids seemed to be influenced by total fatty acid composition. These results introduce a concept of mutual interaction between fatty acids to the investigation of positional distribution of fatty acids.     

Soybean oil triacylglycerol analysis by reversed-phase high-performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

Soybean oil triacylglycerols from genetically modified soybean lines were conclusively identified by reversed-phase high-performance liquid chromatography coupled with mass spectrometry with atmospheric pressure chemical ionization. Atmospheric pressure chemical ionization is a soft ionization technique which gives simple spectra for triacylglycerols. Spectral identification of the triacylglycerols was based on the molecular [M+1]⁺ ion and the 1(2)-, 2(3)- and 1(3)-diacylglycerol fragments. Triacylglycerols identified in high-stearic and high-palmitic soybean varieties were quantitated by reversed-phase high-performance liquid chromatography with flame-ionization detection. There was excellent agreement between the fatty acid composition calculated from the triacylglycerol composition and the fatty acid composition obtained by gas chromatography of the transmethylated oils. The oils of the modified soybean varieties, compared to typical soybean oil, contained increased content of triacylglycerols known to be more oxidatively stable, such as linoleoyloleoylstearoyl, linoleoylpalmitoylstearoyl, and linoleoyldipalmitoyl glycerols, and less triacylglycerols like trilinoleoylglycerol, known to decrease oxidative stability. This study showed that the atmospheric pressure chemical ionization technique is suitable for mass spectral identification of neutral molecules, such as triacylglycerols, which do not contain a chargeable functional group.     

Fractionation of soybean phospholipids by high-performance liquid chromatography with an evaporative light-scattering detector

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from 23 soybean lines with a wide range of fatty acid compositions were resolved into seven fractions by high-performance liquid chromatography (HPLC). Fraction identities were assigned from fatty acid compositions determined by gas chromatography (GC). A mass detector, i.e., an evaporative light-scattering detector, was used for HPLC quantification. The detector response was a power function of PC and PE concentrations. Various correction methods were applied to the detector response to obtain the best agreement between phospholipid (PL) fatty acid compositions determined by GC and that calculated from the corrected HPLC fraction percentages. The corrected HPLC fraction composition also was compared with that calculated from stereospecific distribution data using a 1-random-2-random hypothesis. Correlation between PL-fatty acid and HPLC-fraction percentages showed that genetic modification of soybean oil composition caused changes in PL species, which alter physical properties and may alter the physiological functions of PL in biomembranes.     

Separation of monoacylglycerols by high-performance liquid chromatography on nitrile-bonded phase

Monoacylglycerol molecular species, as their di-3,5-dinitrophenylurethane derivatives, were well separated by normalphase high-performance liquid chromatography on nitrilebonded phase. The peaks emerged in the order 20∶0, 18∶0, 16∶0, 18∶1, 16∶1, 18∶2, and 18∶3. The peaks of 1- and 2-monoacylglycerols with the same acyl group showed complete overlapping. This method could be applied to get acyl compositions in the three positions of triacyl-sn-glycerols in their stereospecific analysis. Presented partially at the 83rd AOCS Annual Meeting held in Toronto, Canada, May 10–14, 1992.     

Determination of molecular species of oil triacylglycerols by reversed-phase and chiral-phase high-performance liquid chromatography

A method is described for the determination of molecular species of oil triacylglycerols. The method is based on the analytical separation of the enantiomericsn-1,2-andsn-2,3-diacylglycerols, derived from triacylglycerols, by high-performance liquid-chromatography (HPLC) on a chiral column containing N-(R)-1-(α-naphthyl)ethylaminocarbonyl-(S)-valinecarbonyl-(S)-valine as stationary phase. Model triacyl-glycerol molecules comprising three known fatty acids were isolated from peanut oil and cottonseed oil by a combination of argentation-TLC and reversed-phase HPLC and submitted to partial chemical deacylation. The derivedsn-1,2(2,3)-diacylglycerols were analyzed and fractionated as 3,5-dinitrophenyl urethane derivatives by reversed-phase HPLC according to chainlength and unsaturation. From thesn-1,2(2,3)-diacylglycerol composition and the diacylglycerolsn-1,2-andsn-2,3-enantiomer composition, the individual molecular species of four peanut oil triacylglycerols and one cottonseed oil triacylglycerol were identified and quantitated. The method can be applied to triacylglycerols of any other oil or fat.     

Regioselective analysis of the fatty acid composition of triacylglycerols with conventional high-performance liquid chromatography

A new method for regioselective analysis of triacyglycerols via conventional high-performance liquid chromatography (HPLC) has been developed. The method is simple and avoids the time-consuming purification processes normally characteristics of regioselective analyses. The procedure utilizes an sn-1,3-specific lipase from Rhizopus arrhizus to deacylate the fatty acid residues located at the sn-1 and sn-3 positions of triacylglycerols. The fatty acid residues esterified at the sn-2 position are determined by subtraction of the results of the sn-1,3 analysis from an overall composition analysis based on complete saponification of the original sample. The fatty acid mixtures are converted to p-bromophenacyl esters and analyzed using conventional HPLC techniques. The analytical procedure has been verified using a standard structured triacylglycerol. The analytical results for three edible vegetable oils are compared with those obtained via the method proposed by P.J. Williams and co-workers.     

Stereospecific analysis of triacylglycerols from vegetable oils by two procedures—II: Normal and high-oleic sunflower oils

Triacylglycerol stereospecific analysis of normal (NOS) and high-oleic sunflower (HOS) oils was carried out by two procedures to study the influence of variety and growing conditions. Four cultural varieties, two NOS and two HOS, were grown in seven different places of Italy. Three of the four varieties were grown both in dry conditions and with irrigation. Concerning the triacylglycerol fatty acid compositions, the results showed no significant differences between irrigated and nonirrigated samples (P>0.05), between the two NOS, and between the two HOS varieties. Between NOS and HOS varieties, only stearic acid showed no significant differences (P>0.05). The fatty acid compositions of the sn-2 position of NOS and HOS samples showed different percentage abundances (P<0.01), especially for oleic and linoleic acids. Fatty acid distributions in the sn-1 and sn-3 positions indicated a certain asymmetry. The relationships between the percentage intrapositional content of each acid (one sn-position at a time) and its percentage content in the original triacylglycerol matrix were studied. A general regression model was used to verify if the content of each acid at the three stereospecific positions changed at the same rate as the content in the intact triacylglycerols. The interpositional compositions of all varieties of NOS and HOS oils showed analogous trends for each acid.     

Enantiomeric resolution of diacylglycerol derivatives by high-performance liquid chromatography on a chiral stationary phase at low temperatures

Separation of 1,2-diacylglycerol (DG) enantiomers as their 3,5-dinitrophenylurethane derivatives by high-performance liquid chromatography on a chiral column (Sumichiral OA-4100) was significantly improved at low temperature,e.g., at −30°C. A linear relationship between logarithmic retention volumes and the number of carbons and olefinic bonds of the DG enantiomers was obtained. The effects of temperature on the separation are discussed.     

Stereospecific analysis of triacyl-sn-glycerols by chiral high-performance liquid chromatography

A method for the stereospecific analysis of triacyl-sn-glycerols by high-performance liquid chromatography (HPLC) on a chiral column is described. Triacyl-sn-glycerols were partially degraded with ethyl magnesium bromide, and the monoacylglycerols produced were separated as 1- and 2-monoacylglycerol fractions by thin-layer chromatography on boric acid-impregnated silica gel plates. The 1-monoacylglycerols were resolved intosn-1 andsn-3 monoacylglycerol fractions by HPLC on a chiral column (Sumichiral OA-4100) after derivatization with 3,5-dinitrophenyl isocyanate. Fatty acid methyl esters obtained from the original triacyl-sn-glycerols, 1- and 2-monoacylglycerol fractions, andsn-1 andsn-3 monoacylglycerol fractions were analyzed by gas chromatography on open-tubular columns. Stereospecific acyl distributions in triacyl-sn-glycerols were calculated from the data. The acyl distributions of several oils were obtained. The method is rapid, simple and gave reproducible results.     

Silver ion high-performance liquid chromatography of the triacylglycerols ofCrepis alpina seed oil

The triacylglycerols ofCrepis alpina oil were characterized because this oil has a high concentration of crepenynic (cis-9-octadecen-12-ynoic) acid, a fatty acid useful in the chemical synthesis of deuterated fats for human metabolism studies. The triacylglycerols were separated from the crude oil by solid-phase extraction. Resolution, quantitation and isolation of the individual triacylglycerols were performed by silver ion high-performance liquid chromatography on a commercial column, an acetonitrile in hexane isocratic mobile phase and flame-ionization detection. Isolated triacylglycerols were identified by capillary gas chromatography of their fatty acid methyl esters. Of the eleven eluted triacylglycerols ofCrepis alpina oil, 85% included 35% tricrepenynoyl, 34% linoleoyldicrepenynoyl and 16% dilinoleoylcrepenynoyl glycerols. Triacylglycerols eluted according to the numbers of alkene and alkyne bonds. Elution times, resolution and quantitation were reproducible over a three-month period. The flame-ionization detector response required no response factors for quantitation of the triacylglycerols present inCrepis alpina oil. The silver ion chromatography system permitted the identification of 95% of the triacylglycerols compared to 70% of the triacylglycerols previously identified with reversed-phase high-performance liquid chromatography.     

Separation and purification of six isoflavones from Iris tectorum Maxim by macroporous resin-based column chromatography coupled with preparative high-performance liquid chromatography

ABSTRACT

A rapid and robust preparation method of six isoflavones from ethanol aqueous extract of Iris tectorum Maxim (I. tectorum) was established by using macroporous resin column chromatography and preparative high-performance liquid chromatography (Prep-HPLC). After separation by AB-8 resins, total flavonoids content increased from 10.60% in the crude extract to 54.20% with a recovery yield of 75.12%. Subsequently, the extracts were purified by Prep-HPLC, and the purities were more than 82.2% after assessment by analytical HPLC and characterization by mass spectrometry. The established method is expected to be used for preparing available quantities of pure isoflavones from I. tectorum.     

Separation of wax esters from olive oils by high-performance liquid chromatography

To detect adulteration of olive oil with solvent-extracted oils, the determination of the wax ester content has become more important during recent years. Hence, a greater number of wax ester analyses need to be performed by quality control laboratories. The most common method in use requires a liquid chromatographic (LC) separation of the less polar fraction, which contains the wax esters, from the glyceride matter on a hand-filled silica gel column. The aim of this project was to verify the possibility of replacing LC with high-performance liquid chromatography by taking advantage of the greater reliability and repeatability of this technique, as well as of the possibility of making the separation automatic. The paper describes how to perform the analysis and the statistical test that was carried out; furthermore, a comparison has been made with the usual method and results are in good agreement.     

Comparison of analyses of surfactants in cosmetics using high-performance liquid chromatography and high-performance capillary electrophoresis

Separation of anionic, cationic, and amphoteric surfactants containing n-dodecyl groups and hydrophilic moieties was done by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE), and an ultraviolet-visible detector. Quantitation of surfactants in commercial cosmetic and toiletry products was also done using similar methods. Conditions used to separate mixtures of surfactants by HPLC are as follows: stationary phase: ODS 2; mobile phase: MeOH/H₂O (80∶20, vol/vol) containing 1.0M NH₄Cl, 0.003 M tetrabutylammonium hydrogen sulfate, and 0.005 M diammonium phosphate buffer solution at pH=6.0. Conditions for HPCE are as follows: an uncoated capillary (100 μm i.d., 110 cm in length, effective length of 75 cm) with 25 kV of applied voltage, and an aqueous buffer containing 80 mM sodium borate/20 mM NaOH at pH=9.2. Surfactants were eluted within 15 min. The accuracy of both techniques was evaluated by analyzing the recovery ratio of surfactants in the mixture.     

Quantitative analysis of phospholipids from whey protein concentrates by high-performance liquid chromatography with a narrow-bore column and an evaporative light-scattering detector

A procedure for separation and quantitative determination of phospholipid classes by high-performance liquid chromatography with a narrow-bore column and a light-scattering detector was developed. Cerebrosides (CER), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomeyelin (SPH), and lysophosphatidylcholine (LPC) were base-line resolved, and column life was improved due to low back pressure and low alkalinity of the solvents. Solvent consumption was reduced by 80%, and the detection limit was improved more than tenfold, compared to an analytical column. A gradient elution with pH modifier was necessary for good resolution of acidic phospholipids. A binary solvent system, consisting of A: chloroform/methanol, 80:20 and B: chloroform/methanol/water/ammonium hydroxide (20%), 60:34:6:0.25, was used. Analysis was completed in 36 min, and repeated injections of the samples were possible. The method was applied to the analysis of phospholipids from whey protein concentrates (WPC). Phospholipids in WPC (75% protein) contained (%, w/w) 3.57±0.13 CER; 18.13±1.23 Pl; 4.45±0.21 PE; 7.45±0.58 PS; 30.54±1.84 PC; 35.82±1.16 SPH; and no detectable LPC.     

Nuclear magnetic resonance spectroscopy and reversed-phase high-performance liquid chromatography of peracetylated digalactosyldiacylglycerols

A simple method for the peracetylation of digalactosyldiacylglycerols is described. The peracetylated compounds were isolated by thin-layer chromatography and subjected to nuclear magnetic resonance spectroscopy for structure elucidation. The proton signals at acetylated sites are shifted downfield compared to the nonderivatized compounds, resulting in improved resolution. The peracetylated compounds were also subjected to molecular species analysis by reversed-phase high-performance liquid chromatography. The chromatography behavior of the peracetylated compounds was similar to that of triacylglycerols, displaying narrow and symmetrical peaks yielding a highly resolved molecular species profile.     

Separation of five bioactive compounds from Glycyrrhiza uralensis Fisch using a general three-liquid-phase flotation followed by preparative high-performance liquid chromatography

ABSTRACT

Glycyrrhiza uralensis

Fisch is homologous medicine for food plant and health-protective affection in the human body system. To further understand the functions of G. uralensis Fisch, separation of the bioactive compounds from this natural product is very important. A general three-liquid-phase flotation (TLPF) technique was established to separate and concentrate five bioactive compounds from G. uralensis Fisch. The three-liquid-phase system, pH of initial aqueous phase, (NH₄)₂SO₄ concentration, nitrogen flow rate, and flotation time were investigated. The flotation efficiency of liquiritin apioside, liquiritin, and glycyrrhizic acid in the middle phase and semilicoisoflavone B and licoisoflavone B in the upper phase could reach 91.24%, 94.23%, 88.04%, 94.67%, and 96.44%, respectively. Furthermore, the established TLPF was used for separation of four kinds of anthraquinones from Cassiae Semen. The results showed that TLPF is feasible for separating the compounds with quite a different polarity from natural products.     

薄层色谱分析在层析分离过程中的应用

介绍了薄层色谱分析方法对层析分离过程的控制性分析和优化作用。