Separation and quantitation of plasma free fatty acids as phenacyl esters by HPLC

We have developed a rapid method for the separation of plasma free fatty acids as their phenacyl esters by high-performance liquid chromatography (HPLC) using a reversed-phase (C18) column. The derivatives of series of both saturated and unsaturated fatty acids (C12:0-C22:6) are simultaneously separated within 45 min and detected with ultraviolet at 241 nm. The limit of detection of fatty acids was approximately 0.5 nmol in 20 microL injected volume of extracts, and the coefficient of variation of the present method did not exceed 3.0%. Comparison of the results of the present HPLC method with those of gas chromatography, gave very good correlations for all fatty acids in human plasma.     

Rapid assay of vitamin E in vegetable oils by reversed-phase capillary electrochromatography

A rapid capillary electrochromatographic (CEC) method for the analysis of vitamin E in vegetable oils is reported. Vitamin E consists of a group of eight isomers, tocopherols (TOHs) and tocotrienols. The separation of four TOHs (alpha-, gamma-, delta-TOH), alpha-tocopherol acetate (alpha-TOH-Ac), and an antioxidant compound, butylated hydroxytoluene (BHT) used to prevent TOH autoxidation, was optimized. The CEC experiments were carried out in a 75 microm inner diameter (ID) fused-silica capillary, partially packed with 3 microm C(18 )stationary phase (33 cm total length, 8.4 cm and 7 cm effective and packed lengths, respectively). The optimum mobile phase was a polar organic phase composed of a mixture of methanol-acetonitrile in the ratio 50/50 v/v containing 0.01% ammonium acetate, applying a voltage and temperature set at -25 kV and 20 degrees C, respectively. The tocopherols and the BHT were successfully separated within 2.5 min using the short-end injection method. Under these experimental conditions, repeatability of retention time and peak area, analyte detection and quantitation limits, linearity, precision, and accuracy were studied. The CEC method was applied to determine the content of TOHs in different commercially available oils of virgin olive, hazelnut, sunflower, and soybean. The extraction of vitamin E isomers from oil samples was achieved using methanol and a methanol-isopropanol mixture.     

Analysis of free and esterified sterols in vegetable oil methyl esters by capillary GC

The introduction of quality standards for vegetable oil methyl esters is gaining in importance due to their increased use as diesel fuel substitutes and as technical products. Free and esterified sterols, the main constituents of the unsaponifiable matter in vegetable oils, are recovered in vegetable oil methyl esters and may influence the technical properties of vegetable oil methyl ester products. A rapid gas chromatographic method for the qualitative and quantitative determination of free and esterified sterols in vegetable oil methyl esters has therefore been developed. The concentration of the free sterols as well as their qualitative and quantitative composition and the concentration of the sterol esters have been determined in rape seed oil methyl ester samples by GC–FID. Prior to analysis, the free sterols were silylated with N,O-bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane; betulinol was used as an internal standard. Calibration was performed by analysis of standard solutions containing β-sitosterol, cholesteryl stearate, and betulinol. The reproducibility of the quantitative results has been evaluated by repeated injections of the same test solution and by repeated complete analysis of the same sample.     

Capillary electrochromatography of sterols and related steryl esters derived from vegetable oils

Capillary electrochromatographic (CEC) separations of plant sterols and related esters were evaluated under various conditions. Stationary phases included octadecylsilica (C18) and triacontylsilica (C30). Mobile phases comprised acetonitrile, tetrahydrofuran, and tris(hydroxymethyl) aminomethane buffers in aqueous or non-aqueous systems. Apart from notable differences in component resolution, both C18 and C30 phases had dramatic influence on the elution behavior of the title compounds. Generally, C18 had greater selectivity for most components with elution patterns in consistence with the hydrophobicity of side chain structures, while no predictable trend of analyte elution was observed in CEC with C30. In the latter column systems, analyte separations appeared to be improved by conversion to benzoates or ferulates. Twenty-four-epimers of campesterol acetate and 7-campestenol acetate as well as the campesterol-stigmasterol pair were readily resolved by CEC with either phase. However, the cholesterol-stigmasterol pair was barely resolved and had an elution order opposite to that of their acetates or benzoates. Potential applicability of the CEC technique in the analysis of sterols and sterol ferulates in vegetable oil is demonstrated.     

Quantitation of phenacyl esters of retinal fatty acids by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the separation and quantitation of retinal fatty acids containing long-chain polyunsaturated fatty acids is described. Fatty acids from frog retinal lipids were converted to the corresponding phenacyl derivatives which were separated on a C18 reversed-phase column and detected at 242 nm. Molar absorptivities (peak area units/nmol) of up to seventeen fatty acid phenacyl derivatives were determined and used for quantitation of fatty acids separated by HPLC. Compared with gas chromatography, the HPLC method gave a similar molar percent distribution of the fatty acids and was twenty to fifty times more sensitive. This HPLC method provides a useful means for the study of chemistry and metabolism of long-chain polyunsaturated fatty acids in retina and other tissues where amounts of material may be limited or recovery of individual components desirable.     

Acrylate ester-based monolithic columns for capillary electrochromatography separation of triacylglycerols in vegetable oils

A simple and reliable method for the evaluation of triacylglycerols (TAGs) in vegetable oils by capillary electrochromatography (CEC) with UV-Vis detection, using octadecyl acrylate (ODA) ester-based monolithic columns, has been developed. The percentages of the porogenic solvents in the polymerization mixture, and the mobile phase composition, were optimized. The optimum monolith was obtained at the following ratios: 40:60% (wt/wt) monomers/porogens, 60:40% (wt/wt) ODA/1,3-butanediol diacrylate and 23:77% (wt/wt) 1,4-butanediol/1-propanol (14 wt% 1,4-butanediol in the polymerization mixture). A satisfactory resolution between TAGs was achieved in less than 12 min with a 65:35 (v/v) acetonitrile/2-propanol mixture containing 5 mM ammonium acetate. The method was applied to the analysis of TAGs of vegetable oil samples. Using linear discriminant analysis of the CEC TAG profiles, the vegetable oils belonging to six different botanical origins (corn, extra virgin olive, hazelnut, peanut, soybean and sunflower) were correctly classified with an excellent resolution among all the categories.     

Determination of free fatty acids by capillary zone electrophoresis

Capillary zone electrophoresis was investigated for the separation of free fatty acids as an alternative to well established techniques such as GC or HPLC. The analysis was performed with indirect UV detection in a counterelectroosmotic flow mode using a diethylbarbiturate carrier electrolyte at a pH between 10 and 11 in a mixed aqueous-organic solvent. Separation of saturated and unsaturated fatty acids could be achieved after bromination of the double bonds. Problems with wall adsorption of fatty acids could be overcome by increasing the temperature and using a high concentration of a zwitterionic reagent to inactivate the silica surface. Increased sensitivity could be achieved after preconcentration on alumina. The method was applied to the determination of free fatty acids in dairy products. The advantages compared to traditional methods include short analysis times and simple preparation steps.Dedicated to Univ. Prof. Dr. Karl Winsauer on the occasion of his 70th birthday     

Flow injection determination of free fatty acids in vegetable oils using capacitively coupled contactless conductivity detection

A single line flow injection analysis (FIA) method that incorporated a preconcentrator column packed with C(18) particles and capacitively coupled contactless conductivity detector (C(4)D) was developed for the determination of free fatty acid (FFA) in vegetable oils. The carrier stream was methanol/1.5 mM sodium acetate (pH 8) 80:20 (v/v) at a flow rate of 1.0 mL min(-1). Calibration curve was well correlated (r(2)=0.9995) within the range of 1-200 mg L(-1) FFA (expressed as palmitic acid). Sampling rate of 40-60 h(-1) was achieved. Good agreement was found between the standard non-aqueous titrimetry method and the proposed method when applied to the determination of FFA in palm (crude, olein, and refined, bleached and deodorised) and other vegetable (soybean, rice bran, walnut, corn and olive) oils. The proposed method offers distinct advantages over the official method, especially in terms of simplicity, high sampling rate, economy of solvents and sample, offering considerable promise as a low cost automated system that needs minimum human intervention over long periods of time.     

Separation of the insecticidal pyrethrin esters by capillary electrochromatography

A simple, rapid technique for the direct separation and quantification of the six insecticidally active pyrethrin esters in typical extracts and commercial formulations by capillary electrochromatography (CEC) has been described. The separation of the pyrethrin esters was achieved by optimizing several parameters including the length of stationary phase, the mobile phase composition and column temperature. The mobile phase composition had the most pronounced effect toward resolving these structurally similar compounds. A ternary mobile phase composed of acetonitrile-aqueous buffer-tetrahydrofuran (55:35:10) provided the elutropic solvent strength needed to resolve the six esters from an extract mixture in under 16 min. A 25 cm packed bed of Hypersil 3 microm C18 stationary phase was used with the ternary mobile phase at 25 degrees C and 30 kV voltage. These conditions also yielded excellent separation of the pyrethrin esters in two different commercially available insecticidal formulations. In addition, the developed CEC method was shown to be a fast and easy way of quantifying the amount of these esters in typical pyrethrin formulations.     

Specific process features of epoxidation of fatty acid methyl esters of vegetable oils with performic acid generated in situ

Physicochemical relationships of the phase distribution of the reactants in epoxidation of fatty acid methyl esters of vegetable oils with performic acid generated in situ were determined.. The progress of epoxidation is accompanied by redistribution of the organic and aqueous phase volumes. The major cause of changes in the phase volumes is the transfer of peroxide oxygen from the aqueous phase into the organic phase upon formation of epoxy groups. A comparable change in the phase volumes is due to reactive extraction of aqueous phase components with the epoxy compounds formed.     

A microfluidic device for the automated derivatization of free fatty acids to fatty acid methyl esters

Free fatty acid (FFA) compositions are examined in feedstock for biodiesel production, as source-specific markers in soil, and because of their role in cellular signaling. However, sample preparation of FFAs for gas chromatography-mass spectrometry (GC-MS) analysis can be time and labor intensive. Therefore, to increase sample preparation throughput, a glass microfluidic device was developed to automate derivatization of FFAs to fatty acid methyl esters (FAMEs). FFAs were delivered to one input of the device and methanolic-HCl was delivered to a second input. FAME products were produced as the reagents traversed a 29 μL reaction channel held at 55 °C. A Design of Experiment protocol was used to determine the combination of derivatization time (T(der)) and ratio of methanolic-HCl:FFA (R(der)) that maximized the derivatization efficiencies of tridecanoic acid and stearic acid to their methyl ester forms. The combination of T(der) = 0.8 min and R(der) = 4.9 that produced optimal derivatization conditions for both FFAs within a 5 min total sample preparation time was determined. This combination of T(der) and R(der) was used to derivatize 12 FFAs with a range of derivatization efficiencies from 18% to 93% with efficiencies of 61% for tridecanoic acid and 84% for stearic acid. As compared to a conventional macroscale derivatization of FFA to FAME, the microfluidic device decreased the volume of methanolic-HCl and FFA by 20- and 1300-fold, respectively. The developed microfluidic device can be used for automated preparation of FAMEs to analyze the FFA compositions of volume-limited samples.     

Separation of phenylthiohydantoin amino acids by capillary electrochromatography

Capillary electrochromatography (CEC) was employed as a rapid and high-efficiency method for the isocratic separation of all 20 important phenylthiohydantoin (PTH) amino acids, the end products of Edman degradation during N-terminal protein sequencing. For this purpose, 75 microm ID fused-silica capillaries were packed with standard 3 microm Hypersil octadecyl silica (ODS) particles using a two-step column fabrication process, which represents a fast, reliable and efficient means of producing long-term stable columns. The influence of solvent composition, pH, type of buffer cation, buffer concentration, and temperature on retention behavior of PTH amino acids was investigated. Same-day and day-to-day reproducibility of the retention times (over a period of two months) were found to be better than 3%. When comparing this new technique with traditional reversed phase-high performance liquid chromatography (RP-HPLC) methods applied in automated protein sequenators, CEC shows essentially shorter separation times and superior resolution.     

Quantitative analysis of fatty acid methyl esters and dimethyl acetals on a polar (free fatty acid phase) capillary column

Separation of fatty acid methyl esters and dimethyl acetals from complex biological samples has been achieved by gas-liquid chromatography on a capillary column coated with free fatty acid phase. Response-correcting factors were determined, showing rather large variations with fatty acid length. Polyunsaturated fatty acid methyl esters were shown to have lower responses than saturated species, whereas dimethyl acetals and equivalent methyl esters were found to give similar responses. Total fatty acid and aldehyde compositions of human and simian erythrocytes were determined and compared, showing a somewhat higher level of linoleate and arachidonate, and a lower level of plasmalogens in simian erythrocytes.     

超高效合相色谱-串联质谱法测定食用植物油和油条中15种3-氯-1,2-丙二醇脂肪酸酯

建立了超高效合相色谱-三重四极杆质谱(UPC²-MS/MS)测定食用植物油和油条中15种3-氯-1,2-丙二醇脂肪酸酯(3-MCPDE)含量的方法。3-MCPDE的结构与甘油酯极为相似,因此很难将其从植物油中分离出来。为降低基质干扰,实验以不同极性的溶剂依次洗脱载有样品的氨基填料层析柱,用UPC²和ACQUITY QDa质谱检测器分析每单元洗脱液以绘制洗脱曲线。分段收集的洗脱液经混合、浓缩和过滤后,以Viridis HSS C18 SB色谱柱(150 mm×2.1 mm, 1.8μm)为分析柱,超临界CO₂和40%乙腈甲醇溶液(含0.1%甲酸)为流动相梯度洗脱,以97%异丙醇水溶液(含0.2%氨水)为补偿液,用配备电喷雾电离源的三重四极杆质谱在正离子、多级反应监测模式下测定,外标法定量。结果表明:15种3-MCPDE在各自范围内线性关系良好(相关系数(r²)≥0.997 3),检出限为0.01～0.68μg/L(S/N=3),定量限为0.04～1.74μg/L(S/N=10);在3个加标水平下的平均...     

Analysis of oxygen heterocyclic compounds in citrus essential oils by capillary electrochromatography and comparison with HPLC

Summary A new method for the analysis of oxygen heterocyclic compounds in bergamot, mandarin and sweet organge oils by capillary electrochromatography (CEC) is reported. The fused silica capillaries employed were slurry-packed in house with 3 μm ODS1 stationary phase. The repeatability of the technique has been investigated with two neutral standard mixtures. Conditions for the analysis have been optimised by varying several parameters such as applied voltage, temperature, composition of the buffer and percentage of organic solvent. The best separations were obtained using the buffer acetonitrile/TRIS 10 mM at pH 7.8 in the ratio 80∶20. The results were compared with those obtained by high performance liquid chromatography (HPLC) in terms of time of analysis, resolution and efficiency. A study on the behaviour of oxygen heterocyclic compounds analysed with a “non-aqueous” mobile phase has also been carried out.     

超高效合相色谱-质谱法快速分析食用植物油中常见脂肪酸

建立了超高效合相色谱-质谱(UPC²-MS)快速分析6种食用植物油(玉米油、葵花籽油、大豆油、茶油、菜籽油、花生油)中棕榈酸、硬脂酸、油酸、亚油酸、亚麻酸等5种常见脂肪酸的方法,并比较了这6种食用油中上述5种脂肪酸的含量差异。采用皂化反应对植物油进行前处理,以ACQUITY UPC² BEH 2-EP色谱柱(100 mm×2.1 mm, 1.7 μm)为分析柱,以超临界CO₂-甲醇/乙腈(1:1, v/v)为流动相进行梯度洗脱,流速为0.8 mL/min。在电喷雾负离子模式下进行检测,外标法定量。结果表明:5种脂肪酸标准物质在0.5~100 mg/L范围内呈现良好的线性关系,相关系数为0.9985~0.9998,定量限(S/N≥10)为0.15~0.50 mg/L;在3个添加水平下,样品的加标回收率为89.61%~108.50%;方法重复性的相对标准偏差(RSD)为0.69%~3.01%。该方法简单、快速、分离效果好,无需对脂肪酸样品进行衍生化,已成功地用于玉米油、葵花籽油、橄榄油、茶油、大豆油和花生油等6种食用油中常见脂肪酸含量的测定。     

Determination of short-chain free fatty acids in lipolyzed milk fat by capillary electrophoresis

The objective of this study was to monitor the release of short-chain free fatty acids (FFA) from milk fat during hydrolysis with lipase using capillary electrophoresis. Sample and run buffer allowed FFA to be maintained in solution by using cyclodextrin and methanol. Indirect UV detection at 270 nm was used, employing p-anisate as a chromophore. Calibration curves constructed for each individual FFA followed linear relationships with highly significant (p < 0.01) correlation coefficients. Electrophoretic FFA profiles of fresh milk fat and lipolyzed milk fat showed marked qualitative and quantitative differences. Butanoic acid (C4) was found in a concentration of 64 ppm, while hexanoic (C6) and octanoic (C8) acids were found in concentrations of 3.8 ppm in fresh milk fat. After a 60-min hydrolysis with commercial lipase, FFA released from milk fat consisted mainly of high concentrations (ppm) of butanoic (C4) (900), followed by hexanoic (C6) (427), octanoic (C8) (282), decanoic (C10) (92), pentanoic (C5) (47), and dodecanoic (C12) (37.5) acids. Ratios of FFA that were associated with flavor balance were calculated. The application of CE for lipolysis monitoring in milk fat offers a simple and fast method for the determination of FFA. Quantitative data can be obtained in 20 min, including sample preparation. The lengthy and laborious steps required in traditional chromatographic techniques, such as lipid extraction, FFA isolation, and derivatization, were not required in this CE method. The implementation of CE for milk fat lipolysis monitoring may be a useful quality control tool for dairy flavor development and production.     

Profiling fatty acids in vegetable oils by reactive pyrolysis-gas chromatography with dimethyl carbonate and titanium silicate

A novel methodology in on-line pyrolysis-gas chromatography (Py-GC) for the fast analysis of fatty acids in vegetable oils with minimal sample treatment and the use of non-toxic reagents is described. Pyrolysis at 500 degrees C for 10 s of sub-microgram quantity of vegetable oil dissolved in dimethyl carbonate (DMC) and in the presence of nanopowder titanium silicon oxide resulted in the production of fatty acid methyl esters (FAMEs) as unique products. Pyrolysis performed by means of a resistively heated filament pyrolyser interfaced to a GC-MS apparatus enabled the direct analysis of evolved FAMEs. The DMC/Py-GC-MS analysis was tested on soybean, coconut, linseed, walnut and olive oil and the results compared to the classical BF(3)-methanol as reference methodology. The DMC method exhibited a lower precision and was biased towards lower levels of polyunsaturated fatty acids (PUFA) in comparison to the BF(3)-methanol method, but was more advantageous in terms of reduced sample treatment, waste generation and risk factors of employed chemicals.     

On-line hydrogenation of fatty acid methyl esters in capillary gas chromatography

An injection splitter in front of a glass capillary column was used for the hydrogenation of fatty acid methyl esters (FAME) mixtures. Hydrogenation followed by gas chromatographic analysis on capillary columns permitted detection and identification in complicated natural mixtures of branched fatty acids, showing minor structural differences, in quantities down to 10^?8g. The technique described, apart from its suitability for FAME analysis, shows promise for structure determination studies of other classes of compounds.