Preparation of fatty acid methyl esters by direct transesterification of lipids with aluminium chloride-methanol

A new and simple procedure has been developed that allows the direct transesterification of lipids, using aluminium chloride as a catalyst and methanol as the esterifying alcohol. The concentration of the salt and reaction conditions have been investigated for the different lipid classes. Comparative studies, performed with boron trifluoride-methanol, indicate that the same values are obtained when using either reagent. In addition, the method has been adapted for transesterification in the presence of silica gel and other adsorbents, thus allowing the preparation of fatty acid methyl or ethyl esters directly from samples previously fractionated by thin-layer chromatography. This new reagent is very stable and easy to handle, the fatty acids being generated in the same tube without further purification steps.     

Determination of lipids in infant formula powder by direct extraction methylation of lipids and fatty acid methyl esters (FAME) analysis by gas chromatography.

Fatty acid methyl esters (FAMEs) of pure triglyceride standards, oils, and fat from dry matrixes were formed by transesterification using sodium methoxide in methanol-hexane. FAMEs were produced by direct addition of sodium methoxide-hexane to samples and heating to simultaneously extract and transesterify acyl lipids. FAMEs were quantitated by capillary gas chromatography (GC) over a fatty acid concentration range of 0 to 1.7 mg/mL (r > or = 0.9997). Total fat was calculated as the sum of individual fatty acids expressed as triglyceride equivalents, in accordance with nutrition labeling guidelines. Saturated, polyunsaturated, and monounsaturated fats were calculated as sums of individual free fatty acids. Absolute recoveries determined from individual fatty acids in test samples ranged from 69.7 to 106%. Recoveries (relative to the C13:0 internal standard) for individual fatty acids in test samples ranged from 95 to 106%. Reproducibility was constant at each fatty acid level in the reaction mixture (n = 5, coefficient of variation [CV] < 2%). Absolute recovery determined from the sum of total fatty acids in standard reference material (SRM) 1846 (powdered infant formula) was 96.4%. Analysis of SRM 1846 gave results that agreed closely with the certified fat and fatty acid values. Analysis of commercial infant formula gave results that were comparable to those obtained with AOAC Method 996.01. The direct extraction methylation procedure is rapid, and the transesterification of acyl lipids to form FAMEs is complete within 15 min. Classical saponification and refluxing are not required. This method provides FAMEs free of interferences and easily quantitated by GC or confirmed by GC/mass spectrometry (MS). Unambiguous MS identification of individual FAMEs derived from pure standards, SRM 1846, and powdered infant formula product was obtained.     

Characterization of fatty acid methyl esters by thermal analysis

The thermal stability of selected straight-chain (C₆-C₁₄) esters of fatty acids has been studied by TG-DTG and DTA analysis. In DTG, a peak is detected between 84° and 125° C followed by a main effect in the range 105°–215°C, whereas in DTA only an exothermic peak appears in the range of 126.5° to 187°C (onset temperatures). The temperatures of these effects have been related with ignition points, molecular weights and boiling points. The characteristics of melting and recrystallization of the above fatty acid methyl esters and those with carbon numbers between C₁₄ and C₂₄ have been established by DSC along the melting range between ?83° and 50°C. Polymorphism appears in caproic, heptanoic, palmitic and stearic acid methyl esters.     

Enantiomeric separation of various lipoxygenase derived monohydroxy polyunsaturated fatty acid methyl esters by high-performance liquid chromatography

Lipoxygenase derived monohydroxy polyunsaturated fatty acid methyl esters were separated by chiral high-performance liquid chromatography (HPLC) with a Chiralcel OD-H column in the normal-phase mode. Major lipoxygenase derivatives of linoleic, -linolenic and arachidonic acids are well resolved by this column, provided they have been individually purified. Our method allows an easy and rapid determination of lipoxygenases enantioselectivity. In all cases tested the R enantiomer is eluted first.     

Separation of triacylglycerols and free fatty acids in microalgal lipids by solid-phase extraction for separate fatty acid profiling analysis by gas chromatography

Microalgal lipids were separated into two fractions, triacylglycerols (TAGs) and free fatty acids (FFAs), by solid-phase extraction employing sodium carbonate as the sorbent and dichloromethane (20% by volume) in n-hexane as the extracting solvent. The TAG fraction was then saponified, followed by acidification, extraction and tert-butyldimethylsilyl esterification. The FFA fraction was directly acidified, extracted and derivatized. From the lipid extracts of eight microalgal species examined, a total of 13 fatty acids were detected in the TAG fractions and nine were found in the FFA fractions, with at much higher total TAG content in all microalgae. Oleic acid was the most prominent fatty acid in three species, α-linolenic acid was more abundant in two others, and palmitic acid was present in highest concentration in the remaining three species.     

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.     

Identification of fatty acid methyl esters as minor components of fish oil by multidimensional GC-MSD: New furan fatty acids

The separation and analysis of furan fatty acids and other minor component fatty acids present at very low concentrations in complex sample matrices, such as fish oil or lipids derived from liver and testes, require several pre-analytical separation steps if single column gas chromatography is to furnish sufficient resolution: after extraction and transesterification hydrogenation, urea complex precipitation and argentation TLC have been applied prior to GC analysis of furan fatty acids. By using multidimensional GC-MSD with cooled injection and flow-controlled column switching with intermediate cold trapping, it has been possible to identify directly the methyl esters of furan fatty acids without further pre-analytical separation. The most common of the furan fatty acids can be subdivided into two groups depending on whether they bear a propyl or pentyl side group in the 5-position of the furan ring. In addition to the eight furan fatty acids known to be present in fish oil, six new ones were identified, four with propyl substitution and two with pentyl substitution. Four have earlier been reported to be present in the hepatopancreas of crayfish and in fish tissue, whereas the propyl-substituted 16,19-epoxy-17,18-dimethyldocosa-16,18-dienoic acid and the pentyl-substituted furan fatty acid 6,9-epoxy-7-methyltetradeca-6,8-dienoic acid were hitherto unknown.     

气相色谱法测定生物柴油中脂肪酸甲酯含量

生物柴油是利用动植物油脂等可再生资源通过酯交换技术制造的可以替代石化柴油的新型清洁安全燃料[1-3]它的主要成分是脂肪酸甲酯。由于不同油脂原料所生产的生物柴油的脂肪酸甲脂组成不同因而测定时所需的气相色谱条件与方法也不尽相同[4-6]。本文采用HP-innowax毛细管色谱柱,     

Separation characteristics of fatty acid methyl esters using SLB-IL111, a new ionic liquid coated capillary gas chromatographic column

The ionic liquid SLB-IL111 column, available from Supelco Inc., is a novel fused capillary gas chromatography (GC) column capable of providing enhanced separations of fatty acid methyl esters (FAMEs) compared to the highly polar cyanopropyl siloxane columns currently recommended for the separation of cis- and trans isomers of fatty acids (FAs), and marketed as SP-2560 and CP-Sil 88. The SLB-IL111 column was operated isothermal at 168°C, with hydrogen as carrier gas at 1.0 mL/min, and the elution profile was characterized using authentic GC standards and synthetic mono-unsaturated fatty acids (MUFAs) and conjugated linoleic acid (CLA) isomers as test mixtures. The SLB-IL111 column provided an improved separation of cis- and trans-18:1 and cis/trans CLA isomers. This is the first direct GC separation of c9,t11- from t7,c9-CLA, and t15-18:1 from c9-18:1, both of which previously required complimentary techniques for their analysis using cyanopropyl siloxane columns. The SLB-IL111 column also provided partial resolution of t13/t14-18:1, c8- from c6/c7-18:1, and for several t,t-CLA isomer pairs. This column also provided elution profiles of the geometric and positional isomers of the 16:1, 20:1 and 18:3 FAMEs that were complementary to those obtained using the cyanopropyl siloxane columns. However, on the SLB-IL111 column the saturated FAs eluted between the cis- and trans MUFAs unlike cyanopropyl siloxane columns that gave a clear separation of most saturated FAs. These differences in elution pattern can be exploited to obtain a more complete analysis of complex lipid mixtures present in ruminant fats.     

Quantification of primary fatty acid amides in commercial tallow and tallow fatty acid methyl esters by HPLC-APCI-MS

Primary fatty acid amides are a group of biologically highly active compounds which were already identified in nature. Here, these substances were determined in tallow and tallow fatty acid methyl esters for the first time. As tallow is growing in importance as an oleochemical feedstock for the soap manufacturing, the surfactant as well as the biodiesel industry, the amounts of primary fatty acid amides have to be considered. As these compounds are insoluble in tallow as well as in the corresponding product e.g. tallow fatty acid methyl esters, filter plugging can occur. For the quantification in these matrices a purification step and a LC-APCI-MS method were developed. Although quantification of these compounds can be performed by GC-MS, the presented approach omitted any derivatization and increased the sensitivity by two orders of magnitude. Internal standard calibration using heptadecanoic acid amide and validation of the method yielded a limit of detection of 18.5 fmol and recoveries for the tallow and fatty acid methyl ester matrices of 93% and 95%, respectively. A group of commercially available samples were investigated for their content of fatty acid amides resulting in an amount of up to 0.54%m/m (g per 100 g) in tallow and up to 0.16%m/m (g per 100 g) in fatty acid methyl esters.     

Separation of polychlorinated biphenyls from chlorinated pesticides using aminopropyl bonded-phase cartridge and determination by GC-ECD

Summary Gas chromatography of polychlorinated biphenyls and chlorinated pesticides in water samples is carried out after adsorption from a 25–500 mL sample, on a cartridge containing 100 mg aminopropyl-bonded porous silica. The clean-up step in which the PCBs and chlorinated pesticides are separated in different eluates is achieved by passing 25 mL of 40% aqueous methanol through the NH₂ Sep-Pak cartridge. The PCBs are desorbed with 500 μL ethylacetate, which is concentrated and analysis by GC-ECD. The average recovery, at 1 ppb is >97% with a standard deviation <2. The limits of detection are 0.1 ng μL⁻¹ and 5 pg μL⁻¹ respectively for Cl₃-PCB and Cl₈-PCB congeners. In the separation of PCBs from the chlorinated pesticides tested in this work, only the Aldrin is adsorbed for 60% with the PCBs by the NH₂ Sep-Pak cartridge. The method described is rapid, simple and reproducible.     

The production of artifacts during preparation of fatty acid methyl esters from fish oils,food products and pathological samples

Methyl esters were synthesized from lipid extracted by a modified Bligh and Dyer technique. The lipid was saponified and the free fatty acids methylated using boron trifluoride in methanol. Butylated hydroxytoluene (BHT; 2,6-di-tert-butyl-4-methylphenol) was added to the initial sample and to the extracted lipid prior to methyl ester synthesis. Under these conditions, the BHT was derivatized to a range of compounds, some of which can result in misinterpretation of the GC trace. Three components have been characterized by mass spectroscopy. Two of these, which eluted slightly before 16:0 on a polar column, were shown to be 2,6-di-tert-butyl-4-methoxyphenol and 2,6-di-tert-butyl-4-methoxy-methylphenol. The third component, which coeluted with 15:0 on the same column, is 2,6-di-tert-butyl-4-methoxy-5-hydroxyphenol.     

超高效液相色谱法测定生物柴油中的11种脂肪酸及脂肪酸甲酯

建立了采用超高效液相色谱(UPLC)-蒸发光散射检测器(ELSD)测定生物柴油中11种常见的脂肪酸及脂肪酸甲酯含量的方法。这11种常见的脂肪酸及脂肪酸甲酯为豆蔻酸、亚油酸、棕榈酸、油酸、亚麻酸甲酯、硬脂酸、亚油酸甲酯、棕榈酸甲酯、油酸甲酯、芥酸和硬脂酸甲酯。样品经提取后用甲醇溶解,采用Acquity UPLC BEH Phenyl C18柱(100 mm×2.1 mm,1.7 μm)分离,乙腈-水（体积比为3∶1)混合液为流动相进行等度洗脱,采用的ELSD条件为增益80,漂移管温度为45 ℃,载气压力为172 kPa,雾化器为冷却模式,并用外标法进行定量分析。结果表明,在一定的质量浓度范围内,峰面积的对数和质量浓度的对数线性关系良好。与其他检测生物柴油成分的方法相比,该方法简单,分离效果好,速度快,特别是此方法可以同时实现脂肪酸及脂肪酸甲酯的分离,并进行定量分析,能有效测定反应的进行程度,从而满足生物柴油工艺研究的需要。     

Rapid determination of chlorinated pesticides in fish by freezing-lipid filtration, solid-phase extraction and gas chromatography-mass spectrometry

An analytical method has been developed for measuring 24 chlorinated pesticides in fish tissue samples. Extraction of chlorinated pesticides was carried out by ultrasonication using an acetone-n-hexane (5:2, v/v) mixture. Most of the lipids in the extract were eliminated by freezing-lipid filtration, prior to solid-phase extraction (SPE) cleanup. During freezing-lipid filtration, about 90% of the lipids extracted from the fish samples were easily removed without any significant losses of chlorinated pesticides. For purification, SPE using Florisil was shown to be more effective than silica. Quantification was performed using gas chromatography-mass spectrometry in the selected ion monitoring mode. Spiking experiments were carried out to determine the recovery, precision, and limits of detection (LODs) of the method. The overall recovery was above 80% in the spiked fish tissue sample at 100 ng/g level. The detection limits for chlorinated pesticides were ranged from 0.5 to 5 ppb, except for endosulfan I and II which was 20 ppb. The newly developed method is demonstrated to give efficient recoveries and LODs for detecting chlorinated pesticides spiked into fish tissue with high lipid content.     

Determination of potassium in fatty acid methyl esters applying an ion-selective potassium electrode

An easy and inexpensive method of potassium determination in fatty acid methyl esters (FAME) applying an ion-selective potassium electrode (ISE-K) is presented. FAME are considered alternative fuel for diesel engines. Simple apparatus and procedure were developed to avoid contact of ester samples with the sensitive ISE-K membrane, which can damage the ISE membrane surface in long time operation. Using different FAME samples with a wide range of known potassium content a calibration curve was constructed. Among all samples, ISE-K satisfactory predicted potassium content in FAME as identified by the AAS reference method.     

Rapid identification of fatty acid methyl esters using a multidimensional gas chromatography-mass spectrometry database

A multidimensional approach for the identification of fatty acid methyl esters (FAME) based on GC/MS analysis is described. Mass spectra and retention data of more than 130 FAME from various sources (chain lengths in the range from 4 to 24 carbon atoms) were collected in a database. Hints for the interpretation of FAME mass spectra are given and relevant diagnostic marker ions are deduced indicating specific groups of fatty acids. To verify the identity of single species and to ensure an optimized chromatographic resolution, the database was compiled with retention data libraries acquired on columns of different polarity (HP-5, DB-23, and HP-88). For a combined use of mass spectra and retention data standardized methods of measurement for each of these columns are required. Such master methods were developed and always applied under the conditions of retention time locking (RTL) which allowed an excellent reproducibility and comparability of absolute retention times. Moreover, as a relative retention index system, equivalent chain lengths (ECL) of FAME were determined by linear interpolation. To compare and to predict ECL values by means of structural features, fractional chain lengths (FCL) were calculated and fitted as well. As shown in an example, the use of retention data and mass spectral information together in a database search leads to an improved and reliable identification of FAME (including positional and geometrical isomers) without further derivatizations.     

氨基甲酸酯型脱氧胆酸分子钳对氨基酸甲酯的手性识别 研究

以脱氧胆酸为spacer,通过三光气桥连各种芳胺,合成了新的氨基甲酸酯型分子钳受体1～4,这些化合物的结构经IR,^1HNMR和元素分析所证实。利用差光谱滴定法考察了其与D/L氨基酸甲酯的对映选择性识别性能。结果表明,分子钳1～4对所考察的氨基酸甲酯均具有识别能力,其对D-氨基酸甲酯的识别优于对L-氨基酸甲酯的识别。从主客体间的大小形状匹配及几何互补关系等方面对这些受体的识别能力及对映选择性进行了讨论。