Evaluation of ionic liquid stationary phases for one dimensional gas chromatography-mass spectrometry and comprehensive two dimensional gas chromatographic analyses of fatty acids in marine biota

Ionic liquid stationary phases were tested for one dimensional gas chromatography-mass spectrometry (GC-MS) and comprehensive two dimensional gas chromatography (GC×GC) of fatty acid methyl esters from algae. In comparison with polyethylene glycol and cyanopropyl substituted polar stationary phases, ionic liquid stationary phases SLB-IL 82 and SLB-IL 100 showed comparable resolution, but lower column bleeding with MS detection, resulting in better sensitivity. The selectivity and polarity of the ionic liquid phases are similar to a highly polar biscyanopropyl-silicone phase (e.g. HP-88). In GC×GC, using an apolar polydimethyl siloxane×polar ionic liquid column combination, an excellent group-type separation of fatty acids with different carbon numbers and number of unsaturations was obtained, providing information that is complementary to GC-MS identification.     

Application of a new capsule-type silica gel coated with hydrophobic polymer to preparative liquid chromatography on an industrial scale

Summary A new type of column packing material designed for preparative liquid chromatography, silicone polymer-coated silica gel modified with octadecyl group (S/S-C₁₈), was applied to chromatographic purification of a lipophilic biotechnological product. Triglycerides containing γ-linolenic acid were separated from the curde oil that consisted of triglycerides, diglycerides, free fatty acids, sterols and other polar substances, using a S/S-C₁₈ packed column (150 mm I.D. × 1000 mm). No column deterioration was observed after more than 1500 times of sample introductions.     

Gas-liquid chromatographic determination of fatty acid composition of cholesteryl esters in human serum using silica Sep-Pak cartridges

A simple and fast analytical procedure for separation and purification of cholesteryl esters of human serum is described. A single lipid extract, together with spiked cholesteryl pentadecanoate, as an internal standard, was passed through a Silica Sep-Pak cartridge. 1.5% diethyl ester in light petroleum was used to elute cholesteryl esters from the column. The separation was verified with thin-layer chromatography on silica gel using light petroleum-diethyl ether-glacial acetic acid (80:20:1) as a solvent. A very clean thin-layer chromatogram of cholesteryl esters without any additional spots of other lipids was obtained. The cholesteryl esters were quantitated by analyzing their fatty acid composition as methyl esters by gas-liquid chromatography. The coefficients of variation were 0.8--4.9% for the major fatty acids (C16:0, C16:1, C18:1, C18:2, C20:4) and 6.7--30.8% for the minor fatty acids (C18:0 and C20:0). The recoveries for cholesteryl palmitate, cholesteryl oleate and cholesteryl linoleate were 90.7, 92.3 and 91.0%, respectively.     

In-house validation of an improved sample extraction and clean-up method for GC determination of isomers of nervonic acid in meat products

An improved extraction and clean-up method for determination of brain-specific fatty acids, in particular lignoceric acid (C24:0) and the cis/ trans isomers of nervonic acid (15 c-t C24:1), in meat products has been developed. The method is based on isolation of the polar lipids of interest from the bulk lipids by solid-phase extraction. The fatty acids, derivatised to their fatty acid methyl esters, are quantified by GC in a DB5 column. Fresh meat samples were extracted by using a mixture of n-butanol:hexane (1:9) as solvent. The extract was loaded in a silica gel cartridge column previously equilibrated with hexane. The first fraction containing the major part of the fat was eluted with hexane while acetone and methanol allowed the elution of fatty acids bound to polar moieties such as nervonic and lignoceric acids. This second fraction containing the analyte was methylated and injected into the GC for quantification after addition octacosane (C(28)) as internal standard.     

Identification of ω-oxocarboxylic acids as acetal esters in aerosols using capillary gas chromatography-mass spectrometry

An homologous series of ω,ω-dimethoxycarboxylic acid methyl esters (acetal esters) has been identified in the most polar methyl ester fraction separated from remote aerosol samples by liquid chromatography. These compounds were separated by silica gel column chromatography and their structures determined by capillary gas chromatography (GC) and GC—mass spectrometry with a synthesized standard. The acetal esters are originally present in the samples as ω-oxocarboxylic acids, which are derivatized to acetal esters during treatment with boron trifluoride in methanol. This method enables the determination of ω-oxocarboxylic acids in environmental samples as their acetal esters.     

Determination of Trace Levels of Fatty Acid Methyl Esters in Aviation Fuel by GC × GC–FID and Comparison with the Reference GC–MS Method

Due to the negative impact of the presence of fatty acid methyl esters in kerosene for the aviation industry, in this work a comprehensive two-dimensional gas chromatography method with flame ionization detector for the determination of trace levels of fatty acid methyl esters in kerosene was developed. It is based on the use of a first dimension polar column and a second dimension non-polar column. Identification of fatty acid methyl esters is based on retention times and the external standard calibration is used for quantitation. Results were compared with those obtained from the GC–MS reference method; good recoveries, close to 100 %, and limit of detection in a range from 3 to 6 μg kg⁻¹ were found without requiring a specific detector.

     

Tandem Solid-Phase Extraction Columns for Simultaneous Aroma Extraction and Fractionation of Wuliangye and Other Baijiu

Wuliangye baijiu is one of the most famous baijiu in China, with a rich, harmonic aroma profile highly appreciated by consumers. Thousands of volatiles have been identified for the unique aroma profile. Among them, fatty acid esters have been identified as the main contributors to the aroma profile. In addition, many non-ester minor compounds, many of which are more polar than the esters, have been identified to contribute to the characteristic aroma unique to Wuliangye baijiu. The analysis of these minor compounds has been challenging due to the dominance of esters in the sample. Thus, it is desirable to fractionate the aroma extract into subgroups based on functional group or polarity to simplify the analysis. This study attempts a new approach to achieve simultaneous volatile extraction and fractionation using tandem LiChrolut EN and silica gel solid-phase extraction (SPE) columns. A baijiu sample (10 mL, diluted in 40 mL of water) was first passed through the LiChrolut EN (1.0 g) column. The loaded LiChrolut EN column was then dried with air and coupled with a silica gel (5.0 g) SPE column with anhydrous Na₂SO₄ (10.0 g) in between. The volatile compounds were eluted from the LiChrolut EN column and simultaneously fractionated on the silica gel column based on polarity. The simultaneous extraction and fractionation technique enabled the fractionations of all fatty acid esters into less polar fractions. Fatty acids, alcohols, pyrazines, furans, phenols, hydroxy esters, and other polar compounds were collected in more polar fractions. This technique was used to study the volatile compounds in Wuliangye, Moutai, and Fengjiu baijiu. In addition to fatty acid esters, many minor polar compounds, including 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, p-cresol, and 2-acetylpyrrole, were unequivocally identified in the samples. The procedure is fast and straightforward, with low solvent consumption.     

气相色谱/质谱法分析孔石莼中的脂肪酸

建立了孔石莼脂肪酸的气相色谱/质谱(GC/MS)测定方法。使用Folch法提取了孔石莼中的总脂,经过2 mol/L HCl-甲醇溶液的甲酯化处理后,采用GC/MS法对其脂肪酸组成进行了分离分析,同时结合有机质谱学规律,分别对饱和脂肪酸甲酯、单不饱和脂肪酸甲酯和多不饱和脂肪酸甲酯的裂解规律和质谱特征进行了分析归纳。通过质谱数据库检索和标准品对照,鉴定出孔石莼中的24种脂肪酸,其中9,12,15-十八碳三烯酸、4,7,10,13-十六碳四烯酸和6,9,2,15-十八碳四烯酸3种主要多不饱和脂肪酸占总脂肪酸含量的45.14%。通过对孔石莼中脂肪酸的分析,表明特征离子在脂肪酸甲酯尤其是多不饱和脂肪酸甲酯的定性方面具有很好的应用价值。     

A new procedure to analyze free fatty acids. Application to 20-mg brain tissue samples

Fatty acids were analyzed by a new method which involved their isolation from hexane extracts of serum or brain tissue in aqueous potassium hydroxide (10 microliter) and methylation directly in this solution with methyl iodide. The resulting fatty acid methyl esters were partitioned into ethylene chloride (25 microliter) and were quantitated by gas-liquid chromatography. The procedure was documented by comparison with conventional methylation reactions on serum fatty acids. This method, which avoids thin-layer chromatography and which measures individual free fatty acid concentrations in 20-mg brain tissue samples, should be of particular value for examining regional free fatty acids in brain following ischemia and trauma.     

Method for evaluating the bioconversion of radioactive polyunsaturated fatty acids by use of reversed-phase liquid chromatography

Reversed-phase high-performance liquid chromatography on a thermostatted octadecylsilyl column was used to separate mixtures of labelled polyunsaturated fatty acids (as their methyl esters) formed by successive desaturations and elongations of labelled linoleic (18:2 n - 6) or linolenic (18:3 n - 3) acid by rat liver microsomes. Acetonitrile-water mixtures were used for elution of the esters. Unsaturated and saturated esters were detected by their refractive indices. The order of elution of fatty acid methyl esters in complex mixtures varies as a function of the chain length and unsaturation, analysis temperature, water concentration and solvent flow-rate. The peak areas vary as a function of the unsaturation. Specific radioactivities of 14C-labelled fatty acids and the percentage distribution of radioactivity among fatty acids from complex mixtures can be efficiently determined by collection and direct measurement of the radioactivity in the solvent by liquid scintillation counting. The method can be applied to complete compositional analysis, but is especially useful for determination of specific radioactivities during studies on the metabolic conversion of labelled polyunsaturated fatty acids.     

气相色谱法在生物柴油生产工艺研究中的应用

综述了气相色谱法在生物柴油生产工艺研究中的应用,包括反应产物和生物柴油产品中脂肪酸甲酯含量和分布的测定,单脂肪酸甘油酯、二脂肪酸甘油酯和三脂肪酸甘油酯含量的测定,游离脂肪酸含量的测定以及微量甲醇含量的测定等。讨论了进样方式、色谱柱类型、硅烷化等因素对反应产物组成测定的影响;提出了一种采用双柱压力反吹的方式测定生物柴油产品中微量甲醇含量的方法：采用正丙醇作内标,甲醇与内标通过预切柱进入分析柱后,通过压力变化,将其余组分通过分流出口反吹出色谱系统;采用极性聚乙二醇色谱柱测定了8种不同植物油中脂肪酸甲酯的含量和分布。     

13C nuclear magnetic resonance spectroscopy for determining the different components of epicuticular waxes of olive fruit (Olea europaea) Dritta cultivar

¹³C nuclear magnetic resonance spectroscopy (NMR) was applied to determine the different components of apolar and polar fractions which were isolated by column chromatography from the crude chloroform-soluble waxes of olive fruits (Olea europaea) Dritta cultivar.¹³C NMR enabled the determination in the wax apolar fraction, of aliphatic aldehydes, and of benzyl, alkyl and glyceryl esters. In particular, the fatty acid composition of alkyl esters, comprising saturated and unsaturated oleic and linoleic acids, was determined. Acyl chain composition and the chain composition of 1,3- and 2-glycerol positions were also determined for triacylglycerols of olive fruit waxes.Oleanolic and maslinic acids were confirmed to be the major components of wax polar fraction. Complete assignments of ¹³C NMR chemical shifts of oleanolic and maslinic acids as a mixture were achieved by using homonuclear correlation spectroscopy with gradient (g-COSY), attached proton test (APT), inverse-detected heteronuclear single-quantum coherence with gradient (g-HSQC), high-resolution heteronuclear correlation spectroscopy (HETCOR) for C-H directly attached pairs and C-H long-range-coupled experiments.     

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.     

Capillary column gas chromatography-mass spectrometry for the determination of the fatty acid composition of human adipose tissue

A procedure for determining the fatty acid composition of human adipose tissue using gas chromatography-mass spectrometry was developed. Adipose tissue was obtained from the lateral upper aspect of the right thigh by needle biopsy and prepared for analysis by lyophilisation, total lipid extraction and base-catalysed transesterification of the complexed fatty acids to form fatty acid methyl esters. Capillary column gas chromatography resolved thirty different peaks, ranging in carbon length from 12 to 24. Provisional identification of the peaks was by cochromatography with authentic standards and confirmed by gas chromatography-mass spectrometry using electron-impact ionisation. Fatty acid methyl esters were quantified in absolute amounts with respect to dry tissue weight and as a percentage of the total fat. Statistical analysis of the results from twenty healthy subjects using the two-tailed unpaired Student's t-test demonstrated women had significantly higher levels of myristoleic and palmitoleic acids (p less than 0.001) and lower levels of palmitic acid (p less than 0.05) in adipose tissue when compared with the male group. Similarly total saturated fatty acids was lower (p less than 0.05) and total monounsaturated fatty acids was higher in women than in men.     

Simple thin-layer chromatographic purification procedure for the determination of cholesterol ester fatty acid compositions

A mild procedure for the purification of methyl esters of the fatty acid components of cholesterol esters, from interfering free cholesterol and other contaminating residues, is described. Methyl esters and free cholesterol are formed during the methylation of cholesterol esters. When co-extracted, cholesterol and other contaminating residues interfere with the methyl esters because minute proportions of these residues tend to elute at the same retention times as palmitoleic and stearic acids, to yield unreliable but significantly higher values for palmitoleic (p less than 0.001) and stearic acids (p less than 0.0001), and correspondingly lower values for oleic acid (p less than 0.0001). Purification of methyl esters by thin-layer chromatography eliminates this problem and yields reliable analysis of cholesterol ester fatty acids, without measurable oxidation of unsaturated fatty acids.     

Analysis of conjugated linoleic acids as 9-anthrylmethyl esters by reversed-phase high-performance liquid chromatography with fluorescence detection

A simple and highly sensitive method for determining the fatty acid composition of food lipids containing conjugated linoleic acid (CLA) is described. The method is based on the separation of the 9-anthrylmethyl ester derivatives of saturated and unsaturated (conjugated and non-conjugated) fatty acids by reversed-phase high-performance liquid chromatography with fluorescence detection. Just like the other fatty acids, CLA reacts readily with 9-anthryldiazomethane at room temperature to produce 9-anthrylmethyl esters without isomerization and decomposition of the conjugated double bonds. Clear resolution of the individual fatty acids as their 9-anthrylmethyl esters is achieved on a highly efficient octadecylsilylated silica column (150- x 3-mm i.d., 3-microm particle size) using a stepwise gradient elution with methanol-water. The method is standardized with commercially available CLA isomers (cis-9, trans-11 and trans-10, cis-12-octadecadienoic acids, and their cis,cis and trans,trans isomers) and applied for determination of the fatty acid compositions of milk and sdairy products.     

Gas chromatography of fatty acids.

Lipids in foods contain a wide variety of fatty acids differing in chain length, degree of unsaturation, position and configuration of double bonds and the presence of special functional groups. Modern capillary gas chromatography offers excellent separation of fatty acids. Fused-silica capillary columns with stationary phases of medium polarity and non-polar methylsilicone stationary phases successfully separate most of the natural fatty acids. Special applications, such as the separation of complex cis-trans fatty acid mixtures and cyclic fatty acids, required particular chromatographic conditions, including the use of very long capillary columns or more polar stationary phases. The derivatization methods for the preparation of fatty acid esters also need to be optimized to obtain accurate quantitative results. This paper reviews the derivatization techniques, capillary columns and stationary phases commonly used in the gas chromatography of fatty acids in food.     

Separation of carotenol fatty acid esters by high-performance liquid chromatography

Employing isocratic and gradient-elution high-performance liquid chromatography (HPLC) a number of straight-chain fatty acid esters (decanoate, laurate, myristate, palmitate) of violaxanthin, auroxanthin, lutein, zeaxanthin, isozeaxanthin, and beta-cryptoxanthin, prepared by partial synthesis, have been separated on a C18 reversed-phase column. Several chromatographic conditions were developed that separated a mixture of di-fatty acid esters (dimyristate, myristate palmitate mixed ester, dipalmitate) of violaxanthin, auroxanthin, lutein, and zeaxanthin in a single chromatographic run. Hydroxycarotenoids such as lutein, zeaxanthin, and isozeaxanthin that are not easily separated by HPLC on C18 reversed-phase columns, can be readily separated after derivatization with fatty acids and chromatography of their esters. Chromatographic conditions for optimum separation of carotenoids from various classes are discussed.     

Assay of lipids in dog myocardium using capillary gas chromatography and derivatization with boron trifluoride and methanol

The fatty acids of three lipid classes (free fatty acids, triglycerides, and cholesteryl esters) from dog heart were analysed by gas chromatography. Samples of the left ventricle were homogenized and total lipids were extracted. After separation by thin-layer chromatography, the bands of the lipid classes studied were scraped off, transmethylated according to the boron trifluoride-methanol procedure, and the fatty acid methyl esters were extracted and analysed. The problems related to the quantitation of fatty acids were investigated, namely transmethylation procedure, thin-layer chromatography, and gas chromatographic conditions. Fatty acid methyl esters were separated on capillary columns coated in the laboratory with SP 2340 stationary phase. The high performance of the separation ensured the reliability and the precision of the analysis.     

大鼠肝过氧化物酶体中脂肪酸的分析

 用双 (2 乙基己基 )酚酞酸酯 (DEHP)诱导大鼠肝过氧化物酶体增殖 ,再用蔗糖密度梯度离心法分离大鼠肝细胞过氧化物酶体 ,并用十七烷酸作内标 ,以毛细管气相色谱法在非极性SPB 1石英毛细管柱上对其中的 11种脂肪酸进行分离测定。正常组和诱导组的大鼠肝过氧化物酶体中的不饱和脂肪酸和长链脂肪酸所占总脂肪酸的比例及总脂肪酸的统计结果是 :诱导组的不饱和脂肪酸的含量高于正常组的 (P <0 0 5 ) ,而两个组的脂肪酸总量及长链脂肪酸的含量无明显差别。结果提示 :诱导组的大鼠肝过氧化物酶体的脂肪酸成分发生了变化 ,其膜结构与正常组的不相同。