Analysis of mixtures containing free fatty acids and mono-, di- and triglycerides by high-performance liquid chromatography coupled with evaporative light-scattering detection

Commercial monostearates of glycerol, generally used as antistatic agents for thermoplastic polymers, consist of a complex mixture of mono-, di- and trisubstituted glycerides and the corresponding fatty acids. Thus far, the glycerides and the fatty acids have been analyzed separately by high-performance liquid chromatography (HPLC). In fact, the simultaneous analysis of glycerides and fatty acids requires esterification of the acids in order to avoid overlapping of the chromatographic peaks. This paper describes an HPLC method which allows the direct separation and identification, simultaneously and in a single run, of the variously substituted glycerides, and also the corresponding saturated fatty acids that are found as by-products in commercial glycerol monostearates. The procedure is based on the use of a ternary gradient HPLC instrument equipped with an evaporative light-scattering detector; the stationary phase was a reversed-phase RP-8 end-capped (Merck) column; the mobile phase was two consecutive binary gradients consisting of acetonitile-water plus acetic acid (0.1%, v/v) and acetonitrile-methylene chloride. The method is fast and shows high sensitivity and selectivity, being able to separate also the positional isomers of mono- and digycerides in addition to the mixed di- and triglycerides.     

Overview of methods for the determination of trans fatty acids by gas chromatography, silver-ion thin-layer chromatography, silver-ion liquid chromatography, and gas chromatography/mass spectrometry

trans Isomers of naturally occurring cis-unsaturated fatty acids are produced when liquid vegetable oils or marine oils are partially hydrogenated to produce margarine, shortenings, and other hardened-fat products. Isomeric trans fatty acids are also formed in the intestinal tract of ruminants, and they appear in small amounts in dairy products and ruminant meat. Currently, satisfactory analyses for the fatty acid profiles of fats containing trans fatty acids are obtained by gas chromatography (GC) using capillary columns coated with highly polar cyanosilicone stationary phases. In capillary GC methods, the key limitation has been the incomplete separation of trans-monoenoic acid isomers from their cis isomers; however, recent reports have demonstrated that improvements in separation are attainable with the use of 100 m columns. In these columns, there is very little overlap of cis and trans isomers. More accurate trans fatty acid analyses can be obtained by coupling GC with either silver-nitrate thin-layer chromatography or silver-nitrate liquid chromatography.     

Utilisation of reversed-phase high-performance liquid chromatography as an alternative to silver-ion chromatography for the separation of cis- and trans-C18:1 fatty acid isomers

Silver ion-high-performance liquid chromatography (HPLC) has been commonly used for the separation and the analysis of trans-18:1 isomers in partially hydrogenated oils and milk fat. This paper describes an easy HPLC method using two reversed-phase columns. The cis- and trans-18:1 fatty acids isomers as methyl esters were eluted as two separate fractions. The collected fractions were analysed by gas chromatography (GC). The purity of the two fractions were tested by GC-MS and GC-Fourier transform IR.     

全二维气相色谱-四极杆质谱法检测植物油脂中脂肪酸

建立了植物油脂中31种脂肪酸成分的全二维气相色谱-四极杆质谱(GC×GC-qMS)分析方法。样品经甲酯化衍生后,以DB-1柱(30 m×0.25 mm×0.25 μm)作为一维柱、DB-Wax柱(3.2 m×0.1 mm×0.1 μm)作为二维柱组成柱系统进行分离,在调制周期为3.5 s、四极杆质量扫描范围为m/z 40～350的条件下,植物油脂中31种脂肪酸成分可以在50 min内得到准确和灵敏的检测。将本方法应用于实际样品的分析,灵敏度较传统的气相色谱-质谱法提高了100倍以上,一些植物油中微量的脂肪酸成分也因此被检出。该研究不仅为植物油中脂肪酸成分的分析提供了新的技术手段,同时对于确保食用植物油的质量安全、消除食用植物油的掺假伪劣等均有重要意义。     

Polyunsaturated but not conjugated linoleic acid supplementation of leukemic U937 cells can act as an amplification factor for photofrin-mediated photodynamic therapy

Polyunsaturated fatty acids located in leukemia cell membranes are excellent targets for peroxidation. They can significantly enhance the effectiveness of Photofrin-mediated photodynamic therapy (PDT)-induced cell killing. In this study, the peroxidizability of conjugated fatty acid isomers (9c,11t-linoleic acid and 9c,11c-linoleic acid) and polyunsaturated fatty acids (PUFAs; linoleic acid, gamma-linolenic acid and arachidonic acid) with 2,2'-azo-bis(2-amidinpropane)dihydrochloride, soybean lipoxygenase and photomediated peroxidation are compared with each other. Peroxidation was determined using different methods: by means of gas chromatography to estimate the fatty acid (FA) consumption, by photometry for the level of FA peroxides or phospholipid peroxides and by definition of the content of malondialdehyde for thiobarbituric acid reactive substances (TBARS). The results suggest that the generation of oxidation products from individual FAs indicate a different formation rate of oxidation products. Radical FA peroxides were produced most by polyunsaturated arachidonic acid, followed by linoleic acid and gamma-linolenic acid, whereas conjugated FA isomers did not generate peroxides. Accordingly, the levels of lipid peroxides and TBARS were substantially increased after incorporation and oxidation of polyunsaturated FAs into U937 cells and could significantly enhance the effectiveness of Photofrin-PDT-induced cytotoxicity. The results showed that PUFA, but not conjugated FA supplementation of U937 cells, can act as a PDT amplification factor.     

The use of (S)-(−)-1-(1-naphthyl)ethylamine as a resolving agent for α-methoxy fatty acids

A general method was developed for the diastereoselective resolution of α-methoxy fatty acids utilizing (S)-(−)-1-(1-naphthyl)ethylamine as resolving agent. The diastereomeric amides can be easily separated by silica gel column chromatography and/or capillary gas chromatography, thus allowing for a preparative and analytical method for determining the enantiomeric purity of naturally occurring and/or synthetic α-methoxy fatty acids. The first synthesis of the naturally occurring (R)-2-methoxyhexadecanoic acid was also accomplished in four steps starting from commercially available (±)-2-hydroxyhexadecanoic acid.     

脂肪酸的色谱保留时间规律与质谱特征研究及其在食品分析中的应用

用石油醚提取食品中的脂肪,经甲酯化反应后,采用HP-88(100m×0.25mm,0.33μm)弹性石英毛细管柱分离脂肪酸甲酯的同系物及异构体,GC/MS法测定。研究了不同链长脂肪酸的同系物及异构体的气相色谱出峰顺序,得到其保留时间规律;研究了不同脂肪酸的质谱断裂规律,选择3个特征离子来鉴定脂肪酸成分。建立了3个特征离子确定脂肪酸碳数及双键数目,色谱保留时间规律确定脂肪酸顺反异构体及双键位置异构体的方法。本法无需标准品即可快速测定脂肪酸同系物及异构体的含量,适用于脂肪酸组成的研究;及油脂、食品中脂肪酸,特别是反式脂肪酸的测定。     

气相色谱全分析海水和淡水养殖南美白对虾组织中脂肪酸组成与含量

采用超声提取-毛细管气相色谱法分析测定了海水养殖和淡水养殖的南美白对虾的肌肉和肝胰腺(中肠腺)中脂肪酸的组成与含量,以用于对虾繁殖与营养的研究。采用Folch 液超声提取对虾组织中的脂质,脂质经氢氧化钾-三氟化硼法甲酯化后上机分析。采用BPX70(60 m×0.25 mm i.d.)石英毛细管柱分离,火焰离子化检测器检测。以线性程序升温等当碳长度(ECLT)值结合标准品对照的方法对17种脂肪酸定性,用面积归一化法定量。结果表明,两种对虾的脂肪酸组成均以不饱和脂肪酸为主,而且不饱和多烯酸的含量明显高于不饱和单烯酸。两种对虾的肌肉脂肪酸中高度不饱和脂肪酸,如二十碳五烯酸(EPA)与二十二碳六烯酸(DHA)的总含量均较高,达20%以上;肝胰腺中EPA和DHA的总含量较低,为11%～17%。淡水虾中n-6族不饱和脂肪酸含量比海水虾高,海水虾中n-3族与n-6族脂肪酸含量之比值比淡水虾高。     

Two methods for the separation of monounsaturated octadecenoic acid isomers

The identification and quantification of complex mixtures of cis and trans octadecenoic (18:1) fatty acid isomers presents a major challenge for conventional one-dimensional GC/FID analysis of their methyl esters. We have compared the use of two methods to achieve optimized separations of positional and geometrical octadecenoic fatty acid isomers—comprehensive two-dimensional gas chromatography (GC × GC), and silver ion high performance liquid chromatography interfaced to atmospheric pressure photoionization (APPI) mass spectrometry. Nine isomers of octadecenoic acid methyl ester were well separated on a single silver ion column with a mobile phase of 0.018% acetonitrile and 0.18% isopropanol in hexane. Reproducible retention times were obtained with relative standard deviations of around 1% over 5 injections. The extra selectivity and reproducibility afforded by APPI-MS, together with the wide separation of cis and trans isomers by silver ion chromatography, resulted in a promising method for measurement of octadecenoic acid FAME. The GC × GC separation was performed using various column combinations, and optimal separation was obtained by coupling an ionic liquid column (Supelco SLB-IL100 [1,9-di(3-vinyl-imidazolium) nonane bis(trifluoromethyl) sulfonyl imidate]) in the first dimension with a SGE BPX50 (50% phenyl polysilphenylene-siloxane) in the second dimension. These methods have been applied to the analysis of octadecenoic acid in milk and beef fat.     

Identification of very-long-chain fatty acids in rat and mouse harderian gland lipids by capillary gas chromatography-mass spectrometry

Lipids of Harderian ophthalmic gland were separated by means of thin-layer chromatography with flame ionization detection in an latroscan apparatus. Wax ester and polar lipids (phosphatidylethanolamine and phosphatidylcholine) were detected as the main lipids in rats and glyceryl ether diester and both polar lipids were the main lipids in mice. Fatty acids were determined in individual lipid classes by means of gas chromatography and gas chromatography-mass spectrometry on capillary columns. The content of fatty acids, the positional isomers of monoenoic acids being predominantly C18, C20 and C22, is most interesting. Very-long-chain fatty acids, saturated fatty acids up to C30 and even monoenoic acids up to C28 were detected. Branched-chain fatty acids, predominantly iso and anteiso, are minority components, although their chain length distribution (C15-C27) is broad.     

Analysis of sorbitan ester surfactants. Part II: Capillary supercritical fluid chromatography

A rapid, simple and quantitative approach to the separation and identification of sorbitan ester surfactants has been developed using capillary supercritical fluid chromatography (SFC). The sorbitan ester surfactants were well separated into five groups: starting materials and mono-, di-, tri-, and tetraesters, with each group consisting of a number of peaks representing different isomers. High purity glycerides of fatty acids were employed to estimate the relative response factors of sorbitan esters, and reliable group-wise integration served for quantitation of the distribution of sorbitan fatty acid esters. A very important parameter, hydrophilic–lipophilic balance (HLB), which describes the hydrophilic and hydrophobic characteristics of surfactants, could be correlated with the distribution of the sorbitan esters. A combination of solid-phase extraction (SPE) and SFC was used to separate, concentrate, and analyze Span-20 from salt-water samples. In comparison with the HPLC method, capillary SFC broadens the scope of the technique to encompass high molecular weight sorbitan polyesters while maintaining high separation efficiency.     

Fatty-acid composition of rat brain lipids. Determined by support-coated open-tubular gas chromatography.

The nonhydroxy fatty acid composition of rat brain lipids (except gangliosides) was determined by support-coated open-tubular (SCOT) gas chromatography. Fatty acids of both odd and even chain lengths ranging from C14 to C26 were detected. Brain lipids contained 49% saturated, 29% monounsaturated, and 22% polyunsaturated fatty acids. Monoenoic fatty acids were mainly of the omega-9 and omega-7 series with minor amounts of omega-10 and amega-11 isomers. Dienes and trienes consisted of omega-6, amega-7, and omega-9 series. Tetraenes were of the omega-6 series. Small amounts of omega-6 and omega-3 pentaenes were detected. The most abundant polyunsaturated fatty acid was 22:6omega-3. The advantages of support-coated open-tubular columns over wall-coated open-tubular columns for the analysis of brain lipid fatty acids are discussed.     

Altitudinal Variation of Metabolites,Mineral Elements and Antioxidant Activities of Rhodiola crenulata (Hook.f. & Thomson) H.Ohba

Rhodiolacrenulata (Hook.f. & Thomson) H.Ohba is an alpine medicinal plant that can survive in extreme high altitude environments. However, its changes to extreme high altitude are not yet clear. In this study, the response of Rhodiola crenulata to differences in altitude gradients was investigated through chemical, ICP-MS and metabolomic methods. A targeted study of Rhodiola crenulata growing at three vertical altitudes revealed that the contents of seven elements Ca, Sr, B, Mn, Ni, Cu, and Cd, the phenolic components, the ascorbic acid, the ascorbic acid/dehydroascorbate ratio, and the antioxidant capacity were positively correlated with altitude, while the opposite was true for total ascorbic acid content. Furthermore, 1165 metabolites were identified: flavonoids (200), gallic acids (30), phenylpropanoids (237), amino acids (100), free fatty acids and glycerides (56), nucleotides (60), as well as other metabolites (482). The differential metabolite and biomarker analyses suggested that, with an increasing altitude: (1) the shikimic acid-phenylalanine-phenylpropanoids-flavonoids pathway was enhanced, with phenylpropanoids upregulating biomarkers much more than flavonoids; phenylpropanes and phenylmethanes upregulated, and phenylethanes downregulated; the upregulation of quercetin was especially significant in flavonoids; upregulation of condensed tannins and downregulation of hydrolyzed tannins; upregulation of shikimic acids and amino acids including phenylalanine. (2) significant upregulation of free fatty acids and downregulation of glycerides; and (3) upregulation of adenosine phosphates. Our findings provide new insights on the responses of Rhodiola crenulata to extreme high altitude adversity.     

Seed Oil Fatty Acids of Mongolian Compositae: The trans‐Fatty Acids of Heteropappus hispidus,Asterothamnus centrali‐asiaticus and Artemisia palustris

Seed oils from the Compositae plant family are known to contain a variety of unusual fatty acids. Subsequent to the recent discovery of γ‐linolenic acid in Saussurea and Youngia, further Mongolian Compositae species were investigated for their seed oil fatty acid composition. A number of δ3trans‐fatty acids (16 : 1δ3t, 18 : 1δ3t and 18 : 3δ3t, 9c, 12c) were found in the seed oils of Heteropappus hispidus and Asterothamnus centrali‐asiaticus. The latter fatty acid, but not the trans‐monoenes, was also found in one species of Artemisia. These unusual fatty acid isomers were characterized by capillary gas‐liquid chromatographic (GLC) separations in combination with other chromatographic techniques (analytical thin layer chromatography, TLC and preparative argentation TLC), and infrared spectrocsopy (IR). Their identity was further confirmed by co‐chromatography with other seed oils known to contain these trans‐fatty acids. The fact that within the Compositae plant family there are apparently two or three distinct groups of genera containing δ3trans‐fatty acids is discussed.     

Structure-antibacterial activity relationship of secondary metabolites from Ajuga pseudoiva Rob. leaves

A selection of steroids, glycerides, clerodane diterpenoids, and a beta-hydroxy fatty acid methyl ester, all previously isolated from Ajuga pseudoiva leaves, were tested for their antibacterial activity toward three Gram- rods and one Gram+ coccus using the dilution method; MIC values were determined. The results suggested some importance for a free beta-hydroxy group in the fatty acid ester and also in the glycerides and clerodane derivatives; the absolute configurations of the latter, notably at C2, had little influence on activity.     

薄层色谱-热辅助水解甲基化-气相色谱法测定生物柴油中的甘油酯

建立了薄层色谱-热辅助水解甲基化-气相色谱法测定生物柴油中残余甘油酯含量的方法.样品中的甘油酯经薄层色谱分离,萃取后与三甲基氢氧化硫(0.1 mol/L)各3 μL先后加入到样品杯中,在350℃下,于裂解器中进行衍生化反应,气相色谱测定生成的脂肪酸甲酯,确定甘油酯的含量.生物柴油中常见的甘油一酯、二酯、三酯在60～20...     

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.     

Alkali-katalysierte Umesterungsmethoden für die gas-chromatographische Analyse der Fettsäuremethylester aus Lipoiden

Sodium methylate splits the ester linkage of glycerides, glycerophosphatides, cholesterin esters, waxes, and estercerebrosides under formation of fatty acid methylesters. Sodium methylate neither attacks the acid-amide linkage nor esterifies free fatty acids. Patty acid methylesters can be easily prepared for gas-liquid chromatography by the described alkali-catalyzed transesterification methods. The advantages of these methods are: 1. the simplicity of the technique, 2. the rapidity of the trans-esterification reaction, 3. the various combinations with thin-layer chromatography, 4. the application for the analysis of μg-quantities, and 5. the rapid parallel preparation of many samples on silicagel plates.     

Amino acid, fatty acid, and carbohydrate content of Artocarpus altilis (breadfruit)

A study is conducted to determine the amino acid, fatty acid, and carbohydrate content of breadfruit using high-performance liquid chromatography (HPLC) and gas chromatography (GC). An HPLC method is used for the determination of amino acids and fatty acids in breadfruit. Representative amino acid samples are derivatized with phenylisothiocianate and the resulting phenylthiocarbamyl derivatives are separated on a reversed-phase column by gradient elution with a 0.05M ammonium acetate buffer and 0.01M ammonium acetate in acetonitrile-methanol-water (44:10:46, v/v). Representative fatty acid samples are derivatized with phenacyl bromide and the resulting fatty acid phenacyl esters are separated on a reversed-phase column by gradient elution with acetonitrile and water. Amino acid and fatty acid derivatives are detected by ultraviolet detection at 254 nm. The analysis of the carbohydrates in breadfruit employs a GC method. Carbohydrates are derivatized using trimethylchlorosilane and hexamethyldisilazane to form trimethylsilyl ethers. Compounds in the samples are separated by the temperature programming of a GC using nitrogen as the carrier gas. Percent recoveries of amino acids, fatty acids, and carbohydrates are 72.5%, 68.2%, and 81.4%, respectively. The starch content of the breadfruit is 15.52 g/100 g fresh weight.     

Gas chromatography of volatile fatty acids. Method involving separation from biological material by vacuum distillation.

A method is described for the quantitation of C2-C5 volatile fatty acids present in biological tissues. It involved recovery of the acids from their biological matrix by vacuum micro-distillation at room temperature, followed by gas phase separation of aqueous solutions on orthophosphoric acid-modified Phasepak Q columns. The subsequent gas chromatographic procedure resolved iso from normal isomers and showed a linear response for each volatile acid over the range 10-400 ng. There was no evidence of ghosting, isomer peak broadening, or peak tailing. Relative molar response values were shown to be linear with carbon number for all the volatile fatty acids studied.