FABES and FAPES – transesterification of triglycerides with higher alcohols. Quantitation and mass spectrometric evaluation

In order to facilitate the gas chromatographic determination of butyric as well as other short chain fatty acids, triglycerides containing these fatty acids were transbutylated and transpentylated to give FABES (fatty acid butyl esters) and FAPES (fatty acid pentyl esters), respectively. This method allows the molecular weight of the compounds to be increased while their polarity is decreased. Short chain esters elute sufficiently separated from the solvent; due to the decreased polarity, elution of long chain fatty acids is only slightly retarded compared to the corresponding methyl esters. Quantitative evaluation over a large series of injections proved the linearity of correction factors in both cases, FABES and FAPES, with a slight deviation for 16:0 and 18:0, respectively. Structural characterization was performed by GC/MS. It was shown that there are characterstic fragmentations for FABES as well as for FAPES. The El-mass spectra give structural information about the alcohol and acid moieties, the ester molecule and the Cl-spectra indicate the molecular weight. The absence of abundant highly characteristic peaks in the spectra of these esters makes the detection of FABES or FAPES in complex mixtures more difficult than the detection of FAMES with their very characteristic ions at m/z 74 and 87.     

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.     

Reversed-phase high-performance liquid chromatography purification of methyl esters of C(16)-C(28) polyunsaturated fatty acids in microalgae, including octacosaoctaenoic acid [28:8(n-3)

A preparative reversed-phase (RP; C(18)) high-performance liquid chromatography (HPLC) method with gradient elution using acetonitrile (MeCN)-chloroform (CHCl(3)) (or dichloromethane (DCM)) and evaporative light-scattering detection (ELSD) with automatic multiple injection and fraction collection was used to purify milligram quantities of microalgal polyunsaturated fatty acids (PUFA), separated as methyl esters (ME). PUFA-ME purified included methyl esters of docosahexaenoic acid (DHA; 22:6(n-3)), eicosapentaenoic acid (EPA; 20:5(n-3)) and the unusual very long-chain (C(28)) highly unsaturated fatty acid (VLC-HUFA), octacosaoctaenoic acid [28:8(n-3)(4, 7, 10, 13, 16, 19, 22, 25)] from the marine dinoflagellate Scrippsiella sp. CS-295/c. Other PUFA purified from various microalgae using this RP-HPLC method to greater than 95% purity included 16:3(n-4), 16:4(n-3), 16:4(n-1) and 18:5(n-3). The number of injections required was variable and depended on the abundance of the desired PUFA-ME, and resolution from closely eluting PUFA-ME, which determined the maximum loading. The purity of these fatty acids was determined by electron impact (EI) GC-MS and the chain length and location of double bonds was determined by EI GC-MS of 4,4-dimethyl oxazoline (DMOX) derivatives formed using a low temperature method. Advantages over silver-ion HPLC for purifying PUFA-ME is that separation occurs according to chain length as well as degree of unsaturation enabling separation of PUFA-ME with the same degree of unsaturation but different chain length (i.e. between 18:5(n-3) and 20:5(n-3)). In addition, PUFA-ME are not strongly adsorbed, but elute earlier than their more saturated corresponding FAME of the same chain length. This method is robust, simple, and requires only a short re-equilibration time. It is a useful tool for preparing milligram quantities of pure PUFA-ME for bioactive screening (as free fatty acids), although many multiple injections may be required for minor PUFA-ME. It also enabled dose-response and structure-activity studies to be carried out. It can be used for the enrichment of low levels of VLC-HUFA-ME to facilitate elucidation of their chemical structure and so is a useful adjunct to EI GC-MS of DMOX derivatives and other techniques such as NMR, which requires milligram quantities of purified compounds.     

High-performance liquid chromatography-thermospray mass spectrometry of hydroperoxy polyunsaturated fatty acid acetyl derivatives.

A method for the analysis of hydroperoxy polyunsaturated fatty acids was developed. The hydroperoxy groups were acetylated by acetic anhydride, and the mixture was partially purified on a Sep-Pak C18 cartridge and analysed by high-performance liquid chromatography with thermospray mass spectrometry. Generally, the base ion, [M+H - n(60)]+ or [M+H - n(60) - n(H2O)]+, is produced through elimination of acetic acid or water (n = number of hydroperoxy groups). The detection limit for these derivatives was ca. 1 pmol at concentrations of hydroperoxy polyenoic acids prior to derivatization. Using this method, many hydroxy and hydroperoxy polyunsaturated fatty acid derivatives could be detected simultaneously within 30 min on a selected-ion monitoring detection chromatogram without a gradient system. The assay was successfully applied to hydroxy and hydroperoxy polyunsaturated fatty acids from an incubation mixture of rat brain homogenate to which polyunsaturated fatty acids had been added.     

Efficient lipase-catalyzed synthesis of new lipid antioxidants based on a catechol structure

Lipid antioxidants phenolic saturated fatty acid esters were synthesized in high yields and short reaction times using the corresponding ethyl fatty acid esters, lipase from Candida Antarctica, vacuum and no solvent. Phenolic esters with mono- and polyunsaturated fatty acids (EPA and DHA) were also prepared.     

Capillary electrochromatographic enantioseparations using a packed capillary with a 3 microm OD-type chiral packing

A technique for separating methyl esters of monounsaturated fatty acids by argentation chromatography using silver nitrate-impregnated TLC plates is described. Monounsaturated fatty acid methyl esters are separated from polyunsaturated and saturated fatty acid methyl esters and the monounsaturated fatty methyl esters are resolved according to chain length. cis isomers are well resolved from the corresponding trans isomers. R(F) values for individual monounsaturated fatty acids are very reproducible. The potential of the technique in metabolic studies is demonstrated in the chain elongation of [14C]-18:1(n-9) and delta-9 desaturation of [14C]-18:0 by human skin fibroblasts. Recoveries of individual [14C]-fatty acids for scintillation counting exceed 94%.     

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.     

In vivo and in vitro lipid peroxidation of arachidonate esters: the effect of fish oil omega-3 lipids on product distribution

The effect of lipid composition on the distribution of free radical oxidation products derived from arachidonic acid (20:4) esters has been studied in vitro and in vivo. Pro-inflammatory prostaglandin (PG) F2-like compounds, termed F2-isoprostanes (IsoPs), are produced in vivo and in vitro by the free radical-catalyzed peroxidation of arachidonic acid. Controlled free radical oxidation of mixtures of fatty acid esters in vitro showed that the formation of IsoPs from arachidonate is dramatically influenced by the presence of other fatty acid esters in the reaction mixture. Thus, three lipid mixtures containing the same arachidonate concentration but different amounts of other fatty esters (16:0; 18:1; 18:2; 20:5, and 22:6) were oxidized, and the product yields were determined by GC and LC/MS/MS analysis. The yield of F2-IsoP formed after 1 h of oxidation was 18% (based on arachidonate consumed) for mixtures containing arachidonate as the only oxidizable PUFA, but yields of these biologically active compounds dropped to 6% in polyunsaturated fatty acid (PUFA) mixtures typical of those found in tissues of fish oil-fed animals. F2-IsoP levels were also monitored in the livers of mice on diets supplemented with eicosapentaenoic acid (C20:5 omega-3; EPA), the PUFA most abundant in fish oil. While the level of arachidonic acid present in livers was not significantly different from that in control animals, levels of IsoPs in the liver were reduced in the EPA-fed mice compared to those in controls under conditions of oxidative stress (60 +/- 25% reduction, n = 5) or at baseline (48 +/- 14% reduction, n = 5). These results suggest that dietary omega-3 PUFAs may influence the formation of bio-active peroxidation products derived from omega-6 PUFAs by channeling the free radical pathway away from the F2-IsoPs.     

Characterization and purification of polyunsaturated fatty acids from microalgae by gas chromatography-mass spectrometry and countercurrent chromatography

Summary The present study was undertaken in order to characterize then to purify fatty acids from marine phytoplankton. From a crude mixture of fatty acid methyl esters it was possible to isolate by countercurrent chromatography a mixture of four polyunsaturated fatty acid methyl ester identified as being hexadecatrienoic acid methyl ester, octadecatetraenoic acid methyl ester, eicosapentaenoic acid methyl ester and docosahexaenoic acid methyl ester by gas chromatography coupled with mass spectrometry in electron impact and in positive-ion chemical ionization mode. The four polyunsaturated fatty acids are in different ratios in mixtures from the two microorganisms:Skeletonema costatum andIsochrysis galbana.     

Isocratic Separation of Fatty Acid Derivatives by Reversed Phase Liquid Chromatography. Influence of the Solvent on Selectivity and Rules for Elution Order

Abstract

The p-bromophenacyl esters of 16 fatty acids (C₁₂-C₂₂) have been separated by isocratic chromatography on a Radial Pak A cartridge (Reverse phase C₁₈ material). The separation factors α were measured using two solvent mixtures of comparable strength and the superiority of methanol-water to acetonitrile-water becomes evident.

Five precise rules are established, which indicates the retention of every fatty acid. They explain the chromatographic process i.e. elution order, resolution and selectivity.     

生物柴油树种油脂脂肪酸组成对燃料特性的影响

以目前中国主要开发或具有开发潜能的10种生物柴油树种为研究对象,分析其果实或种子油脂脂肪酸组成对合成生物柴油燃料特性的影响。结果表明,木本植物生物柴油产品十六烷值、碘值、氧化安定性等燃料特性主要由原料油脂肪酸的不饱和度决定,脂肪酸不饱和度低于133.13,十六烷值(GB/T 20828-2007)和碘值(EN 14214)就可以达标。生物柴油产品冷滤点随着长碳链饱和脂肪酸的增加而升高,脂肪酸饱和碳链长度因子分别小于8.41和2.72时,可以满足冷滤点0℃和-10℃的要求。高品质生物柴油的原料中应该具有较高的单元不饱和脂肪酸含量。通过油脂脂肪酸单不饱和脂肪酸、多不饱和脂肪酸和饱和脂肪酸的组成绘制出生物柴油特性三角预测图,为预测生物柴油产品燃料特性提供参考依据。     

Phase Behavior and Polymorphism of Saturated and Unsaturated Phytosterol Esters

This study investigated how the physicochemical characteristics of phytosterol esters are influenced by the chain length and degree of unsaturation of the fatty acid ester moiety. Saturated and unsaturated phytosterol esters (PEs) were synthesized by the esterification of different types of fatty acids (stearic, palmitic, lauric, oleic, and linoleic acid) to β-sitosterol. The non-isothermal crystallization and melting behavior of the pure PEs were analyzed. It was proven by X-ray diffraction that saturated β-sitosteryl esters and β-sitosteryl oleate formed a bilayer crystal structure. The lamellar spacings of the bilayer structure decreased with decreasing fatty acid chain length and with an increasing degree in unsaturation. The degree of unsaturation of the fatty acid chain of the β-sitosteryl esters also influenced the type of subcell packing of the fatty acid moieties in the bilayer structure, whether or not a metastable or stable liquid crystalline phase was formed during cooling. Furthermore, it was found that the melting temperature and enthalpy of the β-sitosteryl esters increased with an increasing fatty acid chain length while they decreased with an increasing degree of unsaturation. The microscopic analyses demonstrated that β-sitosteryl oleate formed much smaller spherulites than their saturated β-sitosteryl analogues.     

Reaction efficiency of organic alkalis with various classes of lipids during thermally assisted hydrolysis and methylation

Reaction efficiencies of two organic alkalis, tetramethylammonium hydroxide (TMAH) and trimethylsulfonium hydroxide (TMSH), with lipids during thermally assisted hydrolysis and methylation (THM) were examined focusing on (1) the types of lipids and (2) degree of unsaturation of fatty acid moieties. Different types of lipids such as triglycerides, phospholipids, free fatty acids and cholesteryl esters containing saturated, monounsaturated or polyunsaturated fatty acid (PUFA) residues were subjected to THM-gas chromatography (GC) in the presence of TMAH or TMSH. The obtained results revealed that the THM reaction using TMAH allowed almost quantitative methylation of saturated and monounsaturated fatty acid components independently of the classes of lipids. However, strong alkalinity of TMAH brought about isomerization and/or degradation of PUFA components. In contrast, the use of TMSH was effective to highly sensitive detection of PUFA as well as saturated and monounsaturated fatty acid components contained in triglycerides, phospholipids (phosphatidylcholines) and free fatty acids. On the other hand, TMSH was proved to react hardly with any kind of fatty acid residues in cholesteryl esters due to their steric hindrance.     

Fast high performance liquid chromatography analysis in lipidomics: Separation of radiolabelled fatty acids and phosphatidylcholine molecular species using a monolithic C18 silica column

HPLC procedures using conventional C₁₈ columns are usually used to separate simple and complex lipid mixtures but these methods of separation remain often laborious and very slow. Here, monolithic columns were successfully applied to separate lipids - radiolabelled fatty acid mixtures and individual phosphatidylcholine (PC) molecular species. For that, isocratic elution was performed using two Chromolith™ Performance RP-18e columns connected in series. Detection was achieved by online measurement of radioactivity for radiolabelled fatty acids and by UV absorbance at 205 nm for PC molecular species. The performances of such silica rods were compared to conventional reverse-phase silica columns. Monolithic stationary phase separated radiolabelled fatty acids and PC molecular species two times and four times faster, respectively. In each analysis, monolithic columns allowed better separation efficiency per unit of time, with lower inlet pressure. The main advantages of this method for lipid separation are that, under isocratic conditions, it is simpler and much faster, while remaining accurate and selective when compared to conventional methods. Therefore, monolithic columns may represent a powerful tool for the near future in the field of lipidomics.     

Improved identification of conjugated linoleic acid isomers using silver-ion HPLC separations

Silver-ion high-performance liquid chromatography (Ag+-HPLC) has been shown to be effective in the resolution of most of the isomers of conjugated octadecadienoic acids (18:2), also known as conjugated linoleic acid (CLA). The CLA isomers identified in natural fats from ruminants are a mixture of numerous positional and geometric isomers from 7,9- to 12,14-18:2. Ag+-HPLC separates both geometric (trans,trans < cis/trans < cis,cis) and positional CLA isomers using the mobile phase hexane/acetonitrile (99.9:0.1). The elution volumes for the CLA isomers were not only affected by the concentration of acetonitrile (in the prepared mobile phase) but also with successive runs during the day using a prepared mobile phase batch, due to the partial solubility of acetonitrile in hexane. However, this drift does not affect the relative resolution of the CLA isomers. The addition of diethyl ether to the mobile phase partly stabilizes the solvent mixture. In order to facilitate the interpretation of Ag-+HPLC chromatograms, the relative retention volumes (RRV) were calculated for each CLA isomer. Toluene was added to all the test portions and served as an estimator of dead volume, whereas the elution of the ubiquitous 9c,11t-CLA isomer was chosen as unity (1.00). Expressing the elution of all the CLA isomers as their RRV greatly helped to standardize each CLA isomer, resulting in relatively small coefficients of variation (% CV) for the trans,trans (<1.5%) and cis/trans (<0.5%) CLA isomers. The identification of the CLA isomers was further facilitated by synthesis of authentic CLA isomers. All the geometric CLA fatty acid methyl esters (FAME) from positions 6,8- to 13,15-CLA were commercially available or synthesized by a combination of partial hydrazine reduction of known polyunsaturated fatty acids followed by alkali isomerization, isolation of products, and further iodine-catalyzed geometric isomerization. Based on expressing the elution volume as RRV and the availability of the synthetic CLA isomers, a unique reversal of the elution order of the c/t CLA isomers was found. It is also proposed that the retention times of CLA isomers by gas chromatography (GC) should be expressed as their relative retention times (RRT) relative to methyl gamma-linoleneate. The availability of CLA reference materials and the application of RRV and RRT to Ag+-HPLC and GC separations, respectively, will greatly improve in the identifications of CLA isomers.     

Fatty acid composition of glycerophospholipids in seven tissues of cod (Gadus morhua), determined by combined high-performance liquid chromatography and gas chromatography

A method for the separation from fish tissues of the four main glycerophospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, using adsorption high-performance liquid chromatography with ultraviolet detection and consecutive gas chromatographic analysis, based on one injection for their fatty acid compositions, is described. Fatty acid 19:0 was used as an internal standard for the calculation of the relative concentrations of the phospholipids. The patterns of fatty acid distribution within each of the phospholipids from seven cod organs had some general similarities. Phosphatidylcholine had the highest levels of 16:0 and 18:2 n - 6, and the ratio of 20:5 to 22:6 varied between 0.5 and 0.9. Phosphatidylethanolamine had the highest total polyunsaturated fatty acids, (n -3) polyunsaturated fatty acids, and 22:6 n -3, and the ratio of 20:5 to 22:6 varied between 0.2 and 0.5. Phosphatidylinositol showed the highest level of 18:0 and 20:4 n - 6 and had the lowest ratio of (n - 3) to (n - 6). Phosphatidylserine had the highest ratio of (n - 3) to (n - 6) and the lowest ratio of 20:5 to 22:6. A generally low level (less than 1.5%) of the long-chain monoene, 22:1, was found in the phospholipids in all tissues.     

HPLC of fatty acid esters of mono- and polyhydric alcohols. Part 2: Preparative separation

This paper demonstrates how simply and rapidly fatty acid esters of mono- and polyhydric alcohols can be separated quantitatively in preparative quantities on Lobar® RP-8 packed columns. After the separation of unknown mixtures, the isolated esters are identified from spectroscopic data (IR/NMR) and, after saponification of the ester components (fatty acids and alcohols), from the retention times of gas and high-pressure liquid chromatographic separations. Thus, in particular, sparingly volatile or nonvolatile partial esters of polyhydric alcohols, as well as the long-chained full esters, can be determined qualitatively and quantitatively. The following fatty acid esters of mono- and polyhydric alcohols have been separated: the i-propylesters of the laurinic and myristinic acids, the i-butyl-, i-octyl- and i-octadecyl-esters of the palmitinic and stearic acids, the mono- and di-fatty acid esters of the 1,3-bis-(2-hydroxyethyl)-5,5-dimethylhydantoins, the mono-, di- and tri-esters of the trimethylalpropane and the full esters of the pentaerythrite.     

Perfluoroalkyl ketones: novel derivatization products for the sensitive determination of fatty acids by gas chromatography/mass spectrometry in electron impact and negative chemical ionization modes

Analytically useful pentafluoro ketone derivatives of fatty acids are described. The gas chromatographic/mass spectrometric characteristics of these new derivatives are compared with those of methyl, trimethylsilyl and pentafluorobenzyl esters. Pentafluoro ketones exhibit excellent chromatographic properties and significantly shorter chromatographic retention times than these other esters. The electron impact mass spectra of these new compounds show informative acylium ions, whose intensity decreases with the degree of unsaturation of the parent fatty acid. The formation of strong and informative fragment ions in negative chemical ionization (CH(4)) mass spectra of pentafluoro ketone derivatives allows the detection and the characterization (length of the chain and number of double bonds) of fatty acids at trace levels (femtomole), even in the case of polyunsaturated compounds. The scope and limitations of this new derivatization technique are also discussed.     

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 chromatographic analysis of infant formulas for total fatty acids, including trans fatty acids

Twelve powdered and 13 liquid infant formulas were analyzed by using an extension of AOAC Official Method 996.01 for fat analysis in cereal products. Samples were hydrolyzed with 8 N HCl and extracted with ethyl and petroleum ethers. Fatty acid methyl esters were prepared by refluxing the mixed ether extracts with methanolic sodium hydroxide in the presence of 14% boron trifluoride in methanol. The extracts were analyzed by gas chromatography. In powdered formulas, saturated fatty acid (SFA) content (mean +/- SD; n = 12) was 41.05 +/- 3.94%, monounsaturated fatty acid (MUFA) content was 36.97 +/- 3.38%, polyunsaturated fatty acid (PUFA) content was 20.07 +/- 3.08%, and total trans fatty acid content was 1.30 +/- 1.27%. In liquid formulas, SFA content (mean +/- SD; n = 13) was 42.29 +/- 2.98%, MUFA content was 36.05 +/- 2.47%, PUFA content was 20.65 +/- 2.40%, and total trans fatty acid content was 0.88 +/- 0.54%. Total fat content in powdered formulas ranged from 4.4 to 5.5 g/100 kcal and linoleic acid content ranged from 868 to 1166 mg/100 kcal. In liquid formulas, total fat content ranged from 4.1 to 5.1 g/100 kcal and linoleic acid content ranged from 820 to 1100 mg/100 kcal. There were no significant differences between powdered and liquid infant formulas in concentrations of total fat, SFA, MUFA, PUFA, or trans fatty acids.