Separation Of Fatty Acid Methyl Esters Research Articles

Automated sample preparation and fast GC-MS determination of fatty acids in blood samples and dietary supplements.

The present research is focused on the optimization of an automatized sample preparation and fast gas chromatography-mass spectrometry (GC-MS) method for the analysis of fatty acid methyl esters (FAMEs) in blood samples and dietary supplements, with the primary objective being a significant reduction of the analysis time and, hence, an enhanced sample throughput. The mass spectrometer was operated in the scan/selected ion monitoring (SIM) acquisition method, thus enabling the obtainment of qualitative and (highly sensitive) quantitative data. The separation of FAMEs was obtained in about 11min by using a micro-bore column of dimensions 15m × 0.10mm ID × 0.10µm df with a polyethylene glycol stationary phase. The novelty of the research involves reducing analysis time by using the novel fast GC-MS method with increased identification reliability and sensitivity in a single chromatographic run. With regard to the figures of merit, linearity, accuracy, and limits of detection (LoD) and quantification (LoQ) were determined. Specifically, regression coefficients were between 0.9901 and 0.9996; the LoDs ranged from 0.05 to 1.02µgg-1 for the blood analysis method, and from 0.05 to 0.26mgg-1 in the case of the dietary supplement approach. With respect to LoQs, the values were in the ranges of 0.15-3.39µgg-1 and 0.15-0.86mgg-1 for blood and dietary supplements analysis methods, respectively. Accuracy was evaluated by analyzing certified reference materials (human plasma, fish oil).

Microwave-assisted protocol for squalene isolation and conversion from oil-deodoriser distillates

Abstract Aiming to design a green and efficient protocol for squalane production from low-cost biomasses, a practical and scalable procedure for squalene extraction and hydrogenation to squalane is presented herein. The oil-deodoriser distillates that are produced by the vegetal-oil production chain are a renewable and cheap source of squalene. We were able to isolate an enriched fraction containing 89.0% of pure squalene (yield 55.4%) from a matrix containing about 2% squalene. Efficient microwave- assisted esterification under heterogeneous catalysis enabled the separation of fatty-acid methyl esters (FAME) via vacuum distillation. The residue was purified by flash-chromatography on a C-18 silica column using MeOH/H2O/2-propanol as the mobile phase. Finally, squalene was hydrogenated to the more stable squalane in a pressure-resistant microwave reactor. The reaction was performed over a Pd/C catalyst in EtOH, and even in solvent-free conditions, and was optimised using commercial squalene (5 bar of H2 at 100°C for 1 h).

Ionic liquid phases with comprehensive two-dimensional gas chromatography of fatty acid methyl esters.

New generation inert ionic liquid (iIL) GC columns IL60i, IL76i and IL111i, comprising phosphonium or imidazolium cationic species, were investigated for separation of fatty acid methyl esters (FAME). In general, the iIL phases provide comparable retention times to their corresponding conventional columns, with only minor selectivity differences. The average tailing factors and peak widths were noticeably improved (reduced) for IL60i and IL76i, while they were slightly improved for IL111i. Inert IL phase columns were coupled with conventional IL columns in comprehensive two-dimensional GC (GC × GC) with a solid-state modulator which offers variable modulation temperature (TM), programmable TM during analysis and trapping stationary phase material during the trap/release (modulation) process, independent ofoven T and column sets. Although IL phases are classified as polar, relative polarity of the two phases comprising individual GC × GC column sets permits combination of less-polar IL/polar IL and polar IL/less-polar IL column sets; it was observed that a polar/less-polar column set provided better separation of FAME. A higher first dimension (1D) phase polarity combined with a lower 2D phase polarity, for instance 1D IL111i with 2D IL59 gave the best result; the greater difference in 1D/2D phase polarity results in increasing occupancy of peak area in the 2D space. The IL111i/IL59 column set was selected for analysis of fatty acids in fat and oil products (butter, margarine, fish oil and canola oil). Compared with the conventional IL111, IL111i showed reduced column bleed which makes this more suited to GC × GC analysis of FAME. The proposed method offers a fast profiling approach with good repeatability of analysis of FAME.

Characterization of biodiesel and biodiesel blends using comprehensive two‐dimensional gas chromatography

In this work the development of a comprehensive 2-D GC flame ionization detection (GC x GC FID) method for biodiesel fuels is reported. This method is used for the analysis of fatty acid methyl esters (FAMEs) in both biodiesel (B100) and biodiesel blend (B5) samples. The separation of FAME was based on component boiling point in the first dimension and polarity in the second dimension by using a BPX5/BP20 column set to provide a measure of 'orthogonality' in the 2-D space. Here the columns are coupled with a cryogenic modulator operating in a novel temperature programmed mode (T(M)) whereby the cryotrap is progressively incremented in temperature as the oven temperature is increased. The final method employs eight cryotrap temperature settings. The developed GC x GC method is able to successfully characterize and identify both B100 and B5 FAME components, which are produced from a variety of vegetable oils, animal fats and waste cooking oils, with high precision. The method is capable of analysing FAME with carbon numbers C4-C24, and is particularly suitable to characterize various types of biodiesel, making it possible to differentiate the origin and type of FAME used in the biodiesel samples.

Interplay between lipoproteins and bee venom phospholipase A2 in relation to their anti-plasmodium toxicity

We previously showed that the in vitro intraerythrocytic development of the malarial agent Plasmodium falciparum is strongly inhibited by secreted phospholipases A(2) (sPLA(2)s) from animal venoms. Inhibition is dependent on enzymatic activity and requires the presence of serum lipoproteins in the parasite culture medium. To evaluate the potential involvement of host lipoproteins and sPLA(2)s in malaria, we investigated the interactions between bee venom phospholipase A(2) (bvPLA(2)), human triglyceride-rich lipoproteins, and infected erythrocytes. Even at high enzyme concentration (100x IC(50)), bvPLA(2) binding to Plasmodium-infected or normal erythrocytes was not detected. On the contrary, tight association with lipoproteins was observed through the formation of buoyant bvPLA(2)/lipoprotein complexes. Direct involvement of the hydrolysis lipid products in toxicity was demonstrated. Arachidonic acid (C20:4), linoleic acid (C18:2), and, to a lesser extent, docosahexaenoic acid (C22:6) appeared as the main actors in toxicity. Minimal oxidation of lipoproteins enhanced toxicity of the lipolyzed particles and induced their interaction with infected or normal erythrocytes. Fresh or oxidized lipolyzed lipoproteins induced the parasite degeneration without host cell membrane disruption, ruling out a possible membranolytic action of fatty acids or peroxidation products in the death process. In conclusion, our data enlighten on the capability of secreted PLA(2)s to exert cytotoxicity via the extracellular generation of toxic lipids, and raise the question of whether such mechanisms could be at play in pathophysiological situations such as malaria.

Quantitative Deuterium Isotopic Profiling at Natural Abundance Indicates Mechanistic Differences for Δ12-Epoxidase and Δ12-Desaturase in Vernonia galamensis

Quantitative (2)H NMR spectroscopy can determine the natural abundance ((2)H/(1)H) ratio at each site of a molecule. In natural products, variation in these values is related to the reaction mechanisms in the pertinent biosynthetic pathway. For the first time, this novel approach has been exploited to probe for mechanistic differences in the introduction of different functionalities into a long-chain fatty acid. Vernolic acid, a major component of the seed oil of Vernonia galamensis, contains both an epoxide and a desaturation. The site-specific isotopic distribution ((2)H/(1)H)(i) has been determined for both vernolic acid and linoleic acid isolated from the same V. galamensis oil. It is found that the ((2)H/(1)H) ratio of vernolic acid shows a pattern along the entire length of the chain, consistent with linoleic acid being its immediate precursor. Notably, the C13 relates to the C13 of linoleic acid but not to the C13 of oleic acid. Furthermore, the C12 and C13 positions in vernolic acid are less depleted, consistent with a change in hybridization state from sp(2) to sp(3). However, the C11 position shows a marked relative enrichment in the vernolic acid, implying that it plays a role in the epoxidase but not the desaturase mechanism. Thus, although it can be concluded that the catalytic mechanisms for the epoxidase and desaturase activities are similar, marked differences in the residual ((2)H/(1)H) patterns indicate that the reaction mechanisms are not identical.

Gas chromatographic analysis of fatty acid methyl esters.

The full process of fatty acid methyl ester (FAME) analysis consists of esterification of lipids, and of injection, separation, identification and quantitation of the FAMEs. In order for the required accuracy and precision to be attained, each of these steps has to be optimized. Esterification of lipids can be carried out with several reagents based on acid-catalysed or base-catalysed reactions. The advantages and disadvantages of these reagents are discussed. The most critical step in the gas chromatographic analysis of FAMEs is sample introduction. The classical split injection technique, which is the most widely used technique in the analysis of FAMEs, has the potential disadvantage of boiling-point-dependent sample discrimination. Cold injection of the sample, either on-column or by programmed-temperature vaporization, does not present this problem and should therefore be preferred. Modern, commercially available fused-silica capillary columns offer excellent separation of FAMEs from biological samples. Very polar stationary phases give excellent separation of all FAMEs but have relatively low thermal stability, resulting in long retention times. Non-polar phases have a much greater thermal stability but inferior selectivity. For many analyses, phases of intermediate polarity, which combine the advantages of a relatively high resolution capability with relatively high thermal stability, are the most suitable. FAMEs can be identified by comparison of their retention times with those of individual purified standards or secondary standards based on lipids that have been well characterized in literature. Relative retention times and equivalent chain-length values also provide useful information. FAMEs can be quantitated by peak areas via calibration factors, and absolute concentrations can be determined by adding an internal standard. Among numerous applications in biomedical research, the analysis of fatty acids from body tissues may contribute to the understanding of the link between the dietary intake of fatty acids and the diseases with which these acids are associated.

Silver‐complexed dicyanobiphenyl‐substituted polymethylsiloxane encapsulated particles for packed capillary column supercritical fluid chromatography

AbstractHigh nitrile content cyano‐substituted polymethylsiloxane encapsulated particles were prepared for double bond selectivity in packed capillary column supercritical fluid chromatography (SFC). Silica particles were deactivated with 50% cyanopropyl‐substituted polymethylhydrosiloxane, and coated with 25% o,p‐dicyano‐p‐biphenyl‐substituted polymethylsiloxane (o,p,p‐DCBP), and 25% m,m‐dicyano‐p‐phenyl‐substituted polymethylsiloxane (m,m,p‐DCBP) stationary phases. Argentation of the particles was carried out by complex formation between silver ions and the cyano groups on the particles. Unsaturated fatty acid methylesters (FAMEs) were used to investigate the selectivity of these packing materials to double bonds using packed capillary column SFC. Separations were performed according to the degree, position, and geometrical configuration of the double bonds. The effect of temperature on selectivity was also investigated. The stability of the packing materials was evaluated by monitoring the loss in selectivity of the oleate/elaidate isomers after washing the column with supercritical CO2 at 300 atm and 85°C. The separation of FAMEs in a commercial fish oil was compared using capillary columns packed with DCBP coated and silver‐complexed particles.

Performance of silica‐bonded quinolinol as a selective stationary phase for packed capillary supercritical fluid chromatography

AbstractSelective stationary phases are of crucial importance in packed column supercritical fluid chromatography (SFC), and a potentially powerful selective stationary phase, silica‐bonded 8‐quinolinol, has been evaluated. Its performance was compared with that of an aminopropyl modified silica. The 8‐quinolinol columns could be used at temperatures up to 150°C, while the aminopropyl columns were degraded above 90°C. The general performance of the quinolinol column was very good when utilizing a mobile phase containing a polar modifier. Reduced plate heights of 4.8 to 3.8 and symmetrical peaks were obtained. Further, the quinolinol phase showed excellent selectivities under supercritical conditions. This was demonstrated by the separation of fatty acid methylesters and azaarenes. In addition, changes in selectivity could readily be made by loading with metal ions.

Temperature dependence of equivalent chain length values on capillary columns of different polarity

AbstractThe influence of temperature and capillary column stationary phase polarity on the equivalent chain length (ECL) values of unsaturated fatty acid methyl esters (FAMEs) is discussed. Comparisons are made of a bonded, nonpolar methyl silicone, bonded and nonbonded polyethylene glycols, and a highly polar, stabilized cyanosilicone stationary phase. The change in the ECL values over a 20° temperature range is used to demonstrate selectivity shifts and the influence of temperature on the separation of FAMEs on these phases. The effect of the degree of unsaturation of the FAME components, on the various stationary phases is also investigated.