A Versatile GC Method for the Analysis of Alkylglycerols and Other Neutral Lipid Classes

The present study focuses on the GC analysis of alkylglycerols and other neutral lipid classes. For that matter, a simple and rapid method based on the direct on-column injection has been developed for the simultaneous analysis of numerous fatty samples from different sources. Chemical transformation or treatment of the sample before the injection of samples is not required. In addition, quantification of different lipid classes was also evaluated with satisfactory results. Excellent results regarding reproducibility and resolution were observed. This method can be utilized for multiple purposes such as analyses of edible oils, shark liver oils, deodorizer distillates, time course analyses of lipase-catalyzed reactions, etc.     

Direct flow injection profiling of acyl glycerols from food products using isopropanol as solvent

This work proposes a novel method for the direct flow injection profiling of acylglycerols in edible oils and fats without preliminary extraction and consequent reconstitution in the injection solvent. The work exploits the outstanding performance of high‐resolution mass spectrometry to target unique elemental compositions even in the most complex matrices. The performance of isopropanol as the unique solvent for both the solubilization and analysis of acylglycerols was investigated in comparison with other classical methods involving preliminary extractions, sample recovery, and analysis. The calibrations of two triglyceride standards (triolein and trilinolenin) were successfully performed in presence and absence of oil matrix. As final application, the effects on the acylglycerol fraction of a heat treatment on three different fat samples (extra virgin olive oil, lard oil, and fat from dry cured ham—speck) were monitored. The proposed method is therefore suitable for a rapid evaluation of acylglycerol fractions in food lipid samples.     

Lipophilicity of oils and fats estimated by TLC

A representative series of natural toxins belonging to alkaloids and mycotoxins classes was investigated by TLC on classical chemically bonded plates and also on oils‐ and fats‐impregnated plates. Their lipophilicity indices are employed in the characterization and comparison of oils and fats. The retention results allowed an accurate indirect estimation of oils and fats lipophilicity. The investigated fats and oils near classical chemically bonded phases are classified and compared by means of multivariate exploratory techniques, such as cluster analysis, principal component analysis, or fuzzy‐principal component analysis. Additionally, a concrete hierarchy of oils and fats derived from the observed lipophilic character is suggested. Human fat seems to be very similar to animal fats, but also possess RP‐18, RP‐18W, and RP‐8.     

Fast GC for the analysis of fats and oils

Fast and conventional GC techniques were both applied to ten different lipidic matrices and the results then compared. The fats and oils were of fish, animal, and vegetable origin and were all simultaneously transesterified with acidic methanol before performing batch analysis of the fatty acid methyl esters (FAMEs) obtained. All FAMEs samples were consecutively analyzed three times by each method. The fast method significantly reduced the time required for analysis by a factor of 5 while maintaining a similar resolution. Furthermore, the reproducibility of relative quantitative data was measured on going from one method to the other. Peak identification was achieved through conventional GC‐MS in combination with linear retention index values contained in a home library and information derived from comprehensive 2D GC group patterns.     

High Resolution Separation of Non-Polar Lipid Classes by HPLC-ELSD Using Alumina as Stationary Phase

This study describes the performance and capacity of alumina as stationary phase in an HPLC-ELSD (evaporative light-scattering detection) method optimized for the separation of the non-polar lipid classes hydrocarbons, wax esters, sterol esters, triacylglycerols, and sterols, including quantitative determination of these lipid classes in natural samples. By using gradient elution and constant equilibration times between injections, highly reproducible separations of triacontane, stearyl oleate, and cholesterol oleate were accomplished with a binary mobile phase system. Phase A contained 0.5% tetrahydrofuran in hexane and phase B 20% isopropanol and 20% tetrahydrofuran in hexane. The same system was also used to determine the non-polar lipid classes in a zooplankton sample, the major lipid class being wax esters, followed by triacylglycerols, sterol esters, sterols, and hydrocarbons. Substantial amounts of an unknown compound, possibly acylated glyceryl ethers, were also found. The equilibration time of alumina was relatively slow compared to a polyvinyl alcohol stationary phase used earlier by the authors and calibration curves for different lipid classes were more uniform and linear with alumina.     

1-alkoxyvinyl esters: renaissance of half-century-old acyl donors with potential applicability

Among the various kinds of acyl donors, the 1-alkoxyvinyl esters have characteristic features, such as a high reactivity under nearly neutral conditions and the generation of neutral and volatile esters as single coproducts. Although their use in organic syntheses began in the middle of the 1950s, no significant progress has been seen. This is probably because the existing method of preparing alkoxyvinyl esters used toxic mercuric salts and was not totally applicable for those esters having functionalized acyl moieties. We have discovered that the use of a catalytic amount of the less toxic [RuCl2(p-cymene)]2 effectively accelerates the addition of carboxylic acids to ethoxyacetylene to give ethoxyvinyl esters bearing a variety of functionalized acyl groups in high yields. This discovery has opened a new avenue for developing new reactions and new synthetic methodologies based on the design and use of these acyl donors with suitable functional groups. Such examples include (i) the installation of hydrophilic acyl moieties on biologically active compounds, (ii) asymmetric Pummerer-type reactions, (iii) aromatic Pummerer-type reactions, (iv) the lipase-catalyzed desymmetrization of symmetrical 1,3-diols, and (v) lipase-catalyzed domino reactions. Future possibilities for these acyl donors are also discussed.     

Separation of fatty acid methyl esters by comprehensive two-dimensional supercritical fluid chromatography with packed columns and programming of sampling duration

Fatty acid methyl esters from various fats and oils were separated by comprehensive two-dimensional supercritical fluid chromatography with conventional packed columns and FID detection. The first dimension was a silica gel column and the second dimension was an ODS column. This combination was largely orthogonal for the separation of fatty acid methyl esters. The first dimension separations were primarily based on the number of double bonds while the second dimension separations were based on the chain length. The highly-ordered chromatograms and improved resolution allowed the easy detection and identification of minor components. Although the first dimension separations were performed under isobaric conditions where the peak width increased in proportion to the retention, the programming of the sampling duration allowed us to maintain the optimum re-injection frequency (3–4 times) per peak into the second dimension and so to minimize the total analysis time without deteriorating the resolution.     

Monitoring lipase-catalyzed methanolysis of sunflower oil by reversed-phase high-performance liquid chromatography: elucidation of the mechanisms of lipases

Reversed-phase high-performance liquid chromatography (RP-HPLC) with UV detection at 210 nm was used to monitor the formation of the major compounds during the lipase-catalyzed transesterification reaction of sunflower oil with methanol. Individual triacylglycerols, diacylglycerols, monoacylglycerols as well as fatty acids and their corresponding methyl esters were separated using acetonitrile/acetone as a mobile phase and a combined linear gradient-isocratic-step gradient-isocratic elution procedure. Another relatively short method consisting of a linear gradient elution followed by an isocratic elution gave similar results, yet with lower resolution. HPLC/mass spectrometry with an ion trap analyzer and atmospheric pressure chemical ionization source was used for the identification of the individual compounds. Individual calibration curves obtained with UV detection at 210 nm were found to be of use for quantitative analyses of double-bond containing methyl esters and acylglycerols. The use of the RP-HPLC methods in the elucidation of the mechanisms of three immobilized lipases, namely Lipozyme TL IM, Lipozyme RM IM and Novozym 435, in biodiesel production was described.     

Major lipid classes separation of buttermilk,and cows,goats and ewes milk by high performance liquid chromatography with an evaporative light scattering detector focused on the phospholipid fraction

An improved HPLC–ELSD method has been developed for the analysis of the lipid classes of buttermilk and milk from different species, focused in the phospholipids fraction without a prior fractionation step and in a single run. The total lipid profile analysis showed the major and minor lipid compounds as cholesterol esters, triacylglycerides, cholesterol, diacylglycerides, free fatty acids, monoacylglycerides, and also the polar compounds as glucosylceramide, lactosylceramide, phosphatidyl-ethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine. The identification and quantification of the different compounds, using calibration curves made with individual standards and the low coefficients of variation obtained in the inter- and intra-assays showed the suitability of the developed method. In this study, we optimized and validated a quantitative HPLC–ELSD method at a concentration level suitable for routine analysis of the major lipid classes in milk and dairy products.     

Application of high-performance liquid chromatography diode array detection and mass spectrometry to the analysis of characteristic compounds in various essential oils

A high-performance liquid chromatography (HPLC) method was established using an analytical reversed-phase column and gradient elution to achieve chromatographic separation of typical compounds in essential oils. For detection, a diode array detector monitoring different wavelengths simultaneously as well as a mass spectrometer (MS) were used. Atmospheric pressure chemical ionization operating in the positive mode turned out to be a suitable tool to detect volatiles of different chemical classes and to identify them in essential oil matrices. Characteristic fingerprints of eucalyptus, lavender, may chang, pine, rosemary, thyme, and turpentine essential oils monitored at a representative wavelength (220 nm) demonstrated the suitability of HPLC in essential oil analysis. Additional monitoring wavelengths (210, 250, and 280 nm) provided useful information about the identity of the specific component and opened the possibility to differentiate presumably coeluting compounds by means of their distinct absorption behavior. Finally, peak assignment in seven essential oils was performed on the basis of characteristic retention times and UV and MS data of a broad set of reference volatiles.     

New opportunity for enzymatic modification of fats and oils with industrial potentials

Novozym 435 (Candida antarctica lipase)-catalyzed glycerolysis of commercial oils and fats to produce monoglycerides (MGs) was investigated using a tetraammonium-based ionic liquid (IL) as a reaction medium. A 90% yield of MGs and nearly 100% conversion of triglycerides in this ionic liquid were achieved, markedly higher than in normal solvents. The amphiphilic structure of cocosalkyl pentaethoxi methyl ammonium methosulfate (CPMA.MS) was suggested to be capable of creating a compatible system for glycerol, oils and fats, as well as inducing the shift of reaction equilibrium to the formation of MG. Interestingly, over a wide range of solvent dosage, the higher yields of MG were observed, indicating the good bulky substrate-tolerating capacity of the IL. The universal validity of the protocol was verified by being successfully applied to different commercial oils and fats. Excellent operational stability of the lipase and the reusability of IL were also observed in consecutive batchwise reactions. The results indicate that the protocol developed in this work provides a new environmentally benign "solution" to the enzymatic modification of fats and oils with industrial potentials.     

Comprehensive two-dimensional liquid chromatography x gas chromatography: evaluation of the applicability for the analysis of edible oils and fats

Edible fats and oils are complex mixtures containing a wide range of (classes of) compounds. The most important group of compounds are the triglycerides (triacylglycerides, TAGs). Because of the large number of possible fatty acid combinations, an enormous number of TAGs is possible. In the present feasibility study, the applicability of different modes of comprehensive two-dimensional LCXGC for detailed oil and fat analysis is evaluated. Comprehensive LCXGC was found to be an extremely powerful analytical method for the analysis of complex TAG samples. Using the new comprehensive set-ups, TAGs can be separated according to two independent parameters: carbon number vs. number of double bonds, or fatty acid composition vs. number of double bonds. The information content of comprehensive separations by far exceeds that of the current generation of analytical methods. The quantitative results of the separations show a good agreement with data obtained from standard analytical methods. The comprehensive methods studied can also be used for fingerprinting of oil samples, as well as for the analysis of target compounds or compound groups. Highly detailed separations of olive oil samples were obtained. Zooming in on one region of the chromatogram allowed reliable analysis of wax esters without interferences of sterol esters.     

Comprehensive two-dimensional liquid chromatography×gas chromatography: evaluation of the applicability for the analysis of edible oils and fats

Edible fats and oils are complex mixtures containing a wide range of (classes of) compounds. The most important group of compounds are the triglycerides (triacylglycerides, TAGs). Because of the large number of possible fatty acid combinations, an enormous number of TAGs is possible. In the present feasibility study, the applicability of different modes of comprehensive two-dimensional LC×GC for detailed oil and fat analysis is evaluated. Comprehensive LC×GC was found to be an extremely powerful analytical method for the analysis of complex TAG samples. Using the new comprehensive set-ups, TAGs can be separated according to two independent parameters: carbon number vs. number of double bonds, or fatty acid composition vs. number of double bonds. The information content of comprehensive separations by far exceeds that of the current generation of analytical methods. The quantitative results of the separations show a good agreement with data obtained from standard analytical methods. The comprehensive methods studied can also be used for fingerprinting of oil samples, as well as for the analysis of target compounds or compound groups. Highly detailed separations of olive oil samples were obtained. Zooming in on one region of the chromatogram allowed reliable analysis of wax esters without interferences of sterol esters.     

Update on solid-phase extraction for the analysis of lipid classes and related compounds

This article provides information on the different procedures and methodologies developed when solid-phase extraction (SPE) is used for lipid component separation. The analytical systematics, established by different authors and designed to separate groups of compounds and also specific components by using a combination of chromatographic supports and solvents are presented. The review has been divided into three parts, which we consider well defined: edible fats and oils, fatty foods and biological samples. Separations of non-polar and polar lipids is the most extensive systematic, although many other published methods have been established to isolate specific components or a reduced number of components from edible fats and oils, fatty foods or biological samples susceptible to further analysis by other quantitative techniques.     

Autooxidation of Unsaturated Fatty Acids and Their Esters

The shape of DSC curves of non-isothermal oxidation of fats was explained. Two main exothermic effects overlapped partially are caused by hydroperoxide formation (first peak) and by further oxidation of peroxides (second peak). The oxidation of oils and lipid analogues of various peroxide concentration showed that only the start of the oxidation process is affected by initial concentration of peroxides, other temperatures determined from DSC curves are not connected with this parameter. The computer simulations gave the best agreement of theoretical and experimental data for kinetic scheme of a two-step consecutive reaction with autocatalytic start. The comparison of activation energies calculated for isothermal and non-isothermal autooxidation of unsaturated fatty acids and their esters also confirmed this interpretation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Predicting vapor–liquid equilibria of fatty systems

In the present work, a group contribution method is proposed for the estimation of the vapor pressure of fatty compounds. For the major components involved in the vegetable oil industry, such as fatty acids, esters and alcohols, triacylglycerols (TAGs) and partial acylglycerols, the optimized parameters are reported. The method is shown to be accurate when it is used together with the UNIFAC model for estimating vapor–liquid equilibria (VLE) of binary and multicomponent fatty mixtures comprised in industrial processes such as stripping of hexane, deodorization and physical refining. The results achieved show that the group contribution approach is a valuable tool for the design of distillation and stripping units since it permits to take into account all the complexity of the mixtures involved. This is particularly important for the evaluation of the loss of distillative neutral oil that occurs during the processing of edible oils.The combination of the vapor pressure model suggested in the present work with the UNIFAC equation gives results similar to those already reported in the literature for fatty acid mixtures and oil–hexane mixtures. However, it is a better tool for predicting vapor–liquid equilibria of a large range of fatty systems, also involving unsaturated compounds, fatty esters and acylglycerols, not contemplated by other methodologies. The approach suggested in this work generates more realistic results concerning vapor–liquid equilibria of systems encountered in the edible oil industry.     

Evaluation of a rapid method for preparation of fatty acid methyl esters for analysis by gas-liquid chromatography

The major limitation to fatty acid analysis by gas-liquid chromatography is associated with preparation of fatty acid methyl esters (FAME). In the present study, FAME preparations were made from plant oils (corn, olive, sunflower), sunflower oil margarine, lard and various animal tissue fats by a rapid transesterification involving tetramethylammonium hydroxide in methanol, and also by a longer conventional saponification-esterification method. Fats from animal (beef, mutton, pork) adipose tissues were extracted by a simpler modified procedure and also by the Folch method prior to the rapid and the conventional FAME preparations, respectively. FAME analysis on a gas-liquid chromatograph equipped with a Silar 10C glass capillary column indicated similar fatty acid composition of a given fat or oil, whether FAME was prepared by the rapid or the longer conventional method. The data obtained by both methods were very highly correlated for all the fats (r = 0.9895 - 0.9999). However, the rapid method showed a tendency for enhanced recoveries of lower chain fatty acids (e.g. 14:0), and also of unsaturated C18 isomers. Possibly, losses of fatty acids that occurred during the lengthy fat extraction, fatty acid esterification or ether-evaporation FAME concentration steps (conventional method) were minimised by the single transesterification step (rapid method). This rapid transesterification method appears to be an attractive alternative to FAME preparation from a wide variety of different fats for gas-liquid chromatographic analysis.     

Synthesis of deoxy sugar esters: a chemoenzymatic stereoselective approach affording deoxy sugar derivatives also in the form of aldehyde

A chemoenzymatic synthesis of deoxy sugar esters is described. The synthesis is based on the O-alkylation of carboxylic acid with 2-bromo-5-acetoxypentanal. The method allows treatment of hydroxy carboxylic acids without protection of alcoholic hydroxyl groups. Several stereoisomeric deoxy sugar esters were resolved (up to ee or de > 98%) using a lipase-catalyzed acetylation of hemiacetals that in certain cases afforded deoxy sugar derivatives in the form of aldehydes. The stereochemistry of the reactions was determined by the NMR spectra of mandelic acid derivatives.     

Determination of Biodiesel and Triacylglycerols in Diesel Fuel by LC

A high performance liquid chromatographic method was developed for quantifying blends of biodiesel (simple alkyl esters of fatty acids) in petrodiesel. The method uses a silica column with an isocratic mobile phase consisting of hexane and methyl t-butyl ether. Separated components were quantitated using either an evaporative light scattering detector (ELSD) or UV detector. Precision of injection and linearity of response of the ELSD and UV detectors over a range of biodiesel-petrodiesel blends [1–30 v/v %] were established by use of standards. The method also can be used for quantitating similar levels of oils or fats (triacylglycerols) in petrodiesel.     

Identification and Quantitation of Reaction Intermediates and Residuals in Lipase-Catalyzed Transesterified Oils by HPLC

A high-performance liquid chromatography (HPLC) unit equipped with size exclusion column and a refractive index detector was used for simultaneous monitoring, identification, and quantitation of the reaction components from lipase-catalyzed transesterification of three oils. The procedure simultaneously separated and detected the unreacted triacylglycerols (TAG), diacyl-, and monoacyl-glycerol (DAG and MAG) co-products, residual alcohol as well as free fatty acid (FFA) based on retention times. The chromatograms showed well separated and resolved peaks. The elution of the components from the transesterification reaction in increasing order was: TAG < DAG < FFA < MAG. Generally, higher alcohol ratios decreased the conversion of TAG in all the oils studied with between 14% and 94% of TAG remaining at all the treatment combinations. Higher amount of salmon skin oil (SSO) TAG was generally converted to DAG than Rothsay composite (RC) and olive oil (OO) TAG. Relatively higher amount of OO DAG was converted to MAG than SSO and RC with only 5–14% DAG remaining in OO. RC and OO generally accumulated less MAG, and this was reflected as lower MAG levels in RC (<6%) and OO (<14%) compared with SSO (<27%). For the various treatment combinations and the three oils used in this study, the least amount of FFA was recorded in transesterified OO with a maximum of approximately 4%. This HPLC method can be used as a simple and fast technique to analyze the reaction components and products of transesterification reactions without the need for additional derivatization steps.