13C nuclear magnetic resonance spectroscopy to determine olive oil grades

¹³C nuclear magnetic resonance spectroscopy was used in a first attempt to differentiate olive oil samples by grades. High resolution ¹³C NMR Distortionless Enhancement by Polarization Transfer (DEPT) spectra of 137 olive oil samples from the four grades, extra virgin olive oils, olive oils, olive pomace oils and lampante olive oils, were measured. The data relative to the resonance intensities (variables) of the unsaturated carbons of oleate (C-9 and C-10) and linoleate (L-9, L-10 and L-12) chains attached at the 1,3- and 2-positions of triacylglycerols were analyzed by linear discriminant analysis. The 1,3- and 2- carbons of the glycerol moiety of triacylglycerols along with the C-2, C-16 and C-18 resonance intensities of saturated, oleate and linoleate chains were also analyzed by linear discriminant analysis. The three discriminanting functions, which were calculated by using a stepwise variable selection algorithm, classified in the true group by cross-validation procedure, respectively, 76.9, 70.0, 94.4 and 100% of the extra virgin, olive oil, olive pomace oil and lampante olive oil grades.     

Comparative study of the determination of triacylglycerol in vegetable oils using chromatographic techniques

The triacylglycerols of some vegetable oil samples were determined using isocratic HPLC with refractive index (RI) detection, gradient solvent HPLC with evaporative light scattering detection (ELSD), capillary GC and theoretical calculations from FAME analysis in order to establish the suitability of these techniques. The response factors and the repeatability were investigated. Generally, the HPLC-RI detection technique can be used without application of response factors. HPLC-ELSD yields inaccurate results for low concentrations. Calculations assuming a 1,3-random 2-random distribution of fatty acids gave good results for olive oil and acceptable results for sunflower oil. The GC analysis requires the use of response factors.     

Composition of the triacylglycerols of the seeds of some representatives of the family labiatae

The fatty acid compositions of five species and the compositions of the triacylglycerols of 22 species of the family labiatae have been studied for the first time. Octadeca-ε12,13-dienoic acid has been detected in five species. The typical compositions of the triacylglycerols differs from those of known plant oils with a similar set of fatty acids by the absence of triacylglycerols of the S₃ type and the presence of the S₂U type (0.1–1.6%). The main types are SU₂ (5–24%) and U₃ (74–95%). In a comparison of the position-species composition of the oils studied it was found that the oils of the plants of this family are distinguished by a greater diversity of species of triacylglycerols and also by the nature of the distribution of the unsaturated acyl residues between the 1,3- and 2-positions. In the majority of oils studied, the 2- position is enriched with the 18:1 acid, while the 18:2 acid is distributed predominantly in the 1,3- positions, and the nature of the distribution of the 18:3 acid is determined by its proportion in the total.     

Capillary gas chromatography combined with high performance liquid chromatography for the structural analysis of olive oil triacylglycerols

The triacylglycerol composition of olive oil samples has been determined by stereospecific analysis after partial hydrolysis with ethyl magnesium bromide, derivatization, preparative chiral HPLC, transesterification, and GC quantitation of fatty acid methyl esters. The data obtained for position sn-2 were compared with those from capillary GC analysis of monoacyl sn-2-glycerols after enzymatic lipolysis of triacylglycerols. The determination of triacylglycerols collected by silver ion HPLC and quantified (as fatty acid methyl esters) by GC, together with direct GC analysis on a polar column, have then furnished a comprehensive picture of the triacylglycerol content of olive oil.     

Composition of the triacylglycerols of the seeds of some representatives of the family labiatae

The fatty acid compositions of five species and the compositions of the triacylglycerols of 22 species of the family labiatae have been studied for the first time. Octadeca-12,13-dienoic acid has been detected in five species. The typical compositions of the triacylglycerols differs from those of known plant oils with a similar set of fatty acids by the absence of triacylglycerols of the S₃ type and the presence of the S₂U type (0.1–1.6%). The main types are SU₂ (5–24%) and U₃ (74–95%). In a comparison of the position-species composition of the oils studied it was found that the oils of the plants of this family are distinguished by a greater diversity of species of triacylglycerols and also by the nature of the distribution of the unsaturated acyl residues between the 1,3- and 2-positions. In the majority of oils studied, the 2- position is enriched with the 18:1 acid, while the 18:2 acid is distributed predominantly in the 1,3- positions, and the nature of the distribution of the 18:3 acid is determined by its proportion in the total.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 618–625, September–October, 1979.     

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.     

A nuclear magnetic resonance (1H and 13C) and isotope ratio mass spectrometry (δ13C, δ2H and δ18O) study of Andalusian olive oils

We have determined δ¹³C, δ²H and δ¹⁸O isotopic abundances in Andalusian olive oils. In addition, the fatty acid composition and the distribution of isomers at positions 1,3 and 2 of glycerol were determined by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy, respectively. Isotopic results obtained for four series of oil samples extracted from olives harvested in the 2004/05 and 2005/06 seasons are discussed in terms of olive variety, ripeness, geographical origin, fatty acid composition and growing altitude. A distinction was also established between olives grown in irrigated and in dry land by studying selected samples of the previous series and others from the 2005/06, 2006/07, 2007/08 and 2008/09 seasons. The results showed that olive ripeness does not influence the abundance of any of the three isotopes studied. On the other hand, the olive variety influences the abundance of the oxygen and hydrogen isotopes, and also, less markedly, that of carbon. No clear‐cut effect of height or latitude on isotope values is observed, probably because the olive variety also changes with height and latitude, thus masking such influences. The oil samples from dryland‐grown olives had increased δ¹³C values relative to irrigation‐grown olives. In addition, no definite relationship appears to exist between isotope distribution and fatty acid composition. Finally, oil samples from olives harvested in the 2005/06 season in Italy could be distinguished from those from Spain in terms of their isotopic values (δ²H mainly). Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of fatty acid and triacylglycerol composition of human adipose tissue.

The fatty acid composition of adipose tissue was studied in a population in western Andalusia with a diet in which the fat contribution is mainly from olive oil. The lipid composition of adipose tissue, including the fatty acid composition of triacylglycerols, was examined by capillary gas chromatography. Thirty-five peaks were resolved, ranging in chain length from 12 to 24 carbon atoms, including geometric and positional isomers. The major triacylglycerol was POO, followed by PLO and OOO.     

Triacylglycerol determination based on fatty acid composition using chemometrics

Multivariate statistics were applied to analyse the relationships between fatty acids and triacylglycerols in virgin olive oil. The chemical significance of factors obtained by applying principal components analysis to both sets of variables was established, and this method is proposed as a useful tool for obtaining information about the biosynthetic route of fatty acids and its regulation. Finally, because both sets of data present the same latent structure, multiple regression analysis is proposed for determining the triacylglycerol composition of an oil according to its fatty acid composition.     

Mass-spectrometric behavior of oxidized triacylglycerols and their TMS derivatives

The mass spectrometric fragmentation of oxidized triacylglycerols of castor oil and their TMS derivatives have been studied for the first time. Three types of fragmentary ions have been detected: 1) products of the successive elimination of TMSOH; 2) ions characteristic for unoxidized triacylglycerols; and 3) products of the cleavage of C-C bonds present in the α-positions to the TMSO groups, which are characteristic for these hydroxy acid derivatives. The origin of these ions has been confirmed by measurements of elementary composition and by the analysis of MD spectra. The specific cleavage of the C-C bonds in the α-positions to the OTMS (OH) groups permits the mass-spectrometric method to be proposed for the analysis of mixtures of oxidized triacylglycerols.     

A novel approach to the rapid assignment of 13C NMR spectra of major components of vegetable oils such as avocado,mango kernel and macadamia nut oils

Assignment of ¹³C nuclear magnetic resonance (NMR) spectra of major fatty acid components of South African produced vegetable oils was attempted using a method in which the vegetable oil was spiked with a standard triacylglycerol. This proved to be inadequate and therefore a new rapid and potentially generic graphical linear correlation method is proposed for assignment of the ¹³C NMR spectra of major fatty acid components of apricot kernel, avocado pear, grapeseed, macadamia nut, mango kernel and marula vegetable oils. In this graphical correlation method, chemical shifts of fatty acids present in a known standard triacylglycerol is plotted against the corresponding chemical shifts of fatty acids present in the vegetable oils. This new approach (under carefully defined conditions and concentrations) was found especially useful for spectrally crowded regions where significant peak overlap occurs and was validated with the well‐known ¹³C NMR spectrum of olive oil which has been extensively reported in the literature. In this way, a full assignment of the ¹³C{1H} NMR spectra of the vegetable oils, as well as tripalmitolein was readily achieved and the resonances belonging to the palmitoleic acid component of the triacylglycerols in the case of macadamia nut and avocado pear oil resonances were also assigned for the first time in the ¹³C NMR spectra of these oils. Copyright © 2009 John Wiley & Sons, Ltd.     

Original normal-phase high-performance liquid chromatographic separation of monoacylglycerol classes from extra virgin olive oil triacylglycerols for their stereospecific analysis

An innovative procedure to separate the 3 isomeric sn-monoacylglycerols (MAG) classes (sn-1-, sn-2-, sn-3-MAG) is described. MAGs, obtained by chemical deacylation of triacylglycerols (TAGs), have been derivatized with (S)-(+)-1-(1-naphtyl)ethyl-isocyanate, and the resulting urethane derivatives have been separated by normal-phase high-performance liquid chromatography. This procedure allows resolution as diasteroisomers of the 2 enantiomeric classes (sn-1-MAG and sn-3-MAG), without the need of a chiral column, and to separate also the isomeric sn-2-MAG class; moreover, by introducing a chromophoric moiety, this reagent makes possible the ultraviolet detection of the analyte molecules. This procedure has been used to obtain the stereospecific analysis of the TAG fraction of extra virgin olive oil samples. The use of a nondestructive detector permitted the collection of the individual urethane classes; the fatty acid composition of each was determined by high-resolution gas chromatography, obtaining directly from the data the fatty acid distribution within each sn- position of TAGs. To validate this new method, the results have been compared with those obtained by 2 other procedures for TAG stereospecific analysis, and the obtained results were satisfactory since the proposed method gave data very similar to the other procedures.     

Quantification of the ratio of positional isomer dilinoleoyl-oleoyl glycerols in vegetable oils

The distribution of fatty acids in a triacylglycerol is of great importance from nutritional, biochemical, quality and technological points of view. The ratio of triacylglycerol positional isomers containing two linoleic acid (18:2) and one oleic acid (18:1) moieties--namely, 1(3),2-dilinoleoyl-3(1)-oleoyl glycerol (LLO) and 1,3-dilinoleoyl-2-oleoyl glycerol (LOL)--were quantified in grape seed, olive, pumpkin seed, soybean, sunflower and wheat germ oils by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (HPLC/APCI-MS) in selected ion monitoring (SIM) mode. Relative LOL contents (LOL/(LLO+LOL)) of the oils were calculated from the mass abundances of the [LL]+ and [LO]+ diacylglycerol fragment ions ([M+H-RCOOH]+) using a calibration curve. The calibration curve of the relative diacylglycerol mass abundances was measured in SIM mode. The relative LOL contents were found to be relatively consistent for each oil variety. The relative LOL content in grape seed, sunflower, pumpkin seed, soybean and wheat germ oils accounted for 44.2 +/- 2.6, 26.8 +/- 3.2, 16.7 +/- 4.6, 15.9 +/- 2.9 and 13.9 +/- 4.3%, respectively. Only olive oils contained practically 100% of the LLO isomer. These results indicate that the unsaturated fatty acids such as linoleic and oleic acids have 'non-random' distribution patterns in various oils.     

Production of Human Milk Fat Replacement Rich of 1,3-dioleoyl-2-palmitoilglycerol From Enzymatic Interesterification Tripalmitin,Ethyl Oleate And Mixture of VCO,Soybean Oil And Fish Oil

Human Milk is naturally the only source of food for infant in their early life. It contains 2-6% lipid which provides about 50% of the total energy needed by the infant. Human milk fat (HMF) mainly as TAG with the specific fatty acid composition, palmitic acid (C16:0) (20-25%), which is primary located at sn-2 of glycerol bonds (70%) and oleic acid (C18:1), located at sn-1,3 (35%). HMF also provide fatty acids such as linoleic acid, linolenic acid, EPA, DHA and lauric acid that are very important for infant. The purposes of this research are to synthesize of 1,3-dioleoyl-2-palmitoilglycerol (OPO) and to determine the best composition of OPO, VCO, soybean oil and fish oil for HMFS production for infant formula. Interesterification of tripalmitin and ethyl oleate using immobilized lipase from Rhizomucor miehei (Lipozym RM IM) were used to synthesize of OPO. Interesterification product of mixed VCO, soybean oil and fish oil that are source of lauric acid, linoleic acid, α-linolenic, EPA and DHA, were formulated in mass ratio (58:20:20:2) and (70:18:10:2) for obtaining HMFS which have fatty acids composition similar or close to HMF. Composition of fatty acids from product were analyzed by GCMS. From this research, were obtained HMFS containing palmitic acid as much as 28.89% where 84.49% of that are located at sn-2 while sn-1,3 position are dominated by oleic acid as much as 55.11% from the total 38.7% and 70:18:10:2 w/w is the best composition of interesterification product, VCO, soybean oil and fish oil to obtain HMFS similar to HMF.     

The renaissance of high-energy CID for structural elucidation of complex lipids: MALDI-TOF/RTOF-MS of alkali cationized triacylglycerols

Triacylglycerols were analyzed as cationized species (Li⁺, Na⁺, K⁺) by high-energy CID at 20 keV collisions utilizing MALDI-TOF/RTOF mass spectrometry. Precursor ions, based on [M+Li]⁺-adduct ions exhibited incomplete fragmentation in the high and low m/z region whereas [M+K]⁺-adducts did not show useful fragmentation. Only sodiated precursor ions yielded product ion spectra with structurally diagnostic product ions across the whole m/z range. The high m/z region of the CID spectra is dominated by abundant charge-remote fragmentation of the fatty acid substituents. In favorable cases also positions of double bonds or of hydroxy groups of the fatty acid alkyl chains could be determined. A-type product ions represent the end products of these charge-remote fragmentations. B- and C-type product ions yield the fatty acid composition of individual triacylglycerol species based on loss of either one neutral fatty acid or one sodium carboxylate residue, respectively. Product ions allowing fatty acid substituent positional determination were present in the low m/z range enabling identification of either the sn-1/sn-3 substituents (E-, F-, and G-type ions) or the sn-2 substituent (J-type ion). These findings were demonstrated with synthetic triacylglycerols and plant oils such as cocoa butter, olive oil, and castor bean oil. Typical features of 20 keV CID spectra of sodiated triacylglycerols obtained by MALDI-TOF/RTOF MS were an even distribution of product ions over the entire m/z range and a mass accuracy of ±0.1 to 0.2 u. One limitation of the application of this technique is mainly the insufficient precursor ion gating after MS1 (gating window at 4 u) of species separated by 2 u.     

Neutral lipids of freon extracts of the fruit ofHippophaë rhamnoides

The composition of Freon extracts of the fruit of two samples of sea buckthorn has been studied — from the Western Pamir and from Biisk. The presence of 16 classes of lipids has been shown. The distribution of the fatty acid residues between the 2- and 1,3-positions of the triacyl glycerols of these extracts have been studied by the method of lipase hydrolysis, and their position-species compositions have been established.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 231–234, March–April, 1987.     

Deuterium NMR used to indicate a common mechanism for the biosynthesis of ricinoleic acid by Ricinus communis and Claviceps purpurea

Previous studies have shown that ricinoleic acid from castor bean oil of Ricinus communis is synthesized by the direct hydroxyl substitution of oleate, while it has been proposed that ricinoleate is formed by hydration of linoleate in the ergot fungus Claviceps purpurea. The mechanism of the enzymes specific to ricinoleate synthesis has not yet been established, but hydroxylation and desaturation of fatty acids in plants apparently involve closely related mechanisms. As mechanistic differences in the enzymes involved in the biosynthesis of natural products can lead to different isotopic distributions in the product, we could expect ricinoleate isolated from castor or ergot oil to show distinct (2)H distribution patterns. To obtain information concerning the substrate and isotope effects that occur during the biosynthesis of ricinoleate, the site-specific natural deuterium distributions in methyl ricinoleate isolated from castor oil and in methyl ricinoleate and methyl linoleate isolated from ergot oils have been measured by quantitative (2)H NMR. First, the deuterium profiles for methyl ricinoleate from the plant and fungus are equivalent. Second, the deuterium profile for methyl linoleate from ergot is incompatible with this chemical species being the precursor of methyl ricinoleate. Hence, it is apparent that 12-hydroxylation in C. purpurea is consistent with the biosynthetic mechanisms proposed for R. communis and is compatible with the general fundamental mechanistic similarities between hydroxylation and desaturation previously proposed for plant fatty acid biosynthesis.     

Rapid characterization of edible oils by direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis using triacylglycerols

Direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis of solutions of edible fats/oils yielded spectra useful for their rapid differentiation and classification. Results also reflected the individual fatty acid components and their degree of unsaturation. After dissolution in hexane, MALDI-MS analysis revealed spectra showing characteristic triacylglycerols (TAGs), the main fat/oil components, as sodium adduct ions. The Euclidean distances calculated using the mass and intensity values for 20 TAGs were used to evaluate and compare spectra. With cluster analysis, animal fats grouped together differently than vegetable oils and the individual oils grouped together by type. The ion abundances for the individual TAGs and their presumed compositions were used to approximate the overall fatty acid composition of canola, soybean, corn, olive and peanut oil, as well as lard. Using this approach the calculated fatty acid compositions and degree of unsaturation generally fell within about 4% of literature values. When the degree of saturation was compared with values calculated from the package labeling the differences were about 7%.     

Lipase-Catalyzed Acidolysis of Palm Mid Fraction Oil with Palmitic and Stearic Fatty Acid Mixture for Production of Cocoa Butter Equivalent

Cocoa butter equivalent (CBE) was prepared by enzymatic acidolysis reaction of substrate consisting of refined palm mid fraction oil and palmitic–stearic fatty acid mixture. The reactions were performed in a batch reactor at a temperature of 60 °C in an orbital shaker operated at 160 RPM. Different mass ratios of substrates were explored, and the composition of the five major triacylglycerols (TAGs) of the structured lipids was identified and quantified using cocoa butter certified reference material IRMM-801. The reaction resulted in production of cocoa butter equivalent with the TAGs' composition (1,3-dipalmitoyl-2-oleoyl-glycerol 30.7 %, 1-palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol 40.1%, 1-palmitoy-2,3- dioleoyl glycerol 9.0 %, 1,3-distearoyl-2-oleoyl-glycerol 14.5 %, and 1-stearoyl-2,3-dioleoyl glycerol 5.7 %) and with onset melting temperature of 31.6 °C and peak temperature of 40.4 °C which are close to those of cocoa butter. The proposed kinetics model for the acidolysis reaction presented the experimental data very well. The results of this research showed that palm mid fraction oil TAGs could be restructured to produce value added product such as CBE.     

Chemometric Study of Fatty Acid Composition of Virgin Olive Oil from Four Widespread Greek Cultivars

Virgin olive oil (VOO) is one of the key components of the Mediterranean diet owing to the presence of monounsaturated fatty acids and various bioactive compounds. These beneficial traits, which are usually associated with the cultivar genotype, are highlighting the demand of identifying characteristics of olive oil that will ensure its authenticity. In this work, the fatty acid (FA) composition of 199 VOO samples from Koroneiki, Megaritiki, Amfissis, and Manaki cultivars was determined and studied by chemometrics. Olive cultivar greatly influenced the FA composition, namely, oleic acid (from 75.36% for Amfissis to 65.81% for Megaritiki) and linoleic acid (from 13.35% for Manaki to 6.70% for Koroneiki). Spearman’s rho correlation coefficients revealed differences and similarities among the olive oil cultivars. The use of the forward stepwise algorithm identified the FAs arachidonic acid, gadoleic acid, linoleic acid, α-linolenic acid, palmitoleic acid, and palmitic acid as the most significant for the differentiation of samples. The application of linear and quadratic cross-validation discriminant analysis resulted in the correct classification of 100.00% and 99.37% of samples, respectively. The findings demonstrated the special characteristics of the VOO samples derived from the four cultivars and their successful botanical differentiation based on FA composition.