Quantitative high resolution13C nuclear magnetic resonance of the olefinic and carbonyl carbons of edible vegetable oils

The acyl distribution and acyl positional distribution (1,3-acyl and 2-acyl) of triacylglycerols derived from edible vegetable oils has been examined by¹³C nuclear magnetic resonance (NMR) spectroscopy. The acyl profile of three natural oils (corn, peanut, canola) and one specialty oil (high oleic sunflower oil, Trisun^® 80) has been defined from the high resolution (medium field 75.4 MHz) spectrum of the carbonyl and olefinic regions. The quantitative integrity of the NMR derived acyl profile is substantiated by gas chromatographic (GC) analysis. The positional distribution data of the three natural oils indicates that polyunsaturates are replaced in the 1,3-glycerol position exclusively by saturates, while the oley distribution remains, for the most part, randomly distributed. The same is not true for Trisun^® 80, which shows a more random distribution of the linoleyl groups as well.     

Quantitative high-resolution13C and1H nuclear magnetic resonance of ω3 fatty acids from white muscle of atlantic salmon (Salmo salar)

High-resolution¹³C nuclear magnetic resonance (NMR) spectra have been obtained and used to define the ω3 (n-3) fatty acid distribution in lipid extract and white muscle from Atlantic salmon (Salmo salar). The¹³C spectrum of lipid extracted from muscle gives quantitative information about the individual n-3 fatty acids, 18:2n-6, 20:1/22:1 and groups of fatty acids. The quantitative data compare favorably with those obtained by gas-liquid chromatography. The¹H NMR spectrum of the lipid extract gives information about the amount of 22:6n-3 and the total content of n-3 fatty acids. The¹³C NMR technique also revealed the positional distribution (1,3- and 2-acyl) of the important 20:5n-3 and 22:6n-3 acids in the triacylglycerol molecules. In the quantitative¹³C NMR spectrum of white muscle, the methyl region of the acyl chains of triacylglycerols gave rise to sufficiently resolved signals to permit estimation of the total concentration of lipids and the n-3 fatty acid content. The NMR data are in good agreement with corresponding data obtained by traditional methods.     

A novel analytical method to detect adulteration of virgin olive oil by other oils

The present study focuses on the olefinic region of the ¹³C nuclear magnetic resonance (¹³C NMR) spectrum of virgin olive oil which shows 12 peaks resonating between 127.5 and 130 ppm. These peaks are assigned to the most abundant unsaturated fatty acid moieties of the olive oil, oleic and linoleic acids, which are present in α and β positions of the glycerol backbone. With the use of an internal reference pyrazine, the 12 peaks were integrated and their areas were expressed in mmol/g of virgin olive oil. The intensities of the 12 observed peaks were affected when an authentic virgin olive oil was mixed with a seed oil. This observation was used to develop a semiquantitative method to detect adulteration of virgin olive oil by other oils based on ¹³C NMR spectroscopy.     

Characterization of sacha inchi (<Emphasis Type="Italic">Plukenetia volubilis</Emphasis> L.) oil by FTIR spectroscopy and <Superscript>1</Superscript>H NMR. Comparison with linseed oil

Three oil samples obtained from sacha inchi (Plukenetia volubilis L.) seeds were studied by means of FTIR and ¹H NMR. Frequency data of the most significant bands of the IR spectrum of this oil are given. These data show that sacha inchi oil has a high degree of unsaturation. The same fact is deduced from the ratio between the absorbance of the bands due to the stretching vibrations of the cis olefinic CH double bonds at 3010.5 cm⁻¹ and to the methylene symmetrical stretching vibrations at 2855.1 cm⁻¹. The proportions of monounsaturated, polyunsaturated, and saturated acyl groups were predicted from the frequency of some IR bands, and these were in satisfactory agreement with the values obtained through FAME generation and their quantification by GC. Likewise, simple observation of the ¹H NMR spectra provided a great deal of information about sacha inchi oil, with regard not only to the relative proportions of the different acyl groups but also to their nature. Thus, the presence of γ-linolenic acyl groups was discounted. Furthermore, the area of some ¹H NMR signals was used to determine the proportion of saturated and mono-, di-, and triunsaturated acyl groups, which also were in satisfactory agreement with the values obtained by classical methods. IR and ¹H NMR determinations take very little time in comparison with classical methods and do not require chemical manipulation or transformation of the sample. A comparison was also made between the compositions of sacha inchi and linseed oil. Both oils are important sources of the healthful n−3 linolenic acyl groups, and sacha inchi also contains high proportions of the n−6 linoleic acyl groups.     

Nondestructive NMR determination of oil composition in transformed canola seeds

Magic-angle spinning (MAS) ¹³C nuclear magnetic resonance (NMR) spectroscopy is a convenient method for nondestructive, quantitative characterization of seed oil composition. We describe results for intact hybrid and transformed canola seeds. The MAS ¹³C NMR technique complements and agrees with gas chromatography results. The spectral resolution approaches that of neat, liquid oils. MAS ¹³C NMR data allow quantitative analysis of major oil components, including saturates and oleic, linoleic, and linolenic acyl chains. ¹³C NMR directly and quantitatively elucidates, triglyceride regiochemistry and acyl chain cis-trans isomers that cannot be quickly detected by other methods. MAS ¹³C NMR can serve as the primary method for development of near-infrared seed oil calibrations. These NMR methods are nondestructive and attractive for plant-breeding programs or other studies (e.g., functional genomics) where loss of seed viability is inconvenient.     

Occurrence of conjugated fatty acids in the seed oil ofcouepia longipendula (chrysobalanaceae)

The seeds ofCouepia longipendula contain 74.2% oil. Gas chromatography (GC) and gas chromatography/ mass spectrometry (GC/MS) of the methyl esters and oxazoline derivatives of the fatty acids and ultraviolet (UV), infrared (IR),¹H-nuclear magnetic resonance (NMR) and¹³C-NMR spectra of the oil showed the presence of palmitic (25.2%), palmitoleic (0.9%), stearic (6.2%), oleic (26.5%), vaccenic (0.4%), linoleic (7.4%), arachidic (0.3%), α-eleostearic (11.3%) and α-licanic (21.8%) acids. Licanic acid methyl ester was isolated by thin-layer chromatography (TLC) and the¹³C-NMR and¹H-NMR data are presented.     

JAOCS News Feature biosynthesis of saturated and unsaturated fatty acids by maturingcarthamus tinctorius L. seeds

Recent advances in the biosynthesis of fatty acids by maturing safflower seeds are described. We now can define the systems responsible for (a) the conversion of CO₂ to sucrose to acetyl CoA; (b) the condensation of acetyl CoA via the de novo acyl carrier protein pathway to the terminal product, palmityl acyl carrier protein; (c) the elongation of palmityl acyl carrier protein to stearyl acyl carrier protein by a specific malonyl acyl carrier proteinelongation system; (d) the desaturation of stearyl acyl carrier protein to oleic acid by a highly specific, soluble stearyl acyl carrier protein desaturase; (e) the further desaturation of oleyl CoA to linoleyl CoA; and (f) the transfer of acyl CoAs to glycerol-3-phosphate to the final product, a triacylglycerol and its coalescence to the typical high lipid containing mature seed, engorged with oil droplets. Future research should be directed to a fuller understanding of how the maturing seed triggers these complicated series of reactions, how all these enzymes are coordinated to allow a smooth flow of substrates for the formation of oil droplets, and, finally, how biochemical engineering can further improve oil crops in the ever growing demand for high yielding systems. Winner of the Award in Lipid Chemistry presented at the 48th Annual Fall Meeting, September, 1974, Philadelphia.     

General properties and the fatty acid composition of the oil from the mophane caterpillar, Imbrasia belina

A preliminary investigation of the bulk properties of the oil from the edible mophane caterpillar (phane), Imbrasia belina, showed a significant difference in the iodine values of the oils from mature and young phane. Detailed analysis of the fatty acid composition of the two oil samples was thus carried out by capillary gas chromatography (GC) and complemented with ¹H and ¹³C nuclear magnetic resonance (NMR) studies to investigate the degree of unstauration in the two oil samples. While these studies showed that the oil samples from the mature and young mophane caterpillar were much the same in fatty acid composition, the data revealed a significant divergence from a literature report on phane oil. This earlier report puts the ratio of total saturated to total unsaturated fatty acids at approximately 1:1 (48.2:48.8, in percentages) and estimates the fatty acid composition for the major fatty acids as 16:0 (31.9%), 18:0 (15.2%), 18:1 (20.4%), 18:2 (9.9%), and 18:3 (19%). The data collected from the present work, however, showed the fatty acid composition for total saturated and total unsaturated fatty acids to be 40.5 and 57.0%, respectively. This work estimated the fatty acid composition for the major fatty acids as 16:0 (27.2%), 18:0 (12.3%), 18:1 (16.1%), 18.2 (10.7%), and 18:3 (29.0%). Thus, linolenic acid was the most abundant fatty acid in the phane oil. The GC results of the present analysis were largely corroborated by studies of the composition of fatty acid classes in the phane oil estimated from integrals of ¹H and ¹³C NMR signals. Oils from other edible Lepidoptera larvae are also known to be much richer in unsaturated than saturated fatty acids.     

Carbon-13 nuclear magnetic resonance analysis of intact oilseeds

High resolution natural abundance¹³C nuclear magnetic resonance (NMR) spectra of intact oilseeds have been obtained by Fourier transform techniques. The spectra can be interpreted in terms of the relative concentrations of the major fatty acids in the oilseed. The¹³C NMR spectra are well resolved despite the fact that¹H NMR spectra of the same seeds are poorly resolved. The difference in resolution can be attributed to the simplicity of the¹³C NMR spectra in which all spin-spin coupling can be removed, in which the separation of lines is increased by a factor of 30, and in which line broadening due to intermolecular dipolar interactions is not important. The¹³C NMR Fourier transform technique is sufficiently sensitive that a high quality spectrum can be obtained from a single soybean, for example, in about 10 minutes. Similar spectra of single seeds can be obtained in comparable times for corn, castor bean, peanut, sunflower seed, and rapeseed. Because the NMR technique is nondestructive, it can be used to select individual oilseeds for use in breeding programs designed to improve oil quality. By employing some special experimental NMR line narrowing techniques, it also appears feasible to obtain moderately well resolved, natural abundance¹³C NMR spectra of the immobile, rigid protein, and carbohydrate components of an intact oilseed, as well as the more mobile oil components.     

Fullerenoid lipids: first synthesis of structured triacylglycerols containing an Aza-[60]fullerene unit

Some 1,2- and 1,3-diacyl glycerols (with acyl groups as stearyl, oleyl, linoleyl, or stearolyl) were synthesized by conventional methods. The diacyl glycerols were esterified with 6-bromo-hexanoic acid to give the corresponding bromotriacylglycerols (of the type AAB and ABA containing a bromo group at the distal part of the hexanoate chain). The bromo function was transformed to an azide group by reaction of the bromotriacylglycerols with sodium azide. The resulting azido-triacylglycerols were then reacted with [60]fullerence to give the requisite aza-fullerenoid triacylglycerol of the type ABA or AAB (45–62% yield based on the amount of [60]fullerence reacted). The nitrogen atom attached to the carbon cage formed a “[5,6]-open” type aza substructure, which was confirmed by the appearance of 31–32 signals in the region of δ_C 133–148 (carbon shifts of sp ² carbons of the cage) in the ¹³C nuclear magnetic resonance spectra. The spectroscopic and mass spectrometric properties of these novel fullerenoid triacylglycerols are reported.     

Acyl Migration Kinetics of 2-Monoacylglycerols from Soybean Oil via <Superscript>1</Superscript>H NMR

The acyl migration kinetics of neat 2-monoacylglycerol (2-MAG) to form 1-MAG was determined using ¹H NMR spectroscopy to monitor the β-proton integration ratios of the two species over time. 2-MAG was synthesized by the Novozym 435-catalyzed alcoholysis of soybean oil and isolated by solvent extraction or molecular distillation at a mole fraction (X _2-MAG) of 0.94 relative to total MAG. The kinetics parameters of the neat 2-MAG acyl migration were investigated over the temperature range of 23–80 °C. The 2-MAG mol fraction remained unchanged at 23 °C over the course of 168 h and reached an equilibrium of X _2-MAG = 0.09 at only 80 °C. Modeling of the kinetics data revealed a 2-MAG half life (t _1/2) of 3,500 and 22.8 h at 23 and 80 °C, respectively, with an activation energy of 79.0 ± 6.5 kJ mol⁻¹. The use of ¹H NMR spectroscopy proved an expedient method for monitoring the acyl migration in 2-MAG compared to other reported methods (e.g. GC, HPLC, and ¹³C-NMR spectroscopy), requiring no sample manipulation and minimizing the deleterious effects of high temperatures and solvent exposure. Product names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may be suitable.     

The triglyceride composition,structure, and presence of estolides in the oils ofLesquerella and related species

Members of the genusLesquerella, native to North America, have oils containing large amounts of hydroxy fatty acids and are under investigation as potential new crops. The triglyceride structure of oils from twenty-fiveLesquerella species in the seed collection at our research center has been examined after being hydrolysis-catalyzed by reverse micellar-encapsulated lipase and alcoholysis-catalyzed by immobilized lipase. These reactions, when coupled with supercritical-fluid chromatographic analysis, provide a powerful, labor-saving method for oil triglyceride analysis. A comprehensive analysis of overall fatty acid composition of these oils has been conducted as well.Lesquerella oils (along with oils from two other Brassicaceae:Physaria floribunda andHeliophilia amplexicaulis) have been grouped into five categories: densipolic acid-rich (Class I); auricolic acid-rich (Class II); lesquerolic acid-rich (Class III); an oil containing a mixture of hydroxy acids (Class IV); and lesquerolic and erucic acid-rich (Class V). The majority of Class I and II triglycerides contain one or two monoestolides at the 1- and 3-glycerol positions and a C₁₈ polyunsaturated acyl group at the 2-position. Most Class III and IV oil triglycerides contain one or two hydroxy acids at the 1- and 3-positions and C₁₈ unsaturated acid at the 2-position. A few of the Class III oils have trace amounts of estolides. The Class V oil triglycerides are mostly pentaacyl triglycerides and contain monestolide and small amounts of diestolide. Our triglyceride structure assignments were supported by¹H nuclear magnetic resonance data and mass balances.     

13C nuclear magnetic resonance spectroscopic analysis of the triacylglycerol composition ofBiota orientalis and carrot seed oil

¹³C nuclear magnetic resonance (NMR) spectroscopic analysis of the whole oil (triacylglycerols) ofBiota orientalis seeds confirms the presence of oleate [18:1(9Z)], linoleate [18:2(9Z, 12Z)], linolenate [18:3((9Z, 12Z, 15Z)], 20:3 (5Z, 11Z, 14Z), 20:4(5Z, 11Z, 14Z, 17Z), and saturated fatty acids in the acyl groups by comparing the observed carbon shifts with previously established shift data for model triacylglycerols. This technique shows that the saturated, 20:3 and 20:4 fatty acids are distributed mainly in the α-acyl positions, whereas oleate, linoleate, and linolenate are randomly acylated to the α- and β-positions of the glycerol “backbone”. Stereospecific hydrolysis of theBiota oil with pancreatic lipase, followed by chromatographic analysis of fatty esters, reveals the presence of trace amounts of 16:0(0.7%), 18:0(0.5%), 20:3 (0.4%), and 20:4 (1.3%) in the β-position of the glycerol “backbone”, which are undetectable by¹³C NMR technique on the whole oil. Semiquantitative assessment of the¹³C NMR signal intensities gives the relative percentages of the fatty acid distribution as: saturated 16:0, 18:0 (12.0% α-acyl), oleate (7.7% α-acyl 8.7% β-acyl), total linoleate and linolenate (31.7% α-acyl; 24.2% βacyl), total 20:3 and 20:4 (15.7% α-acyl). The¹³C NMR spectroscopic analysis of carrot seed oil identifies the presence of saturated (18:0), 18:1(6Z), 18:1(9Z), and 18:2(9Z, 12Z). The saturated fatty acid is found in the α-acyl positions. Semi-quantitative assessment of the signal intensities gives the relative percentages of the fatty acids as: 18:0 (4.5% α-acyl), 18:1(6Z) (49.6% α-acyl; 19.7% β-acyl), oleate (6.5% α-acyl; 8.6% β-acyl) and linoleate (5.2% α-acyl; 6.9% β-acyl).     

High-Resolution NMR Spectroscopy: An Alternative Fast Tool for Qualitative and Quantitative Analysis of Diacylglycerol (DAG) Oil

Multinuclear (¹H, ¹³C, ³¹P) and multidimensional NMR spectroscopy was employed for the analysis of diacylglycerol (DAG) oil and the quantification of its acylglycerols and acyl chains composition. A number of gradient selected two dimensional NMR techniques (TOCSY, HSQC-DEPT, HSQC-TOCSY, and HMBC) facilitated the assignment of the complex one dimensional ¹H- and ¹³C-NMR spectra. In several cases, the aforementioned 2D-NMR techniques offered solid proof of earlier assignments based on chemical shift changes induced by the presence and relative positions of double bonds within the acyl chains. Integration of the appropriate signals in the NMR spectra of the three nuclei allowed the determination of DAG oil composition, which was found to be within the limits accepted for this oil, namely 1-monoacylglycerols 0.40–0.60%; 2-monoacylglycerols 0.40–0.50%; 1,3-diacylglycerols 57–62%; 1,2-diacylglycerols 28–32%; triacylglycerols 9–11%; saturated fatty acids 3–5%; oleic acid 37–45%; linoleic acid 49–53%; and linolenic acid 5–6.5%; tocopherols 0.24–0.27%. The compositional results obtained from the NMR spectra of the three nuclei were compared and discussed in terms of repeatability and ease of performance.     

Strain-induced crystallization of natural rubber as studied by high-resolution solid-state C NMR spectroscopy

A series of high-resolution solid-state ¹³C NMR experiments were performed on both unstretched and in situ stretched natural rubber samples. From the ¹³C CP/MAS spectra, it was found that natural rubber does form small crystals at room temperature though the degree of crystallinity is very small. Furthermore, from the ¹³C DD/MAS spectra, the crystalline signals were found to increase with the increase of draw ratio. ¹³C spin-lattice relaxation time (T₁) and ¹H spin-spin relaxation time (T₂) of in situ stretched natural rubber were measured for the first time, which provided further evidences for the conclusion that there exist crystals in both stretched and unstretched natural rubber samples. Quantitative ¹³C NMR measurements indicated that strain-induced crystallization occurs when the draw ratio reaches about 2.0 and the maximum crystallinity of our natural rubber samples can be as high as 19.3% upon stretching.     

13C NMR spectra of TAG: An easy way to distinguish milks from different animal species

In order to differentiate milks from different species, we carried out a comparative analysis of TAG from cow, buffalo, goat, and sheep milk fat based on ¹³C NMR experiments. NMR spectroscopy, although less sensitive than other techniques, does not require an extensive chemical manipulation of samples and can easily highlight the differences in the content of short-chain acyl groups in the four milk species. The resonances were assigned and quantified, and by using only three NMR parameters in data clustering with fuzzy logic analysis, we were able to distinguish goats' milk from sheep's milk, and both of these milks from cows' and buffaloes' milks. This appears to be an important result, considering the ease and rapidity with which milk identification can be obtained. From ¹³C NMR spectra of TAG, the positional distribution of FA chains on the glycerol backbone can also be easily evaluated. In particular, analysis of the positional distribution of monounsaturated FA revealed that it may be species-specific, and we are currently analyzing larger data sets in order to evaluate the use of this parameter as a suitable approach to address the issue of milk authenticity.     

Determination of positional distribution of butyryl groups in milkfat triacylglycerols,triacylglycerol mixtures,and isolated positional isomers of triacylglycerols by gas chromatography and1H nuclear magnetic resonance spectroscopy

Positional isomers (1-butyryl-2X-3Y-rac-glycerol and 2-butyryl-1X-3Y-rac-glycerol;X,Y=long-chain acyls) of saturated triacylglycerols (TAG) with 34 and 40 acyl carbons were shown to separate in two chromatographic peaks on immobilized phenyl(65%) methylsilicone column by gas-liquid chromatography, and on reversed-phase ODS-1 column by high-performance liquid chromatography. The analysis of 500-MHz¹H nuclear magnetic resonance (NMR) spectra showed distinct differences between 2-butyryl-1X-3Y-rac-glycerol and 1-butyryl-2X-3Y-rac-glycerol isomers in the resonance signals of methylene and methine protons of glycerol backbone, and carbon-2 methylene of acyl groups, and methyl protons of butyryl group. The¹H NMR spectra of three interesterified mixtures of three monoacid TAG containing saturated butyrate and caproate TAG and unsaturated butyrate TAG showed that triplets of methyl protons of butyryl groups atsn-1(3)- andsn-2-positions in saturated and unsaturated TAG had similar chemical shifts and that the chemical shift of caproyl methyl protons was different from those of butyryl methyl protons. The positional distribution of butyryl groups in isolated positional isomers of butyrate TAG, interesterified TAG mixtures, and natural and interesterified butteroil can be determined by integration of these signals.     

Use of 13C nuclear magnetic resonance distortionless enhancement by polarization transfer pulse sequence and multivariate analysis to discriminate olive oil cultivars

Distortionless enhancement by polarization transfer (DEPT) pulse sequence was used to set up a quantitative high-resolution ¹³C nuclear magnetic resonance (NMR) method to discriminate olive oils by cultivars and geographical origin. DEPT pulse sequence enhances the intensity of NMR signals from nuclei of low magnetogyric ratio. The nuclear spin polarization is transferred from spins with large Boltzmann population differences (usually protons) to nuclear species characterized by low Boltzmann factors, e.g., ¹³C. The signal enhancement of ¹³C spectra ensures the accuracy of resonance integration, which is a major task when the resonance intensities of different spectra must be compared. The resonances of triglyceride acyl chains C _n:0, C_18:1, C_18:2, and C_18:3, were also assigned. Multivariate analysis was carried out on the 35 carbon signals obtained. By using variable reduction techniques, coupled with standard statistical methods—partial least squares and principal components analysis—it was largely possible to separate the samples according to their variety and region of origin. With one problem variety removed, 100% prediction of the three remaining varieties was achieved. Similarly, by using the three regions with greatest representation in the data, all but one of a test set of 34 samples were correctly predicted. Thus, the composition of olive oils from different cultivars and of different geographical origin were compared and successfully studied by multivariate analysis. These considerations in conjunction with the structural elucidations of triglyceride molecules demonstrated that ¹³C NMR is among the most powerful techniques yet described for analysis of olive oils.     

Chemometric classification of olive cultivars based on compositional data of oils

European Union regulations provide important guidelines for maintaining the Protected Designation of Origin (PDO) of olive oil and other foods. This includes characterization of foods based on variety (cultivar) and geographical origin, as this may be used as a criterion for determining authenticity and quality. Therefore, analytical method standards need to be established to ensure these criteria. This study describes how cultivar differences can be established between Italian oils, obtained from single varieties, based on acid, sterol, and TAG differences determined by chemometrics. TAG and FA composition provided the best basis for differentiation of olive oils among cultivars. The results were compared with those obtained using ¹³C NMR analysis, and a similar differentiation between oils of different cultivars was achieved. ¹³C NMR provides useful information on the acyl composition and on the positional distribution of the glycerol moiety and can be used for classification of cultivars based on oil composition. Furthermore, the advantages of this technique come from the rapidity with which information can be obtained and from the very simple preparation procedure required for analysis.     

Determination of unsaturated fatty acid composition by high-resolution nuclear magnetic resonance spectroscopy

High-resolution nuclear magnetic resonance (NMR) spectroscopy provides useful data for analyzing fatty acid compositions of edible vegetable oils. Quantitation of each fatty acid was carried out by evaluation of particular peaks. According to the ¹H NMR method, terminal methyl protons, divinyl protons, and allyl protons are useful to calculate linolenic acid, linoleic acid, and oleic acid, respectively. The ω-2 carbon, divinyl carbon, and allylic carbons were used for calculation of these acids by the ¹³C NMR method. Compositional results obtained by NMR coincided well with those of the conventional gas chromatography (GC) method. Results from ¹³C NMR were in better agreement with those from GC than were the results obtained by the ¹H NMR method.