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.     

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.     

Determination of Cis- and Trans-18:1 fatty acid isomers in hydrogenated vegetable oils by high-resolution carbon nuclear magnetic resonance

Carbon nuclear magnetic resonance (¹³C NMR) methods for determining the composition of cis-/trans- and positional isomers in hydrogenated vegetable oils were developed to reduce analytical time. By selecting appropriate olefinic carbon peaks and by measuring individual peak areas subsequent to the identification of isomeric peaks on the NMR spectrum, compositional results of the isomers coincided well with those obtained by conventional gas chromatography (GC). Therefore, it is highly beneficial to choose the ¹³C NMR method when analysis time is limited. Though the proposed ¹³C NMR method is promising, further development is needed. For the time being, combination with the traditional GC method is still encouraged for precise compositional analysis of cis-/trans- and positional isomers.     

Discrimination of Cod Liver Oil According to Wild/Farmed and Geographical Origins by GC and <Superscript>13</Superscript>C NMR

The objective of this study was to test the possibility of using lipid profiles obtained by gas chromatography (GC) and ¹³C nuclear magnetic resonance (NMR) in authentication of cod liver oils according to wild/farmed and geographical origin. GC and ¹³C-NMR data of cod liver oil from wild and farmed fish from different locations in Norway and Scotland were obtained, and analyzed by principal component analysis (PCA) and linear discriminant analysis (LDA) to test if it was possible to differentiate oil from wild and cultured cod (Gadus morhua L.), and to further elucidate differences between fish from the different farms/catch area. Cod liver oils of wild and farmed origin were clearly separated in the PCA score plot both from GC and NMR data. From NMR data it was also possible to observe groupings based on geographical origin (farm/catch area) of the different samples. Using LDA with cross validation the wild/farmed classification rates were 97% for GC data and 100% for NMR data. In the classification of cod liver oils according to geographical origin (38 samples from six different farms/catch area), the correct classification rate was 63% for GC data and 95% for NMR data.     

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.     

NMR,GC–MS and ESI-FTICR-MS Profiling of Fatty Acids and Triacylglycerols in Some Botswana Seed Oils

In the search for non-traditional seed oils, physicochemical parameters, fatty acid (FA) and triacylglycerol (TAG) profiles for five Botswana seed oils, obtained by Soxhlet extraction, were determined. GC–MS and ¹H-NMR analyses showed the FA profiles for mkukubuyo, Sterculia africana, and manketti, Ricinodendron rautanenii, seed oils dominated by linoleic and oleic acids, 26.1, 16.7 and 51.9, 24.4%, respectively, with S. africana containing significant amounts of cyclic FAs (19.9%). Mokolwane, Hyphaene petersiana, seed oil was typically lauric; 12:0 and 14:0 acids were 25.9 and 13.4%, respectively. Morama, Tylosema esculentum, seed oil resembled olive oil; 18:1 (47.3%) and 18:2 (23.4%) acids dominated. Moretologa-kgomo, Ximenia caffra, seed oil had 45.8% of 18:1 FA, plus significant amounts of very long chain FAs: 26:1 (5.8%), 28:1 (13.9%), 30:1 (3.9%), and acetylenic acids, 9a-18:1 (1.5%) and 9a, 11t-18:2 (16.0%). TAG classes and regiochemistry were determined with ESI-FTICR-MS, and ¹³C-NMR spectra, respectively. Morama showed seven major TAG classes with C54:4 and C54:3 dominating; mokolwane had 16 major classes with C32:0, C38:0 and C42:2 dominating; manketti had 11 major classes with C54:7, C54:6 and C54:4 dominating; mkukubuyo had 12 major classes with C52:4, C52:3 and C54:4 dominating; moretologa-kgomo had 30 major TAG classes with C64:5, C64:3 and C62:3 dominating. Saturated FAs were generally distributed over the sn-1(3) position for morama, manketti, and moretologa-kgomo but at the sn-2 position for mokolwane and mkukubuyo. These findings indicate that morama and manketti seed oils can be developed for food uses, whilst moretologa-kgomo and mkukubuyo seed oils only for nonfood uses.     

Determination of cyclopropenoid fatty acids inSterculia seed oils from senegal

Seed oils ofSterculia tomentosa andS. tragacantha (Sterculiaceae) were found to contain malvalic (5.8 and 5.1%), sterculic (11.3 and 30.2%) and dihydrosterculic (0.9 and 0.5%) acids. The total amount of these two cyclopropenoid fatty acids was established by¹H nuclear magnetic resonance and their cooccurrence by gas chromatography. Besides these unusual compounds, the main common fatty acids were palmitic (20 and 24%), oleic (21 and 15%) and linoleic (30 and 16%) acids.     

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.     

Nondestructive quantitative determination of docosahexaenoic acid and n−3 fatty acids in fish oils by high-resolution 1H nuclear magnetic resonance spectroscopy

By using a 500 MHz proton nuclear magnetic resonance (¹H NMR) spectrometer we have developed a quantitative method for determining the contents of docosahexaenoic acid (DHA) in fish oils (mg/g), the molar proportions (mol%) of DHA to all other fatty acids composing the fish oils, and the molar proportions of total n-3 fatty acids to all other non-n-3 fatty acids in the fish oils. After examining the suitability of ethylene glycol dimethyl ether (EGDM), methanol, and 1,4-dioxane as internal standards, experimental conditions were optimized by mainly using EGDM as an internal standard. By setting the pulse repetition time at 30 s, five times longer than the longest T ₁ of the ¹H NMR signals of fish oils, good reproductibility of data and analytical times less than 10 min were achieved. The use of the internal standard also allowed us to quantify DHA on a weight basis (mg/g). Verification of the method was carried out in an interlaboratory study between Japan and Norway on bonito, tuna, and salmon oils. The relative errors in the ¹H NMR data between Japan and Norway were 0.57–5.29% for quantification of DHA, 0.7–2.09% for the molar proportion of DHA, and 0.1–1.41% for the molar proportion of total n-3 fatty acids. Good agreement was observed between the ¹H NMR data and those obtained by gas chromatography (GC). The sample preparation before ¹H NMR measurements required only two steps: sample weighing and preparation of an internal standard solution. Based on the high reproducibility, simplicity of the procedure, and clarity of principle, the proposed ¹H NMR method was judged to be a promising alternative to the GC method in quantification of DHA and n-3 fatty acids in fish oils.     

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.     

New preparation of pure petroselinic acid from fennel oil (Foeniculum vulgare)

Pure petroselinic acid (cis-6-octadecenoic acid) has been isolated from fennel oil by acid soap crystallization at 4°C in methanol, followed by two urea segregations at room temperature and crystallization at −30°C in acetone. The purity control of petroselinic acid was effected by combined gas chromatography, and¹³C nuclear magnetic resonance. This petroselinic acid preparation was compared to other previous crystallization or enzymatic methods, showing that this method is both short (four steps) and easy to apply.     

Purity assessments of major vegetable oils based on δ13C values of individual fatty acids

The fatty acid compositions and δ¹³C values of the major fatty acids of more than 150 vegetable oils were determined to provide a database of isotopic information for use in the authentication of commercial maize oil. After extraction of oils from seeds, nuts or kernels, and methylation, fatty acid compositions were determined by capillary gas chromatography. All compositions were within the ranges specified by the Codex Alimentarius. Gas chromatography combustion-isotope ratio mass spectrometry was employed to determine the δ¹³C values of the major fatty acids of the oils. A large number of pure maize oils and potential adulterant oils from various parts of the world were studied to assess the sources of variability in δ¹³C values. Such information is vital to establishing the compound specific isotope technique as a reliable means of assessing vegetable oil purity. Variability in δ¹³C values was related to the geographical origin of the oil, year of harvest, and the particular variety of oil. This suggests that the ultimate δ¹³C values of fatty acids are determined by a combination of environmental and genetic factors.     

Evaluation of ethanolysis with immobilized Candida antarctica lipase for regiospecific analysis of triacylglycerols containing highly unsaturated fatty acids

The suitability of a recently proposed method based on ethanolysis with immobilized Candida antarctica lipase for regiospecific analysis of oils containing long-chain PUFA such as [PA and DHA has been evaluated using selected marine oils and regio-isomerically enriched synthetic TAG substrates. 1,3-Regios-electivity of the lipase was enhanced when the ethanolysis was conducted in a high excess of ethanol, typically 10–50 times by weight of the oil. This enabled the reaction to be conducted on a milligram scale. However, irrespective of the ethanol-to-oil ratio, C. antarctica lipase released FA from TAG at different rates depending on the degree of unsaturation and/or chain length of the FA. Differences in lipolysis rates were particularly significant for EPA and DHA, with EPA released faster than DHA. Although DHA can be measured with reasonable accuracy by ethanolysis with C. antarctica, the method requires further optimization before it can be adopted for reliable regiospecific analyses that are as accurate as those obtainable by ¹³C NMR analysis for all major FA occurring in oils rich in long-chain PUFA.     

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.     

The seed oil ofBernardia pulchella (Euphorbiaceae) —A rich source of vernolic acid

The fatty acids from the seed oil ofBernardia pulchella (Euphorbiaceae) have been analyzed by gas chromatography (GC) and GC-mass spectrometry (MS) analysis of their methyl esters. Vernolic acid is the main compound (91%), along with other usual fatty acids. In addition to the quantitation by GC analysis,¹H-nuclear magnetic resonance (NMR) signals from the seed oil have been used to estimate the total epoxy fatty acid content. The structure of vernolic acid has been proven by spectroscopic methods (infrared,¹H, and¹³C-NMR) and by GC-MS analysis of the corresponding silylated hydroxy-methoxy derivative. The 4,4-dimethyloxazoline derivatives of the fatty acid mixture have also been examined by GC-MS, and it was shown that this derivazation reaction is not suitable for the structure analysis of vernolic acid.     

Quantitative triacylglycerol analysis of whole vegetable seeds by1H and13C magic angle sample spinning NMR spectroscopy

High-resolution¹³C and¹H magic angle sample spinning nuclear magnetic resonance (NMR) spectra have been obtained and used to define the relative unsaturated acyl distribution of triacylglycerols in whole oil seeds. Inverse gated proton decoupled¹³C and¹H NMR spectra permit the quantitative analysis of seeds containing simple oils,e.g., sunflower seeds containing oleyl and linoleyl unsaturates only. More sensitive¹³C NMR techniques are necessary for the analysis of specific seed classes. One such class is the rapeseed, which is especially difficult due to its low oil content (≈ 2 mg oil/seed) and complex unsaturated acyl profile of oleyl, linoleyl, linolenyl, erucyl, and eicosenoyl. The Distortionless Enhancement by Polarization Transfer technique significantly improves sensitivity to the extent that single rapeseeds can be examined within an hour of acquisition time. Furthermore, some positional (1,3- or 2-glycerol attachment) groups can be identified leading to a partial estimation of the 1,3-, 2-acyl distribution.     

FA composition and regiospecific analysis of Acer saccharum (sugar maple tree) and Acer saccharinum (silver maple tree) seed oils

GC analysis was performed to determine regiospecific distribution and FA composition in seed oils of the Aceraceae species, Acer saccharum and A. saccharinum. The oil content in the seeds was low at 5.0% in A. saccharum and 5.8% in A. saccharinum, and the main FA were linoleic (30.8 and 29.4%), oleic (21.3 and 27.6%), palmitic (10.1 and 10.5%), and cis-vaccenic (9.4 and 7.9%) acids, respectively. In addition, both oils contained long-chain monoenes of the n−9 and n−7 groups, including 11-eicosenoic, 13-docosenoic, 15-tetracosenoic, 13-eicosenoic, and 15-docosenoic acids, whereas γ-linolenic acid accounted for 0.8% of total FA in A. saccharum, and 0.5% in A. saccharinum. Regiospecific analysis, performed using the methodology of dibutyroyl derivatives of MAG, indicated that linoleic, oleic, and linolenic acids were mainly esterified at the internal position of TAG in both seed oils, whereas long-chain monoenes of the n−7 group were almost exclusively esterified on the external positions.     

Characterization and Authentication of Significant Chinese Edible Oilseed Oils by Stable Carbon Isotope Analysis

The ratios of stable carbon isotopes (δ¹³C) of 12 oils extracted from Chinese edible oilseed samples and their individual fatty acids were determined by elemental analysis-isotope ratio mass spectrometry (EA-IRMS) and gas chromatography-isotope ratio mass spectrometry (GC-IRMS). The results have demonstrated that the δ¹³C ratios of the oils from C3-plant seeds range from ?26.8 to ?30.7‰, while the δ¹³C ratios of C4-plant maize oil are in the interval of ?14.1 to ?16.2‰. Eighteen fatty acids were identified and their abundances were measured by gas chromatography–mass spectrometry (GC–MS) in these oils with C_16:0, C_18:0, C_18:1 and C_18:2 as the major constituents. From the data on fatty acids and stable carbon isotopes, several sensitive markers were developed to detect the adulteration of Chinese edible oilseed oils. Examples are provided with pre-blended samples to illustrate the discrimination procedures and corresponding sensitive markers with emphasis on camellia seed oil, flax seed oil and perilla seed oil.     

A Comparative Study of the Properties of Selected Melon Seed Oils as Potential Candidates for Development into Commercial Edible Vegetable Oils

A comprehensive compositional and characterization study was carried out on five seed oils from varieties of the melons Citrullus lanatus and C. colocynth in order to evaluate their suitability for large-scale exploitation as edible vegetable oils. The oils were extracted by Soxhlet with a 3:1 mixture of n-hexane/2-propanol with yields that ranged from 24.8 to 30.0% (wt/wt). The refractive indices and relative densities of the oils fell within the narrow ranges of 1.465–1.469 and 0.874–0.954 g/cm³, respectively. Saponification values ranged between 182.1 and 193.8 mg KOH/g, whilst iodine values (IV) ranged from 95.8 to 124.0 (Wijs). The ranges of the values for free fatty acid (AV), 1.2–4.0 mg KOH/g, peroxide (PV), 1.1–10.9 meq/kg and p-anisidine (p-AV), 0.2–9.0, indicated that secondary oxidation products were barely present. GC analysis gave total unsaturation contents of 67.93–82.36%, with linoleic acid (18:2) being the dominant fatty acid (55.21–66.85%). The GC results agreed closely with those from proton NMR analysis of the fatty acid classes. The physicochemical and compositional properties determined in this study show that the qualities of the test Cucurbitacea seed oils are highly comparable to those of soybean, sunflower and groundnut seed oils. Therefore, the test melon seed oils could be developed into commercial products to serve as alternate vegetable oils in Southern and West Africa, the regions where these melons grow.     

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).