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.     

CE- and HPLC-TOF-MS for the characterization of phenolic compounds in olive oil

We present an easy and rapid method for the analysis of phenolic compounds in extra-virgin olive oil by CZE coupled with ESI-TOF-MS. Optimum electrophoretic separation was obtained using a basic carbonate electrolyte. We thus achieved the determination of several important families (phenyl alcohols, phenyl acids, lignans, flavonoids, and secoiridoids) of the polar fraction of the olive oil. Furthermore, other "unknown" compounds were also identified. In addition to the CZE method, HPLC analyses were made, separating compounds belonging to the main families present in this polyphenolic fraction, as well as other new compounds. We compared the results obtained with both techniques and found it was possible to determine more than 45 compounds with both methods. The sensitivity, together with mass accuracy and true isotopic pattern of the TOF-MS, allowed the identification of a broad series of known and so far not described phenolic compounds present in extra-virgin olive oil.     

Nano and rapid resolution liquid chromatography–electrospray ionization–time of flight mass spectrometry to identify and quantify phenolic compounds in olive oil

The applicability of nanoLC‐ESI‐TOF MS for the analysis of phenolic compounds in olive oil was studied and compared with a HPLC method. After the injection, the compounds were focused on a short capillary trapping column (100 μm id, effective length 20 mm, 5 μm particle size) and then nanoLC analysis was carried out in a fused silica capillary column (75 μm id, effective length 10 μm, 3 μm particle size) packed with C18 stationary phase. The mobile phase was a mixture of water + 0.5% acetic acid and ACN eluting at 300 nL/min in a gradient mode. Phenolic compounds from different families were identified and quantified. The quality parameters of the nanoLC method (linearity, LODs and LOQs, repeatability) were evaluated and compared with those obtained with HPLC. The new methodology presents better sensitivity (reaching LOD values below 1 ppb) with less consumption of mobile phases, but worse repeatability, especially inter‐day repeatability, resulting in more difficulties to get highly accurate quantification. The results described in this article open up the application fields of this technique to cover a larger variety of compounds and its advantages will make it especially useful for the analysis of samples containing low concentration of phenolic compounds, as for instance, in biological samples.     

Identification and quantification of the main organic components of vinegars by high resolution 1H NMR spectroscopy

A detailed analysis of the proton high-field NMR spectra of vinegars (in particular of Italian balsamic vinegars) is reported. A large number of organic substances belonging to different classes, such as carbohydrates, alcohols, organic acids, volatile compounds and amino acids, were assigned. The possibility of quantification of the substances identified in the whole vinegar sample, without extraction or pre-concentration steps, was also tested. The data validity was demonstrated in terms of precision, accuracy, repeatability and inter-day reproducibility. The effects of the most critical experimental parameters (sample concentration, water suppression and relaxation time) on the analysis response were also discussed. ¹H NMR results were compared with those obtained by traditional techniques (GC-MS, titrations), and good correlations were obtained. The results showed that ¹H NMR with water suppression allows a rapid, simultaneous determination of carbohydrates (glucose and fructose), organic acids (acetic, formic, lactic, malic, citric, succinic and tartaric acids), alcohols and polyols (ethanol, acetoin, 2,3-butanediol, hydroxymethylfurfural), and volatile substances (ethyl acetate) in vinegar samples. On the contrary, the amino acid determination without sample pre-concentration was critical. The ¹H NMR method proposed was applied to different samples of vinegars, allowing, in particular, the discrimination of vinegars and balsamic vinegars.     

In situ complexation of rhodium(II) tetracarboxylates with some derivatives of cysteine and related ligands studied by 1H and 13C nuclear magnetic resonance spectroscopy

The complexation of N-phthaloyl, N-formyl, and N,N-dimethyl derivatives of S-methylcysteine methyl ester (both racemic and optically pure) with three dimeric rhodium(II) salts, acetate Rh₂AcO₄, trifluoroacetate Rh₂TFA₄, and (R)-(+)-α-methoxy-α-trifluoromethylphenylacetate Rh₂Mosh₄ was investigated by nuclear magnetic resonance spectroscopy (NMR) at room and lower temperatures. The complexation was carried out in situ, in CDCl₃ solution using titration procedure; the results were examined by the analysis of ¹H and ¹³C NMR chemical shift change (Δδ). The complexation of free S-methyl cysteine and hydrochloride salt of its methyl ester was performed in D₂O solution. For comparison, complexation of some derivatives of leucine, phenylalanine, and proline was examined.

N-phthaloyl and N-formyl derivatives of cysteine formed 1 : 1 and 1 : 2 axial complexes with all dirhodium salts. Rhodium substrates were bonded via sulfur. In one case, the complexation of Rh₂TFA₄ by both sulfur and N-formyl oxygen was noted. Similar complexation of Rh₂TFA₄, via CHO group, was found for N-formyl derivatives of leucine, phenylalanine, and proline. For N,N-dimethyl derivative of cysteine, both N and S atoms were involved in bonding. At room temperature, in all cases, ligand exchange was fast on the NMR timescale.     

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.     

Gas chromatography-atmospheric pressure chemical ionization-time of flight mass spectrometry for profiling of phenolic compounds in extra virgin olive oil

A new analytical approach based on gas chromatography coupled to atmospheric pressure chemical ionization-time of flight mass spectrometry was evaluated for its applicability for the analysis of phenolic compounds from extra-virgin olive oil. Both chromatographic and MS parameters were optimized in order to improve the sensitivity and to maximize the number of phenolic compounds detected. We performed a complete analytical validation of the method with respect to its linearity, sensitivity, precision, accuracy and possible matrix effects. The LODs ranged from 0.13 to 1.05ppm for the different tested compounds depending on their properties. The RSDs for repeatability test did not exceed 6.07% and the accuracy ranged from 95.4% to 101.5%. To demonstrate the feasibility of our method for analysis of real samples, we analyzed the extracts of three different commercial extra-virgin olive oils. We have identified unequivocally a number of phenolic compounds and obtained quantitative information for 21 of them. In general, our results show that GC-APCI-TOF MS is a flexible platform which can be considered as an interesting tool for screening, structural assignment and quantitative determination of phenolic compounds from virgin olive oil.     

A metabonomics investigation of multiple sclerosis by nuclear magnetic resonance

Multiple sclerosis (MS) is a nervous system disease that affects the fatty myelin sheaths around the axons of the brain and spinal cord, leading to demyelination and a broad range of signs and symptoms. MS can be difficult to diagnose because its signs and symptoms may be similar to other medical problems. To find out which metabolites in serum are effective for the diagnosis of MS, we utilized metabolic profiling using proton nuclear magnetic resonance spectroscopy (¹H‐NMR). Random forest (RF) was used to classify the MS patients and healthy subjects. Atomic absorption spectroscopy was used to measure the serum levels of selenium. The results showed that the levels of selenium were lower in the MS group, when compared with the control group. RF was used to identify the metabolites that caused selenium changes in people with MS by building a correlation model between these metabolites and serum levels of selenium. For the external test set, the obtained classification model showed a 93% correct classification of MS and healthy subjects. The regression model of levels of selenium and metabolites showed the correlation (R²) value of 0.88 for the external test set. The results indicate the suitability of NMR as a screen for identifying MS patients and healthy subjects. A novel model with good prediction outcomes was constructed between serum levels of selenium and NMR data. Copyright © 2012 John Wiley & Sons, Ltd.     

A set of triple-resonance nuclear magnetic resonance experiments for structural characterization of organophosphorus compounds in mixture samples

The ¹H, ¹³C correlation NMR spectroscopy utilizes J_CH couplings in molecules, and provides important structural information from small organic molecules in the form of carbon chemical shifts and carbon-proton connectivities. The full potential of the ¹H, ¹³C correlation NMR spectroscopy has not been realized in the Chemical Weapons Convention (CWC) related verification analyses due to the sample matrix, which usually contains a high amount of non-related compounds obscuring the correlations of the relevant compounds. Here, the results of the application of ¹H, ¹³C, ³¹P triple-resonance NMR spectroscopy in characterization of OP compounds related to the CWC are presented. With a set of two-dimensional triple-resonance experiments the J_HP, J_CH and J_PC couplings are utilized to map the connectivities of the atoms in OP compounds and to extract the carbon chemical shift information. With the use of the proposed pulse sequences the correlations from the OP compounds can be recorded without significant artifacts from the non-OP compound impurities in the sample. Further selectivity of the observed correlations is achieved with the application of phosphorus band-selective pulse in the pulse sequences to assist the analysis of multiple OP compounds in mixture samples. The use of the triple-resonance experiments in the analysis of a complex sample is shown with a test mixture containing typical scheduled OP compounds, including the characteristic degradation products of nerve agents sarin, soman, and VX. The viability of the approach in verification analysis is demonstrated in the analysis of the 30th OPCW Proficiency Test sample.     

Structural changes from heating amber and copal as observed by nuclear magnetic resonance spectroscopy

Structural changes caused by heating of fossilized (amber) and semifossilized (copal) resins have been examined by nuclear magnetic resonance spectroscopy. A set of 28 samples was constituted to include different geographical sources, degrees of maturation, colors, and structural groupings. The onset of structural alterations was determined by observation of the lowest temperature at which spectral changes occurred. Both proton spectra in solution and carbon-13? spectra in the solid state then were recorded of cooled samples after heating for 12 hr at temperature increments, until liquification of the sample began. The spectra of both nuclides exhibit loss of a few peaks, broadening of most peaks, and enhancement of the unsaturated or aromatic region at the expense of saturated resonances. Such changes are irreversible and lead to a harder and less soluble material on cooling. The changes parallel those that occur with maturation of fossil resins or materials that lead to coal.     

Quantitative analysis of malic and citric acids in fruit juices using proton nuclear magnetic resonance spectroscopy

¹H NMR spectroscopy was applied to the quantitative determination of malic and citric acids in apple, apricot, pear, kiwi, orange, strawberry and pineapple juices. Aspartic acid was studied as a potential interference. The effect of the sample pH on the chemical shifts of signals from malic, citric and aspartic acids was examined and a value of 1.0 was selected to carry out the determination. Integration of NMR signals at 2.89-2.95 and 3.00-3.04 ppm were used for calculating the concentration of malic and citric acids, respectively. At this pH the integrated signals were not overlapped. Sodium 3-(trimethylsilyl)tetradeuteropropionate (TSP) was used as an internal reference. The obtained results applying NMR procedures to analyze the juices from different fruits were compared to those obtained using enzymatic methods and both were in close agreement. The intra- and inter-day repeatability was tested for apple juice (7.86 g l⁻¹ malic acid, 0.32 g l⁻¹ citric acid) and apricot juice (5.06 g l⁻¹ malic acid, 4.79 g l⁻¹ citric acid) obtaining coefficients of variation lower than 3.4% for intra-day measures (n = 10) and lower than 3.8% for inter-day measures (n = 20).     

Monitoring of the insecticide trichlorfon by phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy

Trichlorfon is an organophosphorus insecticide, which is extensively being used for protection of fruit crops. Trichlorfon is a thermal labile compound, which cannot be easily determined by gas chromatography (GC) and has no suitable group for sensitive detection by high performance liquid chromatography (HPLC). In this study, a ³¹P nuclear magnetic resonance (³¹P NMR) has been described for monitoring of trichlorfon without any separation step. The quantitative works of ³¹P NMR spectroscopy has been performed in the presence of an internal standard (hexamethylphosphoramide). Limit of detection (LOD) for this method has been found to be 55 mg L⁻¹, without any sample preparation, and the linear working range was 150-5500 mg L⁻¹. Relative standard deviation (R.S.D.%) of the method for three replicates within and between days was obtained ≤9%. The average recovery efficiency was approximately 99-112%. This method was applied for monitoring trichlorfon in a commercial insecticide sample and tomato sample.     

Analysis of glycans in glycoproteins by diffusion-ordered nuclear magnetic resonance spectroscopy

Enzymatically cleaved glycans from sub-milligram quantities of erythropoietin (EPO) and ovalbumin have been analyzed, without further purification, by two-dimensional diffusion-ordered nuclear magnetic resonance spectroscopy. At NMR sample concentrations below 50 μmol L⁻¹ the major components of the oligosaccharide fractions could be distinguished by their anomeric proton chemical shift and their size-dependent diffusion coefficients. Figure ¹H NMR diffusion decay curves of anomeric protons in the EPO glycan fraction     

Investigation of the inclusion of the herbicide cycluron in native cyclodextrins by X-ray diffraction,nuclear magnetic resonance spectroscopy and isothermal titration calorimetry

The formation of inclusion complexes between the native cyclodextrins (CDs) and the urea herbicide cycluron has been investigated both in solution and in the solid state. Single-crystal X-ray structures of both the uncomplexed guest and the β-CD·cycluron complex were determined while powder X-ray diffraction was used to confirm complexation between γ-CD and cycluron in the solid state. Solution-state complexation between the herbicide and α-, β- and γ-CD was established using ¹H NMR spectroscopy and isothermal titration calorimetry (ITC). From the ¹H NMR spectroscopic studies 1:1 complex stoichiometry was indicated in all cases and association constant values (K) were determined as 228, 3254 and 155 for the complexes α-CD·cycluron, β-CD·cycluron and γ-CD·cycluron, respectively. Assigning a 1:1 host–guest ratio, the ITC technique produced K values of the same order as those determined using the spectroscopic method. The thermodynamic parameters ΔH, ΔS and ΔG obtained using ITC provide insights into the driving forces involved during complex formation.     

Soybean biodiesel methyl esters,free glycerin and acid number quantification by 1H nuclear magnetic resonance spectroscopy

Production of alternative fuels, such as biodiesel, from transesterification of vegetable oil driven by heterogeneous catalysts is a promising alternative to fossil diesel. However, achieving a successful substitution for a new renewable fuel depends on several quality parameters. ¹H NMR spectroscopy was used to determine the amount of methyl esters, free glycerin and acid number in the transesterification of soybean oil with methanol in the presence of hydrotalcite‐type catalyst to produce biodiesel. Reaction parameters, such as temperature and time, were used to evaluate soybean oil methyl esters rate conversion. Temperatures of 100 to 180 °C and times of 20 to 240 min were tested on a 1 : 12 molar ratio soybean oil/methanol reaction. At 180 °C/240 min conditions, a rate of 94.5 wt% of methyl esters was obtained, where free glycerin and free fatty acids were not detected. Copyright © 2012 John Wiley & Sons, Ltd.     

Quality assessment of Radix Codonopsis by quantitative nuclear magnetic resonance

Radix Codonopsis (Dangshen) is a famous traditional Chinese medicine and has long been used for replenishing energy deficiency, strengthening the immune system, lowering blood pressure and improving appetite in China, Japan and Korea. A highly specific quantification method using ¹H NMR has been developed for the simultaneous determination of novel quaternary ammonium alkaloids codotubulosine A and B, adenosine and 5-(hydroxymethyl)furfural in Radix Codonopsis materials Codonopsis pilosula, C. pilosula var. modesta, C. tangshen, C. tubulosa, C. subglobosa, C. clematidea, C. lanceolota and Campanumoea javanica collected from different regions of China and Taiwan. A solid-phase extraction with C-18 cartridge followed by elution with water can easily remove sugars the major components that may affect the determination of target constituents. In the ¹H NMR spectrum, the signals of N-CH₃ of codotubulosine A (δ 2.75) and B (δ 2.83), H-8 of adenosine (δ 8.15), and CHO signal of 5-(hydroxymethyl)furfural (δ 9.49) are well separated from other signals in [²H₄]methanol. The quantity of the compounds was calculated by the relative ratio of the integral values of the target peaks of each compound to the known amount of internal standard pyrazine. The described NMR method is found to be relatively simple, specific, precise and accurate for the quality control of Radix Codonopsis herbs and no reference compounds are required for calibration curves, in comparison to conventional HPLC methods, for instance.     

Characterizing phosphorus in environmental and agricultural samples by 31P nuclear magnetic resonance spectroscopy

Phosphorus nuclear magnetic resonance (³¹P-NMR) spectroscopy has advanced our knowledge of organic phosphorus (P) in soils and environmental samples more than any other technique. This paper reviews the use of ³¹P-NMR spectroscopy for soil, water and other environmental samples. The requirements for a successful solid-state or solution ³¹P-NMR experiment are described, including experimental set-up, sample preparation, extractants, experimental conditions, and post-experimental processing. Next, the literature on solid-state and solution ³¹P-NMR spectroscopy in environmental samples is reviewed, including papers on: methods; P transformations; agricultural, forest and natural ecosystem soil studies; humic acid and particle size separations; manure, compost and sludge studies; and water research, including freshwater, estuary and marine studies. Future research needs are also discussed as well as suggestions to improve results, such as increased standardization among research groups.     

Molecular signal suppression by in situ microextraction in nuclear magnetic resonance spectroscopy

The detailed characterization of complex mixtures by NMR is often hampered by the presence of signals from uninformative compounds, the resonances of which overlap with those of the molecules of interest. We provide here a proof of principle for an approach to NMR signal suppression in complex samples using Molecularly Imprinted Polymers (MIPS). Addition of a few milligrams of polymer to a solution traps the target molecule in typical micromolar to millimolar concentration, thus achieving in situ signal suppression, without altering any other spectral features. This method minimized any manipulation or perturbation of the spectrum and was applied to a complex mixture of known compounds and to a plant extract, in both cases spiked with a compound (bisphenol A), which was subsequently removed by selective binding to a complementary MIP. What is described in this report is comparable with microextraction and may in due course be applied to a large number of analytical challenges. Copyright © 2014 John Wiley & Sons, Ltd.     

Prediction of phenolic compounds in red wine fermentations by visible and near infrared spectroscopy

Near infrared (NIR) spectroscopy was used to simultaneously predict the concentrations of malvidin-3-glucoside (M3G), pigmented polymers (PP) and tannins (T) in red wine. A total of 495 samples from 32 commercial scale red wine fermentations over two vintages using two grape varieties (Cabernet Sauvignon and Shiraz), and also including as additional variables two types of fermenters, two different yeasts, and three fermentation temperatures were used. Samples were scanned in transmission mode (400-2500 nm) using a monochromator instrument (NIRSystems6500). Calibration equations were developed from high performance liquid chromatography (HPLC) and NIR data using partial least squares (PLS) regression with internal cross validation. Using PLS regression, very good calibration statistics (R_cal²>0.80) were obtained for the prediction of M3G, PP and T with standard deviation (S.D.)/standard error in cross validation (SECV) ratio (residual predictive deviation, RPD)) ranging from 1.8 to 5.8. It was concluded that near infrared spectroscopy could be used as rapid alternative method for the prediction of the concentration of phenolic compounds in red wine fermentations.     

Formation of ionic liquid submicron particles. 1H and 19F nuclear magnetic resonance spectroscopic studies

We prepared novel ionic liquid submicron particles (ILSPs) in water by emulsifying the ionic liquid (IL) N-(2-methoxyethyl)-N-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide (Pyr_12O1TFSI), which is immiscible with water, with the nonionic surfactants Tween 20 and Span 80. The mean particle size and zeta potential of the ILSPs were about 580?nm and ?30?mV, respectively. The ILSPs were characterized using ¹H and ¹⁹F nuclear magnetic resonance (NMR) spectroscopic methods. The chemical shifts of the Pyr_12O1⁺ cation and TFSI^? anion in the ¹H and ¹⁹F NMR spectra of the ILSP suspension were consistent with those corresponding to pure Pyr_12O1TFSI. This indicated that most of the Pyr_12O1TFSI was still in the IL state in the ILSP suspension. In addition, ¹H–¹H nuclear Overhauser effect correlated spectroscopic measurements showed that the ILSP droplets contained Pyr_12O1TFSI in the neat IL steric state even in the ILSP suspension, and Pyr_12O1TFSI species localized in the droplet surfaces interacted with the hydrophobic acyl side chains of the surfactants. These NMR spectroscopic results show that the ILSPs formed an IL-in-water microemulsion in which droplets of neat Pyr_12O1TFSI were surrounded by the two surfactants, that is, Tween 20 and Span 80.