Resolution Nuclear Magnetic Resonance Spectroscopy Research Articles

A screening method for the quantitative determination of selective androgen receptor modulators (SARMs) in capsules by high resolution 19F- and 1H-NMR spectroscopy.

A new method for rapid determination of the content of selective androgenic receptor modulators (SARMs) andarine, cardarine, ligandrol, ostarine and S-23 in capsules by 1H- and 19F-high resolution nuclear magnetic resonance spectroscopy was described and validated. Specificity, linearity, accuracy, precision, detection and quantification limits were considered as validation parameters. Full 1H-, 13C- and 19F-NMR structural assignment of the SARMs is provided as a tool for self-standing identification without a reference standard. Amounts of 7-15 mg of SARMs/capsule were detected in different products with an intermediate precision of 0.8-1.7% in 4 to 20 minutes of analysis time. The validation results and rapidity of analysis confirm the applicability of the method for large-scale screening. The statistical analysis of the results from 19F- and 1H-quantitative NMR showed that both approaches were equally effective, thus expanding the potential use of the methodology to non-fluorinated SARMs. At present, no SARM has been approved for human consumption; however, SARMs are actually used by bodybuilders and recreational athletes, who purchase them even though the risk-benefit ratio of these molecules has not been definitively established.

Silage for upcycling oil from saithe (Pollachius virens) viscera – Effect of raw material freshness on the oil quality

The main objective of this study was to investigate how the freshness of saithe (Pollachius virens) viscera affected the quality, composition and yield of oil obtained by silaging. Minced viscera with and without liver were stored separately for up to 3 days at 4 °C before silaging at pH 3.8 for 6 days at 10 °C. An antioxidant mixture was added to evaluate the effect on the lipid oxidation. Oil was extracted thermally from untreated raw material during storage (day 0–3) and after silaging. For oil obtained after silaging of viscera with liver, the oil yields increased significantly when the raw material was stored for more than one day prior to the treatment. Use of fresh raw material (collected at day 0) led to significantly lower oxidation compared to longer raw material storage. After one day of storage, the oxidation was less dependent on the freshness. Silaging with antioxidants resulted in significantly lower formation of oxidation products compared to acid without antioxidants and the most significant differences were observed after one day of storage. Contents of docosahexaenoic acid (DHA) and total omega-3 fatty acids decreased significantly when the raw material was stored for 1–3 days prior to silaging compared to fresh raw material. Results obtained by high resolution nuclear magnetic resonance (NMR) spectroscopy indicated that oxidation of esterified DHA might explain the DHA decrease. The free fatty acid content was highest when fresh raw material was used and was most likely affected by the formation of cholesteryl esters observed in NMR spectra after longer storage. The study shows that although the oil quality is reduced during silaging, processing shortly after catch and use of antioxidants can optimize the quality resulting in less oxidized oil richer in omega-3 fatty acids.

Optical detection of Copper and Cadmium from Aqueous solution using Arylidenemalanonitriles

We have synthesized new fluorescent organic molecules namely arylidene malanonitriles, through Knovenegal condensation reaction of aryl aldehydes and malanonitrile, which are capable of detectingCd2+and Cu2+ ions in water with sensitivity. The synthesized molecules are characterized through infrared spectrometry, high resolution Mass spectrometry and Nuclear Magnetic Resonance spectroscopy. The fluorescent organic molecules exhibited a fluorescent emission and significant UV-Vis absorption, the intensity of which is increased proportional to the addition of Cd2+ and Cu2+ ions. The lowest detection limit for the Cd2+and Cu2+ were found to be 2.0×10-10 M and 4×10-12 M, respectively.

Force degradation studies of relugolix: identification, isolation and structure characterization of stress degradation products by using liquid chromatography-mass spectrometry, auto purification mass mediated preparative-high performance liquid chromatography, high resolution mass spectrometry, nuclear magnetic resonance spectroscopy

To assess the stability of Relugolix under stress conditions, identify and characterize the degradation products, it was subjected to hydrolytic and oxidative stress conditions, according to the International Council for Harmonization guideline Q1A (R2). The drug showed degradation in basic and oxidative conditions, while it was stable in acid conditions. Four degradation products were obtained, two from each basic and oxidative degradation, which was separated on a C18 column by using Auto Purification Mass Spectrometer. The four degradant products have not been reported earlier in any of the literature and are being reported here for the first time. HIGHLIGHTS Present work captures force degradation of Relugolix under acidic, basic, oxidative, photolytic, and thermal conditions as per International Council for Harmonization guidelines. Two degradation products were observed in basic condition marked as Degradation Product-1, Degradation Product-2 and two degradation products were formed in oxidative condition marked as Degradation Product-3, Degradation Product-4. All the four degradants are novel and are not published in any of the literature. Four degradation products were identified and characterized using various high-end analytical techniques such as Liquid Chromatography and Mass Spectrometer, Reversed Phase–High Performance Liquid Chromatography, High Resolution Mass Spectrometer and Nuclear Magnetic Resonance spectroscopy.

Analysis of thermal degradation of Brazilian palm oil by quantitative 1H NMR and chemometrics

Palm oil (Elaeis guineensis Jacq.) is an edible vegetable oil widely used in the processing industries and Afro-Brazilian culture. Due to its chemical composition rich in lipids, it is susceptible to hydrolytic and oxidative degradation. In this context, the present study describes an analytical method based on the high resolution nuclear magnetic resonance spectroscopy and the statistical analysis for the qualitative and quantitative monitoring of the thermostability of palm oil, subjected to heat treatment at temperatures 80, 100, 120, 140, 160, 180, and 200 °C. The palm oil profiles were characterized by 1H NMR spectra, which were used as data matrices for the statistical analysis. The NMR-based PCA allowed the identification of lipid descriptors related to the degradation mechanisms that were preponderant during the heating of palm oil. The NMR-based PCA allowed identifying the chemical shifts of lipid descriptors related to the mechanisms of degradation and secondary oxidation products observed in samples heated from 140 °C. The results showed the restrictions on the use of palm oil, commonly classified as a stable edible oil, given that the secondary oxidation products identified may present health risks.

NMR-based metabolomics of human cerebrospinal fluid identifies signature of brain death.

Brain death (BD) is the irreversible cessation of all functions of the entire brain, including the brainstem. Cerebrospinal fluid (CSF) is a biological liquid that circulates in brain and spine. Metabolomics is able to reveal the response of biological systems to diverse factors in a specific moment or condition. Therefore, the study of this neurological condition through metabolic profiling using high resolution Nuclear Magnetic Resonance (NMR) spectroscopy is important for understanding biochemical events. The aim of the current study is to identify the metabolomics signature of BD using 1H-NMR spectroscopy in human CSF. 1H-NMR spectroscopy has been employed for metabolomic untargeted analysis in 46 CSF samples: 22 control and 24 with BD. Spectral data were further subjected to multivariate analysis. Statistically significant multivariate models separated subject's samples with BD from controls and revealed twenty one discriminatory metabolites. The statistical analysis of control and BD subjects using Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) model resulted in R2X of 0.733 and Q2 of 0.635. An elevation in the concentration of statistically discriminant metabolites in BD was observed. This study identifies a metabolic signature associated with BD and the most relevant enriched selected metabolic pathways.

In vitro Photoprotective Evaluation and Development of Novel Nanoemulsion with Chromone Derivative

Chromone derivatives exhibiting high absorbance values in the UVA/UVB region were synthesized, and their photoprotective properties were evaluated. Chromones were prepared according to known literature procedures and characterized by high resolution mass spectrometry, infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy. The in vitro solar protection factor (SPF) was determined by the Mansur method and cytotoxicity was evaluated using the sulforhodamine B assay. Two of the chromones synthesized demonstrated suitable SPF values and displayed no cytotoxic effect towards MRC-5 human fibroblasts at the tested concentrations, indicating great potential for future in vivo assays and clinical trials. Finally, the lead compound was incorporated into a nanoemulsion. Nanoemulsions showed high droplet size homogeneity and excellent stability. Chromones bearing methoxy substituents were found to be the most promising compounds with ideal photoprotective properties desirable for utilization and incorporation in sunscreen formulations.

On the Rational Drug Design for Hypertension through NMR Spectroscopy.

Antagonists of the AT1receptor (AT1R) are beneficial molecules that can prevent the peptide hormone angiotensin II from binding and activating the specific receptor causing hypertension in pathological states. This review article summarizes the multifaced applications of solid and liquid state high resolution nuclear magnetic resonance (NMR) spectroscopy in antihypertensive commercial drugs that act as AT1R antagonists. The 3D architecture of these compounds is explored through 2D NOESY spectroscopy and their interactions with micelles and lipid bilayers are described using solid state 13CP/MAS, 31P and 2H static solid state NMR spectroscopy. Due to their hydrophobic character, AT1R antagonists do not exert their optimum profile on the AT1R. Therefore, various vehicles are explored so as to effectively deliver these molecules to the site of action and to enhance their pharmaceutical efficacy. Cyclodextrins and polymers comprise successful examples of effective drug delivery vehicles, widely used for the delivery of hydrophobic drugs to the active site of the receptor. High resolution NMR spectroscopy provides valuable information on the physical-chemical forces that govern these drug:vehicle interactions, knowledge required to get a deeper understanding on the stability of the formed complexes and therefore the appropriateness and usefulness of the drug delivery system. In addition, it provides valuable information on the rational design towards the synthesis of more stable and efficient drug formulations.

Identification of potential urine biomarkers in idiopathic parkinson's disease using NMR

IntroductionParkinson’s disease (PD) is the most common neurodegenerative disease caused by the loss of dopamine chemicals resulting in urinary incontinence, gastrointestinal dysfunction, gait impairment and mitochondrial dysfunction. Study investigated urinary metabolic profiles of patients with idiopathic PD as compared to healthy controls (HC) to identify the potential biomarkers. MethodsUrine samples were collected from 100 PD subjects and 50 HC using standard protocol. Metabolomic analyses were performed using high resolution nuclear magnetic resonance (NMR) spectroscopy. The integral values of 17 significant metabolites were estimated and concentration values were calculated, which were subjected to univariate and multivariate statistical analysis. ResultsWe found significantly increased levels of ornithine, phenylalanine, isoleucine, β-hydroxybutyrate, tyrosine and succinate in the urine of patients with PD in comparison with HC. These metabolites exhibited area under the curve greater than 0.60 on ROC curve analysis. We also observed a significant association between succinate concentration and UPDRS motor scores of PD. DiscussionMetabolic pathway alterations were observed in aromatic amino acid metabolism, ketone bodies synthesis, branched chain amino acid metabolism and ornithine metabolism. Comprehensive metabolomic profiling revealed variations in urinary signatures associated with severity of idiopathic PD. This profiling relies on non-invasive sampling and is complementary to existing clinical modalities.

Compositional identification and authentication of Chinese honeys by 1H NMR combined with multivariate analysis.

Honey authentication has been becoming more and more important and necessary to the honey producers, the consumers and the market regulatory authority due to its favorite organoleptic and healthy properties, high value and increasing export but prevalent falsification practice for economic motivation in China and the potential health risk of adulterated honey. In this study, we obtained the spectral profiles of 90 authentic and 75 adulterated Chinese honey samples by means of high resolution nuclear magnetic resonance (NMR) spectroscopy, and 65 kinds of major and minor components in honey were identified and quantified from their NMR spectra. Combining with the multivariate statistical analyses including principal component analysis (PCA), linear discriminant analysis (LDA), and orthogonal partial least squared-discriminant analysis (OPLS-DA), the discrimination models were successfully established to identify the adulterated honeys from the authentic ones with an accurate rate of 97.6%. Furthermore, the corresponding volcano plot was used to screen out 8 components including proline, xylobiose, uridine, β-glucose, melezitose, turanose, lysine and an unknown component, which are responsible for the differentiation between the authentic and adulterated honeys and will help to control Chinese domestic honey market.

High Resolution Nuclear Magnetic Resonance Spectroscopy on Biological Tissue and Metabolomics.

High-resolution nuclear magnetic resonance (NMR) spectroscopy is a universal analytical tool. It can provide detailed information on chemical shifts, J coupling constants, multiplet patterns, and relative peak areas. It plays an important role in the fields of chemistry, biology, medicine, and pharmacy. A highly homogeneous magnetic field is a prerequisite for excellent spectral resolution. However, in some cases, such as in vivo and ex vivo biological tissues, the magnetic field inhomogeneity due to magnetic susceptibility variation in samples is unavoidable and hard to eliminate by conventional methods. The techniques based on intermolecular multiple quantum coherences and conventional single quantum coherence can remove the influence of the field inhomogeneity effects and be applied to obtain highresolution NMR spectra of biological tissues, including in vivo animal and human tissues. Broadband 1H homo-decoupled NMR spectroscopy displays J coupled resonances as collapsed singlets, resulting in highly resolved spectra. It can be used to acquire high-resolution spectra of some pharmaceuticals. The J-difference edited spectra can be used to detect J coupled metabolites, such as γ-aminobutyric acid, the detection of which is interfered by intense neighboring peaks. High-resolution 1H NMR spectroscopy has been widely utilized for the identification and characterization of biological fluids, constituting an important tool in drug discovery, drug development, and disease diagnosis.

Promotion of mammalian angiogenesis by neolignans derived from soybean extracellular fluids.

Excessive or insufficient angiogenesis is associated with major classes of chronic disease. Although less studied, small molecules which can promote angiogenesis are being sought as potential therapeutics for cardiovascular and peripheral arterial disease and stroke. Here we describe a bioassay-directed discovery approach utilising size exclusion and liquid chromatography to purify components of soybean xylem sap that have pro-angiogenic activity. Using high resolution accurate mass spectrometry and nuclear magnetic resonance spectroscopy, the structure of two pro-angiogenic molecules (FK1 and FK2) were identified as erythro-guaiacylglycerol-8-O-4'-(coniferyl alcohol) ether (eGGCE), and threo-guaiacylglycerol-8-O-4'-(coniferyl alcohol) ether (tGGCE). These two molecules, which are coniferyl neolignan stereoisomers, promoted in vitro angiogenesis in the μM to nM range. Independently sourced samples of eGGCE and tGGCE exhibited comparable pro-angiogenic activity to the soybean derived molecules. The cellular mode of action of these molecules was investigated by studying their effect on endothelial cell proliferation, migration, tube formation and adhesion to the extracellular matrix (ECM) components, fibronectin and vitronectin. They were found to enhance endothelial cell proliferation and endothelial cell tube formation on Matrigel, but did not affect endothelial cell migration or adhesion to fibronectin and vitronectin. Thus, this study has identified two coniferyl neolignan stereoisomers, eGGCE and tGGCE, as pro-angiogenic molecules, with eGGCE being less active than tGGCE.

Quality of Filleted Atlantic Mackerel (Scomber Scombrus) During Chilled and Frozen Storage: Changes in Lipids, Vitamin D, Proteins, and Small Metabolites, including Biogenic Amines

ABSTRACTQuality changes of vacuum-packed Atlantic mackerel (Scomber scombrus) fillets during 12 months’ frozen storage at −27°C and 9 days’ chilled storage at +4°C were evaluated. Freezing at −27°C preserved the long chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs), both in light and dark muscle, vitamin D, and the low molecular weight metabolites (LMW) (studied by high resolution nuclear magnetic resonance spectroscopy, HR NMR). Protein oxidation took place, especially between 1 and 7 months, decreasing water holding capacity and protein extractability. During chilled storage, no lipid or protein oxidation was observed, but lipolysis increased, and several LMW metabolites relevant for sensory and nutritional quality degraded into non-favorable compounds. The content of biogenic amines was high at day 9 (e.g., 18 mg histamine/100 g), jeopardizing safety. Preservation of mackerel fillets by freezing at −27°C is thus a better option compared to prolonged chilled storage at +4°C; the quality was well preserved for 12 months’ frozen storage.

Metabolomics insights into the modulatory effects of long-term compound polysaccharide intake in high-fat diet-induced obese rats

BackgroundPolysaccharides can alleviate obesity in mammals; however, studies on mechanism of this alleviation are limited. A few studies have indicated that polysaccharides improve obesity by regulating the metabolism of the body. Therefore, a metabolomics approach, consisting of high resolution nuclear magnetic resonance (NMR) spectroscopy and a multivariate statistical technique, was applied to explore the mechanism of the protective effects of lentinan and Flos Lonicera polysaccharides (LF) on high-fat diet (HFD) induced obesity.MethodsIn this study, rats were randomly divided into three groups: control diet (CD), HFD, and HFD supplemented with a mixture of lentinan and Flos Lonicera polysaccharide. Histopathological and clinical biochemical assessments were also conducted. A combination of a NMR metabolomics study and a multivariable statistical analysis method to distinguish urinary and fecal metabolites was applied.ResultsSignificant obesity symptoms appeared in HFD rats (for example, significant weight gain, epididymal adipose accumulation and lipid deposition in hepatocytes), which was attenuated in the LF group. Additionally, the HFD induced a reduction of choline, citrate, pyruvate and glycerol and increased the levels of trimethylamine oxide (TMAO) and taurine. Of note, these metabolic disorders were reversed by LF intervention mainly through pathways of energy metabolism, choline metabolism and gut microbiota metabolism.ConclusionsLF supplementation had a re-balancing effect on the disturbed metabolic pathways in the obese body. The results of this study validate the therapeutic effect of the compound polysaccharide--LF in obesity and described the biochemical and metabolic mechanisms involved.

A combination of NMR and liquid chromatography to characterize the protective effects of Rhus tripartita extracts on ethanol-induced toxicity and inflammation on intestinal cells

Consumption of ethanol may have severe effects on human organs and tissues and lead to acute and chronic inflammation of internal organs. The present study aims at investigating the potential protective effects of three different extracts prepared from the leaves, root, and stem of the sumac, Rhus tripartita, against ethanol-induced toxicity and inflammation using intestinal cells as a cell culture system, in vitro model of the intestinal mucosa. The results showed an induction of cytotoxicity by ethanol, which was partially reversed by co-administration of the plant extracts. As part of investigating the cellular response and the mechanism of toxicity, the role of reduced thiols and glutathione-S-transferases were assessed. In addition, intestinal cells were artificially imposed to an inflammation state and the anti-inflammatory effect of the extracts was estimated by determination of interleukin-8. Finally, a detailed characterization of the contents of the three plant extracts by high resolution Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry revealed significant differences in their chemical compositions.

Authentication of animal origin of heparin and low molecular weight heparin including ovine, porcine and bovine species using 1D NMR spectroscopy and chemometric tools

High resolution (600MHz) nuclear magnetic resonance (NMR) spectroscopy is used to distinguish heparin and low-molecular weight heparins (LMWHs) produced from porcine, bovine and ovine mucosal tissues as well as their blends. For multivariate analysis several statistical methods such as principal component analysis (PCA), factor discriminant analysis (FDA), partial least squares – discriminant analysis (PLS-DA), linear discriminant analysis (LDA) were utilized for the modeling of NMR data of more than 100 authentic samples. Heparin and LMWH samples from the independent test set (n=15) were 100% correctly classified according to its animal origin. Moreover, by using 1H NMR coupled with chemometrics and several batches of bovine heparins from two producers were differentiated. Thus, NMR spectroscopy combined with chemometrics is an efficient tool for simultaneous identification of animal origin and process based manufacturing difference in heparin products.

JoVE Monthly Highlights: May 2017

Here's a look at what's coming up in the May 2017 issue of JoVE: The World's Premier Video Journal Mindfulness is at the heart of our first featured article this month, from JoVE Behavior. Here, our Authors use a combination of electroencephalography*(EEG) and electrocardiography*(ECG) to investigate traditional spiritual stress-reduction techniques. Examining the EEG and ECG's of participants before and after an 8-week Mindfulness-based Stress Reduction course, the authors recorded changes in the wavelet entropy of these data based on the training level or meditation type used. This provides a quantifiable methodology for the study of the effects of mindfulness training on the links between the brain and the heart. This slides nicely into our next highlighted article on another heart- and brain- healthy subject - fish oils. In JoVE Chemistry, our Authors present a protocol for the rapid evaluation of the lipid profiles in fish oil supplements. Using high resolution Nuclear Magnetic Resonance spectroscopy, individual fatty acids can be identified, as well as their positional distribution on the glycerol backbone. This method is a powerful analytical tool for the screening encapsulated supplements, and can help protect consumers from any fishy products... From wellness and fish oils to well-oiled fish, our next article in JoVE Biology looks at the effects of alcohol exposure on zebrafish. To investigate Alcoholic Liver Disease (ALD)*, our Authors exposed zebrafish larvae to acute ethanol treatment, and then visualized their livers using a specifically optimized Hematoxylin and Eosin (H&E)* staining protocol. The authors show that steatosis and swelling of the hepatic blood vessels is apparent after just 24 hours of exposure to high ethanol levels. This versatile protocol can also be applied to other models of toxin- and virus-induced injury in the increasingly used zebrafish liver model. Finally this month, let's chill out with a novel freezing method from JoVE Biology. Here, our Authors present time-resolved electron microscopy, dubbed flash-and-freeze, which gives a snapshot of membrane dynamics on the order of milliseconds and nanometers. The flash is an optogenetic stimulation of neuronal activity, and induces neurotransmitter release. A high-pressure freeze then swiftly preserves the cells at the resultant stages, allowing researchers to follow morphological changes after synaptic transmission. That's super cool! You've just had a sneak peek of the May 2017 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The World's Premier Video Journal.

Visualizing Structural Details of Disordered Domain Phase Separation Associated with ALS and Cancers

Phase separation of RNA-binding proteins via multivalent interactions between aromatic/polar-rich disordered domains contributes to the formation of functional cytoplasmic granules and nuclear puncta. These domains have also been identified as the nucleators of neuronal inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We use atomic resolution nuclear magnetic resonance spectroscopy approaches for visualizing low complexity domain structure and interactions along the pathway from monomer, to liquid-liquid phase separated state, to static aggregates and hydrogels. We show that the low complexity domain of RNA-binding protein Fused in Sarcoma (FUS LC) remains disordered even within liquid phase separated states and recruits unphosphorylated RNA-polymerase II C-terminal domain into the liquid phase separated state, adding a potential explanation for FUS LC transcriptional activation in cancer. Importantly, phase separation is reversible and is modulated by ionic strength and interaction with RNA, distinguishing these assemblies from static inclusions. In contrast, we show that liquid-liquid phase separation of TDP-43 is mediated in part by α-helical assembly and extension. Some ALS-associated mutations disrupt helix-helix interaction inhibiting liquid-liquid phase separation while leading to enhanced aggregation.

Authentication of butter from lard adulteration using high-resolution of nuclear magnetic resonance spectroscopy and high-performance liquid chromatography

ABSTRACTFood authentication is an interesting issue for all parties in the food industry, including the fats and oils industry. Some unethical players try to blend high-quality foods, such as butter, with lower ones like lard, therefore, the analytical methods capable of analyzing the adulteration practices must be developed. This study used proton nuclear magnetic resonance spectroscopy in combination with high-performance liquid chromatography for the authentication of butter from lard adulteration. The identification of triacylglycerol composition of lard as a chemical marker for halal authentication is analyzed using high-performance liquid chromatography and high resolution nuclear magnetic resonance spectroscopy. The suitability of proton nuclear magnetic resonance provides a high-performance approach for determination butter adulterated with lard in their entirety of all proton bearing components. Peaks in the region of 2.60–2.84 ppm show special characteristics only present in lard. Only lard has its own unique characteristics which only polyunsaturated fatty acids would give signals 7 at δ 2.63, that corresponded to the chemical shift of the double-allylic methylene protons. In the same way, the intensity of signal at 2.63 ppm, due to methylenic protons in a position α to two double bonds, that is to say, due to the linoleic group. Furthermore, we also correlate some signals between 1H and 13C-NMR spectra for the confirmation of signals.

Enhanced detection of aldehydes in Extra-Virgin Olive Oil by means of band selective NMR spectroscopy

High resolution Nuclear Magnetic Resonance (NMR) spectroscopy is a very powerful tool for comprehensive food analyses and especially for Extra-Virgin Olive Oils (EVOOs). We use the NMR technique to study the spectral region of aldehydes (8–10 ppm) for EVOOs coming from the south part of Italy. We perform novel experiments by using mono and bidimensional band selective spin-echo pulse sequences and identify four structural classes of aldehydes in EVOOs. For the first time such species are identified in EVOOs without any chemical treatment; only dilution with CDCl3 is employed.This would allow the discrimination of different EVOOs for the aldehydes content increasing the potentiality of the NMR technique in the screening of metabolites for geographical characterization of EVOOs.