NMR Analysis of Extra Virgin Olive Oil of the Epirus Region of Greece with Emphasis on Selected Phenolic Compounds.

Extra virgin olive oil (EVOO) is recognized for its numerous health benefits, attributed to its rich phenolic components. NMR has emerged as a prevalent technique for precisely identifying these compounds. Among Mediterranean countries, Greece stands as the third-largest producer of olives, with the Epirus region notably advancing in olive cultivation, contributing significantly to the dynamic growth of the region. In this study, an NMR method was employed based on the acquisition of a 1H NMR spectrum along with multiple resonant suppression in order to increase the sensitivity. Using the above method, 198 samples of extra virgin olive oil, primarily sourced from the Epirus region, were analyzed, and both the qualitative and quantitative aspects of the phenolic compounds were obtained. In addition, we examined the effects of various factors such as variety, harvest month, and region origin on the phenolic compounds' concentration. The results revealed an average total phenolic content of 246 mg/kg, closely approaching the EU health claim limit of 250 mg/kg. Approximately 15% of the samples were confidently characterized as high-phenolic olive oil. The highest concentrations were observed in the Thesprotia samples, with several Lianolia varieties exceeding the total phenolic content of 400 mg/kg. Statistical tests demonstrated a significant influence of the olive variety and the month of fruit harvest on phenolic component concentration, followed by the region of origin. A very strong correlation was noted between the total phenolics content and the levels of oleocanthal and oleacein, with a correlation coefficient (r) of 0.924. Upon optimization of all factors affecting olive oil quality, the majority of the EVOOs from the Epirus region have the potential to be characterized as high in phenolic content.

Nuclear magnetic resonance analysis of extra virgin olive oil: classification through secoiridoids.

BACKGROUND: Extra virgin olive oil (EVOO), a natural product with a multidisciplinary role, has been and is continuing to be studied from several points of view. Among them, its chemical analysis is of major importance and several methods have been used. Nuclear magnetic resonance (NMR) spectroscopy has inherent advantages, among them monitoring the chemical constituents without the need for a separation technique and without, for instance, possible carry-over effects. Additionally, several magnetic resonance spectroscopic techniques can provide a novel powered insight into the nature and properties of a sample under study. Moreover, -omics procedure can reveal new information and can lead to the classification of populations under study. The main objective of the present work was the possible classification of the EVOO samples based on their aldehyde content using a proposed unreferenced 1 H-NMR spectroscopic quantification method combined with a metabolomic approach. Moreover, the study of the impact of such elevated aldehyde content on several spectra regions of importance in the proton NMR spectra led to the proposal of a possible new isomer indicator. RESULTS: Univariate analysis of 12 EVOO samples showed that oleacein, oleocanthal, elenolic acid, hydroxytyrosol/hydroxytyrosol derivatives and tyrosol/tyrosol derivatives strongly differentiated two classes of EVOO: OEH (for high aldehyde EVOO content) and OE (for non-high aldehyde content). Moreover, we pointed out the 'impact' of such elevated secoiridoid and derivatives content, through their moieties' units, on a range of several resonances of the 1 H-NMR spectrum. The metabolomic approach demonstrated the classification of EVOO samples based on their secoiridoid and derivatives content. Multivariate analysis showed a strong influence on the discrimination of the EVOO classes based on the protons resonating at the aldehyde region of the 1 H-NMR spectrum; the aldehyde protons corresponding to 5S,4R-ligstrodial and 5S,4R-oleuropeindial, oleacein, oleocanthal, elenolic acid, p-HPEA-EA, 3,4-DHPEA-EA, 5S,4R- and 5S,4S-ligstrodial and the proton corresponding to a new compound were reported for the first time. This isomer compound, reported for the first time, could serve as a possible indicator for EVOO classification. CONCLUSIONS: An unreferenced quantification method was proposed and EVOO samples were classified into two classes: OEH and OE, according to their aldehyde content, gaining thus probably higher nutrient and possible pharmacological value. Moreover, we point out the 'impact' of such elevated aldehyde content on several spectral regions of the 1 H spectrum. Finally, a new compound was detected in the OEH samples and is reported for the first time. This compound could possibly be an indicator. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Synthesis, Structural, and Physicochemical Characterization of a Ti6 and a Unique Type of Zr6 Oxo Clusters Bearing an Electron-Rich Unsymmetrical {OON} Catecholate/Oxime Ligand and Exhibiting Metalloaromaticity.

The chelating catechol/oxime ligand 2,3-dihydroxybenzaldehyde oxime (H3dihybo) has been used to synthesize one titanium(IV) and two zirconium(IV) compounds that have been characterized by single-crystal X-ray diffraction and 1H and 13C NMR, solid-state UV-vis, and ESI-MS spectroscopy. The reaction of TiCl4 with H3dihybo and KOH in methanol, at ambient temperature, yielded the hexanuclear titanium(IV) compound K2[TiIV6(µ3-O)2(µ-O)3(OCH3)4(CH3OH)2(µ-Hdihybo)6]·CH3OH (1), while the reaction of ZrCl4 with H3dihybo and either nBu4NOH or KOH also gave the hexanuclear zirconium(IV) compounds 2 and 3, respectively. Compounds 1-3 have the same structural motif [MIV6(µ3-Ο)2(µ-Ο)3] (M = Ti, Zr), which constitutes a unique example with a trigonal-prismatic arrangement of the six zirconium atoms, in marked contrast to the octahedral arrangement of the six zirconium atoms in all the Zr6 clusters reported thus far, and a unique Zr6 core structure. Multinuclear NMR solution measurements in methanol and water proved that the hexanuclear clusters 1 and 3 retain their integrity. The marriage of the catechol moiety with the oxime group in the ligand H3dihybo proved to be quite efficient in substantially reducing the band gaps of TiO2 and ZrO2 to 1.48 and 2.34 eV for the titanium and zirconium compounds 1 and 3, respectively. The application of 1 and 3 in photocurrent responses was investigated. ESI-MS measurements of the clusters 1 and 3 revealed the existence of the hexanuclear metal core and also the initial formation of trinuclear M3 (M = Ti, Zr) building blocks prior to their self-assembly into the hexanuclear M6 (M = Ti, Zr) species. Density functional theory (DFT) calculations of the NICSzz scan curves of these systems revealed that the triangular M3 (M = Ti, Zr) metallic ring cores exhibit pronounced metalloaromaticity. The latter depends upon the nature of the metallic center with NICSzz(1) values equal to -30 and -42 ppm for the Ti (compound 1) and Zr (compound 2) systems, respectively, comparable to the NICSzz(1) value of the benzene ring of -29.7 ppm calculated at the same level of theory.

NMR-Based Μetabolomics of the Lipid Fraction of Organic and Conventional Bovine Milk.

Origin and quality identification in dairy products is an important issue and also an extremely challenging and complex experimental procedure. The objective of the present work was to compare the metabolite profile of the lipid fraction of organic and conventional bovine milk using NMR metabolomics analysis. ¹H-NMR and 1D TOCSY NMR methods of analysis were performed on extracted lipid fraction of lyophilized milk. For this purpose, 14 organic and 16 conventional retail milk samples were collected monthly, and 64 bulk-tank (58 conventional and 6 organics) milk samples were collected over a 14-month longitudinal study in Cyprus. Data were treated with multivariate methods (PCA, PLS-DA). Minor components were identified and quantified, and modification of the currently used equations is proposed. A significantly increased % content of conjugated (9-cis, 11-trans)18:2 linoleic acid (CLA), α-linolenic acid, linoleic acid, allylic protons and total unsaturated fatty acids (UFA) and decreased % content for caproleic acid were observed in the organic samples compared to the conventional ones. The present work confirms that lipid profile is affected by contrasting management system (organic vs. conventional), and supports the potential of NMR-based metabolomics for the rapid analysis and authentication of the milk from its lipid profile.

High Resolution NMR Spectroscopy as a Structural and Analytical Tool for Unsaturated Lipids in Solution.

Mono- and polyunsaturated lipids are widely distributed in Nature, and are structurally and functionally a diverse class of molecules with a variety of physicochemical, biological, medicinal and nutritional properties. High resolution NMR spectroscopic techniques including 1H-, 13C- and 31P-NMR have been successfully employed as a structural and analytical tool for unsaturated lipids. The objective of this review article is to provide: (i) an overview of the critical 1H-, 13C- and 31P-NMR parameters for structural and analytical investigations; (ii) an overview of various 1D and 2D NMR techniques that have been used for resonance assignments; (iii) selected analytical and structural studies with emphasis in the identification of major and minor unsaturated fatty acids in complex lipid extracts without the need for the isolation of the individual components; (iv) selected investigations of oxidation products of lipids; (v) applications in the emerging field of lipidomics; (vi) studies of protein-lipid interactions at a molecular level; (vii) practical considerations and (viii) an overview of future developments in the field.

Synthesis, Bonding, and Reactivity of Vanadium(IV) Oxido-Fluorido Compounds with Neutral Chelate Ligands of the General Formula cis-[V(IV)(═O)(F)(L(N-N))2](+).

Reaction of the oxidovanadium(IV)-L(N-N) species (L(N-N) is bipy = 2,2'-bipyridine or bipy-like molecules) with either BF4(-) or HF and/or KF results in the formation of compounds of the general formula cis-[V(IV)(═O)(F)(L(N-N))2](+). Structural and spectroscopic (electron paramagnetic resonance) characterization shows that these compounds are in the tetravalent oxidation state containing a terminal fluorido ligand. Density functional theory calculations reveal that the V(IV)-F bond is mainly electrostatic, which is reinforced by reactivity studies that demonstrate the nucleophilicity of the fluoride ligand in a halogen exchange reaction and in fluorination of various organic substrates.

Oxidovanadium(IV/V) complexes as new redox mediators in dye-sensitized solar cells: a combined experimental and theoretical study.

Corrosiveness is one of the main drawbacks of using the iodide/triiodide redox couple in dye-sensitized solar cells (DSSCs). Alternative redox couples including transition metal complexes have been investigated where surprisingly high efficiencies for the conversion of solar to electrical energy have been achieved. In this paper, we examined the development of a DSSC using an electrolyte based on square pyramidal oxidovanadium(IV/V) complexes. The oxidovanadium(IV) complex (Ph4P)2[V(IV)O(hybeb)] was combined with its oxidized analogue (Ph4P)[V(V)O(hybeb)] {where hybeb(4-) is the tetradentate diamidodiphenolate ligand [1-(2-hydroxybenzamido)-2-(2-pyridinecarboxamido)benzenato}and applied as a redox couple in the electrolyte of DSSCs. The complexes exhibit large electron exchange and transfer rates, which are evident from electron paramagnetic resonance spectroscopy and electrochemistry, rendering the oxidovanadium(IV/V) compounds suitable for redox mediators in DSSCs. The very large self-exchange rate constant offered an insight into the mechanism of the exchange reaction most likely mediated through an outer-sphere exchange mechanism. The [V(IV)O(hybeb)](2-)/[V(V)O(hybeb)](-) redox potential and the energy of highest occupied molecular orbital (HOMO) of the sensitizing dye N719 and the HOMO of [V(IV)O(hybeb)](2-) were calculated by means of density functional theory electronic structure calculation methods. The complexes were applied as a new redox mediator in DSSCs, while the cell performance was studied in terms of the concentration of the reduced and oxidized form of the complexes. These studies were performed with the commercial Ru-based sensitizer N719 absorbed on a TiO2 semiconducting film in the DSSC. Maximum energy conversion efficiencies of 2% at simulated solar light (AM 1.5; 1000 W m(-2)) with an open circuit voltage of 660 mV, a short-circuit current of 5.2 mA cm(-2), and a fill factor of 0.58 were recorded without the presence of any additives in the electrolyte.

Interaction of chromium(III) with a N,N'-disubstituted hydroxylamine-(diamido) ligand: a combined experimental and theoretical study.

Reaction of hydroxylamine hydrochloride with prop-2-enamide in dichloromethane in the presence of triethylamine resulted in the isolation of the N,N'-disubstituted hydroxylamine-(diamido) ligand, 3,3'-(hydroxyazanediyl)dipropanamide (Hhydia). The ligand Hhydia was characterized by multinuclear NMR, high-resolution electrospray ionization mass spectrometry (ESI-MS), and X-ray structure analysis. Interaction of Hhydia with trans-[Cr(III)Cl2(H2O)4]Cl·2H2O in ethanol yields the ionization isomers [Cr(III)(Hhydia)2]Cl3·2H2O(1·2H2O) and cis/trans-[Cr(III)Cl2(Hhydia)2]Cl·2H2O (2·2H2O). The X-ray structure analysis of 1 revealed that the chromium atom in [Cr(III)(Hhydia)2](3+) is bonded to two neutral tridentate O,N,O-Hhydia ligands. The twist angle, θ, in [Cr(III)(Hhydia)2](3+) is 54.5(6)(0), that is, very close to an ideal octahedron. The intramolecular hydrogen bonds developed between the N-OH group of the first ligand and the amidic oxygen atom of the second ligand and vice versa contribute to the overall stability of the cation [Cr(III)(Hhydia)2](3+). The reaction rate constant of the formation of Cr(III) complexes 1·2H2O and 2·2H2O was found to be 8.7(±0.8) × 10(-5) M(-1) s(-1) at 25 °C in methyl alcohol and follows a first-order law kinetics based on the biologically relevant ligand Hhydia. The reaction rate constant is considerably faster in comparison with the corresponding water exchange rate constant for the hydrated chromium(III). The modification of the kinetics is of fundamental importance for the chromium(III) chemistry in biological systems. Ultraviolet-visible and electron paramagnetic resonance studies, both in solution and in the solid state, ESI-MS, and conductivity measurements support the fact that, irrespective of the solvent used in the interaction of Hhydia with trans-[Cr(III)Cl2(H2O)4]Cl·2H2O, the ionization isomers[Cr(III)(Hhydia)2]Cl3·2H2O (1·2H2O) and cis/trans-[Cr(III)Cl2(Hhydia)2]Cl·2H2O (2·2H2O) are produced.The reaction medium affects only the relevant percentage of the isomers in the solid state. The thermodynamic stability of the ionization isomers 1·2H2O and cis/trans-2·2H2O, their molecular structures as well as the vibrational spectra and the energetics of the Cr(III)- Hhydia/hydia(-) were studied by means of density functional theory calculations and found to be in excellent agreement with our experimental observations.

A novel NMR method for the determination and monitoring of evolution of hydrogen peroxide in aqueous solutions.

A novel NMR method that allowed the rapid and direct quantitative analysis of hydrogen peroxide in protic solvents was developed. The method was based on the highly deshielded (1)H NMR signal of the H2O2 protons (δ ∼ 11.15 ppm at 298 K) in H2O and the combined use of cryoprotective (antifreeze) mixtures of H2O-DMSO-d6, low temperatures (∼260 K), and pH effects in order to achieve minimum proton exchange rate and, thus, sharp (1)H line widths. Extremely broad resonances with line widths above 550 Hz at room temperature in H2O were observed in a wide range of pH values, which were reduced below 2 Hz with the use of the above method which resulted in a detection limit of 20.0 µmol L(-1) (in tube) even when using very short total experimental time of 10 min. The method was applied in aqueous extract of Greek oregano and in aqueous instant coffee. Line widths below 10 Hz for oregano samples and 17 Hz for instant coffee samples were obtained which resulted (i) in the unequivocal assignment of H2O2 with spiking experiments precluding any confusion with interferences from intrinsic phenolics in the extracts and (ii) in the quantitative investigation of the evolution of H2O2 in real time with parameters easily accessible experimentally.

Dynamic changes in composition of extracts of natural products as monitored by in situ NMR.

The direct in situ NMR observation and quantification, based on the aldehyde -CH chemical shift region, of the inter-conversion of secoiridoid derivatives due to temperature and solvent effects is demonstrated in complex extracts of natural products without prior isolation of the individual components. The equilibrium between the aldehyde hydrate form and the dialdehyde form of the oleuropein aglycon of an olive leaf aqueous extract in D(2)O was shown to be temperature dependent. The resulting thermodynamic values of the Van't Hoff plot with ΔH(o) = -26.34 ± 1.00 kJ mol(-1) and TΔS° (298 K) = -24.70 ± 1.00 kJ mol(-1) demonstrate a significant entropy term which nearly compensates the effect of enthalpy at room temperature. The equilibrium between the two diastereomeric hemiacetal forms and the dialdehyde form of the oleuropein 6-O-ß-d-glucopyranoside aglycon of an olive leaf aqueous extract in CD(3) OD was also shown to be strongly temperature dependent again because of the significant entropy term (TΔS° (298 K) = -26.50 ± 1.39 kJ mol(-1)) compared with that of the enthalpy term (ΔH(o) = -36.64 ± 1.46 kJ mol(-1)). This is the first demonstration of the significant role of the entropy parameter in determining the equilibrium of chemical transformations in complex mixtures of natural products due to solvent and temperature effects.

1H-NMR as a structural and analytical tool of intra- and intermolecular hydrogen bonds of phenol-containing natural products and model compounds.

Experimental parameters that influence the resolution of 1H-NMR phenol OH signals are critically evaluated with emphasis on the effects of pH, temperature and nature of the solvents. Extremely sharp peaks (Δν1/2≤2 Hz) can be obtained under optimized experimental conditions which allow the application of 1H-13C HMBC-NMR experiments to reveal long range coupling constants of hydroxyl protons and, thus, to provide unequivocal assignment of the OH signals even in cases of complex polyphenol natural products. Intramolecular and intermolecular hydrogen bonds have a very significant effect on 1H OH chemical shifts which cover a region from 4.5 up to 19 ppm. Solvent effects on -OH proton chemical shifts, temperature coefficients (Δδ/ΔT), OH diffusion coefficients, and nJ(13C, O1H) coupling constants are evaluated as indicators of hydrogen bonding and solvation state of phenol -OH groups. Accurate 1H chemical shifts of the OH groups can be calculated using a combination of DFT and discrete solute-solvent hydrogen bond interaction at relatively inexpensive levels of theory, namely, DFT/B3LYP/6-311++G (2d,p). Excellent correlations between experimental 1H chemical shifts and those calculated at the ab initio level can provide a method of primary interest in order to obtain structural and conformational description of solute-solvent interactions at a molecular level. The use of the high resolution phenol hydroxyl group 1H-NMR spectral region provides a general method for the analysis of complex plant extracts without the need for the isolation of the individual components.

Investigation of solute-solvent interactions in phenol compounds: accurate ab initio calculations of solvent effects on 1H NMR chemical shifts.

Accurate (1)H chemical shifts of the -OH groups of polyphenol compounds can be calculated, compared to experimental values, using a combination of DFT, polarizable continuum model (PCM) and discrete solute-solvent hydrogen bond interactions. The study focuses on three molecular solutes: phenol, 4-methylcatechol and the natural product genkwanin in DMSO, acetone, acetonitrile, and chloroform. Excellent linear correlation between experimental and computed chemical shifts (with the GIAO method at the DFT/B3LYP/6-311++G(2d,p) level) was obtained with minimization of the solvation complexes at the DFT/B3LYP/6-31+G(d) and DFT/B3LYP/6-311++G(d,p) level of theory with a correlation coefficient of 0.991. The use of the DFT/B3LYP/6-31+G(d) level of theory for minimization could provide an excellent means for the accurate prediction of the experimental OH chemical shift range of over 8 ppm due to: (i) strong intramolecular and solute-solvent intermolecular hydrogen bonds, (ii) flip-flop intramolecular hydrogen bonds, and (iii) conformational effects of substituents of genkwanin. The combined use of ab initio calculations and experimental (1)H chemical shifts of phenol -OH groups provides a method of primary interest in order to obtain detailed structural, conformation and electronic description of solute-solvent interactions at a molecular level.

A new method for the determination of free L-carnitine in serum samples based on high field single quantum coherence filtering 1H-NMR spectroscopy.

The rapid and accurate determination of specific metabolites present in biofluids is a very demanding task which is essential in both medicine and chemistry. L-carnitine (3-hydroxy-4-N-trimethylammonium butyrate) is an important metabolite which participates in a series of biological paths and therefore its determination is of diagnostic importance. A single quantum coherence filtering (1)H NMR methodology was used for the accurate and rapid determination of L-carnitine in human serum samples. The methodology is based on spectral simplification, and specifically on the distinction of the N-methyl proton signal of L-carnitine that is greatly overlapped in the (1)H-NMR spectrum of serum. The quantitative results provided by the proposed method are in excellent agreement with those obtained by the enzymatic method, which is widely used. The proposed method is rapid (~20 min of experimental time), selective, sensitive, and has good analytical characteristics (accuracy, reproducibility). Selected protein precipitation methods were also investigated and sample pretreatment with EtOH is suggested.

Altered RBC membrane lipidome: A possible etiopathogenic link for the microvascular impairment in Type 2 diabetes.

AIMS: Disturbances in red blood cells' (RBCs) membrane structure, that result in altered rheological properties, have been implicated in the pathogenesis of microvascular complications of diabetes mellitus(T2DM). However, the compositional alterations in RBCs membranes of T2DM patients have not been characterized in detail. METHODS: NMR-based lipidomic approach used for the global investigation of the lipidome of RBCs membrane in 20 newly diagnosed T2DM patients. Twenty healthy individuals served as controls. RESULTS: In the lipidomic analysis, the discrimination power among the two groups was of high significance. T2DM patients characterized by an increased content of cholesterol, total sphingolipids, sphingomyelin and glycolipids, and decreased total phospholipids, mainly due to phosphatidylethanolamine, total ether glycerolipids and plasmalogen-phospholipids, and higher cholesterol-to-phospholipids molecular ratio compared to controls. In T2DM, lipids were esterified with saturated rather than unsaturated fatty acids, an atherogenic pattern that may be involved in the impairment of membrane fluidity and rigidity. CONCLUSIONS: NMR-based lipidomic analysis of RBCs can provide insights into molecular lipid features of membrane microenvironment that influence their vital function and rheological behavior in microvascular network in T2DM.Early identification of these disturbances, even before the onset of diabetes, could critically help to the development of novel preventative and curative therapies for reducing the risk of microvascular dysfunction.

Conjugation of Penicillin-G with Silver(I) Ions Expands Its Antimicrobial Activity against Gram Negative Bacteria.

Conjugation of penicillin G (PenH) with silver(I) ions forms a new CoMeD (conjugate of metal with a drug) with formula [Ag(pen)(CH3OH)]2 (PenAg). PenAg was characterized by a plethora of physical and spectroscopic techniques, which include in the solid state m.p.; elemental analysis; X-ray fluorescence (XRF) spectroscopy; scanning electron microscopy (SEM); energy-dispersive X-ray spectroscopy (EDX); FT-IR; and in solution: attenuated total reflection spectroscopy (FT-IR-ATR), UV-Vis, 1H NMR, and atomic absorption (AA). The structure of PenAg was determined by NMR spectroscopy. Silver(I) ions coordinate to the carboxylic group of PenH, while secondary intra-molecular interactions are developed through (i) the nitrogen atom of the amide group in MeOD-d4 or (ii) the sulfur atom in the thietane ring in deuterated dimethyl sulfoxide DMSO-d6. The antibacterial activities of PenAg and the sodium salt of penicillin (PenNa) (the formulation which is clinically used) against Gram positive (Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus)) and Gram negative (Pseudomonas aeruginosa (P. aeuroginosa PAO1)) bacteria were evaluated by the means of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and inhibition zone (IZ). PenAg inhibits the growth of the Gram negative bacterial strain P. aeuroginosa with a MIC value of 23.00 ± 2.29 µM, in contrast to PenNa, which shows no such activity (>2 mM). The corresponding antimicrobial activities of PenAg against the Gram positive bacteria S. epidermidis and S. aureus are even better than those of PenNa. Moreover, PenAg exhibits no in vivo toxicity against Artemia salina at concentration up to 300 µΜ. The wide therapeutic window and the low toxicity, make PenAg a possible candidate for the development of a new antibiotic.

Selective One-Dimensional Total Correlation Spectroscopy Nuclear Magnetic Resonance Experiments for a Rapid Identification of Minor Components in the Lipid Fraction of Milk and Dairy Products: Toward Spin Chromatography?

We report a rapid, direct, and unequivocal spin-chromatographic separation and identification of minor components in the lipid fraction of milk and common dairy products with the use of selective one-dimensional (1D) total correlation spectroscopy (TOCSY) nuclear magnetic resonance (NMR) experiments. The method allows for the complete backbone spin-coupling network to be elucidated even in strongly overlapped regions and in the presence of major components from 4 × 10(2) to 3 × 10(3) stronger NMR signal intensities. The proposed spin-chromatography method does not require any derivatization steps for the lipid fraction, is selective with excellent resolution, is sensitive with quantitation capability, and compares favorably to two-dimensional (2D) TOCSY and gas chromatography-mass spectrometry (GC-MS) methods of analysis. The results of the present study demonstrated that the 1D TOCSY NMR spin-chromatography method can become a procedure of primary interest in food analysis and generally in complex mixture analysis.

Development of an amperometric biosensing method for the determination of L-fucose in pretreated urine.

The first amperometric biosensing method for the determination of L-fucose is described. L-Fucose is the objective of much current research, as it is considered as a potential marker for various pathologic disorders. Recombinant L-fucose dehydrogenase, having as cofactor beta-nicotinamide adenine dinucleotide phosphate (NAD+P), was cross-linked in a water-soluble photosensitive polymer matrix, that is, polyvinyl alcohol (PVA) modified with styrylpyridinium (SbQ), in the presence of BSA and glutaraldehyde. The resulting membrane was sandwiched between two polycarbonate membranes and was mounted in an amperometric cell. The oxidation of the enzymatically produced NADPH was monitored at a platinum anode at +0.25 V versus a silver pseudoreference electrode in the presence of ferricyanide. The system was fully optimized with respect to various analytical parameters. Regarding to the mechanical properties of the membrane and the storage stability of the immobilized enzyme, various parameters were also optimized. Several methods for the pretreatment of urine samples were investigated. Treatment of the samples with PbO2 found to eliminate the interference effect of various electroactive species exist in urine; optimum incubation time was determined since at prolonged incubation times L-fucose is also affected. Calibration curves for the direct and the mediated monitoring of NADPH were liner over the concentration ranges 0.04-1.0 mM (r2=0.9995) and 0.03-3.0 mM (r2=0.9997) fucose, respectively. The detection limits (S/N 3) were 2 and 1.5 microM fucose, respectively. The R.S.D. of the mediated biosensor is better than 1.5% (n=10, 0.5 mM fucose). The proposed biosensor correlates well with a reference enzymatic method and exhibits very good working and storage stability.

Direct nuclear magnetic resonance identification and quantification of geometric isomers of conjugated linoleic acid in milk lipid fraction without derivatization steps: overcoming sensitivity and resolution barriers.

We report the first successful direct and unequivocal identification and quantification of four minor geometric (9-cis, 11-trans) 18:2, (9-trans, 11-cis) 18:2, (9-cis, 11-cis) 18:2 and (9-trans, 11-trans) 18:2 conjugated linoleic acid (CLA) isomers in lipid fractions of lyophilized milk samples with the combined use of 1D (1)H-NMR, 2D (1)H-(1)H TOCSY and 2D (1)H-(13)C HSQC NMR. The significant sensitivity barrier has been successfully overcome under selective suppression of the major resonances, with over 10(4) greater equilibrium magnetization of the -(CH2)n-(1)H spins compared to that of the (1)H spins of the conjugated bonds of the CLA isomers. The resolution barrier has been significantly increased using reduced (13)C spectral width in the 2D (1)H-(13)C HSQC experiment. The assignment was confirmed with spiking experiments with CLA standard compounds and the method does not require any derivatization steps for the lipid fraction. The proposed method is selective, sensitive and compares favorably with the GS-MS method of analysis.

Rapid and direct low micromolar NMR method for the simultaneous detection of hydrogen peroxide and phenolics in plant extracts.

A rapid and direct low micromolar ¹H NMR method for the simultaneous identification and quantification of hydrogen peroxide and phenolic compounds in plant extracts was developed. The method is based on the highly deshielded ¹H NMR signal of H2O2 at ∼10.30 ppm in DMSO-d6 and the combined use of picric acid and low temperature, near the freezing point of the solution, in order to achieve the minimum proton exchange rate. Line widths of H2O2 below 3.8 Hz were obtained for several Greek oregano extracts which resulted in a detection limit of 0.7 µmol L⁻¹. Application of an array of NMR experiments, including 2D ¹H-¹³C HMBC, spiking of the samples with H2O2, and variable temperature experiments, resulted in the unequivocal assignment of H2O2 precluding any confusion with interferences from intrinsic phenolics in the extract.