Constantinos G. Tsiafoulis

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.

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.

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.

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.

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.

Determination of Polyphenolic Phytochemicals using Highly Deshielded –OH 1H-NMR Signals

Introduction Quantitative analysis of plant extracts is a tedious and time consuming process requiring chromatographic techniques and/or the use of specilised hyphenated spectroscopic methods. Thus, investigation of a rapid method capable for the determination of bioactive constituents is of major importance. Objective To employ high resolution 1H-NMR spectroscopy of the –OH spectral region, as a rapid method for the simultaneous determination and quantification of polyphenolic phytochemicals in crude oregano and olive leaf extracts without any prior isolation step. Methodology A critical overview of experimental parameters that influence the resolution of the –OH groups is provided with emphasis on the nature of the extraction solvent, effects of dilution, pH, temperature and nature of the NMR solvent. The compounds were assigned with the use of 1H-NMR, 2D 1H-13C HMBC NMR and spiking experiments with model compounds. Results The method was applied for the NMR analysis of natural product extracts and the results were found to be in good agreement with those obtained with the use of the time consuming HPLC-DAD, preparative LC and LC-SPE-NMR. Conclusion The method is rapid, selective, with very good analytical performance characteristics, which support the efficiency of the suggested methodology. Copyright

Selective 1D TOCSY NMR method for the determination of glutathione in white wine

Glutathione, a naturally occurring tripeptide, is the main thiol in must and wine which inhibits must oxidative browning and prevents the loss of wine aroma. In the present work a direct quantification of glutathione in fresh white wine with the use of selective 1D TOCSY NMR experiments is demonstrated. The method is based on the selective excitation of the βCH2SH protons of the Cys residue resulting in the magnetization transfer to the αCH proton followed by its quantification with the use of the standard addition method. The method: (i) does not involve any derivatization and/or extensive sample preparation procedures; (ii) it allows the spin-coupling network of the L-cysteine residue to be elucidated even in the presence of strongly overlapped abundant analytes; (iii) has good NMR analytical performance characteristics and (iv) allows the simultaneous identification and quantification of both reduced and oxidized glutathione.