Forensic application of position-specific isotopic analysis of trinitrotoluene (TNT) by NMR to determine 13C and 15N intramolecular isotopic profiles.

2,4,6-trinitrotoluene (TNT) is a molecule which is easily identified with current instrumental techniques but it is generally impossible to distinguish between sources of the same substance (TNT). To overcome this difficulty, we present a multi stable isotope approach using isotope ratio monitoring by mass spectrometry (irm-MS) and Nuclear Magnetic Resonance spectrometry (irm-NMR). In the one hand, irm-MS provides bulk isotopic composition at natural abundance in 13C and 15N. The range of variation between samples is rather small particularly for 13C. In the other hand, irm-13C NMR and irm-15N NMR enable the determination of positional intramolecular 13C/12C ratios (δ13Ci) and 15N/14N ratios (δ15Ni) with high precision that lead to larger variation between samples. The present work reports an application of the recent methodology using irm-15N NMR to determine position-specific 15N isotope content of TNT. The interest of this methodology is compared to irm-13C NMR and irm-MS (13C and 15N) in terms of TNT samples discrimination. Thanks to the use of irm-NMR the results show a unique isotopic fingerprint for each TNT which enable origin discrimination between the samples without ambiguity.

Refinement of the inversion-transfer NMR experiment for faster characterization of chemical exchange.

It is shown theoretically that the inversion-transfer experiment used to estimate the value of unidirectional rate constants in chemical exchange systems can be performed faster via a reduction of the recovery delay. The chemical exchange rate constants can then be estimated accurately with a formula close to that of standard inversion transfer and easy to use, after a justified approximation. A function was developed to determine the optimal value of the recovery delay for an optimal inversion-transfer sequence. The validity of these theoretical results was checked experimentally with a solution of N,N-dimethylacetamide in which chemical exchange arises from internal hindered rotation.

Structural features and hydrogen-bond properties of galanthamine and codeine: an experimental and theoretical study.

Structural features of galanthamine and codeine, two allosteric potentiating ligands of nicotinic acetylcholine receptors (nAChRs), have been investigated through experimental studies in solution by FTIR and NMR spectroscopy and by quantum chemical calculations in the isolated state. The infrared spectra accumulated in solvents of various polarities show that the intramolecular OH···O hydrogen bond in galanthamine is stronger than the corresponding interaction in codeine. Molecular electrostatic potential calculations allow rationalisation of the experimental trends. NOE measurements on the two ligands in the same solvent range show significant differences. In apolar solvents, the NMR spectroscopic data indicate the occurrence of CH···O hydrogen-bond interactions, whereas in the more polar solvents, a trans orientation of the methoxy group with respect to the furanyl oxygen atom is favoured. A natural bond orbital (NBO) analysis provides evidence that these stabilising interactions originate in the hyperconjugation between the lone pairs of the furanyl oxygen atoms, n(O), and the methoxy antibonding σ*(C-H) orbitals within the two molecules. Despite the strong structural similarities between the two allosteric modulators, FTIR equilibrium constants measurements of hydrogen-bond complexation combined with quantum chemistry calculations point out the significant increase of hydrogen-bond accepting strength of galanthamine relative to codeine. This increase is mainly assignable to the stronger hydrogen-bond basicity of the hydroxyl group, and to a lesser extent to the higher hydrogen-bond accepting strength of the amino nitrogen of galanthamine in comparison with the corresponding groups of codeine. An analysis of the interactions that occur between the two ligands and acetylcholine esterase (AChE) suggests significant differences with Trp84, a key component of the AChE catalytic active site. In contrast, both ligands appear to interact similarly with acetylcholine binding protein (AChBP).

Optimization of homonuclear 2D NMR for fast quantitative analysis: application to tropine-nortropine mixtures.

Quantitative analysis by (1)H NMR is often hampered by heavily overlapping signals that may occur for complex mixtures, especially those containing similar compounds. Bidimensional homonuclear NMR spectroscopy can overcome this difficulty. A thorough review of acquisition and post-processing parameters was carried out to obtain accurate and precise, quantitative 2D J-resolved and DQF-COSY spectra in a much reduced time, thus limiting the spectrometer instabilities in the course of time. The number of t(1) increments was reduced as much as possible, and standard deviation was improved by optimization of spectral width, number of transients, phase cycling and apodization function. Localized polynomial baseline corrections were applied to the relevant chemical shift areas. Our method was applied to tropine-nortropine mixtures. Quantitative J-resolved spectra were obtained in less than 3 min and quantitative DQF-COSY spectra in 12 min, with an accuracy of 3% for J-spectroscopy and 2% for DQF-COSY, and a standard deviation smaller than 1%.

Improvement of the inverse-gated-decoupling sequence for a faster quantitative analysis of various samples by 13C NMR spectroscopy.

The inverse-gated-decoupling sequence enables quantitative (1)H decoupled (13)C spectra to be obtained. We modified this sequence so as to obtain the same result in less time for molecules containing carbons with various relaxation properties. For that, we determined the optimal (13)C longitudinal-magnetization initial value for a faster relaxation while (1)H decoupler is stopped. This value can be calculated precisely via the nuclear Overhauser effects, the longitudinal relaxation times, together with the determination of the relaxation rate constants of carbons while (1)H are out of equilibrium. A supplementary delay of (1)H decoupling and/or a series of selective pulses applied at the beginning of the recovery delay allow an acceleration of (13)C longitudinal relaxation. We applied this method to the molecule of vanillin. The simultaneous quantification of all carbons was carried out with a recovery delay divided by two compared to the usual sequence.

Evidence for the involvement of tetrahydrofolate in the demethylation of nicotine by Nicotiana plumbaginifolia cell-suspension cultures.

The conversion of nicotine to nornicotine by Nicotiana plumbaginifolia Viv. cells was investigated by analysing the redistribution of label during feeding experiments with (R,S)-[2H- methyl]nicotine, (R,S)-[13C- methyl]nicotine and (R,S)-[14C- methyl]nicotine, and the results show that the N-methyl group of nicotine can be recycled into primary metabolism. Nuclear magnetic resonance (NMR) analysis of ethanolic extracts of cells grown in the presence of (R,S)-[13C- methyl]nicotine, using 1H-13C correlation spectroscopy (HMQC, HMBC), revealed the presence of [3-13C]serine and [13C- methyl]methionine. Label was also identified in a cysteinyl derivative and in several methoxylated compounds, but no evidence was obtained with either NMR or ion-trap mass spectrometry for the presence of any intermediate between nicotine and nornicotine. However, experiments with (R,S)-[14C- methyl]nicotine indicated that 70-75% of the metabolised label was released as carbon dioxide. These results are consistent with a pathway in which the oxidative hydrolysis of the nicotine methyl produces an unstable intermediate, N'-hydroxymethylnornicotine, that breaks down spontaneously to nornicotine and formaldehyde, with the formaldehyde being metabolised either directly to formate and carbon dioxide, or through the tetrahydrofolate-mediated pathways of one-carbon metabolism. However since the key intermediate, N-hydroxymethylnornicotine, could not be detected, the possibility of a direct methyl group transfer to tetrahydrofolate cannot be excluded.

Natural deuterium distribution in branched-chain medium-length fatty acids is nonstatistical: a site-specific study by quantitative 2H NMR spectroscopy of the fatty acids of capsaicinoids.

Quantitative 2H NMR spectroscopy has been used to determine the natural abundance site-specific 2H isotopic content of 6,7-dihydrocapsaicin (1) and capsaicin (2). Prior to analysis, the fatty acyl moieties were released as methyl 8-methylnonanoate (3) and methyl E-8-methylnon-6-enoate (4), respectively. A marked and similar nonstatistical isotopic distribution of (2)H is observed for both fatty acids. Notably, it can be seen that: 1) the isobutyl portion of 3 is more impoverished in 2H than the methylenic portion; 2) the isobutyl portion of 4 is more impoverished than that of 3; 3) an alternating pattern occurs in the (2H/1H)i between the C3 to C7 positions; and 4) the ethylenic hydrogens at C6 and C7 of 4 are, respectively, impoverished and unchanged relative to these positions in 3. These observations are compatible with the proposed biosynthetic origins of the different parts of 1 and 2, and with the view that 1 is a proximal precursor of 2. Furthermore, it can be suggested that, firstly, the hydrogen atoms at C3 to C7 originate alternatively from the substrate and from the environment and, secondly, that the Delta6-E desaturation is introduced by a mechanism closely mimicking that of the Z desaturation of higher plants.