High-throughput authentication of edible oils with benchtop Ultrafast 2D NMR.

We report the use of an Ultrafast 2D NMR approach applied on a benchtop NMR system (43 MHz) for the authentication of edible oils. Our results demonstrate that a profiling strategy based on fast 2D NMR spectra recorded in 2.4 min is more efficient than the standard 1D experiments to classify oils from different botanical origins, since 1D spectra on the same samples suffer from strong peak overlaps. Six edible oils with different botanical origins (olive, hazelnut, sesame, rapeseed, corn and sunflower) have been clearly discriminated by PCA analysis. Furthermore, we show how this approach combined with a PLS model can detect adulteration processes such as the addition of hazelnut oil into olive oil, a common fraud in food industry.

5-Fluorouracil-related severe toxicity: a comparison of different methods for the pretherapeutic detection of dihydropyrimidine dehydrogenase deficiency.

UNLABELLED: 5-Fluorouracil (5-FU)-related early toxicity, due to a metabolic deficiency, is rare but is potentially severe and even lethal (0.1%). It is due to dihydropyrimidine dehydrogenase (DPYD) gene polymorphism or some epigenetic factors. The detection of metabolic change could prevent severe toxicity, but until now it has not been carried out in clinical practice. PURPOSE: To find the simplest and most accurate pretherapeutic test to predict DPD deficiency in patients treated with 5-FU by comparing different approaches. RESULTS: Two hundred and fifty two French Caucasian patients treated by 5-FU infusion were studied. A two-step strategy, combining firstly SNP detection and uracil plasma measurement, followed, in cases where metabolic deficiency was suspected, by dihydrouracil/uracil ratio determination to confirm deficiency and to determine the optimum 5-FU dosage, appeared the best approach, with 83% and 82% sensitivity and specificity, respectively. CONCLUSION: These data support the future use of this approach, suitable to clinical practice, as screening test to identify DPD deficiency before 5-FU-based therapy.

Sensitive MS/MS-liquid chromatography assay for simultaneous determination of tegafur, 5-fluorouracil and 5-fluorodihydrouracil in plasma.

Ftorafur (FT), an oral prodrug of 5-FU, is part of UFT and S1, two oral prodrugs widely used in digestive tract cancer. We set up a liquid chromatography tandem mass spectrometry (LC/MS-MS) method, chosen for its specificity of detection, for simultaneously measuring in human plasma FT, 5-FU and 5-FUH2. Separation was performed on a Hypercarb column. Linearity, precision and accuracy were validated in the concentration range studied for each compound. This simple and reliable LC/MS-MS method allows specific, sensitive and reproducible quantification of FT, 5-FU and FUH2 in human plasma and can be applied to further pharmacokinetic studies in patients treated with FT-based prodrugs.

Measurement of the main photooxidation products of 2'-deoxyguanosine using chromatographic methods coupled to mass spectrometry.

Analytical methods were developed for the measurement of the main photooxidation products of 2'-deoxyguanosine (dGuo), arising from either the type I (electron transfer) or the type II (singlet oxygen) photosensitization mechanism. Oxidation of dGuo by a type I mechanism leads to the predominant formation of 2, 2-diamino-5-[2-deoxy-beta-d-erythro-pentofuranosyl)amino]-5(2H)-oxazo lone. On the other hand, the two 4R and 4S diastereomers of 4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine are the main singlet oxygen-mediated dGuo oxidation products. The modified nucleosides were measured by either gas chromatography coupled to mass spectrometry after silylation of the sample or by high-performance liquid chromatography associated to tandem mass spectrometry. In order to improve the accuracy of the assays, isotopically labeled internal standards were synthesized for an isotope dilution mass spectrometry quantitation. The methods were successfully applied to the measurement of methylene blue- and riboflavin-mediated 2'-deoxyguanosine photooxidation reactions. The advantages of the two above-mentioned methods are discussed on the basis of comparative sensitivity and accuracy.

Specific natural isotope profile studied by isotope ratio mass spectrometry (SNIP-IRMS): (13)C/(12)C ratios of fructose, glucose, and sucrose for improved detection of sugar addition to pineapple juices and concentrates.

The delta(13)C values of fructose, glucose, and sucrose have been determined in authentic pineapple juices. The sugar fraction is separated from the organic acids by an anionic exchange process. Then the individual components (fructose, glucose, and sucrose) are isolated on a preparative HPLC device using a NH(2)-type column. It is demonstrated that no significant isotope fractionation occurs when close to 100% of material is recovered and when the hydrolysis of sucrose is avoided. The control of the recovery rates and of the sucrose hydrolysis rate after purification is recommended for a reliable interpretation of the results. Correlations between the delta(13)C values of fructose (delta(13)Cf), glucose (delta(13)Cg), and sucrose (delta(13)Csu) can be characterized by systematic differences between these values. For the set of measurements on authentic pineapple juices and concentrates, the mean and the standard deviation of the differences are delta(13)Cf - delta(13)Cg = -0.6 +/- 0.6 per thousand, delta(13)Cf - delta(13)Csu = -1.3 +/- 0. 6 per thousand, and delta(13)Cf - delta(13)Csu = -0.7 +/- 0.5 per thousand. The determinations of the (13)C content of fructose, glucose, and sucrose enable a refinement of the detection of added sugars in fruit juices, re-enforcing the SNIP-IRMS method.

Why do all lariat RNA introns have adenosine as the branch-point nucleotide? Conformational study of naturally-occurring branched trinucleotides and its eleven analogues by 1H-, 31P-NMR and CD spectroscopy.

1H-NMR conformational studies of six branched triribonucleotides where the branch-point nucleotide was either U, C or G (4-9) have been carried out by assigning 1H resonances through 2D NMR and then observing the temperature-dependent (i) chemical shifts of the aromatic and the anomeric protons, and (ii) shifts of the equilibrium of N and S pseudorotamer populations of each sugar moiety. The data have been compared with those of 2'----5' dimers (1-3) and other branched trimers (10-16). It emerged that all the branched trimers (4-16) adopt a conformational state closer to the corresponding 2'----5' dimers than the corresponding 3'----5' dimers. A temperature-dependent 31P chemical shift study confirmed that the conformational constraint is mainly associated with the 2'----5' phosphate linkage. Although, it appeared with the CD data that when C or especially when U is at the branch-point the overall constraint is weak. This suggests that even if these trimers adopt a 2'----5' dimer geometry, there is a lack of stabilization by strong stackings within the molecule. This is in sharp contrast with the results found for A (10-16) and to a smaller extent for G (8, 9) at the branch-point.

Structure and toxicity of a peptide hepatotoxin from the cyanobacterium Oscillatoria agardhii.

A peptide hepatotoxin was isolated by reversed phase liquid chromatography from the cyanobacterium Oscillatoria agardhii and characterized structurally and toxicologically. Amino acid analyses, proton nuclear magnetic resonance and fast atom bombardment mass spectrometry showed that the toxin is a cyclic heptapeptide (mol. wt 1023.5) with the structure cyclo-(Ala-Arg-Asp-Arg-Adda-Glu-N-methyldehydroAla) (Adda: 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid). In mice the toxic effects were restricted mainly to the liver where the toxin induced massive hemorrhages and a disruption of the lobular and sinusoidal structure. The i.p. LD50 of the toxin was 250 micrograms/kg. The structural and toxic properties of this peptide are very close to those of microcystins, cyclic peptide toxins produced by the cyanobacterium Microcystis aeruginosa.

Practical aspects of 2D NMR for assigning the non-exchangeable protons in DNA-RNA fragments.

Using the branched trimer A2'-5'A3'-5'A as an example, different 2D NMR experiments such as homonuclear correlations via single-quantum coherence, double-quantum filtration via double-quantum coherence, isotropic mixing, relayed connectivities, cross relaxation and 31P/1H correlations are presented together with the corresponding spectra. The discussion pointed out the advantages and the difficulties of 14 pulse sequences, used for assigning non-exchangeable protons in DNA or RNA fragments. The emphasis has been put on the methodological aspects of 2D NMR as a simple technique, allowing an easier assimilation of the new coming 2D NMR pulse sequences.

Inhibition of the reverse transcriptase from HIV by 3'-azido-3'-deoxythymidine triphosphate and its threo analogue.

3'-Azido-3'-deoxythymidine triphosphate (erythro) and its threo isomer were synthesized and investigated for their inhibition of HIV reverse transcriptase from virus isolate U 937/HTLV-III. The erythro isomer was a competitive inhibitor of the reaction directed by (rA)n(dT)12-18 and the Ki value was 0.0022 microM. The threo isomer was at least 100-fold less active. The inhibition was specific for dTMP incorporation, and dGMP incorporation using (rC)n(dG)12-18 as template/primer was not affected. The Km value for dTTP varied between 0.7 microM and 1.7 microM. Reverse transcriptase from nine HIV isolates were tested for inhibition by the erythro isomer and only slight differences in sensitivity were observed.

The influence of N6-protecting groups on the acid-catalyzed depurination of 2'-deoxyadenosine.

Kinetics for the acidic hydrolysis of several N6-substituted 2'-deoxyadenosines were studied in a wide pH-range. The proportions of the partial reactions proceeding via mono- and di-protonated substrates were estimated on the bases of the rate profiles obtained and the acidity constants determined spectrophotometrically for the monocations. The site of the initial protonation was established by the effects that trifluoroacetic acid exerted on the 15N NMR chemical shifts. The exceptional lability of the monocations of N6-acyl protected compounds is suggested to result from the preferred N7 protonation.

Syntheses of branched trinucleotides and their conformational analysis by 1H-NMR spectroscopy.

Unambiguous chemical synthesis of branched ribonucleotides, which are products of splicing reactions in eukaryotic cells, and their analogues are reported, subsequently, their secondary structures have been determined by a 270 MHz 1H-NMR spectroscopy.

The application of 15N-NMR spectroscopy in the problems of nucleoside and nucleotide chemistry.

15N-NMR spectroscopy has been shown to be useful to understand the chemical nature of nitrogen in a biomolecule. We herein report the use of 15N-NMR as a tool for the study of exact location of a substitution on the guanine or uracil moieties in a nucleoside, to understand the implication of a protecting group on the reactivity and the basicity of nitrogen atoms in pyrimidines or in purines and to assign the structure of isomeric N7 and N9 substituted purines.

The 15N-NMR spectroscopy of wyosine-triacetate and its congener.

Assignments of the 15N-NMR resonances of wyosine-triacetate 1 and a new congener 2 are reported using NOE and INEPT modes.