Serge Akoka

Concentration Measurement by Proton NMR Using the ERETIC Method

The ERETIC method (Electronic REference To access In vivo Concentrations) provides a reference signal, synthesized by an electronic device, which can be used for the determination of absolute concentrations. The results presented here demonstrate the accuracy and precision of the method in the case of (1)H high resolution NMR. Five tubes were filled with D(2)O solutions of trimethylamine hydrochloride (TMA) 3.84 mM and sodium lactate at concentrations ranging from 5.25 to 54.11 mM. Results obtained with the ERETIC method were compared to those obtained by using TMA as an internal reference. The standard deviations were the same for the two methods and always lower than 1% of the mean. The accuracy (difference between true value and measured value) was slightly better for the ERETIC method than for the internal reference. No significant variation was observed when the experiments were performed over 56 h. Measurements were repeated once a month during three months. As the values obtained showed a standard deviation of only 3%, we can conclude that the ERETIC method has a good stability and only requires monthly calibration. Furthermore, it must be noted that nothing is added to the sample and that the reference signal frequency can be freely chosen to fall within a transparent region of the spectrum.

Isotopic 13C NMR spectrometry to assess counterfeiting of active pharmaceutical ingredients: Site-specific 13C content of aspirin and paracetamol

Isotope profiling is a well-established technique to obtain information about the chemical history of a given compound. However, the current methodology using IRMS can only determine the global (13)C content, leading to the loss of much valuable data. The development of quantitative isotopic (13)C NMR spectrometry at natural abundance enables the measurement of the (13)C content of each carbon within a molecule, thus giving simultaneous access to a number of isotopic parameters. When it is applied to active pharmaceutical ingredients, each manufactured batch can be characterized better than by IRMS. Here, quantitative isotopic (13)C NMR is shown to be a very promising and effective tool for assessing the counterfeiting of medicines, as exemplified by an analysis of aspirin (acetylsalicylic acid) and paracetamol (acetaminophen) samples collected from pharmacies in different countries. It is proposed as an essential complement to (2)H NMR and IRMS.

Ultrafast Quantitative 2D NMR: An Efficient Tool for the Measurement of Specific Isotopic Enrichments in Complex Biological Mixtures

Two-dimensional nuclear magnetic resonance (2D NMR) is a promising tool for studying metabolic fluxes by measuring (13)C-enrichments in complex mixtures of (13)C-labeled metabolites. However, the methods reported so far are hampered by very long acquisition durations limiting the use of 2D NMR as a quantitative tool for fluxomics. In this paper, we propose a new approach for measuring specific (13)C-enrichments in a very fast way, by using new experiments based on ultrafast 2D NMR. Two homonuclear 2D experiments (ultrafast COSY and zTOCSY) are proposed to measure (13)C-enrichments in a single scan. Their advantages and limitations are discussed, and their high analytical potentialities are highlighted. Both methods are characterized by an accuracy of 1-2%, an average precision of 3%, and an excellent linearity. The analytical performance is equivalent or better than any of the conventional methods previously reported. The two ultrafast experiments are applied to the measurement of (13)C-enrichments on a biomass hydrolyzate, showing the first known application of ultrafast 2D NMR to a real biological extract. The experiment duration is divided by 200 compared to the conventional methods, while preserving 80% of the quantitative information. This new approach opens new perspectives of application for fluxomics and metabonomics.

Absolute quantification of metabolites in breast cancer cell extracts by quantitative 2D 1H INADEQUATE NMR

Metabolomic studies by NMR spectroscopy are increasingly employed for a variety of biomedical applications. A very standardized 1D proton NMR protocol is generally employed for data acquisition, associated with multivariate statistical tests. Even if targeted approaches have been proposed to quantify metabolites from such experiments, quantification is often made difficult by the high degree of overlap characterizing 1H NMR spectra of biological samples. Two‐dimensional spectroscopy presents a high potential for accurately measuring concentrations in complex samples, as it offers a much higher discrimination between metabolite resonances. We have recently proposed an original approach relying on the 1H 2D INADEQUATE pulse sequence, optimized for fast quantitative analysis of complex metabolic mixtures. Here, the first application of the quantitative 1H 2D INADEQUATE experiment to a real metabonomic study is presented. Absolute metabolite concentrations are determined for different breast cancer cell line extracts, by a standard addition procedure. The protocol is characterized by high analytical performances (accuracy better than 1%, excellent linearity), even if it is affected by relatively long acquisition durations (15 min to 1 h per spectrum). It is applied to three different cell lines, expressing different hormonal and tyrosine kinase receptors. The absolute concentrations of 15 metabolites are determined, revealing significant differences between cell lines. The metabolite concentrations measured are in good agreement with previous studies regarding metabolic profile changes of breast cancer. While providing a high degree of discrimination, this methodology offers a powerful tool for the determination of relevant biomarkers. Copyright

Fast and precise quantitative analysis of metabolic mixtures by 2D 1H INADEQUATE NMR.

Quantitative analysis of metabolic mixtures by (1)H 1D NMR offers a limited potential for precise quantification of biomarkers, due to strong overlap between the peaks. Two-dimensional spectroscopy is a powerful tool to unambiguously and simultaneously measure a larger number of metabolite contributions. However, it is still rarely used for quantification, first because quantitative analysis by 2D NMR requires a calibration procedure due to the multi-impulsional nature of 2D NMR experiments, and above all because of the prohibitive experiment duration that is necessary to obtain such a calibration curve. In this work, we develop and evaluate a 2D (1)H INADEQUATE protocol for a fast determination of metabolite concentrations in complex mixtures. The 2D pulse sequence is carefully optimized and evaluated in terms of precision and linearity. Quantitative (1)H INADEQUATE 2D spectra of metabolic mixtures are obtained in 7 min with a repeatability better than 2% for metabolite concentrations as small as 100 μM and an excellent linearity. The method described in this work allows a fast and precise quantification of metabolic mixtures, and it forms a promising tool for metabonomic studies.

Evaluation of ultrafast 2D NMR for quantitative analysis.

Recent ultrafast methods make it possible to obtain two-dimensional (2D) nuclear magnetic resonance (NMR) spectra in a fraction of a second. This paper presents the first evaluation of ultrafast 2D NMR for quantitative analysis. On the basis of optimized conditions presented in recent studies, two homonuclear ultrafast techniques, J-resolved spectroscopy and TOCSY, are evaluated on model mixtures in terms of repeatability, long time stability, and linearity. The results are compared to conventional 1D (1)H NMR spectroscopy. Repeatabilities better than 1% for ultrafast J-resolved spectra and better than 7% for TOCSY spectra are obtained. The long-term stability is better than 4% for J-resolved spectroscopy and between 2% and 11% for TOCSY. Moreover, both methods are characterized by excellent linearities. This new analytical method opens important perspectives for fast, precise, and accurate quantitative analysis of complex mixtures and for the quantitative study of short time scale phenomena.

Correction of intensity nonuniformity in spin-echo T1-weighted images

This paper presents a method to correct intensity nonuniformity in spin-echo T(1)-weighted images and particularly the inhomogeneities due to RF transmission imperfections which have tissue-dependent effects through the T(1) relaxation times. This method is based on the use of a uniform phantom, first for classic normalization by division by the phantom images, and second for T(1)-correction using the RF transmitted cartography. We present experimental results from a bi-phasic (oil/water) phantom and from a salmon with a 0.2 T imager. The results demonstrate the efficiency of the method in the two cases and its ability to cope with partial volume effect.

Strategy for choosing extraction procedures for NMR-based metabolomic analysis of mammalian cells

Metabolomic analysis of mammalian cells can be applied across multiple fields including medicine and toxicology. It requires the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Particular attention must be paid to the efficiency and reliability of the extraction procedure. Even though a number of recent studies have dealt with optimizing a particular protocol for specific matrices and analytical techniques, there is no universal method to allow the detection of the entire cellular metabolome. Here, we present a strategy for choosing extraction procedures from adherent mammalian cells for the global NMR analysis of the metabolome. After the quenching of cells, intracellular metabolites are extracted from the cells using one of the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol, methanol/water, and methanol/chloroform/water. The hydrophilic metabolite profiles are analysed using 1H nuclear magnetic resonance (NMR) spectroscopy. We propose an original geometric representation of metabolites reflecting the efficiency of extraction methods. In the case of NMR-based analysis of mammalian cells, this methodology demonstrates that a higher portion of intracellular metabolites are extracted by using methanol or methanol/chloroform/water. The preferred method is evaluated in terms of biological variability for studying metabolic changes caused by the phenotype of four different human breast cancer cell lines, showing that the selected extraction procedure is a promising tool for metabolomic and metabonomic studies of mammalian cells. The strategy proposed in this paper to compare extraction procedures is applicable to NMR-based metabolomic studies of various systems.

Sources of sensitivity losses in ultrafast 2D NMR.

Recent ultrafast techniques make it possible to obtain nD NMR spectra in a single scan. However, an important sensitivity decrease is observed when the excitation duration is increased, which is necessary to improve resolution. A detailed, theoretical and experimental study of sensitivity losses in ultrafast experiments is carried out on the example of J-resolved spectroscopy. The importance of molecular diffusion effects during both encoding and acquisition phases is shown by numerical simulations and experimental results. Other possible sources of signal-to-noise decrease are also considered, such as transverse relaxation, homonuclear J-couplings or chemical shift effects.

Sensitivity and lineshape improvement in ultrafast 2D NMR by optimized apodization in the spatially encoded dimension.

Ultrafast 2D NMR allows the acquisition of a 2D spectrum in a single scan. However, even when the acquisition of ultrafast spectra is carried out under optimized conditions, the appearance and the sensitivity of 2D spectra are often not satisfactory compared with what one could expect from this promising methodology. This is due to limitations in terms of sensitivity, spectral width and resolution, and also to non‐ideal lineshapes characterized by asymmetric sinc wiggles. Here, we identify the origin of these distortions by means of numerical simulations compared with experimental data. We then propose a processing approach to improve lineshapes while increasing the sensitivity of ultrafast experiments. The method consists in multiplying the Fourier transform of ultrafast echoes by an optimized apodization function. The principles of the method are described, and a variety of window functions are tested to determine optimum processing conditions. The approach is finally applied to ultrafast 2D spectra, leading to symmetric lineshapes with a sensitivity increased by a factor of 2. Copyright