MIMM : Méthodologie RMN – Laboratoire CEISAM // CEISAM Institute

À partir de son expertise historique en RMN quantitative de haute précision, MIMM a développé une expérience unique dans le développement de séquences d’impulsions originales pour l’analyse de mélanges complexes.

Nos principaux axes de recherche incluent le développement de méthodes quantitatives très précises basées sur des expériences multi-impulsionnelles et multi-dimensionnelles, et la conception de séquences d’impulsions mettant en œuvre un codage spatial de l’échantillon, telles que la RMN 2D ultrarapide, les expériences pure-shift ou la RMN ultrasélective pour l’analyse des mélanges hors équilibre.

Ces séquences d’impulsions sont implantées sur les spectromètres RMN haut champ de la plateforme RMN CEISAM, mais également sur des spectromètres RMN compacts récents équipés de bobines de gradients. Les développements incluent également des méthodes d’analyse des mélanges hyperpolarisés, à travers un système de polarisation nucléaire dynamique par dissolution.

1. Methodological Developments and Experimental Validation for NMR Isotope Analysis

Current team members: Margot Sanchez, Gérald Remaud, Evelyne Baguet, Serge Akoka*.

Past team members: Sophie Renou, Tangi Jezequel, Valentin Joubert, Kévin Bayle, Ugo Bussy.

Collaboration (past and present): Gaëtan Assemat (RS²D), Klemens Kessler (QUAD Systems)

R2D3: A series of DEFT spectra are performed with τr that varies from one increment to another (a). The acquired data (b) are Fourier Transformed (c) and 1 dimensional spectrum is obtained by extraction and addition of a few central rows (d). Figure 5: Single pulse, DEFT and R2D3 vinyl acetate spectra (e).

This project is on-going as it is a permanent objective of the team from the beginnings. Isotopic ¹³C-NMR at natural abundance requires highly precise measurements (a few per mil), that have already been achieved by one-pulse ¹³C acquisitions thank to an original ¹H-decoupling scheme. However, it is quite time consuming, even for a relatively simple molecular structure. Using optimized adiabatic multipulse sequences (1D or 2D) a dramatic reduction is gained in the experimental time without deterioration in short time or long-time stability. This approach permits to envisage for the first time ¹⁵N PSIA by NMR.

Our efforts are now focused on increasing the SNR per unit time in ²H, so that we can perform ²H and ¹³C isotopic NMR measurements on the same instrument and probe. In order to reach this goal, we propose a new acquisition strategy called R²D³. A precision of 1% or less was obtained in almost all the cases, showing that the R²D³ approach can drastically decrease the experimental time, while retaining the key aspects of a quantitative experiment. A high time gain factor can be achieved, close to that of INEPT and without its drawbacks, when trueness is less critical than precision. The R²D³ method will particularly benefit qNMR applications based on observing heteronuclei and analysing large sample series.

The oxygen isotopes content is also of interest. No PSIA information are available on organic molecules. We currently working on ¹⁷O NMR to evaluate its capability.

Key References:

C. Thibaudeau, G. Remaud, V. Silvestre, S. Akoka. Performance Evaluation of Quantitative Adiabatic ¹³C NMR Pulse Sequences for Site-specific Isotopic Measurements. Analytical Chemistry, 2010, 82 (13), 5582–5590.
E. Martineau, S. Akoka, R. Boisseau, B Delanoue, P. Giraudeau. Fast quantitative ¹H-¹³C 2D NMR with very high precision. Analytical Chemistry, 2013, 85(9) 4777-4783.
S. Akoka, G. Remaud. NMR-based isotopic and isotopomic analysis. Progress in NMR Spectroscopy, 2020, 120–121.
L. Haddad, S. Renou, G. S. Remaud, T. Rizk, J. Bejjani, S. Akoka. A precise and rapid isotopomic analysis of small quantities of cholesterol at natural abundance by optimized ¹H-¹³C 2D NMR. Analytical and Bioanalytical Chemistry 2021, 413, 1521–1532.
M. Sanchez, T. Paris, A. Martinez G. Assemat, S. Akoka. The R²D³ approach towards fast quantitative NMR: maintaining accuracy and reducing the experimental time. Analyst 2025,150,9,1939-1951.

Supported by: National Council for Scientific research and Nantes Université, ANRT, RS²D, QUAD Systems

2. Advanced methods in benchtop NMR spectroscopy

Current team members: Benoît Charrier, Patrick Giraudeau*, Jonathan Farjon*

Past team members: Joris Mandral , Shrikant Kunjir, Boris Gouilleux, Maxime Tharaud, Thomas Castaing-Cordier, Dylan Bouillaud, Jean-Nicolas Dumez

Collaborations (past and present): Fabrice Besacier and Virginie Ladroue (INPS Lyon), Paul Bowyer (Magritek USA), Ernesto Danieli and Juan Perlo (Magritek Germany), Myriam Malet-Martino (Univ. Toulouse), Kerstin Münnemann (Kaiserslautern)

High-field NMR has been constantly improving with superconducting magnets and cold probes, but it is associated with high purchase and operating costs and the need for dedicated staff. In the last few years, a new generation of transportable and cryogen-free low-field spectrometers has emerged as a promising alternative. These permanent magnets have reached a recognized analytical potential with stable and homogeneous static magnetic fields.

However, those spectrometers operating at ¹H resonance frequencies between 40 and 80 MHz involve a loss of analytical performance. In addition to the low sensitivity, the reduction of the spectral resolution leads to overcrowded spectra with huge signal overlaps, especially in the case of complex mixtures. Moreover, ubiquitous second order couplings make identification and quantification very difficult.

Thanks to the pulse sequence programming capabilities of benchtop spectrometers and to their instrumental improvement with a gradient coil, advanced high-field NMR methods have been successfully implemented to improve their analytical capabilities. Among them, we are particularly exploring spatially-encoded experiments such as ultrafast 2D NMR and pure-shift experiments. We have also demonstrated the potential of benchtop NMR DOSY to investigate complex mixtures. Gradient coils have also enabled the implementation of modern solvent suppression schemes. Applications to reaction and process monitoring are currently explored.

Key references:

B. Gouilleux, B. Charrier, S. Akoka, F.-X. Felpin, M. Rodriguez-Zubiri, P. Giraudeau, Ultrafast 2D NMR on a benchtop spectrometer: Applications and perspectives, Trends Anal. Chem. 83, 65 (2016)
B. Gouilleux, B. Charrier, S. Akoka, P. Giraudeau, Gradient-based solvent suppression methods on a benchtop spectrometer, Magn. Reson. Chem. 55, 91 (2017)
T. Castaing-Cordier, D. Bouillaud, P. Bowyer, O. Gonçalves, P. Giraudeau, J. Farjon, Highly-resolved pure-shift spectra on a compact NMR spectrometer, ChemPhysChem, 20, 736 (2019)
B. Gouilleux, J. Farjon, P. Giraudeau, Gradient-based pulse sequences for benchtop NMR spectroscopy J. Magn. Reson., 319, 106810 (2020)
T. Castaing-Cordier, A. Benavides Restrepo, D. Dubois, V. Ladroue, F. Besacier, A. Buleté, C. Charvoz, A. Goupille, D. Jacquemin, P. Giraudeau, J. Farjon, Drug Test. Anal., 14, 1629 (2022).
T. Castaing-Cordier, S. Crasnier, D. Dubois, V. Ladroue, A. Buleté, C. Prudhomme, C. Charvoz, F. Besacier, D. Jacquemin, P. Giraudeau, J. Farjon, Magn. Reson. Chem., 62, 378 (2024).

Supported by Région Pays de la Loire (Pari scientifique AMER-METAL 2017-2020), CNRS (Projet interdisciplinarité RMN-(ME)2-TAL 2017-2020), ANR (DEVIL-INSID 2019-2023, ChemAI 2025-2029) European Space Agency (SPACE_ALG 2024-2027), PEPR B-BEST (ALGADVANCE 2023 - 2028)

3. Fast quantitative 2D NMR methods

Current team members: Estelle Martineau, Jérémy Marchand, Jonathan Farjon, Patrick Giraudeau*

Past team members: Tangi Jézéquel, Bertrand Plainchont, Jean-Nicolas Dumez, Boris Gouilleux, Adrien Le Guennec, Meerakhan Pathan, Laetitia Rouger

Collaboration (past and present): Stefano Caldarelli (Aix-Marseille Université), Philippe Lesot (ICMMO Orsay), Hassan Oulyadi (Rouen), Patricia Sepulcri (Sanofi Pasteur), Stanislav Sokolenko (Univ. Dalhousie), Christina Thiele (TU Darmstadt)

Measuring the accurate concentration of analytes in chemical or biochemical mixtures is, together with the identification of these components, one of the main goals of analytical chemistry. Nuclear Magnetic Resonance (NMR) is a widely used quantitative approach, known for its high reproducibility and robustness.

However, 1D proton NMR suffers from large overlap between peaks that restrains its quantitative use, particularly when complex mixtures are studied. In order to overcome these drawbacks, we are developing new quantitative 2D NMR approaches, since multidimensional NMR offers a better discrimination between resonances in complex samples with overlapped peaks.

Still, conventional 2D NMR experiments are affected by long experiment durations that, beyond timetable constraints, limit their use for quantitative analysis (since long experiments are highly sensitive to spectrometer instabilities) or for studying fast processes (kinetics, dynamics, etc.). For this reason, we focus on developing fast and precise 2D NMR experiments and we apply them to a variety of quantitative studies. A large part of our projects concerns the development of quantitative methods based on fast 2D NMR methods, which makes it possible to record 2D NMR spectra in a fraction of a second to a few minutes.

Key References:

J. Farjon, C. Milande, E. Martineau, S. Akoka, P. Giraudeau, The FAQUIRE Approach: FAst, QUantitative, hIghly Resolved and sEnsitivity Enhanced ¹H, ¹³C Data, Anal. Chem. 90, 1845 (2018)
P. M. Le, C. Milande, E. Martineau, P. Giraudeau, J. Farjon, Quantification of natural products in herbal supplements: A combined NMR approach applied on goldenseal, J. Pharm. Biomed. Anal., 165, 155 (2019).
P. Giraudeau, Quantitative NMR spectroscopy of complex mixtures, Commun. 59, 6627 (2023).
L. Botros, Y. Liu, C. Corbett, D. Sorensen, C. Szabo, A. Bzhelyansky, M. Niemitz, P. Korhonen, G. Pauli, P. Giraudeau, G. J. Ray. Connecting the Practice of Modern Qualitative and Quantitative NMR Analysis with Its Theoretical Foundation. J. Nat. Prod. 88, 877 (2025)
J. Marchand, E. Martineau, J. Farjon, P. Giraudeau. Analytical Comparison of Two Quantitative HSQC Methods for the Absolute Quantitation of Metabolites. Magn. Reson. Chem. 63, 434 (2025)

Supported by: ERC (CoG SUMMIT 814747), ANR (QUANTUM, T-ERC SUMMIT, MetaHyp), MetaboHub, IUF, industrial funding

4. Hyperpolarized NMR for complex mixtures

Current team members: Wiktor Adamski, Benoît Charrier, Yuliia Horbenko, Marine Letertre, Patrick Giraudeau*

Past team members: Arnab Dey, Jean-Nicolas Dumez, Clément Praud, Bertrand Plainchont, Elodie Lesquin, Victor Ribay

Collaborations (past and present): Geoffrey Bodenhausen (ENS Paris), Catherine Deborde and Annick Moing (INRAe Bordeaux), Lucio Frydman (Weizmann Institute, Rehovot), Sami Jannin (Univ. Lyon), James Kempf, Dmitry Eshchenko and Roberto Melzi (Bruker Biospin)

First d-DNP-enhanced 13C NMR analysis of a biofluid -urine- at natural abundance, offering unprecedented resolution an sensitivity for this challenging type of sample

Hyperpolarization methods can boost the sensitivity of NMR by several orders of magnitude. In particular, dissolution dynamic nuclear polarization (D-DNP) can yield sensitivity improvements by 4 to 5 orders of magnitude in liquid-state NMR. While D-DNP has been widely applied to in vivo situations, its application to analytical chemistry, and in particular to the analysis of complex mixtures, remains limited. We are exploring the potential of D-DNP in this context, with the aim to improve its applicability to metabolomics.

Thanks to a careful optimization of a D-DNP prototype installed at CEISAM in 2019 (in collaboration with Bruker Biospin and Universite de Lyon), we recently demonstrated that D-DNP assisted by cross-polarization can provide major sensitivity enhancements in samples as complex as extracts or biofluids (urine), with a repeatability of a few percent. We also showed that D-DNP can be used in untargeted metabolomics workflows or for targeted quantitative analysis (when combined with standard additions). The prototype instrument was replaced at the end of 2025 by a commercial Dyamis d-DNP instrument (cryogen-consumption free and automated), whose potential for the analysis of complex mixtures is currently being explored.

Key References:

A. Dey, B. Charrier, E. Martineau, C. Deborde, E. Gandriau, A. Moing, D. Jacob, D. Eschenko, M. Schnell, R. Melzi, D. Kurzbach, M. Ceillier, Q. Chappuis, S.F. Cousin, J.G. Kempf, S. Jannin, J.-N. Dumez, and P. Giraudeau, Hyperpolarized NMR Metabolomics at Natural ¹³C Abundance, Anal. Chem. 92, 14867 (2020)
A. Dey, B. Charrier, K. Lemaitre, V. Ribay, D. Eshchenko, M. Schnell, R. Melzi, Q. Stern, S. F. Cousin, J. G. Kempf, S. Jannin, J. N. Dumez, P. Giraudeau, Fine optimization of a dissolution dynamic nuclear polarization experimental setting for 13C NMR of metabolic samples, Magn. Reson., 3, 183 (2022).
V. Ribay, A. Dey, B. Charrier, C. Praud, J. Mandral, J.-N. Dumez, M. P. M. Letertre, P. Giraudeau, Hyperpolarized 13C NMR Spectroscopy of Urine Samples at Natural Abundance by Quantitative Dissolution Dynamic Nuclear Polarization, 62, e202302110 (2023).
C. Praud, V. Ribay, A. Dey, B. Charrier, J. Mandral, J. Farjon, J.-N. Dumez, P. Giraudeau, Optimization of heteronuclear ultrafast 2D NMR for the study of complex mixtures hyperpolarized by dynamic nuclear polarization, Anal Methods, 2023, 15, 6209 (2023).
A. Dey, B. Charrier, V. Ribay, J.-N. Dumez, P. Giraudeau, Hyperpolarized 1H and 13C NMR Spectroscopy in a Single Experiment for Metabolomics., Anal. Chem, 2023, 95, 16861 (2023)

Supported by: ERC (CoG SUMMIT 814747), ANR (T-ERC SUMMIT, MetaHyp), IUF, MetaboHub, PRC CNRS-MOST

5. High-resolution NMR methodology for complex mixture analysis

Current team members: Wiktor Adamski, Yuliia Horbenko, Armand Régheasse, Lamia Grib, Yaroslava Niderer, Serge Akoka,* Jonathan Farjon,* Aurélie Bernard,* Patrick Giraudeau.*

Research axis led by Jean-Nicolas Dumez until 2025

Past team members: Margherita Bazzoni, Marion André, Maria Grazia Concilio, Boris Gouilleux, Ludmilla Guduff, Adrien Le Guennec, Arnab Dey, Ludmilla Guduff, Ghanem Hamdoun , Corentin Jacquemmoz, Célia Lhoste, Benjamin Lorandel, Achille Marchand, Rituraj Mishra, Clément Praud, Bertrand Plainchont, Laetitia Rouger.

Collaborations (past and present): Daniel Abergel (ENS, Paris), Mohammed Boujtita (Univ. Nantes), Luiz Keng Queiroz and Antonio Gilberto Ferreira (Univ. Sao Carlos), François-Xavier Felpin (Nantes Université), Lucio Frydman (Weizmann Institute), Vincent Gandon (Université Paris-Saclay), Gaspard Huber (CEA, Saclay), Sami Jannin (Université de Lyon), Dennis Kurzbach (University of Vienna), Géraldine Masson (ICSN, Univ. Paris-Saclay), Anna Parker (NVision Imaging), Damien Jeannerat (Université de Genève), Ilya Kuprov (University of Southampton), Vincent Sarou-Kanian and Franck Fayon (CNRS Orléans), Kerstin Münnemann (Kaiserslautern), Ralph Adams, Matthias Nilsson and Gareth Morris (Manchester Univ.)

Spatial parallelisation accelerates 2D NMR by up to three orders of magnitude

The potential of NMR spectroscopy can be greatly enhanced by introducing concepts and methods that exploit spatial (in addition to spectral) dimensions, often building on magnetic resonance imaging (MRI) knowledge and tools. Spatial parallelisation strategies notably yield 2D NMR spectra in a single-scan, and provide a powerful route to the acquisition of ultrahigh resolution ¹H spectra. It can also accelerate the acquisition of ultraselective experiments, that target ¹H multiplets that overlap with other multiplets. We develop novel strategies for the spatial encoding of NMR interactions and other physical properties, such as translational diffusion coefficients.

These developments jointly rely on the experimental, theoretical and numerical characterisation of the underlying spin dynamics. Specifically, we develop pulse sequence elements for spatial encoding (SPEN) (of, e.g., chemical shifts or diffusion coefficients), and integrate them into accelerated experiments for mixture analysis, with the goals of broadening the range of systems that can be analysed, and increasing the information content of NMR spectra. These basic ingredients are then used in all of our mixture analysis projects.

Special focus is brought to the analysis of mixtures that evolve in time, such as the monitoring of organic and catalytic chemical reactions or the sensitive detection of components in hyperpolarized samples. We develop fast multidimensional NMR methods that are compatible with the analysis of time-evolving samples, with three complementary objectives: i/ reducing the duration needed to collect a complete ND data set (using notably single-scan “ultrafast” 2D NMR; ii/ optimising the frequency with which a time-evolving system can be sampled (with polarisation saving methods and original hardware); iii/ making each measurement more robust against sources of errors, such as convection of flow. The resulting methods open new perspective for mechanistic studies in chemical synthesis and metabolomics applications.

Recent references:

B. Plainchont, P. Giraudeau and J.-N. Dumez, Interleaved spatial/spectral encoding in ultrafast 2D NMR spectroscopy, J. Magn. Reson.305, 112 (2019).
J-N. Dumez, Frequency-swept pulses for ultrafast spatially encoded NMR, J. Magn. Reson. 323, 106817 (2020).
C. Jacquemmoz, F. Giraud and J.-N. Dumez, Online reaction monitoring by single-scan 2D NMR under flow conditions, Analyst145, 478 (2020).
R. Mishra, A. Marchand, C. Jacquemmoz, and J.-N. Dumez, Ultrafast diffusion-based unmixing of ¹H NMR spectra, Chem. Commun. 57, 2384 (2021).
K. Singh, C. Jacquemmoz, P. Giraudeau, L. Frydman, and J.-N. Dumez, Ultrafast 2D ¹H–¹H NMR spectroscopy of DNP-hyperpolarised substrates for the analysis of mixtures, Chem. Commun. 57, 8035 (2021).
R. Mishra and J.-N. Dumez, Quadratic spacing of the effective gradient area for spatially encoded diffusion NMR, J. Magn. Reson. 334, 107114 (2022).
J. Marchand, R. Mishra, A. Bernard and J.-N. Dumez, Online Reaction Monitoring with Fast and Flow-Compatible Diffusion NMR Spectroscopy, Chem. Eur. J. e202201175 (2022).
C. Jacquemmoz, R. Mishra, L. Guduff, C. van Heijenoort, and J.-N. Dumez, Optimisation of spatially-encoded diffusion-ordered NMR spectroscopy for the analysis of mixtures, Magn Reson. Chem. 60, 121 (2022).
C. Lhoste, B. Lorandel, C. Praud, A. Marchand, R. Mishra, A. Dey, A. Bernard, J.-N. Dumez, P. Giraudeau, Ultrafast 2D NMR for the analysis of complex mixtures, Prog. Nucl. Magn. Reson. Spectrosc. 130, 1 (2022).
R. Mishra and J.-N. Dumez, Theoretical analysis of flow effects in spatially encoded diffusion NMR, J. Chem. Phys. 158, 014204 (2023)
B. Lorandel, R. Mishra, O. Cazimajou, A. Marchand, A. Bernard, and J.-N. Dumez, Accounting for gradient non-uniformity in spatially-encoded diffusion-ordered NMR spectroscopy, J. Magn. Reson. 355, 207543 (2023)
M. Bazzoni, R. Mishra, and J.-N. Dumez, Single-Scan Ultraselective NMR Experiments with Preserved Sensitivity, Angew. Chem. Intl. Ed. e202314598 (2023)
C. Lhoste, M. Bazzoni, J. Bonnet, A. Bernard, F.-X. Felpin, P. Giraudeau, and J.-N. Dumez, Broadband ultrafast 2D NMR spectroscopy for online monitoring in continuous flow, Analyst 148, 5255 (2023).
C. Praud, V. Ribay, B. Charrier, J. Mandral, J. Farjon, J.-N. Dumez, and P. Giraudeau, Optimization of heteronuclear ultrafast 2D NMR for the study of complex mixtures hyperpolarized by dynamic nuclear polarization, Anal. Methods 15, 6209 (2023).
A. J. Parker, A. Dey, M. U. Qureshi, J. M. Steiner, J. W. Blanchard, J. Scheuer, N. Tomek, S. Knecht, F. Josten, C. Müller, P. Hautle, I. Schwartz, P.Giraudeau, T. R. Eichhorn, and J.-N. Dumez, Solution-State 2D NMR Spectroscopy of Mixtures Hyperpolarized Using Optically Polarized Crystals, Angew. Chem. Intl. Ed., z202312302 (2023)
B. Lorandel, H. Rocha, O. Cazimajou, R. Mishra, A. Bernard, P. Bowyer, M. Nilsson, J.-N. Dumez. Speedy component resolution using spatially encoded diffusion NMR data. Magn. Reson. Chem., 63(1):49–61, 2024.
J. Mandral, J. Phuong, J. Farjon, P. Giraudeau, K. Münnemann, J.-N. Dumez. Ultrafast 2D benchtop NMR spectroscopy enhanced by flow Overhauser dynamic nuclear polarization. J. Magn. Reson. Open., 23:100195, 2025.
M. Bazzoni, A. Régheasse, E. Caytan, F.-X. Felpin, P. Giraudeau, A. Bernard, R. Adams, G. Morris, M. Nilsson, J.-N. Dumez. Pure-Shift NMR in Continuous Flow. Chemistry - A European Journal, 31(1):e202403385, 2025.
Y. Horbenko, M. Jaudronnet, N. El Sabbagh, M. Bazzoni, A. Bernard, M.Nilsson, P. Giraudeau, F.-X. Felpin, J.-N. Dumez. Clean 1H NMR Spectra of Products Directly from Batch and Flow Reaction Mixtures. ChemistryEurope, 3(3):e202500038, 2025.
J. Mandral, S. Roques, J.-N. Dumez, P. Giraudeau, J. Farjon. Evaluation of pure shift NMR methods for the analysis of complex metabolite mixtures with a benchtop NMR spectrometer. Anal. Methods, 17(16):3171–3182, 2025.

Supported by: Agence Nationale de la Recherche (PRC DigitalChem, PRC SOFTNMR, PRC ChemAI, PRC MEtaHyp), Région Pays de la Loire (Connect Talent HPNMR), Royal Society, and European Research Council (STG DINAMIX, CoG SUMMIT, CoG UNMIX)

6. Multiple peak suppression methods