Breaking the Sensitivity Barrier of NMR Spectroscopy

Complex mixtures are a fundamental part of the analytical chemistry field, including applications in medical fields, pharmaceutical development, environmental analysis, or food sciences.^[i]
Understanding complex mixtures, and providing unambiguous structural and quantitative information, has been described as a “pivotal challenge in modern sciences”.^[ii]

In the field of metabolomics – which involves the quantitative analysis of biological samples to better understand metabolic processes – accurate quantification is particularly important because of the growing clinical need to improve understanding of metabolic diseases.

“Metabolomics is an area of analytical chemistry with one of the highest mixture complexities,” says Prof. Dr. Patrick Giraudeau, Professor of Analytical Chemistry at Nantes Université. “Whereas untargeted methods aim at capturing the broadest possible snapshot of metabolites to identify potential biomarkers of a particular phenotype, targeted approaches focus on the quantification of known metabolites, for instance, to validate a biomarker or to obtain further insight into metabolic pathways.”

For analytical chemists, accurate quantitative determination of mixture components is one of the biggest challenges, requiring the determination of often heavily overlapped signals from compounds in very diverse concentrations. “In synthetic chemistry, molecular structures can be extremely similar, leading to the overlapping of signals,” Prof. Dr. Giraudeau explains, “But biological samples can be even more complex, because you may want to detect analytes at low levels or quantify molecules in micromolar concentrations. Here, we are dealing with a very high dynamic range.”

The area of metabolomics – and the analysis of complex mixtures in particular – is driven by technology, developing alongside new instrumentation and analytical methods. There is no singular methodology that is capable of comprehensive quantification of the metabolites in a sample; however many analytical chemists, such as Prof. Dr. Giraudeau, develop and use a range of technologies to maximize the sensitivity and throughput of their analyses.

Introducing MIMM

The MIMM team focuses on the analysis of complex mixtures, with a special focus on quantitative analysis.

"We study complex mixtures with a two-fold and complementary approach,” Prof. Dr. Giraudeau states. “Some of my colleagues are experimenting with the development of new methods that may be useful for certain applications, and there are others who are focused on applications in metabolomics and reaction monitoring – but there is interaction between these two areas.”

Formed originally to measure variations in isotopic abundance by NMR spectroscopy, the team has since developed a whole portfolio of highly accurate quantitative NMR methodology. “Our aim is essentially to take existing concepts and try to make them applicable to real life samples,” Prof. Dr. Giraudeau says. “This requires extensive and tailored optimization to get the precision that’s needed in every instance.”

NMR: a powerful quantitative tool

Along with gas chromatography or liquid chromatography coupled with mass spectrometry (GC-MS or LC-MS), NMR spectroscopy is now recognized as a key technique in metabolomic analysis, allowing accurate molecular identification, elucidation, and quantification. ^vi

NMR is robust, stable, and highly reproducible and, unlike LC-MS, is non-destructive and requires minimal sample preparation.ⁱⁱⁱ As a quantitative method, it also allows both relative and absolute metabolite concentrations to be obtained, and creates distinct spectra, making it a powerful tool for detecting novel biomarkers.

One limitation of NMR, however, is its low sensitivity compared to chromatographic methods. “Sensitivity is crucial for the analysis of the complex mixtures we are working with,” says Prof. Dr. Giraudeau. “Of course, we can increase sensitivity by relying on a higher magnetic field, or by using cryoprobes to reduce noise in the NMR signal, but these are only likely to enhance sensitivity by a factor of 3 or 4.”

Shifting paradigms

Three NMR techniques more recently developed by the team offer increased analytical capabilities for complex mixture analysis: quantitative 2D NMR, ultrafast 2D NMR and dissolution-dynamic nuclear polarization (d-DNP) NMR.

"Quantitative 2D NMR has been shown to be highly effective in the analysis of complex samples, particularly in pharmaceutical applications,” says Prof. Dr. Giraudeau. “For example, with the anticoagulant heparin, 2D NMR was found to enable the most efficient separation and quantification of contaminated samples – something that proton nuclear magnetic resonance (1H NMR) struggled to do as effectively.” ^{vii . viii}

In the MIMM lab, one of the challenges the team faces is the strong overlapping of signals. “What we don’t want to do is to physically separate signals as in chromatography. 2D NMR gives the ability to resolve peaks that overlap in 1D spectra, while providing both structural and quantitative information,” Prof. Dr. Giraudeau says.

As Prof. Dr. Giraudeau’s research has highlighted, ^ix data acquisition time can be a limitation in the analysis of complex mixtures. “We can analyze several hundreds of samples at a time. With traditional multi-dimensional methods, obtaining the spectra can take up to a few hours – a challenge when there could be ongoing chemical reactions,” he explains. The team develops and uses ultrafast 2D NMR for high throughput analysis. “We needed to develop methods that were quick enough to monitor in real time, and ultrafast 2D NMR is the most versatile approach for complex molecule analysis,” he continues.

The technique that has had the biggest impact on Prof. Dr. Giraudeau’s research, however, is d-DNP NMR. “DNP creates hyperpolarized nuclear spin states – allowing us to produce enhanced NMR signals for detection, and ultimately to detect and quantify minute metabolite concentrations in biological samples,” Prof. Dr. Giraudeau says.

d-DNP NMR provides a unique way to detect ¹³C NMR signals with a sensitivity enhanced by several orders of magnitude,^x in vivo metabolic studies using DNP reported a sensitivity increase of >10,000.^xi

“d-DNP is not new – it was invented by Ardenkjaer-Larsen and colleagues 20 years ago – but I believe we are the first to explore its potential in NMR metabolomics, especially at natural abundance,” Prof. Dr. Giraudeau says. “We are seeing results that no one has seen before – it’s a real change of paradigm.”^xii 

Inter-field collaboration

The methodology being developed in the MIMM team has helped the team form collaborations within the metabolomics community. For the last two years, the team has been a member of MetaboHUB, a French research infrastructure that focuses on the development and application of metabolomics, which includes groups working in applied science, health, nutrition, and plant sciences.

Prof. Dr. Giraudeau says that this is highly beneficial to all involved. “We really appreciate these mutual interactions. They allow us access to new challenges in their respective fields, and we apply our NMR-based methods to the different kinds of biological questions these groups seek to answer.”

New biofuels – the future of sustainable energy?
The team also has a long-standing collaboration with the GEPEA Institute in Saint-Nazaire, France, which specializes in bioprocesses and the potential of biofuel development from microalgae. “The team there is seeking to understand the metabolism of microalgae, as it can produce a very broad chemical diversity and has many applications. It can be used in biofuels, to develop a future sustainable energy source, as well as to treat water, boost the immune system, and provide nutrition for humans,” Prof. Dr. Giraudeau explains. The MIMM team has been working closely with the GEPEA to improve analytical techniques.

“When microalgae are subject to nitrogen starvation, they start to develop lipids, and our colleagues at GEPEA wanted to find a way to monitor their lipid metabolism in real-time – something which is not easy to do with conventional techniques such as chromatography,” he explains. The MIMM team has used benchtop NMR technology to monitor the metabolism of microalgae. “We have already done a proof-of-concept in our team with a small photobioreactor, but now we are running tests on a large photobioreactor with a capacity of hundreds of liters.”

The GEPEA and MIMM teams have now also obtained funding from the European Space Agency to work on the development of food supplements for astronauts. “In some ways, the work we’re doing feels a long way from the traditional chemical analysis that NMR was originally used for,” he says. “But whether in biology, the medical field, for food specialists or for environmental experts, complex mixtures is our common ground. NMR opens up many possibilities for us to explore.”

d-DNP NMR: the clinical possibilities

Prof. Dr. Giraudeau is excited about the broad potential of d-DNP NMR in the pre-clinical arena. MIMM will soon purchase new instrumentation that will be dedicated to support those in medical research. “Although our focus is not on clinical application, we have begun a collaboration with medical doctors, with the goal of making our methodologies available to those in the pre-clinical research field,” he adds.

He also believes that NMR has vast potential in the field of precision medicine. “In contrast to mass spectrometry, for example, NMR is highly stable and reproducible, allowing for the same results on different instruments at different sites. Not only does that make NMR unique, but it also means we can, for instance, follow the metabolomic signature of a patient over weeks, months, and even years – and here the implications for personalized medicine are huge.”

Prof. Dr. Giraudeau says he became convinced of NMR’s potential when he was a Masters student, and he is now determined to have a broader impact. “At the beginning of my career, I was really just experimenting with different methods, but now I'm trying to make them applicable to real life, where we can really make a difference. “This is made possible by the people in the team, the funding we have received, and our collaboration with Bruker, which provided the prototype d-DNP machine that allowed us to drive our research forward.”

Prof. Dr. Patrick Giraudeau,
Full Professor of Analytical Chemistry at Nantes Université and Head of the Magnetic Resonance, Isotopomics, Metabolomics and Monitoring (MIMM) research team.

Prof. Dr. Giraudeau leads the MIMM research team and the NMR platform of the CEISAM research institute, part of the Corsaire multi-site metabolomics platform and the MetaboHUB metabolomics and fluxomics infrastructure. His teaching disciplines include NMR spectroscopy and other spectroscopic methods, and his research specialism is NMR methodological developments for quantitative analysis. He is Vice President of the Ampere Society, a member of the Euromar Board of Trustees, and Associate Editor of Magnetic Resonance in Chemistry and Magnetic Resonance.

About the MIMM Research team, Nantes Université

The MIMM team comprises 25 people with complementary expertise in analytical and physical chemistry, particularly in the fields of solution-state NMR spectroscopy and isotope analysis at natural abundance, and their application to a broad variety of fields such as metabolism, authentication, forensics, and reaction monitoring. With numerous major grants in recent years, the MIMM team has become an international leading research team in the fast and high precision quantitative NMR of complex mixtures and its applications to metabolomics, isotopomics and monitoring.

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