Compatibility study for metabolomics: Combined sample preparation for LC/MS and NMR

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  • Published: Jul 23, 2012
  • Author: Steve Down
  • Channels: Sample Preparation
thumbnail image: Compatibility study for metabolomics: Combined sample preparation for LC/MS and NMR

Mating metabolomics techniques

A comprehensive study of the extraction of liver tissue for metabolomics analysis by NMR and LC/MS has shown that one solvent system is suitable for both techniques, with solvent choice the most important factor.


The two principal methods for studying the metabolome involve the complementary techniques of NMR and mass spectrometry. Between them, they reveal a far broader range of metabolites than either technique alone but this advantage is not one that scientists generally take advantage of. Most metabolomics studies involve one or the other.

There are three key reasons for this apparent deficiency. Firstly, lab personnel are usually trained in only one of the technologies, so extra resources are required. However, the main restraint is the difference in the sample preparation protocols and the amount of sample that is required for successful analysis.

NMR is often carried out directly on biofluids without any preliminary extraction. However, its inherent lack of sensitivity compared with mass spectrometry often results in overlapping signals for complex mixtures of metabolites, hindering the identification and measurement of individual components. This situation can be improved by a preliminary extraction procedure.

For LC/MS, which is gaining in popularity compared with GC/MS, far smaller sample volumes are adequate (typically 20 µL compared with 500 µL for NMR) but extraction of the sample is a prerequisite in order to remove proteins and other interfering components.

The third factor which makes it difficult to integrate the two technologies is the use of deuterated solvents in NMR spectroscopy. They are employed to simplify the proton regions of the spectra, since any hydrogen atoms that are replaced by deuterium are shifted to a different region of the spectrum. However, the use of deuterated solvents in mass spectrometry will cause problems. The substitution of labile hydrogen atoms with deuterium can prevent the correct structural assignment.

It seems that an opportunity is being missed when NMR and LC/MS are used independently for metabolomics studies, so a team of scientists in Spain has attempted to simplify the procedures by systematically examining extraction protocols for both techniques to try and find common ground. Their aim was to find conditions for preparing one extract which can be used directly for NMR and LC/MS without solvent exchange or any other manipulations.

Solvent studies

The study was carried out by Antoni Beltran, Oscar Yanes and colleagues from the Rovira i Virgili University, Tarragona, the Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM), Barcelona, the Pere Virgili Health Research Institute (IISPV), Reus, and the Technological Center of Nutrition and Health (CTNS), Reus. They experimented with the homogenised liver of a single rat, varying the solvent polarity and temperature to give 12 different protocols.

The four solvent combinations were 50% aqueous methanol, 50% aqueous acetonitrile, methanol-chloroform-water (70:20:10%) and acetonitrile-chloroform-water (70:20:10%), each applied at -20, 25 and 60°C. The extracts were redissolved in a mixture of deuterated acetonitrile and deuterated water for the analyses.  

Both NMR and high-resolution magic angle spinning NMR spectra were acquired, along with mass spectra in positive- and negative-ion modes on a quadrupole-time-of-flight instrument, which is capable of handling the large amounts of data produced.

Compatibility of NMR and LC/MS

The NMR data were subjected to principal components analysis, which revealed that most of the variance in the results was due to the composition of the solvent, rather than the temperature.

The best extraction for complementary NMR and LC/MS experiments was the methanol-chloroform-water mixture followed by 50% aqueous methanol. They provided the most efficient extraction yields while giving the broadest range of metabolites between the two techniques, with the least repetition.

Methanol was more efficient than acetonitrile for precipitating proteins from the liver extract, demonstrated by the reduced number and abundances of multiply charged species in the mass spectra.

However, dissolution of the extracts in deuterated solvents did not lead to the complications that were anticipated from the exchange of hydrogen and deuterium atoms in the metabolites. The researchers attributed this to the rapid back exchange of deuterium in the presence of water in the HPLC mobile phase. That means that the final extract can be used for LC/MS and NMR. Good results were obtained from both techniques by the use of one solvent system.

The complementary nature of the techniques was illustrated by the detection of metabolites such as glycogen, cholesterol, glucose, ascorbic acid, pyruvic acid and lactic acid by NMR. These substances are difficult to detect by LC/MS in an untargeted analysis.

Given the proven compatibility of the two techniques, the team remarked that “exploration of compatible extraction procedures for both NMR and MS-based analyses opens up new opportunities to expand coverage of the metabolome by implementing multiplatform approaches based on NMR and MS.” However, they acknowledge that the optimum extraction procedure has yet to be found.

Related Links

Analytical Chemistry 2012, 84, 5838-5844: "Assessment of compatibility between extraction methods for NMR- and LC/MS-based metabolomics"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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