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Metabolomics Sample Pre-treatment Methods

Metabolomics Sample Pre-treatment Methods

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The choice of sample pre-treatment strategy contributes to the success of a given experiment as it affects the quality of the observed metabolite profiles and data. The capabilities and limitations of sample preparation methods in a given study can affect the accuracy of biological interpretation.

Pre-treatment of metabolomics samples must meet the following conditions: 1) non-selective; 2) few and rapid steps; 3) reproducible; 4) include a metabolic termination process. Currently, a single pre-treatment does not meet all of these requirements.

One of the most paradoxical needs from the point of view of biomarker discovery is metabolic termination, including samples from cells, plants and tissues. The aim of metabolic termination is to stop the metabolic process using low temperature, addition of acid or rapid heating. However, metabolic processes are very fast and often have time scales of less than 1 s, such as ATP, 6-phospho-glucose and adenosine. It may be very difficult to implement an appropriate termination step within the appropriate time scale, and adding a termination step may result in unintentional degradation or loss of certain metabolites.

For stable metabolites, metabolic termination is of little significance, but for unstable metabolites that are easily degraded or converted, the step of metabolic termination is important. Although the proportion of unstable metabolites is low, it is also possible that these low proportions of metabolites are precisely the important biomarkers. Therefore, determining which metabolites are not affected by different termination/storage conditions has an important role in the maturation of the field of metabolomics and biomarker discovery.

Different metabolomics studies have different requirements for sample pre-processing. In discovery-oriented untargeted metabolomics studies, the fewer pre-treatment steps a biological sample undergoes before analysis, the better, in order to detect as many metabolites as possible in the sample. Therefore, researchers usually use non-selective pre-treatment methods such as protein precipitation with organic solvents (e.g. serum or plasma sample pre-treatment), sample dilution (e.g. urine sample pre-treatment). In targeted metabolomics studies, sample pretreatment is followed by liquid-liquid extraction or solid-phase extraction to remove matrix interferences after protein precipitation with organic solvents for further separation and enrichment of target metabolites. These operations greatly improve the sensitivity and dynamic range of the assay.

Metabolomics Sample Pre-treatment MethodsMetabolomics workflow (Alseekh et al., 2021).

Pre-treatment methods for biological fluid samples

  • Direct dilution injection

It is generally applied to urine samples, and a typical dilution ratio of 1:10 times with pure water is used.

  • Solvent precipitation

The addition of organic solvents (methanol, acetonitrile, ethanol, acetone or a combination thereof) to biological fluid samples such as serum, plasma, etc. can remove proteins and disrupt the cross-linking between proteins and metabolites, and the resulting metabolite concentration is representative of the total metabolite concentration. The best metabolite coverage is obtained with acetone/methanol mixtures and methanol precipitation.

  • Ultrafiltration

Ultrafiltration is a common method for the preparation and release of biological samples, which are separated based on the molecular mass of the analytes. For example, a 3000 Da membrane can separate small molecular mass metabolites from proteins or other large molecules. Ultrafiltration has a bias toward polar molecules and results in a significant loss of hydrophobic metabolite species compared to solvent precipitation.

  • Solid-phase extraction (SPE)

SPE is widely used for targeted bioanalysis and the steps are as follows: 1. the analyte is adsorbed on the solvent surface; 2. interferents weaker than the analyte adsorption capacity are removed by drenching; 3. the analyte is eluted off using the solvent. Currently, C18 and polystyrene-divinylbenzene adsorbent combinations have been used for non-targeted phase metabolomics studies.

  • Solid phase microextraction (SPME)

In vivo sampling avoids the metabolic profile changes caused by the oxygen, solvent, and pH conditions to which the sample may be exposed during sampling, and the corresponding activation of various biological processes. The SPEM technique provides a good balance between hydrophilic and hydrophobic metabolite extraction. This technique can be applied to the in vivo sampling of circulating blood metabolites in mice.

  • Turbulent flow chromatography (TFC)

The TFC method allows direct injection of untreated serum into the LC-MS. TFC meets the conditions of large particles (25-50um) and small particle size (0.5-1.0um), where large molecules cannot be retained and small molecules can be detected. the potential advantages of TFC are high volume, high automation and minimal sample handling, which can reduce the introduction of foreign contaminants in the sample handling process and Unintentional sample loss.

Metabolomics Sample Pre-treatment MethodsSample-pretreatment techniques for mass spectrometry-based metabolomics (Raterink et al., 2014)


  1. Alseekh, S., Aharoni, A., Brotman, Y., et al. (2021). Mass spectrometry-based metabolomics: A guide for annotation, quantification and best reporting practices. Nature methods, 18(7), 747-756.
  2. Raterink, R. J., Lindenburg, P. W., et al. (2014). Recent developments in sample-pretreatment techniques for mass spectrometry-based metabolomics. TrAC Trends in Analytical Chemistry, 61, 157-167.

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