I want to do a metabolite study using GC- without derivatization and UPLC-MS (RP and NP) on samples of plasma and urine. My hypothesis is agnostic about which metabolite will correlate with the disease picture that I study. So I want to separate as many compounds as possible to see what correlates with the disease. There is a step called ‘metabolite extraction’ in metabolite identification literature. I have questions about it.

It seems in plasma for example, some authors say there is exactly one step in metabolite extraction, often involving a so-called ‘extraction solvent’. For example this one: https://youtu.be/MSZXsNBpWf8 [Video summary: (1) The scientist says that ‘the extraction protocol for all sample types are the same’. (2) The scientist simply adds a so-called extraction solvent to sample aliquots and collected the supernatant after cooled centrifugation]. Here is a reference on so-called extraction solvents in the context of so-called metabolomics:

  • Zeki, Ö. C., Eylem, C. C., Reçber, T., Kır, S., & Nemutlu, E. (2020). Integration of GC–MS and LC–MS for untargeted metabolomics profiling. Journal of Pharmaceutical and Biomedical Analysis, 190, 113509.

In contrast, I can see some authors want to study a single compound type, for example phospholipids, organic acids or sterols, and they all use different protocols to do extraction (and many solvents). Even within the same type of compound, the authors use very different protocols. For example the sample preparation-sections of:

  • McDonald, J. G., Smith, D. D., Stiles, A. R., & Russell, D. W. (2012). A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma. Journal of lipid research, 53(7), 1399-1409.
  • Amaral, C., Gallardo, E., Rodrigues, R., Leite, R. P., Quelhas, D., Tomaz, C., & Cardoso, M. L. (2010). Quantitative analysis of five sterols in amniotic fluid by GC–MS: Application to the diagnosis of cholesterol biosynthesis defects. Journal of Chromatography B, 878(23), 2130-2136.
  • Castillo-Peinado, L. S., López-Bascón, M. A., Mena-Bravo, A., de Castro, M. L., & Priego-Capote, F. (2019). Determination of primary fatty acid amides in different biological fluids by LC–MS/MS in MRM mode with synthetic deuterated standards: Influence of biofluid matrix on sample preparation. Talanta, 193, 29-36.


  1. I take it the theory is to use LLE. Are all metabolites methanol-soluble? What isn’t methanol-soluble in plasma?
  2. If it is within your time budget to explain from a high level of abstraction why some of these protocols are elaborate, it would be appreciated. I worry that I won’t be extracting the maximal number of metabolites. If it's too elaborate, can you suggest targeted literature?
  • $\begingroup$ Can you provide a summary of the relevant content of the youtube video? Or provide a reference that describes the extraction solvent for metabolites? The main question is probably one of solubility. The metabolites might have a rather high aqueous solubility compared to some lipids. Some sterols are very insoluble and present in aggregates (LDL fraction etc), therefore the need for more nonpolar solvents. $\endgroup$
    – Buck Thorn
    Mar 9, 2022 at 12:11

1 Answer 1


It is not generally true to say that one solvent can extract all metabolites from a fluid such as blood or urine, this would be an over-generalization. In a urine sample, there are thousands of known compounds, each with varying polarities and solubilities, same with blood. Extraction with a solvent, say 50% acetonitrile (ACN) in water, may capture a large number of metabolites, but it won't capture all of them. When you spin blood down you get a number of layers which is evidence of the broad diversity of matrix components in blood. Another challenge may be solvent miscibility. For instance, methanol is quite miscible in water so LLE is difficult because you cannot separate the methanol from water, therefore you'd have to use a less miscible solvent such as hexane or chloroform.

Metabolomics sample preparation is often overlooked as a means to an end, rather than an important experimental parameter that can provide different insights into the same sample with differential extraction. Sometimes an easy sample preparation such as chloroform is sufficient for metabolomics, but other times the matrix is too complex following extraction, or there is a loss of metabolites or low extraction yield, so it is necessary to optimize the sample preparation, often adding complexity.

I'll also add that GC has a number of sample introduction techniques. I assume you want to do a liquid injection, but approaches such as headspace and SPME can be very insightful.


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