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.
- I take it the theory is to use LLE. Are all metabolites methanol-soluble? What isn’t methanol-soluble in plasma?
- 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?