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The blood in our bodies comprises three major components: blood plasma, formed elements, and the extracellular matrix. Blood plasma is a yellowish fluid that constitutes 55% of the total blood volume. It is primarily made up of water and essential substances such as electrolytes and proteins. Blood plasma serves as a medium for transporting blood cells and also contains nutrients, enzymes, hormones, antibodies, and gases.
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RPLC- and HILIC-based non-targeted metabolomics workflow for blood microsamples.

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Summary

Blood microsampling (BμS) offers a less invasive alternative for metabolomics. Optimized extraction and LC-MS methods enhance the detection of diverse metabolites from dried blood spots and volumetric absorptive microsampling, improving coverage for various metabolic pathways.

Keywords:
Blood microsamplingDBSHILIC-MSNon-targeted metabolomicsRPLC-MSVAMS

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Metabolomics

Background:

  • Blood microsampling (BμS) is a minimally invasive technique for analyzing endogenous metabolites.
  • Dried blood spots (DBS) and volumetric absorptive microsampling (VAMS) are key BμS formats.
  • BμS is suitable for metabolomics, offering an alternative to traditional blood and plasma sampling.

Purpose of the Study:

  • To develop and optimize an extraction protocol for BμS for non-targeted metabolomics.
  • To establish robust reversed-phase liquid chromatography/mass spectrometry (RPLC-MS) and hydrophilic interaction chromatography/mass spectrometry (HILIC-MS) methods.
  • To enhance metabolite coverage for both mid- to non-polar and polar compounds.

Main Methods:

  • Developed and optimized two new HILIC-MS/MS methods for polar metabolites.
  • Utilized an in-house RPLC-MS method for mid- to non-polar metabolites.
  • Investigated five extraction procedures and optimized the workflow for DBS and VAMS.

Main Results:

  • A 15-minute HILIC-MS method with column re-equilibration was successfully developed.
  • A 20% H2O/80% MeOH (v/v) mixture with sample rehydration proved effective for metabolite extraction.
  • The methodology successfully detected metabolites across various pathways, including amino acids, acylcarnitines, and bile acids.

Conclusions:

  • The developed extraction and LC-MS methodology provides comprehensive metabolite profiling from BμS.
  • This approach enhances metabolite coverage and is applicable to both DBS and VAMS.
  • The optimized protocol facilitates non-targeted metabolomics analysis using minimally invasive blood samples.