RPLC- and HILIC-based non-targeted metabolomics workflow for blood microsamples

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.