A method for comparative metabolomics in urine using high resolution mass spectrometry.

Area of Science:

• Analytical Chemistry
• Metabolomics
• Clinical Chemistry

Background:

• Metabolite profiling of biological fluids is crucial for accurate data extraction.
• Standardized workflows are needed for reliable metabolomic analysis.

Purpose of the Study:

• To develop and validate a comprehensive workflow for urine metabolite profiling.
• To assess the utility of the workflow in differentiating between smokers and nonsmokers.

Main Methods:

• Urine samples from smokers and nonsmokers analyzed using UHPLC-HRMS.
• Employed both Reversed Phase Chromatography (RPC) and Hydrophilic Interaction Liquid Chromatography (HILIC) with positive and negative ionization modes.
• Utilized spiked standards for data normalization and pooled quality controls (QCs) for reproducibility assessment.

Main Results:

• Identified a significant number of unique metabolites across four analytical modes (RPC/HILIC, positive/negative ionization).
• Statistical analysis demonstrated clear clustering between smokers and nonsmokers.
• Variable Importance in Projection (VIP) scores highlighted key differentiating metabolites.

Conclusions:

• The developed UHPLC-HRMS workflow provides a reproducible and effective method for urine metabolomic profiling.
• This workflow is applicable for comparative metabolomic studies in various disease models, advancing clinical metabolomics.
• The method successfully distinguished metabolic profiles between smokers and nonsmokers.