Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS Metabolomics

Area of Science:

• Metabolomics
• Analytical Chemistry
• Biochemistry

Background:

• Untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics requires optimized sample preparation.
• Selection of appropriate injection concentration is critical but often undescribed for new sample matrices.

Purpose of the Study:

• To develop and validate a workflow for determining optimal injection concentrations for untargeted LC-MS metabolomics.
• To address the challenge of sample preparation for novel matrices, specifically pig adipose and liver tissues.

Main Methods:

• Bi-phasic extraction was employed for simultaneous lipid and polar metabolite analysis.
• Serial dilutions of pooled samples were analyzed by LC-MS to assess signal linearity.
• Optimal concentrations were determined by evaluating reproducible and linear features.

Main Results:

• Optimum lipid fraction concentrations: 125 mg/mL (liver) and 7.81 mg/mL (adipose).
• Optimum polar fraction concentration: 250 mg/mL (both tissues).
• Deviations from optimal concentrations led to signal overloading, poor linearity, or reduced feature reproducibility.

Conclusions:

• A simple, matrix-agnostic workflow for optimizing injection concentration in untargeted LC-MS metabolomics was established.
• Properly determining injection concentration is critical for robust and reproducible metabolomic profiling.
• The workflow is applicable across various matrices and LC-MS systems.