High-throughput extraction and quantification method for targeted metabolomics in murine tissues.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Tissue metabolomics offers insights into disease mechanisms, complementing body fluid analyses.
• Challenges in tissue sample handling, homogenization, and metabolite extraction hinder accurate quantification.
• Direct tissue analysis is advantageous for understanding disease pathology.

Purpose of the Study:

• To establish an optimized metabolite extraction protocol for targeted metabolomics using the AbsoluteIDQ™ p180 Kit.
• The protocol aims for non-selectivity, broad applicability to various tissues and amounts, and high throughput.
• Key requirements include reproducibility and monophasic solvent systems.

Main Methods:

• Quantified metabolites in eleven murine tissue types.
• Evaluated three extraction solvents: methanol, phosphate buffer, and an ethanol/phosphate buffer mixture.
• Assessed extraction yield, ionization efficiency, and reproducibility at two tissue-to-solvent ratios.

Main Results:

• Methanol and ethanol/phosphate buffer demonstrated superior extraction yield, reproducibility, and ionization efficiency compared to phosphate buffer for most metabolites.
• Phosphate buffer excelled for amino acids and biogenic amines but was suboptimal for lipids.
• Tissue extract matrix effects were comparable to or smaller than those observed in human plasma.

Conclusions:

• Developed optimized, high-throughput metabolite extraction protocols for each murine tissue type.
• The protocols maximize extraction, reproducibility, and quantification for the p180 kit.
• The developed extraction methods are adaptable for other targeted metabolomics assays beyond the p180 kit.