Comprehensive Targeted Metabolomic Assay for Urine Analysis.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Chemistry

Background:

• Urine is a valuable biological fluid for disease biomarker discovery due to its noninvasive collection and chemical complexity.
• Comprehensive metabolomic analysis of urine is less studied compared to blood, with existing assays often optimized for serum or plasma.
• There is a need for robust, quantitative assays for high-throughput urine metabolomics.

Purpose of the Study:

• To develop and validate a comprehensive, quantitative mass spectrometry-based assay for urine analysis.
• To enable robust detection and quantification of a wide range of urinary metabolites.
• To facilitate high-throughput analysis of human urine samples for biomarker discovery.

Main Methods:

• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) in both positive and negative modes using multiple reaction monitoring (MRM).
• Targeted metabolomic assay designed for 96-well plate format enabling automated, high-throughput sample analysis.
• Validation using spiked urine samples with recovery rates of 80-120% and precision <20%.

Main Results:

• The assay robustly detects and quantifies 142 urinary metabolites, including amino acids, organic acids, biogenic amines, acylcarnitines, lipids, and glucose/hexose.
• 67 metabolites were absolutely quantified and 75 were semiquantified.
• Successful application to over 1800 human urine samples, with results consistent with literature and orthogonal NMR spectroscopy analysis.

Conclusions:

• The developed LC-MS/MS assay provides a comprehensive and quantitative approach for urine metabolomics.
• The assay's high-throughput capability and robustness make it suitable for large-scale biomarker discovery studies.
• This method enhances the utility of urine as a biological fluid for clinical chemistry and disease research.