Targeted Metabolomics Using the UPLC/MS-based AbsoluteIDQ p180 Kit

Importance of the Topic

Targeted metabolomics provides a focused approach to quantifying predefined sets of metabolites with high sensitivity and throughput, overcoming limitations of untargeted profiling such as extensive data processing and low identification rates. By applying a validated assay for over 180 endogenous compounds, researchers can rapidly detect biochemical alterations and identify biomarkers in diverse fields including toxicology, drug development, and clinical diagnostics.

Objectives and Overview of the Study

This study demonstrates the use of the commercially available AbsoluteIDQ p180 Kit in combination with Waters ACQUITY UPLC and Xevo TQ/TQ-S tandem quadrupole mass spectrometers to measure and quantify more than 180 endogenous metabolites in mouse serum. The primary goal was to characterize metabolic changes induced by 8 Gy gamma radiation exposure and to evaluate the workflow’s robustness, reproducibility, and suitability for biomarker discovery.

Methodology

Male C57Bl/6 mice were exposed to a single 8 Gy dose of gamma radiation and serum samples collected 24 hours later. Metabolites were extracted using the p180 Kit’s 96-well plate format, which integrates protein removal, derivatization of amino acids and biogenic amines with phenylisothiocyanate, and internal standard normalization. Lipophilic compounds (acylcarnitines, phosphatidylcholines, sphingomyelins, and hexoses) were analyzed by flow injection analysis (FIA-MS/MS), while polar analytes (amino acids and biogenic amines) were separated by UPLC and quantified by multiple reaction monitoring (MRM). Data capture and quantification were automated via the MetIDQ software and Waters TargetLynx application.

Used Instrumentation

• Waters ACQUITY UPLC System with BEH C18 VanGuard pre-column and BEH C18 analytical column
• Agilent Zorbax Eclipse XDB C18 HPLC column (3.0 × 100 mm, 3.5 μm)
• Waters Xevo TQ and Xevo TQ-S tandem quadrupole mass spectrometers
• Flow injection analysis module for FIA-MS/MS experiments
• Nitrogen evaporator for sample drying and derivatization

Main Results and Discussion

The optimized UPLC-based method achieved a total run time of 4.35 minutes at 0.9 mL/min, compared to 7.3 minutes for the HPLC approach at 0.5 mL/min. Principal component analysis clearly separated irradiated and control groups, confirming distinct metabolic signatures. Significant alterations were observed in amino acids (notably arginine and serine), lysophosphatidylcholines, phosphatidylcholines, and several acylcarnitines. Quality-control samples demonstrated median CVs below 15% across metabolite classes, and all measurements above the limit of detection were included in statistical analyses.

Benefits and Practical Applications

By standardizing sample preparation and data processing in a high-throughput format, the AbsoluteIDQ p180 Kit enables reproducible quantification of a broad metabolite panel. Its integration with robust UPLC/MS instrumentation supports rapid turnaround, minimal inter-laboratory variation, and straightforward biomarker discovery workflows. This approach is particularly valuable for radiation biodosimetry, clinical metabolomics studies, and regulatory safety assessment.

Future Trends and Applications

Advancements may include expanded targeted panels covering additional metabolic pathways, integration of ion mobility separation for isomer resolution, and coupling with high-resolution mass spectrometry for enhanced specificity. Improved software pipelines will further automate data curation, multivariate analysis, and pathway mapping, facilitating real-time decision making in translational research and precision medicine.

Conclusion

The combination of the AbsoluteIDQ p180 Kit with Waters UPLC and Xevo TQ/TQ-S instruments delivers a fast, sensitive, and reproducible workflow for targeted metabolomics. This platform effectively measures a comprehensive set of endogenous metabolites, supports robust biomarker identification, and is readily adaptable to diverse applications in biological and clinical research.

References

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