High-resolution Quantitative Metabolomics with biocrates’ AbsoluteIDQ® p400 HR kit on Thermo Scientific Orbitrap Exploris Hybrid Mass Spectrometry Platforms

Importance of the Topic

Metabolomics offers a detailed view of small-molecule and lipid profiles that reflect biological states and disease processes. Standardized, high-throughput workflows are essential to ensure reproducibility, inter-laboratory comparability, and accurate quantitative results in research, clinical diagnostics, and quality control settings.

Objectives and Overview of the Study

This work evaluates the performance of the biocrates AbsoluteIDQ® p400 HR kit on Thermo Scientific Orbitrap Exploris series mass spectrometers (OE120, OE240, OE480). Key goals include optimizing the kit protocol for accurate quantitation of 43 small molecules and 365 lipids, assessing reproducibility, detectability, and accuracy against NIST SRM 1950 reference material and pooled human plasma samples.

Methods and Instrumentation

Sample preparation followed the manufacturer’s protocol for calibration standards, three levels of plasma-based quality controls, and NIST SRM 1950. Analytical measurements combined liquid chromatography (LC) and flow injection analysis (FIA) on a Thermo Vanquish UHPLC system coupled to Orbitrap Exploris instruments.
Data acquisition:

• FIA-MS in full scan mode for lipids
• LC-MS in scheduled full scan and PRM modes for small molecules

Data processing employed WebIDQ software with downstream analysis in Excel and R.

Main Results and Discussion

Reproducibility:

• Intra-instrument median CVs: 9.3% (OE120, OE240), 7.6% (OE480)
• Inter-instrument median CV: 9.7%, with average CV < 15% for all detected metabolites

Detectability:

• Male and female plasma: 230–250 metabolites above LOD per instrument
• NIST SRM 1950: 209 metabolites on OE120/240, 224 on OE480

Accuracy:

• Measured concentrations of amino acids and biogenic amines differed by < 6% from NIST SRM 1950 reference values

Correlation analysis across platforms demonstrated strong concordance for all compound classes.

Benefits and Practical Application

The p400 HR kit provides:

• Standardized sample preparation with built-in calibration and QC materials
• Comprehensive coverage of key metabolite and lipid classes
• Robust quantitative performance across multiple Orbitrap Exploris platforms
• Rapid implementation in multi-instrument laboratories

Future Trends and Potential Applications

Expanding targeted metabolomics to include additional biomarkers and biofluids, integration with automated sample handling, and cross-platform harmonization through international ring trials will further strengthen data comparability and accelerate adoption in clinical and industrial environments.

Conclusion

The AbsoluteIDQ® p400 HR kit, when deployed on Orbitrap Exploris mass spectrometers, delivers reproducible, accurate, and high-coverage quantitative metabolomics data, supporting reliable biomarker discovery and routine laboratory workflows.

Reference

• Phinney KW, et al. Development of a Standard Reference Material for metabolomics research. Anal Chem. 2013;85(24):11732–11738.
• Thompson JW, et al. International Ring Trial of a High Resolution Targeted Metabolomics and Lipidomics Platform for Serum and Plasma Analysis. Anal Chem. 2019;91(22):14407–14416.