High-resolution quantitative metabolomics with the biocrates AbsoluteIDQ® p400 HR kit on Orbitrap Exploris mass spectrometers

Significance of the Topic

The development of standardized workflows in targeted metabolomics is essential to overcome variability in sample preparation, data acquisition, and inter-laboratory comparability. High-resolution quantitative profiling of small molecules and lipids supports deeper insights into cellular processes and disease biomarkers, making reliable analytical kits a cornerstone for academic and industrial research.

Objectives and Study Overview

This evaluation focused on the biocrates AbsoluteIDQ p400 HR kit applied to Thermo Scientific Orbitrap Exploris mass spectrometers (Exploris 120, 240, and 480). The goals were to assess:

• Detectability and coverage of targeted metabolites across diverse plasma samples.
• Precision and reproducibility within and across instrument platforms.
• Accuracy relative to NIST SRM 1950 reference values.

Methodology and Instrumentation

The biocrates AbsoluteIDQ p400 HR kit provides a 96-well filter plate preloaded with internal standards and includes:

• Seven lyophilized calibration levels for quantitative calibration.
• Three plasma-based quality control (QC) levels, each run in quintuplicate for precision assessment.
• A zero sample (PBS) for limits of detection determination.

Sample types comprised pooled male, female, lipemic plasmas (15 replicates each) and NIST SRM 1950 (3 replicates). Analytical workflows combined liquid chromatography and flow injection analysis on a Thermo Scientific Vanquish UHPLC coupled to Orbitrap Exploris 120, 240, and 480 instruments. Data processing and normalization were conducted using the biocrates WebIDQ workflow manager, leveraging 123 preconfigured Metabolism Indicators.

Main Results and Discussion

Detectability across all sample groups averaged 230–250 metabolites above the limit of detection out of 408 targeted analytes, with slightly higher coverage on the Exploris 480 platform. Class-specific coverage remained consistent for amino acids, biogenic amines, lipids, and sugars.

Precision and reproducibility were evaluated by coefficient of variation (CV):

• Intra-instrument median CVs were 9.3% on Exploris 120 and 240, and 7.6% on Exploris 480 across QC levels.
• Inter-instrument median CV was 9.7% for all QC levels combined, with most analytes below 30% CV.

Inter-laboratory correlation of averaged plasma concentrations demonstrated strong linearity across platforms. Accuracy assessments against NIST SRM 1950 reference values showed deviations below 6% for amino acids and biogenic amines, confirming quantitative reliability.

Benefits and Practical Applications

The AbsoluteIDQ p400 HR kit offers:

• Streamlined sample preparation with preloaded standards.
• Robust quantitative results across multiple high-resolution MS platforms.
• Simplified data processing via WebIDQ and built-in metabolism indicators.
• Enhanced comparability for biomarker discovery and clinical research.

Future Trends and Opportunities

Advancements and potential applications include:

• Extension of targeted panels to cover emerging metabolites and lipid subclasses.
• Integration with proteomics and genomics for multi-omics workflows.
• Automation of sample handling and real-time data quality feedback.
• Machine learning-driven data interpretation for clinical diagnostics and personalized medicine.

Conclusion

The biocrates AbsoluteIDQ p400 HR kit was successfully adapted to Thermo Scientific Orbitrap Exploris series, delivering high detectability, precise quantitation, and strong inter-instrument reproducibility. This standardized solution supports consistent metabolomic profiling across laboratories and accelerates research in systems biology and translational studies.

References

• 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.
• Phinney KW et al. Development of a Standard Reference Material for Metabolomics Research. Anal Chem. 2013;85(24):11732–11738.