System suitability testing of LC-IMS-HRMS for metabolomics applications

Significance of the topic

The reliability of large-scale metabolomics studies depends on robust analytical platforms that deliver consistent retention times, accurate masses, characteristic isotope distributions, reproducible MS/MS fragmentation, and stable ion mobility separations. Monitoring instrument performance during extended acquisition sequences is critical to ensure data quality, avoid batch effects, and enable confident compound annotation and quantification across diverse biological sample cohorts.

Objectives and Study Overview

This work presents the development and validation of a System Suitability Test (SST) tailored for liquid chromatography–ion mobility–high-resolution mass spectrometry (LC-IMS-HRMS) workflows in metabolomics. The primary goals were to:

• Design a reference mixture of 18 endogenous, water-soluble, non-hazardous metabolites spanning m/z ~100–550.
• Establish a rapid 5 min chromatographic gradient compatible with high aqueous content reversed-phase separation.
• Implement a targeted parallel reaction monitoring trapped-ion mobility spectrometry (prm-PASEF) acquisition to capture both MS1 and MS/MS data in positive and negative ion modes.
• Evaluate precision and accuracy metrics across 50 injections per ion polarity using automated quality control software.

Methodology and Instrumentation

The SST protocol comprised:

• Reference mixture: 18 isotopically defined metabolites (e.g., adenosine monophosphate, leucine/isoleucine, riboflavin) prepared at defined concentrations in water.
• Chromatography: Agilent 1290 Infinity II LC with an Avantor ACE Excel AQ (2.1 × 100 mm, 2 µm) column at 50 °C; 600 µL/min flow; mobile phases 0.1% formic acid in water (A) and acetonitrile (B); gradient from 99% A to 40% A over 2.5 min and return to 99% A by 5 min.
• Mass spectrometry: Bruker timsTOF Pro 2 with VIP-HESI source; ESI in positive and negative modes; m/z 20–1300 scan range; 1/K0 ion mobility 0.1–1.5; prm-PASEF acquisition in a ~90 min sequence combining both polarities.
• Data processing: TASQ RealTimeQC 2023b employed for automated peak detection, retention time evaluation, mass and mobility accuracy, isotope pattern matching, MS/MS spectral comparison to NIST libraries, and collisional cross section (CCS) verification.

Main Results and Discussion

The SST demonstrated:

• Excellent chromatographic resolution of isomeric leucine and isoleucine within 7 min total runtime.
• Effective mobility separation of co-eluting trisaccharides melezitose and maltotriose.
• Consistent MS1 signal intensities and high-quality MS/MS fragmentation spectra across 10 replicate sequences (50 injections per polarity).
• Stable retention times, mass accuracies within ±5 ppm, CCS reproducibility, and isotope pattern fidelity, exemplified by riboflavin performance metrics plotted over the full batch.

Benefits and Practical Applications

This SST offers a standardized, rapid QC solution that:

• Validates the entire LC-IMS-HRMS setup before and during long sample batches.
• Detects gradual performance drifts or acute hardware issues in advance.
• Consolidates batch setup, data processing, and reporting within a single software tool.
• Facilitates cross-laboratory method transfer and instrument qualification after installation.

Future Trends and Opportunities

Further enhancements may include:

• Integration of chromatographic peak shape and column backpressure monitoring.
• Automated long-term visualization dashboards for trend analysis.
• Extension of SST concepts to alternative instrument platforms and acquisition modes.

Conclusion

The proposed System Suitability Test provides a comprehensive, efficient approach to qualify LC-IMS-HRMS platforms for metabolomics applications, ensuring high data integrity and facilitating routine instrument performance surveillance.