Selective and sensitive quantitation of 18 steroids in human serum using Stellar mass spectrometer

Importance of the Topic

The accurate measurement of steroid hormones in human serum is critical for clinical diagnostics, endocrinology research, and pharmaceutical development. Due to their low concentrations and similar structures, steroids require highly sensitive and selective analytical techniques to avoid interference and ensure reliable biomarker quantification.

Objectives and Study Overview

This study aims to demonstrate a robust method for the selective and sensitive quantification of 18 serum steroids using a hybrid quadrupole–linear ion trap mass spectrometer (Stellar MS). Key goals include:

• Comparing multiple fragmentation modes (HCD, CID, MS3)
• Optimizing collision energies for each steroid
• Establishing limits of quantification (LOQ) and method reproducibility
• Implementing fast scan scheduling for high throughput

Methodology

Sample Preparation:

• Protein precipitation of 160 µL serum with cold acetonitrile and acetic acid
• Reconstitution in 100 µL 50% methanol; 5 µL injection volume

Chromatographic Separation:

• Kinetex C18 column (2.1 x 150 mm, 2.6 µm) at 50 °C
• 16-minute gradient from 50% to 100% methanol with 0.05 mM NH4F modifier

Data Acquisition and Processing:

• Stellar MS operated in positive and negative polarity with fast scan rates
• Optimization of MS2 collision energies via ramped CE and TraceFinder group function
• Evaluation of potential MS3 transitions for additional selectivity
• Scheduled acquisition windows (5 min per analyte) with ~115 total scans per cycle
• Calibration over four orders of magnitude; LOQs determined by 1/x weighting (R² > 0.99)

Instrumentation Used

• Thermo Scientific Vanquish Horizon UHPLC system
• Thermo Scientific Stellar mass spectrometer (quadrupole, dual-pressure linear ion trap)
• OptaMax Plus ion source
• Kinetex C18 column
• Thermo Scientific Xcalibur 4.7 and TraceFinder 5.2 software

Key Results and Discussion

The hybrid approach enabled selection of the optimal scan mode for each steroid to maximize sensitivity and specificity:

• MS2-HCD provided QqQ-like performance for many analytes
• MS2-CID and MS3 fragmentation improved signal-to-background for challenging targets such as dihydrotestosterone (DHT)
• LOQs ranged from 0.05 to 2 pg on-column, with inter-day precision (%RSD) below 20%
• Fast scan speed supported simultaneous optimization of 18 analytes plus 16 internal standards within a single run

The DHT example highlighted a significant reduction in background using MS3 qualifier ions, enhancing quantitation accuracy at sub-pg levels.

Practical Benefits and Applications

This method offers:

• Improved specificity in complex serum matrices
• High-throughput capability via scheduled multiple scan modes
• Streamlined method development using automated CE optimization
• Robust quantitative performance suitable for clinical research and QA/QC workflows

Future Trends and Opportunities

Potential developments include:

• Shorter retention-time windows to increase analyte throughput
• Expansion to additional steroid panels and biological matrices
• Integration of advanced data analytics and machine learning for automated peak identification
• Further miniaturization and ambient ionization strategies to reduce sample volume requirements

Conclusion

The Stellar MS platform, combined with optimized HCD, CID, and MS3 modes, enables sensitive and selective quantification of 18 serum steroids. Fast scanning and targeted scheduling deliver robust performance, making this workflow well-suited for high-throughput clinical and research applications.

References

• Remes P. M., et al. J. Proteome Res. 2024, 5476.