Extending the Interval between Full-Range Mass Calibrations on Orbitrap Exploris Mass Spectrometers

Importance of the Topic

The accuracy of mass measurements in high-resolution Orbitrap instruments underpins reliable identification and quantification in proteomics, biopharma quality control and small molecule analysis. Over time, thermal and mechanical drifts can exceed the correction range of built-in lock-mass features, requiring full-range calibrations that interrupt workflows and reduce instrument availability.

Objectives and Study Overview

This work presents the development and validation of a streamlined 1-Point Mass Calibration for the Thermo Scientific Orbitrap Exploris family. The goal was to extend full-range calibration intervals beyond one month, minimize downtime, and complement the existing EASY-IC lock-mass correction. Calibration performance was assessed over weeks to months in Multi-Attribute Method (MAM) protein digests and in targeted small molecule assays for sulfonamides in complex matrices.

Methodology

Twice weekly over nine weeks, full-range mass calibrations were performed with standard FlexMix and AHFP solutions on an Orbitrap Exploris MX and on an Exploris 240. A dedicated 1-Point procedure was implemented in the Instrument Control Software: ionization in the analytical source is switched off, the integrated EASY-IC source is activated and conditioned to generate fluoranthene radical ions, and a single mass point is acquired in positive and negative modes. The resulting offset is merged with existing calibration curves and applied to all subsequent runs until the next calibration. Long-term drift was monitored by tracking mass deviations of nine BSA digest peptides, seven NIST mAb digest peptides, and 15 sulfonamides over periods up to 14 weeks. Data processing used Thermo Chromeleon CDS, TraceFinder and Origin for statistical evaluation.

Instrumentation

• Orbitrap Exploris MX with BioPharma option
• Orbitrap Exploris 240 with FAIMS Pro Duo
• Thermo Scientific Pierce FlexMix™ and AHFP calibration solutions

Main Results and Discussion

Without altering the analytical setup, the 1-Point Mass Calibration reduced long-term drifts of more than 25 ppm to residual mass errors within ±1.3 ppm over 14 weeks. In MAM workflows, mass deviations for BSA and mAb peptides remained tightly clustered and below 1 ppm RMS after calibration, even following instrument maintenance events. In the sulfonamide assay, pooled mass errors stayed within ±3 ppm for 14 days, matching expectations for the built-in EASY-IC correction. Daily execution of the 1-Point routine reset drift and maintained the EASY-IC offset near zero, preserving accurate extracted-ion chromatograms in aged calibration scenarios.

Benefits and Practical Applications

• Extends full-range calibration validity to at least two months, reducing maintenance frequency
• Requires no modification of existing analytical methods or hardware
• Minimizes instrument downtime by completing in under one minute
• Ensures consistent mass accuracy for MAM QC and targeted small molecule assays
• Complements EASY-IC and lock-mass approaches for drifts beyond ±15 ppm

Future Trends and Possibilities

Scheduling the 1-Point Mass Calibration at defined intervals or embedding it in automated system performance tests could further streamline QC workflows. Integration with remote monitoring and AI-driven calibration recommendations may enable predictive maintenance. Extending this approach to other Orbitrap platforms and combining it with advanced lock-mass schemes promises even longer calibration intervals and enhanced robustness in diverse analytical applications.

Conclusion

The 1-Point Mass Calibration strategy effectively maintains sub-ppm mass accuracy over extended periods without disrupting analytical methods. By leveraging the integrated EASY-IC source and merging a single calibration point into existing curves, laboratories can reduce full-range calibration frequency, increase instrument uptime and ensure reliable results in proteomics and small molecule QC.