Ion mobility separation of recombinant and synthetic insulin variants on the Cyclic IMS mass spectrometer

Importance of the Topic

Accurate differentiation of synthetic insulin analogs from human insulin is essential in anti-doping control and forensic investigations. Due to nearly identical amino acid sequences and molecular masses, conventional chromatographic and mass spectrometric techniques struggle to resolve these isobaric peptides. Cyclic ion mobility–mass spectrometry (IM–MS) promises higher separation power based on collisional cross-section differences, offering rapid and reliable analysis of prohibited insulin variants.

Study Objectives and Overview

This study evaluates the performance of a cyclic ion mobility QToF mass spectrometer in separating recombinant human insulin and three synthetic analogs (lispro, glulisine, aspart). The goals were to determine whether IM alone, or combined with liquid chromatography (LC), could resolve isobaric species at low concentration levels relevant to anti-doping screening.

Methodology and Instrumentation

All experiments were performed on a Cyclic IMS QToF platform. Direct infusion used gold-coated capillaries, and LC–MS employed an ACQUITY UPLC system with CSH C18 peptide column. Low-flow analyses utilized an IonKey source coupled to UPLC M-Class. IM separation was optimized on the +5 charge state by varying the number of passes (5 to 9) around the cyclic device racetrack. High-energy fragmentation scans (HDMSe) were recorded throughout acquisitions to capture precursor and fragment information.

Main Results and Discussion

– Direct infusion IM–MS achieved clear drift time separation of human insulin and lispro, despite identical precursor m/z, with lispro exhibiting shorter drift time.
– Introducing LC ahead of MS did not resolve recHum/lispro by retention time alone, but subsequent IM separation distinguished these isobars within a single LC–IM–MS run.
– In serum extracts spiked with all four analogs at 1.25–5 ng/mL, the combination of LC, IM, and high-resolution MS enabled unambiguous identification of each peptide based on unique m/z, drift time, or both.
– The cyclic IM device’s enhanced resolution surpasses previous instruments, supporting confident detection of minute insulin lispro levels in complex matrices.

Benefits and Practical Applications

• Rapid separation of isobaric insulin analogs without extended chromatographic gradients or tandem MS workflows.
• Improved confidence in identifying illicit insulin use in sports, clinical, and forensic laboratories.
• Integration of drift time, retention time, precursor m/z, and fragment m/z in a single 15-minute protocol enhances throughput and analytical robustness.

Future Trends and Opportunities

Further work will focus on lowering detection limits to 0.5 ng/mL or below, expanding cyclic IM–MS applications to other peptide hormones, and integrating machine-learning tools within software platforms for automated variant assignment. Continued improvements in cyclic device design may yield higher mobility resolution and shorter analysis times.

Conclusion

Cyclic ion mobility separation combined with high-resolution MS offers a powerful strategy to resolve isobaric insulin analogs in anti-doping and forensic contexts. By eliminating the need for long gradients and complex MS/MS methods, this approach enhances efficiency and analytical certainty for routine testing.

Reference

• Thomas A, Schänzer W, Thevis M. Determination of human insulin and its analogues in human blood using liquid chromatography–ion mobility–mass spectrometry. Drug Test Anal. 2014;6(11–12):1125–1132.