Increasing Throughput for the Analysis of Human Insulin and Related Biotherapeutic Analogs using the ionKey/MS System

Importance of the Topic

Recombinant human insulin and its analogs represent the standard of care for insulin dependent diabetes and their bioanalysis is crucial for drug development, clinical monitoring and quality control. Microflow LC-MS methods can achieve high sensitivity but often suffer from longer analysis times, creating a need for techniques that balance sensitivity with higher throughput.

Objectives and Study Overview

The primary goal of this study was to enhance the throughput of microflow LC-MS quantification of human insulin and five biotherapeutic analogs while maintaining picogram per milliliter sensitivity. The work demonstrates the performance of the Waters ionKey/MS System coupled with a 300 µm iKey HT Separation Device for plasma-based analysis.

Methodology

A two-dimensional trap-and-elute microflow LC method was employed, including protein precipitation, Oasis MAX µElution SPE cleanup, and chromatographic separation on iKey devices. Calibration curves were prepared over 25–10 000 pg/mL with bovine insulin as the internal standard. A linear gradient (15–55% B) was applied at 6 µL/min with a 7 min total cycle time.

Used Instrumentation

• ACQUITY UPLC M-Class System with trap and back-flush configuration
• Waters ionKey/MS System
• iKey HT Separation Device, BEH C18, 1.7 µm, 300 µm x 50 mm
• Symmetry C18 trap column, 5 µm, 300 µm x 50 mm
• Xevo TQ-XS Tandem Quadrupole Mass Spectrometer

Main Results and Discussion

Calibration curves for insulin and analogs were linear across three orders of magnitude (R2 > 0.99) with LLOQs between 25 and 100 pg/mL and accuracy > 93%. The 300 µm iKey HT method achieved a 7 min run time with only a two-fold sensitivity compromise compared to the ultra-sensitive 150 µm method (13.5 min). Compared with a conventional 2.1 mm UPLC approach, the iKey HT method halved sample injection volume while doubling sensitivity.

Benefits and Practical Applications

• High throughput analysis compatible with routine bioanalytical workflows
• Picogram-level sensitivity for trace quantification of insulin analogs
• Reduced system dispersion and simplified microflow setup
• Flexibility to switch between ultra-sensitive and high-throughput methods

Future Trends and Potential Applications

Advancements in microfluidic LC-MS are expected to support multiplexed peptide quantification, integration with high-resolution mass spectrometry, and further reduction in sample volumes. Automated workflows and expanded iKey designs may enable broader applications in biotherapeutic and biomarker analysis.

Conclusion

The ionKey/MS System with a 300 µm iKey HT Separation Device delivers a robust solution that balances high sensitivity and accelerated throughput for insulin and analog quantification. Its integrated microfluidic design streamlines bioanalytical assays and enables seamless transition between analytical regimes.

Reference

1. E E Chambers et al. Reducing Sample Volume and Increasing Sensitivity for the Quantification of Human Insulin and 5 Analogs in Human Plasma using ionKey/MS. Waters Application Note 720005119EN (2016).
2. M Donegan et al. High Throughput Microflow LC-MS: Sensitivity Gains on a Practical Timescale. Waters White Paper 720005765EN (2016).
3. E E Chambers et al. Multidimensional LC-MS/MS Enables Simultaneous Quantification of Intact Human Insulin and 5 Recombinant Analogs. Analytical Chemistry 86(1):694–702 (2014).
4. Y W Alelyunas et al. High Throughput Analysis at Microscale: Performance of ionKey/MS with Xevo G2-XS QTof Under Rapid Gradient Conditions. J Appl Bioanal 1(4):128–135 (2015).
5. P D Rainville et al. Integration of Microfluidic LC with HRMS for the Analysis of Analytes in Biofluids. Bioanalysis 7(11):1397–1411 (2015).