Improving Sensitivity of GLP-1 Analogues Quantitation Using Multiple Spray ESI Technology on a Microflow LC-MS/MS

Importance of the Topic

Glucagon-like peptide-1 (GLP-1) receptor agonists are key biotherapeutics for type 2 diabetes and obesity management. Their large size poses ionization and carryover challenges in LC-MS/MS analysis.

Objectives and Study Overview

This study aims to enhance quantitation sensitivity for semaglutide and liraglutide by implementing multiple-spray electrospray ionization (ESI) technology on a microflow LC-MS/MS platform, comparing performance to conventional single-nozzle systems.

Methodology and Instrumentation

A trap-and-elute microflow LC system (Shimadzu Nexera Mikros) was coupled to a Shimadzu LCMS-8060 triple quadrupole mass spectrometer. Mobile phases were water and acetonitrile with 0.1% formic acid. A C8 trap column (0.3×5 mm, 5 µm) and C8 analytical column (0.2×50 mm, 3 µm) ran gradients at a 5 µL/min elution flow. A Newomics DuoESI source with M3 multi-nozzle emitter provided microflow ionization. Source parameters—voltage, probe position, gas flows, and temperatures—were optimized for maximal sensitivity.

Main Results and Discussion

Optimal precursors for both peptides were the [M+4H]4+ charge states at m/z 1029.30 (semaglutide) and 938.75 (liraglutide). Multiple-spray ESI yielded a 4–8× increase in peak area versus single-nozzle ionization. Systematic tuning of interface voltage, nebulizer gas, and temperature showed signal improvements of 10–200% per parameter. Trap-and-elute with internal/external rinsing minimized carryover to below 0.1% at 200 ng/mL while maintaining throughput.

Benefits and Practical Applications

This workflow enables robust, high-sensitivity quantitation of large therapeutic peptides on existing microflow LC-MS/MS setups, supporting routine analysis in drug development and quality control laboratories.

Future Trends and Opportunities

Further improvements may involve adapting multiple-spray emitters for broader peptide classes, integrating high-throughput autosamplers, and combining with high-resolution MS for enhanced structural characterization.

Conclusion

Employing multiple-spray ESI on a microflow LC-MS/MS platform significantly boosts detection sensitivity and reduces carryover for GLP-1 analogues, demonstrating compatibility with current mass spectrometers and potential for widespread bioanalytical applications.

Instrumentation Used

• Shimadzu Nexera Mikros trap-and-elute microflow LC
• Newomics DuoESI source with M3 multi-nozzle emitter
• Shimadzu LCMS-8060 triple quadrupole MS
• CERI L-column2 C8 trap column (0.3×5 mm, 5 µm) and analytical column (0.2×50 mm, 3 µm)