Supercritical Fluid Chromatography MS/MS Method Development for the Analysis of GLP-1 Analogues

Supercritical Fluid Chromatography MS/MS Method Development for the Analysis of GLP‑1 Analogues — Summary

Significance of the topic

Supercritical fluid chromatography (SFC) combines chromatographic selectivity with low solvent consumption and rapid separations, making it attractive for the analysis of complex biotherapeutics. GLP‑1 receptor agonists (e.g., liraglutide and semaglutide) are hybrid molecules with peptide backbones and lipidic modifications that pose chromatographic and ionization challenges. Reliable analytical workflows for these therapeutics are required across pharmaceutical development, quality control, and stability testing. Establishing SFC–MS/MS methods and practical guidance for column and modifier selection can help laboratories adopt faster, greener assays for GLP‑1 analogue quantitation.

Objectives and study overview

The work aimed to demonstrate an SFC–MS/MS approach for GLP‑1 analogues by:

• screening commonly used SFC stationary phases to identify a suitable column for liraglutide and semaglutide;
• optimizing SFC conditions that are compatible with LC–MS (ionization, modifiers, makeup flow);
• demonstrating quantitation and linearity for the two GLP‑1 standards.

Methodology

• Standards and sample prep: Liraglutide and semaglutide standards were obtained commercially and prepared in 100% methanol.
• Analytical strategy: Mass spectrometer source and MRM transitions were optimized first using flow injection analysis (FIA) to maximize signal at the chosen instrument flow conditions; this ensured subsequent chromatographic changes could be interpreted without confounding ionization effects.
• Chromatography: Supercritical CO2 was used as the primary mobile phase with methanol‑based modifiers. A screening campaign evaluated multiple stationary phases; the final quantitative method used a poly(4‑vinylpyridine) (P4VP) DCPak column. Typical conditions included 1.1 mL/min total flow, an analytical gradient from ca. 5% to 85% modifier over ~3–4.5 min, column oven ~68 °C, and a backpressure regulator setting of ~150 bar at 50 °C. A makeup pump delivered methanol with 0.1% formic acid to aid electrospray ionization.
• Mass spectrometry: A triple‑quadrupole instrument operated in MRM mode. Representative precursor ions and transitions for liraglutide and semaglutide were monitored (e.g., liraglutide precursor m/z ~938.8 with product ions at m/z 1128.6, 1064.3; semaglutide precursor m/z ~1029 with associated fragments). Desolvation and gas settings were tuned during FIA prior to chromatographic experiments.

Instrumention used

• Shimadzu Nexera Analytical SFC system.
• Shimadzu LCMS‑8050 triple‑quadrupole mass spectrometer.
• Columns screened included multiple chemistries (C18, phenyl, cyanopropyl, diol, cholesteryl, pyridinyl, aminopropyl, Scepter C18, and a DCPak poly(4‑vinylpyridine) column among others).

Key results and discussion

• Column selection: The poly(4‑vinylpyridine) (P4VP) column delivered the best chromatographic performance for the GLP‑1 analogues tested, providing improved peak shape and retention behavior relative to standard reversed‑phase and several polar bonded phases screened.
• Modifier effects: Methanol‑based modifiers provided a robust baseline performance and were chosen as the primary modifier. Addition of 2‑propanol and small amounts of water altered retention and peak shape in some cases; while mixed modifiers could increase signal intensity for certain transitions, they tended to raise system backpressure significantly (noted ~100 psi increase with IPA additions), so trade‑offs between signal improvement and operability were important.
• MS behavior and method sensitivity: Charge states and MRM transitions observed under SFC–MS conditions were consistent with those previously reported in HPLC–MS workflows. Optimizing source/desolvation parameters by FIA prior to chromatography accelerated method development and ensured reliable signal attribution to chromatographic changes. Quantitation of liraglutide and semaglutide showed good linearity across the tested range with correlation coefficients around R2 ≈ 0.989, demonstrating the potential for quantitative application.
• Practical considerations: The makeup flow of methanol with 0.1% formic acid improved ionization efficiency; operating temperatures and BPR settings were important to maintain adequate supercritical conditions and reproducible retention.

Benefits and practical applications of the method

• Faster analysis times and reduced organic solvent use compared with conventional HPLC methods, supporting higher throughput and improved sustainability.
• Compatibility with triple‑quadrupole MS in MRM mode enables targeted quantitation suitable for QC and method development workflows for peptide therapeutics.
• Column screening approach and MS‑first optimization strategy provide a practical roadmap for laboratories adopting SFC for peptide‑like molecules.

Future trends and potential uses

• Wider adoption of SFC for peptide and peptide–lipid conjugates as instrument availability and method guidance increase.
• Development of more systematic stationary phase selection guidelines for peptide analytes to reduce empirical screening burden.
• Integration with high‑resolution MS for structural characterization, impurity profiling, and stability‑indicating assays.
• Method validation and transfer studies to support regulatory use in QC environments, including robustness against modifier composition and pressure variations.

Conclusion

This study demonstrates that SFC coupled to triple‑quadrupole MS can be a viable alternative to conventional LC–MS for the analysis and quantitation of GLP‑1 analogues. A poly(4‑vinylpyridine) stationary phase provided superior chromatographic performance for liraglutide and semaglutide under the tested conditions. Methanol‑based modifier systems, combined with careful source optimization via FIA and methanol/formic acid makeup flow, enabled reproducible MRM quantitation with good linearity. The approach highlights SFC’s potential for faster, greener, LC–MS‑compatible assays for peptide therapeutics.

References

• Shimadzu Scientific Instruments. Technical overview: General overview of SFC. Shimadzu internal technical material (web resource used by authors), accessed by authors in 2026.
• Shimadzu Scientific Instruments. Peptide therapeutics application notes and product materials (company application resources), accessed by authors in 2026.
• West C., Schad G. Supercritical Fluid Chromatography for Dummies. 1st ed., May 2025.

Note: The original work and authors are affiliated with Shimadzu Corporation; the presented applications are intended for research use only and not for diagnostic procedures.