The Quantification of Glucagon-like Peptide Receptor Agonists (GLP-1 RAs) in Plasma Extracts Using Capillary UHPLC-MS/MS

Importance of the topic

The ongoing clinical success of GLP‑1 and dual GIP/GLP‑1 receptor agonists for obesity and metabolic disease has driven demand for highly sensitive bioanalytical assays. These peptides have long plasma half‑lives and reach sub‑ng/mL concentrations in the elimination phase, necessitating methods capable of reliable quantification at pg/mL levels to support pharmacokinetics, DMPK, and safety studies. Improving sensitivity while retaining throughput, robustness and manageable solvent consumption is therefore a critical analytical challenge.

Objectives and study overview

This application note evaluates whether capillary‑scale UHPLC (300 µm i.d.) coupled with tandem quadrupole MS in MRM mode can (i) improve detection sensitivity for GLP‑1 receptor agonists (liraglutide, exenatide, semaglutide, tirzepatide), (ii) maintain chromatographic performance and robustness suitable for routine bioanalysis, and (iii) enable practical sample loading strategies (large volume injections) without resorting to complex nanospray sources. The work demonstrates sensitivity gains, limits of detection, chromatographic metrics and multi‑hundred injection robustness for human plasma extracts prepared by SPE or protein precipitation.

Methodology and sample preparation

• Analytes: liraglutide, exenatide, semaglutide, tirzepatide; calibration ranges included 20 pg/mL to 1000 pg/mL (semaglutide, tirzepatide), and higher ranges for liraglutide/exenatide.
• Sample prep: two workflows were evaluated—µElution SPE (50 µL plasma; methanol protein precipitation followed by Oasis MAX µElution and elution with water:ACN + 5% formic acid; final dilution 1:1 with 0.1% plasma in water) and simple protein precipitation (50 µL plasma + 100 µL ACN, centrifuge, dilute supernatant 1:1).
• Chromatography: capillary ACQUITY peptide CSH C18 (300 µm × 50 mm, 1.7 µm, 300 Å) at 65 °C, flow 15 µL/min, mobile phases 0.1% FA (A) and 0.1% FA in ACN (B). Gradient from 20% to 80% B (either 3 or 10 min), with large volume loading enabled by a short Symmetry C18 trap (300 µm × 20–25 mm, 5 µm) on a 6‑port valve arrangement.
• Injection/loading: trapping strategy allowed 5–10 µL sample loads (10 µL loading at 20 µL/min for 2 min), avoiding overloading of the analytical capillary column (approx. 1.8 µL column volume).
• Mass spectrometry: Xevo TQ‑XS triple quadrupole with microflow probe in positive ESI; unit mass resolution; capillary voltage 3.0 kV; cone voltage 32 V; cone gas 50 L/h; desolvation gas 300 L/h at 600 °C. MRM transitions monitored included semaglutide (1029.2 → 1238.1) and tirzepatide (1204.2 → 396.2), among others.

Used instrumentation

• ACQUITY UPLC M‑Class (capillary UHPLC) equipped with microflow electrospray probe and trapping valve manager.
• Xevo TQ‑XS triple quadrupole mass spectrometer (microflow probe).
• Analytical column: ACQUITY Premier Peptide CSH C18, 300 µm × 50 mm, 1.7 µm, 300 Å (capillary scale).
• Trapping column: Symmetry C18 trap, 300 µm × 20–25 mm, 5 µm porous silica.
• Consumables: QuanRecovery vials with MaxPeak HPS surfaces for low adsorption; fused silica transfer capillaries for valve/trap connections.
• Software: waters_connect for acquisition and MS Quan for data processing; waters_connect for Quantitation for 21 CFR Part 11 compliance and workflow automation.

Main results and discussion

• Sensitivity gains: capillary (300 µm) separations increased peak responses by ~8–14× versus matched 2.1 mm × 50 mm UHPLC for 1 µL injections (example: semaglutide peak intensity rose from 2.5e4 to 1.96e5; tirzepatide from 8e3 to 1.24e5).
• Limits of detection: with the in‑line trapping approach and 10 µL loads, a lower limit of detection (LLD) of 20 pg/mL was demonstrated for tirzepatide in SPE extracts; protein precipitation extracts gave LLD ~50 pg/mL for tirzepatide. Dynamic ranges extended from low‑pg/mL to high‑pg/ng levels depending on analyte and prep.
• Chromatography: peak widths 6–7.5 s (base) and calculated peak capacities of ~82–99 for 10‑min separations—comparable or slightly improved over 2.1 mm scale. Peptides eluted in the order semaglutide, liraglutide, exenatide, tirzepatide under the tested gradient.
• Robustness and throughput: the capillary method delivered robust performance across >500 injections (five batches of 100 plasma extract replicates), with no significant column backpressure increases and acceptable reproducibility. %CV across 500 injections ranged from ~4.7% (tirzepatide) to 20.1% (liraglutide), reflecting analyte‑dependent variability but overall suitability for routine analytics.
• Operational advantages: the microflow method reduced solvent consumption ~26‑fold relative to conventional 2.1 mm UHPLC (15 µL/min vs 400 µL/min), reducing cost and waste, while avoiding the complexity of nanospray LC‑MS workflows.

Benefits and practical applications

• Substantial sensitivity improvement (up to ~10×) without the delicate instrumentation and optimization required for nanospray LC‑MS/MS.
• Enables reliable quantification of long‑acting peptide therapeutics at low pg/mL levels to support late‑phase PK characterization and DMPK studies.
• Compatibility with routine laboratory throughput and robustness demands (hundreds of injections) and straightforward trapping strategies for large sample volumes.
• Lower solvent consumption and reduced operational costs, beneficial for high-throughput bioanalysis and sustainability.
• Seamless integration with regulated quantitation software (waters_connect) facilitates compliant data handling and workflow scalability.

Future trends and potential applications

• Wider adoption of microflow/capillary LC‑MS for bioanalysis of increasingly potent biologics, oligonucleotides and low‑dose small molecules where pg/mL sensitivity is required.
• Further integration of automated trapping/loading strategies and valve managers to couple large volume sample handling with narrow‑bore columns for high sensitivity without sample cleanup penalties.
• Combining capillary LC‑MS with enhanced data processing, laboratory automation, and miniaturized sample prep (micro‑SPE, DBS, microsampling) to enable sparse sampling in clinical studies and preclinical micro‑sampling workflows.
• Potential method refinement through optimized ion source designs for microflow, improved column stationary phases tailored for peptides, and standardized robustness studies to support regulatory submissions.

Conclusion

Capillary UHPLC (300 µm i.d.) coupled to triple quadrupole MS with an in‑line trapping approach provides a pragmatic route to achieve up to tenfold improvements in sensitivity for GLP‑1 receptor agonist bioanalysis. The approach demonstrated low‑pg/mL detection for tirzepatide, robust performance across >500 injections, and substantial solvent savings while avoiding the operational complexity of nanospray systems. This workflow is well suited to contemporary DMPK and clinical studies requiring high sensitivity, routine throughput and regulatory compliance.

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