Automated and Assisted Solid-Phase Extraction (SPE) Sample Preparation for Bioanalysis of GLP-1 Analogs from Plasma

Importance of the topic

Quantitative bioanalysis of GLP-1 analogs is essential for drug development, pharmacokinetics, safety assessment, and expanding therapeutic indications (obesity, cardiovascular disease, neurodegeneration). These peptides are chemically modified to increase in vivo stability, which increases lipophilicity and non-specific binding (NSB) and complicates extraction from plasma. Reliable, high-throughput sample preparation that minimizes peptide loss and matrix effects is therefore critical to produce accurate, reproducible LC-MS/MS data for PK and clinical studies.

Objectives and overview of the study

This application study evaluated a rapid, two-step solid-phase extraction (SPE) workflow for simultaneous extraction and quantification of five GLP-1 analogs (semaglutide, exenatide, liraglutide, lixisenatide, tirzepatide) from rat plasma. Goals included maximizing recovery and cleanliness, minimizing NSB and matrix effects, demonstrating linearity and sensitivity across a practical range (5–1000 ng/mL), and comparing fully automated (Andrew+ Robot) and assisted semi-automated (Pipette+ device) workflows versus manual preparation in terms of reproducibility, throughput, and traceability.

Methodology

• Sample preparation: 200 µL rat plasma spiked with analytes; protein precipitation with 200 µL cold, acidified acetonitrile; centrifugation; supernatant diluted with 400 µL acidified water.
• SPE cleanup: Oasis PRiME HLB µElution 96-well plates used in a two-step elution scheme (two 25 µL aliquots of 3% NH4OH in 75% acetonitrile). µElution format removed the need for evaporation and reconstitution, limiting peptide loss.
• NSB mitigation: QuanRecovery collection plates and MaxPeak High Performance Surfaces (HPS) were incorporated to reduce adsorption to plastics and metal surfaces; Protein LoBind tubes were used for stock and intermediate storage.
• Automation comparison: Three workflows were tested—fully manual, assisted (Pipette+ with Extraction+), and fully automated (Andrew+ Robot with Extraction+). OneLab software provided scripted, auditable workflows for the automated and assisted approaches.
• LC-MS/MS analysis: Direct injection (no reconstitution) onto ACQUITY Premier LC coupled to a Xevo TQ Absolute triple quadrupole MS; ACQUITY Premier Peptide CSH C18 column (2.1 × 50 mm, 1.7 µm) at 65 °C; positive ESI MRM acquisition. Calibration range evaluated from 5 to 1000 ng/mL.

Instrumentation used

• Oasis PRiME HLB µElution 96-well plates
• QuanRecovery Collection Plates with MaxPeak HPS technology
• Andrew+ Robot with Extraction+ Connected Device (fully automated workflow)
• Pipette+ Smart Pipette and Extraction+ Connected Device (assisted workflow)
• OneLab software for method control, traceability and audit trails
• ACQUITY Premier System with Flow-Through Needle
• ACQUITY Premier Peptide CSH C18 column (130 Å, 1.7 µm, 2.1 × 50 mm)
• Xevo TQ Absolute triple quadrupole mass spectrometer
• MassLynx and TargetLynx software for data acquisition and processing

Main results and discussion

• Recovery and cleanliness: The two-step Oasis PRiME HLB µElution protocol provided reproducible extraction for all five GLP-1 analogs while achieving effective phospholipid cleanup. Use of QuanRecovery plates and HPS surfaces substantially reduced NSB-related losses.
• Matrix effects: Measured matrix effects were below 20% for all analytes, indicating acceptable ion suppression/enhancement for robust quantification by MRM LC-MS/MS.
• Linearity and sensitivity: Calibration curves were linear across 5–1000 ng/mL with R2 > 0.99 for both automated and assisted workflows, demonstrating suitability for typical PK concentration ranges.
• Throughput and labor savings: For 48 samples, the fully automated Andrew+ workflow reduced total protocol time by ~42% relative to manual execution (approx. 2 h 1 min vs 3 h 30 min). The assisted Pipette+ approach was fastest (~1 h 36 min), giving up to ~54% time reduction versus manual processing because human operators can speed selected steps while minimizing solvent exposure.
• Practical challenges: Evaporation of the small-volume elution solvent during automated handling was identified as a reproducibility risk. Temperature-controlled platforms (e.g., a Peltier+ device) are proposed to reduce evaporation, though adaptation and volume scaling would be required.

Benefits and practical applications

• Reduced peptide loss: µElution SPE format avoids evaporation/reconstitution steps that commonly cause peptide adsorption and loss.
• Lower NSB and improved sensitivity: QuanRecovery plates and HPS surfaces, together with LoBind consumables, limit surface adsorption and increase assay recovery and signal consistency.
• High throughput and traceability: Automation via Andrew+ or assisted Pipette+ reduces hands-on time, increases reproducibility, and provides electronic records for regulatory compliance when controlled with OneLab software.
• Generalizability: The streamlined two-step SPE approach is broadly applicable to diverse GLP-1 analogs and adaptable to typical bioanalytical pipelines in discovery, preclinical PK, and clinical support labs.

Future trends and potential applications

• Integration of temperature-controlled modules in automated platforms to prevent solvent evaporation during small-volume elution steps.
• Further minimization of NSB through improved surface chemistries and consumable designs, enabling quantitation of more hydrophobic or highly modified peptides at lower limits.
• Method transfer and validation across species and matrices (human, nonclinical species, CSF) and expansion to other peptide therapeutics and conjugates.
• Increased adoption of semi-automated hybrid workflows that combine human speed with automation traceability for laboratories seeking rapid throughput without full robotic investment.
• Workflow standardization for regulated environments, including implementation of electronic audit trails, automated calibration generation, and QC tracking for streamlined submission packages.

Conclusion

A rapid two-step SPE procedure using Oasis PRiME HLB µElution plates, combined with QuanRecovery collection plates and automation (Andrew+ or Pipette+), yields a sensitive, reproducible, and high-throughput workflow for quantifying multiple GLP-1 analogs in plasma by LC-MS/MS. The approach minimizes peptide loss and matrix effects, provides robust linearity across 5–1000 ng/mL, and reduces hands-on time while preserving traceability and compliance. Addressing solvent-evaporation during automated elution and adopting temperature control will further enhance reproducibility for routine bioanalysis.

References

1. Trudeau M., Scumaci A. SPE-LC/MS Bioanalytical Quantification of the Biotherapeutic Peptide, Semaglutide from Plasma. Waters Application Note; 2023. (Application Note 720008097)
2. Trudeau M., Lauber M.A. Improved GLP-1 Receptor Agonist Peptide Recovery Using a QuanRecovery with MaxPeak High Performance Surfaces (HPS) Collection Plate. Waters Application Note; 2025. (Application Note 720008717)