Automation of Peptide SPE for Bioanalytical Method Development

Importance of the Topic

Advancing peptide bioanalysis hinges on efficient and reproducible sample preparation. Solid-phase extraction (SPE) is pivotal for removing complex biological matrices and ensuring accurate quantitation. Automating SPE workflows enhances throughput, reduces human error, and accelerates therapeutic peptide development.

Objectives and Study Overview

This work aims to demonstrate an end-to-end automated SPE method for therapeutic peptides using the Andrew+ pipetting robot. Key goals include optimizing mixed-mode sorbent selection, validating recoveries across diverse peptides, and comparing automated performance with manual protocols.

Methodology and Instrumentation

Automation was achieved with:

• Andrew+ pipetting robot controlled by OneLab software to script all SPE steps: sample pretreatment, equilibration, wash, elution, post-spike addition, and vacuum control.
• Oasis Peptide Method Development 96-well μElution Plate featuring mixed-mode weak cation exchanger (WCX) and mixed-mode anion exchanger (MAX) chemistries.
• Instrumentation: Waters ACQUITY Premier UPLC system, Xevo TQ-XS triple quadrupole mass spectrometer, IKA VACSTAR pump with a microplate vacuum manifold for gradient pressure control.

Critical reagents included 4% phosphoric acid for pretreatment; 5% ammonium hydroxide and 20% acetonitrile for sequential washes; and 75/25 acetonitrile/water with 1% trifluoroacetic acid for elution.

Results and Discussion

Four peptides—Leuprolide, Goserelin, Pramlintide, and Bivalirudin—were extracted from spiked human plasma:

• Peptides with pI >8 showed optimal recovery on WCX; Bivalirudin (pI ~3.9) favored MAX.
• Comparing manual versus automated workflows yielded <10% difference in mean recovery and RSDs below 5%, underscoring robotic precision.
• Gradient vacuum control (stepwise pressure decrease) outperformed single-step vacuum, delivering higher recoveries and lower variability.

Benefits and Practical Applications

Automation provides:

• Consistent high recoveries across peptide chemistries.
• Reduced hands-on time and operator variability.
• Rapid testing of wash, elution solvents, and vacuum parameters in a logical, reproducible fashion.
• Simplified method transfer and secure protocol sharing via OneLab.

Future Trends and Opportunities

Emerging directions include:

• Integration of machine-learning algorithms for solvent composition and sorbent selection.
• Expanded plate chemistries to accommodate a broader range of biomolecules.
• Miniaturized, high-density formats for ultra-high throughput screening.
• Cloud-based protocol libraries enabling real-time collaboration across laboratories.

Conclusion

Automating peptide SPE with the Andrew+ robot and OneLab software achieves robust, reproducible, and high-throughput sample preparation. Precise liquid handling, controlled vacuum gradients, and seamless integration with UPLC-MS platforms establish a reliable workflow for bioanalytical method development.

References

1. Waters Corporation. Oasis Peptide Bioanalysis Reference Card. Literature No. 720006298, 2018.
2. Andrew Alliance. OneLab protocol: Peptide SPE Method Development, 2022.
3. Dunning CM, Lame M, Wrona MD, Haynes K. Development of an SPE LC-MS/MS Method Utilizing QuanRecovery Sample Plates With MaxPeak Performance Surfaces for the Bioanalytical Quantification of Pramlintide from Serum. Waters App. Note 720006527, 2019.