DEVELOPMENT OF A SPE LC-MS/MS METHOD FOR THE BIOANALYTICAL QUANTIFICATION OF PRAMLINTIDE FROM SERUM

Bioanalytical Quantification of Pramlintide in Serum via SPE-LC-MS/MS

Significance of the Topic

Pramlintide acetate is a synthetic analogue of human amylin used in adjunctive therapy for diabetes and emerging research suggests wider applications in neurodegenerative disorders. Reliable quantification in biological matrices supports pharmacokinetic studies and therapeutic monitoring.

Study Objectives and Overview

The study aimed to develop a selective and sensitive sample preparation and UPLC tandem mass spectrometry method to quantify pramlintide in human and rat serum. Key goals included achieving a lower limit of quantification of 25 pg mL from a 100 µL sample and reducing nonspecific peptide binding.

Methodology and Instrumentation

• Sample preparation used weak cation exchange microelution SPE in 96-well format with QuanRecovery plates featuring high performance surfaces to minimize adsorption losses.
• SPE protocol: conditioning with methanol, equilibration with water, sequential washing and elution with trifluoroacetic acid in acetonitrile:water mixture.
• Chromatography employed an ACQUITY I-Class UPLC PLUS system and Peptide CSH C18 column (1.7 µm, 2.1×50 mm) at 0.4 mL/min with an optimized linear gradient starting at 20% organic, shallow to 22–27% over 3 minutes.
• Mass spectrometry was performed on a Xevo TQ-XS tandem quadrupole in positive electrospray mode using MRM transitions 988.36>968.11 and 988.36>930.78.

Key Results and Discussion

• Lower limit of quantification was established at 25 pg/mL with accuracies above 92% and RSDs below 5%.
• Dynamic calibration range extended from 25 to 50,000 pg/mL with linear correlation coefficients exceeding 0.995.
• Optimized SPE recovery reached approximately 75%, and use of specialized surfaces yielded a 36-fold increase in peptide peak area compared to polypropylene.
• Chromatographic adjustments significantly reduced matrix suppression and interferences, improving assay selectivity.

Benefits and Practical Applications

• The method combines simple sample preparation with high-throughput SPE plates, enabling robust bioanalysis in pharmacokinetic and preclinical studies.
• High sensitivity and broad dynamic range support monitoring of low-abundance peptides in limited-volume samples.
• Minimization of nonspecific binding enhances data reliability across laboratories.

Future Trends and Potential Uses

• Adoption of novel surface treatments and microelution formats may further improve peptide recoveries.
• Integration with automated platforms could enhance throughput for large-scale studies.
• Application of the workflow to other challenging peptides and biomolecules in clinical research.

Conclusion

The first reported SPE-LC-MS/MS method for pramlintide in serum achieves high sensitivity, precision, and accuracy using a streamlined weak cation exchange extraction and optimized UPLC-MS/MS conditions. This approach offers a reliable tool for peptide quantification in biomedical research.

Reference

• Center for Drug Evaluation and Research Approval Package for Application Number 21-332 Clinical Pharmacology and Biopharmaceutics Review; FDA, 2019.
• SYMLIN Product Information, drugs.com, 2019.
• Mohamed et al. Amylin Enhances Amyloid-β Peptide Brain to Blood Efflux Across the Blood-Brain Barrier. J Alzheimers Dis, 2017, 56(3),1087–1099.
• Rabe et al. Understanding Protein Adsorption Phenomena at Solid Surfaces. Adv Colloid Interface Sci, 2011,162(1-2),87–106.