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

Significance of the Topic

Pramlintide is a synthetic amylin analogue used as adjunct therapy in type 1 and type 2 diabetes. Recent evidence links amylin pathways to Alzheimer’s disease models, driving interest in precise quantification of pramlintide in biological matrices. Its hydrophobic nature poses challenges due to nonspecific adsorption during sample handling, necessitating optimized bioanalytical approaches.

Objectives and Study Overview

This study aimed to develop and validate a selective solid-phase extraction (SPE) coupled with UPLC-MS/MS method capable of quantifying pramlintide at low picogram levels from 100 µL of human and rat serum. Key goals included minimizing matrix effects, improving recovery, achieving a lower limit of quantification (LLOQ) of 25 pg/mL, and ensuring robust calibration over a wide dynamic range.

Methodology and Instrumentation

Sample preparation employed an OASIS weak cation exchange (WCX) µElution 96-well SPE plate. After spiking serum with pramlintide and diluting with water, samples were loaded onto SPE, washed with water and 20% acetonitrile, and eluted with 1% TFA in 75:25 acetonitrile:water. Eluates were collected in QuanRecovery plates with MaxPeak High Performance Surfaces (HPS) to mitigate adsorption and then diluted prior to analysis.

Instrumentation:

• UPLC System: ACQUITY UPLC I-Class PLUS with Peptide CSH C18 column (2.1 x 50 mm, 1.7 µm) at 60°C
• Mobile Phases: 0.1% formic acid in water (A) and acetonitrile (B)
• Gradient: Shallow 22–27% B over 3 minutes
• MS System: Xevo TQ-XS triple quadrupole, capillary voltage 1.0 kV, cone 15 V, desolvation 600°C
• MRM transitions: 988.36→968.11 and 988.36→930.78
• Injection Volume: 10 µL, 7.0 min cycle time

Main Results and Discussion

Recovery of pramlintide improved to ~96% with the optimized SPE protocol and MaxPeak plates. Matrix suppression was significantly reduced by adjusting the chromatographic gradient. Calibration curves from 25 to 50,000 pg/mL were linear (r2 > 0.995) with accuracies of 91–111% in human serum and 92–106% in rat serum. QC samples at four levels (25, 75, 2,500, 40,000 pg/mL) showed accuracies between 92–105% and RSDs below 5%.

Benefits and Practical Applications of the Method

• First reported method for pramlintide quantification in serum at pg/mL sensitivity.
• Low sample volume requirement (100 µL) and rapid 96-well plate workflow support high throughput.
• Enhanced recovery and reduced nonspecific binding enable reliable pharmacokinetic and biomarker studies.

Future Trends and Potential Applications

Emerging applications include clinical pharmacokinetic profiling, Alzheimer’s research on amylin analogues, and expansion to other hydrophobic peptide hormones. Integration with automated SPE platforms and multiplexed assays may further increase throughput and data robustness.

Conclusion

The described SPE-LC-MS/MS method delivers high sensitivity, selectivity, and reproducibility for pramlintide quantification in serum. It overcomes adsorption challenges and provides a reliable platform for both preclinical and clinical studies.

References

• Dunning CM, Lame ME, Wrona MD, Haynes K. Development of a SPE LC-MS/MS Method Utilizing QuanRecovery Sample Plates with MaxPeak Performance Surfaces for the Bioanalytical Quantification of Pramlintide from Serum. Waters Application Note 720006527en, March 2019.
• Center for Drug Evaluation and Research. Approval Package for Application Number 21-332 Symlin Injection Biopharmaceutics Review. FDA, 2019.
• Rabe M, Verdes D, Seeger S. Understanding Protein Adsorption Phenomena at Solid Surfaces. Adv Colloid Interface Sci. 2011;162(1-2):87-106.