Rapid Determination of Drug Protein Binding Affinity using Solid Phase Microextraction

Significance of the Topic

Accurate measurement of the unbound fraction of drugs in plasma is critical for understanding their pharmacokinetic behavior and therapeutic efficacy. Traditional techniques such as ultrafiltration, ultracentrifugation and equilibrium dialysis are time-consuming and may require extensive sample processing. A rapid, reliable method to assess drug-protein binding can accelerate drug discovery, therapeutic monitoring and QA/QC workflows.

Study Objectives and Overview

This study compares a novel BioSPME (solid phase microextraction) device using C18-coated LC-Tips against the Thermo Scientific Rapid Equilibrium Dialysis (RED) system. Key goals were to evaluate speed, simplicity and accuracy of binding affinity measurements for a set of model drugs in rat plasma, and to benchmark results against reference binding ranges (20–99%).

Methodology and Instrumentation

The BioSPME technique relies on diffusion-driven partitioning of free analyte onto a C18-functionalized fiber, achieving equilibrium between sample matrix and coating. High-molecular-weight proteins are excluded by the polymeric binder, enabling selective extraction of unbound drug.

Sample Preparation:

• Model drugs (codeine, quinidine, propranolol, warfarin, diclofenac, diazepam) spiked into rat plasma at 200 ng/mL and equilibrated for 3 h at 37 °C.
• Parallel spiked PBS controls prepared at identical concentration.

SPME Protocol:

• Condition fibers in methanol (10 min) then water (10 min).
• Agitate SPME tips in 800 µL sample at 500 rpm for 30 min.
• Desorb analytes in 300 µL ACN with internal standard (10 min), then analyze directly by LC-MS/MS.

RED Protocol:

• Load 200 µL plasma and 350 µL buffer per well, incubate at 37 °C, 250 rpm for 4 h.
• Collect aliquots, perform protein precipitation with ACN + internal standard, centrifuge, and analyze supernatant.

Instrumentation Used:

• Agilent 1290 Infinity II UHPLC with Agilent 6460 QQQ MS (ESI+, MRM).
• Thermo Scientific Rapid Equilibrium Dialysis device (RED).
• Corning LSE Digital Microplate Shaker.
• Eppendorf ThermoMixer C.

Main Results and Discussion

Both BioSPME and RED methods produced protein binding values closely matching reference data for five of six analytes; quinidine showed slightly lower binding in both assays, likely due to specific matrix interactions. BioSPME reduced total assay time to under 60 minutes versus ≥4 hours for RED, representing a four-fold time saving. Calibration curves demonstrated excellent linearity (R² > 0.994) across all compounds.

Benefits and Practical Applications

BioSPME offers direct sampling of plasma without protein precipitation, minimizing matrix effects and streamlining workflow. Its rapid equilibration accelerates throughput in drug development, therapeutic drug monitoring and routine QA/QC analyses.

Future Trends and Potential Applications

Ongoing work aims to optimize fiber chemistries and reduce extraction time further. Integration with automated platforms and expansion to additional drug classes and biological matrices could broaden the method’s utility in clinical and pharmaceutical laboratories.

Conclusion

The BioSPME LC-Tip approach provides a fast, simple and reliable alternative to equilibrium dialysis for assessing drug-protein binding in plasma. It achieves comparable accuracy with significantly reduced assay time and simplified sample handling.

Reference

1. Journal of Laboratory Automation, February 2011, Vol. 16, pp. 56–67.