Ultrafast Plasma Protein Binding Analysis

Importance of the Topic

The measurement of plasma protein binding (PPB) is a critical parameter in drug discovery and development as it directly affects pharmacokinetics and pharmacodynamics. By quantifying the fraction of drug bound to plasma proteins, researchers can predict bioavailability, distribution, and clearance, which supports dose optimization and safety assessment.

Goals and Study Overview

This study aimed to develop a single, ultrafast PPB assay using an Agilent RapidFire high-throughput mass spectrometry platform coupled with a 6550 iFunnel Q-TOF mass spectrometer. A comparison was made against a conventional LC–MS/MS approach using a 1260 Infinity LC system with a 6460 Triple Quadrupole to evaluate speed, throughput, and analytical performance across a diverse set of compounds.

Methodology and Instrumentation

Sample Preparation:

• Human plasma spiked with 5 µM test compounds and incubated in Rapid Equilibrium Dialysis (RED) plates at 37 °C for 5 hours.
• Post-dialysis, protein precipitation with cold acetonitrile containing 0.1% formic acid and an internal standard.
• Supernatants diluted and analyzed in parallel by rapid SPE/TOF/MS and LC/MS/MS.

Instrumentation Used

• Agilent RapidFire 360 high-throughput SPE system with reversed-phase C18 cartridges.
• Agilent 6550 iFunnel Q-TOF Mass Spectrometer operating in positive ESI mode, 125–1 000 m/z, 5 spectra/s.
• Agilent 1260 Infinity Binary LC with Poroshell 120 EC-C18 column (2.1×50 mm, 2.7 µm).
• Agilent 6460 Triple Quadrupole with JetStream ESI in MRM mode.

Main Results and Discussion

• More than 40 compounds spanning xLogP from –0.4 to 7.1 and molecular weights of 160–733 g/mol were tested.
• The SPE/TOF/MS assay achieved a cycle time of 9 seconds per sample (~400 samples/hour), a 20-fold increase over the 3-minute LC/MS/MS method.
• Excellent agreement between methods with R² = 0.977 for percentage bound values.
• SPE/TOF/MS results also aligned well with literature RED values for selected compounds.

Benefits and Practical Applications

The ultrafast SPE/TOF/MS workflow eliminates the need for individual MRM method development, significantly reducing assay setup time. High-resolution accurate mass detection enables simultaneous analysis of structurally diverse compounds without method reconfiguration. The throughput enhancement supports larger sample sets in early ADME screening and streamlines lead optimization.

Future Trends and Potential Applications

• Extension of ultrafast SPE/TOF strategies to other in vitro ADME assays such as metabolic stability and drug–drug interaction screening.
• Integration with automated sample handling and data-processing pipelines for end-to-end high-throughput screening.
• Further improvements in mass spectrometer scan speeds and resolution to support multiplexed assays.

Conclusion

An ultrafast SPE/TOF/MS method using the Agilent RapidFire–Q-TOF system was demonstrated to match conventional LC/MS/MS in accuracy for PPB assays while delivering a 20-fold increase in throughput. This high-resolution, high-speed approach offers a powerful platform for rapid ADME profiling in drug discovery.

Reference

1. van Liempd S. et al. Development and Validation of a Higher-Throughput Equilibrium Dialysis Assay for Plasma Protein Binding. Journal of the Association for Laboratory Automation. 2011;16(1):56–67.
2. Waters NJ et al. Validation of a Rapid Equilibrium Dialysis Approach for the Measurement of Plasma Protein Binding. Journal of Pharmaceutical Sciences. 2008;97(10):4586–95.
3. Shanler MS et al. Validation of an Automated High Throughput Plasma Protein Binding Assay. BD Biosciences Application Note #474. 2009.