Table 2: Summary of advantages and disadvantages of commonly used protein binding determination methods.10,
| Method | Advantages | Disadvantages |
|---|---|---|
| Equilibrium dialysis | - High throughput - Relatively straight forward method - Reliable results, gold standard |
- Experiment duration (drug stability) - Less physiological - Technical concerns (volume shifts) |
| Ultrafiltration | - High throughput - Rapid - Relatively straight forward method - Ease of use |
- Technical considerations (binding to membrane, leakage, volume shifts) |
| Ultracentrifugation | - Simple - No membrane-related technicalities |
- Low throughput - May overestimate binding - Not favorable for large molecules |
| Microdialysis | - Can use in nearly any tissue of interest - Versatile - Can use in vivo - No concern of volume shifts - Ease of continuous sampling |
- Semi-invasive - Low throughput - Technically more complex (binding to membrane/tubing, equilibration and assay development) |
| Charcoal adsorption | - No membrane adsorption concerns - Good for drugs bound to lipoproteins - Characterize full binding profile |
- Binding underestimation - Extensive sampling |
| Chromatographic methods |
- Accurate - Fewer practical considerations (binding to membrane, volume gradients, membrane leakage) - Rapid - Small sample volume needed |
- Less physiological - More complex development and execution - Low sensitivity |
| Solid phase microextraction |
- Simple - High throughput - Highly sensitive |
- Experiment duration - Technically sensitive |