TABLE 2 

Advantages and disadvantages of determining unbound fraction by different dialysis methods

(+) and (−) symbols indicated attributes of relative advantage or disadvantage, respectively.

Eq. Dialysis Method (Matrix vs. Buffer)Eq. Dilution Method (Diluted-Matrix vs. Buffer)Eq. Competition Method (Matrix1 vs. Matrix2)Flux Dialysis Method (Matrix vs. Matrix)
Minimal dialysis time(−) Establishment of equilibrium, (HTDialysis ≤4 h assuming minimal nonspecific binding)(−) Establishment of equilibrium, (HTDialysis ≤4 h assuming minimal nonspecific binding)(−) Establishment equilibrium, (hours, days, weeks, or months depending on fu)(+) Receiver conc >> LLOQ, (always faster than equilibrium dialysis assuming same LLOQ
Impact of device nonspecific binding(−) Increased time to equilibrium, (although no impact on fu)(−) Increased time to equilibrium (although no impact on fu)(+) Nonspecific binding is minimized(+) Nonspecific binding is minimized
Impact of compound matrix instability(−) Invalid fu(−) Invalid fu(−) Invalid fu if degradation rate is different between matrix1 and matrix2(+) No impact on fu
Sample composition(−) Matrix and buffer, two standard curves or sample blanking/matching require; Additional GLP validation and lower LLOQ may be required(−) Matrix and buffer, two standard curves or sample blanking/matching require; additional GLP validation and lower LLOQ may be required(−) Matrix1 and matrix2, two standard curves or sample blanking/matching require; Additional GLP validation may be required(+) All samples identical matrix, single standard curve; preexisting GLP assay likely can be used
LLOQ requirement(−) LLOQ ≤ free buffer(−) LLOQ ≤ free buffer, although free buffer increases roughly proportional with dilution factor(+) LLOQ ≤ initial matrix conc assuming similar fu between matrix1 and matrix2(+) LLOQ ≤ 1/2 initial donor concentration
Additional advantage/
disadvantages(+) Most commonly used dialysis method(−) Risk for saturating protein binding; (−) dilution does not reduce equilibrium time(−) Discrete fu not determined, only fu ratio between matrix1 and matrix2(−) Unconventional time point(s) and data may require nonlinear curve fitting
  • GLP, Good Laboratory Practice.