OPTIMIZATION OF DEFINITIVE PPB USING THE RED DEVICE FOR A HIGH BINDING COMPOUND AT A LOW CONCENTRATION

Introduction: Definitive plasma protein binding (PPB) determinations in rat, dog and human plasma are needed for compound A. Due to low systemic exposure and extremely high binding, the measurement of extremely low free fraction poses great analytical challenge.

Methods: The following conditions were optimized using rapid equilibrium dialysis device (RED).

• Detection
• Screening blank plasma for ketamine
• pH control with CO₂ level
• Incubation volume and sampling
• Incubation time

To achieve proper sensitivity, different sample volumes and extraction techniques were used for extracting the plasma and buffer samples.

Results: Experimental conditions were optimized for in vitro definitive PPB of high binding compound at low concentration and were applied to compound A PPB assay. Aliquots (500 µL) of plasma samples spiked compound A were transferred to the donor side and aliquots (750 µL) of 10 mM PBS pH 7.4 were transferred to the receiver side. The samples in the RED devices were incubated for 6 hours at 37^oC with 5% CO₂. All samples were analyzed by LC‑MS/MS. An overestimation of fu for high binders was observed when ketamine containing dog plasma was used for PPB via equilibrium dialysis.

Conclusions: Compound A was very highly protein bound (>99.4%) in the plasma of all species examined. The recoveries of compound A in rat, dog, and human plasma were above 82% at all concentrations after 6 hours of incubation at 37^oC. Ketamine containing plasma should be avoided for PPB assay.