Rapid equilibrium dialysis device system

The plasma protein fraction was determined using the Rapid Equilibrium Dialysis Device System (Life Technologies, Grand Island, NY) according to the manufacturer’s protocol. Membrane dialysis was performed with 500 μM ¹⁹F–ATRi in serum (500 μL) on one side of the membrane and PBS (700 μL) on the other side. The system was sealed with parafilm and incubated for 4 h at 37 °C on an orbital shaker set to 100 rpm. Thereafter, 50 μL of solution was taken from both sides, and samples were treated with 300 μL of precipitation buffer (90/10 acetonitrile/water with 0.1% formic acid) and vortexed to remove protein before HPLC analysis. After injection, the [¹⁸F]-ATRi peaks from each sample were integrated, and the protein bound fraction determined as % bound = [1 – (Concentration buffer chamber/Concentration plasma chamber)]×100. The final albumin binding was calculated as the mean ± SD.

Plasma protein binding was assessed using the Rapid Equilibrium Dialysis Device system (Thermo Scientific, Rockford IL). The incubation was performed as described previously.(Haney et al., 2019 (link)) Briefly, mouse plasma (500 μL) was spiked separately with 20–223 at two concentrations (1 μg/mL and 10 μg/mL).The spiked 300 μL plasma sample was added to the donor chamber and 500 μL of phosphate-buffered saline (containing 100 mM sodium phosphate and 150 mM sodium chloride, pH 7.4) added to the buffer chamber. The plate was sealed and incubated at 37 °C on an orbital shaker at 100 rpm for 5 h. Aliquots (50 μL) were removed from the sample and buffer chambers and mixed with an equal volume of buffer or blank plasma, respectively. Calibration and QCs were prepared using equal volume of blank plasma and buffer. All matrix-matched samples were prepared as per described and analyzed by LC-MS/MS. Propranolol and atenolol were used as high and low protein bound positive controls. The stability, device recovery, and percent bound were calculated using following equation:
$$\text{\%}\text{Bound}\text{=}\frac{{\text{Concentration}}_{\text{donor}\text{cell}}-{\text{Conentration}}_{\text{receiver}\text{cell}}}{{\text{Concentration}}_{\text{donor}\text{cell}}}\times 100$$