Protein Binding by Equilibrium Dialysis

Instrument. The high throughput dialysis apparatus, model HTD96b, was used (HTDialysis LLC, Gales Ferry, Connecticut). Prior to assembly, dialysis membrane strips (molecular weight cutoff of 6–8 kDa) were hydrated according to manufacturer’s recommendations. The Teflon bars were assembled according to manufacturer’s instructions, with dialysis membrane strips laid between bars creating two compartments per well. The assembled unit was locked in place in the steel base plate. Samples were immediately added to each compartment to prevent dehydration of the membranes.

Equilibrium Dialysis Procedure. Fortified matrix (plasma) was added to the donor side, DPBS was added to the receiver side of the HTD wells, and the plate was sealed. Samples were incubated at 37°C and rotated at 300 rpm for the designated time. After incubation, the seal was removed and a sample from each plasma and dialysate chamber was analyzed by LC-MS/MS. All protein binding determinations were performed in quadruplicate. Time to Equilibrium Determination. Dialysis was performed according to the equilibrium dialysis procedure for 3, 5, 6, 7, and 8 h to determine the minimum time to achieve equilibrium. This experiment was conducted at 50 ng/ml of 6-beta naltrexol in human plasma. Equilibrium was obtained when the percentage of 6-beta naltrexol bound to the proteins in the plasma remained constant over time.

Concentration Dependence. The protein binding in mouse, rat, guinea pig, dog, and human plasma was determined at concentrations of 0.5, 1.5, 5, 15, and 50 ng/ml of 6-beta naltrexol. The dialysis time for the test article was 8 h as determined in the time to equilibrium experiment.

Sample Analysis. Samples were processed prior to LC-MS/MS analysis as follows. The donor side samples (plasma) were diluted with DPBS and the receiver side samples (DPBS) were diluted with blank control plasma at the appropriate volumes to provide a common analytical mixed matrix of 90% DPBS and 10% plasma (90:10 DPBS:plasma, v:v) in 100 µL total volume. Samples were prepared for analysis and analyzed for 6-beta naltrexol using a quantitative LC MS/MS method developed at Covance and modified as appropriate for study optimization.