A 96-well equilibrium dialysis block was designed and constructed that is compatible with most standard 96-well format laboratory supplies and instruments.
Automated conduct of the incubations using a 96-well equilibrium dialysis device as well as high-throughput liquid chromatography mass spectrometry quantitation has now made this level of rigor accessible and useful.
In vitro blood and brain unbound fractions were determined incubating GSK1059865 at 1 µg/mL in a 96-well equilibrium dialysis apparatus (HTDialysis, Gales Ferry, CT) using the method described by Kalvass and colleagues [44].
In brief, a 96-well equilibrium dialysis apparatus (HT Dialysis LLC, Gales Ferry, CT) was used to determine the free fraction in plasma for -U50,488.
Plasma protein binding experiments: In brief, a 96-well equilibrium dialysis apparatus was used to determine the free fraction in plasma for each compound (HT Dialysis LLC, Gales Ferry, CT, USA).
Frozen female mouse plasma was thawed, centrifuged (2100 × g, 10 min), spiked with -U50,488 (10 μg g−1), and 150-μl aliquots (n = 6 replicate determinations) loaded into the 96-well equilibrium dialysis plate.
Thawed plasma was then centrifuged (Allegra X12-R, Beckman Coulter, USA), spiked with test compound (10 μg g−1), and 150 μL aliquots (n=6 replicate determinations) loaded into the 96-well equilibrium dialysis plate.
(1) In vitro plasma protein binding experiments: In brief, a 96-well equilibrium dialysis apparatus was used to determine the free fraction in plasma for each compound (HT Dialysis LLC, Gales Ferry, CT, USA).
Thawed plasma was then centrifuged (Allegra X12-R, Beckman Coulter, USA, 10 min, 5 °C, 3270 g), spiked with test compound (10 μg g−1), and 150 μL aliquots (n=6 replicate determinations) loaded into the 96-well equilibrium dialysis plate.
Diluted plasma (50 75 μL) and nicotine tracer (50 75 μL) in PBS were added to the sample chamber of a 5 kDa MWCO 96-well Equilibrium Dialyzer (Harvard Apparatus), and unlabeled -nicotine (100 150 μL) at varying concentrations in 1% BSA was added to the buffer chamber.
Ritonavir was added to a concentration of 1 µmol/l, and aliquots (n = 3) were loaded into a 96-well equilibrium dialysis apparatus (HTDialysis, LLC; Gales Ferry, CT) and dialyzed against phosphate buffer for 6 h with shaking at 37 °C.
