A 96 well equilibrium dialysis apparatus was used to determine the free fraction in plasma for each compound (HT Dialysis LLC, Gales Ferry, CT). Membranes (12-14 kDA cut-off) were conditioned in deionised water for 60 minutes, followed by conditioning in 80:20 deionised water:ethanol for 20 minutes, and then rinsed in isotonic buffer before use. Female CD1 mouse, Male rat Han Wistar and human plasma was removed from the freezer and allowed to thaw on the day of experiment. Thawed plasma was then centrifuged (Allegra X12-R, Beckman Coulter, USA), spiked with test compound (final concentration 10 μg/mL), and 150 μL aliquots (n=6 replicate determinations) loaded into the 96-well equilibrium dialysis plate. Dialysis vs isotonic buffer (150μL) was carried out for 5 hours in a temperature-controlled incubator at ca. 37ºC (Barworld scientific Ltd, UK) using an orbital microplate shaker at 100 revolutions/minute (Barworld scientific Ltd, UK). At the end of the incubation period, 50 μL aliquots of plasma or buffer were transferred to a 96 well 2.1mL Waters TM plate and the composition in each well balanced with control fluid (50 μL), such that the volume of buffer to plasma is the same. Sample extraction was performed by the addition of 200μL of acetonitrile containing an appropriate internal standard. Samples were allowed to mix for 1 minute and then centrifuged at 3000rpm in 96-well blocks for 15 minutes (Allegra X12-R, Beckman Coulter, USA) after which 150 μL of supernatant was removed to 50 uL of water. All samples were analysed by UPLC-MS/MS. The unbound fraction was determined as the ratio of the peak area in buffer to that in plasma.