Acquity h class hplc

To confirm and compare unbound drug concentrations, plasma protein and medium protein binding were evaluated for each drug utilizing rapid equilibrium dialysis (RED) as described elsewhere (Wetmore et al., 2012 (link); Waters et al., 2008 (link)). The RED assay was conducted using single use RED inserts (cat. no. 90006, Pierce Biotechnology, Rockford, IL) according to instructions, with protocol modification to incorporate “no protein” equilibrium controls. Equilibrium controls comprising of PBS buffer in both sample and buffer chambers were used to ensure drugs are fully equilibrated within the device in the absence of proteins. All RED assays were completed in triplicate. Drug concentrations were measured using an Agilent (Santa Clara, CA) 6470 triple quadrupole mass spectrometer operating in positive ion mode with a Waters Acquity H class HPLC (Milford, MA). Samples were spiked with a known amount of internal standard sotalol (CAS: 959–24-0) prior to analysis. Chromatographic separation was achieved using a linear acetonitrile gradient on a C18 column (Agilent Zorbex Eclipse Plus C18 3.0 X 50 mm, 1.8 μm) with a C18 guard column (Agilent). The mobile phase consisted of 0.1% formic acid, the flow rate was kept at a constant 0.4 μl/min. Complete mass spec conditions for the three drugs are listed in Table S1¹.