Frozen water phase method for log D measurement using a 96-well plate

In this study, a frozen water phase method for log D measurement using a 96-well plate was developed. In the case of log D measurement of compounds, the problem of octanol contamination often occurs; in lipophilic compounds, the concentration of the octanol phase is much higher than that of the water phase. When the water phase is separated from the octanol phase, a small amount of octanol phase contamination could strongly influence the concentration of the water phase. To avoid this problem, the frozen water phase method was developed. The water phase was frozen in liquid nitrogen and then the unfrozen octanol phase was removed. To remove the portion of the octanol remaining on the frozen water phase, the surface of the frozen water phase was washed with octanol and water/ethanol (50/50, v/v). The validity of the method was confirmed by results of commercially available drugs at the log D range from 0 to 4. Further, it was found that this method had the ability to evaluate the pH–log D profile of compounds in the range from pH 2 to pH 12. As a result, we developed the convenient and accurate method that is effective in preventing contamination with a wide dynamic range.