The log P value was determined by the miniaturized shake flask method.28 (link) Stock solutions (200 μL) of KRM-II-81
and the KRM-II-81·HCl salt (1000 μg/mL prepared in n-octanol) were taken in Eppendorf tubes. Upon addition
of 300 μL of presaturated n-octanol and 500
μL of presaturated water, the volume was made up to 1 mL. Eppendorf
tubes were shaken overnight at 500 rpm and centrifuged at 16 000
RCF (G-force) for 20 min to separate aqueous and
organic layers. Three aliquots of 10 μL of the octanol layer
were combined with 70 μL of octanol, and this was followed by
the determination of absorbance at 300 nm using a 384-well plate (Coring
UV star) and a Tecan M1000 microplate reader. The absorbance of the
water layer was determined at 300 nm using a 384-well plate (Coring
UV star) and the Tecan M1000. Calibration curves for octanol and water
were prepared to determine the concentrations of the free base and
also the salt. The assays were carried out in triplicate.
and the KRM-II-81·HCl salt (1000 μg/mL prepared in n-octanol) were taken in Eppendorf tubes. Upon addition
of 300 μL of presaturated n-octanol and 500
μL of presaturated water, the volume was made up to 1 mL. Eppendorf
tubes were shaken overnight at 500 rpm and centrifuged at 16 000
RCF (G-force) for 20 min to separate aqueous and
organic layers. Three aliquots of 10 μL of the octanol layer
were combined with 70 μL of octanol, and this was followed by
the determination of absorbance at 300 nm using a 384-well plate (Coring
UV star) and a Tecan M1000 microplate reader. The absorbance of the
water layer was determined at 300 nm using a 384-well plate (Coring
UV star) and the Tecan M1000. Calibration curves for octanol and water
were prepared to determine the concentrations of the free base and
also the salt. The assays were carried out in triplicate.