3.5. Determination of Partition Coefficient and Solublity

The experimental octanol/water partition coefficient of compound 1 was determined by quadruplicate measurements at two concentrations (1 and 0.25 mg compound/mL of octanol) using the shake-flask method [20], in which the compound was fractionated between octanol (1 mL) and 100 mL of deionized water into glass bottles of 150 mL. Both phases were pre-saturated before the determination. The glass bottles were placed in a shaker for 1 h and then centrifuged for 30 min at 20 °C and 5000 rpm. For the quantitation 1 mL aliquotes from the aqueous phase were evaporated under nitrogen the residue was then reconstituted in a 200 µL glass insert with methylene chloride.

To determine the solubility compounds 1 and 2, supersaturated mixtures of the compounds, in phosphate buffered (11.9 mM) saline (pH 7.4) were sonicated (30 min at 25 °C), vortexed (90 min at 25 °C), and centrifuged (30 min at 13,600 G). Aliquots of the supernatants were passed through a syringe filter, and the amounts of compounds 1 and 2 were determined. The experiments were conducted in triplicate.

The analytical determination of the partition coefficient compound 1 and the solubility of compounds 1 and 2 were performed using single ion monitoring gas chromatography/mass spectrometry (GC/MS) with an Agilent 5975 GC/MSD instrument. The solubility determinations were conducted using a 30 m DB-5MS column (0.250 mm 10 μm, J&W, Folson, CA, USA) with a helium flow rate of 1.8 mL/min, the injector temperature was 200 °C, the transfer line was maintained at 280 °C, and column temperature was maintained at 170 °C for 1 min followed by a gradient of 35 °C/min to 270 °C, and then a gradient of 100 °C/min to 300 °C. The mass spectrometer conditions were electron impact, ion source temperature 230 °C, and ionization voltage 70 eV. The injection volume was 0.2 uL for compound 1, and 0.5 uL for compound 2. A calibration curve for the analytical method was developed with five points, plotting the average ratio of the peak area of the respective compound to the peak area of parthenin internal standard (R² = 0.999).

The partition coefficient determination was conducted using a 15 m DB-1 column (0.250 mm 10 μm, J&W, Folson, CA, USA) with a helium flow rate of 1.8 mL/min, the injector temperature was 200 °C, the transfer line were maintained at 300 °C, the column temperature was maintained at 120 °C for 5 min followed by a gradient of 17 °C/min to 300 °C. The mass spectrometer conditions were electron impact, ion source temperature 200 °C, and ionization voltage 70 eV. A five-point calibration curve (R² = 0.999) was used to calculate the concentration of 1 in the aqueous phase using calaxin as the internal standard. The milligrams of compound 1 in the octanol phase were then indirectly calculated and the concentrations of each compound in each phase were used to determine the partition coefficient. The average of the measurements is reported as the logarithm of the octanol/water partition coefficient (Log P).