2.4. Solubility Measurement

Solubility was measured in pH 7.4, pH 6.5, and pH 2.0 aqueous buffers in a two-tier system via LC-MSMS. In Tier 1 testing, 1 μL of DMSO stock (20 mM) was measured with a Hamilton syringe and diluted to 400 μL with the respective buffer, giving a final concentration of 50 μM test compound with 0.25% DMSO. The buffer solutions were capped and incubated while shaking at 37 °C for 24 h until equilibrium was reached. Buffer solutions were centrifuged at 14,000× g for 15 min and two aliquots are taken from the supernatant. The concentration of the test compound in each aliquot was determined by liquid chromatography-mass spectrometry/mass-spectrometry analysis and by calculations using a linear regression of the test compound standards made over a range of known concentrations. Solubility was reported as the final concentration in the supernatant. If the concentration in the supernatant was determined to be 50 μM (maximum solubility for Tier 1), then a Tier 2 test was carried out. In Tier 2 testing, 5 μL of the test compound’s 20 mM DMSO stock was transferred to a microcentrifuge tube with a Hamilton syringe. The DMSO was then removed in a Speed-Vac concentrator and the test compound was diluted with 100 μL of the respective buffer, giving a final concentration of 1 mM test compound with negligible DMSO. The sample was heated and agitated by vortexing and by pipetting up and down to ensure the test compound was completely exposed to the buffer. The sample was then capped and incubated for 24 h while shaking at 37 °C. Buffer solutions were centrifuged at 14,000× g for 15 min and two aliquots were taken from the supernatant. The concentration of the test compound in the aliquots was determined by LC-MSMS, as described above.