Effects of Different Centrifugation Parameters on Equilibrium Solubility Measurements

The bioavailability of a drug is closely linked to its solubility, making its early determination essential in drug development. The saturation shake-flask (SSF) method is the gold standard protocol for this, which includes a phase separation step-either by sedimentation, filtration, or centrifugation. This step is critical, as it can directly influence the accuracy of the results. This study investigated the impact of centrifugation parameters-time and rotation speed-on solubility measurements. Additionally, we compared two sample preparation protocols: continuous stirring for 24 h versus 6 h of stirring followed by 18 h of sedimentation before centrifugation. Four model compounds were tested at three pH values using Britton-Robinson buffers. Centrifugation was conducted for 5, 10, or 20 min at either 5000 or 10,000 rpm. Results showed that pre-sedimented samples yielded solubility values closer to sedimentation-only references, while continuous stirring often led to overestimated values, particularly at higher speeds and longer durations. One such example was papaverine hydrochloride, that showed solubility values 60-70% higher than the reference after centrifugation at 10,000 rpm for 20 min without prior sedimentation. Lower standard deviations were observed with shorter, slower centrifugation, with 5 min and 5000 rpm yielding results closest to the reference values.