Determination of Thermodynamic Solubility by Saturation Shake-Flask Method

The thermodynamic solubility of baicalin in various media was determined by saturation shake-flask method based on the method of Baka et al. [23]. Briefly, 10 mg of baicalin was added to 5 ml of each solvent in sealed vials. All samples were stirred (approx. 500 rpm) and thermostated (IKA RT-5 power heatable magnetic stirrer, IKA Work Inc., USA) at 37 ± 0.5°C for 24 h allowing it to achieve thermodynamic equilibrium. After this period, 24 h of sedimentation cycle was adopted without stirring at 37 ± 0.5°C. To improve the efficacy of phase-separation, the saturated supernatant of samples was centrifuged at 14,000 rpm for 15 min (Herolab MicroGen 16, Herolab GmbH, Wiesloch, Germany). The aliquots (250 μl) taken for solubility experiments were suitably diluted with blank buffers and the absorbance was measured at λ = 316 nm by UV spectroscopy (Agilent 8453 UV-Visible Spectrophotometer, Agilent Technologies Ltd., USA). The drug exhibited absorption maxima (λ_max) at 214, 279 and 316 nm in distilled water. Ideally, λ_max selected for the analysis of drug should not show any interference due to solvents and excipients present in the dissolution medium. At 279 nm, baicalin showed higher molar absorptivity; however, this wavelength was not selected for the quantification of the drug because there was interference of excipients in biorelevant media at this wavelength. In the solubility study, thus, λ_max 316 nm was selected for the quantification of baicalin. In case of the distribution study, the above mentioned obstacle did not appear, so λ_max 279 nm could be easily used. All experiments were repeated three times and results were calculated from the linear calibration curve of baicalin in each media. Data are expressed as mean ± SD (μg/ml).