**2.1 Determination of thermodynamic solubility**

Solubility determination of drugs in a liquid could be classified as analytical and synthetic methods. The main advantage of the analytical (shake flask) method is the possibility of measuring a large number of samples simultaneously however this method is tedious and time-consuming.

#### **2.1.1 Shake flask method**

The shake-flask method of Higuchi and Connors (1965) is the most reliable method for low soluble compounds and widely used solubility measurement method. In this method, an excess amount of drug is added to the solubility medium. The added amount should be enough to make a saturated solution in equilibrium with the solid phase. In case of acidic or basic drugs dissolved in an un-buffered solubility medium, further addition of the solid could change pH of the solution and consequently the solubility of the drug (Wang et al., 2002; Kawakami et al., 2005; Jouyban and Soltanpour, 2010). Depending on the dissolution rate and type of agitation used, the equilibration time between the dissolved drug and the excess solid could be varied. Equilibration is often achieved within 24 hours. To ensure the equilibration condition, the dissolution profile of drug should be investigated. The shortest time needed for reaching the plateau of drug concentration against time could be considered as a suitable equilibration time. Any significant variation on dissolution profile after reaching the equilibration should be inspected, since there are a number of possibilities including degradation of the drug and also its polymorphic transformation. Both these affect the solubility values of a drug dissolved in the dissolution media. Heating, vortexing or sonicating the sample prior to equilibration could shorten the equilibration time. To overcome the poor wettability of low soluble drugs, one may use small glass microspheres or sonication. Then the two phases, solid and solution phases, are separated using two common methods of filtration and/or centrifugation. Filteration is the easiest method, however, the possible sorption of the solute on the filter should be considered as a source of error in solubility determination, especially for very low soluble drugs. Pre-rinsing the filter with the saturated solution could reduce the sorption of the solute on the filter by saturating the adsorption sites. Centrifugation or ultra-centrifugation is preferred in some cases, and the higher viscosity of the saturated solutions, e.g. in mixed solvents, should be kept in mind as a limitation. A combination of filtration and centrifugation is also could be used. The UV spectrophotometric analysis is the most common and the easiest analytical method. The next is the HPLC methods both in isocratic and gradient elution modes. The HPLC analysis could also detect the possible impurities or degradation products if a highly selective method was used. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) of the residual solid separated from the saturated solution confirm the possible solid phase transformations during equilibration.
