**2.2.2.2 UV/Vis-spectroscopic method 2**

In this method, sample dilution in range of 7 10-9 to 5 10-4 molar is performed. But after precipitation of the stock solution by the aqueous solution, the samples are filtered to another plate. And in this part, 20% acetonitrile is added to the filtered samples for prevention of solute precipitation during analysis. Then the plate is read with a 96-well plate UV/Vis-spectroscopy apparatus and the recorded data changed to molar concentration (determined by calibration curve obtained by standard solutions using another plate) (Pan et al., 2001; Hoelke et al. 2009).

#### **2.2.3 HPLC method**

The sample preparation for this method is the same as UV/Vis-spectroscopic method 2 and the transferring of samples to the 96-well plate is not required. However, filtration of samples is done prior to injection to the HPLC or online filtration is applied. A calibration curve is required for the determination of the concentrations of the prepared samples. This method is the most accurate one in comparison with other mentioned methods (limit of detection < 10-8 molar). But it must be considered that it consumes much more time (around 6 hours for 96 samples) (Pan et al., 2001; Hoelke et al. 2009). A comparison between the mentioned methods is given in Table 1.


Table 1. The comparison between four kinetic solubility determination methods

Kinetic solubility values are valuable source in early stage of drug discovery in place of thermodynamic solubility values where there is good correlation between trends of these two values for a set of compounds (Hoelke et al. 2009). However, because of the amorphic nature of the solutes, in most of the cases the kinetic solubility is higher than thermodynamic solubility values. The effect of 5% DMSO as a cosolvent on the solubility value in kinetic solubility determination methods also should be considered. This is very important where most of the drugs have very low aqueous solubility and very small amounts of solubilizing agents such as cosolvents (e.g. DMSO) enhance their solubility largely.

Also the effect of time after dilution is important, especially in turbidimetry methods. Hoelke et al. have shown that by increasing the time after dilution and precipitation, the determined solubility become smaller (Hoelke et al. 2009).
