**2.3 Data validation**

196 Toxicity and Drug Testing

used. This is because of the fact that most of organic compounds which have UV absorbance (e.g. contain a benzene ring) also have fluorescence property and might interfere with turbidimetry which reads the amount of reflected light (or fluorescence emission light) (Pan et al., 2001). An example of this is phenol red which has light absorption in 430 and 560 nm and is exited by these wavelengths which results in fluorescence emission (Pan et al., 2001). Another limitation is the UV absorbance of the most plates which are made of plastics (Pan

**Concentration (logarithmic scale)**

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

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

et al., 2001).


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

mentioned methods is given in Table 1.

et al., 2001; Hoelke et al. 2009).

**2.2.3 HPLC method** 

500000

1000000

1500000

**Turbidity signal**

2000000

2500000

3000000

0

Fig. 3. The method for finding kinetic solubility.

The collected data could be compared with the previously reported data in order to ensure the accuracy of the experimental procedure employed. Any mistake in the dilution steps, and miscalculations, or using un-calibrated instruments, such as un-calibrated balances, temperature variation and some other factors could be resulted in different solubility values for a given drug dissolved in a solvent at a fixed temperature.
