**2.2.1 Nephelometric method**

194 Toxicity and Drug Testing

perforated rubber cover) was fitted to reduce the solvent's evaporation. A thermometer with an uncertainty of 0.01 K was used to determine the temperature of the system. A laser beam was used as a tool to observe dissolving the solid in liquid. The signal transmitted through the vessel was collected by a detector that decided the rate of temperature rise and estimated the equilibrium point of the given system on the basis of the signal change. The solute and the solvent were prepared using an electronic balance with the estimated uncertainty in the mole fraction of less than 0.001. A predetermined quantity of drug and solvent was placed into the jacketed vessel. The system was slowly heated (heating rate increase is 0.5 to 2 K·hr-1) with continuous stirring. When the solute particles disappeared thoroughly, the signal approached a maximum value. The workstation judged the signal difference at 10-min intervals; if the interval was less than 10, then the workstation gave an order to stop heating and record the temperature. The temperature recorded was the liquid temperature of a given composition upon the complete dissolution of the drug (Ren et al., 2005). In another version of this set up, predetermined masses of drug and solvent were placed in the vessel and the contents were stirred continuously at a constant temperature. As the particles of the drug are dissolved, the intensity of the laser beam increased gradually and reaches to the maximum value when the drug is dissolved completely. Then an additional known mass of the drug is introduced to the vessel and the procedure is repeated until the laser beam could not return to the maximum value which means the last addition could not be dissolved. The total amount of the added drug is recorded and used to calculate the solubility value (Yang et al., 2008). The synthetic method is preferred over shake flask method for solubility determination of drugs in viscous solvents where separation of the excess solids from saturated solutions is not achievable (Grant and

In drug discovery and development, one of the rationalized methods is high-throughput screening (HTS) which includes the design and synthesis of a large set(s) of chemicals to find hit compounds based on specific physicochemical properties (PCPs) and to develop lead compound. One of the important PCPs in determination of hit and lead compounds is aqueous solubility (Pan et al., 2001; Alsenz and Kansy, 2007; Hoelke et al. 2009). However, in practice it is not possible to experimentally determine thermodynamic solubility value in HTS approaches. This is because of large number of compounds which might be more than 1000 compounds in each HTS experiment or little amount of synthesized compounds which is around a few milligrams and is another limiting factor (Pan et al., 2001; Alsenz and Kansy,

Kinetic solubility determination methods were used for covering this problem. The advantages of the kinetics solubility determination in comparison with thermodynamic solubility determination methods are capability to being easily automated, accuracy, rapidity and requiring less amount of the solute (Pan et al., 2001; Alsenz and Kansy, 2007; Hoelke et al. 2009). Its disadvantages might include not assessing the crystal effect on the solubility, the cosolvent action of the dimethyl sulfoxide (DMSO), and its applicability is good for compounds which have solubility more than 10-6 molar. Some of the well established approaches include: nephelometric, UV-Spectroscopic, and HPLC methods

Abougela, 1983).

2007; Hoelke et al. 2009).

which are discussed in the following.

**2.2 Determination of kinetic solubility** 

The nephelometry is based on turbidimetry. Figure 2 shows a schematic view of the mechanism of turbidimetry. For sample preparation in this method, a 10 millimolar concentration of a solute was prepared by dissolving suitable amounts of the solute in DMSO. Then, this stock solution is used to prepare sample solutions in the range of 5 10-7 to 5 10-4 molar. For concentrations above the 10-4 molar, the solutions prepared by direct dilution of the stock solution and for the lower concentrations, serial dilutions were used where the dilutant is a buffer. These dilutions are directly take place in a 96-well plate with the total 5% concentration of DMSO and the final volume of ≈200 μL (Pan et al., 2001; Hoelke et al. 2009). This optimum volume is based on the fact that light scattering (for a specific condition) is nearly constant for a range of particle sizes (Pan et al., 2001) which make the process reproducible and accurate.

For sample analyzing after the preparation section, the 96-well plate is placed in a nephelometer apparatus for measurement of the light scattering. It uses a laser beam (with a fixed wavelength in the range of 550-750 nm) as the light source, and a detector which is placed with a specific angle to the light source. Based on plotting turbidity against prepared concentrations, and drawing its asymptotes and finding their meeting point x coordination, gives the kinetic solubility (see Figure 3) (Pan et al., 2001; Hoelke et al. 2009).

With this method, the kinetic solubility for a plate of 96 samples can be measured in a few minutes.
