**2.1.2 Synthetic method**

192 Toxicity and Drug Testing

Also low solubility is useful when stability of a pharmaceutical compound is low in its solubilized form (Sinko and Martin, 2006). Hence, suspension formulations (i.e. ready to use

In the recent decade, emerging technologies such as micro-formulation, microencapsulation, nano-formulation, and nano-encapsulation are using solubility decreasing principals as a part of their processes. This is usually done by addition of antisolvent and

The solubility of a drug could be measured experimentally using two procedures, namely the thermodynamic and kinetic solubility methods. The thermodynamic solubility determination methods are not feasible at the early discovery stage because of the large sample requirement, low throughput and laborious sample preparation. The kinetic

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

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

and lyophilized powder for suspension preparation) might be a useful strategy.

fine particle stabilizers to gain a suspension with micro/nano-sized particles.

solubility determinations could be used as an alternative method at this stage.

**2. Experimental methods for determination of solubility** 

**2.1 Determination of thermodynamic solubility** 

time-consuming.

**2.1.1 Shake flask method** 

The synthetic method (Hankinson and Thompson, 1965; Ren et al., 2005; Yang et al., 2008; Yu et al., 2009) which is so called laser monitoring technique (Li et al., 2006), last crystal disappearance method (Hao et al., 2005) and dynamic method (Peisheng and Qing, 2001; Weiwei et al., 2007; Wang et al., 2008) is based on disappearance of the solid drug (from the mixture of solvent and drug) monitored by a laser beam. The history of this method backs to 1886 and first introduced by Alexejew and then modified by other research groups (Ward, 1926). The disappearance of drugs could be achieved either by changing the temperature or by addition of a known amount of the solvent. It is claimed that the synthetic method is much faster and more reliable than analytical method (Yang et al., 2008). Figure 1 illustrates a schematic representation of the most completed set up used in the synthetic method.

Fig. 1. Schematic representation of the synthetic method for determination of solubility of drugs; 1, magnetic stirrer; 2, laser generator; 3, jacketed glass vessel; 4, condenser pipe; 5, thermometer; 6, thermocouple; 7, rotor; 8, photoelectric transducer; 9, controller; 10, laser strength display; 11, constant temperature bath; 12, workstation. (Figure is reproduced from Ren et al., 2005).

The solubility apparatus consisted of a jacketed glass vessel (varying from 60 to 250 mL) maintained at the desired temperature by circulating water that was provided by a constanttemperature bath. The water temperature was controlled by a workstation with a temperature accuracy of (0.1 K) achieved continuous stirring, and a condenser (or a

Experimental and Computational Methods Pertaining to Drug Solubility 195

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

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,

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

There are two methods using UV/Vis-spectroscopy for kinetic solubility determination: Method 1 is based on turbidimetry and the other is based on light absorbance intensity as a

The sample preparation is like nephelometry method, but the analyzing is with a 96-well plate UV/Vis-spectroscopy apparatus. This provides a wider range of wavelength to choose for reading the samples turbidity (190-1000 nm) (Pan et al., 2001). The lower the wavelength, the smaller particle is detected. However, in practice, wavelengths greater than 500 nm is

**96-well plate Turbidimetry** 

**Light source beam** 

**Precipitated particles**

**Transmitted light** 

**Photo detector** 

**Reflected light** 

gives the kinetic solubility (see Figure 3) (Pan et al., 2001; Hoelke et al. 2009).

**H G F E D C B A** 

**2.2.1 Nephelometric method** 

minutes.

make the process reproducible and accurate.

**2.2.2 UV/Vis-spectroscopic method** 

function of concentration (Pan et al., 2001).

Fig. 2. Schematic representation of turbidimetry.

**Dilution**

**2.2.2.1 UV/Vis-spectroscopic method 1** 

**10 mM Stock solution** 

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 Abougela, 1983).

#### **2.2 Determination of kinetic solubility**

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, 2007; Hoelke et al. 2009).

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 which are discussed in the following.
