Solubility, determination

Solubility curves have traditionally been determined by either crystallization of a supersaturated solution or by dissolution of crystals in an undersaturated solution. Suitable solution conditions, which produce crystals, must be known in advance for both methods. Protein solubility can be determined as a function of many parameters including temperature, salt concentration, salt type, buffer and pH. For the crystallization method, a grid is made of samples of two or three different initial protein concentrations and at least four different values of a given variable (e.g., three protein concentrations at four different temperatures = 12 samples). Aliquots are removed periodically for concentration determination for up to 6-12 weeks after crystals appear. In the case of dissolution experiments, a batch of crystals previously grown in the appropriate buffer is needed. Crystals are placed in undersaturated solutions and allowed to dissolve. Again, for six to 

---

All solubility determinations for Group tests are carried out at the laboratory temperature in small test-tubes (e.g., 100 X 12 mm.) but of suflScient size to permit of vigorous shaking of the solvent and the solute. 

Practice in solubility determinations. It is suggested that the student should carry out solubility determinations with a selection from the following compounds ... 

Bowman BT, Sans WW. 1983. Further water solubility determination of insecticidal compounds. J Environ Sci Health B 18 221-227. 

Avdeef, A., Berger, C. M. pH-metric solubility. 3. Dissolution titration template method for solubility determination. Eur. J. Pharm. Sci. 2001, 14, 281-291. 

Ofstad EB, Sletten T. 1985. Composition and water solubility determination of a commercial tricresylphosphate. Science of the Total Environment 43 233-241. 

Fig. 5 Dissolution profiles obtained from the solubility determination of two polymorphic forms of the same drug substance. A is the stable form with solubility 31 mg/mL. B is the profile of the metastable form with solubility 46 mg/mL. This solubility (circles) is not achieved in many instances, and precipitation of the stable form occurs at a point beyond the solubility of A, and the trace becomes B. C is the hypothetical profile of the metastable form. 

A reference solution is prepared by a dilution method. A known quantity of sample is dissolved in a known volume of the system buffer of known pH the amount of sample is X times less than in the above case in order to avoid precipitation in the formed solution. The spectrum is immediately taken by the UV spectrophotometer, to take advantage of the possibility that solution may be supersaturated (i.e., solid should have precipitated, but because not enough time was allowed for the solid to precipitate, the solution was temporarily clear and free of solid). Mathematical treatment of the spectral data yields the AUC of the reference sample solution, AUQ . The ratio R = AUCS/AUCS is used to automatically recognize the right conditions for solubility determination when the reference has no precipitate, and the sample solution is saturated with precipitate. Under these conditions, solubility is determined from the expression... 

Results of Aqueous Solubility Determined from A Shifts... 

TABLE 6.2 True Aqueous Solubility Determined from pKa Shifts of Monoprotic Compounds... 

Figure 11 Dissolution of two particle size distribution fractions of a water-insoluble drug (solubility in water at 37°C < 10 g/mL). Equation (41) was used to calculate the values shown by the dashed line, which represents the estimated dissolution of the drug based on the particle size distribution and solubility determined experimentally. 

Conditions used for solubility determinations Pressure PSI Temp. °C... 

The solubility profile for acetazolamide, after 24, 48 and 72 hours from dissolution in water at 25°C is shown in Fig. 17.4. Equilibrium is reached only after 72 hours from dissolution, and any solubility measurements made before that time may lead to major errors in solubility determination. Hence, it is unclear whether all the solubility data reported in the literature has been obtained with careful checking of such phenomena. 

Figure 6.24 compares the amounts of solvent required to analyze 24 compounds for solubility determination employing a four-point calibration and duplicate analysis when traditional HPLC and... 

FIGURE 6.24 Amount of solvent needed for solubility determinations employing HPLC and /iPLC (24 compounds, 4-point calibration curve, duplicate analysis). 

FIGURE 6.25 Comparison of techniques employed for solubility determination. 

Other methods employ a microplate format followed by fast HPLC. Some researchers approach the determination from a different perspective. For example, an alternative method for ionizable substances is the pSol determination based on an acid-base titration.25 26 Kinetic solubility determinations involve determining the concentration of the compound in the buffer of interest when an induced precipitate first appears. 

Bhattachar et al.45 used CLND for solubility determinations and compared results to those obtained from UV spectrophotometry and HPLC. CLND has a throughput of 96 compounds per day with a reduced compound consumption of approximately 3 mg. The sensitivity of the instrument is approximately 6.25 pg/mL for a compound with a molecular weight of 350 and 4 nitrogens per molecule. 

Bhattachar, S.N., Wesley, J.A., and Seadeek, C. 2006. Evaluation of the chemiluminescent nitrogen detector for solubility determinations to support drug discovery. J. Pharm. Biomed. Anal. 41 152. 

Though some workers ensure scrupulously anhydrous conditions in their solubility determinations [e.g., Saad and Soliman <221)], others may have been less careful. 

The solubility should be measured at all of these pH values with a suitable, validated method such as shake-flask or pSol (2) at 37°C to determine whether the (envisaged) dose of the drug can be completely dissolved at all points of interest in the GI tract (see Chapter 11 for more discussion of solubility determination). Typically, this would be the upper GI pH (stomach and proximal small intestine) for immediate release (IR) products, the pH in the small intestine for enteric-coated products and, additionally for MR dosage forms intended to release over a period of six hours or more, the pH in the proximal colon. 

A review of the commonly used experimental methods for solubility determinations is presented in Table 1. Briefly, batch equilibration is the conventional method of preparing saturated solutions for solubility determinations, where an excess amount of solute chemical is added to water and equilibrium is achieved... 

Lipid solubility determines the readiness of drug molecules to cross the gastrointestinal tract, blood-brain barrier, and other tissues. Molecular size is another factor that determines the diffusion of drugs across the membrane, with the smaller molecules able to diffuse more readily. Exhibit 5.2 describes the kinetics for diffusion of drug molecules across the cell membrane. 

---