using as little sample as necessary to cause precipitation near the mid-point. For compounds extremely insoluble in water, cosolvents such as methanol, ethanol, DMSO, or acetonitrile may be used, with the solubility constant determined by extrapolation to zero cosolvent [43].

Usually, the solubility of the salt is determined from separate, more concentrated solutions. To conserve on sample, the titration of the salt may be performed with an excess of the counterion concentration [479]. Also, some amount of sample salt may be conserved by titrating in cosolvent mixtures, where salts are often less soluble.

The graphically deduced constants are subsequently refined by a weighted nonlinear least squares procedure [472]. Although the potentiometric method can be used in discovery settings to calibrate high-throughput solubility methods and computational procedures, it is too slow for HTS applications. It is more at home in a preformulation lab.

6.4.5 Fast UV Plate Spectrophotometer Method

A high-throughput method using a 96-well microtiter plate format and plate UV spectrophotometry has been described [26]. Solubilities at a single pH, or at <12 pH values can be determined, using one of two methods. Aqueous Dilution Method

A known quantity of sample is added to a known volume of a universal buffer solution of sufficient capacity and of known pH. The amount of sample must be sufficient to cause precipitation to occur in the formed saturated solution. After waiting for a period of time to allow the saturated solution to reach the desired steady state, the solution is filtered to remove the solid and obtain a clear solution, whose spectrum is then taken by the UV spectrophotometer. Mathematical treatment of the spectral data yields the area-under-the-curve of the filtered sample solution, AUCs.

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, AUCR. The ratio R = AUCR/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

TABLE 6.1 Intrinsic Solubility S0, Corrected for the Drug DMSO/Drug Aggregation Effects

SAPP Corrected pSOL Shake-Flask Compound pKa (mg/mL) S0 (mg/mL) S0 (mg/mL) S0 (mg/mL)



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