Experimental Methods

Lipinski et al. [12] and Pan et al. [463] compared several commonly used methods of solubility measurement in early discovery, where samples are often introduced as 10 mM DMSO solutions. Turbidity-based and UV plate scanner-based detections systems were found to be useful. The methods most often used in discovery and in preformulation will be briefly summarized below.

6.4.1 Saturation Shake-Flask Methods

Solubility measurement at a single pH [37-39] under equilibrium conditions is largely a labor-intensive procedure, requiring long equilibration times (12h-7 days). It's a simple procedure. The drug is added to a standard buffer solution (in a flask) until saturation occurs, indicated by undissolved excess drug. The thermostated saturated solution is shaken as equilibration between the two phases is established. After microfiltration or centrifugation, the concentration of the substance in the supernatant solution is then determined using HPLC, usually with UV detection. If a solubility-pH profile is required, then the measurement needs to be performed in parallel in several different pH buffers.

6.4.2 Turbidimetric Ranking Assays

Turbidity-detection-based methods [12,459-463], popularized by Lipinski and others, in part have met some high-throughput needs of drug discovery research. The approach, although not thermodynamically rigorous, is an attempt to rank molecules according to expected solubilities. Usually, the measurements are done at one pH. Various implementations of the basic method are practiced at several pharmaceutical companies, using custom-built equipment. Detection systems based on 96-well microtiter plate nephelometers are well established. An automated solubility analyzer incorporating such a detector usually requires the user to develop an appropriate chemistry procedure and to integrate a robotic fluidic system in a customized way. It is important that turbidity methods using an analate addition strategy be designed to keep the DMSO concentration in the buffer solution constant in the course of the additions. The shortcomings of the turbidity methodology are (1) poor reproducibility for very sparingly water-soluble compounds, (2) use of excessive amounts (<5% v/v) of DMSO in the analate addition step, and (3) lack of standardization of practice.

6.4.3 HPLC-Based Assays

In an effort to increase throughput, several pharmaceutical companies have transferred the classical saturation shake-flask method to 96-well plate technology using a robotic liquid dispensing system [463]. Analyses are performed with fast generic gradient reverse-phase HPLC. In some companies, the DMSO is eliminated by a freeze-drying procedure before aqueous buffers are added. This adds to the assay time and can be problematic with volatile samples (e.g., coumarin). Still, the serial chromatographic detection systems are inherently slow. Data handling and report generation are often the rate-limiting steps in the operations.

6.4.4 Potentiometric Methods

Potentiometric methods for solubility measurement have been reported in the literature [467-471]. A novel approach, called dissolution template titration (DTT), has been introduced [472-474]. One publication called it the ''gold standard'' [509].

log S vs.log P for lonizable Compounds

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