Solubility filter plate method

Figure 12 Filter plate method for solubility testing. (1) Add compound dissolved in organic solvent to aqueous buffer. (2) Shake for 90 minutes to allow insoluble compound to precipitate. (3) Apply vacuum to filter solution into collection plate. Precipitates remain on membrane. Analyze filtrate in collection plate to quantify the amount of the compound still in the solution. Source Courtesy of Millipore Corporation, Billerica, Massachusetts, U.S.A.

Figure 4.12 Filter plate method for solubility testing.

The alternative method to turbidimetric detection used for measuring solubility in early discovery is to quantify the aqueous supernatant directly via UV absorbance [13, 20, 21]. Typically, DMSO stock solution is added to aqueous buffer such that the final DMSO composition is kept to a minimum (5% or less) and the resulting precipitate is removed by filtration. A UV plate reader is then used to determine the aqueous solubility by comparing the filtrate absorbance against that of a calibration solution prepared in an identical solvent. It is important to match the sample and calibration solutions to prevent solvochromic effects. Care must also be taken in the selection of the filter plate since nonspecific binding of compound can occur with some filter materials leading to erroneously low solubility values [22], Like nephelometry, the plate-based UV detection approach is amenable to automation. 

The supernatant concentration method uses small volumes of stock solution added to wells containing aqueous buffer in a microtiter filter plate of the type available from Millipore Inc. The solution is incubated for a given amount of time (typically in the range of 1-24 h depending on the requirements of the laboratory) and then filtered or centrifuged. The supernatant is analyzed by UV plate reader or HPLC and the concentration of dissolved species is calculated by reference to a calibration curve. This is often either a three- or a four-point curve prepared from serial dilutions of the stock solution using a solvent such as 80% v/v acetonitrile/water in which the compound is fully soluble. 

Avdeef introduced an alternative approach. Aliquots of DMSO stock solutions are pipetted robotically into incubation well plate containing an aqueous buffer. The concentration of the test compound should be between 50-150 iM in order to keep the DMSO content below 0.5 %. After a time of incubation, the plate is filtered and the solved compound is quantified with an UV plate reader. The method is fast, robust and reported to be reliable (Kerns). 200-300 compounds can be measured per day. Additionally, pH solubility profiles can be set up easily (Kibbey). 

In the direct UV method, compounds are dissolved in DMSO stock solution at 10 mg/mL. A small volume is added to an aqueous buffer and mixed. If the target concentration exceeds the solubility of the compound, the insoluble material will precipitate. The solution is allowed to settle for certain period of time (e.g. overnight) and is then filtered to remove the precipitate. The concentration of the supernatant is determined by using a UV plate reader and the solubility is derived against a single point standard (Avdeef, 2001). 

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