Plate method solubility testing

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 advantages of the capillary assay are its simplicity, quantitative nature, and high sensitivity. Alternative methods for studying chemotaxis such as the swarm plate method of Adler (1966) require that the chemoattractant be metabolized. This is not necessary in the standard capillary assay. In addition, due to the small size of the chemotaxis chamber, only small amounts of compound are required to perform the experiments. The main disadvantage of this method is that the compound tested must be soluble in the chemotaxis medium. 

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). 

This method is used to test the effect of such compounds as soluble CD4 that bind to gpl20 expressed on the surface of infected cells. Similarly, this system could be used to investigate the antibodies or small molecules that may bind to the family of coreceptors (CXR4 and CCR5, and so forth) required for the entry of HIV into cells. Compounds that selectively kill HIV-infected cells may also be investigated with this technique (6). If CPE or cell viability (vital staining) is used as an end-point then plates should be treated as described in Note 6. 

It has recently been demonstrated, that RP-HPLC coupled with a 96-well plate auto-injector can be utilized for the simultaneous determination of log D, log P and pKa of drug molecules. This method is associated with the following advantages low sample requirement, accommodation of low solubility compounds, less restriction on compound purity, higher throughput, precise data and multiple results in one assay. However, the range in log Poet of the test compounds in the dataset was limited from —1.7 to 4.4. 

ASTM F 1769-97, Standard Test Method for Measurement qfDiffusivity, Solubility, and Permeability of Organic Vapor Barriers Using a Flame Ionization Detector (Philadelphia, 1997) ASTM C177-93, Steady-State Thermal Transmission Properties by Means of the Guarded Hot Plate (Philadelphia, 1993)... 

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