Shake-flask method solubility

The shake-flask method has some drawbacks, however. One problem that occurs occasionally is the formation of micro-emulsions which remain stable for hours or days, and prevent the two solvent layers from separating. In this case it is not possible to measure the concentration of analyte in each phase. More generally, the upper and lower range of log D values is limited by problems of solution handling. For very high log D values the sample is much more soluble in octanol than in water. For example, if log D is 4 the sample is 10,000 times more soluble in octa-... 

Aqueous solubility values for the samples analyzed compared favorably with results obtained by traditional methods. The solubility values for amiodarone HC1, reserpine, and benzanthrone were lower than the LOQ of the, uPLC system used for the evaluation. Results of the evaluation of compound solubility employing no-filtration /iPLC were compared with those obtained by two traditional methods (1) multiscreen filtration followed by a UV plate reader, and (2) the shake flask method followed by a UV plate reader. As shown in Figure 6.31, the solubility values determined by the different methods are comparable for most compounds examined. Figure 6.32 shows the results of evaluations of aqueous solubility at four different pH levels for phenazopyridine and piroxicam samples. 

FIGURE 6.31 Comparison of solubility results obtained by no-filtration method followed by PLC detection and those obtained by two traditional methods multiscreen filtration followed by UV plate reader, and shake flask method followed by UV plate reader. (Data provided by Steven Hobbs, Courtney Coyne, and Gregory Kazan.)... 

Groves, F., Jr. Solubility of cycloparaffins in distilled water and saltwater, J. Chem. Eng. Data, 33 (2) 136-138, 1988. Grunewald, G.L., Pleiss, M.A., Gatchell, C.L., Pazhenchevsky, R., and Rafferty, M.F. Gas chromatographic quantitation of underivatized amines in the determination of their octanol-0.1 Msodium hydroxide partition coefficients by the shake-flask method, J. Chromatogr. A, 292(2) 319-331, 1984. 

Zhou, L., Yang, L., Tilton, S. and Wang, J. (2007) Development of a high throughput equilibrium solubility assay using miniaturized shake-flask method in early drug discovery. Journal of Pharmaceutical Sciences, 96, 3052-3071. 

M Company (2001b) Environmental Laboratory Project Number EOO-17 16. Solubility in Octanol—Shake Flask Method. 3M Company, St Paul, MN. US EPA AR 226-0052... 

The basic principle of the method is to measure the dissolution of the substance in two different phases, water and -octanol. In order to determine the partition coefficient, equilibrium between all interacting components of the system must be achieved after which the concentration of the substances dissolved in the two phases is determined. The shake-flask method is applicable when the log Kow value falls within the range from -2 to 4 (OECD 107, 1995). The shake-flask method applies only to essential pure substances soluble in water and -octanol and should be performed at a constant temperature ( 1°C) in the range 20-25 °C. 

The traditional way to measure aqueous solubility is by the shake-flask method, in which the compound is added in solvent and shaken until equilibrium is reached. This is, however, a time-consuming method. Another frequently used technique, the turbidimetric method, is based on aqueous titration of a DMSO solution of the compound under investigation. The solubility corresponds to the concentration at which precipitation occur. However, this method neglects the effect and particular kinetics of the solid state and... 

Figure2.3 The solubility ratios ofa set of43 in-house compounds measured in phosphate buffer at pH 7.4 with and without 1% DMSO by the classic shake-flask method from solid. Compounds span a solubility range from 0.2pM to 5mM.

Classically, thermodynamic solubility is measured using the shake-flask method [21, 30]. This method involves addition of an excess of solid to aqueous buffer at fixed pH. The solution is stirred for a minimum of 24h prior to separation of the supernatant from undissolved material via centrifugation or filtration followed by... 

Many approaches to determine thermodynamic solubility in drug discovery have focused on miniaturizing the classic shake-flask method with solid samples manually weighed and dispensed into 96-well plates or 96-vial arrays [32-34]. The 96-well format enables the use of liquid handling robots to improve throughput. As with the standard shake-flask method, aqueous buffer is added and the solution is agitated for a minimum of 24 h prior to plate filtration or centrifugation to remove the supernatant, which is then analyzed by HPLC-UV or direct UV. 

Table 2.1 The aqueous solubility of 16 drug molecules at pH 7.4 measured from crystalline and amorphous solid materials using the shake-flask method (24 h agitation).

According to HHS-FDA guidances, the determination of the equilibrium solubility should be carried out with the shake-flask method (other methods such as acid or base titration are permitted when their ability to predict the equilibrium solubility is justified). The experiments should be carried out at a temperature of 37 1°C. Further, a sufficient number of pH conditions should be chosen to cover the pH range of 1-7.5 and each determination should be carried out at least in triplicate. The buffer solutions given in the United States Pharmacopeia (USP) are appropriate for the tests, but other buffers are also allowed for these experiments. The pH value of each buffer solution should be checked before and after each experiment. Degradation of the API due to pH or buffer composition should be reported together with other stability data. 

The equilibrium shake flask method is the gold standard for thermodynamic solubility determination (Glomme et al., 2005). The assay is performed by adding a solvent to solid material, and mixing for 24-48 hrs. The solution is filtered and the supernatant is diluted and assayed using LC-UV-MS. This method is low throughput compared to the needs of HTS and early discovery. 

The shake-flask method is based on the phase solubility technique that was developed 40 years ago and is still the most reliable and widely used method for solubility measurement today (Higuchi and Connors, 1965). The method can be divided into hve steps sample preparation, equilibration, separation of phases, analysis of the saturated solution and residual solid, and data analysis and interpretation (Yalkowsky and Banerjee, 1992, Winnike, 2005). 

The effective delivery of a drug to the biological site of action (e.g., oral or intravenous bioavailability) is influenced by the solubility of the drug. For example, a drug must be soluble for intestinal absorption to occur. The solubility of drug candidates can be determined by traditional shake-flask method [76,77], HPLC/UV-absorbance analysis [78], or with LC-MS-based methods [75],... 

Accurate values of Pow are clearly necessary for the ultimate calibration of all surrogate systems, but, in practice, direct measurements of Powby the traditional shake-flask method are seldom used. Particularly for compounds with low water solubility, experimental difficulties may arise from problems in phase separation without carryover, sorption to glass surfaces, or from formation of emulsions. All of these introduce serious uncertainties into the concentrations in the appropriate phases, and may consequently lead to substantial errors in the estimates of partition coefficients. The problem is particularly acute for compounds with extremely low solubility in water such as the chlorinated dibenzo[l,4]dioxins for which widely varying values have been reported (Marple et al. 1986 Shiu et al. 1988). For such compounds, use of a generator column has been advocated (De Voe et al. 1981 Woodburn et al. 1984). In essence, the following steps are carried out (1) a solution of the test substance in octanol is equilibrated with water and the concentration in the octanol phase is determined, (2) the octanol phase is... 

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