The method is called "shake flask method"

1. Take a flask and additions of 10 to 20 ml of solvent

2. Add drug until two phases (liquid: saturated and Solid: Drug undissolved)

3. Carrying ultrasound for 10 minutes (particle size reduction will not increase the solubility but increased of the dissolution rate)

4. Carrying temperature of study - thermodynamic equilibrium (the solubility is constant this is determined by the concentration of the solution steadily until it is a concentration constant -time of equilibrium)-we have shown that a suitable time, so that thermodynamic equilibrium is reached, is three days.

5. Separation of phases: Fitration

6. determination of Concentration (analytical methods-UV, HPLC)

7. Determining of the density of the saturated solution

8. Analysis of the solid phase (determination of possible polymorphisms)

Each data is recommended to be done in triplicate

SHAKE-FLASK METHOD

The shake-fl ask method proposed by Higuchi and Connors is the most reliable and widely used solubility measurement method. This method determines thermodynamic solubility and could be carried out in fi ve steps.

1. Sample preparation: An excess amount of drug is added to the solubility medium. The added amount should be enough to make a saturated solution in equilibrium with the solid phase. In the case of acidic or basic drugs dissolved in an unbuffered solubility medium, further addition of the solid could change the pH of the solution and consequently the solubility of the drug.

2.Equilibration: Depending on the dissolution rate and the type of agitation used, the equilibration time between the dissolved drug and the excess solid could be varied. Equilibration is often achieved within 36 h. To ensure the equilibration condition, the dissolution profi le of the drug should be investigated. The shortest time needed for reaching the plateau of drug concentration against time could be considered a suitable equilibration time. Any signifi cant variation of the dissolution profi le after reaching the equilibration should be inspected, since there are a number of possibilities including degradation of the drug as well as its polymorphic transformation. Both of these affect the solubility values of a drug dissolved in the dissolution media. Vortexing or sonicating the sample prior to equilibration could reduce the equilibration time. To overcome the poor wettability of low soluble drugs, one may use small glass microspheres or sonication.

3. Separation of phases: Two common methods used for phase separations of saturated solutions are fi ltration and centrifugation. Filtration is the easiest method; however, the possible sorption of the solute on the fi lter should

be considered as a source of error in solubility determinations, especially for very low soluble drugs. Prerinsing the fi lter with the saturated solution could reduce the sorption of the solute on the fi lter by saturating the adsorption sites. Centrifugation or ultracentrifugation is preferred in some cases, and the higher viscosity of the saturated solutions, i.e., in mixed solvents, should be considered as a limitation. A combination of fi ltration and centrifugation could also be used

4. Analysis of saturated solution and the excess solid:UV spectrophotometric analysis is the most common and the easiest analytical method in solubility determination experiments. The next is the high-performance liquid chromatography (HPLC) methods in both the isocratic and gradient elution modes. The HPLC analysis could also detect the possible impurities or degradation products if a highly selective method was used. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) of the residual solid separated from the saturated solution confirm the possible solid-phase transformations during equilibration.

5. Data analysis: The collected data could be compared with the previously reported data to ensure the accuracy of the experimental procedure employed. Any mistake in the dilution step, any miscalculations, or using uncalibrated instruments, such as uncalibrated balances, temperature variations, and some other factors, could result in different solubility values for a given drug dissolved in a solvent at a fixed temperature.

You can download and use the OECD guidelines for physic chemical property measurements.

http://www.oecd-ilibrary.org/environment/test-no-107-partition-coefficient-n-octanol-water-shake-flask-method_9789264069626-en

Daniel Ricardo Delgado could you please give me any reference to the procedure you wrote? Thank you very much!

Jelena Vukosavljevic, you could use following one:

T. Huguchi, K. A. Connors, Phase-solubility techniques, Adv. Anal. Chem. Instr. 1965, 4, 117-212.

Currently, developing a new dissolution media for a specific drug product and no reference method is present in any pharmacopoeia. 

Can anyone give more details about the procedure for the quantification of Cs (Concentration of saturation) using Shake--Flask method.  should we do a calibration curve in the different media used or refer to a reference calibration curve in wich we are sure that the API is fully dissolved at very High Cs. Is there any specific formula to use? Is a ratio of area (HPLC) obtained in the reference media (100% organic) and the potential media is enough?

Thanks,,,

Hello Mr. Daniel Ricardo Delgado, can you give a more detailed and elaborated step by step of the procedure you wrote. I mean like an SOP. Currently we are working on a solubility of an API. Thank you very much for your anticipation.

I thank my colleagues Daniel Ricardo Delgado at really very documented and clear answers from which I learned a lot and learned!

My question is how to analyze solutions after solubility studies.  It means we have to make a calibration curve of the compound in the same solvent system in which we have tested its solubility.  Please clarify this point.

@S.M.Farid Hasan

Yes you need to make calibration curves in the same solvent 

Thank you so much Usama for clarifying my question

Making calibration curves in the same solvent can be problematic as the maximum calibrant you could make would be at Cs

You can make a matched matrix by adding some organic solvent to your unknown aqueous samples prior to analysis and making up calibrants in a matched organic/aqueous mixture. This should enable making soluble and stable calibration lines above your measured samples concentration. Then adjust the concentration to account for the dilution of adding the organic.

Usama Ahmad

https://is.muni.cz/th/p9qdf/Appendix_II_Instruction_for_students.pdf

Thanks a lot Ricardo