Claims
- 1. A method for determining the solubility of a compound, said compound having limited solubility in water and in a buffer, comprising:
a. preparing a saturated solution of said compound, after which any insoluble compound is filtered off, b. preparing a reference solution of said compound, under conditions avoiding or suppressing precipitation, c. measuring a spectrophotometric property of said saturated and reference solutions and said buffer, wherein said buffer serves as a blank, and d. analyzing said spectrophotometric property to determine the solubility of said compound.
- 2. The method of claim 1, wherein said spectrophotometric property is selected from the group consisting of UV range spectrophotometry, visible range spectrophotometry, colorimetry, light scattering detection, polarimetry, optical rotation, fluorimetry and circular dichroism detection.
- 3. The method of claim 1 wherein:
a. said reference solution is prepared by dissolving a known quantity of said compound or stock solution containing said compound in buffer to produce a solution free of precipitate, and b. said saturated solution is prepared by dissolving a known quantity of said compound or stock solution containing said compound in buffer to produce a solution with precipitate dispersed therein.
- 4. The method of claim 1 wherein said compound is presented as a stock solution in DMSO and said spectrophotometric property is UV range absorbance.
- 5. The method of claim 4 wherein:
a. said reference solution is prepared by diluting said stock solution with DMSO and buffer, and b. said saturated solution is prepared by diluting said stock solution with buffer, after which any insoluble compound is filtered off.
- 6. The method of claim 5, wherein:
a. said reference solution is prepared by diluting one aliquot of said stock solution with 19 aliquots of DMSO; and one aliquot of said diluted solution is further diluted with 3 aliquots of DMSO and 400 aliquots of buffer, b. said saturated solution is prepared by diluting 1 aliquot of said stock solution with 400 aliquots of buffer, allowing said solution to equilibrate for several hours, after which any insoluble compound is filtered off, and c. said analysis requires that (1) the absorbance of said reference solution be compared to (2) the absorbance of said saturated solution at one or more wavelengths, wherein, all of said solutions are adjusted to the same pH.
- 7. The method of claim 4, wherein
a. said blank is prepared by diluting an aqueous buffer with cosolvent, b. said reference solution is prepared by diluting stock solution with DMSO, buffer, and cosolvent, c. said saturated solution is prepared by diluting stock solution with buffer, filtering off any insoluble compound, and further diluting said solution with cosolvent.
- 8. The method of claim 7 wherein said cosolvent is selected from the group consisting of acetonitrile, methanol, ethanol, iso-propanol, 1-propanol, 1,4-dioxane, dimethylformamide, acetone, ethylene glycol, propylene glycol, polyethylene glycol 400, tetrahydrofuran, DMSO and mixtures thereof.
- 9. The method of claim 8, wherein said cosolvent is 1-propanol.
- 10. The method of claim 9, wherein:
a. said blank is prepared by diluting 1 aliquot of said buffer with 1 aliquot of a cosolvent, b. said reference solution is prepared by diluting 1 aliquot of stock solution with 19 aliquots of DMSO; and 1 aliquot of said diluted solution is further diluted by 15 aliquots of said buffer and 15 aliquots of cosolvent; c. said saturated solution is prepared by diluting 1 aliquot of stock solution with 400 aliquots of buffer, allowing said solution to equilibrate for several hours, after which any insoluble compound is filtered off; and 1 aliquot of said resulting solution is further diluted by 1 aliquot of cosolvent; d. said analysis requires that (1) the absorbance of said reference solution be compared to (2) the absorbance of said saturated solution at one or more wavelengths, wherein, all of said solutions are adjusted to the same pH.
- 11. The method of claim 1, wherein
a. said solubility is intrinsic solubility, b. said compound is ionizable and anomalies are present in solution, and wherein said analysis requires that the log of solubility of said saturated solution be plotted against pH to produce a solubility-pH curve, c. the displacement of the pKa caused by said anomaly can be determined by independently determining the pKa of said compound, and d. said displacement of pKa can be used to removing the effect of the anomaly and determine intrinsic solubility of said compound.
- 12. The method of claim 11, wherein said anomaly is caused by one or more factors selected from the group consisting of:
a. self-association effects, b. presence of bile acids or other surfactants, c. presence of cyclodextrins, d. presence of ion-pair forming counterions, e. presence of non-ionizable polymers, f. presence of phospholipids, and g. presence of DMSO.
- 13. The method of claim 12, wherein said anomaly is caused by the presence of DMSO, wherein:
a. said pKa of said compound in buffer free of anomaly can be determined by using known analytical techniques, b. the pKa of said compound in said solution having said anomaly is obtained by determining the pH of the intersection of horizontal and diagonal asymptotes of said solubility-pH curve, c. the difference between (1) the pKa of said compound in buffer free of anomaly and (2) the pKa of said compound in said solution is determined, and d. said difference is added to the log of the apparent intrinsic solubility to determine the log of the intrinsic solubility of said compound free of anomaly, provided that, (1) for ionizable acids, pKa of said compound in said solution minus the pKa of said compound in buffer free of anomaly is greater than zero, or (2) for ionizable bases, pKa of said compound in said solution minus the pKa of said compound in buffer free of anomaly is less than zero,
- 14. The method of claim 1 wherein said spectrophotometric property is determined at more than one point, and the values at said points are corrected to account for the presence of interferences, said corrections being based on
a. comparing the shape of the spectrophotometric property curve of said reference solution to the shape of a curve of said compound in saturated solution, and b. applying weighting factors, wherein those portions of the curve of said reference solution that differ from the corresponding curve prepared in saturated solution contribute less to said method than those where said shape is similar to said curve relating to said saturated solution.
- 15. The method of claim 14 wherein said spectrophotometric property is UV absorbance.
- 16. The method of claim 15 applied to determination of concentrations of sample solutions when comparing UV absorbance spectra of sample solutions to UV absorbance spectra of reference solutions.
- 17. The method of claim 1, wherein said buffer provides buffering capacity over the range of pH 3-10 and has low-UV absorbance.
- 18. The method of claim 17 wherein said buffer comprises acetic acid, MES, HEPES and boric acid.
- 19. The method of claim 17, wherein said buffer comprises glycolic acid, MES, HEPES, taurine, and acetonitrile.
- 20. An instrument for determining the solubility of a compound that is sparingly soluble in water and in a buffer, said instrument comprising means for:
a. accurately titrating components of a solution, b. collecting components of a solution, c. mixing solutions, d. filtering insoluble residues from solutions, e. adjusting pH values of solutions, f. determining a spectrophotometric property of a solution, and g. performing calculations, including those relating to said UV absorbance spectra.
- 21. The instrument of claim 20 for determining the solubility of a compound that is sparingly soluble in water and in a buffer, wherein said means for determining a spectrophotometric property of a solution is selected from the group consisting of a UV range spectrophotometer, a visible range spectrophotometer, a calorimeter, a light scattering detector, a polarimeter, an optical rotation, a fluorimeter, or a circular dichroism detector.
- 22. A buffer providing buffering capacity over the range of pH 3-10 and having low-UV absorbance.
- 23. The buffer of claim 22 comprising sodium acetate, MES monohydrate, HEPES and boric acid.
- 24. The buffer of claim 22 comprising glycolic acid, MES monohydrate, HEPES, taurine, and acetonitrile.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 60/178,616 filed Jan. 28, 2000, which is incorporated in its entirety herein.
Provisional Applications (1)
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Number |
Date |
Country |
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60178616 |
Jan 2000 |
US |