- Kinetic solubility is assessed by diluting a test compound solution prepared in DMSO into aqueous buffer, SIF, or SGF.¹
- Thermodynamic (or equilibrium) solubility investigates the solubility of a compound as a saturated solution in equilibrium.
- Determining solubility in simulated gastric and intestinal fluids (SGF and SIF, respectively) can give an indication if solubility in the Gl tract is a limiting factor for oral absorption.²
- Since only dissolved drug can permeate the mucosa at the absorptive sites in the GI tract, both the solubility of the drug and its dissolution rate are crucial for its in vivo behavior.
- These data can aid in the classification of substances according to the Biopharmaceutical Classification System (BCS) based upon their solubility and permeability.²,³
Readout: Compound concentration in mg/mL or uM
Controls: Atenolol, Ketoconazole, Tamoxifen
Assay Description – Kinetic Solubility:
A stock solution of compound (50 mM in DMSO) is added to test medium (aqueous buffer, SIF, or SGF). The mixture is shaken at room temperature and then filtered. The liquid is diluted 10x and 30x with DMSO before LC-MS/MS analysis. Standard solutions are prepared as follows: stock solutions are diluted to ten defined concentration points from 60 μM to 0.002 μM with DMSO. Aliquots of samples and standard solutions are filtered and mixed with acetonitrile / H2O, then vortexed and used for LC-MS/MS analysis.
The MS detection is performed by using a SCIEX API 4000 Q trap instrument. Each compound is analyzed by reversed phase HPLC using a Kinetex 2.6u C18 100Å column (3.0 mm X 30 mm, Phenomenex). Mobile phase – solvent A: water with 0.1% formic acid, solvent B: acetonitrile with 0.1% formic acid.
Assay Description – Thermodynamic Solubility:
Test medium (aqueous solvent, SIF, or SGF) is added to solid compound. Excess solid is used and relatively long mixing times are performed to ensure equilibrium is achieved (typically 16-72 h). At the end of the incubation period, the saturated solution is filtered and quantified against a DMSO stock solution.
The MS detection is performed by using a Sciex API 4000 Q trap instrument. Each compound is analyzed by reversed phase HPLC using a Kinetex 2.6u C18 100Å column (3.0 mm X 30 mm, Phenomenex). Mobile phase – solvent A: water with 0.1% formic acid, solvent B: acetonitrile with 0.1% formic acid.
The solubility of the test compound is determined based on the calculated concentration. The final DMSO concentration is 1%, and the maximum test concentration is 0.5 mg / mL.
HPLC High-performance liquid chromatography
LC Liquid chromatography
MS Mass spectrometry
SGF Simulated gastric fluid, pH 1.21
SIF Simulated intestinal fluid, pH 6.81
- United States Pharmacopeia, USP40-NF35 (2017).
- Amidon, G.L.; Lennernäs H.; Shah V.P.; Crison, J.R. “A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability“; Pharm. Res. 12, 413, (1995).
- S. Food and Drug Administration: “The Biopharmaceutics Classification System (BCS) Guidance”; 2016.
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