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LogP and LogD Measurements

 

 

Readout: logD(pH)

Controls: Testosterone; others available on request

 

Assay Description:

1 mL of 1-octanol and 1 mL of buffer are added to a glass vial. Compound stock solution (10 mM in DMSO) is added and the vial is rotated for one hour using a shaker at room temperature. The layers are allowed to separate and aliquots of each layer are taken.

Aliquots of the 1-octanol and aqueous solutions are serially diluted using DMSO to give the final samples for LC-MS/MS analysis. Three sequential dilutions are made of each 1-octanol phase, covering a 2500-fold range in concentration. Two sequential dilutions are made of each final aqueous phase, covering a 100-fold concentration range. The MS peak areas from these solutions are used to generate a log(peak area) against log(relative concentration) calibration line. LogD is calculated from the ratio of the 1-octanol relative concentrations to the interpolated aqueous relative concentrations.

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 100 Å 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.

 

Data Analysis:

LogD is calculated using the following formula:

logD = log {(compound concentration in octanol) / (compound concentration in aqueous phase)}

 

Abbreviations:

DMSO                     Dimethylsulfoxide

HPLC                       High-performance liquid chromatography

LC                             Liquid chromatography

MS                           Mass spectrometry

 

Literature:

  1. Lipinski C. A.; “Drug-like properties and the causes of poor solubility and poor permeability”; J. Pharmacol. Toxicol. Methods. 44, 235, (2000).
  2. Leeson, P. D.; Springthorpe, B.; “The influence of drug-like concepts on decision-making in medicinal chemistry“, Nature Rev. Drug Discov.6, 881, (2007).
  3. Edwards, M. P.; Price, D.A.; “Role of Physicochemical Properties and Ligand Lipophilicity Efficiency in Addressing Drug Safety Risks“, Ann. Rep. Med. Chem. 45, 381, (2010).
  4. Shultz, M. D.; “Setting expectations in molecular optimizations: Strengths and limitations of commonly used composite parameters”, Bioorg. Med. Chem. Lett. 23, 5980, (2013).