Lipophilicity and pKa Assays

Creative Bioarray provides Lipophilicity and pKa Assays to help customers accurately evaluate the drug lipophilicity and pKa, allowing customers to gain more insight into the solubility and permeability of drugs.

Lipophilicity and pKa Assays

Why is it necessary to determine drug lipophilicity and pKa?

  • Lipophilicity and pKa are essential factors in permeability and solubility, and they are frequently tested during drug development.  
  • The assessment of lipophilicity is fundamental to the understanding of molecular properties. In a drug development context, it is also vital to consider about ionization of molecules in the aqueous phase, especially at pH 7.4. Therefore, the distribution coefficient (Log D) is the preferred descriptor of lipophilicity.
  • The distribution of a molecule between a lipid or nonpolar environment and an aqueous or polar environment is affected by lipophilicity. The equilibrium distribution of a chemical between water and octanol is commonly quantified as Log P. Lower aqueous solubility and higher membrane permeability is associated with compounds with a higher Log P.
  • pKa influences the ionization of molecules in an aqueous solution; it is measured by the change of ionization with pH. Basic compounds with pKa of 9 have an even distribution of protonated and neutral molecules at pH9. The lower the pH, the higher the protonation and the more neutral species. Acidic compounds with pKa of 4 have an equal distribution of deprotonated and neutral molecules at pH 4, with more neutral species at low pH and more deprotonated species at high pH.

Brief Protocol

Thermodynamic and kinetic solubility assays are two commonly used laboratory methods to determine drug solubility.

  • Log D7.4, Log P (Shake-flask Method)
    • The test substance is sonicated with octanol (pre-saturated with buffer). After that, a pH 7.4 buffer (pre-saturated with octanol) is added to the octanol.
    • A buffer mixed with octanol is prepared. The system is mixed to ensure that the chemical is distributed evenly throughout the two phases.
    • After separation, the chemical is measured in the aqueous and octanol phases using LC-MS/MS, and the Log D7.4/Log P is calculated using the equation below.

Lipophilicity and pKa Assays

  • pKa

pH-Metric Titration: The pH-metric method is a critical reference method because it can be used to measure all pKas between 2 and 12, with or without a UV chromophore, provided that the sample can be dissolved in water or water/co-solvent over the pH range of interest. While the pH-metric method is not fast enough for rapid screening, it provides a valuable backup for measuring samples without UV absorbance.

Hybrid pH-Metric/UV Method: Multi-wavelength UV absorbance of the sample solution is monitored throughout the titration. Samples must have a chromophore, and the absorbance must change as a function of ionization. During measurement, samples are acid-base titrated across a pH range that includes the pKa(s), and multi-wavelength UV spectra are measured at each pH. The pKas are calculated using a technique based on target factor analysis (TFA)

Quotation and Ordering

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  1. Van de Waterbeemd, Han, H. Lennernäs, and P. Artursson. "Drug bioavailability." Methods and principles in medicinal chemistry 18 (2004).
  2. Meanwell, Nicholas A., ed. Tactics in contemporary drug design. Berlin: Springer, 2015.

For research use only. Not for any other purpose.