Lipophilicity is a crucial physicochemical property in medicinal chemistry which affects drug transporting through lipid membranes as well as drug interacting with the target proteins. Creative Biolabs provides a number of experimental protocols to determine lipophilicity for drugs.
Lipophilicity refers to the ability of a chemical compound dissolving in fats, oils, lipids, and non-polar solvents. On the molecular level, drug lipophilicity affects drug transport through lipid cell membranes as well as drug's interactions with target protein(s). On the organism level, lipophilicity influences several ADME properties, like absorption, distribution into tissues, binding efficiency of a drug, etc.
Lipophilicity is measured by distribution behavior in a biphasic system. The most commonly used measurement parameter of lipophilicity is logD (distribution coefficient, partitioning of neutral and ionized species) or logP (partition coefficient, partitioning of unionized species). It is usually measured by “shake-flask” method in a lipid-lipid system. We also provide other lipophilicity determination methods, including potentiometric titration, reversed-phase high-performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC).
Measurement of LogD by Shake-Flask Method
Shake-flask procedure is a simple extraction in octanol/water system under shaking. After equilibrium between all interacting components is attained, samples are collected from both phases, and concentrations in each phase are quantified with LC-MS/MS. The partition coefficient depends on the relative solubility of a substrate in a polar and non-polar solvent.
Figure 1 Measurement of LogD
Measurement of pKa by the Potentiometric Titration Method
The acid dissociation coefficient (pKa) is the pH at which the molecule is 50% ionized. It predicts the degree of ionization the molecule will have at a particular pH. Ionization of a compound alters its physical behavior and influences its properties such as solubility and lipophilicity. Creative Biolabs provides two methods for evaluating pKa: UV-metric method and pH-metric method. In the UV-metric method, pKa is measured by analyzing changes in UV spectra during acid-base titration. This method is often used for measuring compounds with pH-sensitive chromophores. In the pH-metric method, pKa is measured based on potentiometric acid-base titration. Results are obtained by a calculation program. The ph-metric method is becoming more widespread for determining logP between liposomal membrane and water.
Lipophilicity Determination by Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)
The RP-HPLC is a very popular surrogate to the traditional shake-flask method due to its high throughput, a small amount of required solutes, and a wide applicable range. Components in the different solvent are separated depending on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the stationary phase. We use the chemically bonded hydrocarbon-silica as stationary phase, and the tested compound is in the mobile phase. Silica is a non-polar anisotropic stationary phase, and it is more similar to partitioning into phospholipid bilayers than isotropic property pertained to bulk octanol phase. This method enables to estimate logP values more accurately.
Lipophilicity Determination by Thin Layer Chromatography (TLC)
Thin layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of glass, which is coated with a thin layer C-18 silanized silica gel. The mobile phase is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.
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