Determination of the Partition Coefficient n-octanol/water

The partition coefficient (Pow) is defined as the ratio of the equilibrium concentrations of a dissolved substance in a two-phase system consisting of 2 largely immiscible solvents (n-octanol and water). Being Pow the quotient of 2 concentrations, or the quotient of the fractions of the test substance in the 2 phases multiplied by a fixed volume ratio, is dimensionless and is usually given in the form of its logarithm to base ten.
Based on the range of the test substance log Pow, two methods are available to determine the partition coefficient value:
1) OECD TG 107: Determination of the partition coefficient n-octanol/water: Shake flasks method.
2) OECD TG 117: Determination of the partition coefficient n-octanol/water: HPLC method.

OECD TG 107: Determination of the partition coefficient n-octanol/water: Shake flasks method
Test vessels containing the two-phase system are placed in a mechanical shaker. After a set mixing time, phase separation is carried out usually by centrifugation. The test substance concentrations are determined in both phases and the total quantity of test substance present is calculated and compared to the amount initially added.

Method validations, to determine the substance content in water phase and in n-octanol phase respectively, are performed according to SANCO/3030/99 rev. 4 guidance document.
Validation of the analytical methods for each phase includes:
Linearity with at least 5 concentrations of the ananlytical standard (2 injections/concentration) in the appropriate range of concentrations.
Precision with 5 sample solutions (2 injections/sample solution)
Accuracy with al least 3 fortification levels with 3 samples for each fortification level (2 injections/sample solution) and 1 blank sample for the interference check and specificity.
Confirmatory analyses/specificity by high specific technique or other proper confirmatory technique.
LOQ and LOD determinations.
The test for the determination of Pow is carried out in temperature range (25±1)°C. 2 sample solutions for each volume ratio of the 2 phases are performed except for the 5 sample solutions related to the precision check.
For ionizable substances, the test will be planned to take into account the effects of pH.

: Determination of the partition coefficient n-octanol/water: HPLC method

Reverse phase HPLC is performed on analytical columns packed with a commercially available solid phase containing long hydrocarbon chains (e.g. C8, C18) chemically bound onto silica. A chemical substance injected on such a column is distributed between the mobile solvent phase and the hydrocarbon stationary phase as it is transported along the column by the mobile phase. The chemicals are retained in relation to their hydrocarbon-water partition coefficient, with hydrophilic chemicals eluted first and lipophilic chemicals last. The capacity factor k is described by the retention time tR and the dead time t0 as (tR-t0)/t0. Pow of a test substance can be calculated by an equation containing the capacity factor experimentally determined.

For the setting of the regression equation, at least 7 reference substances with known values of Pow are used including 1 substance for the determination of dead time (t0). The reference substances soultions are individually injected before and after sample solutions injections. The determination of the retention times and the plotting of the corresponding log k as a function of log Pow allow to set the regression equation. After the determination of log k values (2 injections/sample solution), the corresponding Pow is calculated by the regression equation.
For repeatability check log Pow value, derived from repeated measurements made under identical conditions and using the same set of reference substances, should fall within a range of + 0.1 log units. For reproducibility check, if the measurements are repeated with a different set of reference substances, results may differ. Typically, the correlation coefficient R for the relationship between log k and log Pow for a set of test substances is around 0.9, corresponding to an octanol/water partition coefficient of log Pow + 0.5 log units.
Both studies include GLP managment and reporting.

OECD Guideline for Testing of Chemicals, No. 107 (27th July 1995) - Partition Coefficient (n-octanol/water): Shake Flask Method.
OECD Guideline for Testing of Chemicals, No. 117 (13th April 2004) - Estimation of the soil adsorption coefficient (Koc) using High Performance Liquid Chromatography (HPLC).
SANCO/3030/99 rev. 4 (11/07/2000) - Technical Material and Preparations: Guidance for generating and reporting methods of analysis in support of pre- and post-registration data requirements for Annex II (part A, Section 4) and Annex III (part A, Section 5) of Directive 91/414.



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