TITLE: LogP / LogD shake-flask method
AUTHORS: Yurii Kheylik

[DESCRIPTION]
Lipophilicity is possibly the most important physicochemical parameter for any potential drug candidate. Lipophilicity measurements are valuable for understanding how drugs are dissolved in plasma and other aqueous biological fluids. Lipophilicity is typically accessed as the distribution of the tested compound between two solvents - typically non-aqueous organic (1-octanol) and aqueous (pH-buffered water), and then LogP is expressed as a Log of the concentration ratio between two phases. LogP is widely used in cheminformatics and is a component of Lipinski’s “rule of five”, which is a golden standard to evaluate a drug-likeness of a compound. According to this rule, the successful drug candidate should possess LogP value not greater than 5. 

LogD is a distribution coefficient widely used to measure the lipophilicity of ionizable compounds, where the partition is a function of the pH. 
For nonionizable compounds LogP = LogD throughout pH range, whereas for ionizable compounds LogD takes into account the partition of both ionized and non-ionized forms. LogD is more convenient for practical measurements, as it takes into account solution pH, which is important for the analysis of the drug candidate properties in various biological media with different pH values. The shake flask method is considered the gold standard technique for determining log D.

[GUIDELINES]
Calculations of the distribution coefficients (D) for determine LogD:

Calculations of the distribution ratios were carried out using the equation below. 

 

where: 	
So- peak area of the analyte in octanol phase 
Sp- peak area of the analyte in PBS buffer
do- dilution coefficient for octanol phase 
dp- dilution coefficient for aqueous phase 

LogD - it represents the logarithm (log10) of the distribution coefficient (D) of a molecule.

[STEPS]
SECTION: Preparation of auxiliary solutions
1. 1 litre PBS: 0.01 M Phosphate buffer

In a bottle for reagents with a cap of 1L capacity, place:

contents of 1 L sachet with phosphate buffer,
1 L of water, mix thoroughly, and filter through a membrane filter with a pore diameter of 0.45 μm.

 

SECTION: Preparation of auxiliary solutions
2. 100 ml of A40 solution (extract thinner):

In a bottle for reagents with a lid with a capacity of 100 ml, place:

60 mL of H2O,
40 mL of AcN and mix thoroughly.

 

SECTION: Preparation of auxiliary solutions
3. 100 ml of solution A5 (extract thinner):

In a bottle for reagents with a cap of 100 ml, place:

95 mL H2O,	
5 mLof AcN and mix thoroughly.

 

SECTION: Preparation of auxiliary solutions
4. 1-octanol & PBS mix:

The 1-octanol and pH 7.4, 0.01 Molarity (M), shake phosphate buffer together for 1440 min and allow the phases to separate before use. 

SECTION: Preparation of auxiliary solutions
5. Preparation of 10 mM stock compound:

Prepare 10 millimolar (mM) stock solutions of test substances in DMSO. As a rule, 100 µL of solution is enough.

SECTION: Sample preparation
6. 
 

SECTION: Sample preparation
7. Preparation for mixing 1-octanol, PBS, and 10 millimolar (mM) stock solution.

Below, fig. 1 shows the placement of microtubes on a plate to prepare 5 test and reference compounds.

 

SECTION: Sample preparation
8. The plate is installed in position "G" of the robotic station. Test and reference stock compounds should be placed in special holders, as shown in Fig. 2.

 

SECTION: Sample preparation
9. Plastic vials type "Falcon 15 mL" with PBS, 1-Octanol, and A40, and vials for a drain on the automatic station should be placed as shown in Fig. 3.

 

SECTION: Sample preparation
10. Place the box with tips 1000 µl and 200 µl in the indicated positions as shown in Fig. 4.

 

SECTION: Sample preparation
11. In the Lirix3 program Fig. 5, select the pre-created script for sample preparation. 

The first stage includes preparing 1 mLmix solution in a duplicate of 495:5:500 (v/v/v) PBS pH7.4/10mM stock/1-octanol.

 

SECTION: Sample preparation
12. Close the microtubes with lids using a press, after mixing in the rotator for 120 min at 30 revolutions per minute. 

Centrifuge plate for 3000 rpm, 2 min, and put it in the robotics station in place "G".

SECTION: Sample preparation
13. In position "D" of the robotic station, place a rack with microtubes of 0.7 μl at the rate of 6 microtubes per substance in duplicate. Place the microtubes on the plate as shown in Fig. 6. 


 


SECTION: Sample preparation
14. Install a special guide bar on the rack for holding test tubes and positioning tips. The general layout of the boards at the robotic station is shown in Fig. 7.

 

SECTION: Sample preparation
15. Open bottle drain and A40 (see Fig. 3).

SECTION: Sample preparation
16. In the Liric 3 starts script LogD part 2 (see Fig. 5). After that liquid handler provides the next operations:

SECTION: Sample preparation
16.1. The first step is the dilution of 1-octanol in 100 times. In the microtubes "…-о-100" (see Fig. 5) plate position «D» add 990 µL А40 and 10 mL1-octanol phase in the plate position «G», pipetting 5 times. 

SECTION: Sample preparation
16.2. The second step is to remove the excess 1-octanol phase to expose the aqueous phase.

SECTION: Sample preparation
16.3. The next step is to transfer 300 µL PBS phase plate position «G» into microtubes "…-p-1" plate position «D»

SECTION: Sample preparation
16.4. The final, dilution PBS phase is 10 times. Into microtubes "…-p-10" plate position «D» add 225 µL A40 and 25 µL PBS phase in the microtubes "…-p-1" plate position «G», pipetting 5 times.

SECTION: Sample preparation
17. Analyse both the samples (both phases) using an HPLC system coupled with a tandem mass spectrometer.